STUDIES ON THE A5CARI5-BACTERIA RELATIONSHIP 
IN MAN AND PIGLETS
BY
SHITTU OLADEJO ADEDEJI 
A.I.M.L.S. (London) Bacteriology, 
F.I.M.L.S. (London) Parasitology, 
/ M.Sc.(Ibadan).
A Thesis in the Department of 
Veterinary Microbiology and Parasitology.
Submitted to the Faculty of 
Veterinary Medicine in partial fulfilment 
of the requirements for the degree of
DOCTOR OF PHILOSOPHY 
OF THE
UNIVERSITY OF IBADAN.
\
November.
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ABSTRACT
The paucity of knowledge of the inter-relationship 
of bacteria and Ascaris stimulated this study* Very 
little was known about the sources of Ascaris infection 
in man in Ibadan. Investigations conducted on the action 
of intestinal bacteria on the development of Ascaris eggs 
to the infective stages involved the growing of fertile 
eggs of Ascaris lumbricoidea and Ascaris suum in diluted 
and undiluted overnight broth cultures of Escherichia coli, 
Proteus mirabilis. Streptococcus faecalis (Enterococci), 
Bacillus subtilis. Bacillus cereus. Pseudomonas aeruginosa* 
Clostridium welchii and also in sterile nutrient browth.
The bacterial species used in the experiments inhibited 
the cleavage and development of both the human and porcine 
scaris eggs beyond 2-cell stage. The ovostatic action of 
the bacterial species on the eggs was found out to be 
related to respiratory processes of the actively growing 
and multiplying bacteria which consumed all the available
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c :ygen from the environment of the eggs. After the 
removal of bacteria from cultures, the eggs developed 
very well.
The bacterial flora of Ascaris suum and its 
relationship to the host flora was also investigated.
The body surface of the adult worms was cultured on 
selective media. The different parts of the adult 
wornfs gut were cultured for isolation of micro-organisms. 
The faeces collected from pigs were also cultured.
E. coli. Streptococcus faecglis, Staphylococcus aureus. 
Staphylococcus albus. Proteus vulgaris, Proteus mirabilis. 
Pseudomonas aeruginosa. Lactobacillus acidophilus.
Bacillus subtilis and Candida albicans were isolated from 
the cultures but no anaerobic organism was isolated. The 
faeces cultures yielded the same genera of bacteria as in 
Ascaris suum adult worms, bat Clostridium welchii, an 
anaerobic organism was isolated from the faeces. The
results showed that adult Ascaris suum can act as a vehicle
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of bacterial infections in ascariasis where the adult 
worm is active and migratory. In this case the pig 
bacteria which are limited in its ability to penetrate 
the intact epithelium of the animal can often be deposited 
on internal tissue by the migrating worms where untold 
problems could be set up.
The effect of intestinal flora on the establishment, 
development and pathogenicity of Ascaris suum larvae in 
piglets was also investigated.
The results have shown that development of Ascaris 
suum larvae to adult worms took place in the presence of 
a normal intestinal flora in piglets. Furthermore, the 
results have shown that the two agents (Ascaris suum larvae 
and bacterial species) worked together to produce a disease 
condition more severe than the sum total of effect produced 
by either the worm or the bacteria independently. Finally, 
investigations were conducted to find out sources of 
infection with human ascariasis in Ibadan. The results
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have shown that common food items like fruits, 
vegetables, gari and palm-wine are contaminated by 
Ascaris eggs and therefore could serve as sources of 
Ascaris infection to those people who eat these food items 
raw or uncooked. It was observed that the Ascaris 
infection could occur through contaminated fingers of 
egg-passers, through dust and through the activity of 
flies. Ascaris eggs were found on edible vegetables and 
fruits and this observation emphasises the need for strict 
observation of simple hygiene methods aimed at eliminating 
the Ascaris eggs before consumption of the uncooked food 
items.
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ACKNOWLEDGEMENT
I am grateful to God for sparing my life and for 
seeing me through this study.
I am greatly indebted to my first supervisor, 
Professor E. 0. Ogunba of Sub-Department of Medical 
Parasitology and Applied Entomology, Department of 
Medical Microbiology, University of Ibadan for his help 
throughout the course of this study.
I am equally indebted to my second supervisor, 
Professor 0. 0. Dipeolu, Dean, Faculty of Veterinary 
Medicine, University of Ibadan, for his keen interest, 
motivation, innovative ideas and supervision freely given 
to me during the course of this work.
My thanks go to my senior sister Mrs. Adebisi Abeni 
Akintunde for her contributions both morally and finan­
cially to my education and well-being.
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The assistance rendered by the following people is 
greatly acknowledged;
Professor (dr) Z.Przyjalkowski, Institute of 
Parasitology, Polish Academy of Sciences, Warszawa, Poland; 
Professor A,M. Failis, Director of Research in Parasitology, 
Department of Parasitology, Ontario Research Foundation; Dr,
J.Q. Akinyemi and Mr, Nsoro, Department of Pathology, University 
of Ibadan; Messrs, Ayo Odebumni and 3.0, Layiwola of the 
Department of Medical Microbiology, University College Hospital, 
Ibadan and the members of staff of the Departments of Medical 
and Veterinary Microbiology and Parasitology, University 
College Hospital, Ibadan and University of Ibadan
I thank Mr, S, Akinkunmi for assisting with micro­
photograph and all who participated directly or indirectly 
to the success of this study particularly the Medical Labo­
ratory Scientists in various Pathology Departments of the
University College Hospital, Ibadan,
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I am grateful to Messrs C.Q.J. Ikejiora and 
D.C. Arimah for their secretarial services.
Finally, my sincere gratitude and thanks go to 
my wife and children for their support and for exercising 
great patience and understanding throughout the period 
of this study,
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CERTIFICATION BY SUPERVISORS
We certify that this work was carried out by 
Mr. S.O, Adedeji in the Departments of Medical Microbiology 
and Veterinary Microbiology and Parasitology, University 
of Ibadan.
Professor E. 0, Ogunba,
B.Sc(Hull), H.Sc(Liverpool), M.I,Biol., 
F,R.E,S,(Lond.), Ph.D.(Ib), F.N.S.P.(Nig.). 
Department of Medical Microbiology, 
University of Ibadan.
Professor 0.0. Dipeolu, 
Dip.Tierarzt(Giessen), Dip.Biol. (Txwrp.Par) 
Ph.D.(Edin.), F.R.E.S.(Lond.).
Dean, Faculty of Veterinary Medicine, 
University of Ibadan.
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D E D I C A T I O N
To my late Father and Mother
Pa AKAMBI ADEDEJI 
and
Madam ADEOTI ANIKE ADEDEJI
for their contributions to my education.
MAY THEIR SOULS REST IN PERFECT PEACE
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TABLE OF CONTENTS
Page:
ABSTRACT i
ACKNOWLEDGEMENT v
CERTIFICATION BY SUPERVISORS viii
DEDICATION ix
TABLE OF CONTENTS x
LIST OF FIGURES xv
LIST OF TABLES xx
1. INTRODUCTION 1
1.1 General 1
1.2 Sources and consequences of 6
Ascaris infections in man and piglets
1.3 Objective of the study 11
2.00 LITERATURE REVIEW 13
2.10 Introduction 13
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Page:
2.11 General consideration *3I 0! 
2.12 Epidemiology and Prevalence 16 
2.13 Morphology and Biology of Ascaris 24 
2.14 Comparison of Ascaris lumbricoides 31
and Ascaris suum
2.15 Xnfectivity of Ascaris lumbricoides 
to swine
2.16 Developmental cycle 3}
2.17 A. suum - Bionomics and cross infectivity 46
2.18 Ascaris suum - Life cycle. A p
2.19 Diagnosis of Ascaris infection 492.20 Control of human Ascariasis 59
2.21 Identification of bacterial microflora 68
in human alimentary tract
o oo Ascaris lumbricoides and Bacteria 76
2.23 Inter-relationship of bacteria and 79
intestinal nematodes (Ascaris species)
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Page
3.0 EXPERIMENTS (CHAPTERS)
3.1 Development of human and porcine 82
Ascaris eqqs to infective larvae in 
distilled water, in ?.% Formalin at 
25°C and 27°C and under Actual Field 
condition.
