THE TOXIC EFFECT OF SIX COMMONLY OCCURRING
PLANTS IN .NIGERIAN PASTURES
BY
MATTHEW OLUWOLE ABATAN
D.V.M., M.Sc. Ibadan
A THESIS IN THE
DEPARTMENT OF VETERINARY PHYSIOLOGY AND PHARMACOLOGY
.
SUBMITTED
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF THE
DOCTOR OF PHILOSOPHY OF THE
UNIVERSITY OF IBADAN
SEPTEMBER, 1992.
nSADA
ce.
NO. 93-09441 \
LOC.
MARK.
2
ABSTRACT
The toxicology of six suspected poisonous plants Leucaena
leucocephala Benth, Tribulus terrestris Linn Eugenia uniflora Linn,
Dichapetalum madagascasience Poir, Lantana camara Linn, and Solanum
torvum Benth); were evaluated in Sprague Dawley rats weighing
between 200-300 grams. Two of these plants, Lantana camara and ~.
madagascasience were re-evaluated in West African dwarf goats.
Spectrophotometric and titrimetric methods were also used to
determine some inorganic and metallic contents of the plant.
The extracts of all the poisonous plants except ~. torvum and
~. madagascasiense produced dose dependent effects on rat serum
proteins. The extracts of these plants which produced
statistically significant changes (P( 0.05) in serum proteins of
the rat include the 400 and 600mg/kg doses of the extrct of 1..
leucocephala; 400 mg/kg dose level for extract of T. terrestris;
200 mg/kg dose level, for the extract of E. uniflora; 400 and 600
mg/kg dose levels extract of L. camara. The gl&bulin fraction did
not show any significant changes in all the rats treated with the
poisonous plants. No changes were also observed in the serum
dectrolytes analYsed.
I.terrestris, 1.. Camara and E. uniflora produced dose related
increases in activities of serum enzymes, alanine aminotransferase
ALT (EC 2:6.1.2), aspartate aminotransferase (AST) (EC.2.6.1.1.),
- .and.La Lka.Ldne.. .phos.phat-as e (ALP) (EC.3.1.-3.1. ) . All the plants
produced significant increases in ALT except L. Leucocepha!a and ~
torvum whereas E. uniflora, L. camara, and D. madagascasie use
produced significant changes in AST (P<.0.05).
An evaluation of the ha~matological parameters of the treated
rats, revealed that only extracts of T. terrestris, and ~. camara
produced dose dependent decreases in the total red cell blood
count. These decreases were significant for, all the doses of the
extracts of T. terrestris; the 400 and 600 mg/kg dose levels of ~.
camara (P < 0.05). The decreases in erythrocyte counts were
associated with decreases in haemoglobin concentration.
The piosonous plants did not cause changes in the blood
coagulation time. Only the extract of T. terrestris produced
decreases in total leucocyte count.
Si~ilarly, only the extract of T. terrestris caused clinical
symptoms in the rats which included depression and inappetence.
However, all the plan,t extracts except that of ~. torvum produced
gross pathologic lesions in the liver including pin point or
ecchymotic hemorrhages. Histopathologic lesions include necrotic
lesions of the hepatic tissues and vacuolation of some hepatic
lobules. T. terrestris, ~.camara, ~. uniflora and~. leucocephala
produced gross lesions of the renal tissue. Histologically the
affected renal parenchyma was hyperemic and the tubular epithelium
showed degeneration. The extracts of D.madag-gascasience and ~.
-camara produced --gastroint-e-stinal res-ions including superficial
4
necrosis of intestinal epithelial lining and desquamation of
patches of the gastric and ileal mucosa.
In the West African Dwarf goats, 1.. camara and Q. madagas-
casience produced increases in the serum activities of ALT, AST and
ALP. Furthermore, these plants also produced increased total blood
protein level and blood urea nitrogen. Lantana toxicity in the
goats also produced decreased total RBC counts, decreases
hematocrit and increased haemoglobin concentration. Animals with
Lantana toxicity also showed anorexia and were emaciated. Q.
madagascasience did not produce changes in haematological
parameters.
Phytochemical analysis showed different levels of cyanide,
(CN-), nitrate (N03-) nitrite (N02-), oxalate and elements such as
lead )Pb+), zinc+), iron (Fe++), manganese (Mn+), and copper (Cu++)
in all the plants
These studies therefore shows ( that terrestris
,
~. unifora, 1.. leucocephala, 1.. camara but particularly ~ camara
and T. terrestris are hepatotoxic, produce renal lesions and also
affect hemopoiesis. Thus these plants are toxic to livestock and
their growth among normal pasture could post potential risk to
livestock production in Nigeria.
5
ACKNOWLEDGEMENT
My immense gratitude to the Omnipotent Almighty God who
has spared and guided me through thick and thin.
I am thankful to all my supervisors Dr. Arowolo, R.O.A.,
Prof. Oyejide, A. and Prof. Olorunsogo, 0.0. but most
espec i aLly Dr. Arowolo, R.O.A., for all assistance given to
make the completion of this work possible. I am very grateful
to all the members of the department of Veterinary Physiology
and Pharmacology, particularly Mr. Erinle and Olowookorun for
their assistance.
I appreciate the wonderful assistance of following people
who are experts in their own field who have contributed so
immensely to the success of this work that without them my
efforts would have been dwarfed
(i) Mr. Fajinmi of the Department of Chemical Pathology,
Unive,rsity College Hospital, Ibadan.
(i i ) Mr. Abaire of the Natural Products Section of
Department of Chemistry.
(iii) Dr. Gbile, Mr. Aruwodo and others of the Forestry
Research Institute of Nigeria, Jericho, Ibadan.
(iv) Dr. (Mrs) Joyce Lowe of Department of Botany
(v) Dr. Steve Akpavie of the Department of Veterinary
Pathology.
6
CERTIFtCATION BY SUPERVISORS
We certify that this work was carried out
by
MATTHEW OLUWOLE ABATAN
DEPARTMENT OF VETERINARY PHYSIOLOGY
AND PHARMACOLOGY
AROWOLO, R. O. (Ass. Professor)
D.V.M. (Ibadan), M.Sc. (Guelph),
Ph.D . (Ibadan )
.... ~ .
OYEJIDE, A.' (~rofessor) OLORUNSOGO, O.O.(Professor)
D.V.M. (Ibadan), B.Sc., Ph.D. (Ibadan)
M.V.P.M., M.S., Ph.D
(Davis)
7
TABLE OF CONTENTS
PAGE
TITLE OF THESIS . 1
ABSTRACT ........................................... 2 -
ACKNOWLEDGEMENT ................•................... 5
CERTIFICATION . 6
TABLE OF CONTENTS . 7
LIST OF PLATES _,' . H
LIST OF TABLES . 22
LIST OF FIGURES . 26
CHAPTER ONE - GENERAL INTRODUCTION AND LITERATURE
REVIEW
1.0 General Introduction .
1.1 The potentially harmful effects of toxic
materials in plant foodstuffs . 33
1.2 Literature review ...................... 35
1. 2.1 Poisons and types of poisoning . 35
, Types of plant poisoning . 37
1.3 Toxic chemical constituents of plants .. 39
1. 3.1 Cyanogenic Glycosides . 40
1.3.1.1. Plants containing cyanogenetic
glycosides . 41
1.3.1.2. Tissue response to cyanide . 42
1. 3.2 Protease inhibitors . 45
8
1. 3.3 The lectins 46
1.3.3.1 Agglutinins 46
1. 3.4. Polyphenolic:s 49
1.3.5. Alkaloids . . 51
1.3.6. Toxic Amino Acids 53
1.3.7. Saponins 54
1.3.8. Flavones and Isoflavones 55
1.3.9. Oxalate-Bearing Plants 56
1.3.10. Plants which contain dangerous levels
of nitrates 59
1.3.10.1 Nitrates/nitrites and nitrosamine
formation
1. 4. Toxic effects on cellular elements of
Blood 64
1.4.1. Physiologic properties of constituents
of bl()od 64
1. 4.2. Haematopoiesis 64
1.4.3. H~ematopathies associated with poisons .. 65
1.4.3.1. Anaemia . 66
1.4.4. £lassification of Anaemia 67
1.4.4.1 Hemolytic Anaemias associated with plant
poison:'ng
1.4.4.2. Hypoproliferative anaemia associated
with plants 71
9
1.4.4.3. Sensitivity to Drug-ipduced Hemolytic
Anaemia due to Intraerythrocytic
defects 7'2.
1.4.4.4. Deficiency of Glucose-6-Phosphate
Dehydrogenase 74
1.4.4.5 Glutathione reductase Deficiency ....•.. 7f>
1.4.4.6. Glutathione deficiency . 76
1.4.4.7. Phosphogluconic dehydrogenase (PGD)
deficiency 76
1.4.4.8. The unstable Haemoglobins .•............ 76
1.4.4.9. Drug-dependent immune Hemolytic
Anaemia 78
1. 4.5. Hemorrhage . 79
1.4.5.1. Poisonous Plants producing hemorrhages .. 79
1. 4.6. Methaemoglobinaemia and Methaemoglobin
forming substances HO
1.4.6.1 Formation of methaemoglobins . H1
,
1.4.6.2. Chemical interactions and properties of
methaemoglobin H~
1.4.6.3. Effect of methaemoglobinaemia on blood
oxygenation H4
1. 4.7. Coagulation and platelet interaction
during haemostasis H7
1.4.7.1 Acquired coagulopathies ..........•.....
10
1.4.7.2. Destructive lesions of the liver and
blood coagulation 89
1.4.7.3. Hypocalcaemia and blooa coagulation .... 90
1.4.7.4. Platelet Disorders .....•.....•....•.... 90
1.4.7.5. Vitamin K. Deficiency and Antagonism ...
1.4.8. Leucocytes 93
1.4.8.1. The neutrophils
1.4.8.2. The eosinophils
1.4.8.3. The basoph i1s . 95
1.4.8.4. The monocyte and macrophages . 95
1.4.8.5. The lymphocytes . 96
1. 5. Toxic effects on non-cellular consti-
tuents of blood 98
1.5.1. Nitrogenous constituents of the blood ..
1.5.1.1. Blood nonprotein ni t.r oge n ...•..•..•..... 100
1.5.1.2. Nitrogen Balance ....................... 100
1.5.1.3. D,ysprote inaemia . 100
1.5.1.4. Interpretation of serum protein changes.
1.5.1.5. Proteinuria . 104
1. 5.2. Pigment Metabolism-Jaundice ..•......... 105
1. 5.3. The influence of plant poisons on
blood urea nitrogen 109
1. 5.4. Serum enzymes of Diagnostic importance .. llO
1.5.4.1. Creatinine kinase ............•......... III
1 1
1.5.4.2. Alanine Aminotransferase •••.•......•..• 102
1.5.4.3. Aspartate Aminotransferase .........•... 113
1.5.4.4. Gamma-Glutamyltransferase 113
1.5.4.5. Lactate Dehydrogenase 114
1.5.4.6. Sorbitol Dehydrogenase ...•............. 114
1.5.4.7. Alkaline Phosphatase .•................. 115
1.5.5. Serum Enzyme Tests 115
1.5.5.1. Effect of some poisonous plants on serum
enzymes 116
1.5.5.2. Urinary enzymes........................ 119
1. 5.6. Primary Renal Dysfunction.............. 119
1.5.7. Serum calcium and phosphorus 121
1.5.7.1. Toxic plants affecting calcium
and phosphorus metabolism 122
1. 6. Gross and Histologic lesions in
toxicosis 124
1.6.1.
,Death of cell and tissues . 124
1.6.2. Plants producing damage to the gastro-
intestinal tract . 125
1.6.2.1. plants producing stomatitis •........... 125
1.6.2.2. Plants producing gastritis . 126
1.6.2.3. Signs and lesions associated with
other parts of the G.I.T......... 126
1. 6.3. Morphologic lesions in the liver
12
due to toxic agents •.•. .... ...• .....•. 132
1.6.3.1. Mechanism of liver injury . 132
1.6.3.2. Morphologic lesions in the liver due
to toxic agents .•......•.............. 135
1.6.3.3. Classification of Chemical-induced
liver injury •.••..•.....•............. 135
1.6.3.4. Hepatitis . 137
1.6.3.5. Chemical substances and plant toxins
producing hepatitis 13H
1.6.4. Pathological lesions associated with
the kidney 141
1.6.4.1. Toxic tubular nephrosis •.............. 143
1. 6.5. Morphologic lesions associated with
Central Nervous System toxicosis...... 145
1.6.5.1. Symptoms and lesions in the CNS due
to pl~nt poisons 146
1. 6.6. Pl,ants producing teratogenic and
developmental abnormalies 150
1. 6.7. Tumour forming plant agents . 153
1. 7. Aim and scope of study 153
CHAPTER TWO - MATERIALS AND METHOD
2.1 Experimental animals 160
2.2 . The poisonous plants under study . 161
2.2.1. Preparation of the extracts of the
13
poisonous plants ... .•.••...••....•.... 161
2.2.2. Technique for pelleting leaves . 162
2 .3. Technique for administration of
extracts and leaves................... 162
2 • 4 • Technique for obtaining blood and serum
samples ...............••...•.......... 163
2 .5 . Determination of inorganic constituents
of plants 164
G. 5.1. Determination of phytin content . 164
G •5 .2. Determination of oxalate content of
leaves 166
2.5.3. Determination of hydrocyanic acid in
the leaves 16H
2.5.4. Determination of nitrate content of
the leaves 170
2.5.5. Dete~mination of the nitrite contents
,of the leaves 172
2.5.6. Determination of the metallic consti-
tuents of the plant 173
2.5.6.1. .Determination of the copper content .. 173
2.5.6.2. Determination of iron content . 174
G •5 .6. 3 • Determination of zinc content . 174
2.5.6.4. Determination of the manganese
content 175
14
~.5.6.5. Determination of lead content ..••...• 175
~ . 5 • 6 • 6 • Determination of molybdenum content .• 176
~.6. Determination of Hematological
parameters .......................... 176
~. 7 . Determination of serum biochemical
parameters 1 B 1
~.7.1. Measurement of blood urea nitrogen
(BUN) un
z • 7.2. Measurement of total protein . IH~
~ . 7 • 3 • Serum bilirubin . 1H3
z , 7.4. Activity of Aspartate aminotransferase
(AST) IH4
z , 7.5. Activity of alanine aminotransferase
(ALT) IH5
~ • 7 • 6 • Activity of Alkaline Phosphatase .... 1H7
Histo~ogical Technique ..•........•.. IHH
CHAPTER THREE - EXPERIMENT AND RESULTS
3.1 SECTION A.
3.1. Chemical Analysis of the Poisonous
.P1ant s 1!16
3.1.1. Introduction . 1!16
3 . ~ • Experiment one . 1!1!1
3.2.1. Determination of the inorganic consti-
tuents of the plant .......•.....•... 1!1!1
15
3.3. Experiment 2 . 202
3.3.1. Molybdenum levels in the leaves of
the poisonous plants 202
3 .3 .2. Manganese level in the leaves of the
poisonous plants.................... 202
3 . 3 . 3 . Iron level in the leaves of the
poisonous plants 203
3.3.4. Copper level in the leaves of the
poisonous plants. 203
3 .3 .5. Zinc level in the leaves of the
poisonous plants .. 203
3 •3 .6. Lead level in the poisonous plants .. 203
SECTION B.
3.4. Toxicological effects of the poisonous
plants on haematological parameters of
rats and goats .......•........•...... 205
,
3.4.1. Experiment 1. Determination of the
lethal dose 50 (LD 50) of the poisonous
plants in mice 205
3 . 5 • Expe rimerrt 2. Effect of the poisonous
plants on the hematological parameters
of rats and goats 207
3 • 6 . Result ............................... 210
3.6.1. Effect of the extract of Eugenia uniflora
16
on the leucogram and erythron of rats... 210
3 • 6 • 2 • Effect of Tribulus terrestris on hemato-
logical parameters of the rat . z i s
3 .6 .3 . The effect of Leucaena leucocephala
extract on the hematological parameters
in rats................................. 216
3.6.4. The effect of Solanum torvum on the
haematological parameters in the rats 217
3.6.5. The effect of Lantana camara on the
hematological parameters of rats . 218
3 • 6 • 6 • The effect of Dichapetalum madagasca-
siense on hematological parameters in
rats 218
3 • 6 • 7 • The effect of Q. madagascasiense on
hematological parameters in goats ..... 219
3.6.8 . The effect of L. camara on the hemato-
logical parameters in the goat . 219
3.7. 't;onclusion . 219
SECTION C
3.8. Effects of the Poisonous plants on
Serum biochemical parameters of rats
and goats . 236
3 . 9 • Resul t . 239
3.9.1. Jh~ _e_ffect of L. leucocephala on serum
17
biochemical parameters in rats
The effect of T. terrestris on the serum
biochemical parameters in rats 240
The effect of the extract of ~.
uniflora on the serum biochemical
parameters in rats 241
The effect of ~. camara on the serum
biochemical parameters in rats 241
The effect of Q. madagascasiense on the
biochemical parameters of rats 242
Effect of ~. torvum on the serum
biochemical parameters of rats 243
The effect of Q. madagascasiense on
the serum biochemical parameters in
goats . 24~
Effect of ~. camara on serum biochemical
parameters in goats 244
r
SECTION D. ,
Clinical signs and pathological changes
in rats associated with ingestion of
poisonous plants 265
Lesions due to ~. camara in goats 26H
Lesions due to ~. torvum in rats 26H
Lesions due to T. terrestris in rats... 26H
18
3.10.4. Lesions due to 1.. leucocephala in rats.
3.10.5. Lesions due to E· uniflora ............ 269
3.10.6. Lesions due to Q. madagascasiense in
rats .................................. 269
0.10.7. Lesions due to 1.. camara in rats . 270
CHAPTER FOUR - DISCUSSIONS
4.1. Toxic chemical constituents of poisonous
plants 276
4.1.1. Nitrate and nitrite content of the
poisonous plants 277
4.1. 2. Oxalate content of the poisonous plants. 2H2
4.1. 3. Phytin content of the poisonous plants .. 2H4
4.1. 4. Cyanide content of the poisonous plants. 2H5
4.1. 5. Metallic ion concentration in poisonous
plants 2H7
4.1.5.1. Level of molybdenum in the poisonous
plants ' 2H7
4.1.5.2. The level of manganese in poisonous
plants 2HH
4.1.5.3. The level of copper in poisonous plants. 2H9
4.1.5.4. The level of zinc in poisonous plants ..
4.1.5.5. The level of lead in the leaves of
po isonous plants 291
4.2. Effect of poisonous plants on bio-
19
chemical parameters ..••.......••..•••. 292
4.2.1. The effect of the poisonous plants on
serum proteins ..•...•.•.••..........•. 293
4.2.2. Effect of the poisonous plants on blood
urea in nitrogen 297
4 • 2 . 3 . Effect of the poisonous plants on serum
electrolytes ..........•............... 29H
4.2.4. Effect of the poisonous plants on serum
enzyme activities 300
4.2.4.1. Effect of plants on serum activities of
aspartate aminotransferase ...•........ 301
4 • 2 . 4 . 2 . Effect of the poisonous plants on
alkaline phosphatase .•.......•........ 302
4 .2 .4 .3 . Effect of the poisonous plants on
serum alanine aminotransferase 303
4 • 3 . Effect of the poisonous plants on the
hematologic parameters ....•........... 304
4.3.1. , The effect of the poisonous plants on
erythron .........•.................... 304
4 .3 .2. Effect of the poison plants on leucotron
in rats and goats ......•....•..•...... 310
4.4. Clinical signs grosi and histopathologic
effects of poisonous plants ..•........ 311
20
4.4.1. Gross histologic lesions associated
with ~. leucocephala 31~
4.4.2. Gross and histologic changes associated
with ~. torvum. :31:3
4.4.3. Lesions associated with E. uniflora 315
4.4.4. Gross and histologic lesions due to T.
terrestris . 315
4.4.5. Lesions associated with Lantana camara. 316 ,.
4 . 5 • General Discussions and Conclusions . 317
4.5.1. The problem of poisonous plants . 317
4.5.2. The toxic effects of Solanum torvum 31B
4 • 5 • 3 • The toxic effects of Lantana camara 3~O
4 .5 .4. Toxic effects of Leucaena leucocephala. 3~~
4 • 5 . 5 • Toxic effects of Tribulus terrestris ... 3~5
4.5.6. Toxuc effects if Dichapetalum
madagascasiense 3~7
4 • 5 • 7 • ~oxic effect of Eugenia uniflora . 3~B
4.6. Clinical symptoms and suggested treat-
ments of poisoning in animals.......... 329
References •........................... 334
21
LIST OF TABLES
TABLE NO. PAGE
1. Levels of inorganic anions in the poisonous
plants 204
2. Levels of metallic cations in the poisonous
plants 204
3. Acute LD50 values of the poisonous plants
in mice . 200
4. Mean values (+ S.E.M.) of the hematological
parameters following exposure of rats to
ethanolic extract of ~. uniflora
5. Mean values(+ S.E.M.) of total and diffe-
rential leucocytes counts of rats given
extract of ~. uniflora
6. Mean values (+ S.E.M.) of hematological
paramete~s following exposure of rats
to, extract of T. terrestris . 224
7. Mean values (+ S.E.M» of total and differ-
ential leucocytes counts of rats given
extract of Tribulus Terrestris .
8. Mean values (+ S.E.M.) of hematological
parameters following exposure of rats to
extract of Leucaena leucocephala
22
9. Mean values (+ S.E.M.) of total and
differential leucocytes counts of rats
given extract of Leucaena leucocephala
10. Mean values (+ S.E.M.) of hematological
parameters following exposure of rats to
extract of Solanum torvum .
11. Mean values (+ S.E.M.) of total and
differential leucocytes counts of rats
given extracts of Solanum torvum .
12. Mean values (+ S.E.M.) of hematological
parameters following exposure of rats to
extract of ~. camara 230
13. Mean values (+ S.E.M.) of total and
differential leucocyte counts of rats
given extract of Lantana camara . 231
14. Mean values (+ S.E.M.) of hematological
pa~ameters following exposure of rats
to ethanolic extract of n.
madagascasiense .
15. Mean values (+ S.E.M.) of total and
differential leucocyte counts of rats
given extract of D. madagascasiense
23
16. Mean hematological values (+ S.E.M.) of
goats on Q. madagascasiense or ~. camara
for 14 days . 234
17. Mean hematological values (+ S.E.M.) of
goats after feeding with Q. madagascasiense
or Lantana camara for 21 days . 235
18. Mean serum parameters (+ S.E.M.) of rats
following oral administration of ~.
leucocephala . 246
19. Mean serum enzyme values (+ S.E.M.) of rats
following the oral administration of
.1. leucocephala. . . 247
20. Mean serum parameters + S.E.M.) of rats
following the administration of
I. terrestris . 24H
21. Mean serum enzyme values (+ S.E.M.) of
rats following the administration of
extract of T. terrestris 249
22. Mean serum parameters (+ S.E.M.) of rats
following oral administration of extracts
of E.., uniflora.............................. 250
23. Mean serum enzyme values (+ S.E.M.) of rats
following the administration of extract of
24
E. uniflora................................. Zf)l
24. Mean serum parameters (+ S.E.M.) of rats
following the oral administration of extract
of 1. camara................................. ~5~
25. Mean serum enzyme values (+ S.E.M.) of rats
following the administration of extract of
1. camara.................................... ~53
26. Mean serum parameters (+ S.E.M.) of rats
fed with Q. madagascasiense.................. 254
27. Mean serum enzymes values (+ S.E.M.) of rats
fed with Q. madagascasiense................... 255
28. Mean serum parameters (+ S.E.M.) of rats
following administration of extract of
~. torvum..................................... 256
29 Mean serum enzyme activities (+ S.E.M.) of rats
following ~dministration of ~. torvum . ~57
30 Mean serum values (+ S.E.M.) of goats after
feeding~ith Q. madagascasiense and 1. camara
for 14 days.................................... ~5g
31 Mean serum values (+ S.E.M.) of goats after
feeding Q. madagascasiens or 1. camara for
21 days "25~
25
LIST OF PLATES
PLATE PAGE
1. Solanum torvum Benth growing along a cattle
route from the abattoir in Bodija Ibadan ..... l~O
2. Tribulus terrestris Linn showing a cluster of
growth and yellow flower heads............... l~l
3. Leucaena leucocephala with its flowers in
white globose heads. ..... ... ... .... ..... ..... lYZ
4. Eugenia uniflora Linn stands with other plants
growing with it............................ 1~3
5. Lantana camara Linn growing in the grazing
pasture at the Teaching and Research Farm,
University of Ibadan....................... 1~4
6. Dichapetalum madagascasiense Pair on a bush
path ,................................... 195
7. Renal glomerulus of a rat dosed with Tribulus
terre~tris showing moderate messangial proli-
feration and proteinaceus material in
glomerular space ..........••••• ,"\\\\\\\\\\ ~11
8. Sectioning the same kidney as Plate 7 with
protein cast in tubule 272
26
9. Section of the liver of a rat dose with Leucaena
leucocephala showing lymphocytic cuffs around
the portal vessel ~7::3
10. Histological appearance of the liver of a rat
dosed with Lantana camara showing moderate
fatty degeneration 274
..
,
27
LIST OF FIGURES
FIGURE PAGE
1. Log-dose mortality curve of Mice Administered
with extract of Lantana camara orally . 211
2 . Log-dose response curve of Mice Administered
with extract of Solanum torvum orally....... 212
3. Log-dose mortality curve of Mice Administered
~ith extract of Eugenia uniflora orally ..... 213
4. Log-dose mortality curVe of Mice Administered
with extract of Tribulus terrestris orally 214
5. Log-dose mortality curve of Leucaena
Leucocephala . 215
6. Effect of Leucaena leucacephala on serum
enzyme activities in rats....... 260
7 . Effect of,Tribulus terrestris on serum
enzymes in rats 261
8. Eff~ct of Eugenia uniflora on serum enzymes
acti vities in rats 262
9. Effect of Lantana camara on serum enzymes
activities in rats.......................... 263
10. Effect of Solanum torvum on serum enzymes
activities in rats.......................... 264
CHAPTER ONE
GENERAL INTRODUCTION AND LITERATURE REVIEW
The human race and his animals depend on plants as a renewable
source of many foodstuff. However, the harmful effects produced by
certain constituents of these plants in many parts of the world
each year, are not so widely appreciated (Kinghorn, 1979,
Habermehl, 1987, Fernando 1987, Wee, 1987).
Plants constitute the third largest category of poisoning
(Woodward 1985, Fernando 1987). Such plant poisoning are
attributed to the fact that house plant have become more popular
and many of these are unfortunately poisonous. Such poisonous
plants are all around us as potted plants, imported exotics grown
in green house, cultivated hybrids, decorating the gardens and even
as everyday foodstuffs on the larder shelf. Moreover families are
outdoors more with the,renewed interest in the environment. There
is more camping, picnicking, hiking and gardening therefore man and
his animals ar~ constantly in contact with poisonous plants
(Woodward, 1985).
Conditions which favor the occurence of plant poisoning in
livestock are prevalent in Nigeria. They include nomadism,
luxuriant growth of pasture with poisonous plants, movements to
obtain grazing on hoof from the north to the south. (Nwude and
Prason 1977). During the annual dry season and particularly during
29
the periodic droughts animals are exposed by changing from one
environment to the other to strange vegetation. Such cattle are
usually unfamiliar with the plants in the new environment and are
liable to be poisoned. The natural vegetation is in most instances
relatively undisturbed in many areas so that poisonous plants could
still constitute a significant part of the flora. In some areas,
much of the land could be grazed, resulting in- sufficient
palatable forage even during the rainy season (Ewer and Hall 197H).
Poisoning by plants in animals could arise from the
following:-
(i) the seasonal nature of the growth of the poisonous
plants or parts of it such as the seeds flowers or
leave, which invariably are normally poisonous. For
example Sclerocarpus africanus is found as a contaminant of hay and
causes a lot of poisoning (Evans, Evans and Hughes 1954, Rajendran,
Chennakasavalu, Rao Viraraghavan and Damodaran 19B3). Both the
,
leaves rhizomes contain the toxic principle the concentration of
which vary with season (Thomas 1963).
(i i ) Climatic conditions such as high humidity, d~ll or
bright weather and temperature are known to favor the
proliferation of poisonous plants and even the elaboration of the
toxic constituents.
30
(iii) Location of the poisonous plants p~ay a vital role in
terms of how readily accessible they may be to the
grazing animals. Poisonous plants which can only be found in the
high rising forest will,definitely not be a source of worry for
livestock owners. However if these plants can be found in the
grazing areas of the animals then these plants can be grazed along
with the normally grazed plants which will cause toxicity. For
example Holarrhena floribunda and Adenum obesum are trees or high
shrubs and animals may not reach the leaves (Irvine 1961).
'iv} Most ~oisonous ~lants are un~alatable and may therefore
not be grazed. The unpalatability may be associated with
bitterness, presence of spines e.g. Encephalatos bateri, Euphorbia
camerunica; the woodiness of the plant e.g. Solanum dulcamara
(Woody nightshade); the presence of a repulsive aromatic smell e.g.
Lantana camara; or the ability of the plant to cause blistering or
rash around the mout h parts of the animal, e.g. Asclepias
(milkweed) or Ca,lotropis procera (Hutchinson and Dalziel 1954, Ewer
and Hall 1978; Azariah, Azariah, Hall, Kesan, Sumathi and
Sundararaj 1987). Animals will normally not graze such plants
assuming that there were more nutritious species of plants around.
Animals during drought or newly introduced animals will however be
compelled by hunger to browse any available plant.
(v) The species and age of the animal affect plant poisoning
since susceptibility or tolerance to poisoning is
31
species related sheep and goats are known to be more tolerant
whereas young animals pigs and horses are easily poisoned
(Hutchinson and Dalziel 1954).
(vi) Occasionally, the poisoning may not be due to a plant
but to some fungi growing on it. This is especially
common with certain fungi which attack cereal grain e.g.
Aspergillus flavus which cause aflatoxicosis, and
Claviceps purpurea which causes ergotism, by attaching
themselves· to grasses mainly Paspalum species and then
render them toxic (Lim 1987).
(vii) Often animals which were born and grew in a locality
become tolerant to some poisonous plants whereas animals
which are newly introduced will quickly die because they are not
able to tolerate the active principle. Furthermore, animals which
may become familiar with the effect of certain poison plants will
avoid it, whereas newly introduced animals will eat it and get
poisoned. This,situation frequently happen with normadism or
transhumance.
(viii) Sometimes the degree of toxicity varies according to
the soil type e. g many poisonous plants are much more toxic in
limestone soil. High selenium soils may be very clearly marked by
the selenium accumulators growing on them e.g. Astragalus species
such as A. bisculcatus and A. pectinatus which may contain up to
32
300 ppm of Selenium (Avery, Nulo, Kramer, Johnson, Darcel and Leo,
1961).
Many plants have all their parts toxic e.g. r.. aguilinum
whereas some only in one or two parts such as the seeds or the
flowers or the roots. This means that animals can only be poisoned
if the poisonous parts e.g. the seeds are consumed e.g. Ricinus
communis. If it is only the roots which are poisonous then animals
like pigs which are notorious for their rooting habit will easily
be poisoned e.g. Manihot escu- Le nt a , P. aquilinum (Evans, Widdop,
and Harding (1972).
Sometimes the plants will change from the toxic to the non
toxic state or vice versa for no apparent reason. This is as a
result of variability in the toxic principle they contain.
Frequently the intervention of man may lead to poisoning of
livestock by naturally occurring substances which are undou- btedly
potentially dangerous but which are normaly avoided by animals.
For example Eq'uisetum species and P. aquilinum are refused by
grazing animals if other herbage is accessible, but when the
animals are housed and the dried plants are presented to them, in
hay the unfortunate beast have no alternati ve but, to eat them or
starve (Clarke and Clarke 1975.
33
1.1 The Potentially harmful effects of toxic materials in plant
foodstuffs.
Toxic contaminants can gain entry into plants by direct
application of pesticides-to plants that are eaten by animals, from
contamination of the soil by pesticides applied to plants, from
chemicals added to the soil or from the presence in soil of
radionuclides.
Watt and Breyer-Brandwiyk (1962) observed that meat that had
been r-oast.ed or grilled on wooden skewers derived from the
ornamental shrub Nerium oleander has caused death from injestion of
the cardioactive (digitalis glycoside) substance. A great variety
of foreign substances have been demonstrated in cow milk and in
human milk which might have been derived from injestion of plants,
drugs or other chemicals (Sapeike 1969) Plant substances which gain
entry into milk may produce minor or major unwanted effects.
(Leiner 1974).
,
Goi t.r-goen i c substances such as go i trin in cabbage, Kale,
turnips rapeseeds and other plants may pass into milk of cows that
eat these plant. These may cause interference with the thyroid
function in human, who drink such milk (Clement 1960). An infant
that drunk milk from a cow that had eaten the foliage of Nerium
oleander was observed to have died from this cause (Watt and
Breyer-Brandwiyk 1962).
34
The juice from Solanum incanum had been used by the Transkeian
Bantu in South Africa to curdle milk. There is high incidence of
esophageal carcinoma a n these group of people. Since
dimethylnitrosamine (DMNA) has been identified in Solanum fruit, it
is suggested that there may be a causative association between DMNA
and nitrosamines and this neoplasm. (Du PIe iss, Nunn, and Roach
1969) .
McBarron, Walker and Gardiner (1975) observed the death of
twenty out of five hundred lambs who obtained water from a dam
(ground tank) on a farm. The lambs were allowed access to the dam
with concentration of bloom of Anabaena circinalis and Schizothrix
calcicola. The feeding of the seeds of Lathyrus sativus
(Moslehuddin, Hang, and Stoewsand 19H7) or Abrus, precatorius as
sole component of meal for poultry have shown to be deleterious
(Rahman and Mia 1972).
In Nigeria, no e~tensive research effort can be said to have
been done to d~termine whether or not most of the plants which are
known in other part of the world to be very poisonous are actually
equally poisonous also in Nigeria or otherwise.For example Lantana
camara has been reported to be poisonous in other parts of the
world. (Sharma et al 1981, Gujral and Vasudevan 19H3, Arhiredy and
Singh 1984) whereas the fruits of the same plant are reported to be
edible (Gujral and Vasude- Van 19H3) However there are no research
evidence to confirm whether the varieties of this plant in this
35
country are poisonous (Nwude and Parsons 1977). This evidence is
important since environmental factors are mentioned to influence
toxic nature of plants. Furthermore in Nigeria, most of the
reported cases of plant poisoning have been based on the
observation of clinical symptoms after the animal might have
consumed toxic quantities of the plant. Many toxic plants may not
produce toxic symptoms until chronic consumption takes place.
Prolonged exposure to such plants may not be possible in for
example animals under normadism. The non observation of symptoms
does not mean such plants are not toxic.
These studies were therefore carried out to evaluate the
haematological, biochemical, clinical symptoms and the gross and
histopathological effects of the suspected poisonous plants,
Solanum torvum Swart, (Plate 1) Tribulus terrestris Linn (Plate 2)
Leucaena leucocephala Benth, (Plate 3) Eugenia uniflora Linn (Plate
4) Lantana camara Linn (Plate 5) and Dichapetalum madagascasiense
Poir (Plate 6),in rats with a re-evaluation of the latter two
plants in goats. Chemical analysis of the plants were conducted so
as to determine some inorganic and metallic substances which are
known to be found in poisonous plants.
1.2 LITERATURE REVIEW
1.2.1. Poisons and types of poisoning
Poison is defined as any substances which taken inwardly in
a very small dose, or applied in any kind of manner to a living
36
body depraves health, or entirely destroys life (Clarke and Clarke,
1975, Woodward, 1985).
Poisoning can be subdivided into acute, (a sudden violent
syndrome caused by singl~ large dose of poison) and chronic forms
(a persistent, lingering condition brought about by small repeated
doses) with subacute poisoning coming in-between these two. This
subdivision, according to Clarke and Clarke (1975) is not tenable,
considering the fact that there are certain types of poisoning
which would be difficult to fit into any of these categories. For
instance, the symptoms produced by the plant Pteridium aquilinum
(bracken fern) poisoning may not manifest until months after the
plant has been ingested (Evans 1964). Furthermore, there are other
symptoms which could arise from poisoning which cannot be
categorized. Such signs are (i) allergy, an immunological response
due to sensitization of the subject by a previous dose of the
po ison; (ii) carc inogen icity, in which the po ison causes neoplas ia
formation e.g., bracken and cycads (Evans and Mason, 1965). (i i i)
teratogenicity in which the poison produces abnormalities in the
offspring of an animal when ingested at a certain stage of
pregnancy e.g. rionsumption of Veratrum californicum which produces
a teratogenic effect in sheep (Keller and Binns, 1964; Clegg, 1971)
or Conium maculatum (Edmonds et al., 1972).
Plants produce and present toxicity in a multitude of com-
plex ways (Kirigsb~rY~ 1979). Although vertebrates have mechanical
31
and biochemical defenses against these toxins, few systems of the
vertebrate body are immune to damage by some compounds from some
plant source (Kingsbury, 1975). Toxicity, by and large, involves an
interrelationship among. dose, absorption, detoxicification and
excretion (Cassarett and Doull, 1975).
1.2.2. Types of Plant Poisoning:
Poisonous plants as may be with other extraneous poisons
have the propensity to cause the following symptoms:
(a) the plant may produce a local injury to tissues to which it
may corne directly in contact with e.g. the latex from Euphorbia
camerunica is known to be esharotic and irritant (Smith, Jones, and
Hunt 1974, Terblanche, Adelaar and Van Straten 1966.)
(b) some plants cause gastroenteritis as a predominant feature
e.g members of the genus Sinapsis and Pennisetum claudestinum
(Kikuyu grass poisoning) Martinovich and Smith 1973.)
(c) poisonous plahts which are hepatotoxic and may also inva-
riably produce nephrotoxic ity. Such plants include 1.. camara
(Sharma et al 1982, Sharma et al., 1984 Phyllanthus abnormis,
Heliotropium europaeum and members of the genus Lupinus (Gardiner
1966).
(d) plants which are predominantly nephrotoxic.These include
oxalate bearing plants as Halogenton glomeratus (Littledike, James
and Cook 1976) Amaranthus retroflexus (Marshall, Buck, and Bell
1967). --
38
(e) poison plants causing death through cardiac insufficiency
with consequent venous congestion, anoxia and petechiae and other
changes commonly associated with anoxic conditions. Such plants
include selenium accumulators such as Astragalus species, Datura
stramonium, and Strophantus hispidus or ~. sarmentosus.
(f) poison plants which produce extensive hemorrhages.These are
plants which contain dicoumarin e.g. sweet clover and other plants
such as bracken fern, plants of the genus Crotalaria (Rao, Joshi,
and Kumar 1988, Singh et I, 1987).
(g) poison plants which produce anaemia or cause destruction of
the red blood cells e.g. Allium cepa, Abrus precat.orius (McPherson
1979).
(h) poison plants which cause the production of methemoglobin and
chocolate colored blood. These are plants containing nitra- tes
such as A. retrofelxus (Usher and Telling, 1975).
(i) plants which cause the production of carboxyhaemoglobin and
cherry-red bloqd. Such plants include those which contain high
levels of cyanide e.g. Cynodon plectostachyum, Manihot esculenta,
Nerium oleander Phaseolus lunatus and Eleusine indica (Clarke and
Clarke 1975, Liener, 1974).
(j) plant which produce marked and conspicuous pulmonary
emphysema. These include plants of the genus Brassica (Greenhalgh,
Sharman, and Aitken 1969).
39
(k) poison plants which may cause gangrene of the extremities
including the limbs, tail and ears so that after several weeks, of
ingestion of small amounts the most distal parts of the extremities
may drop off • Such po~sons are produced by fungus as Claviceps
purpurea, and grasses Festuca arundinacea (Hore, 1961).
(1) poison plants which cause loss of hair or wool.Such plants
include Leucaena leucocephala (Mullenax 1963).
(m) plants which cause degeneration of cardiac and skeletal
muscle. The plants producing such lesions include Cassia
occidentalis L. (0 Hara, Pierce and Read, 1969).
(n) plants which are teratogenic or produce fetal developmen-
tal abnormalities and include plants as Veratrum californicum
(Edmonds et al 1972).
(0) . Miscellaneous or unclassified poisonous plants.Poisoning by
such plants in animals is infrequent.They include phytoestro- gens
in plants as Medicagd sativa (Kelly, Allison and Shirley, 1976).
1.3. Toxic Chem,ical Constituents of Plants
A number of compounds that occur in plant have been isolated
and suggested as having toxic or antinuitritional effects. Such
substances.are either organic or inorganic. Some of.such compounds
include organic substances as haemagglutinins. trypsin inhibitor,
saponins, goitrogens, glycosides, isoflavones, tannins and others
referred to as antivitamins and antimetals. The inorganic
substances include oxalate, cyanide, nitrate-and nitrite, copper,
40
lead magnesium, manganese, cobalt and selenium. However, some of
the inorganic substances when they occur in trace amounts are
essential for plant growth.
1. 3.1 Cyanogenic Glycosides
It is generally believed that the toxic properties associated
wi th cyano'genic gLycosLdes , such as 1inamarin are due to the
hydrocyanic acid released from the glycosides by the activity of an
enzyme complex. For example, linamarin is glucoside of acetone
cyanhydrin.' It is readily hydrolysd by heat labile B-glucosidase
linamarase to yield acetone cyanhydrin, which can be split by a
hydroxynitrile lyase non enzymatically into acetone and hydrocyanic
acid. The enzyme complex is present in the plant tissue and is
released when cells are broken down. Conn (1969) illustrated the
pathway for the enzymatic degradation of linamarin.
Hydrocyanic acid or prussic acid (HCN) is one of the most
toxic and rapidly ac'tLng of the common poisons; its sodium and
potassium salts, are only slightly less toxic. The complex of
cyanides e.g. ferrocyanides and thiocyanates, are also practically
harmless (Clarke and Clarke, 1975).
..
.yf:
41
1.3.1.1. Plants containing cyanogenetic glycoside:
By far the most important source of hydrocyanic acid to
toxicity animals is plant material. Many species of plants contain
hydrocyanic acid either jree or, more usually, in the form of a
cyanogenetic glycoside, an organic compound containing a sugar, and
capable of yielding cyanide on hydrolysis.
Some species of Brassicae (cabbage) contain relatively large
quantities of 3-butenyl-isothiocyanate. Sudden death in cattle
receiving Brassicae seed cake which was later found to contain
0.33% of volatile isothiocyanate has been reported (Van Etten,
1969) Van Kampen (1970) considers that all the conditions
associated with~. sudanense (Sudan grass) represents a lathyrogen
disease. The HCN in the grass is said to combine with L-cystine to
form.B-cyanoalanine, a precursor of the lathyroyan, L-glutamyl-B-
cyanoalanine.
Bracken {Pteridium aguilinum contains several potentially
harmful agents, amdng which are a cyanogenetic glycoside,a thia-
minase, an aplastic anaemia factor, a factor causing hematuria and
a carcinogen (Thomas, 1963).
Linum catharticum both cultivated and purging. flax contain a
cyanogenetic glycoside, linamarin from which HCN is released under
the action of the enzyme linamarase. The processes used in the
extraction of the oil from linseed and in the preparation of
___ Ld.naeedc cake j c f r-om the residue,-do not+dest r-oy the enzyme; boiling
42
for at least 10 minutes is observed to be required. The available
HCN content of fresh linseed meal appears to be of the order of
0.25 to 0.6 g/kg, ). The level is said to drop on storage.
Under normal conditions Qf feeding insufficient HCN is released to
be harmful. However, should the meal be eaten rapidly, and
voraciously, as by starving animals, there is definite danger of
poisoning (Franklin and Reid, 1944). It is also believed that the
rate of liberation of HCN is the significant factor in determining
the toxicity of the case when fed as a cattle food.
The rose family include many of the most commonly grown
fruit trees.The kernels and leaves of these contain many cyan-
ogenetic glycosides.Amygdalin is the common but prunasin and
prulaurasin also occur.The leaves may contain over o.~% HCN which
is considered as ten times the amount usually seen as dangerous
(Stauffer 1970).
1.3.1.2. Tissue response to cyanide
Cyanide is'believed to exert its toxic effect by inhibition of
the respiratory enzyme cytochrome oxidase. Concentration of cyanide
as low as 3x10-8 M have been shown to produse complete inhibition
of cytochrome oxidase invitro, and lethal doses pf cyanide have
been reported to inhibit this respiratory enzyme invivo.
{Hazzard,Kowanish,and Caughey 198~, Tewe and Maner 198~, Tewe and
Pesu 1982. In cyanide poisoning, oxygen transport and oxygen
tension are usually adequate and only cellular utilization of O~ is
43
depressed. As a consequence,this would mean that the
administration of 02 would not subserve any useful purpose in
antagonizing cyanide intoxication (Way, Gibbon and Shedy I~oo).
Significant amounts.of cyanide can be detoxified in the body.
Routes of detoxication proposed include conversion to thiocyanate
ion (SCN-), incorporation into the I-carbon metabolic pool through
interaction with vitamin B12, and conversion to 2-imino-4-
thiozolidine carboxylic acid (Casarrett and Doull 1~75, Gilman and
Goodman and Gilman, 1980).
Lang (1933) proposed that an enzyme from the kidney and liver
called rhodonase catalyses the reaction of thiosulphate with HCN to
give this cyanate, a relatively less toxic substance which can be
rapidly excreted. The properties of the enzyme rhodanase, now
known as thiosulphate s-transferase and of a second enzyme
mercaptopyruvate s-transferase, also capable of forming thiocyanate
by sulphur transfer to cyanide have been reviewed by
Sorbo (1975). ,
Wokes and Picard (1955) also suggested the involvement of
vitamin B12 in cyanide detoxification. In this pathway of vitamin
B12 in the form of cobalamine (BI2a) reacts with-cyanide to form
cyanocobalamine (B12).The latter then loses some of the cyanide to
form I-carbon fragments for the synthesis of methylgroups and the
resul ting hydroxocobalamine returns to the liver to repeat the
cycle.Also the hydroxocobalamine can combine with the thiocyanate
44
formed via the thiosulphate or mercaptopyruvic acid and proceed to
form cyanocobalamine.
The extreme toxicity of cyanide to most species is a well
known phenomenon. Montgomery (1969) observed that the lethal dose
for human of potassium cyanide taken by mouth is estimated to be
between 1.0 and 70.0 mg/kg body weight and will cause death in few
minutes. Acute toxicity and death from consumption of cyanide
containing food in human e.g. cassava and of plant materials in
animals is ·a common occurrence. However it is pertinent to note
that chronic effects from consumption of sublethal amounts of
cyanide over long periods has been associated with occurence of
human ataxic neuropathy and also goitre in Nigeria. This has been
observed in areas where consumption of a staple food is cassava
(Osuntokun 1973). It is suggested that the neuropathy is caused by
or aggravated by the cyanide or derivative acting on the eNS and
the thyroid enlarged by thiocyanate which is a well known goitrogen
derived from metaboJism of ingested cyanide (Osuntokun 1973 and
Wilson 1973).
Animal experiments give credence to such theories. Lesions in
the eNS were observed when rats were injected repeatedly with
sublethal amounts of cyanide over a period of 22 weeks with some
suggestions of demyelination (Smith et al., 1963). Similarly, the
injection daily for 5 weeks of KeN in rats produced degeneration of
neurons in the cortex and the degeneration of the corpus callosum
45
(Smith and Puckett 1965) Demyelination of the optic nerves and
retina were observed in rats given sublethal doses of eN over
periods of 3 weeks ( Wessel 1971).
1.3.2. PROTEASE INHIBITORS
The first observation that there existed an inhibitor of
trypsin in plant material is credited to Read,and Hass (193~) who
reported that an aqueous extract of a soya been flour inhibited the
ability of trypsin to liquify gelatin. The existence of an
inhibitor of trypsin in Soyabean which could be inactivated by heat
gave a reasonable explanation to the work of Osborne and Mende
(1917) who had observed that heat treatment of soyabean increased
the nutritive value of their proteins.
This observation of protease inhibitors which acted as
antinuitritional factors in plant foodstuffs, particularly in
such an important dietary source of protein stimulated search
for other such factois. The protease inhibitors have now
been observed ~o be present in all parts of some plants examined.
In Ipomoea batatas (sweet potato) a trypsin inhibi tor has been
observed to occur in the leaves as well as the tuber (Honavar and
Sohnie, 1955). Similarly the young leaves as well. as the tuber of
Solanum tuberosum (white potato)have been observed to be rich in
chymotrypsin inhibitor (Ryan and Huisman, 1967). The leaves and
cotyledons of Phaseolus aureus (mungbean are fairly high in
trypsin inhibitor activity (Honavar and Sohnie, 1955).
46
The presence of such factors in plants results in growth
depression in animals consuming them. Such growth depression
according to Lyman and Lepkovsky (1957); Hall (1977), Sharma et al.
(1987) may be as a result of an endogenous loss of essential amino
acids derived from a hyperactive pancreas which is responding in a
compensatory fashion to the effect of the trypsin inhibitor. The
mechanism by which the trypsin inhibitor stimulates the enlargement
of the pancreas is unclear. However Khayambashi and Lyman (1966)
in their experiment suggested that trypsin inhibitors may liberate
a hormone like agent which stimulates the secretory function of the
pancreas. These authors observed that a rat pancreas when perfused
with blood plasma obtained from a rat fed with trypsin inhibitor
secreted increased amounts of the enzyme.
1.3.3. The Lectins
Extracts of many plants which have the property to aggL-
utinate red blood cells have been shown to be caused by some
remarkable pr~tein called phytohaemagglutinin or lectins. They
exist in leaves, bark, roots tubers, latex, seeds etc.
1.3.3.1 Agglutinins
Agglutinin-containing plants have been found in many botanical
groups including monocotyledons, dicotyledons, mould, and lichens
but most frequently they have been detected in legumino- sae and
Euphobiaceae, (Tobiaska, 1964;Liener, 1974). Stilmark (lH59) is
reported to have first observed the effect of phytohaemagglutinin.
47
In observing the toxicity of castor oil he concluded that the
toxicity of Ricinus communis was due to protein fraction which is
called ricin which showed the ability to agglutinate the RBC from
human and animal blood. The agglutinins are said to differ widely
from each other in that they show considerable specificity. Thus
they could be different molecular species with different chemical
and biological characteristics. Orally ingested, ricin is known to
be more toxic for horses and rabbits than for chicks (Bierbaum,
1966). Lethal doses of highly active ricin preparation injected
intramuscularly ranged from O.OOOlmg/kg for rabbits to O.03mg/kg
for goats.Toxic-ity of ricin for mice was observed to be influenced
by strain, age, nutritional status and individual susceptibility.
There exist very large differences in toxicity between the
various plant agglutinins and there is reason to believe that so-me
of them may well be devoid of any toxic action (Liener,1974). For
example Phaseolus vu~garis (kidney bean) contains a hemagglutinin
which is toxic,to chicks (Honavar et al.1962) and rats (Jaffe and
Vegalet te, 1968) . It is detoxicated by heating and certain
cultivars, such as haricot beans may contain negligi-ble amounts.
Lesions arising from intoxication with lectin~ whether ricin,
abrin, crotin are similar and include macroscopic, and microsco-
pic lesions. Most notable lesions are extensive inflammation with
destruction of epithelial cells, oedema, hyperaemia and hemorrha-
ges in the lymphatic tissue.The liver presents fatty degeneration
48
and necrosis. The myocardium may show degenerative lesions and the
capillaries of all organs may be extended and filled with blood
clots. Local hemorrhages are frequently observed at the site of
injection (Brocg-Roussen and Fabre, 1977).
Thomson (1950) observed changes in the quantitative composi-
tion of plasma, liver and urine of rats acutely poisoned with ricin
and a reduction of the respiratory quotient of liver slices from
the same animals. He therefore concluded that toxic action of.
ricin may be due to its interference with toxic metabolic
processes in the liver possibly the Krebs cycle. In the studies of
Ikegwuonu and Bassir (1977) they observed rise in the blood values
of urea, bilirubin, transmaminases, lactic dehydrogenase with ricin
poisoned rats.
A hypothesis relating to hemagglutinating and toxic action of
lectins was advanced by Jaffe (1960) who reasoned that reaction
between the agglutinins and the cell membrane is the resulting
alteration of th& cell function and the production of the toxic
effect. Only those cells bearing the specific receptor groups for
the respective lectins would be affected. Thus the reduced
intestinal absorption produced by orally Lngest.ed lectin could
result from the combinations of the lectins with the cell lining
the intestinal wall which thus interfere with normal activity.
According to Lansteiner (1954) the specificity of the action
of the lectins on blood cells from various animals is comparable to
49
that of antibodies. The hemagglutinating activity of many plant
agglutinins can be inhibited by some sugars and oligosacch-arides
in a similar manner as certain hemagglutinating antibodies are
inhibited. This suggest-a structural relationship between these
inhibitors and the receptors groups for both agglutinating agents
located on the surface of R.B.C.
The black and red seeds of Abrus precatorius (Jequirity pea)
a perennial vine found throughout the tropics contain the very
poisonous phytotoxin, abrin, a substance very similar to ricin.
The powdered seed is toxic by mouth to all species though much less
poisonous than parenteral injections of abrin (Rahman and Mia,
1972). Ricin is a protein with molecular weight of 77,000 and its
solutions are coagulated by heat, the coagulation causing complete
Loss :of toxicity but not of antigenicity. An animal rendered
immune by injection of gradually increasing doses of the toxin can
tolerate amounts of up 800 times the normal lethal dose.
1.3.4. Polyphe~olics
Although tree leaves have a high protein content,tannins, and
other secondary compounds may bind this proteins, thus
rendering it unavailable to the animal. Tannins and related
polyphenolics may have negative effects in palatability and
digestibility and many are also poisonous (Woodward and Reed,
1989) .
50
The term tannin refers to a heterogeneous group of pheno- lic
compounds which precipitate protein to varying degrees, depending
on the type of phenolic and protein presents as well as the
chemical environment. Some tannins may bind proteins in the
neutral pH of the rumen and release them in the acidic abomasum
(Barry and Forss, 1983).
Acacia decurrens and A. salicina may be toxic on account of
their high tannin content of 35 per cent and 16 per cent respect-
ively (McCosker and Hunt, 1966). The clinical signs nclude
salivation, inappetence and ataxia of the hind limbs.The acorns and
leaves of Quercus species are known to contain large amounts of
tannic acid (acorns may contain 7 to 9 per cent) together with
small amounts of a volatile oils. It is generally considered that
it is this high tannic acid content which is responsible for the
toxic properties of acorns, but this is by no means established
(Clarke and Clarke, i975.Clarke and Cotchin (1956) conclude that,
aIthough the C'hief, if not the only toxic factor in acorns is
tannin, something apart from mere oral ingestion of tannin is
concerned in acorn poisoning. Dollahite, Pigeon and Camp (1962)
found that the lesions produced in the rabbit by -2. havardii are
very similar to those produced by multiple doses of tannic acid.
Supplejack (Ventilago viminalis) serves as a useful fodder, but can
cause poisoning in sheep owing to its high tannic acid content if
51
it forms the major part of the diet for too long a period (Pryor et
al., 1972).
1.3.5 Alkaloids
These are basically alkali substances which are mainly
nitrogenous in nature. Certain alkaloids found in lupines have
been identified as the cause of illness in animals.The following
alkaloids have been isolated from lupine species : lupinidine or 1-
sparteine, d-Iupanine, hydroxy-(oxy) lupanide and spathulatine.
The toxic effects of these lupine alkaloids, have been grouped
-under the term lupine poisoning, alkaloidal poisoning or lupine
madness (Gardiner, 1967, Smith, Jones and Hunt, 1974). Gardiner
(1967) proposed that the term lupine poisoning be used to
differentiate the syndrome associated with other, unidentified,
toxins in lupines which produced injury to the liver.
The agents responsible for the teratogenic action by Veratum
californicum are the steroidal alkaloids cyclopamine,jervine and
veratrosine. F~d at other pe~iod of embryogenesis, the plant gives
rise to other malformations for which different alkaloids including
possibly veratranine may also be responsible (Binns et al., 1965;
Keller, 1973).
Argemone mexicana (Mexican prickly poppy) though a native of
Mexico and North America is observed to be almost a cosmopolitan
weed. It contains the alkaloid sanguinarine, which in addition to
being a local irritant, causes depression of pyruvate oxidation by
52
brain tissue. This alkaloid is also found in other genera of the
family Papaveraceae (Hakim et al., 1961a). A. mexicana is said to
be of some importance in human toxicology as the ingestion of
argemone oil has been shown to produce epidemic dropsy associated
with glaucoma. Since sanguinaine is secreted in milk it has been
suggested that, the grazing of this weed may give rise to endemic
primary glaucoma in man (Hakim etal.,1961b).
Laburnum anagyroides (laburnum) is one of the most poisonous
trees.AII the parts of this plant are toxic especially the flowers
and seeds with the active principle being the alkaloid cytisine
which has a similar action as nicotine (Clarke et al., 1971).
Lethal doses of the seeds by mouth are in the horse (the most
susceptible animal) 0.5 g/kg and in the dog 6 g/kg.Cytisus
scoparius is observed to contain small amounts of cytisine together
with another alkaloid, sparteine. Sparteine has similar
physiological effects to coniine but it is less poisonous.
The bark o£ the African Erythrophloeum guineense (sassybark)
is well known as ordeal poison and an ingredient of arrow poisons.
It contains cas saine and other alkaloids and has been used for
malicious poisoning of animals (Hall, 1974).
All the parts of the plant Colchicum autumnale have been shown
to contain alkaloids, colchicine and colchicrine. Both are able to
withstand drying, storage and boiling. Colchicine is the most toxic
and according to Clarke and Clarke (1975) the seeds contain 0.2 to
53
0.4 per cent, the bulbs, 0.8 to 0.2%; the leaves 0.01 to 0.03%. In
doses of 0.25mg/kg body weight, colchicine exerts a purgative
effect. The average lethal dose for most species is shown to be of
the order lmg/kg. Colchicine is absorbed only slowly, and exerts
its toxic effects only after several hours and is also slowly
excreted mainly in the milk and urine so that there is a cumulative
effects from small doses.
Lilies of Gloriosa species which are native to Africa contain
colchicine type alkaloids and are extremely poisonous, giving rise
among other symptoms to generalized alopecia
(Gooneratne,1966).Heliotropium europaeum contains the hepatotoxic
pyrrolizidine alkaloids heliotrine and lasiocarpine and their
N-oxides, has caused large-scale poisoning, particularly of sheep
(Bull et al., 1961). Echium lycopsis which also contains the
pyrrolizidine alkaloids echiumidine and echimidine cause a similar
syndrome as R.europaeum, with symptoms of jaundice and
hemoglobinuria, in sheep. On post-mortem the livers are found to
be enlarged and yellow, and to have a copper content of 900 to
1200ppm (St. George-Grambaur and Rao, 1962).
1.3.6. Toxic Amino Acids
Unusual free amino acids are particularly frequent in
Lathyrus and Vicia such as Lathyri ne , homoarginine and hydroxy-
arginine which may be poisonous in large doses.Mimosine in Leucaena
leucocephala and Mimosa pudica causes hair loss in stock,
54
infertility and cataracts in rats and mice. It may interfere with
metabol ism of tyrosine, phenylalanine, and pyridoxal phosphate
(Nowack, 1980). Other amino acids of legumes whose structure
suggest that they may be. toxins are wallardine and aldizzine in
Acacia, though small amounts are also found in Pisum and Vicia as
stizolobic and stizolobinic acid. Species of Mimosoideae contain
the S- amino acid djenkolic acid (which causes kidney disorders in
human) sometimes accompanied by an acetyl derivative
(Nowacks,1980). Experimental poisoning of sheep has shown that
mimosine is largely broken down in the rumen to 3,4- dihydroxy
pyridine and excreted as such, (Hegarty et al., 1~fj4).A certain
degree of detoxication is brought about by moist heat (Mullenax,
1~63).Linum usitatissimum (linseed) produces toxic effect if it is
fed to chicken. The toxic effect have been shown to be due to
linatine, a peptide formed from glutamic acid and L-amino-D-proline
which has an antipyri~oxine action (Liener, 1~fj9).
1.3.7. Saponins
Both the vegetable parts and seeds of some legumes contain
large amounts of heat stable sapogenic lycosides (Nowack, 1980).
The best known ar-ethe soya sapogenols which are known to be bitter
tasting and decreases intake of fodder.Medicago also contains the
sapogenin medicagenic acid which is toxic. The growth of guinea
pig, mice and Japanese quail is inversely correlated with the
concentration of medicagenic acid. Some members of the
5~
Caryophyllaceae family including Saponaria, Agrostemina, Arenaria
and Stellaria are poisonous and known to contain saponin compounds
widely distributed throughout the plant kingdom and which contains
a sugar in combination with a steroid. Agrostemina githago which
contains the sapogenin, githagenin is probably the most poisonous
of these species. The growing plant is avoided by livestock but the
seeds, if mixed with corn or meal can cause poisoning. Bierer and
Rhodes (1960) reported that chicken fed 5% of corn-cockle seed in
the ration showed no ill-effect but Kotz (1965) observed that a
single dose of the ground seed of 2.5-8.0 g/kg will cause acute
poisoning while continued feeding for seven weeks can cause chronic
poisoning.
1.3.8. Flavones and Isoflavones
Flavones and isoflavones which have oestrogenic effects have
been found in many genera of the Papilionoideae (Nowack,
1980). Some members 'of the family Gramineae including Lolium
,
species (short rotation rye grass) and Lolium perenne (perennial
rye grass) contain significant amounts of oestrogen-like compounds.
There may be sufficient presence to cause sterility in cattle.
Trifolium pratense has been shown to contain appreciable amounts of
substances having oestrogenic activity (Cunningham and Hogan, 1954)
and this has produced reproductive disturbances in sheep grazing
it, (Chang,1958).--I.subterraneum (subterranean clover) has been
responsible for considerable economic loss to the sheep breeding
56
industry in Western Australia (Moule, 1961). The agent responsible
for this loss was isolated to be genistein, an isoflavone
possessing oestrogenic activity.
1.3.9 Oxalate-Bearing Plants
In animals cases of oxalate poisoning are encountered
usually due to ingestion of large amounts of plant containing
oxalates. Salts of oxalic acid are found in many plants.Clarke and
Clarke (1975) gives a list of plants containing oxalates as:
Atriplex spp., Beta vulgaris, Calandrinia spp., Emex australis,
Enchylaena tomentosa, Halogeton glomeratus, Oxal is spp. Portulaca
spp.Rheum rhaponticum Rumex spp.,Salsola kali,Sarcobatus
vermiculatus, Sefaria sphocelata; Threlkeldia proceri flora and
Trianthema spp.Salts of oxal ic acid are found in many plants.
Sodium and Potassium oxalates {and acid oxalates} are soluble, but
calcium oxalate is insoluble and is not assimilated during
digetion. The oxalate content of plants is observed to be
highest at the leafy stage of growth.Another plant demonstrated by
analysis {Marshall, Buck and Bell, 1967)to contain high levels of
oxalate is Amaranthus retroflexus. The leaves of this plant may
contain as much' as 30 per cent of total oxalate o~ a dried weight
basis. This plant is believed to be one of the causes of an entity
called perirenal oedema disease of swine (Buck et al., 1966).
The occurrence of nutritional secondary hyperparathyroidism
(NSH) or oseteodystrophia fibrosia in horses grazing a number of
57
grasses which causes lameness, debility is suggested to be due to
the oxalate present in these grasses, Walthall and Mckenzie, 1976).
Mckenzie et al.(1981) also demonstrated that potassium oxalate (2.6
and 4.3%) added to a di-et of oaten and lucerne chaff produced
negative calcium and phosphorus balances in horses but still
allowed some calcium to be absorbed.Many of the known haza-rdous
grasses (those on which NSH has occurred) however contain less than
2 per cent total oxalate (Blaney et al., 1981).The same authors
however noted in a different experiment that all grasses associated
with field cases of NSH produced negative calcium balances with the
main loss of calcium being in the faeces. The ratio of Ca:P in the
grasses (minimum of 0.69) indicated the little likelihood that
absorption of Calcium was depressed by a large excess of dietary
phosphorus. The pangola, green panic, kikuyi and buffel grasses
are observed to supply a calcium intake equivalent to or greater
than the requirement of 4.5 mg/kg live weight per day. However
they were observed, to produce negative calcium balances consistent
with the effects of oxalate (Mckenzie et aL, , 1981). Other
grasses, parabuffel and the setarias with more than 0.5 per cent
oxalate content were however seen to supply less -ca.Lc i um but the
resulting negative calcium balances were greater than the effects
that could be reasonably attributed to a primary calcium deficiency
alone.
58
Mofatt (1974) reports of hepatopathy with renal oxalosis in
bovine fetuses with a suggestion that the ingestion of toxic plants
could result in the fetal lesions. Acute oxalate toxicity has been
recorded in many herbivores and is principally due to excessive
ingestion of either oxalate rich plants or food cont- aminated with
oxalate producing fungi, (Andrews, 1971). Large amounts of oxalic
acid have been shown to be formed on moist straw infected with the
fungus Aspergillus niger. Such straw which is indistinguishable
from unspotted straw was suggested to be dangerous to horses and
other animals. Wilson and Wilson (1961) have shown that A.flavus
can also produce large quantities of oxalate. Silage infected with
oxalate producing fungi has been responsible for progressive
nephrosclerosis in horses (Andrews, 1971). Nutri tional status, the
ration of calcium to oxalate in the diet as well as the level of
oxalate ingested can be important factors in producing oxalate
toxicity, (Blaney et al,1981) .Oxalo- sis with resultant
hypocalcaemia can ,lead to coagulation defects and neuromuscular
dysfunction with the calcium oxalate crystals due to their
deposi tion in the kidney tubules which can lead also to renal
failure.Death has been observed in a Loala Phacolarctos cinereus)
with the typical lesions of oxalate poisoning which was associated
with its consumption of eucalyptus leaves and the leaves of certain
other plants with oxalates (Canfield and Dickens, 198~).
59
1.3.10 Plants Which Contain Dangerous Levels of Nitrate
The principal hazard to livestock from nitrates arises from
the fact that certain plants, grown on soils containing excess of
nitrate salts, may take· up sufficient to render them toxic to
animals eating them. Clarke and Clarke (1975) gives a list of HH
species of plants which may contain dangerous concentrations of
nitrate. It has been observed that the use of the herbicide
2,4-D( 2,4-dichlorophenoxyacetic acid) sublethal doses of which,
like periods of drought, may result in accumulation of amounts of
nitrate toxic to animals. Air-dried leaves from plants sprayed
with 2,4-D were found to contain 4.5 per cent potassium nitrate on
a dry matter basis, an amount considered to be well above the
minimum lethal concentration in animal fodder (Stahler and
Whitehead, 1950) whereas the untreated plants contained 0.22 per
cent. Weather has also been incriminated to affect the level of
nitrates in plants. Harris and Rhodes (1969) observed that on dull
days or at night,, the nitrate level is higher than it is in
sunlight as conversion to amino acids does not take place.
Subacute and chronic nitrate toxicity in livestock produces
signs of anorexia, dyspnea, grinding of teeth, restlessness,
vasodilatation, reduced blood pressure, abortion and decreased milk
excretion, possible avitaminosis A and possible thyroid malfunction
(Blood and Henderson, 1974). The potential influence of nitrate
toxicity on thyroid dysfunction has been studied in several animal
60
species. In rats a definite correlation is observed to exit by
thyroid hypofunction and nitrate poisoning (Lee et al., 1970) and
it appears that nitrate is an antithyroid substance in this
species. This effect is probably the result of nitrate
interference with the thyroidal iodide-concentrating mec-hanism
(Lee et al.,1970).When nitrate is present higher levels of iodine
are needed for the maintenance of normal thyroid glands. Abortion
in swine, sheep and cattle have been attributed to nitra-te and
nitrite poisoning.Schmitz (1961) postulated a relationship between
high maternal methemoglobin levels and abortion in pregnant
women.Stuart et al.(1968) using guinea pigs observed reproductive
dsst.ur-banecs in the female. Abortion, fetal absorption, fetal
mummification and maternal death were important manifeta-
tion.Hypoxia of fetus due to maternal methemogloin was hypothesized
as a cause of fetal death.
Nitrates per se are relatively non-toxic; their importance as
a cause of poisoning is due to their conversion either in the
foodstuff or wi thin the alimentary tract into nitri te. Nitrite
converts the haemoglobin of the blood into methemoglobin, which is
unable to act as an oxygen carrier (Mcilwain and ,Schipper, 1963;
Sinclair and Jones, 1964; Smith et al., 1974; Clarke and Clarke,
1975; Blood and Henderson, 1976). There has been many reports of
livestock poisoning by nitrate and nitrites. Andrade et aL,
(1971a, b). Hill and Blaney (1980) Rogers and Hopekawd (1980)
b1
reported that fatal intoxication was observed in cattle and
yearlings pastured in Brachiaria species which have high contents
of nitrate. Signs of poisoning reported were
hematuria, semi-pasty faeces, weakness, and pale or yellowish
mucous membranes retraction and intermittent urination with
incoordination when the animals were forced to move. There
were liver and kidney damage. The total nitrate in the serum
of affected cows was 0.468 mg per cent which was more than three
times that in control animals (O.14H mg %).The Brachiaria plants
from the pasture had high nitrate content (0.6%) which was the
cause of intoxication. Miyazaki and Kawachinia (1975) observed that
when as much as 50 per cent of the total haemoglobin in the sheep
was converted to methemoglobin by poisoning with sodium nitrite,
biochemical estimations showed that as the methemoglobin increased,
serum ammonia also increased and serum urea inecreased. These
,
authors suggested that nitrite was probably being reduced to
ammonia in the rumen and its synthesis to urea in the liver may not
be so efficient when methemoglobin is pronounced. A decrease
occurred in bilirubin could not be explained. Blood glucose
increased as methemoglobin increased. Methemoglobin analyses in
sheep fed with various Astragalus species, indicated that nitro-
compounds in A·diversifoluis,A.convallarius,and A·plerocarpus
resembled 3-nitro-I-propanol in toxicity and the rate of absorption
from the digestive tract (Williams and James, 1975).
ptercarpus, A.convallaris,and A·diversifolius produced acute
poisoning in sheep at 100 mg nitrite per kilogram body weight and
are the species most likely to cause livestock losses on the range.
1.3.10.1 Nitrates/nitrites and Nitrosamine formation
Nitrosamines can result from an interaction between nitrite
ion and existing secondary amines. For example N- nitrososcosine
is readily produced from creatine and nitrite ion (Archer et al.,
1971). Creatine is present in the muscular tissue of vertebrates
and is a normal constituent of meat. In vitro studies involving
nitri te and secondary amine have shown that gastric juices of
mammals provides an excellent medium for the production of nitroso
derivatives (Dombrowski and Pratt, 1972). Indeed investigations
revealed the presence of simple alkyl nitrosamines in food products
which had been treated with nitrites, (Fazio et al., 1971). Their
presence in food products has been demonstrated to be detrimental
to human health. The situation becomes more complicated when it is
observed that nitrite and secondary or tertiary amines in food can
combine to form nitrosamines which are potent carcinogens.
Magee and Barnes (1956) first reported the carcinogenicity of
dimethylnitrosamine (DMN) in rats and it has since been established
that many other nitrosamines are carcinogenic to a wide range of
species causing cancer at different sites of the body. Some can
cause cancer after only one dose, and some are powerful mutagens
(Liener, 1974). The first evidence that nitrosamines could be
63
present in food was provided by Norwegian workers who were
investigating a liver disease in fur-bearing animals. A similar
hepatic disorder was later observed in cattle and sheep (Liener,
1974).
R1 and R2 can either be alkyl or aryl group or in some cases
alicyclic.
The nitrosation of amines by nitri te leads to nitrosamines
which are then metabol ized to alky lating agents. Thus in vivo
nitrosation of dietary dimethylamine (DMA) will lead to the
formation of the carcinogen dimethylnitrosamine (DMNA) which causes
the methylation of liver DNA Bratchi et al. (1980) observed this
by administration of some doses of potassium nitrate and 14C-DM
,
hydrochloride in rats. After 6 hours, the liver DNA was isolated
and its specific radioactivity was compared with that obtained
after administration of either labelled DMA or DMNA. It was
observed that about 2-3% of the dose of DMA had been nitrosated to
DMNA.
64
1.4 TOXIC EFFECTS ON CELLULAR ELEMENTS OF BLOOD
1.4.1 Physiologic Properties of Constituents of Blood
The blood in an animal serves as a transport medium.It
carries nutrients from the digestive tract to the tissues,
end-products of metabolism from the cells to the organ of
excretion, oxygen from the lungs to the tissues, carbon-dioxide
from the tissues to the lungs and secretions of the endocrine
glands. Additionally the blood plays a role in regulation of body
temperature, maintain a constant concentration of water and
electrolytes in the cells, as well as the body, s hydrogen ion
concentration and defend against micro-organisms (Schalm, Jain, and
Sarro I , 1975) .Three classes of blood cells or corpuscles are
recognized, namely: the erythrocytes, or red cells,the leucocytes
or whi te cells and the thrombocytes or platelets. The leukocytes
defend the body; the erythrocytes transport oxygen, and carbon-
dioxide and the thrombocytes assist in the coagulation process.
The non-cellular portions of the blood include water electrolytes,
proteins, glucose, amino acids, enzymes and hormonal compounds,
(Jain, 1986).
1.4.2 Hematopoiesis
Hematopoiesis or hemopoiesis, means making blood,
particularly blood cells.The haematopoietic system is widely
distributed and includes organs having functions other than
contributing to blood formation such as the bone marrow, thymus,
65
lymph nodes, and follicles, spleen, mono-nuclear phagocyte system
(reticuloen- dothelial system) liver,stomach and intestine and the
kidney (Jain, 1986).
The mass of circulating red cells and erythropoietic
tissue in the bone marrow constitute a functional unit,call-
ed the erythron. Physiologic and pathologic states may affect the
erythron, resulting in an absolute or relative change above or
below normal level. The erythron in heal th remains in del icate
balance, for the daily production of erythrocytes, equal the daily
loss from destruction of senescent cells. The rate of erythrocyte
replacement in normal individuals is incredibly fast; almost 1
million red cells are replaced every second in a healthy 15 kg dog
for example (Jain, 1986). In response to anaemia, the normal
haematopoietic tissue can undergo a six to eight fold
increase in erythrocyte production.
1.4.3 Haematopathies associated with Poisons
The blood is susceptible to a number of diseases brought
about by infectious and non-infectious agents. The RBC for example
is designed to circulate in the blood stream for a number of days.
After this period presumably because its metabolic machinery or
stroma wears outs, the cell is removed from the circulation.
However certain untoward events can abruptly shorten the life span
of RBC. Prominent among these is the administration of drugs,
chemicals or plant substances. Such substances may produce
66
disturbances of blood circulation such as coagulation defects,
hemorrhages or anaemia and agranulocytosis (Schalm et al.,
1975).
1.4.3.1 Anaemia
Anaemia is rarely a primary diseasejrather it generally
reflects a secondary development. It is a sign and more commonly
only one of the results of a generalized disease process.Anaemia
can be evaluated and classified on the basis of erythrocyte morp-
hology,pathogenetic mechanisms and bone marrow erythroid respon-
ses (Smith,Hunt and Andrews,1974jJain, 19H6).
Chemical substances which produce anaemia are classified into
those which cause anaemia in all subjects e.g. plants containing
agglutinins (Liener, 1974) and those which produce anaemia in only
the occasional unusually susceptible individuals such as occurs
with the plant Vicia fava (favism) (Bowman and Walker, 1961).
Susceptibility to chemically induced anaemia may occur because the
RBC of the vulnerable individual are abnormal or because abnormal
plasma factors are formed in response to the administration of the
chemical substance.
67
1.4.4. Classification of Anaemia
Anaemias can be classified according to Smith,Jones and Hunt
(1974) under (1) pathophysiologic considerations into blood loss or
hemorrhagic anaemias; hemolytic anaemias and; depressive or
hypoproliferative anaemias (2) erythrocyte morphologic classi-
fication employing the two red cell indices, mean corpuscular
volume (MCV) and mean corpuscuar haemoglobin concentration (MCHC).
The size of the erythrocyte in the anaemic state is designated
macrocytic, normocytic or microcytic and the mean haemoglobin
concentration is designated either normochromic or
hypochromic.The macrocytic anaemia can be hypochromic or nor-
mochromic and may be divided into true or transitory macrocy-
tic anaemias.True macrocytic anaemias have normochromic
erythrocytes whereas transitory macrocytic anaemias have below
normal MCHC values.
Macrocytic normochromic anaemia is characteristic of
Vitamin B12 and folate deficiency, administration of certain
antimitotic drugs, severe liver disease, (as occurs with some
plant poisoning) splenectomy, myeloproliferative disorder inv-
olving red cells, and some leukemias.Macrocytic hypochromic anaemia
is observed during remission in acute blood loss or acute hemolytic
anaemia.
Normocytic-normochromic anaemias occur when there is selec-
68
tive depression of erythrogenesis in chronic disease. The
microcytic hypochromic anaemias occur when there is selective
depression of erythrogenesis as in chronic disease, (Jain, 1986).
1.4.4.1 Hemolytic Anaemias associated with Plant Poisoning
Hemolytic anaemia results from excessive destruction of the
circulating erythrocytes, occurring within the blood stream. Other
observations which are noticed to accompany hemolytic anaemia are
hemolytic icterus which may be detected by a high bilirubin level
in the blood or the physical signsjhemoglobiuria in the acute forms
an the presence of nucleated red cells and reticulocytes in blood
when it is chronic (Smith et al., 1974).
The category of drugs and chemicals which can cause hemo-
lytic anaemia includes copper, lead, phenothiazine, methylene blue,
saponins, naphthalenes and drugs such as acetanilide, nitrfuran-
toin, neoarsphenamine, phenacetin and some sulfonamides.
Copper toxicity anaemia starts with the accumulation of
copper in the li,ver of animals receiving copper or feeding on
copper-contaminated pasture or certain plants having high copper
content. For example damage to the 1iver caused by grazLng
Heliotropium europaeum can lead to abnormal accumulation of copper
and death resulting from the hemolytic crisis of chronic copper
poisoning (Clarke and Clarke, 1975). Some climatic conditions are
known to favor the growth of non-grainineous plants which
accumulate copper e.g. (Trifol ium subterraneum) (Hogan et al.,
69
1968) . The accumulation of the copper occurs over prolonged
periods during which animals are usually asymptomatic. Under
conditions of stress, copper is released to the blood stream to
precipitate rapid RBC destruction (Pearson, 1956; Smith, Jones and
Hunt, 1974).
In ruminants, the metabolism of copper,molybdenum and sul-
phate ions is inter-related.Copper-molybdenum complex in the
presence of inorganic sulphate is bel ieved to inhibit urinary
excretion and metabolism of copper in tissues, particularly in the
liver, leading to impaired synthesis of ceruloplasmin. Copper
toxicosis is common when a copper: molybdenum ratio in forage is
greater than 10:1 either because of excessive copper or deficiency
of molybdenum (Jain, 1986).
Ricin the toxic principle of the plant R. conmunis when
administered by various routes,will produce hemolytic anaemia
(Geary, 1950). The anaemia which often accompanies poisoning by
Brassica specie,s is also believed to be hemolytic in origin
(Greenhalgh et al., 1969).
Brassica aleracea has been reported to cause Heinz body
anaemia in cat t Le , sheep, and goats (Dunbar and Chambers, 1963;
Greenhalgh et al., 1969). Sheep developed a relatively mild
anaemia but sheep with low levels of glutathione (GSH) were more
susceptible than those with high levels.Affected animals showed
retarded growth, hemoglobinuria, icterus and hemosiderin deposits
70
in the liver. Brassica species feeding in goats has been reported
to induce hemolytic anaemia but recovery followed even though the
feeding of the plant continued (Greenhalgh et al., 1909).
Allium cepa or onions,(wild and domestic) contain n-propyl
disulfide, which produces Heinz body anaemia in cattle (Hutchinson,
1977), sheep (Van Kampen, Lynn, James, and
Johnson, 1970) and horses (Pierse, Joyce, England and Jones,
1972) . Jain (1986) reported the observation of hemolytic anaemia and
some deaths among steers being fed cannery offals, consisting of
onions and tomato pulp and vines. Both symptoms and lesions can be
summarized as hemolytic anaemia, hemolytic icterus, and
hemoglobinuria. Anaemia as estimated by haemoglobin determination is
said to be extreme.
Horses that ingested Acer ruburum leaves developed acute
hemolytic anaemia from an oxidant present in the leaves (Divers,
George, and George 1982; Tennant, Dill, Gl ickman, Mino et aL, ,
1981). Clinical observation included weakness, polypnea,
tachycardia,depression,icterus,cyanosis and brownish discolora-
tion of the blood and urine.Hematologic findings included
methemoglobinemia, free plasma haemoglobin' decr-eased PCV, and Heinz
bodies in erythrocytes.
In the case of Vicia faba hemolytic anaemia and hemoglo-
71
binuria are also symptoms (Bowman and Walker, 19tH).A specific
hemolytic anaemia, favism in certain human subjects is said to be
dependent upon a genetic factor and the ingestion of Vicia faba.
1.4.4.2. Hypoproliferative anaemia associated with Plants
The excessive ingestion of a wide variety of plants or
their products, have been found to cause hypoproliferative anaemia.
In this situation,the hematopoietic tissue of the bone marrow are
injured in such a way that their ability to produce erythrocytes is
impaired or destroyed.
Hypoproliferative anaemia may result from limited supply
or defective utilization of nutrients essential for red cell
production or from anatomic disruption, functional impairment
or lack of stimulation of hematopoietic tissue. In anaemia
secondary to chemicals, plants or drug toxicity, the bone marrow,
ability to produce red cells is often suppressed or destroyed by
the insulting agent so that anaemia ensues in the face of adequate
essential nutrients. Cellular damage in such cases may be mild,
and reversible upon removal of the primary cause, or it may be
severe and permanent. In most cases of marrow suppression
,secondary to drugs, chemicals or plant poison~ng, the anaemia is
normocytic normochromic (Jain, 19H6).
In Pteridium aguilinum poisoning, the clinical picture
and hematological changes, are characteristic of an aplastic
anaemia. The fundamental lesions appear to be a depression
72
of bone marrow activity leading to leucopenia,and thrombocy-
topenia(Rao,Joshi and Kumar,1988).There is increased capillary
fragility, prolonged bleeding time and defective clot retraction.
Numerous petechial hemorrhages can be seen on the visible mucous
membrane and there may be bleeding from the nostrils and intestinal
and urogenital tract especially at the latter stages. Postmortem
reveals extensive hemorrhages throughout the tissues of the body.
Blood smears are found to streak, a phenomenon associated with
abnormal fibrinogen components and increase in
circulating heparin-like substances (Evans, 1964; Singh et
al., 1987; Rao et al., 1988).
1.4.4.3. Sensitivity to Drug-Induced Hemolytic Anaemia due to
Intra-erythrocytic defects
This may be due to metabolic abnormalities or unstable
haemoglobin.The erythrocyte is a living, metabolically active cell.
It must maintain a gradient of sodium and potassium across its
membranes reduce methemoglobin that has been formed by metabolic
activities (Beutler, 1969).
In red cell metabolism, there are three pathways for
energy in form of ATP formation. When glucose is phosphory-
lated to glucose-6-phosphate in the hexokinase reaction, further
metabolism tak~s place by the (1) Embden-Meyerhof way which is the
major way in which glucose is metabolized anaerobically to pyruvate
or lactic acid. This pathway provides energy in the form of ATP
73
and can reduce nicotinamide adenine dinucleotide (NAD) to NADH for
methemoglobin reduction. (2) the hexose monophosphate shunt in
which no ATP is formed but NADP is reduced to NADPH. The main
function of this route of metabolism appears to be to provide
reduced NADP for the reduction of oxidized glutathione.
Glutathione plays a role in maintaining sulphydryl enzymes within
the red cell in active form and in detoxifying small quantities of
hydrogen peroxides. The levels of hydrogen peroxides that may
appear when certain types of drugs react with oxyhaemoglobin may be
so slow that catalase represents a relatively inefficient means of
their disposal, (Cohen and Hochstein, 1963}.Another system the
glutathione peroxidase system appears to be effective in
detoxifying such small amounts of peroxides.
The glutathione that is oxidized (to oxidized glutathione) in
the glutathione peroxidase reaction, that which may be oxidized in
the process of reducing mixed disulfides, and possibly mixed
disulfides themselves, can be reduced to glutathione through the
mediation of the enzyme glutathione reductase with NADPH as
substrate.Thus the complete pathway requires three groups of
enzymes, the glucose-6-phosphate, dehydrogenase-phosphogluconic
dehydrogenase system, to reduce NADP to NADPH; the glutathione
reductase sYJtem, to oxidize NADPH to reduce oxidized glutathione
to glutathione, and finally the glutathione peroxidase system that
oxidizes glutathione for the reduction of peroxides. The red cell
74
becomes excessively susceptible to drug induced hemolysis when any
of these three enzyme systems is not functioning properly (Beutler,
1969) .
1.4.4.4. Deficiency of Glucose-o-Phosphate Dehydrogenase
Hemolytic anaemia associated with the administration
of 8-aminoquinoline antimalarial compounds such as pamaquine
napthoate, primaquine, isopentaquine has been recognized since
shortly after the introduction of these compounds into medical
practice, (Hockwald et al., 1952). Primaquine was very effective
antimalarial compound, but it produced hemolytic anaemia in
susceptible persons.
Hemolysis of individuals with deficient erythrocyte levels of
glucose-6-phosphate dehydrogenase is known to occur upon exposure
to various drugs.It has been suggested that the oxidative changes
observed during hemolysis e.g. loss of glutathione, (GSH) oxidation
of haemoglobin, are manifestations of the presence of hydrogen
peroxides (Cohen ~nd Hochstein, 1903). Glucose-o-phosphate
deficient red cells are sensitive to hydrogen peroxides by virtue
of diminished generation of NADPH.
Specific hemolytic anaemia,favism in certain human subjects
is said to be dependent upon a genetic factor and the ingestion of
the broad bean Vicia faba (Bowman and Walker, 1901).The defect is
noted to result in the deficiency of the enzyme Glucose-o-phosphate
dehydrogenase.The deficiency in this enzyme (G-o-PD) is reflected
75
in reduced glutathione in the red blood cells. A similar hemolytic
anaemia is observed in horses and cattle which have been fed the
same type of plant (Panciera, Johnson and Osburn, 1900).
Earlier studies by Dern et al.(1955) had observed that not
only do other 8-aminoquinoline derivatives produce lysis of
primaquine-sensitive cells in vivo, a variety of other compounds
such as sulfanilamide (Prontosil(R) album) acetanilid (Antifebrin),
phenacetin and small doses of phenylhydrazine, diphenylsulfon
(Dapsone(R»), furazolidone, nitrofurazone, do so as well.
These drugs have been shown to produce selective destruction
of the older red blood cells while the young cells appeared to have
a capaci ty to protect themselves against destruction. Later
observations show that in some types of G-o-PD def iciency even
quite young cells are destroyed and hemolysis was therefore not
self limited (Salvido, Pannacciulli, Tizianetho and Ajmar, 1907).
1.4.4.5. Glutathione reductase Deficiency
Reports have been made of cases with suffoxone- induced
hemolytic anaemia whose red cells G-o-PD act.Ivi ty was normal,
(Beutler, 1969). Deficiency of glutathione reductase of the red
cells has been associated with pancytopenia,with non-spherocytic,
hemolytic anaemia with or without thrombocytopenia and leukopenia
with thrombocytopenia and even with isolated instances of
76
leukaemia and hemophilia B.
1.4.4.6. Glutathione deficiency
A marked diminution of the content of both reduced and
oxidized glutathione in red cells was first described by Oort and
associates (Beutler, 1969). The condition is said to be very rare.
Glutathione (GSH) deficiency is associated with a mild
non-spherocytic hemolytic anaemia. Ashby survival studies showed
that the mean life span of GSH deficient red cell was only
one-third to one-fourth of the normal red cells. Some evidence
suggest that the plant Vicia faba might induced hemolysis in
glutathione deficient patients (Prins, Oort, Loos, Zurcher and
Berkers, 1966).
1.4.4.7. Phosphogluconic dehydrogenase (PGD) deficiency
There can be the partial as well as the complete absence of
pho sphog Luc oni c dehydrogenase (Parr and Fitch 1964) •Phosphoglu-conic
dehydrogenase is the second enzyme in the hexose monophosp-hate
shunt.Decrease iU its activity would limit reduction of NADP to
NADPH and could also result in the accumulation of 6-phosphog-
luconic acid in red cells.If drug induced hemolysis does occur in
PGD deficiency, the mechanism would be essentially the same as that
in the G-6-PD deficiency (Beutler, 1969).
1.4.4.8 The Unstable Haemoglobin
Abnormalities in the globin portion of haemoglobin
molecule affecting either the alpha or beta chain near the
77
site of attachment of heme gives rise to instability of
haemoglobin and results in sensitivity to the hemolytic
effect of some substance.
Hemoglobinopathy.has resulted in the production of
hemolytic anaemia in patients treated with, for example
..• Sulfisomidine (Elkosm). The feeding of the plant Brassica
oleracea (Kale) to cattle was shown to produce reduced
haemoglobin with relatively severe anaemia and the production
of haemoglobin C by sheep with haemoglobin genotype AB and AA
(Grant et al., 1968; Tucker, 1909). The plant also produced
increased numbers of Heinz bodies in the red blood cells of
sheep with haemoglobin genotypes BB, AB and AA.
In these hemoglobinopathies,the RBC usually contained
inclusion bodies, but investigation of the G-o-PD activity,
glutathione concentration and glutathione stability all resulted in
normal values. Unstable haemoglobin apparently readily becomes
denatured into,insoluble aggregates. This process appears to be
hastened by substances that catalyze the oxidation of haemoglobin.
Oxidative denaturation of haemoglobin is prevented by
methemoglobin reductase,superoxide dismutase(SOD) glutathione
peroxidase, and catalase. Certain oxidants perhaps those with a
mild to moderate oxidative action produce only methemoglobinae-
mia, whereas strong oxidants also react with globin chains to
induce denaturation and precipitation of haemoglobin in the form of
78
Heinz bodies. For example, it has been observed that substances as
acetaminophen in cats produces both methemoglobinemia and
Heinz body formation, while nitrite poisoning in cow and pigs
produces only methemoglobinemia (Jain, 19M6). Development
of significant methemoglobinemia has been observed in an
occasional cat anaesthetized with ketamine hydrochloride.
Cats develop Heinz bodies without metahaemoglobinaemia under
natural condition and after methylene blue administration.
Horses feeding on the leaves of Acer rubrum were observed to
also develop acute hemolytic anaemia from methemoglobinemia and
Heinz body formation (Divers et al., 19MG; and Tennant et aL, ,
1981).
1.4.4.9. Drug-dependent immune Hemolytic Anaemia
The first case studied to implicate immunologic factors in
drug-induced hemolytic anaemia involved stibophen (Fuadin)
(Harris,1956).It was found out that an antibody in the serum was
bound to the r~d cells in the presence of the drug or in one of its
derivatives. A di fferent type of drug- induced hemolytic anaemia
was reported by Ley et aI . (195M) after the administration of
massive amounts of penicillin. In their studies, the antibody was
found to be effective against red cells that merely had been
treated with a drug.
Though cases have not been associated with plant poisonings to
produce immune hemolytic anaemia, this by no means does not mean
79
the situation does not exist since many plant poisonings cause
hemolytic anaemia.
1.4.5. Hemorrhage
Hemorrhage is the escape of blood from a vessel whether it be
to the outside of the body, into a body cavity or into adjacent
tissues. Toxic injury to capillary endothelium culminating in
transient openings of punctiform size is chiefly responsible for
the petechiae and ecchymoses seen on serous and mucous membranes,
as well ~s within the depths of the tissues.
1.4.5.1. Poisonous Plants producing hemorrhages
Poisonous plants such as Melilotus alba (sweet clover) contain
the compound dicoumarin, a potent anticoagulant for the blood
(Fraser and Nelson,1959). Cattle which consume hay containing
M.alba for a month are reported to show uncontrollable hemorrhage
from accidental or operative wounds and slow internal hemorrhages
as a result of bruises and minor injuries. Postmoterm lesions show
hematomas in the, subcutis, between the muscles or beneath the'
capsules of organs. Ecchymotic hemorrhages occur in many places,
commonly beneath the endocardium. The liver lobule show petechiae
(Prier and Derse, 1962; Smith et al., 1974).
The illness that is associated with Pteridium aguilinum is
sudden hemorrhages from many and often several body openings,
delayed clotting time, thrombocytopenia, neutropenia, anaemia and
death. Autopsy lesions include widespread petechiae and
80
ecchymoses, especially on the heart and other serous surfaces, on
mucous membranes and in muscles and the subcutaneous tissues.
Abomasal ecchymoses in cattle is said to lead to ulceration (Evans,
Patel, and Koohy, 1982;Rajendran, Chennakesavalu Vivaraghavan and
Danodaran, 1983).
Poisoning by members of Crotalaria genus which are mainly
leguminous plant, include hemorrhages which appear in the form of
petechiae or large ecchymoses. The hemorrhages are noted to be
characterized by a yet unexplained bright red color, involving the
serous and mucous surfaces. All organs are said to be congested,
many are edematous, especially the abomasum, omasum and gall
bladder (Allen, Carstens, and Knezevic, 1965; Carstens, and Allen,
1970).
Abrus precatorius (Jeguirty pea) produces congestion of
the visceral organs and petechial hemorrhages throughout the
body. Anthoxanthum .odoratum (sweet vernal) is known to produce
hemorrhagic diathesis in cattle fed home produced hay (Clarke and
Clarke, 1975).
1.4.6. Methemoglobinemia and Methemoglobin forming substances.
A great variety of chemical substances are' known to convert
haemoglobin into methemoglobin and thereby impair the blood
capacity to transport oxygen to the tissues. Nitrates and nitrites
in food plants and water which are themselves innocuous may by the
action of intestinal micro flora be converted into nitrite and so
81
form methemoglobinemia when transported into the blood circulation
(Miyazaki and Kawashima, 1975; Andrade et al., 1971). There are
reported instances in which without obvious external cause,
methemoglobin is formed from haemoglobin and constitutes a constant
hazard to the well being of the animal.
Bodansky (1951) gives a review of some of the methemoglobin
forming substances. The substances include nitrobenzene, aniline,
p-aminotoluene (p-AT) paraquinoacetophenone (p-AAP) and
para-aminopropiophenone (p- APP).
1.4.6.1. Formation of methaemoglobins
Methemoglobin is an oxidation product of the normal blood
pigment haemoglobin. The prosthetic group of haemoglobin is
variously known as heme, ferrohaeme (Pauling and Coryell, 1936) and
is a complex of iron and protoporphyrin IX.ln methemoglobin, iron
is present in the ferric state. Methemoglobin is also known as
haemoglobin and ferrihemoglobin.
Methemoglobin may be formed from haemoglobin in a variety of
ways.Essentially the action is an oxidation of the ferrous to the
ferric ion, which is brought about by any of these ways: (a) by the
direct action of oxidants (b) the action of hydrogen donors in the
presence of atmospheric oxygen and (c) autoxidation. The ferrous
ion of haemoglobin may be oxidized directly by ferric tartrate,
ferricyanide, bivalent copper, chlorate, nitrate, quinones,
82
alloxans and dyes of high oxidation-eduction potential (Bodansky,
1951).
Another mode of oxidation is that of the action of hydrogen
donors in the presence-of atmospheric oxygen. Chief representatives
in this group are the dyes e.g phenol indophenol , and methylene
blue. The formation of methemoglobin from oxyhemo-globin by
substances such as aminophenols, phencyhydroxylamines,
phenylhydrazines and hydrazobenzene may be formulated in a similar
manner.
The third important mechanism in the formation of methhemo-
globin from haemoglobin is autoxidation.Bodansky (1~51) reported
that various autoxidazable substances present in turpentine,
linseed oil, the alcohol-soluble substances ofpotato juice and
sterile pneumococcus and other bacterial filtrates were capable of
accelerating the formation of methemoglobin. This type of
methemoglobin formation which is greatly dependent upon the oxygen
tension, is negligible at zero oxygen tension and maximal at
relatively low tension where about half of the haemoglobin is in
the form of oxyhaemoglobin. At higher oxygen tensions, where a
greater proportion of the haemoglobin in the form of
oxyhaemogolbin, the rate of formation of methemoglobin is again
decreased.
1.4.6.2. Chemical interactions and properties of methemoglobin
Methemoglobin can combine with a great variety of substan-
ces.Most of these interactions consist of a combination of the
ferric ion with other ions such as fluoride or cyanide, but changes
in the globin part of the molecule are also possible:
1. Methemoglobin hydroxide-Methemoglobin interacts with hydroxyl
ion by attaching to the sixth bond position of the iron atom. The
addition of sodium hydroxide to methemoglobin may also result in
the denaturation of the globin moiety.
2. Methemoglobin fluoride-Methemoglobin reacts with fluoride ion
to form methemoglobin fluoride also known as fluoro-methemoglobin.
3. Cyanomethaemoglobin-the interaction of methaemoglobin with
cyanide is of considerable interest since it is this compound which
has been considered under the therapeutic effectiveness of
methemoglobin in counteracting the toxic or even lethal action of
cyanide.The formation of cyanmethaemoglobin is the basis of the
therapeutic and prophylactic actions of certain compounds in
cyanide poisoning (Chen et al., 1934)
4. Hydrogen peroxide methemoglobin - the addition of
hydrogen peroxide to oxyhaemoglobin leads to succession of
reactions in equilibrium, mainly the formation of methemoglobin,
the combination of methemoglobin with hydrogen peroxide, the
reversion to methemoglobin itself as well as the partial oxida-
84
tion of methemoglobin through peroxidase reaction to other
compounds.Hydroperoxide methemoglobin is not a stable compound.
The nature of the oxidation products of methemoglobin caused by
hydrogen peroxide is of interest in connection with a study of the
nature of bile pigment (Lemberg et al., 1941). The authors
showed that the green pigment choleglobin can be formed by the
action of hydrogen peroxide directly on methemoglobin or even
indirectly from haemoglobin.
5. Methemoglobin azide-azide forms a definite compound with
methemoglobin similar to that which cyanide ion forms.
6. Methemoglobin hydrosulfide-hydrogen sulfide reacts with
methemoglobin at an acid pH, to form methaemo- globin hydrosulfide
(Bodansky, 1951).
7. Other compounds of methemoglobin-methemoglobin has been shown
to combine with a number of other substances such as fulminate,
thiocyanate, cyanate, ethanol, ammonia, imidazole and oxidation
products of aniline.
1.4.6.3. Effect of methemoglobinemia on blood oxygenation
When oxygen tension in the blood is plotted against the degree
of oxygenation, (or percent of oxyha.e mogLobi n sigmoid curve is
obtained.The affinity of oxygen for haemoglobin is influenced by
temperature, pH, ionic strength, concentration of haemoglobin and
by the presence of other blood pigments such as carboxyhemoglobin
and methemoglobin.
85
Douglas et al. (1912) showed that the presence of carboxyhe-
moglobin shifted the dissociation curve of the residual haemoglobin
to the left and the form of the curve even became more hyperbolic
in the presence of ca r-boxyhaemogLobi n . It can therefore be
observed that the effects of poisoning by carbon monoxide are
two-fold. Some of the haemoglobin is combined with carbon monoxide
and therefore becomes unavailable for transport of oxygen and also
the residual oxyhaemoglobin becomes less capable of dissociation
Darling and Roughton (1942) demonstrated that methemoglo-
bin produces a similar effect. Quanti tati vely, however, the effect of
methemoglobin is said to be less than carboxyhemoglobin. For
example, a content of 23% carboxyhemoglobin shifted the
dissociation curve of the residual oxyhaemoglobin in human blood
about as far to the left as did a content of 43% methemoglobin
(Darling and Roughton, 1942). The effect of methemoglobin on the
oxygen dissociation curve is wholly reversible.
The alteration of the shape and position of the oxygen
dissociation curve in carboxyhaemoglobinaemia or methemoglobi-
nemia implies that the tissues are liable to anoxia, not only
because of l6ss of the oxygen-carrying capacity of the blood but
also because of the residual oxyhaemoglobin is less capable of
dissociating and therefore unloading oxygen in the tissue.
Smith and Gosselin (1964) observed that methemoglobin induced
in mice and other animals protects against the lethal
86
effects of inhaled hydrogen sulfide ·or injected sodium sulfide.
This protective effect was said to be due to a trapping and/or
inactivation of free hydrosulfide anions by methemoglobin.
Cyanmehaemoglobin prepared and injected intraperitoneally into mice
protected the animals against injected sulfide. This suggests that
the molecule must possess binding or inactivation sites. For
suIfide in addition to the ferric heme site already occupied by
cyanide.
The reaction of nitrite with haemoglobin therefore,appears to
produce at least two kinds of changes: (a) oxidation of the heme of
iron which produces one binding site/heme group available to a
variety of ligands and (b) globin alterations which results in the
production of 1 to 3 specific sulfide inactivation site/heme group
(Smith,1967).
The usual source of nitrate and nitrite poisoning in vetrin-
nary practice is in plants, growing or cured, which have derived a
large amount of nitrate. A review of plants containing dangerous
levels of nitrate and nitrite has already been presented under this
review. In presence of moisture and possibly with the aid of
bacteria, the phytogenous nitrates are easily reduced to nitrites
(and eventually to ammonia). This occurs within the stomach, and
especially the rumen, or externally as in stacks of hay that have
become wet (Miyazaki and Kawashima. 1975; Andrade et al., 1971).
87
The outstanding symptom from nitrate and nitrite poisoning is
the dark, brownish color of the blood, the effect of methemoglobin
{Andrade et al., 1971).Clotting is said to be normal but the mucous
membranes are cyanotic except those of the stomach and intestines,
which show more or less hyperaemia and inflammation.
1.4.7. Coagulation and Platelet interaction during Hemostasis
The process of coagulation is simply defined as the conver-
sion of fibrinogen to fibrin by the action of an enzyme thrombin,
the conversion being accelerated by the presence of calcium ions
and heat labile accelerator. The coagulation process is one of
four steps namely the contact reaction, thromboplastinogenesis
thrombin formation and fibrin formation.
The hemostatic response is mediated by the cell surfaces and
collagen exposed at the wound site. Exposure to collagen initiates
activation of the intrinsic coagulation system beginning wi th
conversion of factor XII (Hegeman factor) to XlIa. Prekallikrein
and high molecular weight- kinninogen at the injury site enhances
the activation of the intrinsic pathway. Ruptured endothel ial
cells smooth muscle cells and other damaged cells exude tissue
factor initiating the extrinsic pathway beginning with factor VII.
Simul taneously platelets coming in contact with exposed collagen or
thrombin (factor IIa) become activated i.e. they become spherical
and adherent. Platelets bind to collagen via their surface
receptors form von Willebrands factor complexed to factor VII of
88
endothelial cells. Activated platelets then undergo the release
reaction. Platelets release a variety of substance including ADP
and thromboxane A2 both of which cause more platelets to adhere and
aggregate thus enlarging the platelet plug. Availabili ty of
platelet factor 3 and V (Proaccelerin) on the surface of the
activated platelets further potentiates the clotting process (Jain,
1986) .
During the hemostatic phase, factor V molecules at the
surface of platelets in the hemostatic plug serve as binding
sites for factor Xa. The binding step localizes the clotting
process and markedly enhances the speed of the reaction. In
the fibrin generation phase the localized factor Xa molecules
trigger formation of a path of cross-linked fibrin molecules
in which red cells become trapped. The platelet surface membrane
protects the attached factor Xa and possibly thrombin (factor IIa)
from degradation by the antithrombin III - heparin complex or other
serine protease inhibi tors. Finally in the wound repair phase,
thrombosthenin, within clotted platelets contracts, converting the
bulky plug into a tidy patch.
1.4.7.1. Acquired Coagulopathies
Jain (1986) mentions that the acquired coagulopathies
are more common than the inherited disorders and are usually
associated with multiple coagulation abnormalities.The diagnosis of
such disorders is often indicated by the associated cIinical
89
features and by results.of screening tests such as prothrombin time
(which assays extrinsic (factor VII) and common (factors X, V, II
and I) pathways; act ivated part ial thromboplast in time (assays
intrinsic (factors XII,· XI, IX, and VII) and common factors X, V,
II and I) pathways; thrombin time (assays fibrinogen (factor 1).
Chemical substances which produces coagulation abnormalities
usually cause platelets disorders, vitamin K, deficiency and
antagonism, liver disease dysproteinemia or fibrinolysis.
1.4.7.2. Destructive lesions of the liver and blood coagulation
Destructive lesions of the liver which affect the coagul-ation
process occurs in severe forms of acute (e. g. Heliotropium
europaeum, aflatoxicosis (Doerr, Wyatt, and Hamilton, 1976) or
chronic hepatitis (cirrhosis) where there is failure of the blood
to clot due to deficiency of prothrombin which is produced by the
liver cells.
Aflatoxicosis which is associated with various hemorrhagic
episodes causes the loss of one of the two active sites of tissue
thromboplastin. For example in studies in chicken, it was observed
that factors VII and V activities and fibrinogen were reduced at
2.5 ug/g and above of aflatoxin. Factor X activity was depressed
at 5.0 and 10 ug/g. The clot retraction in the whole blood was
found to increase with growth inhibitory levels of the aflatoxin.
The series of studies suggested the severe impairment of extrinsic
and common clotting pathway functions in chicken and that
90
prothrombin is particularly affected during this coagulopathy
(Doerr et al., 1976). However in similar studies the elevation of
prothrombin times and of leucocyte counts were observed within 24
hours of a. flavus intoxication.