3.2 Action of Intestinal bacteria on the 109
development of Ascaris eqqs in vitro.
3.3, Bacterial flora of Ascaris suum 137
Goeze, 1782 and its relationship to 
the host flora
3.4 The effect of Intestinal flora on the 168
development, infectivity and pathogeni­
city of Ascaris suum larvae in piglets
3.5 Sources of human infection of Ascariasis 223
in Ibadan.
4.0 CONCLUSIONS AND RECOMMENDATIONS 241
4.1 Conclusions 241
4.2 Recommendations 243
REFERENCES 245
APPENDIX 239
1 Calculations of thg.spread of 289
Ascaris lumbricoides eggs on lettuce
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APPENDIX Pag©
2. Calculations of ths spread of 290
Ascaris lumbricoides eggs on cocumber
3. Calculations of the spread of 291
Ascaris lumbricoides eggs on carrots (root)
4. Calculations of the..spread of 292
Ascaris lumbricoides eggs on Mango
5. Calculations of the spread of 293
Ascaris lumbricoides eggs on Tomato
6. Calculations of the spread of 294
Ascaris lumbricoides eggs on Garden eggs
7. Calculations of the spread of 295
Ascaris lumbricoides eggs on Sweet pepper
8. Calculations of the spread of 296
Ascaris lumbricoides eggs on Onions (bulb)
9. Calculations of the spread of 297
Ascaris lumbricoides eggs on Gari
10. Calculations of the spread of 298
Ascaris lumbricoides eggs on Palm wine
11. Direct method of examining faecal material 299
12. Kato cellophane Thick smear method 300
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APPENDIX Page
13. Brine Floatation method 302
14. Magnesium sulphate method 304
15. Formalin-Ether method 304
15. HeMaster egg counting method 306
17. Stoll’s egg counting method 309
18. Beaver egg counting method 310
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LIST OF FIGURES
FIGURE Page
1. Showing different stages of 89
Ascaris development
2. Showing different stages of 90
development. Notice both 
fertile and infertile eggs.
3. Showing fertile egg with 91
advanced morula stage of 
development.
4. Showing Tadpole-like larva 92
and first stage larva inside 
the egg-shell
5. Showing second stage larva 93
coming out of egg-shell.
6. Showing second stage larva which 94
has been expressed out of the
egg-shell.
Notice the sheath of the infective 
larva and the egg-shell.
7 Development of Ascaris lumbricoides 95
eggs in distilled water and in 2% 
formalin at 27°C.
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3.4 FIGURE Page
1. Showing unthriftiness and 191
retarded growth of the infected 
piglets at the end of the
experiment
o• Showing control piglets at the 192
end of the experiment, Notice the 
difference between Fig.1 and Fig.2,
3. Enlarged hepatic lymph node (arrowed) 193
due to synergistic effect of Ascaris
plus E. coll infection.
4. Lung showing area of slight alveolar 194
haemorrhage and congestion of alveolar 
capillaries, Many alveoli are normal.
5. Liver showing areas of fibrous tissue 195
proliferation (repair) of the liver 
. sequel to parasitic infection.6 Lung showing diffuse infiltration by 196
some mononuclear cells indicating that 
the animal is progressing towards 
pneumonia.
7. Lung showing area of necrosis with 197
bacteria colonies and surrounding 
thickened wall. Notice the bacteria 
granuloma formed following initial 
parasitic infection.
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3.4 FIGURE Page
8. Lung showing area of necrosis 198
containing bacteria on the right
with surrounding areas of neutrophilic 
infiltration and an outer sone of 
mononuclear cells infiltration which 
are evidence of parasitic infection.
9. Lung showing peribronchiolara nd 199
alveolar neutrophilic infiltration
due to secondary bacterial infection.
Notice the bronchiole which is intact 
and no infection inside it except 
around it.
10. Liver showing central area of necrosis 200 
and mononuclear infiltration around
the necrotic areas - evidence of 
bacterial infection.
11. Normal liver of P161/83, 201
12. Lung showing cross-section of 202
parasite in a bronchiole. There is
slight peri-b-ronchiolar mononuclear 
cells infiltration around the cross- 
section of the parasite predominantly 
eosinophils - indicating parasitic 
infection.
13. Lung showing parasite sections in 203
bronchi with peribronchial mononuclear
cell infiltration. The intervening 
alveoli are normal.
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4 FIGURE Pago
14. Lung showing longitudinal 
section of parasite in the 
bronchus.
Liver showing area surrounding 
parasitic granuloma; Hepatocellular 
necrosis, infiltration by eosinophils
and few neutrophils.
16. Lung showing area of marked 
mononuclear cell infiltration in 
an area where normal bronchial tissue
has been lo? Miotic© the formation
of fibrous connective tissue.
Lung showing f,3. of parasite in a 90 7
bronchus. Note loss of brinchial 
epithelium (arrowed), haemorrhages 
(arrowed) and marked mononuclear 
cell infiltration into the bronchial 
wall and around the parasite section,
18. Lung showing hyperplastic bronchial 808
epithelium with parasite section in 
the lumen. This is an evidence of 
respiratory ernbarrasment caused by 
the parasite. The area will secrete 
more mucus to wash out the irritant 
(parasite).
%
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3.4 FIGURE Page
19. Lung showing cross-section of the 209
parasite in bronchus with normal 
surrounding alveoli - indicating 
that the parasite was just passing 
through the bronchiole and has not 
destroyed the wall of the bronchiole.
20. Liver showing parasitic granuloma 210
with fibrous tissue capsule surround 
and mononuclear coll (eosinophils) 
infiltration around section of parasite
in centre of the lesion.
21. Liver showing parasitic granuloma 211
surrounded by a zone of cellular 
infiltration and a fibrous connective 
tissue capsule
22. Livor tissue in vicinity of parasitic 212
granuloma showing pseudolobulation.
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LIST OF TABLES
1 TABLE
1. Development of Ascarls
lumbricoides eggs in distilled 
water and in formalin at 27 C.
»
2 TABLE
1. Showing development of Ascaris 
lumbricoides eggs in overnight 
broth cultures of‘different 
bacterial species.
2. Showing the development of Ascaris 
lumbricoides eggs after killing 
the bacterial species with Clorox
3. Showing the effect of different 
concentrations of Escherichia coli 
on the development of Ascaris
lumbricoides eggs
4. Showing the results of the four 
controls set up for each species of 
bacteria used, using Ascaris
lumbricoides eggs.
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TABLE Page
5. Showing development of Ascaris 124
lumbrlcoldes eggs in monoculture 
dilutions of Streptococcus faecalis
during 14 days incubation period
6. Showing development of Asccris 125
lumbricoides eggs in various dilutions
of an overnight broth culture of 
Staphylococcus aureus
?. Showing developing Asccris 126
lumbricoides eggs in various dilutions 
of Pseudomonas aeruginosa
8 . Showing the effect of different 127
concentrations of Proteus mirabilis
on the development of Ascaris 
lumbricoides eggs.