1.4.7.3. Hypocalcaemia and blood coagulation
Hypocalcaemia may result in failure of the blood to clot if
calcium levels required for the coagulation process is below
normal. Oxalates are used routinely in hematology to prevent the
clotting cifblood sample by formation of insoluble calcium oxalate
and thereby remove soluble Ca++ required for the clotting mechanism
(Swenson, 1975).
It is to be expected that the same reaction occurs when an
animal ingests large amounts of oxalate at a time as observed to
occur in poisoning by plants such as Halogenton glomeratus (Shupe
and James, 1969) or Amaranthus retroflexus (Buck et al., 1966;
Marshall, Buck and Bell, 1967). The signs consist of depression,
anorexia, slight to moderate bloating, weakness, in coordination,
restlessness frequent attempts to urinate,occasional reddish brown
urine and brownish black faeces, blood tinged nasal exudate, coma,
followed by death. There is widespread hemorrha~es especially in
the rumen.
1.4.7.4. Platelet Disorders
Platelet disorders include thrombocytopenia thrombasth-
&
91
enia,thrombocytopathy (abnormal pl~telet function) thrombocytosis
and thrombocythemia. Acquired functional disorders of platelets
with or without hemorrhagic manifestation associated with toxicity
have been reported in -therapy with certain drugs particular Iy
nonsteroidal anti-inflammatory drugs notably aspirin. Platelet
abnormalities have also been observed to have some relationship to
the level of blood urea nitrogen (BUN) and duration of uremia. Urea
per se does not seem to be directly detrimental but its metabolites
quanLdi no auccLn i.c acid and phenolic acids are believed to be
involved. Decreased production of prostaglandin endoperxidases by
platelets and increased production of prostacyclin (PGI2) an
inhibitor of platelet adhesion and aggregation by blood vessels of
uremic patients are thought to act synergistically and contribute
to the bleeding tendency (Remuzzi and Cavenagh, 197H j Rao and
Walsh, 1983).
The liver is the principal organ concerned with the produc-
tion of most of the procoagulants and thus it is obvious that a
bleeding diathesis would follow a significant decrease in this
function of the liver. Additional contributory factors include
thrombocytopenia, dysfibrinogenaemia, enhanced -fibrinolysis and
abnormalities of platelet function (Rao and Walsh, 19H3).
A reduction in the number of circulating platelets
results in purpura hemorrhagica owing to the prolongation of the
bleeding time.Oedema and hemorrhages of the subcutaneous tissues,
92
mucous membranes and internal organs are the characteristic
pathological and cl inical findings . Plants such as £. aquil inum
which produce aplastic anaemia are reported to produce their effect
through affecting blood-clotting (Rao et al., 19HH). Anthoxantum
odoratum which produces hemorrhagic diathesis in cattle causes
increased prothrombin and partial thromboplastin times.
1.4.7.5. Vitamin K Deficiency and Antagonism
Vi tamin K is essential in the formation of several coagu-
lation proteins referred to as the vitamin K-dependent coagulation
proteins namely factors II, VII, IX and X.
Vitamin K antagonism is encountered in animals upon ingestion
of substances such as warfarin, indanediones, bromadialone,
and brodifacoum whose mode of action depends on vitamin K
antagonism (Mount and Feldman, 19H3). These substances were
observed to inhibit the enzyme system (epoxide reductase
enzyme) essential for recycling of vitamin K necessary for
activation of precursor forms of coagulation factors produced
by the liver.
Poisoning by Mellilotus alba (sweet clover) is reported to
produce serious coagulopathies because of its content of dicoum-
arin a potent vitamin K antagonists (Fraser and Nelson,1959j Prier
and Derse, 1962). Infact Warfarin is a derivative of dicoumarin.
1. 4.8. Leukocytes
The leucocyte system serves to defend the body against
foreign organisms or extraneous material. They are of greatest
numbers in the peripheral blood and are subdivided as neutrophils,
lymphocytes, monocytes, eosinophils and basophils. Each subtype
has a somewhat different function and each may b have a 8 p rAt@
but related system. Leukocytes dOff r fr m the oth r G ~l YP 8 in
the blood because most of its functions are performed outside the
vascular system where they defend the body by phagocytosis
(neutrophils, monocytes, eosinophils and basophil) or antibody
production as carried out by lymphocytes or their more mature
forms, plasma cells (Swenson, 1975).
Only about half of the leukocytes in peripheral blood circu-
late freely with the remaining being attached to the walls of the
blood vessels (or marginated). Upon tissue injury the marginated
neutrophils pass between vascular epithelium cells by diapedesis
into damaged area where they engage in phagocytosis and are found
in inflammatory exudate.Toxic substances may cause leukopenia by
suppressing granulocytopoiesis or lymphopoiesis.Toxic substances
of plant origin, such as bracken fern,certain drugs as sulfonami-
des and similar chemicals which compete with folic acid utilization
cause leucopenia (Jain, 1986).
94
1.4.8.1. The neutrophils
The neutrophils form the first line of cellular defence
against microbial infection. Mature neutrophils have
characteristically polymorphic segmented nucleus with undulated
nuclear membrane and clumped chromatin. Defective bactericidal
activity of neutrophils may be related to cellular dysfunction of
phagocytosis, postphagocytic events and bactericidal action.
Defective bactericidal activity of neutrophils has been found in
various conditions such as ureamia and jaundice (Wardle and
Williams, 1980); drug therapy (example amphotericin B,
tetracycline, certain sulfa drugs, corticosteroids and some
antineoplastic drugs such as methotrexate and Vinca alkaloids,
(Mandell, 1982). Neutropenia and terminal anaemia accompany the
thrombocytopenia in Pteridium aquilinum poisoning and are due to
destruction of the early myeloid cells (Smith et al., 1974).
1.4.8.2. The eosinophils
The eosinophils have characteristically bright pinkish red
uniformly stained cytoplasmic granules and a polymorphic nucleus
that is smoother and less segmented than that in mature
neutrophils. Eosinopenia of physical and emotional stress is
attributed to elevated levels of catecholamines such as adrenaline
and adrenocorticosterods.Corticosteroid-induced eosinopenia is
attributed to migration of eosinophils into lymphoid organs namely
the spleen,lymph nodes, and thymus while eosinophilolysis or
95
decreased production was not involved (Sabag et aL, , 1978).
Eosinophilia occurs in some allergic diseases and infestation with
large parasites and with poisonous plants such as Lasiosiphon
kraussianus (Nwude, 1976).
1.4.8.3. The basophils
The basophils are numerically insignificant but
functionally important leukocytes. The blood basophils bear
some morphologic resemblance to the tissue mast cell and is
believed to share similar function. The question is often
asked whether basophils and mast cells share a common origin
or whether they are one and the same cells, acquiring
different morphology under the influence of local
microenvironment and different locations in the body
(Swenson, 1975).
1.4.8.4. The Monocyte and Macrophages
The monocytes are known to be derived from hemato-
poietic stem cells in the bone marrow, and shortly after
entering the circulation, they migrate into various tissues
and body cavities and become fixed or free macrophages.
The blood monocyte and tissue macrophages constituted what
was called the reticuloendothelial system (RES) which was
used to describe a collection of widely distributed cells
capable of phagocytosis which, in addition to blood mono-
cytes and tissue macrophages (histiocytes), included the
96
reticular cells of the spleen and lymph nodes, reticulo-
endothelial cells of the lymph and blood sinuses, and the
Kupffer cells of the liver, (Jain, 1~86).
The blood monocytes, the promonocytes and their
precursors in the bone marrow, and the tissue macrophages
are considered components of the mononuclear phagocytes,
system (MPS). Members of the MPS include histiocytes,
in connective tissue, fixed and free macrophages in the
lymph nodes, spleen, and bone marrow; pleural and perito-
neal macrophages, Kupffer cells of the liver, alveolar
macrophages, osteoclasts and microglial cells in the
nervous system.
Corticosteroids depress bactericidal and fungicidal
activities of monocytes (Thompson and van Furth, 1~73).
They affect functional competence of the MPS in a number
of ways (Kimberly and Ralph, 1~83). They inhibit prolifera-
tion and maturation of monocyte precursors in the bone
marrow, exudation of monocytes in inflammatory foci and
antibody and complement- mediated phagocytosis by the
MPS. Corticosteriods inhibit activation of macrophages
by rendering them unresponsive to lymphokines.
1.4.8.5. The lymphocytes
The lymphocytes playa pivotal role in the initia-
tion and execution of the immune response. Lymphocytes are
97
a heterogenous group of cells both morphologically and
functionally.
Morphologically, lymphocytes are classified as small
medium and large or simply as small (0-9 urn) and large
(9-15 urn). The cell size, the degree of cytoplasmic baso-
philia, and, to a certain extent, the nuclear chromatin
pattern indicate the relative age or maturity (Garcia and
Iorio, 1968).
Functionally lymphocytes are subclassed into those
concerned with cell mediated immunity and immunoregulatory
functions referred to as thymus dependent, thymus derived,
thymus-processed or T-cells while those concerned with
the formation of humoral antibody are thymus-independent,
bursa-derived in birds and bursa-equivalent bone marrow-
derived in mammals or B-cells. A minor, third population
of mononuclear cells are referred to as "non-T, non B" or
"null" cells (Ferrarini el al., 1980).
A decreases in lymphopoiesis is associated with
lymphopenia such as after corticosteroid administration
or radiation. Increased concentration of corticosteroids
is known to cause lymphoid and thymic atrophy (Esteban,
1968). Corticosteroid-induced lymphopenia is associated
with lympholysis in blood and lymphoid is associated
with lympholysis in blood and lymphoid tissues (rabbit)
98
or altered distribution of lymphocytes out of the vas-
cular pool into other body compartments such as the bone
marrow (Cohen, 1972).
Nwude (1976) reported of lymphopenia associated
with the consumption of L. kraussianus in goats, sheep
and donkeys. Certain plant lectins have the capacity
to release the development potential of specific
resting or dormant cells in the circulatory system of
man and transform them to an active growth state
(Nowell, 1960). This subgroup of the lectins has been
termed mitogen and the class of cells affected is primarily
that of the immune responsive cells, or lymphocytes, Plants
with such lectins include Phytolacca americana, Abrus
precatorius, Hura crepitans and Robinia pseudoacacia,
McPherson, 1979).
1.5 TOXIC EFFECTS ON NON-CELLULAR CONSTITUENTS OF BLOOD
This involves the study of pathologic changes at
the molecular level. It concerns the changes which occur
in the chemical constituents of the blood and tissues and
it therefore provides for a better understanding'of the
disease process as well as supply information helpful in
differential diagnosis, therapy and prognostication
(Cornelius and Kaneko, 1965).
99
1.5.1. Nitrogenous constituents of the Blood
The blood contains a number of nitrogenous substances
mainly proteins and nonprotein nitrogenous substances. The
blood protein shares an -intimate relation to protein meta-
bolism in the liver as well as interacting with other
tissues throughout the body, thus showing the central posi-
tion of the blood proteins in the general metabolism of
protein. Therefore changes that are observed in the
qualitative and quantitative composition of blood protein
is of significance to demonstrate the state of the
subject under-study at a particular time.
The heterogeneity of the blood proteins is readily
demonstrated in the ultracentrifuge. In addition to
fibrinogen, albumin and a group of globulins sedimenting
at a rate between these two, the ultracentrifugal analysis
of blood reveals a group of very rapidly sedimenting
globulins and lipoproteins, the sedimentation of which
varies markedly with the density of the medium.
From isotopic and other evidence, it has been concluded
that the liver is the chief, if not the sole site of formation of
albumin, fibrinogen, prothrombin and & - and B-globulins (Cantarow
and Trumper, 1956; Jain, 1986). The Y-globulins are believed to be
synthesized mainly in plasma cells.
100
1.5.1.1. Blood Nonprotein Nitrogen
The nonprotein nitrogen (NPN) of the blood, that
portion of the nitrogenous substances not precipitated by
the usual protein precipitants e.g. trichloroacetic acid,
includes uric acid, amino acids, creatine, creatinine,
ammonia and a fraction designated undetermined nitrogen.
The NPN constituents of blood are usually observed to
be of greater clinical interest than the proteins since
they represent the products of the intermediary metabolism
of ingested and tissue protein.
1.5.1.2. Nitrogen Balance
Since most of the nitrogen of the diet represents
protein, and most of the nitrogenous excretory products are
derived from protein catabolism, it is apparent that the
balance between the two will reveal significant features
of protein metabolism. Nitrogen balance is defined as the
quantitative difference between the nitrogen intake and the
nitrogen output.
Positive nitrogen balance exists when intake exceeds
output and this obtains whenever new tissues are'being
synthesized. In negative nitrogen balance, the output
exceeds the intake.
1.5.1.3. Dysproteinaemia
The best method for the overall evaluation of pro-
103
degenerative, metabolic and regenerative processes occurring
in specific liver disease. This therefore makes it
difficult to define a typical plasma protein pattern in
certain types of liver "disease. However changes in certain
types of patterns were observed by Kaneko (19BO) to be
characteristic of specific hepatopathies. Total serum
protein values are observed to be rarely of aid in clinical
interpretations unless their values fall below 5gm\dl which
are usually observed only in late stages of disease
processes.
The fall in serum albumin concentration attributable
to failure of hepatic parenchymal synthesis is usually
not an early change, and is common in chronic conditions
such as diffuse fibrosis as is observed with plants of
the genus Senecio which are distributed world-wide. It is
believed that there exists a correlation between the seve-
rity of the liver lesions associated with Senecio poison-
ing and the extent of changes that may be observed in serum
protein and albumin levels (Fowler, 196B; Thorpe and Ford
1968). Similar observations were made with the "plant
Lupinus (Gardiner, 1965; Shape er al., 196B). Aspergillus
flavus (Gumbmann and Williams, 1969; Cysewsk~ et al., 196B).
In portal fibrosis, the characteristic change is usually
diminution in the serum albumin level and in elevation in the
104
gamma-globulin level. The changes in albumin level are less
conspicuous in acute hepatitis, but an elevation in the
gamma-globulins is a consistent finding. Low albumin levels are
also observed in animals with nephritis, nephrosis, malnutrition
and other chronic diseases causing cachexia.
1.5.1.5. Proteinuria
The urinary loss of protein in excessive quanti
ties is proteinuria. Pathological proteinuria is an
anomally intrinsic to renal disease, but may have pre-
renal or postrenal causation. Ordinarily, proteinuria of
renal origin is associated with defects in either glomerular
or tubular apparatus giving rise to excretory dysfunction.
In glomerulonephritis, proteinuria arises through abnormal
glomerular filtration. Proteinuria is marked whenever
renal membrane function is affected. Proteinuria is a
common occurrence in poisoning with nephrotoxic plants
such as R. glomeratus (Shupe and James, 1969).
Amaranthus retroflexus which is reported to produce peri-
renal oedema syndrome (Buck et al., 1966j Marshall, Buck,
and Bell, 1967).
The accompanying renal lesions associated with such
nephrotoxic plants consists of selective structural
injury in certain segments of the tubuli which leads to
impaired tubular reabsorption. The excretion of low
105
molecular weight proteins is reported to be characteristic
or disrupted tubular function. Affected glomerular filtra-
tion which is associated with the excretion of gross
amounts of proteins of ~redominantly high molecular weight
is brought about by only a few renal toxicants (De Bruin
1976).
Substances from plants which produce hepatotoxicity
invariably also cause nephrotoxicity with the accompanying
proteinuria. Therefore plants such as Lupines which are
hepatotoxin also cause nephrotoxicity with accompanying
proteinuria (Gardiner, 1965; Shupe et al., 1967).
1.5.2. Pigment Metabolism - Jaundice
Bilirubin, the pigment of bile, is derived from hae-
moglobin. It has been rather definitely established that
the cells of the reticulo-endothelial system, especially
those present in the bone marrow, spleen and liver (Kupffer
cells) are concerned with the metabolism and formation of
bile pigment. Bound to albumin; bilirubin is then trans-
ported from the reticulo-endothelial cell via the blood
stream to the liver. In the hepatocyte, bilirubin is
separated from albumin and conjugated with glucuronic acid
and excreted in the bile as bilirubin-diglucuronide. The
conjugated bilirubin in the intestine is reduced by
106
bacteria to urobilinogen is reabsorbed into the portal
circulation and carried to the liver (enterohepatic circulation)
where most of it is converted to a bilirubin-like compound and
re-excreted into the bile.
Three of these pigments are of importance in the diagnosis of
icterus (a) Bilirubin-diglucuronide or conjugated bilirubin or
direct reacting bilirubin which gives a direct reaction to the Van
den Bergh test.
(b) non-conjugated bilirubin also called in direct reacting
bilirubin (indirect reaction to the Van den Bergh test),
bilirubin and haemobilirubin, (c) urobilinogen.
Icterus is an important clinical and postmortem disorder in
which biliburin reaches such a high concentration in the
circulating blood that the tissues of the whole body are tinged
yellow.
Icterus or jaundice may be divided into three types,
mainly the hemolytic, toxic and obstructive icterus.
Hemolytic jaundice is the result of excessive destruction
of RBC ordinarily in the circulating blood.
Granineous plants such as Trifolium subterraneum which
accumulate copper is reported to cause jaundice in animal
consuming it over prolonged periods during which the animal is
asymptomatic (Hogan et al., 196H). It causes RBC destruction under
conditions of stress when the copper is released. Ricinus
107
conmunis, the common castor plant also produces intravascular
hemol ysi s resulting in icterus (Jenkins, 1963) . Divers et aI .
1982}, and Tennant et al. (19H1) have reported similar findings in
horses who ingested Acer rubrum. Hemolytic icterus was also
observed in cattle (Hutchinson, 1977), sheep (Van Kampen et a1.,
1970) and steers (Jain, 1986) which were fed feed containing the
plant Allium cepa.
Toxic jaundice is caused by toxic substances acting
upon the cells of the liver and producing hydropic degene-
ration, fatty change and necrosis. There are two ways in
which destructive changes in the liver result in icterus.
First, the hepatocytes may be damaged to such an extent that they
cannot perform their excretory function. Haemo- bilirubin
(unconjugated bilirubin) then remains in the blood just as
hemolytic jaundice.
Secondly, the toxicant can cause swelling of the hepatocytes
which cause occlusion of the bile canaliculi.
The bile is excreted from the cells but cannot pursue its
course to the gall bladder and intestine. In this case
it is bilirubin diglucuronide or posthepatic bilirubin
which accumulates in the liver from where it is reabsorbed
into the blood. Both processes are reported to go on
simultaneously so that the bilirubin-glucuronide and
unconjugated bilirubin are detected in blood. Hepatotoxic
108
plants are most commonly observed to produced toxic jaun-
dice. For example plant of the genus Senecio, Crotalaria
and Heliotropium europaeum which contain pyrrolizidine
-alkaloids produce characteristic change of the enlargement
of the parenchymal cells throughout the liver, a phenomenon
which is termed megalocytosis (Bull, 1955). Shupe et al.
(1967) also reports of similar observation with plants of
the genus Lupinus.
Plants which cause toxic jaundice also produce
swelling of the hepatocyte which results in the obstrc-
tion of the bile canaliculi or obstructive jaundice.
Sharma et al. (1982) reports of marked increase in the con-
jugated form of bilirubin in guinea pig plasma during L.
camara poisoning characteristic of obstructive jaundice.
It is recognized that discoloration of the sclerae
and the skin and passage of bile pigment into the urine
occur more commonly in obstructive and toxic jaundice than
in hemolytic jaundice. This explained on the basis that
in obstructive jaundice, the bilirubin is more readily dif-
fusible (direct-reacting bilirubin) than in hemolytic
jaundice (non direct-reacting bilirubin). For example it
has been observed that in toxic or obstructive jaundice,
small levels of bilirubin in the blood which persist for
some days, diffuse through the capillaries and appear in
109
the tissues. Greater concentrations are usually necessary
in hemolytic jaundice for the production of frank icterus
(Kaneko, 1980).
1.5.3. The influence of Plant poisons on blood urea nitrogen
Urea is the chief end-product of protein metabolism
and in formed normally largely if not entirely in the
liver from amino acid metabolism. Ammonia derived from the
amino acid by deamination, and Co2, arising from oxidations
(Kreb cycle) are carried by a derivative of glutamic acid
(carbamylglutamic acid) and are transferred to ornithine.
This undergoes amination (transfer of NH from aspartic
acid) to form arginine, which is split by the enzyme
arginase, into urea and a molecule of ornithine. The urea
enters the systemic circulation and is excreted from the
body mainly in the urine.
When liver function is severely impaired, as occurs
in acute hepati,c necrosis, there is rise in the blood
amino acids and a fall in the urea concentration of blood
and urine. Increase in blood urea nitrogen may be due to
decrease in renal excretion due to organic disease of the
kidneys with destruction of a considerable portion of
functioning renal tissue. Glomerulonephritis is a common
cause for abnormally high blood urea nitrogen concentration,
evidence of renal functional impairment in acute and chronic
110
glomerulonephritis. Also conditions in which there is ex-
tensive destruction or inflammation of the kidneys, pyelo-
nephritis, advanced nephrosclerosis, renal cortical
necrosis, renal conditions accompanied by marked oliguria
or anuria as in some poisoning. £. aquilinum was noted to
produce increased BUN in calves (Singh et al. 19H7) and
rats (Rao, Joshi, Kumar, 1986), because of impairment of
renal function. A similar observation with increased BUN was
made by poisoning with the plant R. glomeratus in sheep
(Shupe and James, 1969) which was attributed to reduced renal
function as a result of organic changes in the renal
parenchyma. Crotalaria retusa which contains pyrrolizidine
alkaloids was found as a seed contaminant of grain sorghum
at a rate of about 0.1% body weight in the diet fed at a
piggery. The pigs were reported to show reduced weight
gains and later severe illness with many mortalities. The
dominant syndrome was severe nephroses with ureamia. The
pigs showed elevated BUN between 95 and 275mg/100ml, (Hooper,
1977).
1. 5.4. Serum Enzymes of Diagnostic importance
Each cell in an organ is known to have a specific
function and contains enzymes unique to that function.
With the disruption of the integrity of the cell, the
enzymes escape into the surrounding fluid compartment
111
and into the serum or cerebral spinal fluid, where their activity
can be measured as a useful index of that cell's integrity.
There are three readily assayable liver spec i f i c enzymes
which are arginase ARG), sorbitol dehydrogenase (SDH) and alanine
aminotransferase (ALT). Arginase and SDH are liver specific
enzymes in many animals and are excellent markers of hepatocellular
damage. Alanine amino-transferase is also reported to be specific
and is assayed in place of ARG and SDH because its assay is simple
and SDH is moreover not very stable (Kaneko, 19HO).
When an enzyme lacks sufficient specificity for an organ, a
second enzyme may be combined wi th the first to increase the
diagnostic value. For example serum alkaline phosphatase activity
increases with bone and liver disorders and to assist in
identifying the source of the increase, alanine aminotransferase or
gamma-glutamyltransferase (GGT) is assayed as part of the hepatic
profile.
1.5.4.1. Creatinine Kinase
Creatinine kinase (EC 2.7.3.2) or creatinine phosphokinase is
one of the most organ specific serum enzymes in clinical use. It
catalyzes the reversible phosphorylation of creatinine by ATP to
form creatinine phosphate and ADP. Creatinine phosphate the major
storage form of high energy phosphate required by muscle for
112
contraction. Creatinine kinase is a dimer consisting of two
peptides B (for the brain and M (for the muscle) which makes up the
three isoenzymes CKl' CIS, and CK3 or BB, MB and MM respectively.
Many cells contain creatinine kinase but only the heart and
skeletal muscle contain sufficient amounts of CIS and C~ to alter
serum activity in organ specific disorders. The isoenzyme CK is
found predominantly in the brain.
Creatinine kinase has a short half life in serum and it is
relatively unstable when stored at room refrigerator or freezing
temperature. However the activity of CK can be restored by
incubating serum with sulfhydryl activators e.g. cysteine or
glutathione. Elevations of creatinine kinase activities have been
reported in myodegeneration due to ingestion of toxic plants in
cattle (Henson et al., 1960. Elevations in creatinine kinase
activities have been reported in polioencephalomalacia and focal
symmetrical encephalo~alacia of sheep and it has been suggested
that creatinine kin,ase determinations may be of value in diseases
of the central nervous system (Smith and Healy, 1968).
1.5.4.2. Alanine Aminotransferase
Alanine aminotransferase (ALT) (EC.2.6.1.2) and glutamic
pyruvate transaminase (GPT) catalyzes the reversible transamina-
tion of L-alanine and & -oxoglutarate to pyruvate and glutamate.
ALT is present in plasma and cells. In dogs and cats, there is
sufficient ALT activity in the liver for it to be used as a liver
113
specific enzyme. Increase in plasma activity of GPT in these
animals is associated with hepatocellular disorders. Acute hepatic
diseases causing membrane damage or cell necrosis result
in appreiable increase in plasma activity (Kaneko, l~HO).
1.5.4.3 Aspartate Aminotransferase
Aspartate aminotransferase (AST)(EC.G.6.1.1) and glutamic
oxaloacetate transaminase (GOT) catalyzes the transmination of
L-aspartate and -oxoglutarate to oxaloacetate and glutamate. AST
is present in mitochondria and cytosol of almost all cells and in
plasma. The presence of GOT in so many tissues precludes the use
of this enzyme as an organ specific enzyme but it is useful in
conjunction with other enzymes as an index of hepatic or muscular
cell damage (Kaneko and Cornelius, 1~70).
1.5.4.4. Gamma-Glutamyltransferase
Gamma-glutamyltransferase (GGT)(EC.G.3.G.G.) is a carboxype-
ptidase which cleaves the C-terminal glutamyl group and transfers
them to peptides and other suitable acceptors. Glycylglycine is the
most suitable acceptor. The physiological function of
Y-glutamyltransferase is unknown but it is speculated to be
associated with glutathione metabolism. Cell cytosol and membranes
are known to have Y-glutamyltransferase acticity. Most cells have
GGT activity ranging from the kidney with the highest and the
muscle the lowest.
114
Serum has GGT activity most .o t' which is derived from the
liver. Kidney GGT is detectable in serum and urine contains only
kidney GGT (Bruin et aL, , 1978). Ford (1~74) and Johnson (1~76)
observed that GGT may well be a more specific indicator of
obstructive hepatic disorders than alkaline phosphatase.
1.5.4.5. Lactate Dehydrogenase
Lactate dehydrogenase (LDH) (EC.l.l.l.27) catalyzes rever-
sible oxidation of L-lactate to pyruvate with the co-factor NAD.
A large amount of LDH activity is present in all tissues. In the
blood, there is as much a~ 150 fold greater LDH activity in the red
blood cells than there is in plasma. Thus even minimal hemolysis
alter the plasma LDH activity appreciably. Anticoagulants such as
EDTA and oxalate, indirectly inhibit the enyzmes. There
heparinized plasma or serum is the preferred sample.
LDH is a tetrapeptide made up of two types of peptides:
(heart) and M (muscle).The two types of peptides in various
combinations make up 5 isoenzymes LDH-l, LDH-2 LDH-3, LDH-4 and
LDH-5 which are designated for peptide combinations HHHH, HHHM,
HHMM, and MMMM respectively. Tissues contain various amounts of the
LDH isoenzymes band serum isoenzyme profile are· used to identify
speci fic tissue damage by electrophoretic separation (Prase, 1~6~) .
1.5.4.6 Sorbitol Dehydrogenase
The act ivity of Sorbitol dehydrogenase (EC.1.1.1.14) in plasma
is very low. The major source of it is the hepatocyte. It catalyzes
115
the reversible oxidation of D- Sorbitol to D. fructose with the
co- factor NAD. Sorbi tol dehydrogenase is unstable and at room
temperature appreciable amounts are lost in a few hours. Metal
chelating anticoagulants such as EDTA and oxalate decrease the
amount of sorbitol dehydrogenase activity. Therefore heparinize
plasma may be used for its estimation but serum is preferred.SDH is
liver specific and hepatic injury appears to be the only source of
increased SDH activity.
1.5.4.7. Alkaline Phosphatase
Alkaline phosphatase (EC.3.1.3.1.) is an important enzyme for
characterizing bone and hepatic disorders. Alkaline phosphatase is
a non-specific enzyme which hydrolyses many types of phosphate
esters and is present in multiple molecular forms i.e. isoenzymes.
The endogenous substrate of ALP is unknown, but it catalyzes the
dephosphorylation of ATP and is thought to be associated with
energy requiring membrane pumps.
The term alkaline refers to the optimal alkaline PH of this
class of phosphatases in vitro.The PH optimum for ATP is 10, a PH
unlikely to occur in the body.
1.5.5. Serum Enzyme Tests
The level of serum enzymes which is measured by their
biochemical activity changes as a result of processes involving the
liver. Enzyme activity may be increased from disrupted hepatic
parenchymal cells with necrosis or altered membrane permeability
116
e.g. alanine aminotransferase (SGPT) aspartate aminotransferase
(SGOT), arginase, isocitrate dehydrogenase (SICD), sorbitol
dehydrogenase (SDH), glutamate dehydrogenase (GD) ornithine
carbamyltransferase (OCT), and lactate dehydro-
genase (LDH). Increased levels of enzymes also result from their
over production in cholestasis of obstructive icterus e.g. alkaline
phosphatase. Elevations in serum activity provide little
information regarding the reversibility, type of lesions or
functional state of the liver. However the quantiative estimate of
the extent of necrosis can be estimated.
Enzymes whose concentration is increased following hepatic
necrosis are further divided into the liver specific enzymes
because the liver contains their high concenration (SGPT) in dogs,
cats and primates; SDH and GD in sheep and cattle; SDH in horses;
arginase and OCT in all ureotelic animals and enzymes which are
present in high concentration in other tissues including the liver
(SGOT) LDH, and SICD).
1.5.5.1. Pathological Findings
The clinical-pathologic features of A. flavus poisoning have
been demonstrated in experimentally poisoned swine (Gumbmann and
Williams, 1969; Sisk, Carlton and Curtin, 1~68). The serum levels
of SGOT, OCT, ALP and SICD were markedly elevated. The enzymes
117
were reported to be concomitantly lost from the damaged liver.
Himes and Cornelius (1973) attest to the usefulness of them
Pathology involving the skeletal or cardiac muscle end or
the hepatic parenchyma allows for the leakage of large amounts
of this enzyme into the blood. Since all major tissues contain
high concenrations of SGOT, the finding of significant elevations
in SGOT need not indicate hepatic necrosis unless diseases of other
large organ systems can be ruled out. Elevations of SGOT
activities have been observed in Senecio ,jacobaea intoxication in
calves (Ford and Ritchie and Thorpe l~oH) aflatoxicosis in swine
(Cysewski et al., 1968) in feeding of Brassica oleracea to cattle
(Grant et al.,) and on rams fed on the plant of the genus
Astragalus (James and Binns, 1~o7).
In poisoning, there is more rapid disappearance of arginase
which is a mitochondrial-bound enzyme as compared to the
transaminases which'are in the hyaloplasmic fraction to the cells.
Harvey and Hoe (1971) and Kaneko (19HO) suggest that if both serum
arginase and transminase activities are continuously elevated, a
progressive hepatic necrosis was most likely the cause. However if
the serum arginase returns to normal and elevated transaminase
activities are observed following significant elevations of both
enzymes, prognosis is favorable as hepatic necrosis was likely
subsiding. The estimation of serum arginase activity is a reliable
118
liver-specific enzyme test for a.ctive hepatic necrosis in all
ureotelic species.
Harvey and Obeid (1974) reported that arginase was superior
to alkaline phosphate and bilirubin levels for the diagnosis of
hepatic disease in ruminants and camels in Sudan.
Sorbitol dehydrogenase is a serum enzyme which is been found
to be 1iver-speci fic in large domestic animals. Sharma et aL,
(1982) observed a marked increase in the conjugated form of
bilirubin in guinea pig plasma during L. camara toxicity
characteristic of obstructive jaundice. Also activities of SGOT,
lactate dehydrogenase and sorbitol dehydrogenase in plasma of
guinea pig, were elevated. Elevated levels of sorbitol
dehydrogenase was also observed in sheep with lantana poisoning
(Sharma, et al 1981):
Boyd (1962) has shown that glutamate dehydrogenase is
highly concentrated primarily in ovine and bovine liver and
recommended its use in measuring necrosis in these species.
Similarly GDH is present in high concentration in equine
(Freedland et al., 1965). It has become the enzyme of choice
in measuring hepatic necrosis in ruminants (Kaneko, 19HO).
Animals poisoned by the plants in the Lupine family showed
elevated levels of SGOT in the early stages as are the serum
lactate dehydrogenase and glutamate dehydrogenase (Shupe
eta!., 1968).
1 19
1.5.5.2. Urinary enzymes
Enzymes, ordinarly absent or negligible in the urine, are
excreted significantly under pathological conditions involving
renal cell damage. Enzymuria is a sensitive index of minor tubular
lesion preceeding functional impairment as evidenced by renal
function tests. Enzymes gain access to the urine either by impaired
glomerula filtration or as a result of desquamation of
kidney tubular cells.
Lactate dehydrogenase activity is reported to increase in the
urine and serum of patients with urinary tract disease (Gelderman,
1965). Enzymuria is a more sensitive and early criterion of the
nephrotoxic action of substances than are secretory dysfunction.
As certain enzymes are localized predominantly in specific
segments of the renal tubuli, their urinary assay may delineate
specific structural injury. Thus proximal tubular lesions are
suggested from the rise of alkaline phosphatases while increases in
lactate dehydrogenase or carbonic anhydrase is indicative of
structural anomaly of the distal segments (De Bruin, 1976). Injury
of the glomerula type is revealed by acid phosphatase
assay.
1.5.6. Primary Renal Dysfunction
Acute failure
The kidney is particularly susceptible to noxious agents due
to its high perfusion rate, the numerous enzymes and its role as an
120
excretory organs. Nephrotoxins commonly encountered in domestic
animals include most plants poisons such as H. glomeratus (Buck et
al., 1966 Shupe and James 1969), A.retroflexus (Marshall et al.,
1967), aflatoxins (Cysewski et ai., Sisk et al. ,1968),
Nephrotoxins produce their effect by various mechanisms. Some
remain in the renal lumen and are concentrated as water is
reabsorbed.Others are taken up by tubular cells and concentrated
in them. Such compounds are relatively inert and are toxic upon
modification by host enzymes .Oxalates are for example precipi tat-ed
in the renal tubules during the process of elimination and fatal
outcome may occur from renal insufficiency and ureamia. Such
poisoning has been observed in H. glomeratus and A. retroflexus
which contain high oxalate levels (Buck at al., 1966; Shupe and
James, 1969).
There is no unanimity of opinion on the events that culminate
in acute renal failure (Harrington and Cohen,1975; Levinsky, 1977).
The first opinion assumes it is due to renal vasoconstriction. The
second states that tubular blockage occurs as a consequence of
intraluminal debris and interstitial oedema. The third surmises
that glomerular filtrate forms but is totally reabsorbed (passive
back flow) because of tubular necrosis.
Chronic failure.
Chronic renal failure is the consequence of slow insidous
destruction of renal parenchyma. Due to the reserve capacity of
121
the kidney, clinical signs of disease may not develop for months or
years following the initiation of disease. Morphological
examination are of little value to identify causes of chronic renal
failure because renal responses to injury are limited in variety
and tend to assume a common character regardless of cause as
chronicity develops. Unlike the abrupt sequence of events in acute
anuric failure that reflects manifestations of sudden
dysfunction,most changes take place gradually in chronic failure.
The changes are initially undetectable both clinically and
biochemically. It is only when over two- thirds of the normal
parenchyma has been destroyed that clinical signs become apparent.
For example the ultrastructual features of the hyaline lesions in
glomeruli and renal arteries results deposition of amorphous and
fibrillar material which obliterates lumens of glomerular
capillaries following Crotalaria spectabilis intoxication in rats
yet no clinical signs were obvious (Carstens and Allen, 1970).
1.5.7 Serum calcium and phosphorus
Extracellular calcium is present in free calcium ions,
calcium ions that are ionically bound but uItra filtrable, and
calcium bound to plasma proteins. The ionized calcium is
diffusible; the complexed calcium is diffusible but not ionized;
whereas the protein bound calcium is neither ionized nor
diffusible.Although all these forms to calcium are in equilibrium
122
with one another, in the body flui4s, only the ionized fraction is
under direct control (Copp, 196~).
Most of the phosphorus of blood is present as organic
ester of phosphorus within the red blood cells, these contain
small amount of inorganic phosphorus (pi) at any given moment.
Serum contains about 14-15mg oftotal phosphorus per deciliter but
this 5-8mg are 1ipid phosphorus. A trace of the rest is ester
phosphorus, the most significant portion of the remainder being Pi.
The bone mineral is readily mobilized (regulatory mechanisms by
parathyro id hormone, calc itonin and the active metabol ites (of
vitamin D) to maintain the level of serum calcium but less readily
to maintain that of Pi so that a low serum Pi level is the first
sign of deficiency of phosphorus. Pi level appears to be
intimately tied to carbohydrate metabolism. During increase
carbohydrate utilization, the level tends to decrease and
during fasting and increase usually is observed.Measurement of Pi
concentration (preferably heparinized plasma) provide the most
readily determinable index of the phosphorus status of animals,
(Newman, 1968; Little et al., 1971).
1.5.7.1. Toxic Plants affecting calcium and phosphorus metabolism
A series of so-called plant induced calcinosis in animals has
been describeed. Cattle feeding on Solanum malcoxylon are known to
develop a condition characterized by wasting stiffness,
hypercalcaemia, ,and hyperphosphataemia.The most typical lesions is
123
the calcification of the arteries, which is associated with a
greatly increased calcium absorption from the diet (Samson et al.,
1971). In describing this poisoning by ~. malacoxylon,
Krook et al. (1975) give a list of the different names by
which the condition is called in various conuntries ientegueseco
(Argentina) espichamento (Brazil) naalelia disease (Hawaii)
Manchester wasting disease (Jamaica) i enzootic calcinosis (Germany,
Austria). A similar condition with hypercalcaemia and calcinosis
has been reported caused by the shrub Cestrum diurnum in pigs
(Kasali, 1977) and horses (Krook et al., 1~75).
These plant poisonings show great similarities with vitamin D
poisoning i.e.d i fferent degree of hypercalcaemia hyperphospha-
taemia, and calcifications in the circulatory systems, lungs,
kidneys and flexor tendon. Investigations show that all these
diseases most likely are caused by the presence of potent, active
vitamin D-like substances in the plants and that these substances
are responsible for the abnormalities of mineral metabolism in the
animals (Krook et aL, , 1975' Shupe and James, 1~6~i Wasserman et
aI ;, 1977).
Hypocalcaemia has been reported in sheep poisoned by
H. glomeratus (Littledike, James and Cook, 1976; Marshall et a1.,
1967) and other plants such as Sarcobatus vermiculatus, Oxa1is
cernua and /l. retroflexus (Buck et al., 1966; Shupe and James,
1969> Littledike et al. (1976) report that the plasma concentration
124
of Ca and Ca activity decreased over several hours following H.
glomeratus poisoning, to such low levels that tetany or coma
occurred and death followed. Increases in plasma total inorganic
phosphorus and magnesium concentration also occurred as the
hypocalcaemia progressed.
1.6. GROSS AND HISTOLOGIC LESION IN TOXICOSIS
1.6.1. -Death of cell and tissues
Poisonous substances which produce local injury (necrosis and
inflammation) to the tissues have to come into contact with them.
Obviously an appropriate degree of concentration is essential to
the production of this effect. Some toxicants achieve their
highest concentration at their site of toxic action. For example
carbon monoxide which has a very high affinity for haemologbin, and
paraquat which accumulate in the lung (Sharp et al., 197~). Other
agents concentrate at sites other than the site of toxic action.
Lead for example is stored in the bone while the symptoms of lead
poisoning are due to lead in the soft tissues (Clarke and Clarke,
1975). The toxicant
while it is stored often does no harm to the organism. Storage
depots therefore could be considered as protecting organs
preventing high concentrations of the toxicants from being achieved
at the site of toxic action.
Grossly, a dead tissue is regularly paler than a living one
except where it is well filled with blood, which makes it
125
black. A dead tissues also has little strength, especially
tensile strength. If one finds both living and dead tissues
within the same microscopic sections, then the dead part represents
necrosis.
Microscopically a necrotic tissue has the nucleus of its cells
pyknotic or karyorrhetic.The cell nucleus may also show karyolysis
or there may be no nucleaus at all. The cytoplasm is acidophilic
because its reaction is more basic than during life and hence it
takes the acid stain, which is usually red (eosin) or there may be
cytoplasmolysis. With more advanced changes, there is loss of cell
outline and differential staining.
Necrosis could be coagulative, caseous or liquefactive.
There could also be the necrosis of fat and zenkers necrosis.
1.6.2. PLANTS PRODUCING DAMAGE TO THE GASTROINTESTINAL TRACT
1.6.2.1. Plants producing stomatiti
There are many specific diseases in which stomatitis is a
product sign. The Merck Veterinary manual 1975 ) notes that plants
of the genus Anemone which include A. nemorosa and A. pulsatilla;
and family Amaryllidaceae produce stomatitis with reluctance of the
animal to permit manual examination of the mouth cavity. Animals
stand with their mouth open roll their tongues or chew with their
heads turned sideways. Mycotic stomatitis produced by infections
with Monillia species is also common (Blood andHenderson, 1974).
A specific type of ulcerative stomatitis in dogs and cats caused by
126
Candida albicans and characterized by the appearance of ulcers and
soft, white to gray slightly elevated patches onthe oral mucosa
(Merck Manual 1979.
1.6.2.2. Plants producing gastritis
The feeding of damaged feeds,including mouldy and fermented
hay and ensilage, commonly causes a moderate gastritis. Fungi can
produce diffuse or ulcerative gastritis in new born animals,
especially pigs.Mucormycosis and moniliasis are the two commonly
recorded causes. In all species Mucor species and Aspergillus
species frequently complicate gastric ulcers caused by other agents
(Martins et al., 1957). Pharyngeal paralysis in the horse due to
Aspergillus infection has been reported just as the infection of
the gultural pouches.
Fish feeding on blue-green algae Lyngbya species are not
themselves harmed,but become highly toxic to animals eating them.
Cats have been poisoned showing among others anorexia and excessive
salivation. The condition is known as Ciguatera poisoning (Clarke
and Whitwell, 1968). The burrs of bardock (Archlium lappa) are
known to give rise to granular stomatitis in dog. Small papules
develop on the tongue which enlarge and develop necrotic centres
which slough and become ulcerated (Thwienge, 1973).
1.6.2.3. Signs and lesions associated with other parts of the GIT
Disinclination to eat is one of the common response of
most animals to plant intoxication. Agave lecheguilla gives
127
rise to a condition known as swell head chracterized by
listlessness, and loss of appetite (Clarke and Clarke, 1975).
James and Binnus (1966) reports of loss of appetite and weight in
ewes experimentally fed with Allium validum. Watt and
Breyer-Brandwijk (1962) mentioned that plants of the genus
Aristolochus produced decreased appetite constipation,whereas in
addition, plants of the genus Asclepias also produced weakness and
diarrhoea.
Lobelia berlandieri has been one of the causes of stock
poisoning in Mexico.Fed exerimentally to sheep at a level of 0.5
per cent body weight dialy, it caused inappetence and diarrhoea,
followed by ulceration of the mouth, salivation and death.
Scarisbrick (1954) gives evidence of the presence of an irritant
glycoside on Beta vulgaris sub sp tops which induces indigestion
and purgation in sheep. This he attributed to formation of large
quantities of lactic acid in the rumen. Symptoms of poisoning
produced by Sinapis nigra are those of acute gastroenteri tis,
colic, frothing around the mouth and nose, grunting and diarrhoea.
Amongche clinical symptoms that accompany Pteridium aquilinum
poisoning is "the enteric type which is seen most frequently in
adult cattle, with the animal exhibiting
depression, anorexia, enteri tis with frequent blood clots in the
faeces.
1 28
Clarke and Clarke (1975) reports of the presence of lycorine
in members of the genera Amaryllis,· Crinum, Haemanthus and Nerine,
all of which cause poisoning in sheep and goats producing
salivation, vomiting and diarrhoea in small d ses, The usual
symptoms associated with Nerium oleander poisoning are vomiting,
convulsion, diarrhoea and colic. Lesions were those of severe
catarrhal or haemorrhagic gastroenteritis.
Ricin is the toxic agent obtained from the plant Ricinus
communis which is very toxic when administered by whatever route.
All animals are susceptible to its effects. All animals show
profuse watery diarrhoea but even in the most severely affected,
there was no blood in the faeces (McCunn et al., 1945). Anderson
(1948) has given account of poisoning in cattle with the same plant
in which the main symptoms was bloody diarrhoea. Anderson mentions
that with cattle, diarrhoea and bloody purgation have been held to
be characterisitic. Geary (1950) noted vomiting and diarrhoea (not
bloody) in pigs. The animals were weak and showed some
incoordination and signs of abdominal pain. The skin of the ears,
flanks and hams was cyanotic. In pullets, Geary (1950)
noted dullness, dropping wings, ruffled feathers and greyish-
coloured wattles and comb.
The symptoms of poisoning associated with Fagus sylvatica and
oak (Quercus species) usually commences with acute abdoninal pain
of such severity that the animal becomes unmanageable and delirious
129
(Llewellyn, 1962). Symptoms associated with poisoning by the
leaves of Quercus species may not appear for some days after they
have been eaten. Initially there is dullness. inappetence,
cessation of rumination and constipation. Later there is
persistent diarrhoea, with frequent passage of small amounts of
dark coloured faeces which may contain blood. There is also severe
urination (Towers, 1950).
Sinapis nigra of the family cruciferae causes poisoning, the
symptoms of which are acute gastroenteritis, colic, frothing around
the mouth and nose, grunting and diarrhoea. Postmortem examination
reveals acute inflammation of the stomach, intestine and kidney.
Clarke and Clarke (1975) mentions that black mustard seed has
occasionally found its way into cattle cakes and has given rise to
colic and respiratory distress. Linseed meal of ~. nigra
contaminated with swede and turnip seed has caused fatal
haemorrhagic gastroenteritis in cattle.
With the cycads, there are two forms of poisoning noted in
cattle. These are mainly an acute gastrointestinal disturbance
generally terminating fatally in a few hours associated with
extreme liver 4amage and congestion of other organs whereas the
chronic condition is characterized by a progressive and
irreversible paralysis generally of the hind legs.
Pteridium aquilinium poisoning produces enteric symptoms
which is seen most frequently in adult cattle. These inlude
130
depression, anorexia, enteritis with frequent blood clots in the
faeces. Numerous petechial haemorrages can be seen on the visible
mucous membranes and there may be bleeding from the nostrils,
intestinal and urogenital tracts especially at the later stages of
the poisoning (Evans et al., 1954); Evans et al., 1972).
Poisoning by the plant Iris foetidissima of the family
Iridaceae leads to acute diarrhoea often haemorrhagic with death
resulting from exhaustion. Colchicum autumnale contains colchicine
which exert mainly, purgative effect.It has colchicine which exert
mainly, purgative effect. It has also the peculiar property of
preventing mitosis from proceeding beyond the metaphase (by
inhibiting spindle formation) and has been used experimentally for
inducing polyploidy. In cattle the symptoms arising from C.
autumnale poisoning include abdominal pain, violent purgation with
the passage of foetid green or black faeces with general collapse.
Death occurs from respiratory failure which may be delayed for
several days according to the amount of the plant ingested. The
only notable finding at autopsy is that of gastroenteritis
(Goonerate, 1966).
Symptoms associated with poisoning by veratine and related
alkaloids contained in hellebores such as Veratrum album are
salivation, purgation,vomiting, diuresis; excitability followed by
prostration, weak and irregular pulse,deep and slow respiration and
death in convulsions or consequent upon paralysis.
1~1
Danke et al. (1965) noted that ruminants are generally not
affected by Gossypium species (cotton plant) although young calves
before the rumen is fully functional are susceptible while the
compound is highly toxic if injected. Monogastric animals however,
are readily poisoned, the gossypol having a cummulati ve effect.
The horse is said to be relatively resistant. However, in pigs,
even quite low levels incorporated in the ration of fattening pigs
will cause inappetance, loss of weight and lowered food
utilization, while larger quantities will give rise to
weakness dyspnoea, emaciation and death. Poultry show loss of
weight and anorexia with lowered hatchability and discoloured
yolk (Morgan et al., 1988).
Aconi tine is an alkaloid obtained from Aconi tium napellus
which acts as a gastrointestinal irritant. Symptoms of poisoning
in cattle following ingestion of the plant Rhamnus frangula is
diarrhoea, colic and moderate fever with death supervening in few
hours.
The genus Solanum contains a poisonous fraction, solanine
which is known to produce gastrointestinal signs, sal ivat ions ,
stomati tis, vomiting, tympani tis and diarrhoea. Lesions in the
digestive tract are acute catarrhal or haemorrhagic gastritis and
enteri tis sometimes accompanied by ulcers which extend to or
through the muscularis propria (Peterlik et al., 1976) Peterlik and
Wasserman, 1978).
132
1.6.3. MORPHOLOGIC LESIONS IN THE LIVER DUE TO TOXIC AGENTS
These are also known as hepatotoxins. Hepatotoxins can be
defined as a heterogenous group of naturally occurring and
synthetic chemical agents that produce a variety of hepatic
lesions. According to Casarret and Doull (1975) hepatotoxin
have the following common characteristics. (a) the agents produce
a distinctive lesion, (b) the severity of the lesions seems to be
dose related, (c) while quantitative differences in potency can be
found among individuals generally the same type of lesion can be
produced in all individuals, (d) the lesions are reproducible in
experimental animals, (e) they usually appear after a
predictable, usually brief latent period.
1.6.3.1. Mechanism of liver injury
Generally substances which cause injury to the liver
produce their effect through the following mechanisms: (a) they
produce abnormal amounts of fat in the parenchymal cells i.e. fatty
liver, (b) derailment of normal protein synthesis, (c) cholestatic
reaction and (d) lipid perioxidation.
The lipid that accumlates is predominantly triglyceride. The
general mechan~sm that accounts for triglyceride accumulation is
described by Lombardi (1966) as when the rate of synthesis of
hepatic triglyceride is normal but, the liver cell is unable to
secrete the triglyceride into the plasma. Secondly the secretion
of the hepatic triglyceride may be normal but the rate of synthesis
133
is increased. Thirdly there may be a situation where both an
increase in rate of synthesis and a block in the secretion of
synthesized triglyceride. Lastly a situation arises when the
triglyceride synthesis takes place in a compartment of the cell
other than the endoplasmic reticulum and thus, this pool is not
accessible to the normal secretary pathway (Lombardi.,1966). It
has also been shown that carbon tetrachloride,ethionine,phospho-
rus and puromycin interferee with the synthesis of the protein
moiety of the lipoprotein complex.
Morphologic injuries associated with early hepatic injury
include loss of cytoplasmic basophilic material, vacuolation in the
cytoplasm, abnormal configuration of the endoplasmic reticulum and
loss of ribosome particles from the membrane surfaces. These
observations as associated with the inhibitory effects of the
hepatotoxins on protein synthesis (Magee, 1966). For example
ethionine inhibits a~ino acid incorporation in microsomes due to a
deficiency of available ATP in the cell; by the formation of
s-adenosylethionine by ethionine replacing methionine. This leads
to a trapping of cellular adenine and a dimunition in the rate of
ATP synthsis.
Dimethylnitrosamine affects protein synthesis by causing
a loss of m-RNA from polyribosomes which may be due to methylat-
ion of RNA (Casarrett and Doull, 1975).
Also hepatotoxins may affect protein synthesis by influen-
134
ci.ng single unit ribosomes and not on the polysomes. This is as
observed with the effect of carbon tetrachloride (Farber., 1971).
Certain hepatotoxins are known to produce cholestatic reaction.
Alpha-naphythylisothiocyanate (ANIT), Tauroli thochol ic acid and
2-ethyl-2-phenylbutyramide are known to produce such action.
Goldfarb et al. (1962) produced bile stasis and hyperbilirubinaemia
and sulforbromophythalein (BSP) retention in rats following ANT
administration.
Cholestasis could be due to poor water solubility and
precipitation of the substance in the biliary tract as is the
case with taurolithocholate. Canalicular bile plugs as is
observed after treatment with substances as norethsterone,
.-- .me.t.hvLe st.ost.eone ,-mestranol or norethandrolone also cause
cholestasis.
Slater (1966) speculated a mechanism of action associated with
the necrogenic action of some hepatotoxins such as carbon
tetrachloride, based 0 the activation of the parent compound to a
toxic metabolite (lipid peroxidation). He proposed the homolytic
cleavage on the carbon-chloride bond in the endoplasmic reticulum
and that this Fesulted in the production of free radicals which
interact with neighbouring lipid-rich material causing alterations
in structure and function.