9. Showing the effect of different 128
concentrations of Bacillus cereus on
the development of Asccris lumbricoides
eggs.
TABLE
■s8 • Bacterial flora of the body surface ] 53
of adult Ascari q suum.
9 Bacterial flora of the gut and 154
internal organs orif* male Ascaris suum,
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Cj TABLE Page
3. Bacterial flora of the gut and 155
internal organs of female 
Ascaris suum.
4. Bacterial flora of the crushed 156 
eggs of Ascaris suum.
3.4 TABLE
1. Mean packed cell volumes,. 177 
Total white blood ceils counts 
and differential leucocyte counts 
of piglets infected with Ascaris 
larva and bacterial species.
2. Mean packed cell volumes* 170 
Total white blood cells c aunts 
and differential leucocyte counts 
of piglets infected with Ascaris
only.
3. Mean packed cell volumes#. 179
Total 'white blood cells counts and 
differential leucocyte counts of 
negative control piglets.
4. Summary of data on weight gains of 130
piglets experimentally infected with 
Ascaris and Baotoria.
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3.4 TABLfT Page
5. Summary of data on weight gains of *13  oO  <!j
* ^piglets experimentally infected with
Ascaris only. 101
6. Summary of data on weight gains 182
of control piglets
O0«**? 0 ̂^rf’ TABLE
1. Frequency of Ascaris eggs on 227
edible foods.
2. Effect of Acetic acid and Ethyl ooo
alcohol on bacteria.
 ̂0 Effect of Acetic acid and non
alcohol on development of both 
embryonated and non-embryonated 
Ascaris lumbricoides eggs.
#-h--ss- # x  a--* - a - *•
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INTRODUCTION
General
Microbial inter-relationships are 
involved essentially in the aetiology, patho­
genesis, and pathology of intestinal infection
Qjc ° £
of protozoa and metazoa (Philips ;^195sp. The 
theory that bacteria may participate in the 
aetiology of amoebic dysentery was offered first 
by Losch in 1875, and has been adhered to by other 
investigators including Councilman^tAptjtM/ (1891), 
Walker & Sellards (1913), Cleveland & Saunders 
(1930) and Westphai (1937). However, strong 
clinical evidence in support of the theory was not 
forthcoming until 1946 at which time Ellenberg 
presented data which suggested that symbiosis with 
bacteria may be essential to amoebic pathogenicity.
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Studies on the interaction of host microflora and 
metazoan or protozoan parasites became practical 
with development of gnotobiotic techniques (Reynier> 
1959). In gnotobiotic animals infected with monooultures 
of bacteria the development was much worse (Philips 
et al 1955, Przyjalkowski, 1969, Chang & Wescott, 1972, 
Hungate, 1972, Johnson & Reid, 1973), while in germfree 
animals the parasite either showed no development or 
they developed to a very low degree (Philips _et _al 1955, 
kJescott, 1970, Przyjalkowski, 1973d.) *
However, the nature of the influence of the 
accompanying intestinal flora on the development and 
pathogenicity of parasites in animals is still not 
clear. Possibly nutritional factors come here into 
play, but according to ;Stefanski (1965), a host 
organism plays, with regard to its parasite, the same 
role as the external environment plays for each free- 
living organism. A permanent exchange takes place
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between the milieu of the parasite and the external 
environment and from the parasitological viev/point the 
exchange resulting from the question of food is most 
important. However, the alimentary tract mostly involved 
in this exchange, maintains some stable features such as 
temperature, pH, and Oxygen pressure. This refers mainly 
to the paramucosal environment in which among others, 
the oxygen pressure is greater. . A definite
bacteria flora is present in the alimentary tract and 
it only und4J?goes changes as a result of disease conditions, 
changes in diet and administration of some medicines, in 
particular antibiotics. Each segment of the alimentary 
tract has its own characteristic bacteria^flora and the 
bacteria play an important role in meeting the demands 
for certain vitamins such as vitamins B, E and K groups 
usually produced by commensals of the intestinal tract 
are subsequently absorbed into the blood and 
utilized by the host.
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Both the aerobic, microaerophilic and anaerobic
forms of microflora are seen in the alimentary canal of 
man and domesticated animal. The coliform organisms 
predominate amongst aerobic commensal bacteria, but 
Lactobacillus species excel amongst the microaerophilic, 
Clostridium perfrinqeHS(Clostridium welchii) are, however, 
in the lead amongst the anaerobic ones (Cruickshank, 1965),
Microorganisms alone do not have the exclusive
ability to colonise human and animal body, but large 
organisms as well including members of the group Helminths 
and Arthropoda commonly invade and colonise various 
tissues of man and animal. The nematodes are especially 
an important group because many of their members have 
successfully invaded and established themselves in almost 
all the tissues of man. These nematodes are inside the 
human body and after a period of development, they settle 
down in various parts of the body including the alimentary 
tract to which they have adjusted physiologically and 
morphologically.
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Although both the micro-organisms and the larger 
organisms are individual entities and they are capable 
of independent existence, they ail inhabit a common 
environment of the gut and are subjected at times to 
similar circumstances. Some of them may have entered 
into relationships which are mutually beneficial, others 
may be antagonistic and still others may just have an 
accidental association and are just occuring as transport 
agent. Hutchinson (1965) established a relationship 
between NfrtQscaris egg and Toxonlasm^ transmission in 
which Toxoolasma organisms were incorporated into the 
egg of Nspascaris worm inhabiting the intestinal lumen 
of infected individuals. The excited Ascaris lumbricoides 
has often been accused of being a passive agent of conta­
mination of different tissues of the body with pathogenic 
bacteria from intestinal tract. It will be also infor­
mative to ascertain to what extent the digestive system 
of Ascaris lumbricoides and Ascaris suum are invaded by
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bacteria For instance, Mark-all & Kuritsubo (1967) 
recorded that the trophozoites of Entamoeba coli do
ingest Q.iardia intestinalls and thereby assist in 
keeping down on their population in the intestinal 
lumen.
Sources and consequences of Ascoris infections
in roan and piglets
Gastrointestinal infections of man and domesticated 
animals are still of paramount public health importance 
in all parts of the world in spite of the wide use of 
drugs. The infective agents of gastrointestinal tract
include viruses; bacteria, fungi, protozoa and helminth 
(Banestiva, 1971). In the tropical and sub-tropical 
regions of the world where inadequate personal hygiene 
and environmental sanitation combine with the favourable 
weather conditions to enhance the growth and development 
of infecting agents, the incidence of these infections
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is maximised. The parasite development is enhanced 
because of the favourable weather conditions all the 
year round. The sources of infection with Ascaris 
are generally warm, moist soil, vegetables especially 
leafy vegetables, root vegetables, fruit vegetables and 
water where the eggs flourish well. Ascariasis abound 
among the indegenous populations in many parts of the 
world where personal hygiene and environmental sani­
tation combine to favour embryonation of eggs on the 
polluted soil in the environments.
Intestinal nematodes abound in the rural and urban 
environment in this country because our tropical climate 
offers excellent opportunities for easy and rapid deve­
lopment of different stages of these parasites which 
are readily disseminated in our soil through our gross 
and indiscriminate defaecation habits. The Ibadan city 
presents an ideal environment for the study of the pre­
valence and problems of intestinal nematodes of man and
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animal in Nigeria because of the low standard of 
public sanitation (Cowper & Woodward, 1961; Ogunba, 1974)*. 