135
1.6.3.2. Morphologic lesions in the liver due to toxic agents
Morphologically, chemical-induced injury can manifest itself
in different ways. The acute effects can consist of an accumlat~
tion of lipids (fatty liver) and the appearance of degenerative
processes leading to death of the cell (necrosis). The necrotic
process can affect small groups of isolated parenchymal cell (focal
necrosis); groups of cell located in zones (centrilobular, midzonal
or periportal necrosis) or virtually all the cells within a hepatic
lobule (massive necrosis).
Chemical-induced liver injury resulting from chronic
exposure can produce marked alterations of the entire liver
structure with degenerative and proliferative changes observed in
the different forms of cirrhosis.
1.6.3.3. Classification of Chemical-induced liver injury
On the, basis of morphologic changes produced in the liver,
Popper and Schaffner (1959) and Zimmerman 1968) differently
proposed their own classifications.
Popper and Schaffner (1959) described five major group of
reactions which could take place in the liver as a result of
chemical inju~y. The first was called zonal hepatocellular
alterations without inflammatory reaction example as produced by
carbontetrachloride. The lesion is reproducible and dose-depend-
ent.The second group is called intrahepatic cholestasis. The
important histologic feature associated with this response are the
136
presence of bile stasis, the presence of bile plugs in the
cannaliculi, dilatation of the canaliculi with subsequent loss of
the microvilli and the occurence of focal necrosis. The lesion
produced were not dose dependent nor reproducible e.g .lesions
produced by anablic steroids and oral hypoglycaemics. The third
category was called hepatic necrosis with inflammation. The
prominent feature is progression to a massive necrosis
characteristic of viral hepatitis. The forth group was called the
unclassified group and consisted of hepatic injuries which does not
fit those above. The fifth category consisted of those causing
hepatic cancer such as aflatoxin.
On his part Zimmerman (1968) classified hepatotoxins
ac~ording to the supposed mechanism of action, morphologic changes
observed or according to circumstances 9f exposure.
On the basis of mechanism, all agents were divided into
intrinsic hepatotox~ns and those depending upon host idiosyncracy
.The intrinsic hepatotoxins were further divided into direct agents
which injure many tissues including the liver and indirect agents
which affect a particular metablic pathway.
With agerrt s which produced morphologic changes, Zimmerman
(1969) divided them tinto cytotoxic, cholestatic and mixed agents.
The final consideration concerned the circumstances of the exposure
which were classified into "toxicologic" which consists primarily
of chemical overdosage and the second iatrogenic which are made of
137
lesions produced during the course of therapeutic utilization of
drugs.
1.6.3.4. Hepatitis
There is the view that hydropic degeneration, fatty change and
necrosis are incipient stages of a process which soon becomes
inflammatory. Hepatitis can be infectious or non-infectious (toxic
hepatitis) (Smith,Jones and Hunt, 1974). The non-infectious or
toxic hepatitis can be separated into acute toxic hepatitis, also
known as acute yellow (or red) atrophy and chronic toxic hepatitis,
for which the usual name is cirrhosis.
The acute toxic 'hepatitis is characterized by death of
hepatic cells and changes which precede death mainly hydropic
degeneration, fatty change and necrosis. Microscopially, the
necrosis is coagulative in type followed by des integration and
disappearance of cells.
Regarding location, necrosis in the liver could assume any of
the five forms.Diffuse necrosis occurs when necrosis spreads
without recourse to lobular boundaries and is a more severe form.
Focal necrosis, in which minute necrotic areas, or foci of
sublobular size appear here and there which could be at any part of
a lobule. A necrosis could be peripheral in which case only the
peripheral zones of the lobules are regularly necrotic. This type
of necrosis happens when strong toxic substances get to the liver
lobule through the blood stream without any impairment of
138
circulation of the blood and oxygenation of cells. This is because
circulation gets to the peripheral cells first. Midzonal necrosis
occurs when necrotic changes involve the cells half-way between the
periphery and the centre of the lobule. CentrilobulA~ n i
affects cell nearest to the cenral vein which suffer from blood
borne toxins and from a stagnation of circulaton with consequent
anoxia. This is common with acute hepatitis with primary disorder
being responsible for both the production of toxic substances and
impairment of circulation. Paracentral necrosis has necrotic areas
which adjoins the central vein on one side but does not surround
it, (Smith, Jones, and Hunt 1974.
Typically, but not invariable, acute toxic necrosis is
charactertized by centrilobular necrosis with disappearance
of most of the centrally located cells with blood filling their
place. Peripheral to the lobule, the cells will show fatty change
an hydropic degeneration, As the lesions progress, moderate
infltration of lymphocytes into the periportal connective tissue
(islands of Glisson) usually begins.
Grossly, a liver with acute toxic hepatitis is usually
lighter in colour, even to the tan of severe fatty degeneration
however it is also likely to be redder because of an increased
content of blood. Some show accentuation of the lobular markings.
1.6.3.5. Chemical substances and Plant toxins producing Hepatitis
Some of the chemical poisons known to cause acute toxic
139
hepatitis include copper, arsenic arid the arsenical drugs,
phosphorus (if the patient survives more than three days) chronic
mercury poisoning, chloroform (delayed poisoning developing tow or
three days after anaesthesia) tannic acid (used in the treatment of
burns), tetrachloroathane, trinitrotoluene and tetrachlorathylene.
Gossypol produces hepatic lesions including swollen cells, foci of
necrosis around the cenral veins, congestion of the portal
triads,pykn~sis, karyorrhexis and karyolysis of hepatocytes,
perivascular oedema, bile retention and severe fatty change in
fedder lambs (Morgan et al., 1988).
Ingestion of plant substances containing pyrrolizidine
alkaloids has been shown ~o lead-to profound change in the
anatomy of the liver.Microscopically,there is characteriscally a
combination of haemorrhages, necrosis and cirrhosis.Bull (1955) who
studied a detailed h~stology of the liver of sheep poisoned with
Heliotropium europaeum considers that in this species, the
characteristic change is an enlargement of the parenchymal cells
throughout the liver a phenomenon which he terms megalocytosis.
According to the author, haemorrrhages, cwell necrosis, and
fibrosis are commonly associated changes but are not specific.
Hill et al. (1958) however observed that the pyrrolizidine
alkaloid,causes an intimal overgrowth of connective tissue of the
hepatic veins producing partial or complete occlusion; the
ultimate result is generalized fibrosis of the liver.
140
Markson (1960) who followed the sequence of hepatic events in
calves following Senecio jacoboea intoxication observed first
slight parenchymal steatosis.A week later bile duct proliferation
parenchymal cell changes diffuse fibrosis, and centrilobular
endophlebitis developed concurrently. Smith et al. (1974) lists
Senecio species as one of the stronger hepatotoxic substances.
Lupinosis is a chronic poisoning arising from congestion of
the plant of the species genus Lupinus, and this is observed to
cause progressive liver damage. The liver is bright yellow and
friable at postmortem (Gardine~, 1966).
Manifestation of Lantana toxicity in sheep include enlarge-
ment and dilation of bile canaliculi and injury to microvilli in
the liver (Pass et al., 1978) and intrahepatic cholestatis with
decrease in canalicular membrane ATPase activity (Gopinath,1969).
Plants of the genus Phyllanthus e.g. P. abnormis and £.
drumondii cause postmortem lesions which are examples of hepato-
toxic and nephrotoxic poisoning.Changes of acute toxic hepatitis in
these plants were observed to progress in chronic cases, to
cirrhosis.
Toxicosis,by Agave lechuguilla in sheep and goats showns
marked fatty change of the liver which is principally responsi-
ble for its pale or yellow colour grossly.Centrilobular necrosis is
of moderate degree and cloudy swelling is not conspicuous.
Lymphocytic infiltrations in and around the islands of Glisson are
usually noticeable. There is considerable retention and
inspissation of bile, which forms precipitated masses in some of
the intrah~patic hi~e dUC~G.
With Heliotropium europaeum poisoning in sheep, the outcome is
a slowly progressive toxic hepati tis with symptoms of illness
appearing perhaps months after access to the plant has ceased.
Jaundice was observed as a salient feature with the fatty change
progressing to cirrhos1s and a shrunken liver.
The principal lesion of A. flavus intoxication occur in
the liver and may be classified as toxic hepatitis (Smith et
al., 1974).Newberne and Butler (1969) reported that the most
constant responses to aflatoxin B is proliferation of small bile
ductules at the periphery of hepatic lobules. Changes in
hepatocytes (vacuolization, fatty change, loss of parenchyma,
pyknosis) leading to necrosis were usually localized in one part of
the hepatic lobule.
1.6.4. Pathological lesions associated with the Kidney
Even though the kidney may constitute les3 than one per
cent of the bodY,weight, they normally receive 20 to 25 per cent of
the resting cardiac output indeed the renal cortex has as much
greater blood supply than many other organs (Casarrett and Doull,
1975). This extremely rich perfusion implies that large-amounts of
any circulating drug or poison will quickly reach the kidney.
Moreover renal toxicity is also influenced by ability of the organ
142
to extract substances from the blood and to accumulate them within
the renal parenchyma or in the tubular lumen.
Another aspect of renal function also contributing to
the frequency to toxic effects in the kidney is the fact that
filtered substances may be concentrated in the tubular lumen as a
result of salt and water reabsorption. This salt and water
reabsorption process will also further increase the tubular fluid
over plasma ratio of secreted solutes so that values as high as 500
are readily attained. In addition to high inratubular
concentration of certain solutes significant interstitial
accumulation may also result from counter current concentrating
mechanisms in the renal medulla. This could result in higher
levels of diffusible and potentially toxic molecules such as
cyanide or flouride in the renal medulla than other tissues.
Foreign compounds may thus become especially toxic to the kidney by
virtue of their high ~ntraluminal,intracellular or intersti- tial
concentrations.
For example poisoning by Agave lechuguilla produces dilati-
on of the kidney tubules.The distension is probably due to
obstruction by l~rge albuminous casts in the case of some nephrons
but there is also evidence of hypertrophic dilation and also of
generation of epithelium. Fatty change occurs in the ascending
loops of Henle. Similar observations were made with Phyllanthus.
Equally, normal tubular functions as occurs with maintena-
143
nce of normal acid-base balance and correction of metabolic
acidosis depends on secretion of hydrogen ions in both the proximal
and distal portions of the kidney. This permits toxic interaction
in the kidney, that cannot readily occur in other organs. A
substance whose solubility changes in PH may precipitate in the
acidified tubular fluid and block the normal flow of urine.
Alternatively a toxic effect may be produced by a chemical species
set free from a filtered precursor by the action ,of hydrogen ion.
Such mechanism has been invoked to explain the toxicity of uranyl
ions circulating in plasma as a relatively inert but acid, labile
uranyl-bicarbonate - uranyl complex {Nomiyama and Foulkes, 1968}.
In addtion, the kidney as in the case with any other metabo-
lically active organ, is also sensitive to metabolic ·poisons.For
example cyanide injected into the renal artery of dogs causes
saliuresis by inhibiting normal sodium chloride reabsorption
(Vander, 1963).
1.6.4.1. Toxic Tubular Nephrosis
In this condition various irritant substances act directly and
without any previous hypersensitization to produce fatty change and
necrosis of the, delicate epithelial cells lining the tubules.
There is also hyperaemia very limited infiltration by lymphocytes
and proliferation of fibrous tissues.
The proximal convulated tubules with their large epithelial
cells, suffer most as with the ingestion of the young leaves of
144
Quercus havardi (Llewellyn, 1962). The leaves of this plant is
said to contain large amounts of tannic acid which is generally
believed to be responsible for the toxic properties of Quercus
havardi. Dollahite, Pigeon and Camp (196~) found that the lesions
produced in the rabbit by 2. havarrdii are very similar to those
produced by multiple doses of tannic acid. Lipidosis is observed
to be most extensive in the proximal tubules or it may appear
chiefly oi exclusively in the epithelium of the ascending
loops of He~le. The proximal convulated tubules may also show
albuminous degeneration and calcification, usually in the form of
granules no larger than on or two cells in the tubules (Battifora
and Markowitz, 1969).
Most of the substances which~cause acute toxic hepatitis also
produce toxic nephrosis to some degree. Substances which can also
produce tubular nephrosis also include oxalic acid and plants
containing oxalates, e.g. Amaranthus retrof lexus, alphanaph
thylthiourea and other chemical substances (Jaenike, 1966).
Poisoning by A. retroflexus presents microscropic evidence of toxic
tubular nephrosis with interstitial oedema in the renal cortex.
There is presence of large amount of oedema surrounding the kidney
between the renal capsule and perirenal peritoneum. Sometimes
oedema fluid is tinged with blood but the affected kidneys are
usually pale and normal in size. The renal capsule is not usually
145
affected although the oedema may extend into the renal parenchyma
(Buck et al., 1966; Marshall, Buck and Bell, 1967).
Haemorrhages, usually petechial in size, located just
beneath the capsule and visible through it, or in the intertubu-
lar connective tissues, are frequent in many types of poisoning
including those from crotalarias and oak buds, (Boughton and Hardy,
1963) • Extensive haemorrhages into the pelves, or peripherally
with separation and distention of the capsule has been observed
with poisoning by Melilotus alba.
1.6.5. Morphologic lesions associated with Central Nervous
system toxicosis
The site of action most frequently involved in systemic
toxicity is the central nervous system. Even with many compounds
having a prominent effect elsewhere, the CNS particularly the
brain, can be demonstrated to be affected using appropriate and
sensitive methods. H?wever, in general, the CNS is protected from
toxicants by the blood brain barrier. The barrier is a functional
concept based on observations that some substances that enter and
affect many of the-soft tissues of the body, such as the liver,
kidney and muscle are excluded from the brain. Non Polar, lipid-
soluble compounds usually penetrate the blood-brain barrier, while
highly. polar compounds tend to be excluded.
Even those toxic substances that can penetrate the brain
tissue do not affect equally all of the cell types in the brain.
146
Different brain areas usually have different sensitivities to
toxicants reflecting the unique biochemistry of the cells, the kind
and amount of innervation of neurons as well as difference in
degree of vascularization of vbrain areas. Neurotoxicants can be
divided into those that cause damage subsequent to anoxia and those
that cause damage because of affinity for specific structures.
1.6.5.1. Symptoms and lesions in the CNS due to plant poisons
Under certain conditions, the millet Paspalum scrobiculatum,
widely grown in Africa has been known to cause poisoning in cattle
with symptoms including tremors, clonic convulsions, coma, and
death. The toxic agent is said to be a polycyclic hydrocarb-on
which affects the CNS (Gupta and Bhide, 1967).
Since theobromine from Theobroma cacao is completely absorbed
from the alimentary tract, and only slowly excreted,
small doses are known to have a cummulative effect. Death from
poisoning by T. cacao may thus be delayed until a critical level is
attained.Black and Barron (1943) describe nervous excitabil- ityas
a feature of theobromine poisoning in poultry. In calves given
waste chocolate in the diet, Curtis and Griffiths (197~) noted
excitement, swe~ting increased respiratory rate and rapid pulse,
followed by convulsion and collapse.
In the chronic syndrome produced by Phalaris tuberosa
called Phalaris staggers, there are degenerative changes in the
mitochrondria of nerve cells and sometimes accompanied by
147
demyel ination of the spinal cords (Gallagher et al., 1967). The
acute syndrome associated with this perenial grass are neurological
symptoms not unlike those seen in rye grass staggers while the
chronic syndrome is characterized by head nodding, ataxia and
weakness.
In horses, the symptoms first noticed associated with
pteridium aquilinum poisoning, is almost always some slight
incoordination of movement inan animal in poor to fair condition.
As the disease progresses, staggering becomes more pronounced, the
feet and legs moving and being held in an unnatural manner. The
animal stands with its feet well spread and with the back arched.
Incoordination increases and severe muscular tremor appears. If no
treatment is attempted, the animal goes down and may injure itself
in an attempt to rise. Death is preceded by atonic spasms and the
typical opisthotonus of a thiamine deficiency (Evans et al.,1954).
A paresis associated with spinal cord demyelination has
been reported in pigs newly born of sows receiving very leafy
green irrigated white clover (Trifolium rapens) cut and fed
dialy to the 1imit of appeti te. Farrowing was normal and the
piglets norma~ as regards size and vigour, except that they were
unable to stand and suck; they died in a few days apparently from
starvation and dehydration. Mcclymont (1954) considers the
diet to be the most likely aetiological factor and suggested
among other things that the clover may contain a
148
neurotoxic factor whose concentration varied seasonally or that the
demyelinating lesions could have been due to mild chronic cyanide
I
intoxiccation.
Lathyrus latifolius and ~. sylvestris contain the neuroto-
xic factor L-&, Y-diaminobutyric acid.It is suggested that
lathyrogenic agents act by blocking certain carbonyl group normally
present in collagen, and thus interfering with formation of cross
linkages (Levene, 1962). The action may be retarded by reserpine or
by calcium salts.
Spikes of Abrus precatorius coated with the crushedseeds have
long been used for the malicious killing of cattle, the animal
dying in 2-4 days after exhibiting salivation, stiffness,
incoordination, muscular spasms and convulsions with extensive
painful swelling round the site of the implant (Rahman and Mia,
1972). However, when the whole seed was given by mouth to cattle
at had no effect, although they can kill fowls within a few days.
Rams that had eaten Delphinium hybridium thrown over the
fence showed incoordination and violent tetanic spasms followed by
recumbency the spasms recurring at the animal were disturbed
(Milne, 1966).
Symptoms of poisonig with Atropa belladonna (deadly nightsh-
ade) in pigs were nervous symptoms, dilated pupils,and very often
animals were unable to stand with lesions as catarrhal inflammation
in the stomach and intestine.A sydrome associated with the grazing
149
of Sorghum sudense (Sudan grass) characterized by urinary
incontinence and incoordination of the hind-legs has been described
(Knight 1968). There is ataxia and paresis of the bladder due to
a focal axonal degeneration of the lumbar and sacral segments of
the spinal cord (Adams et al., 1969).
Nerium oleander has been known for its extreme toxicity.
Humans have been fatally poisoned not only by eating a few
leaves, but even when oleander twigs were used as skewers in
meat.Horses, cattle and sheep get poisoned when the leaves are
mixed with hay. Tremors and tetanic stiffness give way to
paralysis and death usually without convulsions (Liener, 1974).
The nervous disorder associated with consumption of plants of
the genues Astragalus e.g. A pubentissimus and A. lentiginosus is
observed to be slow in onset. In the horse, hyperexi tabili t y ,
fright and violent reactions to slight stimuli due to inability to
see clearly were the ~igns observed. In the cow the same imparied
vision and disordered judgement cause the animal to perform all
movements of drinking while her mouth was still above the water
(james, Van Kampen, and Hartley, 1970). The sensory and motor
derangements inc~ease uintil the animal is unable to get food for
itself. A slowly increasing ataxia of the limb which results in
ascending paralysis are the signs which top the nervous disorder
which lead to the death of the animal. Microscop-ically Van Kampen
and James (1969) observed vacuolation of the cytoplasm of cells of
1 50
various tissues which leads to necrosis in the neurons. Damage to
neurons is most significant and, is found in the central and
peripheral nervous system including those in Meissner's and
Auerbach' plexuses of the GIT. Later karyolysis, karyorrhexis or
cytolysis leads to loss of neurons or mineralization of the
necrotic remnants. Perivascular oedema was also observed
throughout the central nervous system.
The basic sign associated with poisoning by A. decembens, A
convallarius and A. hylophilas was nervous weakness and incordin-
ation involving the hind limbs in the cattle, sheep, rabbits and
chicken which were experimentally poisoned (Williams, Van Kampen,
and Norris,1969).Nervous incoordination results in crossing of the
legs and weakness shown by knuckling 'over of the fetlock joints .
1.6.6. Plants producing teratogenic and developmental anomalies
Developmental anomalies ordinarily originate before birth r,e.
in embryonic life when body structures are being formed. However,
malformation are also possible in young growing animals. Errors in
the developmental mechanisms which could produce malformations
include the following: (a) arrest of development in a certain part
of the embryo so that some structures dont develop or are small
(hypoplasia).
(b) some embryonal or fetal structures fail to disappear
when they are expected to.
(c) Some grooves, fissures or openings dont close properly.
1 51
(d) aberrant (ectopic or heterotropic) structures.
(e) duplications of structures.
Among the many causes of congenital anomalies are the
intrauterine effects of poisons ingested by the mother. A
distinctive congenital syndrome known as crooked calf disease
has been observed in range cattle which is associated with
the teratogenic effects of plants of the genus Lupinus. The
cows had consumed the plant between the fortieth and
seventieth day of pregnancy. A similar effect was reproduced
in cows fed dried lupine plants especially Lupinus sericeus
(Shupe, James and Binos, 1967). The affected calves were
born with arthrogryposis, scoliosis, torticolis and cleft
palate. Most of the calves were viable and could survive to
become adults though some abortions were recorded among the
pregnant cows.
Feeding to A. pubentissimus to pregnant sheep was reported to
cause frequent occurrence of congenital anomalies in the offspring
(James Keeler and Binns, 1969). The type of malformation was
observed to be assoc iated with the stage of pregnancy. If the
plant was consumed by the ewes during the twenty-fifth to the
forthyninth days of pregnancy, aplasia of the lower jaw was a
dominant anoma~y in the lambs. Between the 40th and 60th days of
pregnancy often resulted in hypermobility of the hock and stifle
joints whereas feeding between the 60th and 90th days of gestation
1 52
resulted in flexures of carpal joints in the lambs. Abortions were
also recorded in some of the ewes. Shupe et ale (196H) in similar
experiments observed loss of weight of the body and testes but no
changes occurred in the libido or sperm counts of the rams fed with
the plant.
Feeding of the fresh or dried plant Veratrum californicum by
the ewe during her thirteenth or fifteenth days of pregancy is
reported to cause a striking and grotosque congenital malformations
in lambs, (Binns et al., 1964; Keeler and Binns, 1964). The
deformed lambs were born alive singly or with a living or dead twin
which mayor not have been affected. The malformations include
partial or complete cyclopia. A single-eye or two fused eyes
usually occupy the same orbit. The upper jaw may be sIightly
distored with a cleft palate or almost totally agbsent. The nose
is distorted in varying degrees and the lower jaw usually protrudes
drastically. A La.r ge media cutaneous protuberance was often
observed over the single eye. The cerebral hemispheres were often
fused wi th the hydrocephalus involving the lateral ventricles
(Clegg, 1971).
Feeding of Lathyrus odoratus to growing rats is reported
to have produced skeletal deformities and changes in other
mesodermal tissues (Levene, 1962). Periosteal new bone
formation, kyphoscoliosis and dissecting aneurysms of the aorta was
reported. Poisoning in sheep has been shown to result in
153
abortions, intrauterine death, contracted tendons and aplasia of
the laower jaw in the offspring (Keeler et al., 1967).
1.6.7. Tumour forming Plant Agents
Tumour or neoplasm according to Smith, Jone and Hunt (1974) is
a new growth of cells which (1) proliferate continuously without
control (2) bear a considerable resemblance to the healthy cells
from which they arose (3) have no orderly structural arrangment (4)
serve no useful function (5) have no clearly understood cause.
In £. aguilinum poisoning, lesions in the urinary system
involve the bladder, ureters or renal pelvis and appear to
represent a chronic but violently hyperplastic and haemorrhagic
inflammation which leads to frank neoplasia (Schacham, Philip and
Cowden, 1970). The transitional epithelium undergoes localized
proliferation with metaplasia to mucinous columnar or stratified
squamous types. The hyperplastic epithlium may acquire neoplastic
properties developin~ into a squamous cell or adenocarcinoma which
is locally invasive and may matastasize to the regional lymph nodes
and lungs. The capillaries of the inflammatory lesion may form
haemangiomas in the stroma or project from the mucosal surface
(Evans and Man&on, 1965).
1.7. Aim and scope Scope of Study
In describing the importance of toxic plants in our
environment both to livestock and humans. Hall (1977) and Clarke
and Clarke (1975) emphasized that it is obviously impossible in
154
anything other than a work of encyclopedic proportions to mention
all the species of plants which at sometime or the other, have been
suspected of causing poisoning in livestock or man.
There can be little doubt that many instances of general
malaise, inappetence and dullness in grazing animals which
veterinarians are called in to deal with, and the cause of which
are seldom diagnosed are due to consumption of subclinical doses of
some harmful plants. Plant poisoning is essentially a local
problem, occuring in localities where poisonous plants may form
large proportion of the herbage available to grazing animals,
(Achhireddy and Singh, 1984). Some of these toxic plants occur
generally around the country, some occur only in restricted areas
in perhaps only one country, some even well known quite deadly
species in one region will be used as feed in another without any
apparent effect, (Irvine, 1961 j Hall, 1977). The reasons for these
variations in toxicit~ can sometimes be accounted for, sometimes it
is guessed at, and sometimes it is completely unkrtown.
Poisonous plants are often refused by animals (many have a
repulsive smell or ~ontain highly irritant juices e.g. Euphorbia
and Asclepias ~pecies) and are eaten only when other herbage is
scare. Whereas highly poisonous plants as ~ cymosum are reported
to be highly palatable (Van Dijk et. al., 1972). It is however, an
unfortunate fact that many animals develop a tase for poisonous
plants. Clarke and Clarke (1975) reported that there were many
155
instances on record of animals having to be forcibly restrained
from returning to patches of poisonous plants after having
recovered from poisoning by them.
Occasionally animals get ,poisoned when cutting from a garden
are thoughtlessly thrown into a dry lot most especially where
animals are accustomed to having hay placed before them.
In common with all plant life, the presence or absence of a
poisonous plant is very much a matter of environment. Certain
ecological conditions favour certain plant species. Variations in
these conditions will cause variations in prevalence of plants and
in the likelihood of these plants poisoning livestock. For
example, plants of the genera Morinda, Astragalus, Naptunia,
Oonopsis, Xylorrhiza and Stanleya have the ability to take up
inorganic selenium and convert it to organic selenium, thus making
it available to other plants and also as a source of selenium
toxicosis (Avery et al., 1961; Harris et al., 197~; and Janses et
aI., 1970). Plants such as Amaranthus sp, Cucumis sativa,
Brachiaria sp., Astragalus are nitrate accummulators (William and
James, 1975; Andrade et al., 1971). Cynodon plectostachus,
Eleusine indica, Vicia sativa, Phaseolus lunatus are some of the
plants which accummulate cyanide (Herrington et al., 1977; Sidha,
1967; Aletor, 1983). Plants such Xanthscephalum sp. are reported
not to be always poisonous. The differences in degree of toxicity
is said to be related to variations in growing conditions (Shaver,
156
Camp and Dollahi t.e, 1964). Such variations in plant toxicity,
therefore calls for caution in trying to use information from other
areas to evaluate plant usefulness in other localities.
The plants which have been chosen for the present study are
commonly available in Nigeria and they can be found in areas where
they are easily accessible to grazing livestock and to playing
children. some of them belong to plant families which are reported
to be poisonous in other countries.
Lantana camara is known to be notorious for its ability to
escape from gardens where it is frequently grown as an ornamental
and flourishes in grassland, (Achhireddy and Singh, 1984). It
inhibits the growth of other vegetation (Sharma, 1984) and worst of
all, it is said to be spreading very rapidly to areas hitherto free,
of Lantana. The plant is important for consideration because of
the severe intoxication including jaundice and photosensitization
it produces in livestock (Sharma et al., 1982 j Sharma et al.,
1987) •
~. torvum belongs to the group of plants known as solanines.
Most members of this family such as Datura spp. are known to be
very poisonous~ Animals have been seen to avoid ~. torvum but in
periods of drought when forage is scarce they ser~e as easy source
of forage (Hall, 1977).
Luecaena leucocephala contains the toxic substance mimosime.
The ingestion of large quantities of this legume has been reported
157
to cause hair loss in horses, dogs rabbits, pigs and may affect
normal reproductive behaviour (Mullenax, 1963; Letts, 1963; and
Seawright, 1963). Cases of suspected poisoning by~. leucocephala
has been made (Akpokodje and Otesile, 1987). However the authors
did not have experimental proof of their observation. Therefore it
becomes necessary to ascertain such observation.
Tribulus terrestris is reported to cause photosensitization
and animals eating the plant show inability to eat and drink,
fever, oedema, especially of the limbs, blindness, jaundice, a
purulent dermatitis and in advanced stages asphyxiation and death
(Amjadi, Ahonrai and Baharse, 1977).
Eugenia uniflora is widely grown for its sourly sweet fruit
- (Irvine, 1961), wh i-Lethe plant is said to contain over :W per cent
of tannin. Levels of tannins of such in plants have been reported
to be source of poisoning in animals (Barry and Forss, 1983). The
leaves by personal observation have a very pungent smell. This
could be indicative of its level of poisonous constituents.
Dichapetalum madagascasiense belong to a group of plant many
members of which have been reported to be very toxic to livestock.
Such other mem~ers include Q. toxicarium (Irvine, 1961; Vickery and
Vickery, 1971; Nwude, 1977; Watt and Breyer Brandwijk, 196~); Q.
cyamosum (Watt et al., 1962; Van Dijk et al., 197~). Q. barteri
(Nwudu et al., 1977). Q. madagascasiense can widely be located in
pastures, hedges and other places around yet little is known about
158
its poisonous potentialities. Therefore its inclusion in this
study is significant.
Most of the reported cases of plant poisoning have been based
on the clinical symptoms observed after the animals have consumed
toxic quanti ties of the plant. Less effort has been devoted
towards the mechanistic evaluation of such toxic substances. Such
studies are often useful for the development of tests for
prediction of risks, which facilitates the search for safer
substances and for rational treatment of the manifestations of
toxicity (Gilman et al., 1980).
This study was carried out therefore to evaluate the effects
of the suspected poisonous plants, Solanum torvum Swartz, Tribulus
terrestris Linn j Leucaena leucocephala Benth j Eugenia uni flora
Linn; Lantana camara Linn and Dichapetalum madagascasiense Poir in
rats and goats using the following para'IDeters as indices of
evaluation:
1. Haematology
This is an essential part of the studies since some of the
most dreaded side effects of poisons are haematological e.g.
agranulocytosi$, thrombocytomenia, complete marrow aplasia or even
red cell destruction. A complete blood count consisting of
haemoglobin, white cell, red blood cell and di fferential count,
packed cell volume and Winthrobe red cell indices evaluations was
determined from each animal.
159
2. Biochemical estimations
These are important since biochemical changes are the earliest
indicators of organic damage and moreover animals grazing on
poisonous plants could be source of meat or other by products to
man. Since the only way of observing such toxic effects while the
animals are still alive autospsies are not anticipated, thus
biochemical changes are useful. Such biochemical parameters
include serum enzymes alanine aminotransferase, aspartate
aminotransferase, alkaline phosphatase, serum electrolytes, total
protein albumin and globulin fractions and blood urea nitrogen.
3. Gross and histopathological lesions arising from toxicity are
the commonly encountered indices for evaluating toxicity.
4. Clinical signs associated with poisoning.
5. Chemical analysis of the toxic plants were attempted. Such
analysis included etimation of inorganic substances as nitrate,
L
nitrite, oxalate, cyanide, phytin, and metals as calcium, lead,
molybdenum, manganese, iron, zinc and copper.
CHAPTER TWO
2. MATERIALS AND METHOD
2.1 Experimental Animals
The animals used in this study were :-
(a) Mice:
The mice were white albino mice,all male and weighing
between 20-25 grams obtained from the Department of Veterinary
Physiology and Pharmacology animal house. These were used for the
pilot toxicity studies.
(b) Rats:
The rats used were of the Sprague Dawley strain weighing
between 200 and 250 grams. They were all male rats and were
maintained at the Department of Veterinary Physiology and
Pharmacology and the University College Hospital, University
of Ibadan. They were kept in rat cages and fed rat cubes
(Ladokun and Sons 'Livestock Feeds. Nigeria Ltd) and allowed
free access to clean fresh water in bottles.
(c) Goats:
The goats were all male West African dwarf goats about
3-~ years ola purchased, from a flock at Abadin~, University
of Ibadan. They were dewormed with Levamisole 15% (Phenix
Pharm, Belgium) and observed for two weeks housed at the
Veterinary Surgery and Reproduction animal house before their
use.
1 61
2.2 The Poisonous Plants
The plants used in this study included:
i. Dichapetalum madagascasiense Poir Plate 6
ii. Lantana cama.ra Linn Plate 5
iii. Eugenia uniflora Linn Plate 4
iv. Solanum torvum Benth Plate 1
v. Tribulus terrestris Linn Plate 2
vi. Leucaena leucocephala Benth Plate 3
2.2.1 Preparation of the extracts of the Poisonous plants
The leaves of the plants were always harvested fresh from
their stands at different periods of the year. The drying
operation was carried out under controlled conditions in good air
draft. Some of the dried leaves were crushed into a coarse powdery
form and weighed. The extraction procedure is as described by
Harbone (1973). The powdered leaves were continuously extracted
using absolute e~hanol in a Soxhlet extractor until all the green
pigment was in the extract. The extract obtained was clarified by
filtration through celite on a water pump and was then concentrated
in vacuo using a rotation evaporator which concentrates bulky
soluti-ons down to small volumes without bumping at low
temperatures. The ethanol remaining in the extract was finally
removed by placing small volumes in porcelain dishes in the oven
set at low temperatures of 40°. This provided the semi solid
materials which were administered to the rats
162
and mice in doses according to the LD50 of the particular plant.
2.2.2. Technique for Pelleting leave
Only the leaves of Dichapetalum madagascasiense were pelleted.
The dried leaves were ground along with maize grain into coarse
powdery form, in different proportions. Domestic starch was
prepared and added to the mixture of maize and leaves or leaves
alone in a proportions to hold them together i.e. binder. These
were then pelleted using specially designed appliance and the
pellet dried in he oven at a temperature of 40°%.
2.3. Technique for administration of extracts and leave
(a) Mice and rats:
The mice used in this study were separated into ~ groups
for each plant with a group consisting of 10 mice. A group was
then administered with an assigned dose of the extract. The rats
were divided into 4 groups with each cage containing the rats
representing the 3 doses of the extract for each plant along with
a control group.
The different groups of rats were administered with the
different doses of each plant dissolved in propylene glycol daily
by gavage (Po). The control group were g~ven only O. Sml of
propylene glycol by the same route. These were repeated daily for
14 days for each plant extract. The groups of rats which were fed
on the leaves of Q. madagascasiense, had the leaves in pellets.
The pellets consisted of the crushed leaves binder (starch) and
163
ground maize. The crushed leaves were mixed in proportion of only
leaves, 75% and 50% with the maize. The control group were fed on
rat cubes for 14 days.
(b) Experimental goats
The goats (four per plant) were fed with freshly harvest-
ed leaves of Q. madagascasiense and ~. camara daily in pens. The
leaves were supplemented with freshly harvested grass and
concentrates after consumption of the poisonous leaves. They were
allowed ·fresh water ad-libitum. Aqueous extracts of ~. camara
(200-300 grams of the leaves were made latter and administered
orally to the goats which were supposed to feed on ~. camara. This
was after it was observed that the goats did not actively feed on
the leaves. This was by homogenizing the fresh leaves with small
quanti ty of water. The homogenate was administered using a 20ml
syringe in bits until the whole quantity was given to each goat.
2.4. Technique for obtaining blood and Serum samples
Blood was collected by Cardiac puncture from
diethylether anaesthetized rats or the jugular vein in the
goats, into clean bottles containing disodium ethylendiamine
t.e t r-eacet i.c . acid (NaEDTA) 1-2mg/ml of blood into bottles and
allowed to clot. The sera' was separated from the clot and
centrifuged according to groups into clean bottles for the
analysis.
164
2 .5. Determination of inorganic constituents of Plants
2.5.1 Determination of Phytin content
Phytin content was determined by a method described by
Wheeler and Ferrel(1971). The principle behind this procedure is
that the phytate in the plant is extracted with trichloroacetic
acid and precipitated as ferric salt.The iron content of the
precipitate is determined colorimetrically and the phytate
phosphate content calculated from the value.
Reagents
Barium phytate (B~ Phy) standard was prepared by dissol-
ving commercial phytin in 3% trichloroacetic acid (TCA) and
filtered. Ferric phytate (Fe. Phy) was precipitated from the
filtrate by adding concentrated FeCl3 solution. The precipitate
was washed with 3% TeA, slurred in water and converted to sodium
phytate and Fe (OH)3 by addi tion of NaOH. The Fe(OH)3 was
filtered out and the Naphytate solution adjusted to about PH 0 with
HCl and Ba Phy precipitated by addition of BaCl. The B~ Phy was
redissol ved in HCl and the double precipi tate cycle repeated twice.""tYf'j'
Procedure
About 30mg of the finely ground leaf material was placed
in a 125ml flask and extracted with some of 3% trichloroacetic acid
or 30 minutes with mechanical shaking. A suspension of this was
165
centrifuged and 10ml of the aupe r-na.t.t,antransferred to a 40ml
conical centrifuge tube. A 4ml FeCl3 solution (made to contain
2mg FeCl3 per ml in 3% TCA) was added to the aliquot. The tube
and its content were again heated in water bath for 45minutes. The
solution was centri f uged for 10 to 15 min. and the clear
supernatant decanted off. The precipitate was dispersed in 20 to
25ml 3% TCA and heated in boiling water bath for 10 m i ns. and
centrifuged. The precipitate was then dispersed in few ml of water
and 3ml of 1.5N NaOH added by mixing. The volume was
increased with water to 30ml and heated again in water bath
for 30 minutes. The solution was filtered hot. The precipitate
was washed with about 60-70ml hot water and the precipitate from
the paper dissolved with 40ml hot 3.2N NH4N02 into a 100ml
volumetric flask. The filter paper was washed with several
portions of hot water into the flask and the content cooled to room
temperature diluting to volume with water. A 5ml aliquot of this
was then diluted to approximately 70ml with water. A 20ml of 1.5M
potassium thiosulphate was added and diluted to 100ml volume and
the absorption of this read over a spectrophotometer at 480nm.
There was a reagent blank prepared for each sample. The iron
content was calculated from the ferric nitrate standard or
alternatively from a standard curve prepared. The phytate
phosphorus was calculated from the iron resul ts assuming a 4.6
iron: phosphorus molecular ratio.
166
2.5.2. Determination of oxalate content of leaves
The oxalate content of the leaves was determined using the
procedure of Baker (1952). This involves the extraction of the
oxalate in the plant with HCl and its precipitation as calcium
oxalate from the deproteinised extract and subsequent extraction
with KMno4.
Reagent:
Dilute HCl (1+1)
NH. OH solution
Phosphoric-Tungstate reagent - dissolve 24g of sodium tungstate in
water, add 40ml of syrupy phosphoric acid and dilute to 1 litre.
Calcium chloride buffer dissolve 25gm of anhydrous caC~ in
500ml of 50% V/V glacial acetic acid and add this
to a solution of 33Ug of sodium acetate in water
and dilute to 50Uml.
Wash Solution - A5% V/V solution of acetic acid kept over
calr.ium oxalate at room temperature. This
solution was shaken periodically and filtered
before use.
Sulphuric acid - a 10% V/V solution
Potassium permanganate - an U.U2N solution prepared as
required by diluting a U.1N solution
Procedure:
For the determination of total oxalate, 6Ugm of chopped
167
green leaves was homogenized with 100ml of water and this
transferred to a 600ml beaker. 2 volumes of diluted HCl was
added to each 10 volumes of the mixture with 2 drops of capryl
alcohol added and boiled for 15 minutes. After cooling, the
mixture was transferred into a 500ml volumetric flask and
diluted to mark with shaking and this left overnight.
The solution was then filtered through fitter paper after
shaking. A 25ml of the filtrate was transferred into a tube
with a stopper and 5ml of the phosphoric tungstate reagent
added.By inversion,the mixture was mixed and set aside for 5
hours.This was then centrifuged for 10minutes at 3000rpm with the
transfer of 20ml of clear solution to a 50ml centrifuge tube.
Ammonium hydroxide was added drop wise from a burette until the
solution was alkaline indicated by formation of slight precipitate
of phosphotungstate. 5ml CaCl, reagent was added with stirring
using glass rod and the tube left overnight in the refrigerator at
This solution was centrifuged for 10 minutes. The
supernatant was carefully removed and the precipitate washed with
20ml of wash solution stirring vigorously with a fine rod until the
precipi tate was broken up. This solution was- centrifuged for
10minutes and the washings carefully removed. The precipitate was
dissolved in 5ml of 10% H2So4 and this placed in water bath at
100°C for 2 mins. The oxalic acid in this was titrated with 0.02N
potassium permanganate. The oxalate content of the leaves was
168
estimated by assuming that lml of O. 02N KMno4 = O. 00090g of
oxalate.
2.5.3. Determination of hydrocyanic acid in the leaves
The principle behind this method is that the bound hydrocy-
anic acid in form of the glucoside is liberated and along with the
free HeN, converted to the red ferric thiocyanate which is measured
colorimetrically.
Reagents:
The ferric reagent-this consists of 10% solution of ferric
ammonium sulphate made up by dissolving 50gm of ferric ammonium
sulphate in 250ml of distilled water. To this was added 250ml of
concentrated nitric acid and the solution boiled to expel any oxide
of nitrogen and the solution filtered.
Preparation of Sodium Polysulphide
A 20% solution of sodium hydroxide was saturated with sulph-
urretted hydrogen and an excess of finely powdered sulphur
added.The mixture was shaken regularly for 10mins. and filtered.
The filtrate was then diluted with distilled water until the color
was like that of a 3.5% aqueous solution of potassium dichromate.
Preparation of solution of HON from leaves.
10gm specimen of leaves were stored in 200 ml of water
containing 10ml of 5% solution of mecuric chloride in closed
169
containers for 5 days.The mecuric chloride reacts with liberated
HCN from the glucoside formed during storage. Stannous chloride is
added to this solution to liberated HCN before analysis.
Procedure:
This was carried out as described by Van der Walt (1944).
(i) Conversion of HCN to thiocyanic acid
Conversion of HCN to thiocyanic acid was carried out in
stoppered vessel using 1.0ml of the sodium polysulphide solution
per 50ml of the solution containing the hydrogen cyanide The
solution was then placed in water bath for 20 mins. for complete
conversion of the HCN into thiocyanate.
(ii) Removal of the sulphur.
After conversion of the HCN into thiocyanic acid a slight
excess of dilute HCN was added and the solution aerated for one
hour to remove the sulphuretted hydrogen and render the
precipi t.a ted sulphur filterable. This is important since if any
sulphur passes the filter it may dissolve on subsequent evaporation
of the filtrate in the presence of sodium hydroxide to form
thiosulphate.
(iit ) 'Concentration of filtrate
The was done by evaporation of filtrate after addi tion of
slight excess of sodium hydroxide on a boiling water bath. The
solution was then rendered acid with a sLight excess of dilute
170
HNo2. The acidified solution was then brought to 29ml with
distilled water before 1ml of the ferric reagent was added.
(iv) Color intensity of ferric thiocyanate
1ml of the ferric Teagent was used per 30ml of solution.
Color intensity of the resulting ferric thiocyanate was measured
using a spectrophotometer using a standard made of distilled water
but treated the same way as above after the addition of the same
quantity of NaOH at 530nm.
Varying quantities of thiocyanate, representing varying
quantities of HCN were dissolved in 30ml of water containing 1ml of
a 20% solution of NaOH and 7ml dilute HCI and used for the
calibration curve.
2.5.4 Determination of nitrate content of the leaves
This is by reaction of the nitrate in the sample with pheno-
ldisulfonic acid which gives a yellow color.It was performed as
described by Snell Snell (1949).
Reagents:
Alumina cream
This was made of 125grams of alumina per litre of water to
which was added slowly while stirring, 55m~ of concentrated
ammonium hydroxide.
Phenoldisulfonic acid reagent
It was prepared by dissolving 25 gram of colorless phenol in
150ml of concentrated sulfuric acid. To this was added 75ml of
171
fuming sulfuric acid containing 13%.of free sulfur trioxide. This
was stirred well and heated in a flask for ~ hours on a boiling
water bath.
Procedure:
The dried ground sample 1mg was suspended in 10ml distilled
water
and heated on water bath for 30 minutes. The solution was then
rendered slightly ammoniacal and decolorized wi th 3ml alumina
cream. Half of this solution was then evaporated to dryness in a
porcel-ain dish. From a pipette was added rapidly 2ml the
phenoldisulfonic acid. The dish was rotated to ensure that the
acid got into contact with all the residue to dissolve it. When
this was cool 1:2 ammonium hydroxide was added slowly until the
solution was slightly alkaline.The solution was then filtered and
the absorbance read on a spectrophotometer with water as reference
520nm.
Series of Standards:
A solution into which was weighed O.lmg of nitrate nitrogen
was evaporated to dryness on a water bath. The residue was treated
with 2ml of phenoldisulfonic acid reagent as above and dissolved in
15ml of water which was then diluted to 500ml. Each of this
solution contains O.Olmg of nitrate nitrogen.For the calibration
curve, 0.1, 0.3, 0.7,1.0, 3, 5, 10, 20, 30 and 40ml of the solution
were taken and each diluted to 40ml and then with the addition of
172
1:2 ammonium hydroxide until faintly alkaline. These were each
diluted to 50ml. The value of each standard in ppm is 0.02 times
the number of ml of the standard solution used.
2.5.5. Determination of the Nitrite contents of the leaves
The methods of determination generally depend on
diazotization and coupling reactions.
Reagents:
Alumina cream - prepared as with the determination of nitrates
above.
Sulfanilic acid solution.
0.60 gram of the recrystallized sulfanilic acid was dissol-
ved in 70ml of hot water.The solution was cooled with the additi-on
of 20ml of concentrated hydrochloric acid and diluted to 100ml.and
mixed thoroughly.&-naphthylamine hydrochloride reagent. This was
prepared by adding 0.06 gram of the recrystallized material to
water with 1ml of conce trated Hel and made up to 100ml.
Procedure:
This was done as described by Snell & Snell (1949). The 20mg
dried ground sample was suspended in 250ml distilled water and
heated on water bath for 30minutes. 200ml of the"filtered solution
was decolorized by placing in a 250ml glass-stoppered bottle with
the addition of 3ml of alumina cream and mixed thoroughly leaving
it for 15 minutes. 1ml of the sulfanilic acid solution was added to
25ml of the sample solution mixed and allowed to stand for
173
10minutes for diazotization at room temperature in the dark to
prevent decomposition. 1ml of the &-naphthylamine hydrochloride
reagent was then added to the diazotized solution and buffered to
PH 2.0 - 2.5 with about 1ml of a 20 per cent solution of sodium
acetate. This was diluted to 50ml and mixed well. After 10
minutes the intensity of the reddish purple color was measured at
520nm with water as reference. The concentration of nitrite was
read off from a calibration curve prepared similar to that for the
determination of nitrate.
2.5.6. DETERMINATION OF THE METALLIC CONSTITUENTS OF THE PLANT
Metallic contents of the leaves were determined by procedu-
res as adopted at the International Institute for Tropical Agric-
ulture using Model 403 Perkin Elmer Spectrophotometer. These are
as described by Berman .(.1980) .
About 0.5gm of the crushed dried leaves were wet ashed with
20ml of nitric acid and 1ml of sulfuric acid.Anti bumping beads
were added and allowed to stand for about 5 hours. The various
metals were then determined by using aliquots from these
digestants.
2.5.6.1. Determination of the Copper content
An aliquot of the digestant as described above was placed in
flask and heated over a flame with continuous addition of nitric
acid until the digestant was clear.The digestant was then washed
174
into a 25ml volumetric flask and the volume made up to mark with
distilled water.
5ml aliquot of this was taken and the PH adjusted to between
6.5 and 7 with 2.5N sodium hydroxide,lml of a 2 per cent solution
of sodium diethyldithiocarbamate (NDDC) 1ml of 1% Ethylene
diaminetetraacetic acid (EDTA) and 3ml of Methylisobutyl ketone
(MIBK) were then added to the solution. This was well shaken
inorder to extract the chelate. This was allowed to equilibrate
and the solvent layer removed and centrifuged. The samples above
and the standards were hen compared at the 324.7nm resonance line
for copper.
2 .5. 6 •2 • Determination of Iron content
An aliquot of the digestant as prepared above was transferred
into a 50ml volumetric flask and the volume made up with distilled
water.This was allowed to settle and 10ul of the supernatant of
this was compared, at the 24H.3nm line in graphite furnace
programmed to dry at 100 C for 40s, char at BOO c for 40s and
atomize at 2400 C for 6s.
2 •5 .6. 3 • Determination of the zinc content
A 10ml aliquot of the digestant as prepared above,was trans-
ferred into a 25ml volumetric flask and this made up to volume with
distilled water. The standard and the prepared samples were then
compared as the 213.8nm line in an oxidizing flame using flow rates
of 51 per min for acetylene and 71 min for air.
175
2 •5 •6 .4. Determination of manganese content
A 10ml aliquot of the digestant prepared above was trans-
ferred into a 25ml volumetric flask and the volume made up with
distilled water.
20ml of this dilution was transferred into a separatory
funnel and the PH adjusted to 6-7.To this was added 1ml of ~% nDDC
nd 3ml of MIBK and thoroughly mixed and set aside for 10 minutes.
This was shaken to extract the chelate. Aliquots of the standard
and the unknown in the solution were compared i the graphite
furnace.
2.5.6.5. Determination of the lead content
A digestant of the leaves were prepared by the addition
of 2ml of perchloric and 30ml of nitric acid to 19ram of the
crushed dried leaves. An aliquot of the digestant was
transferred into a 25ml volumetric flask and the volume made
up to mark with di~tilled water.
5ml aliquot of this was transferred to a 60ml separatory
funnel and the PH adjusted to 5.5.-6.5 with a 2.5N NaOH. To this
was added 1ml of 2% NDDC and 2ml of MIBK mixed and left to stand
for 10minutes. After equilibration, the solvent. Fhase was removed
and centrifuged. The standard and unknown in solution above were
compared at the 282.3nm line.
176
2.5.6.6. Determination of molybdenum content
A digestant of the crushed leaves were prepared by adding
perchloric acid to a quantity of the leaves.The PH of the solution
was adjusted to PH 1 by.the addition of more perchloric acid. The
sample was then chelated and then extracted into
methylisobutylketone.The level of molybdenum content of this
extract was measured in an air acetylene flame at the 313. 3nmk
resonance line. A standard containing known concentration of
molybdenum was similarly treated as above inorder to determine the
concentration in the unknown sample.
2 • 5 • 7 • Determination of Haematological Parameters
(a) Haemoglobin concentration
This was determined as described by Jain (1986) using the
cyanmethaemoglobin method. After hemolysis of the red cells, the
haemoglobin is converted to cyammethaemoglobin by the cyanide in
the diluting soluti~n (Drabkin; diluent).
Reagent:
Drabkin's diluent
The diluent was prepared by dissolving 19 sodium bicarbonate,
52mg potassi~m cyanide, and 198mg potassium. ferricyanide in
distilled water and this was made up to loooml.
Procedure:
A O.02ml of the blood was added to 4ml Drabkin' s diluent
rising the pipette several times and the solution allowed to
1 77
stand for 10 minutes. The absorbance of the resultant solution was
read in a spectrophotometer at 540nm with the Drabkin's diluent as
reference.
Calculation:
Hb concentration (gm/dl) =
Absorbance of test solution x Hb Concentration x dilution factor
Absorbance of standard solution of standard solu.
where Hb concentration of std. = 0.057Ggm/dl
Dilution factor = G01
(b) Packed Cell Volume (Pcv)
This was done by the conventional method of filling a
capillary tube with blood. One end of the tube was sealed and the
tube centrifuged in a microhaematocrit centrifuge for G5minutes.
The Pcv in per cent was then read directly from a graphic reader.
This is as described by Schalm et al (1975).
(c) Blood coagulat~on time
Blood coagulation time was determined by braking bits of plane
non-heparinised capJ..llary tubes containing blood at 10 seconds
interval until a stringy clot was formed in between the two broken
ends of the tube . Alternati vely blood coagulation time was also
determined by drawing one end of an office pin through a drop of
blood on a clean slide until a stringy dot was drawn at the end of
the pin. This is as described by Jain (19H6).
1 78
(d) Differential Leucocyte Count
Reagent
Giemsa stain - this involved making a dilution of 1 in 10
of the original so.1ution of the stain. This is by
diluting 1ml of stain with 9ml dis. water
Procedure:
Identification of the different types of leukocytes was
enhanced using color plates of Diggs Dorothy, and Ann Bell,
Abbott Laboratories.
The di fferential leucocyte count was made by counting the
different types of WBC mainly eosinophils, basophils, neutrophils,
monocytes and lymphocytes viewed from each of thirty fields of oil
immersion objective of a microscope on Giemsa stained slides.
(e) Total Erythrocyte Count
Reagents.
Hayem's solution - this consisted of mecuric chloride
O.Sgm, sodium chloride, 1.0gm, sodium sulfate. 5gm,
dissolved in 200ml of distilled water.