Furthermore, because of lack of necessary health education 
resulting in our low standard of personal and environ­
mental sanitation, the Ibadan population provides 
opportunities for constant contact between ourselves 
and infective stages of the worms. Some authors 
(Cowper & Woodward, 1961, and Ogunba, 1974) showed 
that infectivity with the trio Ascaxis lumbricoides, 
Hookworm and Trichuris trichiura worms was always 
common amongst the Ibadan population. They also showed 
that in an environment which is highly polluted, oppor­
tunity for infection with one nematode opens the gate 
to several others at the same time. Furthermore, Ogunba, 
(1974) showed that the infectivity with intestinal 
nematodes varied in the ten zones of Ibadan that he 
studied which had varying degrees of sanitation. Infec­
tivity was heaviest in zones with the lowest degree of 
environmental sanitation.
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Ascaris infection hc.sm varied effects on man and 
animals. Therefore the consequences of Ascaris infection 
in man and piglets are great. Both man and animals 
living in areas of low degree of evironmer.tc! sanitation 
are staunted, anaemic and generally in poor health. It 
is a well known fact that adult Ascaris rnay wander up 
and down the intestine and into other organs outside 
their usual route. They may thus enter the stomach 
and may be vomitted, or they may enter the bile-ducts 
and occlude these, causing jaundice and other problems. 
Other symptoms may be caused by their migration elsewhere. 
There is in fact hardly any organ of the body of man and 
pig in which Ascaris has not been found. For instance, 
i^eautyman & Woolf (1951) found Ascaris larva in the 
brain of a child. These wandering ascarids reaching 
abnormal foci- provoke acute symptoms. Cerebral symptoms
may occur in children infected with Ascaris in the
\  ..............
absence ' of other helminthiasis or malaria. The most
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common symptoms attendant on the presence of Ascaris 
in the intestine are vague abdominal discomfort and 
acute pains in the epigastric region, (SV*drtzwelder, 
1946). Ascaris may possibly suck blood from the 
intestinal wall (Brown, 1934). In children, the 
presence and activity of worms are characteristically 
associated with fever. At other times, the symptoms 
may suggest abdominal tumour, gastric or duodenal ulcer. 
In the pigs, there is marked unthriftiness which may be 
so severe that the animals die; where they do not die, 
they have a very reduced market value.
In the early stages of infection with Ascaris, 
the migration of the larvae through the lungs may give 
rise to numerous minute haemorrhages, oedema and 
exudation; in severe infections, the worms may produce 
symptoms resembling lobar pneumonia and may cause death
in both man and animal.
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14 3 Objective of the Study
Very little is known about the role played by 
Ascarls of man and that of pig as a passive transport 
agent of contamination of different tissues of the body “* 
with pathogenic bacteria from intestinal tract. Further­
more, very little is known about the relationships which 
exist between the microflora and nematodes, particu­
larly Ascaris lumbricoides and Ascaris suum in the 
intestinal tract of man and pigs. Apart from the 
work of Philips et al, (1958 and 1964), Wescot$ (1970) 
and Hungate (1972) little is known about the relationship 
which exist between metazoa, protozoa and bacteria 
inhabiting man and animal. The literature in this 
field is scanty and there has been no report on this 
subject in Nigeria. The first report (Adedeji, 1981) 
was based on the microflora and nematodes that occur 
simultaneously in human gut.
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This investigation ^therefore to
(1) determine the actio*! of intestinal 
microorganisms on the developmental 
stages of Ascaris of man and pig;
(2) show what happens to embryonated eggs 
in an environment with different levels 
ef bacterial contamination;
(3) show particularly that migrating larvae 
. of Ascaris can carry with them micro­
organisms and thus transmit and, or 
aggravate infection with micro-organisms;
(4) determine to what extent the digestive 
system of Ascaris suum of pig are invaded 
by bacteria and compare this with the 
bacteria flora of the host;
(5) show the sources_of human infection with 
Ascaris lumbricoides in Ibadan.
It is believed that more information would be 
available to us at the end of this study about hitherto 
unknown aspects of Ascarissie in Nigeria.
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LITERATURE REVIEW
• iSS =S = Z = = SS = = SSS2SV'
2.10 INTRODUCTION
2.11 General Consideration
Ascarls lumbricoides, the largest of the human 
intestinal nematode which resembles the common earth­
worm was first discovered by Linnaeus (1758). Apart 
from being the most common helminth of all intestinal 
nematode Ascaris lumbricoides has a cosmopolitan distri­
bution throughout the world, and is most prevalent in 
the tropics and subtropics especially in rural populations. 
It thrives best in warm moist climates or in moist 
temperate regions where poor standards of personal 
hygiene and environmental conditions combine to favour 
embryonation of the eggs which are very resistant to
adverse conditions
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Ascaris is the cause of Ascariasis among children.
A wide variety of clinical symptoms is met with in such 
cases, often the disturbance is very severe and ends 
fatally. Williams (1938) observed that very little 
attention is paid in text-books of medicine to the 
condition, so that practitioners get little guidance 
in diagnosis and treatment of the subject. Perhbps, 
ono reason for this obvious neglect of an important 
subject is the paucity of literature on clinical studies 
of Ascariasis. A search of the literature reveals a 
wealth of experimental and laboratoary work on the subject 
and also a multitude of references dealing with isolated 
cases of complications including toxicity, but very few 
articles exist in which the subject is treated as a whole or 
which record the results of clinical studies of series 
of cases illustrating the various symptomatology, the 
relative frequency and incidence complication, their 
exciting causes and manifestations, and the mortality rates. 
Such studies are necessary if the importance of Ascariasis 
is to be more generally recognised.
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As early as 1864, Cobbold noted that the human 
ascarid, Ascaris lumbricoides, is a different species 
from the equine ascarid Ascaris meqalocephalG. and 
swine ascarid Ascaris suilla? but it was not until the 
appearance of Neumann's paper "Surl1* Ascaridae des 
Detes Bovine" (1083) that the bovine ascarid was generally 
accepted as a valid species. It was previously described 
as Ascaris lumbricoides or Ascaris megalocephala. Meumann*s 
study of this worm showed, however, that the human Ascaris 
differs in many anatomical characters from the bovine one. 
Later investigators classified both the human ascarid 
and the bovine ascarid under the genus Ascaris. Conse­
quently Stewart, 1916a, 1916b,1916c,1916d, 1917, 1918 
referred to the pig ascarids as Ascaris suilla, (now known 
C!S Ascaris suum). Ransom & Foster (1917, 1919 and 1920^ 
and Ransom (1919) referred to the human ascarid as Ascaris 
lumbricoides, while Martin (1926), Schwarts (1922)
Griffiths (1922), Boulenge^^ Hacfie (1922), referred to 
the bovine ascarid as Ascaris vitulorum. The present
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«•*
systematic position of Ascarls according to Craig &
Faust (1970) is as follows;
Super family,; Ascaridaidea (Railliet & Henry, (1915)
Family; - Ascarididae (Blanchard (1849) or 
Ascaridae (Cobbold, 186̂ J-)
Subfamily; - Toxocarinae (Hartwich, 1954)
Genus: Ascaris (Linnaeus, 1753)
Species; Ascarls lymbricoides (Linnaeus, 1758) 
Ascarls s'u'urn' (Goeze, 1872)*
2.12 Epidemiology and Prevalence
Although Ascaris iumbricoidos affects all ages, 
it is more common in young children. A similar 
occurrence has been observed by Ransom & Foster (1920) 
in a closely related sop,, , Ascaris suum of swine in 
which maximum infection is limited to between the third 
and fifth months of birth, although infection does
extend beyond this period in some cases. However, in
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-17-
tha human Asccris, incidence of infection is
higher in children than in adults (Brown, 1927, Cort 
1929 end 1931 , Cort & Otto 1933 , Otto & Cort 1934 , 
ileadlee , 1936. y Winfield 1937,, Scott ,1939 Weir jet alf 
1952) and Chandler 1954). This is because human 
ascariasis is a household infection, primarilly 
propagatted by seeding of the soil immediately around 
the house with eggs present in the excreta of small 
children who in turn become reinfected from eggs which 
they pick up on their fingers and introduce into their 
mouths. The fact that workers like Grassi (1888),
Lutz (1880) and Clandruccio (1386) cited by Steward 
(1916a) could successfully infect themselves and adult 
volunteers, besides the incidence of adult ascariasis 
in tropical and subtropical countries (Brown ,1927 ,
Cort et al ,1929 and 1930 Headlee v1936 Woir> \f 
i
1952, and Chandler 1954)) shows that unlike the 
bovine and to some extent the swine infection, the 
acquisition of human ascariasis is not limited, to
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-18-
children. The essential difference between neo- 
ascariasis of calves and ascariasis of young pigs 
is that, while in the former infection is mainly 
prenatal, in the latter it is always oral as in 
human.
Martins (1926) who investigated the possibility 
of neonatal infection with Ascaris suum in pigs had 
this to say "from the data presented, it seems 
evident that intrauterine infection in swine is very 
uncommon, and if this phenomenon occurs at all in 
nature, it must be looked upon as being nothing ,more 
than a biological curiosity". Later, Wall (1958) and 
Taf££(1961) have also been unsuccessful in demonstrating 
prenatal infection in pigs with Ascaris suum.
In some countries in Europe and Orient where human 
excrements are utilized as fertilizers, field cultivators 
and vegetations are exposed to the infection (Craig & 
Faust, 1970). The infection rates reported by the
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I'orld Health Organisation (WHO), 1967 are as followss 
Italy, 75$ of 354 children in Rofrano, 40$ of toddlers 
in Naples, 12$ of .9126 children in San Marino, 9% of 
297 School children in iiafera, 2.5$ of 550 University 
students in Pisa; 21$ of 200 persons less than 10 years 
of age in Spain, 40-80$ in Portugal, 7-9% and up to 20$ 
in Yugoslavia and Czechoslovakia respectively. Albania; 
18.3$ of 283 pre-School children, Roumania; 18.3$ of 
124,420 persons surveyed. Significant infection levels 
were found in Poland and in many parts of USSR.
Although ascariasis is uncommon among urban 
population in Belgium, Prance and Germany (Craig & Faust, 
1970) high prevalence, especially among children is 
occasionally reported from rural communities (Craig & 
Faust, 1970). In France overall prevalence among 3,797 
School children was 17.8$; in on® group of 162 School 
children it was 46$. In Japan in 1961, 2- 5$ of the 
population in large cities were found to be infected;
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in rural areas the infection rate averaged 20$. It 
was observed by Craig and Faust, 1970 that ascariasis 
is highly prevalent in China. In most countries in 
central and South America, the average infection rate 
was approximately 45$. Small foci of high prevalence 
were found to persist in the South-eastern United 
States of America. Jeffrey et aJL (1963) reported an 
overall prevalence of 63.7% in rural population of 
South Carolina, the highest prevalence being in the 
six-eleven-year age group.
The Nigerian Experience
In Nigeria, Cowper and Woodward (1961) recorded 
2.6,5% infection rate in 21,700 stool samples examined 
routinely at the University College Hospital, Ibadan 
over a three-year period. Okpala (1956) found an 
infection rate of 73% in 4,700 children in Lagos and 
later (1961) the same authov recorded 71.5$ infection 
rate in 510 Government Workers in Lagos. Nnochiri (1965. )
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found 52/o infection rate in 42 patients seen at 
Lagos University Teaching Hospital outpatient 
department. Narsdon (1960) recorded 53$ of a series 
of 500 infected patients in Lagos, and Fisk (1939) 
found Ascaris in 90 out of a series of 120 post­
mortems in Lagos. In Ibadan, Cowper & Woodward (1960) 
found Ascaris in 38 out of a hundred employees at the 
Floor Plantation Agricultural Centre, Gilles (1964) 
recorded infection rate of 70% of 600 villagers at 
Afeufo near Ibadan. Hins, (1968) recorded infection 
rate of 52.3$ of 230,197 people of nine Southern Nigerian 
States he examined. Ogunba (1970) found 29.8$ 
infection rate of 12,384 hospital patients in Ibadan 
and Abioye and Ogunba (1972) found that 64.6$ of 1,272 
School children examined in Ibadan .harboured Ascaris 
ova while Ogunba (1974) recorded 69.6$ of the 1,130 
School children in Ibadan positive for Ascaris infection.
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Obiamiwe (1977) found 19,5% of 6,213 peopl© of Benin 
City positive for Ascaris infection. Further north, 
the prevalence becomes lower. Ramsay (1934) found only 
6% of over 7,000 persons examined in Jos, Plateau State 
infected and Collard (1962) in a survey in Katsina 
Province found ova of Ascaris on only 2,1$ of 536 Mabe 
and 0*4$ of 236 Fulani examined, Ascaris infection is 
thus significantly lower in the drier north compared 
with the humid climate of Southern Nigeria,
Stoll (1947) estimated Morld incidence of Ascariasis 
as 644.4 million, consisting of 30 million in North America 
42.0million in tropical America, 59.0 million in Africa, 
32*0 million in Europe, 19.9 million in USSR, 488.0 
million in Asia and 0.5 million in Pacific Islands. It 
has been estimated that one out of four people in the 
’.Jorld's population was infected with Ascaris, and that 
this parasite has a very high prevalence in overcrowded
towns of non-industrial centres and rural communities
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whore human faeces are utilized as fertilizers (WHO,1967). 
It is observed that there are differences in the altitude 
of various countries to the problems of ascariasis and 
it has been noted that the public health significance 
of ascariasis has not been assessed. Measures aimed 
at improving general sanitation and the self medication 
of infected individuals had little effect on the pre­
valence of the disease, yet in the USSR and Japan 
energetic measures have been introduced to control the 
infection.
In many parts of Europe, the parasite is still 
widespread, and the infection rate in children in some 
areas of the U.S.A. is still more than 20^ (Otto &
Cort, 1934). In communities when no effort has been 
made to control the disease, it was observed that 
intensity of infection was highest in the 1-4 age 
group (Otto & Cort, 1934).
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2.13 Morphology and Biology of Ascaris
The superfamily Ascariodoidea (Railiiet &
Henry, 1915) AS fairly large or stout worms, having 
mouth provided with three conspicuous lips but lacking 
buccal capsule. The males are without bursa corpulcrtrix 
and usually without candal aloei Human representative is 
Ascarijs jum.bricoidGs, the largest of the human intestinal 
roundworms causing ascariasis. The pig representative 
is Ascaris suum. Like all other nematode species 
present in human gut, Ascaris is typically elongated, 
cylindrical in shape with a body cavity in which the 
organs lie. It is primarily bilaterally symetrical 
but with a secondary tri~radiat® symmetry at the 
exterior end. The alimentary canal is characteristi­
cally a straight tube with a mouth and anus. The 
sexess are separate. The head portion is adorned with 
3 large denticulated lips. The three lips lie, one 
dorsally and the other two sub-ventrally and they
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are finely toothed. It is brownish-yellow in colour.
The male is a little smaller than the female, having 
a length of about 25cm and maximum broadth of 4mm.