Procedure:
This was 'determined by the hemocytometer me.thod as
discribed by Jain (1986). Briefly a 0.5ml of blood containing
anticoagulant (NaEDTA) was drawn into the blood pipette and by
steady suction drawing the erythrocyte diluting fluid to the 101
mark. This gives a 1.200 dilution. With gentle shaking, the blood
179
was mixed and the solution used to fill the improved Neubauer
hemocytometer ensuring no overflow. This was allowed to settle.
Using the objective lens of the microscope (x40) erythrocytes
in 5 of the 25 smaller .squares in the central area were counted.
Calculations.
Cell counted x 10 (0.1mm depth) x 5(1/5 of sqmm) x 200(1:200
dilution) = erythrocytes per cu mm (u/l) or
The sum of the cells in the five small squares multiplied by
10,000 = total erythrocyte per cu mm (u/l).
(f) Total Leucocyte count
Reagent:
Leucocyte diluting fluid.
This was made up of glacial acetic acid, 2ml gentian
violet (1% aqueous 1ml; distilled water 100ml).
Procedure:
The technique described under the erythrocyte count was
followed except for the diluting pipette which has the mark 11
above the bulbs. The blood drawn to the 0.5 mark was diluted to
the 11 mark above the bulbs of the WBC pipette, using the WBC
diluting fluid.
2 or 3 drops was discarded from the pipette before filling the
counting chamber. After allowing 1 minute for the leukocytes to
settle, the cells were counted under the x 16 objective lens of the
microscope from the 4 large corner squares.
180
Calculations:
Cells counted x 20(1:20 dilution) x 10 (0.1mm depth) x
4 number of square millimeters counted = WBC per cm mm.or The
sum of the cells counted x 50 = total leukocytes per cu mm.
( Erythrocyte indices
The erythrocyte-indices define the size and haemoglobin
content of the erythrocytes from values obtained from the RBC
count, haemoglobin concentration and hematocrit.
Calculation:
(i) Mean corpuscular volume (MCV)
This express average volume of the individual RBC
MCV = PCV x 10
RBC in millions/cu mm It is expressed in cubic microns
(u3)
(ii) Mean corpuscular haemoglobin concentration (MCHC)
This expresses. the concentration of haemoglobin in the
average erythrocyte.
MCHC = haemoglobin (gm %) x 100
PCV
It is expressed in per cent
(iii) Mean corpuscular haemoglobin (MCH)
It is the amount of haemoglobin by weight in the
average erythrocyte.
181
MCH = haemoglobin (gm %) x 10
RBC count (million/cu mm
It is expressed in micrograms (u gm)
2 .7. Determination of serum biochemical parameters.
2.7.1. Measurement of blood urea nitrogen (BUN)
This was based on the Fearon reaction which is a direct
interaction of urea with diacetylmonoxime Diacetyl Monoxine +
urea Pink chromogen + hydroxylamine urea concentration is
directly proportional to intensity of the color produced.
Reagents
BUN acid reagent: this contain ferric chloride in phosphoric
and sulfuric acid.
BUN color reagent - Diacetyl monoxime, 0.18%(W/V) and
thiosemicarbazide.
Urea nitrogen standard solution
Urea at a urea N level of 30 mg/dl with benzoic acid
as preservative.
Procedure: ,
The Sigma diagnostic procedure based on the method of
Crocker (1967} was adopted.
To each of three test tubes labelled blank, standard and
test were added 3.0 ml of BUN acid reagent and 2.Oml BUN color
reagent and these mixed by shaking. To the tube labelled standard
was added O.2ml of urea nitrogen standard solution; to tube
182
labelled test was added O.2ml of serum sample. The tubes were
shaken and simultaneously placed in boiling water bath for 10
minutes and these cooled in water for 5 minutes before reading the
absorbance at 540nm with the blank as reference.
The BUN level in mg/dl was read from a prepared
calibration curve.
2.7.2 Measurement of total protein
This is based 0 the biuret reaction copper + Serum
Protein alkaline Ph > copper-protein complexes >(purple)
The copper in biuret reagent react with peptide bands of
serum proteins to form purple color.
Reagents
Biuret reagent: copper sulfate, 0.15 (W/V), sodium hydroxide
3 (W/V) with tartrate and iodide added
Protein standard solution:albumin(5g/dl) and globulin (3gm/dl).
Procedure:
The method of Gornall (1949) as described by the Sigma
diagnostic was used.
To three different test tubes containing the following O.1ml of
water (reagent blank); O.lml protein (standard solution); and O.lml
of serum sample, were added 5ml of Biuret reagent. These were
individually mixed thoroughly and allowed to stand for-15 minutes
at room temperature.
183
Reading of the absorbance were made on a spectrophotometer at
540 nm . The biuret reaction is said to be linear therefore a
calibration curve was not prepared.
Calculations:
Serum Total Protein (gdl) = A TEST x 8.
A standard
where ATEST and A STANDARD represent the absorbance of test and
standard solutions respectively and 8g/dl is the total protein in
the standard solution.
2.7.3. Serum bilirubin
Thi$ is the diazo reaction for bilirubin. According to this
technique, bilirubin is coupled with diazotized sulfanilic acid
(P-diazobezenesulfonic acid) to form azobilirubin.
Reagents
Caffeine reagent accelerating reagent of Caffeine, 25g/ L,and
sodium benzoate, 38g/l in sodium acetate solution.
Alkaline tartrate - sodium potassium tartrate 350g/1 in
sodium hydroxide solution.
HCl - 0.05mol/1 (0.05N)
Diazo reagent ~ dry mixture of sulfanilic acid, ~5 mol, and
sodium nitrite 6.6 mol.
cysteine hydrochloride
184
Procedure:
The procedure used here is based on the work of
Jendrassik and Grof (1938). Nosslin (1960) and Michealsson
(1961) and reviewed by Sigma Diagnostics.
To labelled test tubes were added the following :
Component Serum Total Direct
blank Tube Tube(ml)
(ml) (ml)
Serum 0.2 0.2 0.2
HCl 0.5 1.0
Caffeine reagent 1.0 1.0
Diazo Reagent solu. 0.5 0.5
This is mixed well
Alkaline tartrate 1.5 1.5 1.5
This is mixed well.
The absorbance. of the total and direct bilirubin were
read at 600nm with the blank as reference.
Serum Total bilirubin (mg/dl) = A x 13.2
Serum Direct bilirubin (mg/dl) = A x 13.2
Serum Indirect bilirubin (mg/dl) = Total - Direct.
2.7.4 Activity of Aspartate aminotransferase (Ast)
Also known as glutamic oxaloacetic transaminase (GOT) it
catalyzes the transfer of & - amino group from aspartic acid
to & - ketoglutaric acid (AKG)
185
Aspartic Acid + AKG GOT Oxaloacetic acid + Glutamic acid
Reagents
Prepared substrate - DL - Aspartate, O.2mol/l and &-
Ketoglutaric acid, 1.8 mmol/l in phosphate buffer PH 7.5
Color Reagent - 2,4- Dinitrophenylhydrazine (DNP) approximately
20mg/dl in acid solution.
Sodium hydroxide solution was prepared by dissolving 16 NaOH
anhydrous in 1000ml water.
Procedure:
The procedure used is as described by Reitman and Frankel(1957)
1.0ml of the prepared substrate was pipetted into a test
tube and placed in a water bath at 37°. O.2ml of the serum sample
was added and left in the bath. About an hour later, 1.0ml of the
color reagent was added and shaken leaving it at room temperature.
20minutes after this, 10ml of O.4N NaOH was added and mixed by
inversion.
5 minutes after this, the absorbance was read on a spectrop-
hotometer at 340nm. The aspartate aminotransferase activity was
determined in Sigma - Frankel (SF) units per ml from a calibrati-on
curve prepared.
2.7.5. Activity of alanine aminotransferase (ALT)
Also known as glutamic pyruvic transaminase (GPT) and catal-
yzes the transfer of - amino groups from pyruvic acid to
ketoglutaric acid (AKG).
186
Alanine + - AKG GPT Pyruvic acid + Glutamic acid.
Reagents:
Alanine - &- KG substrate
DL - Alanine 0.2mol/L and &- Ketoglutaric acid
1.8 mmol/L in phosphate buffer, PH 7.5.
Sigma Color reagent:
2,4- Dinitrophenylhydrazine (DNP) 20mg/dl in acid solution
Sodium hydroxide solution O.40N.This was prepared by dissolving
anhydrous sodium hydroxide in lOOOml of water.
Procedure:
The procedure used in determining ALT (SGPT) is as described
by Reitman and Frankel (1957).
Test tubes containing 1.0ml of the Alanine - KG substrate
were placed in a water bath at 37 C to warm.To this were added 0.2
ml of the serum samples. About 30 minutes after adding the serum
1.0ml of the color reagent was added, shaking and leaving the tube
at room temperature. 20 minutes after adding color reagent 10ml of
0.40 N NaOH solution was added and mixed by inversion.Absorbance of
the solutions were then read at 340nm after 5 minutes.
The activity of ALT in the sera was read from a calibrat-
ion, curve in Sigma - Frankel (SF) units/ml.The conventional units
of transaminase (SF) who converted to u by multiplying the value by
0.48 (Kaneko 1980)
187
2.7.6. Activity of Alkaline Phosphatase (ALP)
Serum ALP acti vity can be measured using various phosphate
esters as substrates. The principle is that ALP hydrolyses
P-nitrophenyl phosphate-to p-nitrophenol and inorganic phosphate at
alkaline PH. The nitrophenol formed shows as absorbance maximum at
405nm.
Reagents:
ALP reagent A contains diethanolamine buffer PH
- 9.~ (1.214 mol/L) and magnesium ions 0.607 mmol/L
ALP reagent +B consists of p-nitrophenyl phosphate 60.8
mmol/L
Procedure:
This is as described by Sigma Diagnostics.The ALP reagents A
and-B were maintained at 30 C in a water bath after reconstuition.
To 2.7 ml of the ALP reagent A was added 0.05ml of the serum sample
and mixed immediately by inversion. This was incubated for 1
minute at 30°C. O.25ml of the ALP reagent B was then added,
mixing immediately and taking the initial absorbance at 405nm with
water as reference.
The incubation was then continued at 30 C and the absorbance
taken again exactly after 1, ~, and 3 minutes followin~ the initial
absorbance readings.
188
Calculations:
ALP activity (U/L) = A/mm x TV x 1000
18.45 x LP x SV.
where A per min = chang~ in absorbance per minute at 405 nm
TV = total volume (ML)
SV = Sample volume (mL)
18.45 = millimolar absorptivity of p-nitro-pheno1
at 405 nm
LP' = Light path (I-em)
1000 = Conversion of units per ML to units per litre.
2.8. Histological Technique:
Gross and histologic evaluation of the tissues of the ani-
mals were done to determine toxicity of the plants to the tissues
of the animals. The tissues analyzed include the brain, lungs,
head, liver, spleen, pancreas, gastrointestinal tract, kidney and
the skeletal muscles~
These tissues were observed grossly for any lesions after
opening up the animal through a central incision on the abdomen.
The tissues were examined for color, loss of strength, necrotic
changes, size and hemorrhage.
Reagents:
10% solution of formalin in saline water
70% up to 100% ethanol
Xylene
189
Paraffin melted to 58 - 60GC
Haematoxylin and eosin (H&E) dye
Procedure:
Relevant samples of the tissues were isolated and placed in
containers with 10% buffered formalin solution for theirfixation.
Next the tissues were dehydrated by passing through graded
concentrations of ethanol (ranging from 70% absolute alcohol).
Clearing of the tissues were performed using xylene. This
allows Lmpr-egna t.Lon of the tissues with paraffin. The cleared
tissues were then dropped in liquid paraffin at 58% -60 C for a
period of 30 minutes to 6 hours. This makes the tissue more
resistant to sectioning. Small blocks of paraffin containing the
tissues were then sectioned using the blade of a microtome of
thickness 3-8 um. These sections were placed in warm water before
they were transferred to clean slides and stained with haematoxylin
and counter stained with eosin.
190
Plate 1: Solanum torvum Benth growing along a cattle route from
the abattoir in Bodija cattle market, Ibadan.
191
Plate 2: Tribulus terrestris Linn showing a cluster of growing and
yellow flower heads.
192
Plate 3: Leucaena leucocephala Benth with its flowers in white
globose heads.
193
Plate 4: Eugenia uniflora Linn stands with other plants growing
with it.
194
Plate 5: Lantana camara Linn growing in the grazing pasture at the
Teaching and Research Farm, University of Ibadan.
195
Plate 6: Dichapetalum madagascasience Poir on a bush path.
CHAPTER THREE
EXPERIMENTS AND RESULTS
SECTION A
3.1. CHEMICAL ANALYSIS OF THE POISONOUS PLANTS
3.1.1. INTRODUCTION
Plants contain various chemical constituents. Such chemical
constituents could either be organic or inorganic. Some of these
chemicals are natural constituents of the plants whereas others are
acquired from the soil types on which these plants grow (Clarke and
Clarke 1975, Kingsbury, 1979).
The primary chemical compounds can be defined as those requ-
ired for a plant's basic metabolism. Secondary compounds loosely are
all others (Kingsbury, 1979).Most of these secondary compou- unds
are reported to be toxic and are said to be originally for their
defence against the pervasive pressures of herbivorous insects.
That many of these compounds are also toxic to animals is
accidental (Kingsbury 1978, Ewer and Hall, 197H).
According to Der Manderosian, Giler and Roia (1976) and
Woodward (1985) the poisonous chemical constituents of plants
can be classified into:
1. Glycosides-these contain sugar (pyran) and a non-sugar
(aglycone) portion. There are four types of glycosides mainly:
1 9 'Y
(i) cyanogenic glycosides - many plants contain hydrocyanic
acid either free or more usually in the form of cyanogenetic
glycoside which is an organic compound containing a sugar and
capable of yielding cyanide on hydrolysis.
(ii) saponin glycosides-these contain a sugar in combination
with a steroid.
(iii ) anthraquinone glycosides-these yield anthraquinone on
hydrolysis with the aglycone or glycoside having a purgative
effect.
(iv) cardiac glycoside - these include substances such as
digitalis oleandrin, trophantin, etc.
2. Alkaloids - these are basically alkali substances which are
nitro-genous. They are known to consists of various derivatives
some of which are very toxic whereas others have medicinal
importance and include pyrone e.g. caffeine; indole e.g. yohimbine;
pyridine e.g. nicotine;tropine e.g. hyosine;quinoline e.g. quinine;
phenanthrene
e.g. morphine: phenyl alkylamine e.g. ephedrine and imidazole e.g.
pilocarpine.
3. Proteins - they are phytotoxins such as ricin from Ricinus
communis and abrin from Abrus precatorius (Wee, 1987).
4. Esters - such as oil of mustard (allyiosothicyanate) and oil of
garlic (allylsulphate).
5. Resins - amorphous products of complex chemical structure.They
occur as mixtures with volatile oils (oleoresins) gum (gum resins
and sugars).
6. Oxalic acid - the only organic acid of plants toxic to animals
under natural conditions. They occur in plants as soluble (Na+ and
K+) or insoluble (Ca++) oxalates or acid oxalates (Littledike,James
and Cook, 1976).
7. Nitrates and nitrites-nitrates are reported to be relativ-
ely non toxi~.Their importance as cause of poisoning from plants is
due to their conversion either in the foodstuff or within the
alimentary tract into nitrites (Andrade, Peregrino and Aguiar 1Y71j
Blood and Henderson, 1968).
8. The metallic constituents are reported to be derived from the
environment or soil types on which these plants grow. They include
copper, lead, magnesium manganese, zinc, selenium, arsenic, iron,
cobalt phosphorus,etc~(Hall,lY74).Such metals also form complexes
with other substances inorder to be toxic e.g. phytin is an organic
phosphate defined by most investigators as the mixed calcium and
magnesium salt of inositol hexaphosphoric acid (Harland and Prooky,
1979).
199
3.2. EXPERIMENT 1
3.2.1. Determination of the inorganic constituents of the plants
Procedure:
The average of two estimations of the inorganic constituents
of the leaves of Q. madagascasiense, §..torvum, E.unifl- ora,~.
terrestris,~. camara, ~ leucocephala were made using leaves from
two different plant stands. The two determinations were made from
the leaves of the plants harvested in October 1989 and April 1990.
The leaves were kept under shade at a place with good air
draft after weighing them on a triple beam balance. The drying
took almost 2-3 weeks depending on the moisture content of the
leaves. The dried leaves were then crushed into a coarse form and
weighed before subjecting them to analysis or extraction.
The percentage yield of the extract was determined
by = Dry weight of extract x 100
Wet weight of starting material
The inorganic chemical consti tuents were determined by testing
for the levels of nitrate, nitrite, oxalate, cyanide and phytin in
the leaves using the methods as described on pages 164, 100, 107,
168, 169, 170,.171, 172.
200
Results
The levels of inorganic and the metallic substances deter-
mined from the leaves of the poisonous plants 1. leucocephala; 1.
camara; .§.. torvum; E.uniflora, Q. madagascasiense, and T.
terrestris are as presented in tables 1 and G.
(a) Phytin content of the leaves of the poisonous plants. T.
terrestris had the highest content of phytin of GO.IH percent. The
level of phytin in .§..torvum was UL G3 percent; level in L.
leucocephala was 18.71 percent; level in E. uniflora was IH.66
percent; the level in Q. madagascasiense was 17.07 percent whilst
1. camara had the lowest content of phytin of 15.99 percent.
(b) Oxalate level in the leaves of the poisonous plants
Q. madagascasiense showed the highest concentration of
oxalate in its leaves of 0.24%. in decreasing order of oxalate
concentration in the plants, the other plants showed the following:
.§..torvum, 0.18%, 1., camara, 0.14H%, T. terrestris, 0.14%, 1.
leucocephala, 0.126%, E. uniflora of 0.10G%.
(c) Level of nitrate in the leaves of the poisonous plants.
As seen in table 1, the highest concentration of nitrate was
found in the leaves of .§..torvum.Thelevel of nitrate in 1. camara
was 15.34 mg/g, the level in T. terrestris was 15. GH mg/g; the
level in Q. madagascasiense was 14.99 mg/g; level in E. uniflora
was 14.66 mg/g and the level in 1. leucocephala was 14.17 mg/g.
201
(d) Cyanide level in the leaves of the poisonous plants.
The highest concentration of cyanide in the leaves of the
plants under study was found in Q.madagascasiense of 15.06 ppm.
Cyanide concentration in E. uniflora was 15.0 ppm.; the level in
1.leucocephala was 14.2 ppm; level in 1.terrestris was 14.4
ppm;level in ~.torvum was 13.~ ppm;the level in 1.camara was 1~.7
ppm (Table 1).
(e) Nitrite level in the leaves of the poisonous plants.
1. terrestris had the highest level of nitrite content of
9.71 mg/g. The level of ni tri te in 1. camara was 9.5 mg/g; the
level was 9.38 mg/g in E. uniflora; 1.leucocephala had 9.~ mg/g;
Q.madagascasiense had 8.95 mg/g;S.torvum had H.91 mg/g (Table 1).
Conclusions
The level of the antinutritional factor phytin analyzed
from all the plants except 1.camara was high enough to constitute
a cause of toxicity by the plants. Similarly the level of nitrate
and nitrites determined from all the plants were relatively high
when compared with the minimum lethal dose of the pure compounds in
animals. This implies consumption of these plants by livestock could
produce some level of methaemoglobinaemia.
Furthermore all the plants analyzed contained hydrocyanic
acid. The presence of HCN in these plants indicates they contain
cyanogenetic glycosides or free HCN.
202
The level of oxalates determined from the leaves of the
poisonous plants were very 10w.This implies that oxalate cannot be
a source of poisoning to animals grazing on these plants.
3.3 EXPERIMENT 2
Procedure
Determination of the metallic constituents of the plants.
The leaves of the poisonous plants 1.. leucocephala; E. uni f 1
ora, T terrestris, ~.torvum, 1.. camara, Q. madagascasiense were
harvested weighed and treated as required for the different metals,
iron manganese, molybdenum, copper, zinc and lead as outlined in
materials and method in pages 173, 174, 175, 176 respectively.
3.3.1 Results
3.3.1.1. Molybdenum levels in the leaves of the poisonous plants
Table 2 shows that Q. madagascasiense had the lowest level of
moiybdenum of 0.12 ppm. T. terrestris had 0.26 ppm; 1.. camara 0.25
ppm with 1.. leucocephala and ~. torvum exhibiting equal levels of
0.14 ppm; E. uniflora had the lowest level of 0.12 ppm also.
3.3.2 Manganese level in the leaves of the poisonous plants
The level of manganese in the leaves of the plants from the
plant with highest concentration to that with the lowest are Q.
madagascasiense, 1.11 ppm; T· terrestris, 0.060 pprn; 1..
leucocephala, 0.56 ppm; ~. torvum, 0.052 pprn; E. uniflora, 0.51
ppm; 1.. camara, 0.10 pprn.
203
3.3.3 Iron level in the leaves of the poisonous plants
The order of the level of iron estimated in the leaves of the
poisonous plants from the highest level to the lowest is L.
leucocephala, 5.27 ppm; S. torvum, 4.9~ ppm; E. uniflora, 4.H7 ppm;
T. terrestris, 4.78 ppm; D. madagascasiense, 2.54 ppm; L. camara,
1.55 ppm (Table 2).
3.3.4 Copper level in the leaves of the poisonous plants
The highest level of copper was observed in T.terrestris of
2.25 ppm. The level in 1. leucocephala was 1.52 ppm; the level in
Q. madagascasiense was 1.87 ppm; the level was 1.2H ppm in .s..
torvum; 1.21 ppm in 1. camara and 1.02 ppm in!. uniflora (Table
2) •
3.3.5 Zinc level in the leaves of the poisonous plants
The highest level of zinc was found in Q. madagasca-siense of
4.73 ppm. The level in !.uniflora was 2.~9 ppm; the level in .s..
torvum was 2.35 ppm; ~he level in 1. leucocephala was 1.57 ppm. The
level was 0.49 ppm in 1. camara and O.~~ ppm in T. terrestris.
3.3.6 Lead level in the poisonous plants
The highest level of lead of 0.21 ppm was observed in !.
uniflora. The ~evel in 1. camara was 0.14 ppm, it was 0.12 pm in
.s.. torvum; 0.1 ppm in 1. leucocephala; 0.09 ppm in T. terrestris
and 0.04 ppm in Q. madagascasiense.
204
Table 1
Levels of inorganic anions in the poisonous plants.
PLANTS LEVELS OF INORGANIC SUBSTANCES
Oxalate Nitrate Nitrite Phytin Cyanide
% mg/g mg/g content ppm
%
L. leucocephala 0.126 14.1 9.24 18.71 14.2
T. terrestris 0.140 15.28 9.71 20.18 14.4
S. torvum 0.180 16.10 8.91 19.23 13.2
S. madagascas. 0.24 14.99 8.95 17.07 15.06
L. camara 0.148 15.34 9.50 15.99 12.70
E. uniflora 0.102 14.66 9.38 18.66 15.0
Table 2.
Levels of metallic cations in the poisonous plants
PLANTS LEVELS OF METALLIC IONS IN PARTS PER MILLION
Mo Mn Fe Cu Zn Pb
1. leucocephala 0.14 0.56 5.27 1.52 1.57 0.10
T· terrestris 0.26 0.60 4.78 2.25 0.33 0.09
~. torvum 0.14 0.52 4.93 1.28 2.35 0.12
Q. madagascas. 0.12 1.11 2.54 1.87 4.73 0.04
1· camara 0.25 0.10 1.55 1.21 0.49 0.14
E.. uniflora 0.12 0.51 4.87 1.02 2.39 0.28
Each value represents an average of two de t.e r mi nat i o n s from
leaves of two plants stands.
205
SECTION B
3.4 TOXICOLOGICAL EFFECTS OF THE POISONOUS PLANTS ON
HEMATOLOGICAL PARAMETERS OF RATS AND GOATS
3.4.1 EXPERIMENT 1
Determination of the lethal dose 50(LD50) of the poisonous
plants in mice.
INTRODUCTION
Most plants which have come to be known as poisonous, contain
substances ~hich depending on the rate of consumption or amount
consumed, may not produce symptoms. Thus plants as Eleusine indica
and Zea mays could be browsed by animals without the animal
suffering any adverse effects (Kinghorn, 1979; Woodward, 19HO).
However the same observation cannot be associated with plants
such as Aconitum napellus or Colchicum autumnale which are reported
to contain very toxic substances so that just a leaf in the meal of
an animal could prod~ce adverse reactions (Woodward, 1980).
It is customary in toxicological studies to define the lethal
dose of poisonous substances and their effects on important
functions of the animal. The basic test for the determination of
the relative acute toxicity of a substance is by the observation of
the median lethal dose (LD50) (Litchfield and Wilcoxon. 1949).
Procedure
The pilot toxicity studies on the poisonous plants under study
were conducted in white albino mice weighing between 20-25 gm. For
206
each of the poisonous plants was assigned five groups of
experimental mice with each group consisting of 10 male mice. Each
group of the mice for each plant were then administered with
dosages of the extracts of the plants dissolved in propylene
glycol. A control group was simultaneously also administered with
propylene glycol only. The administrations were orally using a
canula. The different doses of the extracts from each plant ranged
between 200 mg/kg to 1,200 mg/kg.
Immedi~tely the dose of the extract of each plant was
administered to a group they were kept in cages for observation for
120 hours. The number of mortalities observed per group were then
recorded.
Results
The percentage of mortalities for each group under each plant
was estimated and plotted on a graph of log- dose against %
mortalities. The LD5Q obtained for each of the plant extracts are
as stated.
Table 3.
Acute LD50 values of the poisonous plants in mice.
Plant LD50 (mg/kg)
.k. camara 1,460 - figure 1
Q. torvum 780 figure 2
E.. uniflora 720 figure 3
1:. terrestris 1,040 figure 4
207
1. leucocephala 1,400 figure 5
CONCLUSION
1. camara and 1. leucocephala had the highest LD50 (oral)
value among the other plants. T. terrestris produced intermediate
LD50 values, whereas §. torvum and I. uniflora had the lowest LD50
values in mice. This means §. torvum and I. uniflora were more
toxic acutely than the other plants evaluated in this study.
EXPERIMENT 2.
3.5. Effect of the Poisonous plants on the hematological
parameters of rats and goats
Introduction
Toxic substances from poisonous plants affect the cellular
elements of the blood by causing direct hemolysis of the
circulating erythrocytes. Other agents such as from the plant
Vicia faba produce hemolysis only in cells congenitally deficient
in glucose-6-phosphate dehydrogenase (Casarrett and Doull, 1975).
Some plants destroy the hematopoietic powers of the bone marrow or
increase the number of circulating white blood cells by their
mitotic effects· (McPherson, 1979). Certain poisons owe their
effects to or at least reveal their presence by numerous petechiae
or ecchymoses, the result of injury to the endothel ium of blood
vessels (Smith et al., 1974).
208
Poisonous plants could also produce destructive lesions in
the liver (Doerr et al., 1976). This activity may result in the
failure of blood to clot due to the deficiency of essential blood
coagulation factors such as prothrombin which are produced by the
liver cells. Fibrinogen, factor VII, the Stuart Factor (factor x)
and probably the Christmas factor (factor ix) are all synthesized
by the liver. All these factors are important blood coagulatory
factors (Jain, 1986). Some of the poisonous plants for example
Dieffenbac~ia spp by the level of oxalate in their leaves cause the
reduction in the level of blood calcium (Woodward, 19HO). This
results in the decreased ability of the blood to clot (Buck et.
al., 1966; Marshall et. aL, , 1967). Fatal and uncontrollable
hemorrhages in numerous bovine animals fed several weeks or longer
on sweet clover hay have also been reported (Prier and Derse,
1962).
Some of the pQisonous plants also cause the production of
methemoglobin or cyanhaemoglobin. These complexes formation by
haemoglobin of the RBC reduces the oxygen carrying capacity of the
blood and thus results in toxicity to the affected animal consuming
them (Andrade et. al., 1971; Miyazaki and Kanashima, 1975).
Certain of the plant lectins have been reported to have the
capacity to release the developmental potential of specific resting
or dormant cells in the circulating system and transform them to an
active growth state (Nowell, 1960). Such plant lectins have been
209
termed mitrogens and the class of cells ~ffected is primarily that
of the immune responsive or lymphocyte (McPherson, 1979).
This study is an attempt to evaluate the toxic effects of the
plants 1. leucocephala, ~. torvum, i. uniflora, T. terrestris, 1.
camara and Q. madagasca"siense on the hematological parameters of
rats and goats. The parameters that were evaluated were the
following:
(i ) total erythrocyte count
(i i) haemoglobin concentration
(iii) packed cell volume
(iv) blood coagulation time
(v) total and differential leucocyte counts.
Procedure
Four groups each made up of ten rats were used for each plant.
Three of the groups were administered with the three different
doses of the extract of each group for a period of 14 days daily by
the oral route. The fourth group of each set of groups for a plant
were also administered with propylene glycol orally.
The doses of the extract for i. uniflora and ~. torvum were
100, 150, and 200 mg/kg; for 1. camara and 1. leucocephala, they
were 200, 400 and 600 mg/kg; they were 150, 200, bnd 400 mg/kg for
T. terrestris. In case of Q. madagascasiense the leaves were not
extracted but pelleted (cf page IH2) and fed to the experimental
rats.
"
210
Freshly harvested leaves of Q. madagascasiense were fed to a
group of goats whereas the group of goats used for ~. camara were
administered with aqueous extracts of the leaves.
Blood obtained as explained in page 103 were analyzed for
changes in hematological parameters.The determinations of total
erythrocyte and leucocyte counts, packed cell volume, haemoglobin
concentration, blood coagulation time and differential leucocyte
counts are as described in pages respectively.
3.6. Results
3.6.1. Effect of the extract of Eugenia uniflora on the
leucogram and erythron of the rat.
The effects can be observed in tables 4 & 5. The extract
increased inconsistently the total red blood cell counts as well as
the mean corpuscular volume as the doses increased. It produced
increase in the pev which is significant (P<O.05) with the 000
mg/kg dose. The ext~act also produced inconsistent increase in the
haemoglobin concentration which shows significance at the
150 mg/kg dose level of the extract.
The extract did not cause significant changes in the
blood coag~lation time of the rats.
The extract of E. uniflora has not caused any serious
changes in the total white cell count except that a significant
increase was observed with the 150 mg/kg dose (Table 50). The
eosinophil count showed a dose dependent increase.
FIG. 1. LOG -DOSE MORTALITY CURVE OF MICE ADMINISTERED
WITH EXTRACT OF LANTANA CAMARA ORALLY
EACH POINT HAS AN .,n " OF 10. LDso = 1460mg/kg.
90
80
70
60
>-
t-
-;.:J! 50 ------------'--------------------------b
o0:::::E
40
t-
Z
W
u
0:::
Wa. 30
20
10
2·8 2·9 3·0 3·1 3'2 3'3
-- •~. LOG DOSE (mg/kg.)
..
l)1'J~t:. CUkVt:. Ur IVII1,t AUtVlll"iblt.kt::,U
WITH EXTRACT OF SOLANUM TORVUM ORALLY
EACH POINT HAS AN "n" OF 10. LDso = 780mg/kg
100
gO
80
70
60
I- 50 -------------------,
Z
W I
u Io:
W0... 40
r 30
20
10
2·3 2'8 2'9 3-0 3·1 3-2 3·2
--~~ LOG - DOSE (mg/kg)
FIG.3. LOG- DOSE RESPONSE CURVE OF MICE ADMINISTERED
WITH EXTRACT OF EUGENIA UNIFLORA ORALLY
EACH POINT HAS AN' n " OF 10 - LDso= 720 mg/kg
100 8.
90
80
70
60
50 -......:--------------
t-- I
Z I
W
u I
0:::
W 40
I
I
0.... I
I
30
20 8.
10
, , , , ,
2-3 2-6 2-8 2-9 3-0 3-1 3-2 3'2 3-3
------;~- LOG DOSE (mg/kg)
FIG.4. LOG - DOSE MORTALITY CURVE OF TRIBULUS TERRESTRIS
IN MICE. EACH POINT HAS AN II n ,/ OF 10. LDso= 1,040mg/kg
100
90
80
>- 70
t-
...J
«
t-
o::
o 60
L:
t-
Z 50--- - - ---- -------- ----A-----
lLJ
u I
0:: II
olLJ, I
40 AII
I
I
I
r 30 II
20
10
2'3 2'6 2'8 2'9 3'0 3-1 3·2 3·2 3·3
---~~~ LOG DOSE (mg/kg)
FIG. 5. LOSE -DOSE MORTALITY CURVE OF LEUCAENA LEUCOCEPHALA
IN MICE. EACH POINT HA S AN "n " OF 10. LDso = 1,400mg/kg.
100
90
80
70
>-
I-
.J
;:!
0:: 60 A
0
~
zI- 50 --------- ----- -----------------
wu0::
~ 40
r 30
20
10
3'0 3'2 3·2 3'3
---~» LOG DOSE (mg/kg)
3.6.2. Effect of Tribulus terrestris on the hematological
parameters of the rat
The extract of T. terrestris caused a dose dependent
decrease in the level of the total RBC and haemoglobin
concentrat ion (Table 6). These decreases were sIgn i ficant with the
200 mg/kg and 400 mg/kg doses for haemoglobin and RBC respectively
(P 0.01). The extract also produced insignificant decreases in
the level of the PCV as the dose of the extract increase. Changes
in the total RBC count and PCV were reflected as ~light increases
in the mean corpuscular volume.
Though the extract produced increases in the blood
coagulation time in rats, these changes were insignificant when
compared with the control values (P<0.05).
The extract caused a dose dependent decrease in the level of
the total WBC count. The decrease is significant at the 400 mg/kg
dose level of the ext r-act, of T. terrestris. The decrease in the
total WBC is reflected as significant decreases in the lymphocyte,
neutrophil and eosinophil counts (P 0.05) (Table 7).
3.6.3. The effect of Leucaena leucocephala extract on the
hematological parameters in rats
The extract of 1. leucocephala did not produce any
significant changes in the hemogram or leucogram of the rats even
with increasing doses of the extract (P 0.05) (Tables ~ and 9).
However the total WBC counts have shown increases as the dose of
212
the extract shown increases as the dose of the extract administered
increased which is reflected as increase in the differential
lymphocyte and monocyte counts (Table ~).
3.6.4. The effect of Solanum torvum on the hematological
parameters in the rats
The extract of~. torvum produced a dose dependent increase
in the level of the total RBC counts, PCV and haemoglobin
concentration as the dose of the extract administered increased in
the rats. The increase was observed to be sLgn i f icant with the
doses administered t for the PCV; only the 150 mg/kg dose for
haemoglobin concentration as well as only the ~OO mg/kg dose level
for the RBC.
The increases in the total RBC counts, PCV and haemoglobin
concentration did not influence the values of the mean corpuscular
volume (MCV), mean corpuscular haemoglobin concentration (MCHC) or
the mean corpuscular h~emoglobin (MCH) (Table 10).
The extract did not produce any changes in the blood clotting
time of the rats. The e~tract produced inconsistent decreases in
total WBC counts as the dose of the extract of S. torvum
administered Lncreased , The decrease produced by the 100 mg/kg
dose is significant (P 0.05) which was reflected as a decrease in
the lymphocyte and neutrophil counts (Table 11).
2H3
3.6.5. The effect of Lantana camara on the hematological
parameters of rats.
L. camara produced a dose dependent decrease in the total
RBC and WBC counts (Tables 12 and 13). It also caused significant
decreases in the haemoglobin concentration as the dose of the
extract increased.
The decrease of haemoglobin concentration was reflected as
significant decreases in the MCHC and MCH. The effect of the
extract on the PCV was inconsistent.
The extract also produced slight increase in the blood
coagulation time of the rats which was not significant (P<U.U1).
The decrease caused by the extract on the total WBC count
was reflected as decreases in the neutrophil and monocyte counts
(Table 12).
3.6.6. The effect of Dichapetalum madagascasiense on
hematological parameters in rats
The leaves of D. madagascasiense produced inconsistent
increases in the PCV and ,inconsistent decrease of the total RBC.
The leaves did not produce changes in the haemoglobin concentration
and the Winthrobe. indices or the rat blood coagulation time (Table
14).
The leaves of D. madagascasiense did not adversely affect
the leucocyte differential or WBC counts in the rats (Table 15).
3.6.7. The effect of D. madagascasiense on hematological
parameters in goats
After 14 days of feeding the goats on the leaves of Q.
madagascasiense in combination with maize, the hematological
parameters examined did not show significant changes (Table 16).
After 21 days of feeding Q. madagascasiense, the
hematological parameters of the goats still did not show any
serious alterations (Table 17).
3.6.8. The effect of Lantana camara on the hematological
parameters in the goat.
After 14 days of feeding the goats with the aqueous lea!'
extract of 1. camara, there were increases in the haemoglobin
concentration, PCV, and decrease total RBC cgYnts (TabLe 1~ • Th~
total WBC count was increased with the corresponding increase in
the lymphocyte and neutrophil counts (Table 16).
After 21 days. of feeding 1. camara, the haemoglobin
concentration and PCV still remained elevated whilst the total RBC
counts were decreased (~able 17).
The total WBC counts were also increased which were reflected
as increased l~mphocyte and neutrophil counts but decreased
eosinophil, basophil and monocyte counts (Table 17).
3.7. CONCLUSIONS
The extract ~. uniflora produced increases in the total RBC
count or polycythaemia. It produced no adverse effects on the
leucocyte counts nor the blood coagulation of the rat. This
suggest that E. uniflora does not have any destructive effects on
the red blood cells nor any adverse effects on its production in
the bone marrow.
Similarly L. leucocephala did not produce either decreases or
increases in the blood parameters examined. It caused eucocytosis.
This indicates that L. leucocephala has no adverse action on the
hematological parameters of the rats.
T. terrestris reduced the level of circulating erythrocytes
as well as the haemoglobin concentration. This implies that T.
terrestris could produc~ anaemia. The increase produced by the
plant on the blood coagulation time indicates the effect of the
plant on blood coagulation factors. The plant also produced
decrease in the leucocyte counts which may be indicative of the
action of the plant on the hemopoietic system of the rats.
§. torvum pr~duced polycythaemia with its increase of the
circulating RBC and the PCV. Since these changes did not influence
the Winthrobe indices, S. torvum may not have any adverse effect on
the hematological para"meters since it also did not affect the
coagulation time of the rats.
The decreases in the hematological parameters, total RBC
and PCV produced by L. camara implies that the plant can cause
anaemia in rats. The leucopenia also produced by the plant shows
its effect on the hemopoietic system of the rats. The
;'216
insignificant increase in the blood coagulation time, does not rule
out the possibility of the plant produc ing serious coagulation
defect if administered for much more prolonged periods.
A similar effect has been produced in goats by 1. camara
as was in rats. This may mean that the adverse effects of the
plant may not be species specific but that it could produce harmful
changes in blood factors of animals which may consume the plant.
On the other hand Q. madagascasiense has not shown any
serious abnormal changes in the hematological parameters of the
rats and goats. This means that the plant does not contain
compounds with adverse effects on blood factors.
,
2212
TABLE 4.
Mean values (+ S.E.M.) of hematological parameters
following exposure of rats to ethanolic extract of
E. uniflora.
DOSE PCV Hb RBC MCV MCHC MCH COAG.
mg/kg " gm/ u/l u/3 " pg TIME/
100ml MIN.
0 45+2.7 14.1+2.3 6.8+1.9 66.2+4 31.3+1.3 20.7+1.4 1.95
100 58+2.0 18.6+3.5'8.2+0.4 70.2+16 32.1+0.7 22.6+3.1 2.80
150 57+2.5 19.3+2.2 9.7+2.6 58.8+02 33.9+1.2 19.9+0.86 2.0
200 67+1.0 17,.7+1.3 7.7+1.6 86.8+3 26.4+0.6 22.9+2 2.0
n - for each dose is 10
TABLE 5
Mean values (+S.E.M.) of total and differential leucocyte
counts of rats given extract of E. uniflora.
DOSE TOTAL Lympho- Neutro- Eosino- Baso- Mono-
mg/kg WBC cyte phils phils phils cytes
103/ml
,
0 12.4+12.1 6200+662 4960+963 6~0+149 496+113 1~4+69
100 14.2+3.4 7526+992 5B~~+1041 4B3+134 4B3+134 0
150 26.7+7.2 14418+2627 1015+~90 B01+19~ 536+19~ ~71+1~3
200 10.6+5.8 5276+561 40~B+5B7 B46+~49 636+15~ 0
n - for each dose is 10
224
TABLE 6.
Mean values (+S.E.M.) of hematological parameters
following exposure of rats to ethanolic extract of
T. terrestris.
DOSE PCV Hb RBC MCV MGHC MCH COAGU
mg/ % gm/100ml u/l u/3 % pg TIM/
kg MIN
0 44.4:1::3215.4:1::0•65 9.5:1::0•86 48.7:1::4•9 35.7:1::2•4 16.8:1::1.02.0
150 44:1::2•9 13.9:1::0•81 8.4:1::0.83 54.2:1::3.332.7:1::0.3617.3:1::162.14
200 42.4:1::3612.6:1::1.1 8.5:1::0•66 50.9:1::4•330.3:1::2.4 14.7:1::122.25
400 28.2:1::6014.3:1::0•61 7.6:1::0•61 57.9:1::5•935.0:1::2•9 14.4:1::132.30
n, - for each dose is 10.
22~
TABLE 7.
Mean values (+S.E.M.) of total and differential
leucocyte counts of rats given extract Jf Tribulus
terrestris.
DOSE TOTAL Lymph- Neutro- Eosino- Baso- Mono-
mg/kg WBC ocyte phil phil phil cyte
103/ml
0 10.4:.1:0•61 5387:.1:689 4050:.l:HHl 31~:.I:100 ~OH:.I:75 410:.1:111
150 6.9:.1:0•51 4623:t294 1725:.1:~71 69:.1:3H 0 4H3:t115
200 6.4:.1:0•63 4416:.1:103 1536:.1:50 1~H:t54 1~H:.I:74 19~:.I:6~
400 5.8:.1:0.5 4524:.1:146 1102:.1:136 0 58±26 96±31
226
TABLE 8.
Mean values (+S.E.M.) of hematological parameters
following exposure of rats to ethanolic extract of
Leucaena leucocephala.
DOSE PCV Hb RBC MCV MCHC MCH COAG.
mg/ % gm/lOO u/L u/3 % pg TIME/
kg ml MIN.
0 46.4±0.93 14,8±0.7 6.7 0.67 84.7±3.5 25.1.1 20.9±O.64 2.1
200 48.4±1.7 13.9±1.2 6.6±O.53 7G.8±5.3 28.9±25 22.0±2.6 2.3
400 47.1±2.6 15.2±034 6.5±0.58 78.3±8.7 33.2±19 25.0±2.3 2.2
600 47. 5±2. 3 15.4±O45 6.8±0.6 64.6±7.9 33.0±14 20.0±2 2.5
227
TABLE 9.
Mean values (+S. E.M.) of total and differential
leukocytes counts of rats given extract of Leucaena
leucocephala.
DOSE TOTAL Lymph- Neutr- Eosin- Baso- Mono-
mgl WBC ocycte ophil phils phil cyte
kg 1003/ml
o 12.9:0.25 6966:909 5418:8077~58:59 1~9:4~ 0
200 13.6:0.48 8092:667 ~590:115307~:1144~0:1~7500:~0
400 14.0:0.34 7396:943 4488:990 95~:~90408:98 ~77:85
600 15.0:0.39 9300:522 5100:050 0 0 300±08
,
228
TABLE 10
Mean values (+S.E.M.) of hematological parameters
following exposure of rats to ethanolic extract of
Solanum torvum
DOSE pcv Hb RBC MCV MCHC MCH COAGU.
MG/ % gm/ 10/ u/3 % pg TIME/
kg 100ml ml MIN.
o 47.0±0.73 13.6±0.3 5.3±035 90.0±51 29.0±0.57 20.1±1.2 2.0
100 55.5±0.65 15.8±077 6.1±025 90±4.7 28.0±0.31 25.8±1.0 2.4
150 57.3±2.6 16.1±0.4 6.4±027 92.1±44 28.0±1.0 20.0±1.2 2.1
200 61.8±2.2 15.2±0.4 7.3±0.1 84.7±35 25.0±1.1 20.9±0641.96
2?9
TABLE 11.
Mean values (+S.E.M) of total and differential
leukocytes counts of rats given extracts of Solanum
torvum.
DOSE TOTAL Lympho- Neutr- Eosin- Baso- Mono-
mgl WBC cyte ophil ophil phil cyte
kg 103/ml
o 17.6±0.14 10376±1777 5810±913 404±lUo 50±37
100 10.2±3.2 6200±341 3325±445 581±9U 11~±34
150 18.2±1.1 11068±1430 6797±1174 3~~±105 n±59
200 15.6±0.28 10636±797 4555±776 343±93 1~5±59
,
230
TABLE 12.
Mean values (+ S.E.M.) of hematological parameters
following exposure of rats of ethanolic extract of
Lantana camara
DOSE PCV Hb RBC MCV MCHC MCH COAG.
mg/ % gm/100 u/L u/3 % pg TIME/
kg ml MIN.
o 45.9:2.6 14.5:0.61 6.9:0.38 68.8:5.1 32.4:2 21.~±16 2.1
200 44.3:2.5 9.0:0.25 5.6:0.3~ ~0.8:0.3~ 18.5:084 160:11 2.0
400 54.1±2.0 9.8:0.38 5.2:0.46 101.0:140 17.4:1.0 200±1~ 2.5
600 49.2:2.9 8.7:0.45 5.3:0.28 ~3.8:8.5 18.2:1.5 170±12 2.5
231
TABLE 13.
Mean values (+ S.E.M.) of total and differential
leucocyte counts of rats given extract of Lantana
camara.
DOSE TOTAL Lympho- Neutro- Eosin- Baso- Mono-
mgl WBC cyte phil ophil phil cyte
kg 103/ml
o 18.4±0.93 10304±887 7176±720 368±102 184±84 368±132
200 133±0.82 8847±920 3485±72ti 606±136 266±61 o
400 13.9±0.96 9897±921 3475±825 139±45 264±72 139±45
600 16.3±0.62 10129:l:549 5226±534 489±11 7 326±91 163±52
232
TABLE 14.
Mean values (+S.E.M.) of hematological parameters
following exposure of rats to ethanolic extract
of Q. madagascasiense.
TREAT- PCV Hb RBC MCV MCHC MCH COAGU.
MENT gm/100 u/l u/3 % pg TIME/
ml MIN.
o 48.3:3.5 14.2:0.41 7.6:0.21 63.6:4.3 30.1:158 187:066 2.1
A 50.3:3.6 15.4:0.37 8.6:0.46 59.6:5.8 31.6:2.0 182:1.0 2.0
B 51.1:3.2 13.8:0.71 6.5:0.25 78.9:4.6 27.3:1.3 201:1.3 2.1
C 49.0:2.7 14.6:0.46 6.8:0.14 72.4:4.4 30.3:1.6 216:081 2.0
o rats fed rat cubes
A rats fed pelleted leaves and maize in 50.50 ratio
B rats fed pelleted leaves and maize in 75.25 ratio
C rats fed pelleted leaves only.
?33
TABLE 15
Mean values (+S.E.M.) of total and differential
leucocyte counts of rats given extract of
Dichapetalum madagascasiense
TREAT- TOTAL Lympho- Neutro- Eosin Baso- Monocyte
MENT WBC cyte phil ophil phil
103/ml
0 8.5±0.17 3655±391 3485±401 510±137 473±107 340±1:i1
A 9.2±1.2 3772±618 4416±570 350±69 460±132 Hl4±H4
B 9.9±0.92 4514±755 4930± 725 297±90 99±55 0
C 9.5±0.75 3686±561 5130±574 2H5±43 2H5±6H 95±43
0 rats fed rat cubes
A rats fed pelleted leaves and maize in 50.50 ratio
B rats fed pelleted leaves and maize in 75.25 ratio
C rats fed pelleted leaves only.
TABLE 16.
Mean hematological values (+S.E.M.) of goats after
feeding on Q. madagascasiense or L. camara for 14 days
PARAMETERS CONTROL Q.Madags. CONTROL L· camara
Hb gm/lOOml 11.58±0. 4 10.9±1.1 11.5±0.6 13.42±7.9
PCV % 23.5±0.78 23.5±2.1 23.5±0.93 25±3.3
RBC ulL 10.*±1.3 10.44±0.79 10.4±0.7 9.85±1.3
WBC 103/ml 14.75±0.49 15.3±1.3 134.G±1.0 14.6±1.6
Lymphocyte 7578±393 7956±11683 6061±649 6939±1087
Neutrophil 6785±253 6732±1026 5935.7±683 6714±688
Eosinophil 295±37 459±926 1194±180 583±107
Basophil 15+3 153+23 601+95 291+45
Monocyte 0 0 212+50 72.5+60
CONTROL A are the control values for goats fed on
Q.,madagascasiense
CONTROL B are control values for goats fed on
L. camara
TABLE 17.
Mean haemalological values (+S.E.M) of goats after
feeding with Q. madagascasiense or L. camara for 21
days
PARAMETER CONTROL Q.madagascasiense CONTROL L. camara
Hb gm/100ml 11.58:t:0.4 10.8:t:0•93 11.5:t:8.3 15.5:t:1.3
PCV % 23.5 :t:0•78 23 :t:17. 23.5:t:0.78 19.5:t:1.7
RBC u/L 10.8:t:1.3 11.0:t:0.68 10.4:t:0.7 8.9:t:0•43
WBC 103/100ml 14.75:t:0.49 15.0:t:0.80 13.2:t:1.0 16.1:t:1.3
Lymphocyte 7578:t:393 7724.5326 6061:t:649 9791:t:621
Neutrophil 6785:t:253 6673.5:t:854 5935:t:1683 7169:t:917
Eosinophil 295 :t:37 377.5:t:5O 1194:t:180 895:t:56
Basophils 15:t:3 301:t:73 601:t:95 0
Monocytes 0 0 212±50
CONTROL A are the control values for goats fed with
I!. madagascasiense
CONTROL B are the control values for goat fed with L· camara
236
SECTION C
3.8. EFFECTS OF THE POISONOUS PLANTS ON SERUM BIOCHEMICAL
PARAMETERS OF RATS AND GOATS
EXPERIMENT 1
Introduction
The toxicity of any compound in the final analysis is the
sum total of its interactions with cell constituents to produce
chemical alterations and the cell response to these aberrations.
The untoward effects arising from poisons can usually be
traced back to the functional incompetence of organs. Whenever
such dysfunction happens in a tissue, it is traceable to
biochemical derangements at the subcellular level. Therefore
recourse could be made to appropriate function tests to elucidate
subtle effects of this type even when the animal is still alive (De
Bruin 1976). The biochemical abnormality revealed by such
biochemical tests are often obvious long before the genesis of
morphological damage and precedes the development of chronic
degenerative disease (Kaneko, 1980).
Since the liver and the kidney are often the major targets
of poisonous s~bstances, organ function tests are directed at these
two organs. One of the liver function tests is that for measuring
hepatic transport including uptake conjugation and excretion of
organic anions. These include measurement of unconjugated and
conjugated bilirubin in serum. Bile pigments in mammals are waste
·237
products. There are two forms of bilirubin which are
distinguishable in serum of clinical icterus. Using the Van den
Bergh test the two forms can be measured by their requirement
(unconjugated bilirubin or indirect reaction) or non requirement
(conjugated bilirubin or direct reaction) of alcohol to produce
the diazo reaction color (Smith et. al., 1974).
Variation in concentration of certain enzymes as measured by
their biochemical activity occurs primarily as a result of two
processes involving the liver. These are mainly their elevation
due to escape of enzymes from disrupted hepatic parenchymal cells
with necrosis or altered membrane permeability (Kaneko, 1980).
A valuable adjunct to the clinical diagnosis of poisoning is
the utilization of serum enzymes determinations. This involves the
detection of enzymes in the serum or plasma which are normally
confined to the tissues and whose activities or concentrations are
normally low in the serum or plasma (Schmidt and Schmidt, 1967).
Enzymes which are normally measured include alanine minotransferase
(ALT) EC. 2.6.1.2., aspartate aminotransferase (AST) EC. ~.6.1.1.,
alkaline phosphatase (ALP E.C. 3.1.3.1.) Creatinine kinase E.C.
2.7.3.2 Sorbitol dehydrogenase (SD. EC. 1.1.1.14) Lactate
dehydrogenase (LDH E.C .1.1.1. ~7) and Gamma-glutam; 1transferase (GGT
EC .2.3.2.2.).
Specific biochemical tests include protein metabolism test.
Although none of the alteration in the serum proteins are entirely
238
specific for liver damage, the combination of an absolutely low
albumin and or gamma globulin levels is quite typical (Kaneko,
1980) .
In this study, measurements of changes in serum levels of the
following biochemical agents have been used as indices of toxicity
by the suspected poisonous plants ~. torvum, Tribulus terrestris,
Dichapetalum madagascasiense, Lantana camara, Eugenia uniflora and
Leucaena leucocephala.
i. serum proteins (total), albumin and globulin
ii. blood urea nitrogen, direct and indirect bilirubin
iii. activity of some serum enzymes
iv. some serum electrolytes.
Procedure
The experimental rats were randomly separated into four groups
consisting of ten rats per group. Three of these groups received
the different doses,of the extracts while the fourth group received
propylene glycol only (cf page 162). The goats were given the
leaves of Q. madagascasiense and 1. camara as described in page
At the end of the extract or plant leaves administration as
described blood was obtained and treated as described in page 126.
\
The following parameters were then determined by procedures as
described in pages 181, 182, IH3, IH4, IH5, IH6, IH7, IHH.
r-
239
serum total and direct reacting bilirubin.
ii. blood urea nitrogen
iii. serum total protein, albumin and globulin
iv. sodium, calcium, potassium, bicarbonate and chloride
ions
v. The serum activities of aspartate aminotransferase,
alanine aminotransferase and alkaline phosphatase.
3.9. Results
3.9.1. The effect of L. leucocephala on serum biochemical
parameters in rats
The leaf extract of 1. leucocephala produced a dose
dependent decrease in the serum total protein which was reflected
as decreased levels of serum albumin (Table 18). The decrease in
the level of serum total protein is significant with the groups of
rats which were given the 400 and 600 mg/kg doses of the extract
(P<0.05). The globul in fraction did not show any significant
change.
The extract also produced significant increases in the level
of the serum urea nitrogen (P<0.05). This can be observed with the
different groups of rats with the various doses of the extract
(Table 18).
The leaf extract did not produce any changes in the serum
electrolyte sodium and potassium (Table 18).
240
The extract produced increases in the activities of the serum
enzymes alkaline phosphatase and aspartate aminotransferase though
these increases were not statistically significant (P(0.05).
However the 600 mg/kg dose of the extract produced significant
changes in the serum actlvity of aspartate amino- transferase (Fig.
6 and Table 19).
3.9.2. The effect of T. terrestris on the serum biochemical
parameters in rats
The extract of T.. terrestris produced decreases in the
serum total protein of rats. The decreases were significant at the
200 mg/kg and 400 mg/kg dose levels of the leaf extract (P 0.05)
(Table 20). The serum protein decreases were reflected as
decreases in the level of both albumin and globulin fractions.
The leaf extract produced dose dependent increase in the
level of serum urea nitrogen which were significant at the 200 and
400 mg/kg dose levels (P(0.05). The extract produced insignificant
changes in the level of the serum electrolyte sodium. It did not
produce changes in the serum potassium values *Table 20).
The extract of T.. terrestris caused dose dependent
increases in the level of serum alanine aminotransferase and
\
aspartate aminotransferase. These increases were significant at
the dose levels of 200 and 400 mg/kg for SGPT and the 400 mg/kg for
SGOT (Table 21 and Fig. 7). The 150 and 400 mg/kg dose levels
241
produced increases in ALP level with a decrease observed with their
intermediate dose of 200 mg/kg (Fig. 7).
3.9.3 The effect of the extract of E. uniflora on the
serum biochemical parameters in rats
The extract of E.- uniflora produced dose dependent decreases
in the level of serum protein. These decreases were reflected as
dose dependent decreases in serum albumin level. The globulin
fractions showed slight increases. The decreases in serum total
protein and albumin levels were significant in rats which received
the 150 and 200mg/kg doses of the leaf extract (Table GG).
The leaf extract of E. uniflora also produced increased
levels of the serum electrolyte calcium and sodium. The increases
were significant at the 150 and GOO mgZkg dose levels of the
extract (P<0.05) (Table 22).
The extract did not produce significant changes in serum
bicarbonate and chloride levels.
The extract produced dose dependent changes in the serum
activities of SGPT and SGOT (Table G3). The increases were all
significant for the doses of the extract used (P 0.05 and Fig. B).
3.9.4 The effect of L. camara on the serum biochemical
\
parameters in rats.
The leaf extract of L. camara decreased the serum total
protein level significantly at the 400 and 500 mg/kg dose levels.
This was accompanied by decreases in serum albumin level which is
significant only at the 600 mg/kg dose level (P<O.05). The level
of the blood urea nitrogen also increases significantly with the
400 and 600 mg/kg dose levels of the leaf extract (Table Z4).
There were no significant changes in the level of serum
potassium and sodium though there appeared to be slight increases
(Table 24).
There were no significant changes in the level of serum
potassium and sodium though there appeared to be slight increases
{Table 24}.
All the dose levels of 1.. camara produced significant
changes in the level of activities of the serum enzymes GPT, GOT
and ALP {P 0.05} {Table 25 and Figure 4}.
3.9.5 The effect of D. madagascasiense on the biochemical
parameters of rats
Q. madagascasiense caused increased levels of the serum total
protein. This was followed by corresponding increases in the level
of serum albumin and globulin fractions. The increase in the serum
total protein level was significant when only the plant was
pelleted for the animals. The plant also produced decreases in the
blood urea nitrogen {Table 26}. No changes were observed in the
\
level of the serum electrolyte sodium and potassium (Table Z6).
The leaves of Q. madagascasiense when mixed in proportions
with ground maize i.e.50:50 or 75:25 respectively or when fed alone
to the rats in pelleted forms produced increases in the serum
activities of ALP, SGOT, and SGPT. These increases in case of the
serum enzyme ALP were inconsistent because it was observed that
when only the plant was fed to the rats without mixing with the
ground maize, the level of ALP activity was even lower than
obtained in the control group (Table 27).
3.9.6. Effect of S. torvum on the serum biochemical
parameters of rats
The leaf extract of ~. torvum did not produce any
significant changes in the serum level of total protein, albumin,
globulin, potassium or sodium (Table 2H).
~. torvum produced dose dependent increases in the serum
activity of the enzyme alkaline phosphatase. It did not increase
the serum activity of aspartate aminotransferase. It was only the
200mg/kg dose that produced significant increase in the activity of
th~ enzyme alanine aminotransferase (Table 29 and Figure 10).
3.9.7. The effect of D. madagascasiense on the serum
biochemical parameters in goats
After 14 days of the goats consuming the leaves of Q.
,
madagascasiense, the serum total protein was slightly elevated with
a corresponding. increase in the serum albumin. The gLobuLin
fraction was unaffected. The conjugated and total bilirubin levels
did not show changes (Table 22). The serum showed highly elevated
levels of ALP and AST. The slight increase in the level of ALT was
not statistically significant (P<0.05) (Table 30).
244
After 21 days of consuming the leaves, the total serum
protein, albumin and globulin showed increases. The serum levels
of conjugated and unconjugated bilirubin did not show changes
(Table 31). The serum level of ALP was further elevated whereas
the level of AST had slightly dropped though the level was still
higher than the control values. The level of ALT had also slightly
increased further (Table 31).
3.9.8 The effect of L. camara on the serum biochemical
parameters in goats.
At the end of 14 days of the goats consuming the leaves of L.
camara, the serum of the goats showed elevated serum total protein
which was reflected as increased levels of serum globulin fraction.
The total bilirubin and conjugated bilirubin were insignificantly
reduced (Table 30). The goats showed highly elevated levels of the
enzymes AST and ALP. The increase in the level of ALT was slight
and not statistically significant (Table 30).
At the+end of 21 days, the goats which were fed with the leaf
extract of L. camara showed highly increased level of serum total
protein as well as the albumin and globulin fractions. The serum
levels of the total bilirubin and conjugated bilirubin were also
significantly elevated (Table 31). T\he serum of the goats showed
further increases in the serum activities of the enzymes AST, ALT
and ALP (Table 31).
245
CONCLUSIONS
Leucaena leucocephala produced reduced levels of the serum
total protein of rats which is reflected as decreases in the serum
albumin level with a related increase in blood urea nitrogen.
This implies that the plant produced some toxic effect on
the liver cells. This observation is further strengthened by the
observed increases in the serum enzyme acti vities of alkal ine
phosphatase and alanine aminotransferase.
The extracts of T. terrestris, E. uniflora and 1. camara are
also hepat~toxic because they produced decreases in the serum total
protein level of the rats which is reflected as decreases in the
serum albumin. Furthermore they cause elevation of the blood urea
nit.r-goen , the serum enzyme acti vities of alkal ine phosphatase,
alanine aminotransferase and aspartate aminotransferase.
The level of liver insufficiency caused by Q. ~adagascasiense
is not very serious since there were no dysproteinaemia produced
but there were incre~ses in the serum activities of ALT, AST and
ALP.
The plant'S. torvum is not hepatotoxic since it did not
produce any adverse changes in the serum biochemical parameters
utilized in evaluating liver function in this study.
246
TABLE 18.
Mean serum parameters (+S.E.) of rats following the
oral administration of Leucaena leucocephala
DOSE TOTAL ALBU- GLOB- BUN K Na
mg/ PROTEIN MIN ULIN mg/ mmole/ mmole/l
kg g/dL g/dl g/dl 100ml 1
0 6,2±0.1 4.1±0.36 2.0±0.46 9.7±1.4 6.1±0.3H 160.1±~Ll
200 4.8±0.87 2.1±0.52 2.7±0.5H 123±1.9 5.H±0.6H 14H.H±0.51
400 4.1±0.53 1.8±0.27 2.3±0.27 144±043 6.6±0.9 15H.l±0.HH
600 3.3±1. 0 1.4±0~45 1.9±0.51 165±1.3 5.4±0.42 153.H±0.H3
n - for each dose is 10.
24r]
TABLE 19
Mean serum enzyme values (+S.E.) of rats following the
oral administration of Leucaena leucocephala
Activities of Serum enzymes
DOSE ALP AST ALT.
mg/kg u/L u/L u/L
0 98.9:t:1.1 141.2:t:2.5 00:t:4•3
200 114.1:t:5•O 142.2:t:0.6H 74.0:t:3.5
400 103. ~:t:1.65 131.H:t:1.2 70.5:t:U•9H
600 105.3±2.6 14H. 4:t:1.5 6H.6:t:2•2
.p - for each dose is 10.
:248'
TABLE 20
Mean serum parameter (+S.E) of rats following the
oral administration of Tribulus terrestris
DOSE TOTAL ALBU- GLO- BUN K+ Na+
mg/ PROTEIN MIN BULIN mg/100 mmole/ mmole/
kg g/dl g/dl g/dl ml L L
0 8.3±0.77 4.9±0.56 3.4±0.45 10~,1±O.33 4.2±O.18 142.4±2.0
150 7.4±0. 15 4.3±0. 16 3.1±O.~7 13.6±O.28 4.8±O.36 144.3±2.4
200 7.5±0.31 4.6±0.26 2.9±0.0.1 14.0±O.24 3.7±O.21 150.7±2.6
400 6.3±0.25 3.8±1.,2 3.9±0.19 17.4±0.~2 5.2±O.O9 149.4±O67
n - for each dose is 10.
249
TABLE 21
Mean serum enzyme values (+ S.E.) of rats following
the administration of extract of Tribulus terrestris
DOSE Activities of serum enzymes
mg/kg ALT AST ALP
u/L u/L u/L
0 135%3.6 190.3%3.9 120.2%5.3
150 144.1±1.6 192.1%1.1 126.3%0.96
200 149.3%4.7 198.4%2.2 108.6%3.0
400 158.5;t2.1 201.8;t4.7 125.2%0.8
n - for each dose is 10.
,
250
TABLE 22
Mean serum parameters (+S.E.) of rats following oral
administration of extracts of Eugenia uniflora
DOSE TOTAL ALB- GLOB- Ca K Na HCo2 CL
mg/ PROTEIN UMIN ULIN mmole mmole mmole oz mmole/
kg g/dl g/dl g/dl /L /L /L mmole L
L
0 5.6±0.6 2.6±0.8 3.0±0.4 7.9±0.9 43±0.3 148±13192±0685±1.1
100 4.7±3.4 1.6±1. 9 3.1±1.9 8.5±2.7 52±1.0 141±15201±0986±0.7
150 4.1±1.9 0.6±1.4 3.5±1.2 9.1±0.6 63±1.0 145±02216±2084±1.8
200 3.4±0.7 0.2±0.9 3.2±1. 6 9.6±0.7 74±0.4 149±13189±1385±0.9
n - for each dose is 10
2 5~
TABLE 23.
Mean serum enzymes values (+S.E.) of rats following
the administration of extract of Eugenia uniflora
Activities of Serum enzymes
DOSE ALT GOT
mg/kg u/L u/L
0 27.5±4.2 36.8±3.5
100 40.2±3.8 59.2±7.1
150 47.5±9.2 58.1±4.7
200 4~.3±8.4 53.5±3.2
n - for each dose is 10.
252
TABLE 24.
Mean serum parameters (+S.E.) of rats following
the oral administration of extract of Lantana camara
DOSE TOTAL ALBUMIN GLOBU- BUN K+ Na+
mg/ PROTEIN g/dl g/dl mg/100 rnmole/ mmole/
kg g/dl ml /L L
0 9.4±1.1 6.9±0.68 2.5±0.52 10.2±1.6 4.8±0.46 151±2.6
200 8.6±0. 13 6.5±2.21 2.1±0. 19 1~6±0.46 5.5±0.3~ 150.2±2.1
400 8.1±0.9 6.3±0.99 1.8±0. 54 15.2±1.5 5.9±1.2 161.~±2.1
600 6.6±2.4 4.4±0.68 2.2±0.4 18.2±1.9 6.8±0.7~ 169.4±~.9
n - for each dose is 10.
253
TABLE 25.
Mean serum enzyme valued (+S.E.) of rats following
the administration of extract of Lantana camara
DOSE
mg/kg Activities of Serum enzymes
ALP AST ALT
ulL ulL ulL
0 78.3±1.3 15tL 0±3.1 105.6±0.8
200 108.3±3.7 16~.4±~.~ 1~0.7±1.1
400 12~.4±5.2 167.3±6.1 134.1±0.3
600 141.6±0.6 178.1±11.6 130.3±4.3
n '" for each dose is 10.
254
TABLE 26
Mean serum parameters (+S.E.) of rats fed with
Dichapetalum madagascasiense
TREAT- TOTAL ALBU- GLOB- BUN Na+ K
MENT. PROTEIN MIN ULIN mg/dl mmole/ mmole/
g/dl g/dl g/dl L L
A 6.7:0.3 3.4:0.2 3.3:0.2 10.8:0.17 140.8:0.0 4.3:0.13
B 7.5:0.39 40±0.32 3.5±010 10.1±0.0 130.3±0.2 3.8±0.57
C 7.8:0.17 39:0.09 4.0±014 14.2±1.3 143±0.2 4.4:0.32
D 8.1:0.33 4.2:0.2 3.9:021 15.0:0.84 141.3±10 4.0±0.19
A - control fed rat cubes. B fed leaves in ratio of
50:50 with carbohydrate. C fed leaves in proportion of
60:40 with carbohydrate; D - fed only pelleted leaves
255
TABLE 27.
Mean serum enzyme values (+ S.E) of rats fed with
Dichapetalum madagascasiense
TREATMENT Activities of Serum enzymes
ALT AST ALP
u/L u/L u/L
A 72±6.3 139.3±13.9 103±7.H
B 164±7.9 160.H±H.6 12H.H±lH.2
C 174.7±6.0 176.7±3.H 14H.7±6.~
D 17~.3±5.7 1H9.0±3.1 HH.3±16.3
n - for each dose is 10.
2.56
TABLE 28.
Mean serum parameters (+S.E.) of rats following
administration of extract of Solanum torvum
DOSE TOTAL ALBU- GLOBU- K+ Na+
mg/kg PROTEIN MIN LIN mmole/ mmole/
g/dl g/dl g/dl L L
o 7.1+0.21 2.4+0.19 4.7+0.14 5.0+0.1 149.7+1.0
100 7.0+0.15 2.0+0.2 5.0+0.~1 4.4+0.14 14H.6+1.3
150 7.4+0.13 2.1+0.1 5.4+0.014 4.7+0.~1 144+0.HH
200 7.0+0.03 2.5+0.07 4.6+0.06 4.5+0.04 142.~+0.H4
n - for each dose is 10.
,
251
TABLE 29
Mean Serum enzyme actives (+S.E.) of rats followine
administration of Solanum torvum
DOSE Activities of Serum enzlme::>
mg/kg ALP AST ALT
u/L u/L u/L
0 116.0±8.4 194.3±2.1 139.7±3.3
100 141.3±6.2 191.7±1.2 123.7±4.5
150 64.5 ±5.2 165.5±11.6 90.5±2.0
200 52 :t1.8 1H3:t3.9 94.5±0.52
n - for each dose is 10.
,
258
TABLE 30
Mean Serum values (+ S.E) of goats after feeding with
Q. madagascasiense or L. camara for 14 days
PARAMETER CONTROL Q. madagas. CONTROL L·camara
AST U/L 83±9.8 273.5 79.5±5.4 130.512.3
ALT ulL 17±1.2 U:L5±1.6 16.0:!::1.6 19.5:!::2.6
ALP ulL 262±12.7 463.6:!::18.9 522.5:!::38.814875:!::802
Total Protein
g/dl 4.7±0.69 5.0:!::0.84 4.7 ±O. 7 6.2±0.83
Albumin g/dl 2.1±0.39 2.3±0.46 2.45:!::0.39 2.4:!::0.37
Globulin g/dl 2.7±0.43 2.6:!::0.2 2.2±0.42 3.8:!::0.8
Total
Bilirubin
mg/dl 0.7±0.6 0.7±0.2
Conjugated ,
bilirubin
mg/dl 0..35±0.17 0.35:!::0.12 0.35±0.1 O. 3±0. 1
CONTROL A indicates the control values for goats fed
the leaves of Q. madagascasiense
COTTROL B are the control values for goats administered
with extracts of Lantana camara
"
-259
TABLE 31
Mean Serum values (+S.E.) of goats after feeding
Q. madagascasiense or ~. camara for 21 days
PARAMETER CONTROL A Q..madagas. CONTROL ~.camara
AST U/L 83±9.8 262.5±5.0 7~.5±5.4 185.5±3.5
ALT u/L 17.0±1.2 22±1.0 10.0±1.0 33.5 ±2.5
ALP u/L 262±12.7 1003±3.7 522.5±38.8 14~3±351
Total Protein
g/dl 4.7±0.69 5.7±0.1 4.7 ± 0.7 7.7±0.0
Albumin g/dl 2.1±0.39 2.55±0.7 2.45±0.34 3.7±0.55
globulin g/dl 2.7±0.43 3.2±0.35 2.2±0.42 4.05±0.05
Total Bilirub-
in mg/dl 0.75:0.19 0.75±0.15 0.85±0.23 1.35±0.15
Conjugated
bilirubin mg/dl 0.35±0.17 0.3±0.1 0.35±0.1
CONTROL A indicates the control values for goats fed
leaves of Q.. madagascasiense.
CONTROL B are the control value for goats administered
with extracts of Lantana camara.
FIG. 6.
EFFECT OF LEUCAENA LEUCACEPHALA ON SERUM
ENZYME ACTIVITIES IN RATS. t' n "IS 10 FOR
EACH POINT.
IJJ
~ 1.0
>-
N
Z
IJJ
~
:::J
0:
120 ALKALINE PHOSPHATASE·W II.
tf) I
100 I
80
60
1.0
20
200 1.00 600 \
-+ DOSES OF PLANT EXTRACTS in mg/kg. j
-~ -
FIG. 7.
EFFECT OF TRIBULUS TERRESTRIS ON SERUM
ENZYMES IN RATS r'n~ IS 10 FOR EACH POINT.
120
80
ALKALINE PHOSPHATASE
1.0
--I:::J
(/) 21.0
W
t=
>
t=
U ie o«
W 120 SGOT2>:-
~
W 60
2:
:::J
0::
W
(/)
180
$
120 ~
SGPT
60
l I
200 I.00
--+ DOSES' OF PLANT EXTRACT in mg /kg.
FIG. 8.
THE EFFECTS OF EUGENIA UNIFLORA ON SERUM
ENZYME ACTIVITIES IN RATS un"
VALU~ IS 10"
80
-I
:::::>
l/)
W I
l- J.O j
> I,
l- 560T I
u<!. I
/"
w
>z-
N -1
Z
w ,IL: I
:::::> 80
0::
W
l/) i
J
I
t
\,
56PT
1.0 , I
I ,
100 200
---+ DOSES OF PLANT EXTRACTS in mg/ kg
FIG. 9.
EFFECT OF LANTANA CAMARA ON SERUM ENZYMES
ACTIVITIES IN RATS "n" VALUE IS 10 .
120
SGPT
80
1.0
--l:::J
160 -+- -+ tIf)
l1J
t-
> SGOT
t- 120
U«
W 80
>2-:
N
Z
l1J 1.0
~
::>
0::
W
If) 160
120
,
80 ALKALINE PHOSPHATASE
40
200 1.00 600
DOSES OF PLANT EXTRACT in mg{kg-
FIG·lO.
EFFECT OF SOLANUM TORVUM ON SERUM ENZYME
160 ACTIVITIES IN RATS "n" VALUE IS 10.
120
80 SGPT
1.0
--':::J
Vl
W 180.--
.>u--« 120 SGOT
W
>~- 60
N
Z
w
::::E
c::::rJ:
W 160
Vl
120
ALKALINE PHOSPHATASE
80 ,
1.0
, I I i
50 100 150 200
~DOSES OF PLANT EXTRACT in mg/kg.
265
SECTION D
3.10 CLINICAL SIGNS AND PATHOLOGICAL CHANGES IN RATS
ASSOCIATED WITH INGESTION OF POISONOUS PLANTS
EXPERIMENT 1
Introduction
The effect of m~ny poisonous substances often manifest by
the production of typical clinical symptoms and gross and
histopathologic lesions in the tissues. The lesions of poisoning
are reported to be rarely characteristic. Nevertheless, the
findings at autopsy are reported to provide definite clues to the
nature of the poison (Clarke and Clarke, 1975).
The poisons, when in sufficient concentration kill the
tissue which they come in contact or if the action is milder,
injure the tissues and initiate an acute inflammatory reaction. If
the poison has been ingested, it is the alimentary mucous membranes
whiph suffer the necrosis or inflammation. The most powerful ones
destroy the lining of the mouth or oesophagus as they are swallowed
and then carry their effect to the stomach. Others pass through
the stomach with little damage and cause superficial necrosis and
,
inflammation in the upper or lower intestine presumably because
they remain longer in contact with the injured part (Smith et. al.,
1974).
Furthermore, some poisons are known not to have immediate
action but are absorbed to produce their action on the delicate
2E6
epithelial cells of such parenchymatous organs as the liver, or
kidney which they reach by the blood stream.
The skin and visible mucous membranes may have a
characteristic discoloration. Jaundice has been reported as a
frequent sign of hepatic damage by plant poisons (Jenkins, 1~o3j
Hutchinson, 1977, Tennant et. al., 1~H1, Dwers et. al., 1~H2). A
cherry red or pink color is seen in cyanide and carbon monoxide
poisoning. Methaemoglobinaemia due to nitrates, nitrites or
chlorates may impart a brown coloration (Andrade et. al., 1~71).
In toxicology therefore, the most important evidence of
toxicity even to the untrained person will be the observation of
clinical symptoms and gross pathologic lesions in the tissues of
the affected animals. The level of tissue damage can then be
further evaluated with the examination of histologic studies.
This study was therefore carried out to evaluate the
organotropic effects of the poisonous plants, ~. torvum, E.
uni flora, .T. terrestris, Q. madagascasiense, 1.. camara and 1..
leucocephala in rats and the re-examination of the effect of 1..
camara and Q. m,adagascasiense in goats.
Pr0cedure
The extracts of the different plants were administered in
doses to groups of rats daily for 14 days. Each group consisted of
10 rats. After administration the rats were left in their cages
and observed for periods of up to three hours for any obvious
267
clinical signs until the next dosage was administered. The goats
consuming the leaves were similarly observed in their pens.
The extract administration was stopped on the 14th day and
on the 15th day, the rats were sacrificed by a blow to the head and
exsanguination. The experimental goats were fed the leaves of Q.
madagascasiense and L. camara for 21 days and were slaughtered on
the 22nd day.
Gross and histologic evaluation of the tissues of the
animals were performed to determine the toxicity of the plants to
the tissues of the animals. The tissues examined include the
brain, lungs, heart, liver, spleen, pancreas, gastrointestinal
tract, kidneys and the skeletal muscles.
For histopathology, tissue blocks were fixed in 1U% buffered
formal in, processed routinely and stained with hematoxylin and
eosin (page 188).
268
3.10.1 Lesions due to L. camara in goats
Grossly the lesions were those of a pale, swollen, friable
liver and ecchymotic hemorrhages in the kidney.
At histology the liver from this group of animals showed
mild fatty degeneration. The intestinal lesion was characterized
by marked oedema, moderate glandular degeneration and marked
lymphocytic infiltrates into the laminar propria. These
infiltrates were accompanied by few macrophages and plasma cell.
There was also goblet cell hyperplasia. In the spleen, there was
moderate depletion of lymphocytes from the germinal centers and in
addition there was very mild macrophages and plasma cell
hyperplasia.
3.10.2. Lesions due to ~. torvum in rats
Al though gross lesions were not observed, the lungs at
histology showed vasculitis with moderate perivascular lymphocytic
and macrophage infil~ration.
3.10.3 Lesions due to 1. terrestris in rats
At necrops~, petechial hemorrhages were observed in the
kidney and liver. Histology showed a moderately congested heart
while the kidney lesions were characteri by moderate messangial
proliferation, thickened glomerular basement membrane and presence
of protein casts in renal tubules (Plate 7).
?69
3.10.4 Lesions due to L. leucocephala in rats
Grossly the kidney was markedly congested and the liver
pale. Microscopic examination of tissues confirmed renal
congestion, while the liver lesion was that of marked perivascular
lymphocytic cuffs around central veins and portal vessels (Plate
9). Although lesions were not observed grossly in the heart,
histology revealed degenerative and necrotic changes characterized
by cellular swelling and clumping of nuclear chromatin. These
changes were accompanied by moderate lymphocytic infiltrate and
fibroblast proliferation.
3.10.5. Lesions due to E. uniflora in rats
The only gross and histologic lesions observed in rats dosed
with this plant were those of congestion in the kidney and moderate
fatty change in the liver.
3.10.6. Lesions due to D. madagascasiense in rats
Gross lesions were not observed at necropsy but
histological examination of tissues revealed mild pulmonary oedema
associated with perivascular lymphocytic cuffs in the lungs. In
the liver, there was mild fatty change and most of the bile
ductules were distended with bile. The intestinal lesion was that
of marked macrophage, few lymphocytic and eosinophilic infiltrates
into the laminar propria. In addi tion, there was goblet cell
hyperplasia.
270-
3.10.7. L. camara in rats
At necropsy, only animals that received 600 mg/kg doses of
~. camara showed lesions which were characterized by ecchymotic
hemorrhages in the heart, kidney and liver.
Histology of these tissues revealed marked diffuse fatty
degeneration in the liver which also showed moderate periportal
lymphocytic infiltrates (Plate 10).
,
270-
3.10.7. L. camara in rats
At necropsy, only animals that received 600 mg/kg doses of
~. camara showed lesions which were characterized by ecchymotic
hemorrhages in the heart, kidney and liver.
Histology of these tissues revealed marked diffuse fatty
degeneration in the liver which also showed moderate periportal
lymphocytic infiltrates (Plate 10).
,
211
Plate 7:
Renal glomeru,lus of a rat dosed with Tribulus
terrestris showing moderate messangial proliferation
and proteinaceus material in glomerular space
(H & E X 750)
272
Plate 8:
Sectioning the same kidney as Plate 7
with protein cast (arrow) in tubule
(H & E X 750)
273
,
Plate 9: Section of the liver of a rat dosed with
Leucaena Leucocephala showing lymphocytic cuffs
around the portal vessel (H & E X 75d).
274
Plate 10:
Histological appearance of the liver of a rat dosed
with Lantana camara showing moderate fatty
degeneration (H & E X 750).
CHAPTER FOUR
DISCUSSIONS
It is a striking fact that majority of the plants are,at least
to some extent toxic. Caution is necessary even in the consumption
of many crop plants (Ewer and Hall, 1978). In describing the
occurrence of toxic plants in our environment, both to human and
livestock, it has been observed that it is obviously impossible in
anything other than a work of encyclopedic proportions to mention
all the species of plants which sometime or the other have been
suspected to cause poisoning in man and livestock (Clarke and
Clarke, 1975; Hall, 1977 and Kinghorn, 1979).
However because Nigeria has a poor or non existent reporting
system to document the incidence and occurrence of poisoning by
plants, coupled with the system of livestock management, incidence
of phytotoxicology assumes less significance. Taking into
consideration the transhumans nature of livestock management in
Nigeria, it is estimated that about 10 per cent of all grazing
,
livestock are seriously affected by poisonous plants each year
(Nwude , 1975). The nature of the losses from such encounters is
variable. The effect can be either direct, causing death,
debil itation, reduced weight gain or management problems in grazing
animal or indirect causing abortions or congenital deformities in
the unborn offsprings of that animal (Keeler, 1973).
27L6
4.1 TOXIC CHEMICAL CONSTITUENTS OF POISONOUS PLANTS
Poisonous plants produce their toxic effects by virtue of
their secondary chemical constituents such as gums terpenes,
glycosides, alkaloids, tannins and phenolic compounds (Ewer and
Hall, 1978; Kingsbury, 1979). Cocoyam is not only disagreeable to
eat when raw because of the piercing action of raphides, but is
also slightly toxic when cooked because of the calcium oxalate of
which the raphides consist of. Cassava tubers always contain
poisonous cyanogenic glycosides in the peel; bitter varieties
contain them in the flesh also (Osuntokun, 1973; Tewe and Pesu,
1982) . Wild forms of the yam Dioscorea dumetorum contain a
poisonous alkaloid dioscorine and poisoning may result if they are
eaten by mi stake for cuItivated less toxic forms of the same
species.
Poisonous plants may not always be poisonous depending on
certain factors. For,example plants such as Amaranthus retroflexus
are able to acquire dangerous levels of their toxic factor i.e.
nitrates only when they are grown on nitrate soils or when the
pasture is fertilized by artificial manures containing sodium,
potassium or ammonium nitrate (Clarke and Clarke, 1975) i.e.
environmental factors affect poisoning.
Furthermore handling also affects the toxic constituents of
plants. For example a plant like Melilotus alba is reported to
produce toxicity only when spoiled (Frasser and Nelson, 1959; Prier
and Derse, 1962).
4.1.1. Nitrate and nitrite content of the Poisonous plants
The principal hazard to livestock from nitrates or nitrites
arises from the fact that certain plants grown on soils containing
excess of the salt may take up sufficient to render them toxic to
animals eating them. Outbreak of nitrite poisoning have been
reported to have occurred in sheep grazing normally safe plant,
after a p r-o Lorig ed drought. In such circumstances scme normally
safe plants with a capacity for fast growth are said to become
dangerous (Blood and Henderson, 1976).
In this study Tribulus terrestris, Lantana camara, Leucaena
leucocephala, Solanum torvum, Dichapetalum madagascas iense , and
Eugenia uniflora were all observed to contain nitrate and nitrites
(Table 1). The highest concentration of nitrate was analyzed from
~. torvum with an am9unt of 16.10 mg/g. The lowest concentration
was determined in L. leucocephala with an amount of 14.1 ug/g.
Plants which a~cumulate more than 1.5 per cent by dry matter
of nitrate are reported to be potentially toxic (Blood and
Henderson, 1~74). Similarly, Clarke and Clarke (1975) reported
that the minimum lethal dose of nitrate in form of potassium
nitrate was 4.5 per cent. This implies that the level of nitrate
analyzed from the six toxic plants are relatively low to contribute
to acute toxicity which may be produced by such plants. However in
.218
the event of an animal being exposed to pasture containing mainly
a variety of one of these plant, it is possible that the level of
the nitrate in these plants could build up as the quantity of the
plant consumed increases Poisoning by nitrate from these plants
could also be feasible in a situation of prolonged drought as
nitrate and nitrite level are reported to increase under this
circumstances (Blood and Henderson, 1976, The Merck Veterinary
Manual, 1979).
If the minimum lethal dose of nitrate in form of potassium
nitrate is 4.5 per cent (Clarke and Clarke, 1975) then an animal
exposed to a pasture infested only with .§.. torvum will have to
consume as much as 279.5 grams of the leaves inorder to suffer
nitrate poisoning. The animal has to consume as much as 293.4
grams of L. camara leaves to show symptoms of nitrate intoxication;
consume 294.5 grams of T. terrestris leaves; consume 300.2 grams of
the leaves of Q. mada~ascasiense; consume 319.2 grams of the leaves
of L. leucocephala inorder for such animals to accumulate enough
substance to s~ffer from nitrate intoxication.
Nitrate per se are reported to be relatively non toxic.
They become important as toxic factors only when they are converted
either in the plant or within the alimentary can-al into nitrite
(Andrade et. al., 1971; Merck Veterinary Manual, 1979). Nitrates
are reduced by the microflora in the alimentary tract especially
the rumen, to nitrite, hydroxylamine and finally to ammonia. The
279
rate at which this reduction takes place is an important factor
affecting toxicity (Clarke and Clarke, 1975). It is the nitrite
which is then absorbed into the blood circulation to convert
haemoglobin into methemoglobin which then disturbs the oxygen
carrying capacity of the blood. If the level of methemoglobin
conversion is considerable, then the animal suffers tissue anoxia.
Symptoms are reported to be observed when about 20 per cent of the
haemoglobin is converted into methemoglobin. This becomes
increasingly more adverse as the proportion increases and death is
reported to supervene when the conversion reaches 80 per cent
(Harris and Rhodes, 1969; London, Henderson and Cross, 1967).
Poisoning by these poisonous plants resulting from their nitrate
content will therefore also depend on the rate of nitrate
conversion into nitrite or production of ammonia by the microflora.
Environmental factors are also known to influence the amount
of nitrate concentrated by plants. Low temperatures, 1imited
,
sunlight and poor mineral sources are known to contribute to
increased nitrate level (Merck Veterinary Manual, 1979). Harris
and Rhodes (1969) reported that on dull days or at night, the
nitrate level is higher than it is in the sunlight as conversion to
amino acids does not take place. Since the sunshine is no limiting
factor around here, the level of nitrate analyzed in these
poisonous plant could have been affected
280
Similarly, in this study, the highest level of nitrite was
determined in T. terrestris with an amount of 9.71 mg/gm. The
lowest concentration was analyzed in ~. torvum with an amount of
8.91 mg/g (Table 1). It is r-epo r-t.e d that pigs are more
susceptible to nitrite poisoning than are cattle and sheep
(Crawford, Kennedy and Davison, 1966). The minimum lethal dose
that was reported for the pigs was in the order of 70 to 75 mg/kg
in form of sodium nitrite. With the determinations obtained for
the various plants in this study, it means the following quantities
of the plant material has to be in the animals food, for nitrite to
contribute to the animals poisoning. T. terrestris 7.7 kg of the
leaves; 1.. camara 7.9 kg; E.. uniflora 8 kg of the leaves; 1..
leucocephala 8.1 kg of the leaves; Q. madagascasiense 8.4 kg of the
leaves; ~. torvum 8.4 kg of the leaf material.
Sinclair and Jones (1964) further alluded that it is
difficult to make a categorical statement as regards the toxic dose
of nitrate since according to them this depends on the rate at
which substance is co~sumed. A single dose of nitrate of level 220
mg/kg was reported to be lethal to sheep whereas the same dose
consumed over ~ long period (about 24 hours) showed no ill effect.
Thus poisoning by these poisonous plants will have to depend on how
long they were ~xposed to them. Under this circumstances, none of
the plants under study can produce nitrate/nitrite toxicity with
transhumans cattle management.
281
Furthermore, toxic levels of nitrate or nitrite are reported
to be found in common pasture species such as Brachiaria species
during rapid growth (Andrade et. al., 1971; Blood and Henderson,
1976) . The leaves used in these studies were harvested from
already matured stands, therefore the observation that rapidly
growing plants have abil ity to accumulate nitrate may not be a
factor in consideration.
Fasting has been reported to increase the susceptibility of
animals to, poisoning (Clarke and Clarke, 1975). This is an
important observation since livestock in Nigeria are exposed to
reduced availability of pasture during the dry seasons when there
are no rains and they have to trek long distances in search of
pasture. Unfortunately, plants are reported to be able to
concentrate the level of their nitrate and nitrite content during
the dry season (Blood and Henderson, 1976). It is not impossible
that the level of nitrate and nitrite in ~. camara, ~. torvum, Q.
madagascasiense, E. uniflora, ~. leucocephala and T. terrestris
could be speculated to be able to increase during such dry season,
for the level of these inorganic substances to build up to a level
contribute to their toxicity. In such a situation chronic exposure
then becomes a very important factor to be considered in
determining toxicity by these plants. This is because as the
animal continuously consumes the plants, the level of nitrate and
282
nitrite accumulates wi th their subsequent conversion of haemoglobin
into methemoglobin to precipitate symptoms of poisoning.
4.1. 2. Oxalate content of the poisonous plants
The poisoning of animals by pure oxalate is reported to be
very rare. Cases of poisoning by oxalate are reported to be
encountered due to the ingestion of large amounts of plant
containing oxalates (Clarke and Clarke, 1975). In these studies
the highest concentration of oxalate was determined in Q.
madagascas~ense with an amount of 0.24 per cent whilst the lowest
concentration was found in E. uniflora with an amount of 0.102 per
cent. The oxalate content of plants are known to be mainly in form
of sodium and potassium oxalate (Shupe and James, 1969). These
salts of oxalate are reported to be soluble whereas the calcium
oxalate is not soluble and is therefore not assimilated during
digestion. Mckenzie et. al. (1981) reported that potassium oxalate
at the level of 2.6 ~nd 4.3 per cent, when added to a diet of oaten
and lucerne chaff and fed to horses produced typical symptoms of
oxalate toxicity. On the other hand, Stewart and McCallum (1944)
gave two doses of 454 gm of oxalic acid as sodium oxalate or
ammonium oxalate within 24 hours or 200 gram of ammonium oxalate
daily for 5 days in order to kill the animals.
Utilizing the observations of Mckenzie et aI ,(1981) , it is
estimated that the following amounts of the poisonous plants have
to be consumed by a horse inorder for it to have enough oxalate to
283
produce toxicity. 1.. leucocephala 3.4 kg of the leaves; T.
terrestris, 3.07 kg of leaves; ~. torvum 2.39 kg of the leaves, Q.
madagascasiense 1.79 kg of the leaves; 1.. camara, 2.91 kg of the
leaves; E. uniflora 4.22 kg of the leaves.
The oxalate content of plants is reported to be highest at
the leafy stage of growth where plants which have been associated
with oxalate toxicity have been observed to contain high levels of
the substance. Many of such plants are reported not to contain
less than 2~ per cent total oxalate (Blaney et al., 1981). It can
then be inferred that the level of oxalate in the plants under
study are too low to be associated with toxic
effects they may produce.
Nutritional status, the ratio of calcium to oxalate in the
diet, as well as the level of oxalate ingested are reported to be
very important factors in the production of oxalate toxici ty
(Blaney et al., 19811. This observation is important in that a
malnourished and hungry animal which becomes exposed to Q.
madagascasiense, from which was analyzed the highest level of
oxalate-in--thrs study, most e-specially at the leafy stage when
oxalate absorption is known to be highest, may suffer toxicity
associated with oxalate. This is with the assumption that Q.
madagascasiense possesses the ability to accumulate oxalate at the
leafy stage.
284
4.1.3. Phytin content of the poisonous plants
A large part of the phosphorus in plant is reported to be
present as phytin which is the calcium magnesium salt of phytic
acid. Harland and Prosky (1979) reported that as the intake of the
dietary fibre from fruits, nuts, vegetables, and whole grains
increases, then the intake of phytic acid also increase.
These studies have shown that T. terrestris leaves contained
the highest concentration of phytin of 20.18 per cent, whilst the
lowest level was obtained in 1. camara of 15.99 per cent (Table
1). Edman and Forbes (1977) reported that at dietary levels of one
per cent or greater, phytic acid can interfere with mineral
availability. It is reported that phytin decreases the
bioavailability to animals and humans of minerals such as zinc and
iron. Morris and Ellis (1981) observed that the dietary
requirement of zinc for growth was generally higher if seed protein
was taken as source ~f protein. This is because seeds have rather
high level of phytate than animal protein.
The phytat,e contents of all the leaves of the poisonous
plants determined in these studies were rather very high. This is
an indication that phytin in these plants could contribute to the
toxic effects associated with these plants. Consequently a
prolonged exposure of livestock to the leaves of these plants will
affect zinc and iron metabolism in these animals. Such toxic
effects are expected to be most obvious in chronic exposure to the
285
plant considering the restorative ab i Iity of the animal in terms of
the substances that will be lost from the body.
4.1.4. Cyanide content of the poisonous plants
Many species of plants contain hydrocyanic acid either free
or more usually in the form of cyanogenetic glycoside, an organic
compound containing a sugar and capable of yielding cyanide on
hydrolysis (Fernando, 1987). The glycoside itself is reported not
to be poisonous but becomes so when the hydrocyanic acid content is
released by. an enzyme. The release of such enzymes is associated
with the plant damage or decay (Herrington, Elliot and Brown, 1971;
Fernando, 1987). In the poisonous plant assayed in this study the
highest concentration of total cyanide was analyzed in ~.
madagascasiense. Thi s concentrat ion was 15.06 ppm whil st the
lowest level of cyanide was determined in L. camara
with a concentration of 12.7 ppm. The observation of hydrocyanic
acid from these pIant s may be an indication that among other
chemical substances they contain, they also possess cyanogenetic
glycoside. According to Clarke and Clarke (1975) the minimum
lethal dose of free hydrocyanic acid and of potassium cyanide given
per os is about 2.0 to 2.3 mg/kg HCN. Taking into consideration
the level of cyanide determined from these poisonous plants under
study (Table 1) it becomes obvious that the level of cyanide in
them are considerably high enough to participate in producing
toxicity that may be associated with these plants. Infact it is
2.86
reported that materials containing over 20 mg HCN per 100g is
potentially dangerous to stock. However, most authors working on
cyanide poisoning allude that it is not possible to state with any
certainty the toxic dose of cyanide in form of the cyanogenetic
glycoside. This is because the level of cyanide varies according
to the conditions obtaining in the plant and that of the animal at
the time of consuming the plant (Van der Walt 1944; Chen et. al.,
1934; Clarke and Clarke, 1975).
Cyanid~ concentration are highest in the young actively
growing plants. Van der Walt (1944) mentioned that poisoning in
ruminants depends upon the quantity of the plant ingested, the
previous diet of the animal, the pH of the stomach contents, the
percentage of the total hydrocyanic acid present in the free state
in the plant, the concentration of cyanide liberating enzyme
present in the plant, and the total hydrocyanic acid content of the
plant.
In view of the foregoing variables, in determining the
toxicity of cyaftide by cyanogenetic plants, it becomes difficult to
attribute the toxic effects that could be produced by~. torvum, E.
uniflora, Q. madagascasiense, l!. camara, 1.. terrestris, or l!.
leucocephala to their HCN content. Infact it is reported that it
is only those animals which rapidly eat the
plants that die. It is also observed that ruminant are more
susceptible to poisoning by cyanogenetic plants than are horses,
281
and pigs, since the enzymes concerned in the release of hydrocyanic
acid are destroyed by gastric hydrochloric acid. Moreover,
hydrogen cyanide is detoxicated by conversion into thiocyanate and
is excreted in the urine over a period of several days. Owing to
the rapid detoxication of cyanide, it is reported that it is
possible for animals to ingest amounts of cyanide only slightly
less than the lethal dose over extended periods without harm
(Hazzard et al., 1982). In this situation these plants under study
could be con~umed over prolonged periods with their cyanide content
not constituting danger.
4.1.5. Metallic ion concentration in the poisonous plants
As shown in table 2, the metallic ions which were assayed
for their contribution or otherwise in the toxic nature of §..
torvum, Q. madagascasiense, L, camara, E. uniflora, T. terrestris,
L. leucocephala were molybdenum, manganese , iron, copper, zinc and
lead.
4.1.5.1. Level of molybdenum in the poisonous plants
Plants hav~ been reported to concentrate excessive amounts of
molybdenum from the soil on which they grow. Normal herbage are
classed as those which contain 1 to 3 ppm (Clarke and Clarke,
1975). Analysis of the leaves of the plants used in this
study showed that T. terrestris contains the highest concentrati-
ion of molybdenum with an amount of 0.26 ppm. The lowest
concentration was found in E. uniflora and Q. madagascasiense. The
presence of a small excess of molybdenum of up to 2 to 25 ppm in
young growing herbage has been reported to cause deficient bone
metabolism in herbivores consequent upon the effects of molybdenum
on the utilization of copper and of phosphorus (Hall, 1977). If
the lowest level of molybdenum cited above as causing symptoms of
molybdenosis i.e. 2 ppm is used as a measure to determine whether
the toxic plants studied could have enough molybdenum to produce
toxicosis, then about 14.3 kg of L. leucocephala will have to be
consumed; 7 ,,7 kg of T. terrestris; 14.3 kg of Q.. torvum; 16.7 kg of
Q. madagascasiense; 8 kg of L. camara and 16.7 kg of E. uniflora
would have to be consumed. These observations are mainly
presumptive since Cunningham and Hogan (1959) had reported that
high molybdenum levels are not toxic to sheep or cattle unless the
diet contains enough inorganic sulphate. When the intake of
sulphate is low, the excretion of molybdenum in the urine is
negligible consequently the level of molybdenum in the blood rises.
The implication of this is that the level of sulphate in the plants
understudy, hav~ to be manipulated inorder to cause a toxicity.
4.1.5.2. The level of manganese in poisonous plants
The highest concentration of manganese in plants is reported
to be found in the roots as opposed to its occurrence in the leaves
of the plant (Berman, 1980). The level of manganese in diet which
has been observed to interfere with haemoglobin formation is 1~5
ppm whilst supplementation of the diet with 1~50 to ~OOO ppm was
289
reported to have depressed growth (Martone, Hartman, and Clawson,
1959).
The level of manganese that was estimated in the leaves of
the toxic plants was low to be considered to be of any consequence
in the toxic effects produced by the plants. The highest
concentration of manganese was determined in T. terrestris.
Considering the observation of Matrone et al. (1~5~) it means the
plants in this study cannot produce manganese toxicity. The level
of manganes~ in T. terrestris was 0.60 ppm (Table 2).
4.1.5.3. The level of copper in poisonous plants
Poisoning of animals by copper of plant source could take
either the acute form in which case the animal consumes herbage
with high levels of copper or chronic situations in which
successive non-toxic doses of copper from the plant have a
cumulative effect. Cases of animal poisoned by copper in plants
have been associated ~ith situations where there was no growth of
herbage so that animals were forced to consume the grass which was
heavily contami~ated with copper. Poisoning of sheep which grazed
areas previously sprayed with salts of copper has been
attributed to the ability of certain plants to accumulate copper
(Muth 1952, Gracey and Todd, 1960).
The toxic dose of the sulphate of copper by mouth is reported
to be of the order of 25 to 50 mg/kg in lambs, 130 mg/kg in sheep
and in the cow about 200 mg/kg. If these concentrations are
reckoned with then the level of copper determined from §.
torvum,T·terrestris, ~.uniflora;L.camara,madagascasiense,
L.leucocephala are relatively low. This shows that these plants
are not able to accumulate copper which may pose danger for copper
toxicity. The implication of these observations is that copper is
of no consequence in what ever type of toxic symptoms these plants
may produce.
Damage to the liver caused by some plants e.g.Heliotropium
europoeum can lead to abnormal accumulation of copper and death
resulting from the hemolytic crisis of chronic copper poisoning
(Clarke and Clarke, 1975). Gracey and Todd (1960) were able
to find 200 ppm of copper in some grasses sprayed with copper
sulphate. Tait et. al, (1971) estimates that a level of 27 ppm of
copper in the diet is fatal to lambs.
4.1.5.4. The level of zinc in poisonous plants
The metallic zin~ is reported to contaminate pasture which are
found in areas where zinc is produced from its ores. However, it
was observed tha,t symptoms could not be produced in domestic
animals by feeding them on pasture contaminated with zinc chloride
(Clarke and Clar~e, 1975). The average daily intake of the cow and
of the sheep and goats used in the experiments were 527 and 53
grammes respectively. The amount of zinc estimated in the
poisonous plants under this study were relatively low. The highest
concentration of zinc of 4.73 ppm was determined in Q.
291
madagascasiense. Thus in general, it appears relative large
proportions of the leaves of such plants will have to be consumed
inorder to produce zinc toxicity.
4.1.5.4. The level of Lead in the leaves of the poisonous plants
Lead is reported to be the most common cause of poisoning in
animals especially in dogs and cattle (Todd, 1~6G). Contamination
of pasture near open cast or disused lead mines have been reported
to be the cause for serious losses in farm livestock (Harbourne et.
a1., 1968). The herbage in such areas are said to contain up to
580 ppm of lead on a dry matter basis.
The highest concentration of lead was estimated in E.
uniflora with an amount of 0.21 ppm. This represents quite low
concentration so that lead toxicity cannot be connected with the
symptoms that may be associated with these poisonous plants.
Plants which are located near busy streets have been known to
concentrate up to 500,ppm of lead due to their contamination by the
exhaust fumes of cars (Scott, 196:3). The stands from which the
leaves of Q. maaagascasiense, §. torvum and L. 1eucocepha1a
were collected were all along busy streets. This means that the
level of lead analyzed from their leaves could have been acquired
from the exhaust fumes. Therefore depending on how busy the street
is with cars and the length of growth, the level of lead
could increase in such plants.
292
4.2. Effect of the poisonous plants on biochemical parameters
This observation of biochemical changes is very relevant since
some toxic substances take long periods to exhibit any symptoms or
produce any organic damage.
During the process of degeneration leading to necrosis,
several biochemical alteration occur. The changes in capacity of
oxidative phosphorylation is one of the earliest manifestation
measured by. adenosine triphosphate (ATP) level. (Beutler, 1~6S).
Such an assault results in decreased intracellular pH and lack of
energy, for energy dependent cation pump working against normal
electrochemical gradients. Failure of the later and loss of
integrity of cell membrane results in an influx of sodium chloride,
calcium and water culminating in cellular swelling and leakage of
intracellular ions (especially potassium) proteins and enzymes
(Schmidt and Schmidt, ·1967; Smith, Jones and Hunt, 1~74; Morgan et
al., 1988). These leaking enzymes provide an important diagnostic
aid for the recQgnition of dead or dying tissues in living patients
(Kaneko, 1980; Cornelius et. al., 1~65). Measurements only allows
for recognition' of necrosis, and by characterization of these
enzymes by electrophoretic separat ion, exactly local ization of
necrosis to specific tissue is said to be possible. This is the
rational for the use of serum biochemical changes as one of the
parameters for evaluating toxicity of the poisonous plants in this
study taking into consideration that the plants were fed
subacutely.
4.2.1 The effect of the poisonous plants on serum proteins
In this study, E. uniflora, L. camara, T.terrestris and
L. leucocephala have all produced dose dependent decreases of the
total protein level. These decreases in the total protein are
accompanied by decreases also in the level of serum albumin. The
globulin levels also increased in the serum of the rats treated
with T. terrestris and only slightly i.e insignificantly
with rats treated with L. camara (Tables 20 & 24). Globulin levels
for animals treated with E. uniflora and L. cleucocephala did not
show changes (Tables 22 & 18).
Changes in the serum protein are quite significant for these
four plants. With E. uniflora, the 100 mg/kg dose produced 10.1%
decrease; the 150 mg/kg 27% decrease; the 200 mg/kg dose 40.3%
decrease. With L. leucocephala, the 200 mg/kg dose produced 23.0%
decrease; the 400 mg/kg dose a 34.9% decrease; the 000 mg/kg dose
47.8% decrease., In case of T. terrestris, the 150 mg Zkg dose
produced 10.8% decrease; the 200 mg/kg dose 10% decrease; and the
400 mg/kg a 24.1% decrease. With L. camara poisoning the 200 mg/kg
dose produced 8.5%; the 400 mg/kg a
13.8% decrease and the 600 mg/kg dose a 29.B% decrease in the serum
total protein.
294
Factors responsible for decreased level of serum protein
include toxic liver deficiency (Cantarow & Trumpert, 1956; Kaneko,
1980) . This result from defective protein manufacture in the
liver. The response either arise from the direct effects of
hepatotoxicants on the Kupfer cells or are secondary to
hepatocellular insult. Albumin is said to notably decrease though
hypoalbuminaemia may also follow upon excessive loss of protein
into the urine in case of severe kidney damage.
The ~ecreases in the serum protein therefore observed with
the rats treated with ~.camara, ~.leucocephala,T.terrestris and ~.
uniflora indicate that these plants produced hepatocellular toxic
changes and or nephrosis. Decreases in total serum protein levels
were also reported in layer strain after 16, 28 and 56 days of
feeding the plant Brassica oleracea (Pearson et. al., 1983) or ~.
campestris (Fenwick and Curtis, 1980) .Such observation were
associated with hepatic damage.