Its posterior extremity is definitely pointed, and 
curved ventrally in the form of a hook. Two curved 
spicules can often be seen protruding from the orifice 
of the cloaca, around which there are large numbers of 
minute parpilla. The female measures up to 35cm in 
length and has a maximum diameter of about 5mm. The 
posterior extremity, though conical, is not pointed 
or ventrally curved as in the male, but straight. The 
vulva is very minute and situated ventrally at the 
junction of the anterior and middle-thirds of the body. 
It leads into a stout duct, the vagina which divides 
into two branches; these tubes lie anteroposteriorly 
within the worms. That portion of each duct close 
to the common vagina functions as the uterus. The 
middle part is the oviduct and the distal part the ovary
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(Chitwood & Chitwood/ yfc9f74' ; : Clr Aa ti ■g, \ & Faust, 1970
" /-’A  'U,os)J_ . \and, pfjvey & Crew£ 1973). Ascaris. lumbricoides eggT Qr : ....
was first described^ byiiinnaeus (1750) as being round
* * ; • f /
or oval in shape, brownish in colour and it measures
about 60yum in length by aFout"45yum in breadth. The 
egg has a thick transparent shell., Wharton (1915) 
reported that the Ascaris lumbricoides egg is surrounded 
by a shell and an outer albuminous layer. Nelsgp (1851) 
reports^that the number of layers surrounding the egg 
of ascarids is three and in the case of human ascarids 
Christenson (1940); Krenser (1953) and Frenzen (1954) 
disagreed with Nelson (1051) and they stated that 
Ascaris lumbricoides egg has four layers. This report 
was confirmed by RogerS(1956) using electronsicroscope 
in the study of the eggs of Ascaris lumbricoides varj 
suum. The eggs are noted for their great resistance 
to extremes of temperature and chemicals. Davaine 
(1863) found the ova of Ascaris lumbricoides remained 
infective after storage for five years and Bailliet(1866)
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observed that the eggs of A.suum remained viable
after twelve months exposure to heat of summer and to
the cold of winter. The effect of various temperatures
on development of Ascaris eggs has been studied by
Paure-Fremiet (1913) who showed that the ova of A.suum
are capable of deveipment at varying temperatures below
o5°C, but that the optimum temperature is about 30°C.
Wharton (1915), determined that at a temperature around
37°C, the ova of Ascaris lurnbricoides developed to the
eight-cell stage and then died. As reported by Nolf
(1932), the ova of human Ascaris are more resistant to
the effect of high temperature exposure than those of
Trichuris, as shown by the fact that 6Z% of Ascaris ova
eventually became embryonated following exposure for
three minutes at 35 oC whereas no Trichuris ova survived 
similar treatment. Ogata (1925) studied the effect of 
heat in the destruction of the ova of Ascaris lurnbricoides 
and found that no eggs developed following exposure to
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70°C for one minute, and that they were unaffected 
by exposure to temperatures up to 45°C for a period 
of one hour. The eggs remain infective for several 
months in the soil and on contaminated objects. For 
example, infective eggs have on many occasions been 
known to be transferred from contaminated fingers to 
paper money (Dolt & Theme, 1949). Reptiles and amphi­
bians are known to be among the various vessels of 
transmission of infection. Marinkell & Williams!1964) 
showed that reptiles may act as potential mechanical 
vector of A.lumbricoides, as they found 206 <£u rinam 
toads heavily loaded with the eggs.
Although the eggs are resistant to extremes of 
climatic conditions, even with long freezing spells, 
dossication is however, lethal to them.
The fertilized egg undergoes the first pre-parasi
0 o
moult in 14 days to 18 days at 22 - 26 C opt. Lower
temperatures prolong this period. The second moult
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occurs about 1? - 14 days later, when it becomes 
infective. In all the egg requires about 30 days to 
become infective after being passed in the faeces under 
optimum conditions of temperature, oxygen and moisture. 
In effect, the bionomic requirements of the Ascaris egg 
are similar to those of the Xtronavloides.
Favourable seed beds of Ascaris eggs may remain 
infective for many years from which infection can be 
obtained. The indistinguishability of the egg of the 
human species of Ascaris and the porcine variety was 
confirmed by Taff$& Voiler (1963) using fluorescent 
antibody studies. They were also able to establish ; 
that both species share common antigens.
Unfertilized eggs aI<~o ^ccur and are very common 
in human faeces. This may be duo to the production of 
large number of eggs by female Ascaris lumbricoides
and some of these eggs escape fertilization probably due
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to absence of male worms in the host to effect
fertilization. An unfertilized egg is more elongated 
than the normal egg. It has a small protein coat and 
atrophied ovum, marked by numerous highly refractile 
granules. In some cases, the mamillations commonly 
seen in fertilized eggs is lost and the egg is then 
at times difficult to recognise. Hurra & Nishiuchi 
(1902) gave a comprehensive discussion of the appearance 
and occurence of the unfertilized eggs of Ascaris 
lumbricoides. Huira and Nishiuchi (1902) dealing with 
these unfertilized eggs and Fsaterc(1914) /considering 
the unusually large fertilized eggs sometimes seen, 
have pointed out that the diameters of all the different 
types of eggs of Ascaris lumbricoides are essentially 
the same, the variation in size being primarily in 
length. As already mentioned, solitary females or 
several females without males, are found when a person 
passing only unfertilized eggs is treated. Again, this 
may be due to the females never having been in copula 
or the supply of spermatozoa received by copula having
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already been exhausted. Proof that copulation takes 
place more than once during life of the worm is 
equally difficult to obtain. However, Looss (1905) 
and Herrick (1920) both suggest from experimental 
evidence with hookworm that it does take place more 
than once. Thus a female probably does not receive 
and store in the seminal receptacle enough spermatozoa 
from one copulation for the fertilization of all the 
eggs to be produced. Hence if a solitary male were 
to be dislodged after its copulation, the female might 
easi ly use up its supply of spermatozoa after a time 
and produce unfertilized eggs thereafter.
2.14 Comparison of Ascaris lumbricoides 
and Ascaris suum
The human and pig adult Ascaris species are 
almost identical, Sprent (1952) established host 
specific differences within existing species through 
variations in size and shape of the dentigerous ridges.
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He observed that the denticles of the pig Ascaris
form a conspicuous row of more or less equilateral 
triangles, while the denticles of the human Ascaris 
are relatively much smaller and their edges are concave. 
Crewg,& Smith (1971) corroborated Sprent' s observation 
in making morphological distinction between the human 
and the porcine species by examining the teeth on the 
dentigerous ridges on a worm passed by a child. This 
worm was compared with random samples of specimens of 
Ascaris from the teaching collection at the Liverpool 
School of Tropical Medicine which had been obtained from 
pig and'man respectively, and they were identical 
with Sprent*s description, thus proving morphological 
difference between the human and the porcine strain 
based on the denticular arrangements. Dipeolu & Sellers 
(1977) confirmed that the Ascarids retrieved from the 
pigs were human ascarids (Ascaris lumbricoides) which
is highly suggestive of the transmissihility of the
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human strain to pigs, especially those roaming freely 
and foraging for food on dumps of household refuse 
and human excreta.
2.15 inf activity of A. lurnbrlcoides to Swine
Immunity of the pig to ascariasis has been 
regarded by helminthologists for some years as 
problematic. Ransom (1922) was of the opinion that 
the pig attains immunity at the age of 4 months, but 
this was subsequently refuted by Morgan (1931) who 
successfully infected pigs at 10 months of age, and 
Clapham (1934) at the ago of one year. Sandground 
(1929) referred to the difficulty in establishing 
infections under normal conditions, however, the fact 
that almost every pig under field conditions picksup 
an infection at an early age is well known. Clapham 
(1934) bottle-fed 8 pigs from birth and free from 
infection. These were experimentally fed with large
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number of eggs of A.lumbricoldes of pig origin. Only 
4 infections resulted from the experiment.
A number of pigs of various ages and of known 
past history were used in an experiment. The Ascaris 
eggs were of pig origin. Two pigs aged 8 weeks were fed 
with a large number (unestimated) of infective eggs of 
A. lumbricoides in an emulsion ®f Th®r® w®t® n<s
s£gn» of their migration through the liver and lungs, 
and no eggs were demonstrated in their faeces during the 
next four months. At the age of 6 months, these 2 pigs 
were again fed on enormous number of eggs in water as 
before. One pig died 6 days later, following signs of 
pneumonia. Larvae were recovered from both lungs and 
liver in large numbers when teased in physiological 
saline, and the tissues all showed lesions usually asso­
ciated with Ascaris migration. The other pig survived
and later survived a severs attack of diarrhoea lasting
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about a week. With the restoration of the metabolism 
of the host, its general health improved. The pig 
became "clean" again in 23 weeks and a month later was 
successfully infected again with Ascaris eggs.
Subsequently 4 clean pigs 11 weeks old, and reared 
from a clean sow were used in an experiment, and these 
were fed with Ascaris eggs. On this occasion, the 
technique adopted was aimed at approsiunating the method 
to more natural conditions. Therefore, a number of small 
doses were given daily. The eggs were rather old and 
the culture not more than 50% infective. Each individual 
pig was given a feed of 200 infective eggs for 10 days 
giving a total of 4,000 eggs, with an experimental error 
of not more than 5%, Although empty shells were observed 
in the faeces following administration, there was no 
reaction until the 4th day when the pigs were less 
inclined than usual for their food. Between the 6th to
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the 12th day after the first feeding, they were 
lethargic. One pig did not exhibit symptoms, but the 
other three developed a persistent cough and breathing 
was definitely laboured. At the end of a fortnight after 
the first dose and 4 days after the last, the symptoms 
began to moderate and the animals made a recovery.
Ascaris eggs were later demonstrated in the stools 
showing that the infections had been successful.
Later another pig, aged 9 weeks, was fed with 
Ascaris eggs, 200 at a time, once a day in the food 
making a total of 2,000. This animal developed a slight 
oough with respiratory difficulties. The susceptibility 
of the animal regardless of the small dose was ascribable 
at least in part to the fact that it was the smallest one 
of the litter though it was perfectly normal and healthy.
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This recalls ffoino*s experiment published in 1922, 
when a young man 21 years old showed pneumonia symptoms, 
following a dose of 500 eggs of Ascaris suum from the pig. 
Similar symptoms were observed by Koino himself after 
swallowing 2,000 Ascaris eggs of human origin. As early 
as 1888, Lutz fed a human patient with 100 eggs and later 
obtained 35 immature Ascaris 'worms, with a percentage 
closely approximating to that obtained by Koino. The 
experiments of Clapham (1934) would suggest that a very 
hopeful method of inducing an infestation with A.lumbricoides 
in the pig is by means of a series of comparatively small 
insignificant doses.
2,16 Developmental Gycle
The development of Ascaris lumbricoides is at 
present universally considered to be direct (Stewart, 19/7).
It is well known that the eggs passed in the faeces of man, 
undergo development in the outeriDorld up to the formation
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of a motile vermiform embryo. Infection is acquired 
through the ingestion of fully embryonated eggs which 
have been accidentally picked from polluted soil, from 
food or drink contaminated by viable embryonated eggs 
or by children who eat dirt. The second stage larva 
contained in the egg is released in the small intestine, 
the shell having been acted upon by enzymic action of the 
intestinal juices. The released second stage larvae 
reach the liver within 6 hours of infection where they 
moult to the third stag© larvae. Four days post-infection, 
still in the third stage larvae, they leave the liver 
through the blood stream to the lungs, from whence tracheal 
migration commences. The two parasitic moults occur after 
they have reached the small intestine, Douvres (1967) 
states that the moult to the fourth larval stage occurs 
about the 10th day, in the intestine, this being contrary 
to the views of Roberts (1934) who stated that this moult 
occurred in the respiratory system and that only fourth stage
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. 3
larvae were able to survive the acid environment of the 
stomach.
Douvres, Tomba and Matakatis (1969) confirmed the 
view that hatching of the eggs containing second stage 
larvae of A.suum in the swino occurred in the small 
intestine from whence migration proceeds to the liver 
within 6 hours from infection. The first parasitic moult 
occurs in the liver. The third stage larvae leaves the 
liver to the lungs via the blood stream four days post­
infection. The remaining two parasitic moults occur in 
the small intestine after completion of tracheal migration. 
This hypothesis is based on the work of^Davaine,
1863, Grassi, 1887-88, d^tcmdruccio 1986, Lutr, 1887-90, , 
apstein .1892 ,Jammos & Martin,1906 J,& Martin & Wharton 1915
Davaine (]8{p̂ ) administered ripe eggs to rats and 
found that after 12 hours, living larvae were found in 
the lower part of the small intestine. He also introduced
ripe' and unripe eggs in glass capsules closed with linen
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into the alimentary canal of the dog and found that 
after-the lapse of a certain period the ripe eggs had 
disappeared, whereas the unripe eggs remained. He 
concluded that hatching and development occurred in the 
alimentary canal of the definitive host. Grassi, (1880) 
administered ripe eggs to himself and two months later 
found eggs in his stool. Olandruccio (1886) cited by 
Stewartl (1917) successfully infected a child of ten who 
had previously suffered from worms but had been relieved 
of these parasites by antihelminthics. Lutz, (1807-08) 
fed an adult on ripe eggs and found evidence of the 
subsequent appearance of adult worms. Epstein (189.0) 
successfully infected a man. James & Martin (1906-8’'; 
allowed ripe eggs to hatch in artificial and natural 
solutions and found that hatching took place readily 
en masse in 0,8% salt solution at 37°C-40 C. Martin 
hfharton (1915) fon^d that the embryos of the 
ascarids from calf, pig, horse and dog, hatch in alkaline
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solutions and that when developed eggs are introduced
into the alimentary canal of an animal, they pass through
the stomach unaffected and only hatch after they have been
subjected to the action of the alkaline juices of the
intestine. Wharton (1915) states tlhat direct infection
can take place, but that the embryo must be completely
v\,o^
developed. He doesAgive the period necessary to secure 
this complete development.
In spite of the general acceptance of the hypothesis 
that infection takes place by the direct method, there 
is a considerable bulk of evidence against it. Davaine 
(1858) administered three to four hundred ripe eggs to 
an ox, an animal which is stated to harbour Ascaris 
lumbrieoides and found that after four months, no worms 
were present in the intestine. Leuckart (1867) fed a 
rabbit on ripe eggs and found no worms after ten days; a
dog was also treated and was equally unresponsive after
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14 days. He also fed a pig for 3 weeks on several 
thousand of ripe eggs and did not find any worms on 
section.
Although Fulleborn (1922) suggested that certain 
Ascaris parasites of terrestrial mammals may utilise 
intermediate host, this possibility has been overlooked 
since the discovery by Stewart (1917) and (1910a) of the 
life history of Ascaris lumbricoides. Stewart, having 
discovered the lung migration of the larva© in rodent, 
at first concluded that rodents act as intermediate host 
in the spread of this parasite, but later 'in (1918b, 
1919, and 1921) concluded that only one host was used.