The decrease in serum total protein in the rats treated with
the poisonous plants ~. leucocephala, ~. camara. T. terrestris and
~. uniflora were associated with decreases in the serum albumin
levels. Hypoalbuminaemia is reported to be most consistently
demonstrable in acute and subacute hepatic necrosis (Kaneko, 1980).
The liver is the organ chiefly if not entirely responsible for the
formation of the plasma albumin. It would therefore be anticipated
that hepatic function impairment might result in decrease in the
plasma albumin concentration. Since the globulin levels in the
rats treated with these poisonous plants did not show significant
changes it can be inferred that the decrease in serum total protein
in these animals is a result in their reduced serum albumin levels.
Decreases in serum total protein and hypoalbuminaemia have also
been reported in poisoning by A. flavus in pigs (Cysewski et. a1.,
19(8).
In the goats which were poisoned with.1. camara, the
observation made with the serum protein levels was quite different
(Tables 30 and 31) 1. camara increased the serum total protein
level as the days of consumption of the plant was increased. The
goats which consumed the plant Q. madagascasiense also showed
elevated serum total protein levels. The increases in the total
protein observed in the goats were associated with decreased
albumin content but with increased globulin levels. It has been
observed that in so~e liver ,damages, the level of total
protein is increased as a result of increased serum globulin level
to offset the hypoalbuminaemia that occurs (Cantarow and Trumpert,
1956; Kaneko, 1980). In some cases, it is even reported that the
total protein qoncentration may not reflect either the
nature or the extent of an existing abnormality and may indeed even
fail to indicate presence. This is as a result of changes in the
albumin" globulin fractions in opposite directions to offset each
other (De Bruin, 1976).
297
The decrease r n serum protein level observed in the rats
ttreated with ~. camara in this study is not in consonance with the
observations of Sharma et. al. (1981) who reported increases in the
serum total protein in guinea pigs which were poisoned with 1.
camara though this observation is in agreement with the observation
of ~. camara treated goats (Seawright, 1904). Howeve r , the
observation of increase in serum total protein level in the goats
which were fed with ~. camara agrees with the work of Sharma, Vaid
and Dawra (1984) who observed serum protein increases in goats fed
with ~. camara.
Observations made with leucocephala toxicity are
associated with hyperacti vity and incoordination (Falvey, uno j and
Jones et al., 1984) emaciation and drooling of saliva (Jones and
Hegarty, 1984) enlarged thyroid glands and death of offspring
(Jones, 1979) loss of hair, swelling of face and severe bilateral
oedematous palpebral conjunctivitis (Akpokodje and Otesile, 19H7).
All these authors did not include biochemical observations in their
studies. ,
4.2.2 Effect of the poisonous plants on blood urea nitrogen
Increase in blood urea nitrogen in poisoning situation, has
been attributed to decrease renal excretion associated with organic
disease of the kidneys with destruction of a considerable portion
of functional renal tissue. Glomerulonephritis is a common cause
for abnormally high blood urea nitrogen which is an evidence of
renal functional impairment in acute and chronic
glomerulonephritis. Renal conditions accompanied by marked
298
oliguria or anuria, lower nephron nephrosis are associated with
increase BUN (De Bruin, 1976).
As urea is normally formed in the liver, decrease blood urea
nitrogen are observed in conditions associated with acute hepatic
insufficiency (Cantarow & Trumpert, 195o).In this study
.1.leucocephala, T. terrestris and .1. camara were observed to
produce dose dependent increase in the BUN in rats. This is an
indication that the urea nitrogen formed in rats poisoned by these
plants was not being adequately excreted as a result of renal
insufficiency.
This observation agrees with the observation that .1. camara
produces elevated levels of BUN in guinea pigs (Sharma et. al.,
1981). Plants which are poisonous to animals and cause
nephrotoxicity usually raise blood urea nitrogen. For example H..
glomeratus is nephrotoxic and produces increased levels of the
BUN (Shupe and Jane, 1969). Plants as A. retroflexus which produce
perirenal tissue oedema with renal tubular degeneration and
necrosis is r,eported to produce elevated BUN in pigs (Marshall et
al. , 1967). The elevated levels of BUN associated with 1.
leucocephala, .1. camara, E. uniflora and T. terrestris may
therefore be an indication of their nephrotoxic effect.
4.2.3 Effect of the poisonous plants on serum electrolytes
Increased levels of serum calcium and potassium was observed
in the rats treated with E. uniflora. Significant increases were
299
also observed of potassium from the serum of 1.. camara treated
rats.
Potassium is known to be the most abundant cation in the
intracellular fluid of both plants and animals. For this reason
this element is present in high concentration in almost all normal
animals. Changes in serum potassium observed with E. uniflora may
be associated with the tissue damage these plant caused thus
allowing outflow of the elements.
An increase in serum calcium concentration is found during
hyperparathyroidism and may also be induced via the administration
or intake of excessive amounts of vitamin D or vitamin D like
substances A series of so called plant induced calcinoses in
animals has been described: enteque seco (Argentine espichaments
(Brazil), naalelia disease (Hawaii, Manchester wasting disease
(Jamaica); enzootic calcinosis (Germany, Austria) Cestrum diurnum
poisoning (Florida) Krook et al., 1975, Wassermann et al., 1977;
Kasali (1977). However the cause of the hypercalcaemia observed
with rats treated 'with E. uniflora cannot be explained since there
has not been an account of vitamin D-like substance isolated from
this plant. Ipvestigations however indicate that all the diseases
associated with the plant induced calcinosis are caused by the
presence of potent, active vitamin D like substance in the plants
and that these substances are responsible for the abnormalities of
300
mineral metabolism in the animals poisoned by them (Wassermann et.
al., 1977).
4.2.4 Effect of the poisonous plants on serum enzyme activities
Variation in the concentration of certain enzymes as measured
by their biochemical acti vity, occurs primarily as a result of
elevation due to the escape of the enzymes from disrupted
parenchymal cells with necrosis or altered membrane permeability.
In this study, ~. torvum decreased the serum
activities of the enzymes alkaline phosphatase (ALP) and alanine
aminotransferase (SGPT); ~. leucocephala caused inconsistent
increase in ALP, ALT (SGPT) and aspartate aminotransferase AST
(SGPT); ~. uniflora caused increases in SGPT but inconsistent
increase in SGOT; T. terrestris in SGPT and SGOT; ~. camara
produced increases in SGOT, SGPT and ALP; whiles Q.madagascasie-
nse caused increases in SGOT and SGPT (Figures 6-1~).
Although eleyations in serum activity profile provides little
information regarding the type of lesion or functional state of an
organ (Kaneko~ 1980) it can be used to gain a quantitative estimate
of the extent of necrosis. Enzymes which increases in
concentration in the blood following hepatic necrosis are divided
into two groups; enzymes which are liver specific in that high
concentrations are present primarily in hepatic tissue such as SGPT
and enzymes which are high in concentration in other tissues in
301
addition to the liver such as SGOT, lactate dehydrogenase and serum
isocitric dehydrogenase.
Moreover, the activities of the liver specific enzymes are the most
sensitive and reliable test available for detecting mild to severe
hepatic necrosis.
4.2.4.1. Effect of plants on serum activities of aspartate
aminotransferase (SGOT or AST)
All the plants used in this study except L. leucocephala and
~. torvum produced elevated levels of SGOT which were significant
(p< 0.05) compared with untreated controls in the rats. However
the elevations produced by T. terrestris, L. camara and Q.
madagascasiense were the only ones which were dose dependent
(Tables 21, 25 and 27). Elevations in the activity of SGOT can be
associated with alterations in cell necrosis of many tissues. For
example pathology involving the skeletal or cardiac muscle and or
the hepatic parenchyma allows for the leakage of large amounts of
this enzyme into the blood. The elevations in SGOT levels produced
by the plants under study is therefore an indication of tissue
necrosis (Kaneko, 1980).
Similar ~levations in SGOT activ1ties have also been observed
in some plant poisoning such as with members of Senecio (ragworts)
(Ford and Ritchie, 1968); in calves; aflatoxicosis in swine
(Cysewski et. al., 1968). Lantana toxicity in guinea pig
3C"
(Sharma et. al. ,1981). All these observations were associated with
tissue damages such as liver damage as well as nephrotoxicity. Q.
madagascasiense, .1. camara, E. uniflora, and T. terrestris should
have produced tissue damage to have caused elevated levels of SGOT.
in the sera of the rats and/or goats which were fed with these
plants;
4.2.4.2. Effect of the poisonous plants on alkaline phosphatase
.1. leucocephala and .1. camara produced elevations in the
level of alkaline phosphatase (ALP). .1. camara produced dose
dependent changes in ALP activities (Table 25).
ALP is reported to be present in a large number of cells but
only in a few is the act ivity suff icient to be 0 f cLinical
importance. In general ALP activity is associated with the
microvilli of secretory and absorptive cells such as epithelium of
bile duct canaliculus, intestinal tract, renal tubular epithelium
{
and placenta. It is also found in liver cells and in associated
with osteoblastic activity in the bone (Pickrell et. al., 1974;
Hoffman et. ,aL; , 1977; Hoffman and Dorner, 1977; and Saini and
Saini, 1978). When obstruction of duct system'occurs at any level
in the liver, there is increase in hepatic ALP activity .in serum
(Hoffman et. a1., 1977). Liver ALP activity is also reported to
increase in hepatic fibrosis produced by poisonou9 substances.
Increases in the serum activities of ALP associated with the
plants ~{ leucocephala and ~. camara can therefore be adduced to be
303
most likely associated with microvilli of absorptive or secretary
cells in the rats. In similar studies, Sharma et. aI . (1981)
reported elevated levels of ALP in guinea pig poisoned with 1..
camara. In their clinico - pathologic features associated with A.
flavus poisoning in pig~ other authors reported the observation of
elevated levels of ALP among other serum-enzymes and concluded that
the increased levels of the enzymes is related to the hepatic
damage caused by the plant (Gumbmann and Williams 1969; Cysewski
et. al., 1968; and Sisk et al. 1968). The principal lesions occur
in the liver and they conclude it can be classified as toxic
hepatitis. Similarly increases in the serum alkaline phosphatase
were observed in the goats poisoned with Q. madagascasiense and 1..
camara.
4.2.4.3. Effect of the poisonous plants on serum alanine
aminotransferase
All the plants used except §. torvum in this study produced
increases in the serum alanine aminotransferase activity (SGPT) in
the rats. Such ...• increases were dose dependent with only
Q.madagascasiense and T. terrestris, 1. camara, and Q.madagasc-
asiense also produced elevated levels of SGPT in the goats.
SGPT is present in plasma and cells. Acute hepatic diseases
causing membrane damage or cell necrosis resul t in appreciable
increase in plasma activity of the enzyme. Since SGPT is one of
the specific assayable liver enzymes, the elevated levels of SGPT
.504
observed in the rats and goats poisoned by these plants used is an
indication of the hepatic damage produced by the plants.
4.3 Effect of the poisonous plants on hematological parameters
Extraneous poisons often reveal their toxic effects on the
blood circulating system by numerous petechiae or ecchymoses, the
result of injury to the endothelium of capillaries some hemolyze
the circulating erythrocytes; a few destroy the
hematopoietic powers of the bone marrow or the spleen whereas other
produce their effect by blocking vital enzyme systems, usually
without any identifiable lesion (Smith, Jones and Hunt 1~74,
Beutler 1969).
Poisonous plants may affect the formed elements of the blood
by their actions on the production in the bone marrow; by
increasing their rate of peripheral destruction or hemolysis, or by
influencing their distribution in various body portions. Anaemia
can result from a lack of production of the various erythropoietic
factors, inactivation of the factors (as by antibodies) or a
failure of the marrow to respond to erythropoietin. (Swenson 1~75)
if the damage to bone marrow is severe enough, a decrease may be
observed in tQ.e numbers of the major groups of formed elements
(pancytopenia) (Harris and Kellermeyer 1~70, Jain 1~H6) •
.,/
4.3.1. The effect of the poisonous plants on erythron
In this study, T. terrestris, and L. camara were the only
plants which produced a dose dependent decrease in the total RBe
305
counts, packed cell volume and haemoglobin concentration (Tables 6
and 12).
The same decreases in PCV and RBC were observed in the goats
that were fed 1. camara. Q.madagascasiense did not affect the RBC
count in goats. This means that 1. camara and T. terrestris have
adverse effect on the blood circulating system
and can cause anaemia in animals that browse on them. The
observation of anaemia in this study supports the work of seawright
(1964) but is contrary to the work of Sharma, Makkar, Dawra and
Negi (1982) who observed increases in the total RBC
counts and packed cell volume. Sharma et al (19H2) worked with
guinea pigs whereas this work was in rats and goats (Seawright
(1964), worked wiIth sheep)and it may also lend credence to
variations in terms of toxicity of the same plant at different
areas or it may reflect some species variation in susceptibility to
the effect of Lanta~a poisoning. However it is interesting to note
that Sharma, Makkar, Pal and Negi (19Hl) in an earlier work
observed that'1.camara affects erythrocyte fragility. This means
that 1.camara consists of chemical substances which could cause
hemolysis of b~ood cells. It is therefore acceptable to infer that
the anaemia produced by Lantana in this study is hemolytic though
hemorrhages could also contribute to it.Similar works (Seawright
1904, Sharma et al 1982) mention of observation of hemorrhages in
Lantana poisoning.
306
The variation in the response of the RBC in animals to
Lantana poisoning may also be related to environmental differences
in poison accumulation in plants. Environmental conditions as
earlier on reviewed plays significant role in the degree of
toxicity produced by plants.
The observation of anaemia with T. terrestris supports the
earlier work of Tonder, Basson and Va Rensburg (197~) and Amjadi,
Ahonrai, Baharce (1977) who observed anaemia in clinical poisoning
of T. terrestris in animals. Amjadi et al (1977) make
mention of their observation of serious poisoning of a herd of
cattle who fed on the plant and the experimental reproduction of
the anaemia among other adverse clinical signs in sheep.
The clinical implications of the production of anaemia by
those poisonous plants is quite obvious most especially in the
system of livestock management in this country. With the trans
humans in livestock management, the animals are constantly
exposed to different plants which they are not used to and
therefore ar~ unable to differentiate between the poisonous ones
and non poisonous. For example, in this study the locally acquired
goats which w~re used in the experiment refused to eat the freshly
harvested 1.. camara leaves mixed or unmixed with their normal
pasture. They smelled the plants and moved away from it or
extracted the normal pasture among the included Lantana. The
Lantana leaves had to be aqueous extracted and administered to
produce the poisoning. The goats however readily ate the other
freshly harvested plant. The plant is eaten by
animals elsewhere (Amjadi et aL 1977, Sharma et al 19S2). The
anaemia produced by T. terrestris and ~. camara was microcytic and
'hypochromic due to their production of decreased mean corpuscular
volume (MeV) and mean corpuscular haemoglobin concentration (Tables
6 and 12) Literature examined have not mentioned the type of
anaemia produced by T. terrestris.
Anaemia produced by poisonous plants generally can arise if
for any reason, the rate of red cell destruction in the peripheral
blood exceeds the normal rate of production. Such plant poison
could have a direct hemolytic effect as occurs with plants
containing saponin e.g. Agrostemma githago, Stellaria media, or
vicin e.g. in plants as R. communnis (Geary 1950, and Bierer and
Rhodes 1960) Others infact may produce hemolysis in cell
congenitally deficient in glucose - 6 - phosphate dehydrogenase
e.g. Vicia fava (Bowman and Walker 1961) whereas hemorrhagic
anaemia is reRorted in plants like £. aquilinum (Singh, Joshi, and
Kumar 1987), Rao, Joshi and Kumar 19S5).
E. un~flora and §. torvum in this study all increased the
level of circulating erythrocytes (i.e. polycythemia). The
mechanism by which these plants have produced their polycythemia is
difficult to predict since non of them produced the typical
308
symptoms of diarrhea and vomiting observed in plants which produced
such symptoms. For example among the clinical symptoms associated
with Solanum tuberosum was a gastric type, which was characterized
by salivation, vomiting, diarrhea and other sign of
gastrointestinal irritation (Clarke and Clarke 1~75). These are
all symptoms which could produce fluid loss and this decrease blood
volume. Accordirig to Smith et al (1~74) in great majority of cases,
an excessive number of erythrocytes noted in the blood cell count
is purely relative; the total number of cells is not increased but
the total volume of plasma is decreased. This is ordinarily the
result of dehydration which is also produced by the fact that the
animal patient is too weak to get to its source of water.
Dullness, listlessness were some of the symptoms observed in he
rats that were fed with E. uniflora which could imply that these
animals were so weak from intoxication, to be unable to get to
their drinking water which could produce dehydration and
hemoconcentration. It is however, significant to note the
hypoalbuminaemia produced by E. uniflora. Since albumin is
reported to be iillportantin the maintenance of colloid osmotic
pressure of the blood plasma (Kaneko l~HO) the clinical implication
of reduced serum albumin level is obvious. The regulation of the
distribution of water between the intravascular and interstitial
tissue compartments are affected. A marked shift of intravascular
water into the interstitial tissues in the E. uniflora poisoned
309
animals could therefore reduce the plasma volume and therefore the
polycythemia that was noted.
L. leucocephala and ~. madagascasiense did not produce any
significant changes in the erythron (Table H J 14 J ). The
inconsistent changes in the total RBC of the rat produced by ~.
madagascasiense may be due to abnormal responses of some of the
individual rats within group since such inconsistencies were not
observed in the goats which were feeding on the fresh leaves of ~.
madagascasiense.
All the plants did not affect the blood coagulation time in
the rats or in the goats. Plant poisoning generally influence
blood coagulation by causing severe liver damage e.g. H. europauem
or aflatoxicosis (Doerr et a1 1976, Bull et al 1961) Aflatoxicosis
causes loss of two of the active sites of tissue thromboplastin and
reduces the level of prothrombin and fibrinogen which are all
required for the coagulation process (Doerr et al 1976). Plants may
produce hypocalcaemia which removes the calcium required for the
coagulation prQcess e.g. H. glomeratus (Shupe and James 1969 or A.
retrof1exus (Buck et a1 1966, Marshall et al 1967). Coagulopathies
are also obser~ed with plants which cause platelet disorders e.g.
£. aguilinum (Rao et a1 19HH) or vitamin K deficiency and
antagonism as observed with. M. alba poisoning (Prier and Derse
1962). The non observation of coagulation defects with the plants
in this study may imply that they do not produce the effects
310
enumerated above. This observation may not be conclusive since
chronic feeding of the plants were not carried out. Chronic
feeding of the poisonous plants could produce cirrhosis which will
affect blood coagulation by virtue of lowered coagulation factor
synthesis in the liver.
4.3.2 Effects of the poison plants on leucotron in rats and goats
Toxic plants are not reported to produce a direct effect on the
white blood cells, neutrophils, lymphocytes eosinophils, monocytes,
or basophils. However plant substances which produce toxic
aplastic anaemia are known to also produce hypoplasia of the bone
marrow and the depression of the formation of granu Loc-: cytic
leukocytes leading to a more or less severe agronulocytosis (Smith
et 1974). For example E. aquilinum is reported to cause
agranulocytosis because of its toxic action on the bone marrow (Rao
et al 1988, Singh 1987) The plant is reported to have an aplastic
anaemia factor.
In this study, ~. torvum, E. uniflora, Q.madagascasiense, ~.
leucocephal~did not affect the level of WBC in the rats and goats.
~. camara produced decreases in total WBC with some of the doses of
the extract. administered to the rats though such changes were
inconsistent (Table 12; For example the lower doses of ~. camara
(200 and 400 mg/kg) produced changes which were significant when
compared with untreated controls whereas such dose dependent
decrease was not observable with the highest dose (600mg/kg) of ~.
311
camara. Moreover there were no ~ecreases observed within the goats
which fed on the leaves of ~. camara. Seawright (1965) observed no
changes in the level of leukocytes in ruminant and guinea pig
poisoned with ~. camara although Sharma et al (19H2) reported
increases in the total WBC counts.
In the contrary T. terrestris produced dose dependent
changes in the total WBC and neutrophil counts in this study.
Other authors (Brown 1968, Tonder et al 1972) did not mention
the effect of T. terrestris on leukocytes though the plant is
reported to produce photosensitization, anaemia, liver and
kidney lesions. The mechanism of reduction in leucocyte
counts in this study therefore is difficult to predict. The
need for experimentation in other animals may therefore be a
pertinent step in elucidating the leucocyte level changes
associated with feeding of T. terrestris in rats.
4 .4. Clinical signs. gross and histopathologic effects of
poisonous plants
The effec~s of poisons which are most often looked for,
in plant toxicology in animals are their gross and histologic
actions on the tissues. The lesions of poisoning are rarely
characteristics; nevertheless, the finding at autopsy can
provide definite clues to the nature of the poison. (Smith,
et al 1974).
312
The poisonous plants cause an array of different symptoms
since often, the effect of the poison is no more than one body
system. Clinical evidence is reported to be only limited value.
There are only nine systems of organs in the body that are capable
of being affected, so the permutations and combinations of clinical
signs are extremely limited. In addition there is extraordinary
variability shown by different individuals in the symptoms caused
by the same poison and not every symptom is known to appear on each
occasion (Zook and Gilmore 1967).
The absence of lesions may be as important as their presence
as it serves to exclude various toxic agents. The skin and visible
mucous membranes may have a characteristic discoloration. Jaundice
is a frequent sign of hepatic damage which has been noted in
various plant poisoning such as Aspergilus flavus, Heliotropium
europaeum (Smith et al 1974), A cherry red or pink color is seen in
cyanogenetic plant poisoning (Van der Watt 1944).
Methaemoglobinaemia due to nitrates and nitrites impart a brown
coloration. ,
4.4.1. Gross and histologic lesions associated with L.leucocephala
In these st~dies no clinical symptoms were observed in the rats
fed extracts of 1. leucocephala. This contrasts with the findings
of other authors who reported of hyperactivity in cattle (Falvey
1976 emaciation and drooling of saliva (Jones and Hegarty 1984)
Severe bilateral oedematous palpebral conjunctivitis in cattle
(Akpokodje and Otesile 1987). Most of the other authors consulted
gave different symptoms connected with L. leucocephala
intoxication, These variations have been associated with respects
to the rate of consumption and environmental factors related to the
plant. (Jones and Hegarty 1984).
Apart from the cl inical signs that were reported, several
authors did not describe post mortem or histologic lesions. The
congestion of the kidney and the paleness of the liver of rats
which were fed extracts of L. leucocephala was indicative of
pathologic changes in these organs. Akpokodje and Otesile (1987)
reported of swelling of the face with oedema of cattle that
consumed L. leucocephala. The perivascular lymphocytic cuffs that
were observed around the central veins and portal vessels in some
of the rat livers may be associated with a secondary infection
probably resulting from the effect of the plant extract. However
the degenerative and necrotic changes characterized by cellular
swelling and clumping of the cardiac tissue could be associated
with the effect of L. leucocephala. This compares with the
swelling of muscle fibers observed with the poisoning of Cassia
occidental is (,Henson et al 1965) In contrast however, the cardio
myodegeneration observed with Q. occidentalis intoxication also
involved the skeletal muscle (O'Hara et al 1969) .
. .
4.4.2 Gross and histologic changes associated with S. torvum
The Solanaceae, the family of plants to which.~. torvum
) 1 ,
belongs consist of several genera associated with poisoning
due to the nature of the active principles they contain. All
parts of these plants especially ~. dulcamara and ~. nigrum are
reported dangerous (Clarke and Clarke 1975, Woodward 1980) No
ciinical signs were produced in the rats that were fed extracts of
~. torvum though symptoms were observed in experimental animals fed
other species of Solanum. Pienaar et al (1976) in their
experimental studies with Solanum in cattle reported of loss of
balance with transient epileptiform seizures, with these symptoms
being precipitated by exerc ise, handl Lng or fright. Contrary to
these observations, other authors reported of depression,
apathy, narcosis and paralysis in animals poisoned with Solanum
species (Andrade 1960, Buck, Dollahite and Allen 1960).
The vasculitis with moderate perivascular lymphocytic
and macrophage infiltration was not a common occurrence in all
the experimental rats and may thus not be associated with the
toxic effect of ~. torvum.
The non observation of central nervous system effects in
these studies which is associated with the Solanines (Pienaar
et al 1976, Andrade,1960) might be related to the level of
Solanine in the plant. For example the alkaloid content and
hence the toxicity of ~. nigrum is reported to varies enormously
with soil, climate and season. Clarke and Clarke 91975) reports
that in some areas ~. nigrum, is reported to be harmless and deadly
315
in others. Therefore the absence of a serious organic damage
produced by the extract of .s.. torvum may be due to the low
concentration of Solanine in the variety around Nigeria or that the
content of Solanine was low at the season at which the plants were
collected for extraction.
4.4.3 Lesions associated with E. uniflora
E. uniflora represents one of the poisonous plants for which
there is less literature as regard its toxicity. Its toxic effect
is corroborated by local farmers. However the gross and histologic
lesions of congestion of the kidney and moderate fatty change is
indicative of its less severe toxic effect. The restriction of
lesions to the kidney and liver may be due to the rOle there two
organs play in the excretion and metabolism of the poisons from
these plants. The plant is reported to have a high tannin level
(Irvine 1961) The fact that it produced lesions on Subacute
consumption means the plant could adversely affect livestock
production when they are continuously exposed to it.
4.4.4. Gross and histologic lesions due to T. Terrestris
Tribulus terrestris represents an important fodder plant in some
parts of the worl~ (Clarke and Clarke 1975). The depression and
disinclination of the animals treated with T. terrestris to feed
was an indication of the effect of the plant.This was shown by the
post mortem lesions observed on the liver and kidney of
experimental rats terrestris is reported to cause
I~
photosensitization in sheep (Brown 1968, Tonder et al 197~).
However photosensitization was not observed in the rats used in
this studies. The absence of such a symptom in these rats may not
be unconnected with the fact that the rats were not exposed to
direct sunlight. Furthermore the degree of hepatotoxicity might
have not been so severe as to allow the accumulation of the
photodynamic substance phyloerythrin which is what is responsible
for the development of photosensitivity in animals poisoned by such
plants.
The hemorrhages observed on the liver and kidney indicates
that T. terrestris causes damages to the circulatory system. The
absence of histologic lesions in the liver from the rats treated
with T. terrestris may be associated with the level of the active
agent in the plant. Since it is reported that wilting seriously
affects the degree of poisoning from this plant (Tonder et aI
1972) . The leaves used in this study were dried leaves. The
lesions observed in the kidney may be due to the concentrating
ability of the kidney so that the relatively reduced level of
the active substance could be concentrated in the kidney to
produce the l~sions.
4.4.5. Lesions associated with Lantana camara
The ecchymotic hemorrhages observed in the heart kidney and
liver indicates the damage due to L. camara to the blood
circulating ~ystem (Smith et al 1974) However hemorrhages were not
317
observed in the tissues of the goats which fed on the plant. This
observation may be due to the species or to genetic variation in
response to ~. camara toxicity that has been reported (Seawright
1965). The severity of the lesions are also reported to depend on
such factors as starvation and dehydration (Seawright and Allen
1972).
The fatty degeneration of the liver observed in both the
rats and goats supports the observation of other authors (Ahuja and
Skewes 1971), Brown 1968, Sharma et al 19B7). However the
photosensitization which was reported in some animals poisoned with
1. camara (Aluja and Skewes 1971 and Seawright and Allen 1972) was
not deserved in the rats and goats used in these studies. Though
the rats were not exposed to sunlight the goats grazed normally
outside in sunshine after they were exposed to the plants. The non
observation of photosensitivity reactions again may be associated
with such factors as nutritional status and the degree of
dehydration of the animals (Seawright and Allen 1972). Furthermore
it may be due to the degree of hepatic damage and/or the level of
the circulating photodynamic agent in the experimental rats and
goats.
4.5 GENERAL DISCUSSIONS AND CONCLUSIONS
4.5.1 The problem of poisonous plants
The word problems according to Kingsbury (1979) is anthropo-
centric and mainly implies a human point of view. From that vintage
31<3
point, the problem of poisonous plants is not their direct
toxicity to man and animal alone but also the inconvenie- nce and
economic loss associated with the poisoning of domestic animals and
the cost of preventing or reducing such happenings. It is worthy
of note that plant poisons can either be accumulated in the animal
or in certain organs or they are metabolized and excreted with milk
(Liener 1969) By this food chain, toxins or metabolites thereof may
become harmful to man (Habermehl 1987),
4.5.2 The toxic effects of Solanum torvum
Most of the cultivated members of the family Solanaceae
to which~. torvum belongs are reported to be poisonous (Clarke and
Clarke 1975) The leaves of ~. torvum analyzed contained levels of
phytin which was high enough to produce inhibition of essential
minerals such as zinc and iron required for the nutrition of
animals. The level of the nitrate and nitrite could constitute a
source of methemoglo~in formation if animals are exposed to large
quantities of the plant alone. However the ethanolic extraction of
the leaves w,hich was performed to concentrate the toxic
constituents of the plant did not cause expected symptom probably
because the lev~l of the toxic factor was low in the leaves or the
extraction procedure did not achieve the expected outcome. Except
for the histologic lesions in the lungs which could not be
associated with the effect of the plants, no gross or histologic
lesions were observed in the experimental animals dosed with the
319
leaf extract. The absence of gross or histologic lesions is
reflected in the serum biochemical "changes produced in the rats.
The plant only produced dose dependent increase in the serum
activity of alkaline phosphatase. ALP is used in characterizing
bone and hepatic disorders (Kaneko 19HO). Since it is a non liver
specific enzyme it becomes difficult therefore to directly
associate its increase with hepatic damage moreso when the more
liver specific enzymes like alanine aminotransferase did not show
increases in activity.
The Solanaceae family include members with active alkaloids
related to atropine solanine, nicotine which are mostly substances
which affect the central nervous system. Pienaar et al 1976,
Gilman et al 1980) A subgroup however are reported to contain a
compound related to 1, 25- dihydroxycholecalciferol which is
responsible for the calcinosis which are reported in many animals
which were maintain~d on for example ~. malacoxylon and Cestrum
diurnum (Samson et al 1971, Krook et al 1975, Peterlik et al 1976)
It is preposterous to conclude that the species of Q. torvum
used in this study is not poisonous because the glycoalkaloids
which are repoTted to be responsible for the toxic nature of this
group is known to be concentrated especially in the berries
(Woodward 1985) The observation of less toxic effects in this study
might be due to the low level of the glycoalkaloids in the leaves
or environmentally related factors. (Clarke and Clarke 1975).
320
4.5.3. The toxic effects of Lantana camara.
The toxic nature of ~ camara is widely reported and the
spread of the plant into pastures, forest areas and gardens are
also reported (Sharma et al 1971, Sharma et a! 1981, Achhireddly
and Singh 1984). However since the degree of toxicity is
environmentally related and there are no experimental evidence of
toxicity in Nigeria regarding this plant it was auspicious to find
out the toxic nature of the plant in Nigeria.
The ~ camara used in this study was found to contain
relatively high levels of nitrate and nitrite. However since it is
reported that it is difficult to make a categorical statement as to
the toxic dose of nitrate as it depends on the rate at which the
substance is consumed, it is hasty to say that the effects observed
were due to these substances. The production of dose dependent
decrease in the serum total protein in both the rats and goats is
a reflection of the toxic damage to the liver at the cellular level
by the plant. This observation is further augmented with the dose
dependent increases in the activities of the serum enzymes that
were measured mainly alanine aminotrasfe- rase (ALT), aspartate
aminotransferase (AST) and alkaline phosphatase (ALP). ALT among
other enzymes is reported to be one of the excell~nt markers of
hepatocellular damage (Kaneko 1980). With the additional increase
in the serum activities of ALP and AST which though are not liver
321
specific enzyme, it is appropriate to confirm the production of
hepatocellular damage by L. camara.
This observation is supported by the gross and histologic
lesions that were observed in the liver of the rats and goats that
were used in this study.
The level of toxic inorganic substance assayed in this plant
were not very high so that they cannot be associated with the
damages observed in the tissues of the experimental animals. A
substance known as lantadene A, has been connected with the damage
which is usually observed in the animals which consumed ~ camara
(Sharma et al 1980 Alfonso et al 1982, Sharma et al 1988). Using
the guinea pig, Sharma et al (1982) described Lantana toxicity as
an acute occurrence resulting in the death of- the animal. It was
mentioned that the plant produces intrahepatic cholestasis
associated with dispersal and fragmentation of endoplasmic
reticulum. Furthermore, Sharma et al (1982) also observed that
Lantana toxicity caused obstructive jaundice, photo sensitization
and a rise in ~erum bilirubin. In this study no observation was
made of the hypertrophy of the hepatocytes nor intrahepatic
cholestasis. T4e level of the total bilirubin was seen to rise in
the goats used in the experiment though jaundice was not noticed.
Photosensitization in the goats was also not noticed though the
goats were allowed free access to the sunshine after been fed their
ration of the leaves of ~ camara. However since the level of
322
conjugated bilirubin increased in the treated goats it could not be
ruled out that the animals might develop jaundice if the feeding of
the Lantana leaves had continued beyond the twenty one days in the
goats.
The Lesions produced by h camara in other organs of the
animal were non consistent. This is because apart from the pale
kidneys that were observed in the goats used in this study and have
also been mentioned with other animals (Sharma et al 19HO. Alfonso
et al 1982. Achhireddy et al 19H5. Sharma et al 19HH) lesions in
the gastrointestinal tract. spleen. lungs. heart were not a
constant finding in the animals poisoned by h camara.
The variations observed in Lantana toxicity in goats and rats
as compared with the observations in other countries may be
connected with environmental or climatic factors reported to
influence the toxicity of plants from one area to another.
Similarly such variations could be related to the season at which
animals were exposed to Lantana camara.
4.5.4. Toxic ekfects of Leucaena leucocephala
A considerable variation has been mentioned about the toxicity
of h leucocephala in different parts of the world
(Falvey 1976) Mullenax 1963). In this study. the level of phytin
and cyanogenetic glycoside analysed from the leaves of h
leucocephala used. were high enough to be mentioned as one of the
causes of poisoning by this plant. Since the hydrocyanic acid
323
present in form of cyanogenetic glycoside determines the toxic
nature of this compound (Vander Watt 1974) it means the nature of
the HCN from the ·plant will have to be verified inorder to
ascertain its role in influencing ~ leucocephala toxicity.
~ leucocephala in this study, produced decreased level of the
blood total protein which was reflected as a decrease in blood
albumin. This means the plant is capable of damage to the liver.
This observation is further buttressed with the increased level of
the blood area nitrogen. However the effect on the liver may be
mild since the serum enzymes which were assayed to determine tissue
damage in this work, did not show increased activity. It was only
the 200 mg/kg dose of the extract which produced an increase in the
serum activity of alkaline phosphatase. Since ALP is non liver
specific enzyme, the leakage of this enzyme could have taken place
from any tissue in the body of the rats (Kaneko 19HO).
The gross and histologic lesions that were observed further
confirms the fact that ~ leucocephala produces mild toxicity. The
swollen liver ~th the perivascular lymphocytic cuffs around the
central veins and portal vessel which is suspected to be associated
with a secondary, infection could account for the decreased blood
total protein and albumin which were earlier mentioned. Contrary
to these observations Jones and Hegar-t.hy (19H4) observed
emacipation drooling of Saliva and vomiting in animals that were
fed 1.leucocephala. Jones (1977) had earlier mentioned the
324
observation of enlarged thyroid and death of offspring in cattle
that were exposed to L. leucocephala~
A relatively common observation in animals that were
poisoned with L. leucocephala was the loss of hair which did not
occur allover the body. Akpokodje and Otesile (1987) noticed loss
of hair from only the tail switch and swollen face. Hill (1971)
reported of hair loss in a young women who consumed the seeds of
the plant. The experimental rats used in this study did not show
hair loss from any part of the body. The important chemical
component of L. leucocephala which has been incriminated as the
caused of hair loss is mimosine (Jones 1979, Jones and Hegarthy
1984). Since L. leucocephala has been chosen as an important plant
in alley forming as is presently practiced at the International
Institute for Tropical Agriculture, Ibadan, (Akpokodje and Otesile
1987) it is very important that the true toxic nature of L.
leucocephala in this country be thoroughly ascertained. This is
necessary because variations in the effect of the plant have been
noticed from different areas (Jones 1984). Furthermore the level
of mimosine needs, to be evaluated since its excretion in cattle
fed with the pl~nt could be consumed to produce the typical hair
less observed with it (Hill 1971).
The observation of an increase in total white blood cell
count which was reflected as an increase in lymphocyte count
could either be the result of secondary infection or that the
plant contain lectins which act as lymphocyte mitogens (McPherson
1979). If the plant acts as a lymphocyte mitogen, then it may be
important in maintaining the immune status of the animals fed on
the plant most especially during challenge by pathogens.
4.5.5. Toxic effects of Tribulus terrestris
The toxic nature of T. terrestris in animals has long been
documented in other parts of the world most especially in South
Africa where it is given various types of names, though it is
doubtful whether its the plant which solely produces the associated
symptoms or that other factors are equally involved (Clarke and
Clarke 1975, Amjadi et al 1977). Since it is an introduced plant
and it is very widespread in Nigeria, the toxic nature had to be
evaluated in our local livestock.
T. terrestris harvested for this study was observed to
contain the highest levels of nitrate, nitrite and phytin. This
means that these in organic constituents could contribute to the
symptoms of poisoning in animals most especially during the dry
season when the water content of the plant is low and the plant
tissues are able to concentrate more of the secondary chemical
constituents coupled with poor feeding or fasting animals (Winter
and Hokanson 1964).
~. terrestris was the only plant among the other poisonous
plants studied which produced clinical symptoms in the experimen-
tal animals. The hemorrhages which were produced in the liver and
326
kidneys of these animals, is an indication of the toxic injury to
the capillary endothelium or a disorder in the blood clothing
mechanism of the animals (Smith et al 1974, Jain 1980). However
since the plant did not produce significant changes in the blood
coagulation time of the animals it is doubtful whether the cause of
the hemorrhages was due to effect on the coagulation mechanism. The
implication of the hemorrhages produced by T. terrestris is that
prolonged exposure to this plant will result in hemorrhagic
anaemia. The reduced total red cell count encountered in the rats
dosed with the extract of T.terrestris could therefore have a link
with the hemorrhages produced.
The plant also showed liver damage in the rats by producing a
decreased blood total protein as well as increases in the
activities of the liver enzymes that were measured mainly alanine
aminotransferase and aspartate aminotransferase. The damage to the
kidney could be responsible for the increase in blood area
nitrogen. The photophobia, jaundice, swelling and acute dermatitis
that have been ~ssociated with poisoning by this plant (Amjadi et
al 1977) were not observed in this study. Since photophobia,
jaundice and dermatitis may be the aftermath of liver damage by the
plant it could be that these observations were not made in this
study because the degree of liver damage was minimal due to the
poor level of the poison in the plant or that the length of
exposure to the plant was short.
321
4.5.6~ Toxic effect of Dichapetalum madagascasiense
Various authors have reported about the toxic effects of
some of the species of members of the family challetiaceae to which
~. madagascasiense belongs (Irvine 1961, Watt, Breyer Brandwijk
1962, Van Dijk et al 1972, Vickery and Vickery, 1971, Nwude et al
1977. However ~. madagascasiense in this study has produced very
minimal toxic effects.
The level of the inorganic substances analysed in the leaves
of ~. madagascasiense were relatively low and can therefore not be
associated with the poisoning by the plant. This means species of
Dichapetalum contain other factors which could be responsible for
the effects. The substance which was mentioned as possible cause
.of toxicity by other members of the family was fluoroacet- ate (Van
Dijk et al 1972, Nwude et al 1977). However a prolonged exposure
of animals to the plant could increase the amount of phytin which
gets into the gastrointestinal tract inorder for some antinu-
tritional effects to be produced.
~. madagasca•siense did not affect the blood cellular compon-
ents of the rats and goats. This is because the plant did not
produce any cha~ges in the level of the total RBC, WBC and other
hematological parameters. This means the plant in"this study did
not have any direct effect on the blood cellular elements or the
hemopoietic tissue.
Though no gross lesions were observed in the rats and
328
goats used, the histologic lesion seen in the rat tissues but not
observed in the goats is an indication of the mild toxic nature of
Q. madagascasiense. The plant was however observed, to increase the
total blood protein. It also increased the serum activities of the
enzymes alanine aminotransferase and aspartate aminotransferase in
rats and goats. These are indications of the toxic effect of the
plant at the subcellular level (Kaneko 1980). It is therefore
possible that the plant can produce gross and histologic lesions on
chronic exposure in goats. Alternatively it could be that Q.
madagascasiense does not have enough fluoro fatty acids which is
alluded to undergo oxidation into the fluoroacetic acid which is
responsible for the toxic effects of these plants (Vickery and
Vickery 1971). Variations in the degree of toxicity of
fluoroacetate has been reported (Watt and Breyer Brandwijk 1962).
The palatable nature of the leaves of members of the plants in
this family has been reported (Van Dijk et al 1972) This
observation was noted by the voracious nature by which the goats
which were gLven the leaves of Q.madagascasiense consumed them
whenever they were fed.
4.5.7. Toxic effect of Eugenia uniflora
g. uniflora is quite a widespread plant in Southern Nigeria
and it is quite a common occurrence to see groups of animals
browsing on the plant. This means that the plant is potentially
dangerous and the type of symptoms caused by it have to be
329
determined. In this study, the plant did not produce gross lesions
in the tissues of the rats dosed with the extract of the plant
though histology showed moderate fatty change in' the liver.
Except for the level of phytin which was relatively high, and could
be antinutri tional, the level of nitrate, nitrate cyanide or
oxalate analysed from E. uniflora were very low. Toxicity produced
by this plant could therefore be associated with other secondary
chemical factors in the plant.
On the hematology of the rats, E. uniflora produced
increases in the packed cell volume haemoglobin concentration
and total RBC levels which could be an evidence of the increased
requirement for oxygen by the animals due to the effect of the
plant on cellular respiration (Swenson 1975).
The increases in the serum activities of AST and ALT can be
associated with the fatty change observed in the liver of he rats
fed on the extracts o~ E. uniflora.
CLINICAL SYMPTOMS AND SUGGESTED TREATMENTS OF POISONING IN ANIMALS
Clinical sy,mptoms of poisoning by poisonous plants, are hardly
observed in the affected animals (Clarke and Clarke, 1975). This
is because the a~imals do not consume enough quantity, or that by
the time they are observed they are dead or comatose.
Acute toxicity studies in rats using extracts of Q.
madagascasiense, ~. torvum and E. uniflora produced irritability,
330
excitement and tremor until the animal went recumbent and then died
2 - 5 days after administration of the extract.
Tribulus terrestris, 1.. camara and 1.. leuco'cephala produced
anarexia, somnolence with evidence of pain with the animals dying
between 6 - 11 days.
Treatment of plant poisoning is done symptomatically.
Prevention of further absorption can be done using stomach gavage
and if the active component of the plant is known a specific
antidote can be used.
The excitement and tremor produced by Q. madagascasiense, ~.
torvum, and ~. uniflora can be treated with pentobarbitone (10 to
30mg/kg) given intravenously. The somnolence produced by 1..
camara, L. leucocephala and T. terrestris can be treated with
amphetamine or methylamphetamine (0.~5mg/kg, subcutaneouslY).
331
REFERENCES
Achhireddy, N.R., and Singh, M. (1984). Allelopathic
effects of Lantana (Lantana camara) oh milk
weed vine (Morrenia odorata). Weed Science
32, 757-761.
Adams, L. G. , Dollahite, J.W., Romane, W.M., Bullard, T.L.
and Bridges, C.H. (1969). Cystitis and ataxia
associated with Sorghum ingestion by horses.
J. Am. Vet. Med. Assoc. 155, 518-520.
Akpokodje, J.U. and Otesile, E.B. (1987). Leucaena
leucocephala toxicity in Ndama cattle. Bull.
Anim. Hlth. Prod. Afr. 35, 77-78.
Aletor, V.A. (1983). Implications of Lima beans
antinutritional factors for digestion, nutrient
utilization, and physiopathology of the rat.
Ph.D. Th~sis, University of Ibadan.
Allen, J.R. Carstens, L.A. and Knezevic, A.L. (1965).
Crotalaria spectabilis intoxication in Rhesus
monkeys. Am. J. Vet. Res. 26, 753-757.
Alfonso, H.A. Figueredo, J.M. and Merino, H. (1982).
Photodynamic dermatitis caused by Lantana camara
in Cuba - preliminary study. Revta Salud.
Anim. 4. 141-144.
Aluja, A.S. de; Skewes, H.R. (1971). Further investigation
332
regarding toxicity of members of the genus
Lantana in Mexico. Proceedings 19th World Vet.
Congress, Mexico City.
Amjadi, A.R., Ahonrai, P., Baharce, M. (1977). First
report of geeldikkop in sheep in Iran Archwesde
l'Institut Raziza 71 - 78 in The Vet. Bull. 6183
AnderI son, T.S. (1948). Caster poisoning in Ayrshire cattle.
Vet. Res. 60 (B), 28-31.
Andrade, S.O. , Peregrino, C.J.B. and Aguiar, A.A. (1971a).
Studies on Bracchiaria sp (tanner grass) Toxic
effect in cattle Arquivos de Institulo Biologico,
S. Paulo 38(3), 135-150.
Andrade,S.O., Retz, L., Velloso, C.A.C. (1971b). Studies
on Bracchiaria sp (tanner grass). II. Measurement
of nitrate in bovine serum. Arguivos do
Instituto.Biologica, S. Paulo 38(3), 151 - 161.
Andrews, E.J. (1971). Oxalate neuropathy in a horse. J.
Arne. Vet. Med. Assoc. 159, 49-51.
Archer, M.C., Clark, S.D., Thilly, J.E. and Tamnenbaum, S.L.
(197~). Environmental nitroso-compounds-reaction
of nitrite with creatine and creatinine.
Science 174, 1341-1343.
Avery, R.J. Nulo, L., Kramer, T., Johnson, A. and Darcel, C.,
Le Q (1961). Two cases of poisoning in livestock
333
presenting difficulties in diagnosis. Can Vet. J.
2(8), 301-304.
Azariah, J., Azariah, H., Mallikesan, S., Sumathi, M.V. and
Sundararaj (1987). Toxicology of the plant
Calotropis giganteea. In Progress in venom and
toxin research. Proceedings of the first Asia-
Pacific Congress on animal, plant and microbial
toxins, Pp. 633-640.
Barry, T.N. and Forss, D.A. (1983). The condensed tannin
content of vegetative Lotus pedunculatus its
regulation by fertilizer application and effect
upon protein solubility. J. Sci. Food Agric.
34, 1047-1056.
Baker, C.T.L. (1952). The determination of oxalates in
fresh plant materials. Analyst 77, 340-346.
Battifora, H.A. and Markowitz, A.S. (1969). Nephrotoxic
nephritis in monkeys. Am. J. Path. 55, 267-281.
,
Beutler, E. 9(1969). Drug-induced hemolytic anaemia.
Pharmacol. Rev. 21, 73-103.
Bierer, B.W. and, Rhodes, W.H. (1960). Poultry ration con-
. .
taminants, J. Am. Vet. Med. Assoc. 137, 352-353.
Binns, W. James, L.F. and Shupe, J.L. (1964). Toxicosis
of Veratrum californicum in Ewes and its rela-
tionship to congenital deformity in lambs. Ann.
334
New York Acad. Sci.lll: 571-576.
Berman, E. (1980). Toxic metals and their analysis. Heyden
International Topics in Science, Heyden & Son Ltd.,
London, Pp 249-287.
Binns, W., Lynn, F.J. James, L. Shupe, J.L. and George,
M.S. (1963). A congenital cyclopian type mal-
formation in Lambs induced by material ingestion
of a range plant. Veratrum californicum. Am.
J. Vet. Res. 24, 11641175.
Binns, W., Shupe, J.L. Keeler, R.F. and James, l.F. (1965)
Chronologic evaluation of teratogenicity in sheep
fed Veratrum californicum. J. Am Vet. Med. Assoc.
145, 839-842.
Bierbaum, K. (1966). Beitragzur Giftigkeit des Samen yon
Ricinus communis Dissertation, Gotha.
Black, D.J.G. and Bar~on, N.S. (1943). Observations on the
feeding of a Cacao waste product to poultry.
Vet. Rec. 55, 166-167.
Blaney, B.J. Mannion, P.F. , Tudor, G,.D., McKenzie, R.A.
(1981). Examination of the Diplodia-Maydis
infected maize for toxicity of chicken and cattle
Letter Aust. Vet. J. 57(4), 196-197.
Blood, D.C. and Henderson, J.A. (1976). Veterinary Medicine
4th Edition Bailliere Tindall, London.
335
Bodansky, O. (1951). Methemoglobinemia and methemoglobin
producing compounds. Pharmacol. Rev. 3, 144-196.
Bowman, J.E. and Walker, D.G. (1961). Action of Vicia Fava
on erythrocytes! Possible relationship to Favism.
Nature 189, 555-556.
Boyd, J.W. (1962). The comparative activity of some enzymes
in sheep, cattle and rats normal serum and tissue
levels and changes during experimental liver
necrosis. Res. Vet. Sci. 3, 256-268.
Boughton, B. and Hardy, W.T. (1963). Oak poisoning in
range cattle and sheep. J. Am. Vet. Med. Assoc.
89. 157-162.
Braun, R.P. Rico, A.G. Bernard, P., Thouvenot, J.P. and
Bonnefis, M.J. (1978). Tissue and blood distr-
ibution of gamma-glutamyltransferase in lamb and
ewe. Res. Vet. Sci. 25(1) 37-40
Brocg-Roussen, O. and Fabre, R. (1977). Les. toxines vege-
table~. Herrman and Cie, Paris.
Buck, W.B., Dollahite, J.W. and Allen, T.J. (1960).
Sil~er-leafed nightshade poisoning. J. Am. Vet.
Med. Assoc. 137, 348-350.
Brown, J.M.M. (1968). Biochemical studies on Geeldikkop
and enzootic icterus. Onderstepoort. J. Vet. Res.
35(2), 319-576.
336
Buck, W.B., Preston, K.S., Abel, H., Harshall, V.L. (1966).
Perirenal edema in swine, A disease caused by common
weeds. J. Am. Vet. Hed. Assoc. 14H, 15~5-1531.
Bull, L.B., Roggers, E.S. keast, J.C. Les toxines vegetables.
Herrman and Cie., Paris.
Bull, L.B. (1955). The biological evidence of liver damage
from pyrrolizidine alkaloids, megalocytosis of the
liver cells and inclusion globules. Aust. Vet. J.
31, 33-36.
Cantarow, A. and Trumpert, H. (1956). Clinical Biochemistry.
5th Edition. W.B. Saunders Compo Philadelphia.
Canfield, P.J. and Dickens, R.K. (19H~). Oxalate poisoning
in Koala, Phascoloretos cinereus (Goldfuss). Aust.
Vet. J. 59(4), 121-1~~.
Carstens, L.A. and Allen, J.R. (1970). Arterial degeneration
and glom~rular hyalinization in the kidney of
monocrotaline intoxicated rats. Am. J. Path. 60,
75-91.
Cardinet, G.H. Litterel, J. F and Freedland, R.A. 91967).
Comparative investigations of serum, creatinine
phosphokinase and glutamic oxaloacetic transaminase
activities in equine paralytic myoglobinuria,. Res.
Vet. Sci. 8, 219-~~3.
Casarrett, L.J. and Doull, J. (1975). Toxicology, the basic
337
science of poison. MacMillan Publishing Co. ,Inc. New
York.
Chen, KK. Rose, L.L. and Clowers, G.H.A. (1934). Comparative
values of several antidote in cyanide poisoning. Am.J.
M. Science 1881, 767-769.
Chang, T.S. (1958). Reproductive disturbances of Romney ewe
lambs grazed on red clover (Trifolium pratense) pasture.
Nature (London). 182, 1178.
Clarke, E.G.C. and Clarke, M.L. (1975). Veterinary
Toxicology. Bailliere Tindall, New York. 3rd Edition.
Clarke, E.G.C.and Cotchin, E. (1956). A note on the toxicity
of Acorn. Br. Vet. J. 112, 135-136.
Clarke, M.L., Clarke, E.G.C. and King, T. (1971). Fatal
laburnum poisoning in a dog. Vet. Rec. 88 (7), 199-201.
Clarke, L. and Whitwell, G.B.(1968). Ciguatera poisoning in
cats in Brisbane. Aust. Vet. J. 49,81-83.
Clegg, D.J. 91971). Teratology, Ann. Rev. Pharmac. 1, 409.
Clement, F.W. (1960). Naturally occurring goitrogen.
Brit. Med. Bull. 16, 133-135.
Cohen, G. and H~chstein, P. (1963). Glutathione peroxidase,
the primary agent for the elimination of hydrogen peroxides
in erythrocytes. Biochem. 2, 1420-1428.
Cohen, J.J. (1972). Thymus-derived lymphocytes sequestered
in the bone marrow of hydrocortisone treated mice. J.
338
Immunol. 108, 841-843.
Conn, E.E. (1969). Cynogenic glycosides. Agric. Food. Chem.
17(3), 519-529.
Copp. D.H. (1969). Endocrine control of calcium homeostasis,
J. Endocrinol. 43, 137-139.
Cornelius, C.E., Arias, I.M. and Osburn, B.I. (1970).
Hepatic pigmentation with photosensitivity. A syndrome in
Corriedae sheep resembling Dubin-Johnson syndrome in man.
J. Am. Vet. Med, Assooc. 140, 709-710.
Crocker, C.L. (1967). Rapid determination of urea nitrogen
in serum or plasma without deproteinization. Am. J. Med.
Technol. 334, 361-303.
Crawford, R.F., Kennedy, W.K. and Davison, K.L. (1900).
Factors influencing the toxicity of forages that contain
nitrate when fed to cattle. Cornnel. Vet. 50, 3-17.
Cunningham, I.J. and Hogan, K.J. (1954). Oestrogens in New
Zealand pasture plants. N.Z. Vet. J. 2, 128-134.
Cunningham, I.J. Rogan, K.J. and Lawson, B.M. (1959). The
effect of sulphate and molybdenum on copper metabolism in
cattle. N.Z.J. Agric Res. 2, 134-144.
Curtis, P.E. and Griffiths, J.E. (1972). Suspected chocolate
poisoning of calves. Vet. Rec. 90(11), 313-315.
Cysewski, S.J., Pieri, A.C. Engstrom, C.W. Richard, J.L.,
Dougherty, R.W. and Thurston, J.R. (1908). Clinical
339
pathologic features of acute aflatoxicosis in swine.
Am. J. Vet. Res. 29, 1577-1578.
Darling, R.C. and Roughton, F.J.W. (1942). Effect of
methemoglobin on equilibrium between oxygen and
haemoglobin Am. J. Physiol. 1~7, 56-68.
Danke, R.J. Panciera, R.J. and Tillman, A.D. (1965).
Gossypol toxicity studies with sheep. J. Anim. Assoc.
24, 1199-1201.
De Bruin, A. (1976). Biochemical toxicology of Environmental
Agents. 1st Ed. Elsevier/North-Holland Biomedical
Press, New York.
Dee Snell, F. and Snell, C.T. (1954). Colorimetric methods
of analysis. 3rd Ed. Vol. IV D. Van Nostrand Compo
Inc. New York. Pp. 27-28.
Dern,R.J. Beutler,E. and Alving, ,A.S. 91955). The hemolytic
effect of primaquine. V. Primaquine sensitivity as a
manifestati,on of a multiple drug sensitivity. J. Lab.
Clin. Med. 45, 30-39.
Der Marderosia~, A., Giller, F. and Roia, Jr. F.G (1976).
Phytochemical and Toxicological screening of Household
plants potentially toxic to Human. J. Toxicol.
Environ. Health 1,939-953.
340
Divers, T.J .., George, L.W. and George, J.W. (1982). Haemolytic
anaemia in horses after the ingestion of red maple leaves.
J. Am. Vet. Med. Assoc. 180(3), 300-302.
Doerr, J.A., Wyatt, R.D., Hamilton, P.B. (1976). Impairment
of coagulation function during aflatoxicosis in young
chickens. Tox Ap. Pharm. 35(3), 437-446.
Dombrowski, L.J., and Pratt, E.J. (1972). Fluorometric method for
determination of nanogram quantities of nitrite ion.
Analyt 44(14), 2268-2271.
Dollahite, J.W., Pigeon, R.F. and Camp, B.J. (1962). The
toxicity of gallic acid pyrogallol, tannic acid and
Quercus harvardi in the rabbit. Am. J. Vet. Res. 23,
1264-1267.
Dollahite, J.W. Hardy, W.T. and Henson, J.B. (1964). Toxicity of
Helenium microcephalum (smallhead sneezeweed).J. Am. Vet.
Med. Assoc. 145, 694-696.
Doughlas, C.G., Haldane, J.S. and Haldane, J.B.S. (1912).
Laws of ,combination of haemoglobin with carbon monoxide
and oxygen. J. Physiol. 44, 275-004.
Plessis, L.S. N~nn, J.R. and Roach, W,A. (1969). Carcinogen
in a Transkeian Bantu food additive. Nature (Lond.)
22, 1198-1199.
Dunbar, G.M. and Chambers, T.A.M. (1963). Suspected kale
poisoning in dairy cows. Vet. Rec. 75, 566-568.
341
Dwivedi, S.K. Shivnani, G.A. and Joshi, H.C. (1971). Clinical and
biochemical studies in Lantana poisoning in ruminants.
Ind. J. Animal. Sci. 41, 948-951.
Edman, J.W. and Forbes, R.M. (1977). Mineral bioavailability
from phytate containing foods. Food Prod. Dev. 11,46-49.
Edmonds, L.D., Selby, L.A. and Case, A.A. (1972). Poisoning
and congenital malformation associated with consumption of
poison hemlock by sows. J. Am. Vet. Med. Assoc. 160,
1319.
Esteban, J.N. (1968). The differential effect of hydrocortisome on
the short lived small lymphocyte. Ant. Rec. 162, 349-353.
Evans, W.C. (1964). Bracken poisoning in farm animals. Vet.
Rec. 76(13), 365-359.
Evans, W.C. Patel, M.C. and Koohy, Y. (1982). Acute bracken
poisoning in monogastric and ruminant animals. Proc.
Roy. Soc. (E~in) 81, 26-64.
Evans, I.A. and Mason, J. (1965). Carcinogenic activity of
bracken. Nature 208, 913-914.
Evans, W.C., Evans, E.T,P, and Hughes, L.E. (1951). Studies on
bracke~ poisoning in cattle. Br. Vet. J. 110, 295-365.
Evans, I.A. Widdop, B. and Harding, J.D.J. (1972).
Experimental poisoning by bracken rhizome in pigs.
Vet. Rec. 90, 471-475.
Ewer, D.W. and Hall. J.B. (1978) Ecological Biology. Longman
342
Group Ltd., London. 1st Edition.
Falvey, L. (1976). The effects of Leucaena leucocephala on
cattle in the northern territory.Aust.Vet. J. 52, 243-246.
Fazio, T. Damico, J.N. Howard, J.W., White, R.H. ,Watts, J.D.
(1971). Gas chromatographic determination mass
spectrometric confirmation of N-nitrosodimethylamine in
smoke processed marine fish. J. Agric. Food Chern. 19,
250-256.
Farber, E.(1971).Biochemical Pathology.Ann.Rev.Pharmac. 1,71-81.
Fenwick, G.R. and Curtis, R.F. (1980). Rape seed meal and
its uses in poultry diets. A review Anim. Feed. Sci. nd
Tech. 5, 255-298.
Fernando, R. (1987). Plant poisoning in Sri. Lanka. In
Progress in venom and toxin research. Proceedings of the
first Asia-Pacific Congress in animal, plant and microbial
toxins Pp. 62,4-627.
Ferrarini, M., Cadoni, A., Franzi, A.T. , Ghigliot, C., Leprini, A.
Zicca,~. (1980). Utrastructure and cytochemistry of
Human per1pheral blood lymphocytes. Similarities between
the cells of the three population an TG lymphocytes. Eur.
J. Immunol. 10, 562-566.
Fowler, M.E. (1968). Pyrrolizidine alkaloid poisoning in
calves. J. Am. Vet. Med. Assoc. 152 (8), 1131-1144.
Ford, E.J.H. (1974). Activity of gamma-glutamyl
343
,
transpeptidase and other enzymes in serum of sheep with
liver and kidney damage. J; Compo Pathol. 84(2),231-243.
Ford, E.J.H., Ritchie, H.E. and Thorpe, E. (1968). Serum
changes following feeding of ragwort (Senecio jacobea) to
calves. J. Compo Pathol 78, 207.
Forsyth, A.A. (1980). British Poisonous Plants. Ministry of
Agriculture, Fisheries and Food, HMSO No. 161.
Fraser, C.M. and Nelson J. (1959). Sweet clover poisoning
in newborn calves. J.Am. Vet. Med. Assoc. 135,
283-286.
Fernando, R. (1987). Plant poisoning in Sri Lanka. In
Progress in venom and toxin research. Proceedings
of the first Asia-Pacific Congress in animal, plant
and microbial toxins Pp. 624-627.
Freedland, R.A. Hjerpe, C.A. and Cornelius, C.E. (1965).
Comparative .s t.ud Le s on plasma enzyme activities in
experimental hepatic necrosis in horses. Res. Vet. Sci.
6, 16-l9.
Frisch, J.E. 0 Neill, C.J. and Burrow, H>M> (1984). The
incide~ce and effect of poisoning with Lantana camara
in different cattle breeds. J. Agric. Sci. Lamb.
102, 191-194.
Gallagher, C.H., Koch, J.H. and Hoffman, H. (1967). Death
of ruminants grazing Phalaris tuberosa in Australia.
344
Aust. Vet. J. 43, 495-500.
Garcia, A.M. and Iorio, R. (1968). ·Studies on DNA in leuco-
cytes and related cells of mammals. V. ~he fast green
histone and the Feulgen-DNA content of rat leukocytes.
Acta Cyto. 12, 46-47.
Gardiner, M.R. (1965). The pathology of Lupinosis of sheep,
gross and histopathology. Path Vet. 2, 417-445.
Gardiner, M.R. (1966). Fungus-induced toxicity in Lupinosis.
Bri. Vet. J. 122, 508-513.
Geary, T. (1950). castor bean poisoning. Vet. Rec. 62(32),
472-473.
Gelderman, A.M. and Peacock, A.C. (1965). Increased specific
activity and formation of an inhibitor from the LDHs
isoenzymes of lactic dehydrogenase. Biochem. 4, 1511-
1518.
St.George-GramBauer, ,T.D. and Rac, R. (1962). Hepatogenous
chronic copper poisoning in sheep in South Australia
due to,the consumption of Echium plant. Aust. Vet. J.
38, 288-291.
Gilman, G.A., G90dman, L.S. and Gilman, A. (1980). The
pharmacological basis of therapeutics. 6th Ed.
Macmillan Publishing Co., Inc. New York.
Goldfarb, S., Singer, E.J. and Popper, H. (1962). Experi-
mental cholangitis due to alpha-naphthylisothiocyanate
345
(ANIT). Am. J. Pathol. 40, 685-696.
Gooneratne, B.W.M. (1966). Massive "generation alopecia
after poisoning by Gloriosa superba. Br. Med. J. 1,
1023-1026.
Gopinath, C. Jones, R.S. and Ford, E.J.H. (1970). Effect
of repeated administration of halothane on liver of
horses. J. Pathol. 102(2), 107-110.
Gracey, J.F. and Todd, J.R. (1960). Chronic copper poisoning in
sheep following the use of copper sulphate as a
mollusacide. Br. Vet. J. 116, 405-409.
Gornall,A.G., Bardawill, C.J., David, M.M. (1949). Deter-
mination of serum proteins by means of the biuret
reagent. J. BioI. Chern. 177, 751-755.
Grant, C. A., Holtenius, P. Johnson, G. and Thorell, C.B.
(1968). Kale anaemia in ruminants. I. Survey of the
literature and experimental induction of kale anaemia
in lactating cows. Acta Vet. Scand. 9, 126-140.
Greenhalgh, J.~.D., Sharman, G.A.M. Aitken, J. N. (1969).
Kale anaemia I. The toxicity to various species
of animals of three types of kale. Res. Vet. Sci. 10,
64-68.
Gumbman, M.R. and Williams, S.N. (1969). Biochemical - effects of
aflatoxin in pigs. Toxicol. Appl. Pharmac.
15, 393-404.
346
Gujral, G.S. and Vasudevan, P. (1983). Lantana camara L.
a problem weed. J. Scient. undo Res. 4G, G81-G84.
Gupta, I. and Bhide, N. K. (1967). Study of protective
action of some CNS depressants in experimental
Paspalum scrobiculatum poisoning. Indian Vet. 44,
786-793.
Gupta, I. Nanriyal, M.M. (1966). Acacia leucophloea Wild
(Raunja) poisoning in livestock. Indian Vet. J. 43,
538-543.
Habermehl,G.G.(1987).Progress in plant toxins research.
Progress in venom and toxin research. Proceedings of the
first Asiatic Pacific Congress on animal, plant, and
microbial toxins. Pp. 619-6G9.
Hakim,S.A.E.,Mijovic,V.and Walker,J.(1961)Experimental
transmission saguinarine in milk.Detection of metabolic
products. N~ture(London) 189, G01.
Hakim,S.A.E., Mijovic, V.and Walker,J (1961a) Distribution of
certain pop~py fumaria alkaloids and a possible link with
the incidence of glaucoma. Nature (Lond) 189, G01-GOG.
Hall, H.T.B. (19J7). Diseases and parasites of livestock in
the tropics. 1st Edition. Longman Group Ltd., London.
Honavar,P.M. and Sohnie, K. (1955). Distribution of trypsin
inhibitors in different organs of plant of sweet potato
(Ipomea batatas) and green grain (Phaseolus aureus). J.
347
Univ. Bombay 243, 64-69.
Harrington, J.T. and Cohen, J.J. (1975). Current concept of
acute oliguria. New Engl. J. Med. 292, 89-91.
Harris, J.W. (1956). Studies on the mechanism of a drug
induced hemolytic anemia. J. Lab. Clin. Med. 47, 760-
775.
Harris, D.J. and Rhodes, H.A. (1969). Nitrate and nitrite
poisoning in cattle in Victoria. Aust. Vet. J. 45, 590-
593.
Harvey, D.G. and Hoe, C.M. (1971). Application of some liver
function test to sheep dosed with carbon tetrachloride
and hexachlorophene. Vet. Rec. 88 (22), 562-564.
Harvey, G.D. and Obeid, H.M.A. (1974). Application of certain
function tests including serum alkaline phosphatase
estimation to domesticated animals in Sudan. Bri. Vet. J.
130 (66), 544-555.
Harborne, J.B. (1973) Phytochemical methods, Fakenham Press Ltd.,
Fakenham, Nor,folk. G. Britain.
Harris, J.W. and Kellermeyer, R.W. (1970). The red cell
produc~ion, metabolism, destruction, normal and
abnormal. Rev. ed. Harvard Uni. Press. Cambridge,
Mass.
Harris, W.N., Baker, F.P. and Johnston, A. (1972). Outbreak
of locoweed poisoning in horses in South-Western
348
Alberta. Can. V~t. J. 13(3), 141-145.
Harbourne, J.F. McCrea, C.T. and Watkinson, J. (196H). An
unusual outbreak of lead poisoning in calves. Vet. Rec.
83, 515-517.
Harland, B.T. and Prosky, C. (1979). Development of dietary
fibre values for food cereals. Food's World 24, 3H7-3H9.
Hazzard, J.T., Kowanish, S., Caughey, W.S. (19H2). Mechanism
of cyanide inhibition of cytochrome-C oxidase, myoglobin
and haemoglobin-function by hypotensive agent
Nitroprusside. Meeting. Fed. Prod. 4(4), 142H-1430.
Hegarty, M.P. Schinckel, P.G. and Court, R.D. (1964). Reaction of
sheep to the consumption of Leucaena glauca Barth and its
toxic principle mimosine. Aust. J. Agric. Res. 15,
153-165.
Henson, J.B., Dollahite, J.W., Bridges, C.H. and Rao, R.R.
(1965). Myodegeneration in cattle grazing Cassia
species. J. Am. Vet. Med. Assoc. 147, 142-145.
Herrington, M.D., Elli,ott, R.C., Brown, J.E. 91971). Diagnosis and
treatment of thyroid dysfunction occurring in sheep fed on
Cynodon plectostachyum Rhodesian J. Agric. Research 9(2),
87-93.
Hilberg, A.D.R., Waite, P.R. and Mackinnon, J. (1973). Accidental
poisoning in animals. Vet. Rec. 92(IH), 4H4-4H5.
Hill, K.R. and Martin, H.M. (195H). Hepatic venocclussive
349
disease and megalocytosis in Senecio 'poisoning of horse
(Further investigation of the walking disease of North
Western Nebraska). Br. Vet. J. 114, 345-350.
Hill, B.D. and Blaney, B.J. (19HO). Urochloa panicoids (liverseed
grass) as a cause of nitrate poisoning letter. Aust. Vet.
J. 56(5}, 256-260.
Himes, J.A. and Cornelius, C.E. (1973). Hepatic excretion and
storage of sulfobromopthalein sodium in experimental
hepatic necrosis in dogs. Cornell Vet. 63, 424-431.
Hoffmann, W.E. and Dorner, J.L. (1977). Disappearance rates
of intravenously injected canine alkaline phosphatase
isoenzyme. Am. J. Vet. Res. 3H(10}, 15553-1556.
Hoffman, W.E., Reneger, W.E., and Dorner, J.L. (1977)
Serum half life of intravenously injected intestinal
and hepatic alkaline phosphatase isoenzyme in cat. Am.
J. Vet. Res. 38(10}, 1637-1638.
Hogan,K.G., Money, D.F.L. and Blaney, A (1968). The effect
of a molybdate and sulphate supplement on the
accumulation of copper in the livers of penned
sheep. N! 2. J. Agric. Res. 11, 435-444.
Hooper, P. (1977). Crotalaria retusa poisoning of pigs and
poultry. Aust. Vet. J. 53(3), 109-114.
Hockwald, R.S., Arnold, J. Clayman, C,B. and Alving, A.S.
(1952). Status of primaquine IV Toxicity of primaquine
350
in Negroes. J. Am. Med. Assoc. 49, 1568-1570.
Honavar, P.M. Cheng-Ven Shih and Liener, I.E. (1962). The
inhibi tion of growth of rat by purified he'maggLu t.Ln in
fractions isolated from Phaseolus vulgaris. J. Nutr.
77, 113-115.
Hore, D. (1961). Tall Fescue (Festuca arundinaea) Poisoning
in dairy cattle. Aust. Vet. J. 37, 312-317.
Honavar, P.M. and Sohnie K (1955). Distribution of trypsin
inhibitors in different organs of plants of sweet potato
(Ipomoea batata) and green grain (Phaseolus aureus) J.
Univ. Bombay 243, 64-69.
Hutchinson, J and Dalziel J.M. (1954) Flora of West Tropical
African 2nd edition Vol. 1 part 2 Crown Agents London.
Hutchinson, T.W.S. (1977). Onions as a cause of Heinz body
anaemia and death in cattle. Can Vet. J. 18, 358-361.
Humpreys, L.R. (1981). Deficiencies of adaptation of pasture
legumes. Trop. Grassl. 14(3), 153-158.
Ikegwuonu, I, an~ Bassir, O. (1976). Effect of phytohema-
gglutinins from unmatured legume seeds on the function
and enzy~e activities of the liver and on the
histopathological changes of some organs of the rat.
Tox. Appl. Pharmac. 40, 217, 226-228.
Irvine, F.R. (1961). Woody plants of Ghana. Oxford
University Press. London.
351
v
Jaenike, J.R. (1966). The renal lesions associated with
hemoglobinemia. J. Exp. Med." 123, 532-535.
Jaffe, W.G. and Vegalette, C.L. (1968). Heat labile growth
inhibiting factor in beans. J. Nutr. 94, 203-206.
Jaffe, W.G. (1~60). Uber phytotoxine aus Bohnen Arzneimittel
Forsch, 10, 1012-1013.
Jain, N.C. (1986). Schalm's Veterinary Hematology. 4th
Ed. Lead and Febiger, Philadelphia.
James, L.F. and Binns, W. (1967). Blood changes associated
with locoweed poisoning. Am. J. Vet. Res. 28, 1107-
1110.
James, L.F and Binns, W. (1966). Effect of feeding wild
onions (Allium validium) to bred ewes. J. Am. Vet.
Med. Assoc. 149, 512-516.
James, L.F. Van Kampen, K.R. and Hartley A.E., (1970).
Physiophatholo~ic changes in locoweed poisoning
in livestock. Am. J. Vet. Res. 31(4), 663-664.
James, L.F., Keeler, B.F,. and Binns, W. (1969). Sequence in
the abortive and teratogenic effects of locoweed fed to
sheep. ~m. J. Vet. Res. 30, 377-380.
Jenkins, F.B. (1963). Allergenic and toxic components of
castor bean meal. Review of the literature and studies
on the inactivation of these components. J. Sci. Food.
Ag r ic . 14, 773 .
352
Jendrassik, L. and Grof, P. (193B). Vereinfachte photome-
metrische methoden zur bestimmung des blutbiliribins.
Biochem. 297, 81-84.
Jones, R.J. and HIegarthy, M.P. (19B4). Effect of different
proportions of Leucaena leucocephala ~n the diet
of cattle on growth, feed intake, thyroid function
and urinary eicretion of 3-hydroxy-4(IH)-pyridone.
Aust. J. Agric. Res. 35, 317-325.
Jones, R.J. (1979). Value of Leucaena leucocephala as feed
for ruminants in the tropics. World Anim. 1979 (31)
13-23.
Johnson, A.E. (1976). Changes in calves and rats consuming
milk from cow's fed chronic lethal doses of Senecio
Jacobaea (tansy ragwort). Am. J. Vet. Res. 37(1), 107-
110.
Kaneko, J.K. (1980). Clinical biochemistry to domestic
animals. 3rd Ed.Academic Press. Inc., New York
Kaneko, J.J. and Cornelius C.E. (1970) Clinical biochemistry
of domestic Animals 2nd Edition Academic Press New York
Katiyar, R.D. ,(1981). A report on plant poisoning in sheep
a~d goats. Livestock Advisor 6, 51-56.
Kasali, O.B. (1977). Cestrum diurnum intoxication in normal
and hyperparathyroid pigs. Cornell Vet. 67(2),
190-221.
353
Keeler, R.F. James, L.F. Binns, W. and Shupe, J.L. (1967)
An apparent relationship between locoism and Lathyrism.
Canadian J. Compo Med. 31, 334-341.
Keller, R.F. (1973). Teratogenic compounds of Veratrum
calfornicum (Durand) 14 Limb deformities. produced by
cyclopamine. Proc. Soc. expo Biol. Med. 142, 1287.
Keller, R.F. and Binns, W. (1964). Chemical compounds of
Veratrum californicum. Related to congenital ovine
cyclopian malformations. Extraction of active
material. Proc. Soc. Exp. Bio. Med. 16, 123-127.
Kelly, R.W. , Allison, A.J., and Shirley, D.K. (1976).
Interactions between phytooestrogens and steroids in
cervical mucus and uterine weight responses in ewes.
Aust. J. Agr. 27(1), 101-107.
Khayambashi, H. and Lyman, R.L. (1966). Stimulation of
perfused pancreas secretion by plasma of rats fed Soya
bean trypsin inhibitor. Fed. Proc. 25, 276-278.
Kimberly, R.P. and Ralph, P. (1983). Endocytosis by the
mononuclear phagocyte system and autoimmune disease.
Am. J.,Med. 74, 481-483.
Knight, P.R. (1968). Equine cystitis and ataxia associated
with ~razing of pastures dominated by Sorghum species
Aust. Vet. J. 44, 257-259.
Kotz, J. (1965). Morphology and pathogenesis of corn cockle
354
Agrostema githago poisoning poultry 1 An investigations
Medycyna wet 21, 143-150 ..
Kinghorn, A.D. (1979). Toxic plants. Columbia University
Press, New York, Guildford, Surrey.
Kingsbury, J.M. (1975). Phytotoxicology. In. L.J. Casarett
and J. Doull, eds, Toxicology: The Basic Science of
Poisons pp. 591-603, New York Macmillan.
Kingsbury, J.M. (1979). The Problem of Poisonous plants In
A.D. Kinghorn, eds. Toxic plants pp .. 1-0, Columbia
University Press, New York.
Krook, L. Wassermann, L.R., McEntee, K. Brokken, T.D. and
Tergland, M.B. (1975). Cestrum diurnum poisoning
in Florida cattle. Cornell Vet. 05, 557-575.
Lang, K. (1933). Die Rnodambeldung in Tierkopor. Biochem.
2, 259, 243-246.
Landsteiner, K. (1954). Specificity of serological
reactions. Edited by K. Landsteiner, Harvard Univ.
Press ,Cambridge. Massachussetee.
Lee, C., Weiss, R and Horvath, D.J. (1970). Effect of
nitr9gen fertilization on the thyroid function
of rats fed 40 percent orchard grass diets. J. Nurt.,
100, 1121-1120
Lemberg, R. Legg, T.W. and Lockwood, W.H. (1941). Coupled
oxidation of ascorbic acid and hemoglobin; quantitative
395
studies on choleglobin formation. Estimation of
haemoglobin and ascorbic -acid oxidations. Biochem. J.
35, 339-352.
Letts, G.A. (1963). Leucaena glauca and ruminants. Aust.
Vet. J. 39, 287-291.
Ley, A.B., . Harris, J.P. , Brinkley, M., Liles, B. , Jack,
J.A. and Kahan, A. (1958). Circulating antibody
directed against penicillin. Science 127, 1118-1119.
Liener, I.E. (1969). Toxic constituents of animal
foodstuffs. Acad. Press. New York, pp. 15.
Levinsky, N.G. (1977). Pathophysiology of acute renal
failure. New Engl. J.Med. ~96 (~6), 1453-1458.
Levene, C.I. (1962). Studies on the mode of action of
lathyrogenic compounds. J. Exp. Med. 116, 119-133.
Liener, I.E. (1974). Phytohaemagglutinnins. Their nutrition
and significance J. Agric. Food. 22(11} 17-23
Lim, G. (1987). Toxins and fungi. In Progress in venom and
toxin, research. Proceedings of the first Asia-Pacific
Congress on animal, plant and microbial toxins pp.
649-656.
Littledike, E.T. James., E. and Cook,H. (1976). Oxalate
(Halogenton) poisoning of sheep. Certain physiopath-
ologic changes. Am. J. Vet. Res. 37(6}, 661-666.
Little, D.A. Robinson, P.J. Playne, M.J. and Haydak, K.P
(1971). Factors affecting blood inorganic phosphorus
determinations in cattle. Aust. Vet. J. 47(4), 153-156.
Llewellyn, C,A. (1962). A case of oak poisoning Vet. Rec. 74,
1238-1241.
Londonk, W.T. Henderson, W. and Gross, R.F. (1967). An
attempt to produce chronic nitrite toxicosis in
swine. J. Am. Vet. Med. Assoc. 150, 39B-402.
Lombardi, B. (1966). Consideration on the pathogenesis of
fatty liver. Lab. Investigation 15, 1-20.
Lyman, R.L. ~nd Lepkovsky, S. (1957). The effect of raw
soyabean meal and trypsin inhibitor diets on
pancreatic enzyme secretion in rat. J. Nutr. 62,
269-284.
Magee, P.N. (1966). Naturally occurring alkylating agents.
Proc. Roy. S. Med. 59, 751-756.
Mandel, L.A. (1982). Effects of antimicrobial and anti-
neoplastic drugs on phagocytosis and microbicidal
function of the polymorphonuclear leukocytes.
Red. infect. Dis. 4, 6B3-5B5.
Martinovich, D.,and Smith, B. (1973). Kikuyu poisoning of
cattle. L. Clinical and pathological findings.
N.Z. Vet. J. 21(4), 55-53.
Martone, G. Hartman, R.H. and Clawson, A.J. (1959). Studies
of a manganese-iron antagonism on the nutrition of
357
rabbits and baby pigs. J: Nutr. 67, 309-317.
Martins, W.B., Thomas, B.A.C. and Urquart, G.M. '(1957).
Chronic diarrhoea in housed cattle due to atypical
parasitic gastritis. Vet. Res. 69, 736-739.
Markson, L.M. (1960). The pathogenesis of the hepatic lesion
in calves poisoned experimentally with Senecio jacobea
Proc. Roy. Soc. Med. 53, 283-284.
Marshall, V.L. Buck, w.B. and Bell, G.L. (1967). Pigweed
(Amaranthus retroflexus): an oxalate containing plant.
Am. J. Vet. Res. 28, 888-889.
McCunn, J. Andrew, H. and Clough, G.W. (1945). Castor-bean
poisoning in horses. Vet. J. 101, 136-138.
McKenzie, R.H., Blaney, B.J., Connole, M.D. Fitzpatr. L.A.
(1981). The effect of dietary oxalate on calcium
phosphorus,magnesium, balances in horses, J. Agr. Sci.
97, 69-74.
McCosker, P.J. ,and Hunt, S.E. (1966). Suspected poisoning
of cattle with Acacia salcina. Aust. Vet. J.
42, ~55-357.
Mcilwain, P.K. and Schipper, A. (1963). Toxicity of nitrate
nitrogen to cattle. J. Am. Vet. Med. Assoc. 142, 502.
McPherson, A. (1979). Pokeweed and other lymphocyte mitogens
in Toxic Plants Eds. Kinghorn, A.D. Columbia
University Press, New York pp. 83-102.
~58
McBarron, E.J., Walker, R.I., Gardiner, I., Walker, K.H.
(1975). Toxicity to livestock of the blue-green alga
Anabaena circinalis. Aust. Vet. J. 51(1~),
587-588.
McClymont, G.L. (1954). Paresis associated with spinal cord
myelin sheath degeneration in new born pigs. Aust.
Vet. J. 30, 345-346.
The Merck Veterinary Manual 91979). A handbook of Diagnosis
and Therapy for the Veterinarian. 5th Ed. Merck and
Therapy for the Veterinarian. 5th Ed. Merck and Co.
Inc., Rahway, N.J.
Michaelson, M. (1961). Bilirubin determination in serum and
urine. Scand. J. Clin. Lab. Invest. (Suppl. 56)
13,1.
Milne, J.A. (1966). A case of Delphinium poisoning in rats.
N.Z. Vet. J. 14, 1~7-13~.
Miyazaki, A and Kawashima, R. (1975). Effect of methemoglobin
format,ion in blood on some serum constituents related to
the liver function of sheep Jap. J. Zootech. Sci. 46(7),
433-437.
Moffat, J. (1974). Problems in U.S. sheep industry. N.Z. J.
Agr. 129 ( 2 ) I 37-3H •
Montgomery, R.D. (1969). In: Toxic constituents of plant
feedstuffs. Edited by I.E. Liener Acad. Press, New
359
York and London.
Moule G. R. (1961) Fertility of sh~ep grazing estrogenic
pastures. Aust. Vet. J. 37, 10~-114
Morris, E.R., Ellis, R. (1981). Phytate-Zinc molar ratio
of breakfast cereals and bioavailability of zinc
to rats. Cereal Chern. 5S(5), 363-366.
Moslehuddin, A.B.M., Hang, Y.D. and Stoewsand, G.S. (1~S7).
Evaluation of the toxicity of processed Lathyrus
sativus seeds in chicks. Nutr. Reports. Int.
36(4), 851-855.
Mount, M.E. and Feldman, B.F. (1~S3). Mechanisms of
Diphacinon rodenticide toxicosis in the dog and
therapeutic implication. Am. J. Vet. Res. 44, 2009.
Morgan, S., Stair, E.L. Martin, T., Edwards, W,.C. and
Morgan, G.L. (l~SS). Clinical, clinicopathologic,
pathologic and toxicologic alterations associated with
gossypol toxicosis in feeder lambs. Am. J. Vet. Res.
49(4)~ 493-499.
Mowe, G.R. (1961). The fertility of sheep grazing oestrogenic
past4res. Aust. Vet. J. 37, 109-114.
Mullenax, C.H. (1963). Observations on Leucaena glauca
Aust. Vet. J. 39, SS-92.
Newberne, P.M. and Butler, W.H. (1969). kcute and chronic
effects of aflatoxin in the liver of domestic and
360
laboratory animals. A Review. Cancer Res. 29, 236-250.
Newman, D.M.R. (1968). Preservation of bovine blood for
determination of inorganic phosphate. Aust. Vet.
J. 44, 443-446.
Nomiyana, K. and Foulkes, E. C. (196H). Some effects of
uranyl acetate on proximal tubular functions in rabbit
kidney. Tox. Appl. Pharmac. 13, H9-9H.
Nosslin, B. (1960). The direct diazo reaction of bile pigments in
serum. Scand. J. Clin. Lab. Invest. (Suppl. 49) 12, 1-4.
Nowack, E. (1980). In Advances in legume Science. Edited by R.J.
Swoerfield and Bunting, Acad. Press, New York, London.
Nowell, P. (1960). Phytohaemagglutinin: An inhibitor of mitosis
in culture of normal human leukocytes. Cancer Res. 20,
462-466.
Nwude, N. and Parson L.E. (1977). Nigerian plants that may
cause poisoning in livestock. Vet. Bull. 47(11) H11-817
Nwude, N. (1976). Toxicological investigations of Lassiosiphon
kraus,sianus (Reisn). Abstract of Ph.D. Thesis, Ahmadu
Bello University, Zaria, Nigeria, 4pp. Abst. 6731.
Veterinary Bulletin.
Nwude, N. , Parson, L.E. and Adaudi, A.O. (1977). Acute toxicity
of the leaves and extract of D. barteri (Engl.) in mice,
rabbits, and goats. Tox. 7(1) 23-29.
361
Nowell, P. (1960). Phytohemagglutinin: An inhibitor of mitosis in
culture of normal human leukocytes. Cancer Res. 20,
462-466.
Osuntokun, B.O. (1973). Ataxia, neuropathy associated with
high cassava diet in West Africa. Proceeding of an
Interdisciplinary workshop. London, England pp. ~9-30.
Osborne, T.B. and Mendel, L.B. (1917). The uses of soyabean as
food. J. Bio. Chem. 3~, 746-74H.
O'Hara, P.J., Pierce, K.R. and Reed, W.K. (1969). Degenerative
myopathy with ingestion of Cassia occidentalis L.
Clinical and pathologic feature of the experimentally
induced disease. Am J. Vet. Res. 30, 217H3-21HO.
Panciera, R.J., Johnson, L. and Osburn, B.L. (1966). A disease
of cattle grazing hairy retch pasture. J. Am. Vet. Med.
Assoc. 148, 804-HOH.
Parr, C.W. and Fitch, L.I. (1964). Hereditory partial deficiency
of human erythrocytes phosphogluconate dehydrogenase.
Biochem. J. 93, 28-30.
Pauling, L. and Coryell, C.D. (1936). Magnetic properties
and s,tructures of the haemochromogens and related
structures. Proc. Nat. Acad. Sci. 22, 159-163.
Pass, M.A., Gremwell, R.T., Heath, T.J. (197H). Effect of
Lantana camara on the ultrastructure of the liver
of sheep. Tox. and Appl. Pharmac. 43(3). 5H9-596.
Pearson, J.K.J.(1956). Copper poisoning in sheep following
the feeding of a copper supplemented diet. Vet. Rec.
68, 766-769.
Pearson, A.W. , Greewood, N.M, Butler, E.J., Fenwick, G.R.
(1983). Biochemical changes in layer and broiler
chickens when fed on high glucosinolate rapeseed
meat. Bri. Poultry Sci. 24(3), 417-427.
Peters, R.A. (1963). Biochemical lesions and lethal synthesis.
Macmillan, New York, pp. 321.
Peters, R.A. (1969). Biochemical lesions and its historical
development. Bri. Med. Bull. 25, 223-226.
Peterlik, M. and Wassermann, R.H. (1978). Stimulatory effect
of 1 - 25 = dihydroxycholecalciferol like substances
from Solanum malaccxylon and Cestrum diurnum on
phosphate transport in chick jejunum. J. Nutr. 108(10)
1673-1679.
Peterlk, M., Bursac. , K., Haussler, M.R. Hughes, M.R. and
Wasse~mann, R.H. (1976). Further evidence for the 1, 25-
dihydroxycholecalciferol-like activity of Solanum
malacqxylon. Biochem. Biophys. Res. Comm. 70, 797-804.
Pickrell, C.H. and Jones, R.K. (1974). Relationship of age
of normal dogs to blood serum constituents and
reliability of measured single values, Am. J. Vet. Res.
35(7),897-903.
363
Pierce, K.R., Joyce, J.R. England, R.B. and Jones, L.P.
(1972). Acute haemolyticanaemia caused wild onion
in horses. J. Am. Vet. Med. Assoc. 160(3), 323-3~6.
Pienaar, J.G., Kellerman, T.S. Basson, P.A. Jenkins, W.L. and
Vahrecijer, J. (1976). Maldronksiekte in cattle
a neuronopathy caused by S. kowebense. N.E. Br.
Onderstepoort J. Vet. Res. 43(~), 67-74.
Popper, H. and Schaffner, E. (1959). Drug induced hepatic
injury. Ann. Intern Med. 51, 1~30-1~5~.
Prase, K.W. (1969). Lactic dehyrogenase activity and isoenzyme
distribution in serum of normal cattle. Am. J. Vet. Res.
30, 2181-2184.
Prier, R.F. and Derse, P.H. (196~). Evaluation of the harzard of
secondary poisoning by warfarin poisoned rodents. J, am.
Vet. Med. Assoc. 40, 351-354.
Prins, H.K., Oort, M., Loes, J.A. Zurcher, C. and Bekers, T.
(1966). Congenital nonspherocytic haemolytic anaemia,
associa,ted with glutathione deficiency of erythrocytes.
Blood 27, 145-166.
Pryor, W.J., M~Donald, W.J.F. and Seawright, A.A. (197~).
Supplejack (Ventilago viminalis) feeding of sheep
Nutritional and toxicological investigation. Aust.
Vet. J. 48, 339-342.
364
Rahman, A. and Mia, A.S. (1972). Abrus precatorius poisoning in
c a t f'Le . Ind. Vet. J. 49, 1045-1049.
Rajendram, M.P., Chennakesavalu, M., Rao, C.V.N.,
Viraraghaven, K. and Damodaran, S (1983). Experimental
production of enzootic bovine hematuria with bracken
fern. Ind. Vet. J. 60, 173-178.
Rao, D.S. T. Joshi, H.C. and Kamar, M. (1988). Haemogram in
bracken fern (Pteris aquilina) poisoning in rats.
Int. J. Anim. Sci. 3(1~ 39-43.
Rao., A.K. and Walsh, P.N. (1983). Acquired qualitative
~latelet disorders. Clin. Haematol. 12, 201.
Read, J.W. ~nd Hass, L.W. (1938). Studies on the baking
quality of flour as affected by certain enzyme action
Further studies concerning potassium bicarbonate and
enzyme acti~ity. Cereal Chern. 51., 59-68.
Reitman, S., Frankel, S. (1957). A colourimetric method for
the d~termination of serum glutamic oxaloacetic and
glutamic pyruvlc transaminases. Am. J. Clin. Pathol.
28, 5E?-61.
Remuzzi, G., Cavenagh, A.E. (1978). Altered platelet and
,
vas~ular prostaglandin generation in patients with renal
failure and prolonged bleeding time. Thromb. Res.
13(3), 1007-1011.
Ryan, C.A. and Huisman, C.C. (1967). Chymotrypsin inhibitor.
365
L. from potatoes a transient protein component in leaves
of young potato plants. Nature ~14, 1047-1050.
Rogers, P.A.M. and Hopecawd, M.J. (19~0). Monensin ketosis
and nitrate toxicity in cows Note. Vet. Rec. 100(14)
311-312.
Sabago N. , Castrill, M.A. , Tchernit, A. (197~). Cortisol
induced migration of eosinophil leukocytes to lymphoid
organs. Experimentia 34, 000-069.
Salvido, E., Pannacciulli, I., Tizianetho, A., and Ajmar, F.,
(1967). Nature of haemolytic crisis and the fate of
glucose-6-phosphate dehydrogenase deficient, drug
damaged erythrocytes in Sardinians. New Engl. J. Med.
276, 1334-1339.
Samson, B.F., Vagg, M.J. and Dobereiner, J. (1971), Effect
of Solanum Malacoxylon on calcium metabolism in cattle.
Res. Vet. ?ci. 12, 604-005.
Saini, P.K. and Saini, B.K. (197~). Origin of serum alkaline
phosPQatase in the dog. Am. J. Vet. Res. 39, 1510-1513.
Sapeike, N. lI969). Food pharmacology: Thomas Springfield
Illin~is, Symposium on Natural food toxicants. J. Agric.
Chem. 17, 413-538.
Scott, H.M. (1963). Lead poisoning in small animals. Vet. Rec.
75, 830-832.
:; 66
Schalm, O.W., Jain, N.C. and Sarrol, E.J., (1975). Veterinary
Hematology. 3rd Ed. Lea and Febiger, Philadalphia.
Schmidt, E. and Schmidt, F.W. (1967). Release of enzymes
from the liver. Nature (Lond~) 213, 1125-1126.
Schmitz J.T. (1961). Methaemoglobinaemia a cause of
abortions. Preliminary report Obstet. Gynecol, 17,
413-415.
Schacham, P., Philip, R.B. and Cowden, C.W. (1970). Anti-
hemopoietic and carcinogenic effects of bracken fern.
Am. J. Vet. Res. 31, 191-197.
Scarisbrick, R., (1954). Acid indigestion in a sheep fed on
mangolds. Vet. Rec. 66, 131-133.
Schmitz, J.T. (1961). Methaemoglobinaemia - a cause of
abortions. Preliminary report. Obstet. Gynecol.
17, 413-415.
Seawright, A.A. (1963) Electron - microscopic observations of the
hepatocytes of sheep in Lantana poisoning. Path. Vet. 2,
175-196.
Seawright, A.A. and Allen, J.G. (1972) Pathology of the liver and
kidney in ~antana poisoning of cattle. Aust. Vet. J. 4H,
325-330.
Sharma, O.P., Makkar, H.P.S. and Dawra, R.K. (19HH). A review of
the noxious plant Lantana camara. Toxicon 26(11),
975-987.
367
Sharma, O.P., Vaid, J. and Dawra, R:K. (1984). Evaluation
of serum proteins in lantana poisoning, IRCS Med.
Sci. 12, 659-660.
Sharma, O.P., Makkar, H.P.S. Dawra, R.K. and Negi, S.S. 198~).
Changes in blood constituents of guinea pigs in Lantana
toxicity. Toxicol. letts. L, 73-76
Sharma, O.P. Makkar, H.P.S., Dawra, R.K. and Negi, S.S. (1981). A
review of the toxicity of Lantana camara {Linn} in
animals. Clin. Toxicol. 18, 1077-1080.
Sharp, C.W., Ollolenghi, A and Posner, H.S. (197~) Correlation of
paraquat toxicity with tissue concentrations and weight
loss of the rat. Toxicol. Appl. Pharmacol. ~2, 241-~51.
Shaver, T.N., Camp, B.J. and Dollahite, J.W. (1964). The
chemistry of a toxic constituent of Xanthocephalum
species. Ann. New York. Acad. Sci. Ill, 737-743.
Shupe, J.L. anQ James, L. (1969). Additional physiopatholo-
gic changes in Halogenton glomeratus (oxalate)
poiso~ing in sheep. Cornell Vet. 59, 41-55.
Shupe, J.L. Balls, L.F. and James, L.F. (1968). Changes
in blood serum transaminase associated with lupine
and larkspur poisoning in cattle. Cornell Vet.
58, 128-135.
Shupe, J.L. James., L.F. and Binn, W. (1967). Observation
of crooked calf disease. J. Am. Vet. Med. Assoc.
151, 191-197.
Sidha, K.S., Hargus, W.A., and Pfander, W.H. (1967). Meta-
bolic inhibitors in fractions of orcharo grass
(Dactylis glometrata L) Detected by in vitro
rumen fermentation technique. Proc. Soc. expo Bio
Med. 124, 1038-1042.
Sisk, D.B., Carlton, W.W. and Curtin, T.M. (1968). Experi-
mental aflatoxicosis in young swine. Am. J. Vet. Res.
29, 1591-1602.
Sinclair, K.B. and Jones, D.H. (1904). Nitrate toxicity in
sheep. J. Sci. Food. Agr. 15, 717-719.
Singh, R.P., Joshi, H.C. and Kumar, M (1987). Experimental
bracken fern toxicity in calves - changes in blood
and urine. Ind. J. Vet. Med. 7(2), 90-100.
Slater, T.F. (19~6). Necrogenic action of carbon tetrachlo-
ride in the rat. A speculative mechanism based on
acbivation. Nature (Lond.) 209, 36-40.
Smith, A.D.M., Puckett, S. and Waters A.H. (1963). Neuro-
pathological changes in chronic cyanide intoxication.
Nature (Lond.) 200., 179-181.
Smith, A.D. and Puckett, S (1905). Cyanide, vitamin B
experimental demylination and tobacco amblyopia.
Br. J. Exp. Path,. 40, 015-022.
Smith, H.A. Jones, T.C. Hunt, R.D. (1974). Veterinary
Pathology. 4th Edition, Lea and Febiger,
Philadelphia.
Smith, R.P. and Gosselin, R.E. (1964). On mechanism of
,, sulfide inactivation by methaemoglobin. Fed. Proc.
23, 297-299.
Smith, R.P.(1967). Nitrite methaemoglobin complex. Its
significance in methaemoglobin analyses and its
possible role in methaemoglonaemia. Biochem.
Pharmacol. 16, 1655-1694.
Smith, J.B. and Healy P.J. (196H). Elevated creatinine
phosphokinase activity in diseases of central nervous
system in sheep. Clin. Chim. Acta. 21
295.
Sorbo, B.C. ,(1975). Thiosulphate S. transferase and
mercaptopyruvate sulphustransferase In: Metabolic
pathways. Edited by S.M. Greenberg Acad. Press
New York. pp 433-453.
Stauffer, V, .D. (1970). Hydrocyanic acid poisoning from choke
cherry leaves. J. Am. Vet. Med. Assoc. 157(10),
1324-1329.
370
Stahler, L.M. and Whitehead, E.I. (1950·). Effect of 2,
4-D on potassium nitrate levels in leaves of sugar
beets Science 12, 749-751.
Stewart, J. and McCallum, J.W. (1944). The anhydraemia of
oxalate poisoning in horses. Vet. Rec. 56, 77-79.
Swenson, M.J. (1975). Duke's Physiology of Domestic Animals
Constock. Pub. Associate. 9th Ed. Ithaca and Lond.
Tennant, B., Dill, S.G. Glickman, L.T., Mirro, E.V., King,
J.M. Polale, D.M. Smith,. M.C. Kradel, D.C. (19Hl).
Acute hemolytic anaemia. Methaemoglobinaemia and
Heinz body formation. Association with ingestion of
red maple leaves by horses. J. Am. Vet. Med. Assoc.
179(20, 43-46.
Tait, R.M. Krishnamurti, C.R., Gilchrist, E.W. and Mac-
Donald, K. (1971). Chronic copper poisoning in
feeder lambs. Can Vet. J. 12 (3), 73-77.
Tewe, 0.0. and Pesu, E. (19H7). Performance and nutrient
utilization in growing pigs fed cassava peel rations
containing different cyanide levels. Nutr. Rep.
Int. 26(1), 51-58.
Tewe, 0.0. Maner, J.H. (19H2). Cyanide, protein and some
) 71
interaction in the physiology and metabolism of
rats. Food Chern. 9(3), 195-204.
Terblanche, M., Adelaar, T.F. and Vanstraten, A.M. (1966)
Euphorbia mauritanica L. as a poisonous plant in
South Africa. J.S. Afr. Vet. Med. Assoc. 37(3), 311
Thompson, and Van Furth, R. (1973). The effect of gluco-
steroids on the kinetics of promonocytes of the bone
marrow. J. expo Med. 137, 10-14.
Thomas, A.J. (1963). Bracken poisoning of animals.
Biochem. J. 88, 56-57.
Thompson, J.F. (1950). Some observation on the mechanism
of toxic action of ricin, J. Pharmac. Exp. Ther. 100,
370-373.
Thorpe, E. and Ford, E.J.H. (1968). Development of hepatic
lesions in calves fed with ragwort (Senecio jacobea)
J. Compo P~th. 78, 195-204,
Thwierge, G. (1973). Granular stomatitis in dogs due to
burdock. Can Vet. J. 14(4), 96-97.
Tower, K.G. (1950). A corn poisoning in heifers. Vet. Rec.
62, 7:'1-75.
Tobiaska, J. (1964). Die phythaemagglutinine Acta 154.
780-781.
Todd, J.R. (1962). A knackery survey of lead poisoning
incidence in cattle in northern Ireland. Vet. Rec.
372
74(9), 116-118.
Tonder, E.U. Van Basson, P.A. and Van Renburg, I.B.J.
Geeldikkop: experimental induction by feeding the
plant Tribulus terrestris L (Zygophylaceae). J.S.
Afr. Vet. Med. Assoc. 43, 363-375.
Tokarnia, C.H. Dobereiner, J., Lazzari, A.A. and Peixo,
P.V. (1984). Poisoning of cattle by species of Lantana
in Mato Grosso and Rio de Janeiro State Brazil Pesquis
a Vater Brasileira 4, 12~-132.
Tucker, E.M. (1969). The onset of anaemia and the production
of haemoglobin C in sheep fed on Kale. Brit. Vet. J.
125, 472-472.
Usher, C.D. and Telling, G.M. (1~75). Analysis of nitrate
and nitrite in foodstuffs. Critical review.
J. Sci. Food. 26(3), 384-386.
Van Etten, C.M. (1969) In Toxic constituents of plant
Foodstuffs, p. 103, I.E. Gierer ed. New York and
London, Academic Press.
Van Kampen, K.R. Lynn, F., James, J.A.E. , Johnson, A.E.
(19701. Hemolytic anaemia in sheep fed wild onions
(Allium validum) J. Am. Vet. Med. Assoc. 156(2),
328-332.
Vander, A.J. (1963). Effect of zinc, cadmium, and mercury
on renal transport systems. am. J. Physiol. 204, 781-
'373
784.
Van Kampen, K.r. and James, L.F. (1969). Pathology of loco-
weed poisoning in sheep. Path. Vet. 6, 413-423.
Van Dijk, C., Vickery, B., Vickery, M.L. (1972). Poisoning
of livestock by Dichapetalum toxicarium. Nig. J.
Sci. 6(2), 135-138.
Van der Walt, S.J. (1944). Some aspects of the toxicology
of hydrocyanic acid in ruminants. Onderstepoort
J. Vet. Sci. 19, 79-160.
Vickery B., and Vickery, M.L. (1971). Studies on the
Dichapetalaceae. Fluoride metabolism in Dichapetalum
toxicarium Phytochem.
Wardle G.N. and Williams R. (19BO). Polymorphonuclear
function in uraemia and jaundice. Acta Haematol.
64, 157-159.
Wassermann, R.H. Kro?k, L. and Dirksen, G. (1977). Evidence
for anti-rachitic activity in calcinogenic plant .
••
Trisetum flavescens. Cornell Vet. 67(3), 335-350.
Watt, J.M. and Breyer-Brandwijk, M.G. (1962). Medicinal and
poiso,nous plants of Southern and Eastern Africa. 2nd Ed.
Livingstone, London.
Walthall, and McKenzie, (1976). Osteodystrophia fibrosa in
horses at pasture in Queensland Field and Laboratory
observations. Aust. Vet. J. 52(1), 11-16.
374
Way, J.L. Gibbon, S.L. and Sheely, M, (1966). Cyanide
intoxication. Protection with oxygen. Science 152
(3719), 210-211.
Wessel, S. (1971). Experimental cyanide optic neuropathy.
Arch. Opthal. 84, 194-204.
Wee, Y.C. (1987). The role of poisonous plants in the
greening of Singapore. In Progress in Venom and Toxin
Research. Proceedings of the 1st Asia-Pacific Congress
on animal, plant, and microbial toxins, P. 642-64H.
Wheeler, E.L. and Ferrel, R.E. (1971). A method for phytic
acid determination in wheat and wheat fractions.
Cereal Chem. 48(3), 312-320.
Williams, M.C. and James, L.F. (1975). Toxicity of nitro-
containing Astragalus to sheep and chicks.
J. Range Management 2H (4), 260-263.
Wilson, J. (1973). 9yanide and human disease. In
Proceedings of an interdisciplinary workshop. Lond.
Eng. 29-30th Jan. 1973. I.D.R.C. 12~-125.
Williams, M.C., Van Kampen, K.R. and Norris, F.A. (1969),
Timbe.r Milkvetch poisoning in chickens, rabbits and r
cattle. Am. J. Vet. Res. 30, 21H5-2190.
Wilson, B.J. and Wilson, R.N. (1961). Oxalate formation in
moldy feedstuffs as a possible factor in livestock
toxic disease. Am. J. Vet. Res. 22, 961-963.
'>
375
Woodward,L. (198~). Poisonous plants a color field guide.
David and Charles Publishers Ltd., London.
Woodward, A., and Reed, J.D. (19H9). The influence of
polypheno1ics on nutritive value of browse: A
summary of research conducted at ILCA. ILCA Bull.
35, 2-11.
Wokes F and Picard C.W. (1955). The role of vitamin B in human
nutrition. Am. J. Clint Nutr. a, 3H3-3H5.
Zimmerman, H.J. (1968). Toxic hepatopathy. Am. J.Gastro-
entero1. 49, 39-56.
Zook and Gilmore (1967) Thallium poisoning in dogs J. Am.
Vet. Med. 155, 1329-1332.·
,