The observation by Ransom and his co-workers (1917,
1919, 1920, 1921, 1922), Martin (1926), Roberts (1934) 
and de-Boer (1935) have amply confirmed that pigs may
become infected with Ascaris lumbricoides directly by
ingestion of ,embryonated eggs, the larvae migrating through
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the liver and lungs before they reach maturity in the 
intestine. Koina (1922) demonstrated that human strain 
of At lumbricoides makes a similar migration through the 
human body and confirmed earlier reports (See Martin/ 1926) 
that human infection results from the ingestion of embryonated
From the above observation of earlier workers, it is 
apparent that it has been a rather difficult task explaining 
the early migratory course of Ascaris larvae on any basis 
other than its presentation of panoramic view of the 
evolutionary history of these worms. It appears that this 
migration results in the dispersal of the parasites throughout 
the body, inside as well as outside of the hepatic - pulmo­
nary routes, and into organs and tissues from which they 
probably cannot extricate themselves rather than the circui­
tous journey of the larvae from the intestines to the lungs 
and back again to the intestine, and it does not appear to 
be an adaptation that has survival value for the species i.e.
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A suum and A. 1 umbra.coides» Fulleborn (1922) in 
accounting for the "superfluous" migratory route of these 
larvae suggested that Ascaris might have evolved from 
ancestral forms that penetrated the skin. Later, he 
advanced a more logical hypothesis suggesting that this 
larval migration probably points to the derivation of 
these worms from ancestral forms that migrated in an 
intermediate host.
Investigators who reported failures in their 
attempt to develop Ascaris in the intestine of pigs had 
no difficulty in raising the parasites in pigs to the 
stage that they attain in the lungs and, to some extent 
beyond that, in the intestines. There appears to be 
practically no known mammalian host specificity in the 
early development of Ascaris. A.lumbricoides as well as 
A.suum has repeatedly been shown to produce signs of 
pulmonary changes in pigs and other mammals. Host speci­
ficity becomes evident only when the worms return to the
intestines,*. It is observed that in small mammals, e.g, mice, 
rats, guinea-pigs and rabbits, A, suum passes out of the
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body shortly after returning to the intestine.
„ In goats, and more so in sheep and cattle,
A. suum has been found to be capable of developing in 
the intestine and may attain about half its normal size.
Half grown A.suum, the female containing eggs that 
could be cultured to the embryonated stage were found in 
a calf.
The fact that the behaviour of A.suum in pigs often 
parallels its behaviour in a strange host presents an 
anomaliy that is difficult to explain, except by postulating 
the development of a morked resistance to these worms by 
the host. This resistance could occur particularly during 
the wondering period of the larvae through the body.
Schwartz (1959) is of the opinion that the migration 
of newly hatched larvae appear^ to be ^phylogenetic signi­
ficance and probably points to the evolution of these worms 
from ancestral forms that had an indirect life cycle. On 
the basis of this assumption, he concluded that the life 
of A.suum and of related species apparently has become
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compressed into one host, although there is still some 
evidence of an indirect development with one phase 
culminating in the lungs and the other in the intestine.
2.17 A.suum - Bionomics and Cross infectivity
A. suum has a common o c C l i i n  domestic pigs, 
especially amongst free roaming ones foraging on garbage 
heaps in rural areas. 75% of pigs in the United States 
of America and Canada are infected before they are 6 month 
old. The principal effect is stunting of growths. S 
Spindler (1947) found that pigs infected with 20 or more 
worms at 8 weeks of age did not gain weight. One pig 
with 109 worms did not gain weight at all when compared 
with uninfected pigs gaining an average weight of TOOlbs. 
(Chandler & Read, (1961)).
A.suum exhibits sexual dimorphism, the females 
often longer (25-40cm) and stouter than the males (15- 
25cm). The adults are provided with three denticulated
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lips. These large worms inhabit the small intestines of 
the pigs. It is a common parasite of pigs in Nigeria. It 
is morphologically and serologically identical to A. 
lumbricoides of man. Dipeolu and Sellers (1977) observed 
in an investigation conducted in Eruwa, Western Nigeria, 
on the endoparasites of indigenous pigs that out of eleven 
(11) helminth species identified, A.sutfm was in the lead 
with highest incidence of 92$, Although for sometime, the 
specific identification of A.lumbricoides and A.suurn has 
posed a problem to many scientists, Sprent (1952) claims on 
the morphological basis that both are identical, evon 
though epidemiological evidence and experimental infections 
appear to indicate that there is a difference in the two 
parasites, Sprent (1952) carrying out comparative studies 
on human and porcine ascarids showed that the pig ascarid 
form a conspicuous row of more or less equilateral triangles 
and that the edges of the denticles are straight. In 
contrast, the denticles of the human strain are considerably 
less conspicuous, beingsmaJLl.fr and possessing concave edges. 
This observation was confirmed by Crew & Smith (1971) in
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a morphological study of a worm passed by an infant.
As regards the cross infectivity, patent cross 
infection trials were carried out by several workers but 
they did not establish any proof. However, Clapham (1934) 
was able to infect young colostrum deprived piglets with 
human species.
2,18 Ascaris suum - Life Cycle
The ingested eggs hatch in the intestine and the 
larvae burrow into the wall of the gut. They pass through 
into the peritoneal cavity and thence to the liver? but 
majority reach this organ by way of the hepatoportal blood 
stream (Soulsby, 1973), The eggs of A.suum will hatch 
and the larvae migrate in many animals species, including 
man. Much work has been done with A.suum infections in 
guinea-pigs, rabbits, rats, and mice and in these, the 
migratory cycle is much the same as in the pig* There
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are slight differences in si^e of larvae from various 
animals, thus larvae from mice are smaller than those from= 
swine. Normally, A.suum does not mature in such animals 
though Berger jet jal, (1961) have reported this in rabbit.
The mode of migration of A.suum in laboratory animals, 
especially th© white mice, has received attention by 
Sprent,(1956, 1958). Essentially this parasite follows 
the tracheal route of migration. On occasions in infections 
with A,suum in th© pigs, earthworms and beetles have inter­
vened as paratenic hosts. The eggs containing infective 
second stage larva® are ingested by earthworms and these 
beetles. The larvae hatch and remain alive in the tissues, 
fully infective for long periods.
2.19 Diagnosis of Ascaris infection
The diagnosis of the intestinal helminths which are 
found in man can be made readily by any one of the numerous 
methods of faecal examination. The recovery of the adult
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worm, the mature stages or the eggs in an individual 
is confirmation of infection. Host of the eggs which 
are found in the faeces are characteristic for each 
worm. In th © © C3 S  Q O f As caris lumbricoidos, however, 
both fertilized and unfertilized eggs may be found.
This fact introduces a possible source of error in the 
diagnosis of this particular parasite. Fertilized eggs 
are very characteristic, having a thick, clear, inner 
shell, covered by an irregular, warty, albuminous coat 
which is stained yellow or brown in th® intestine. They 
are usually spherical or slightly oval in shape and measure 
from 40 to 75 micro in length by 35-50 micra in width. 
Unfertilized eggs are much more longer, narrower, more 
elliptical in shape. They measure from 70 to 88 micra 
in length by 38-50 micra in width. The inner structure 
is unorganised and frequently contains amorphous granular 
material or it may be vacuolated. The albuminous coat may 
be absent from both fertilized and unfertilised eggs.
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The unfertilised eggs are usually bile-stained but are 
more likely to be colourless, grey, or unstained.
There is not much information available concerning 
the frequency with which fertilized and unfertilized 
Ascaris eggs occur in cases of ascariasis. However,
Keller £t al, (1933) has shown that the percentage of 
fertilized eggs increases with the intensity of infostation. 
It is interesting to note that in the 289 cases, he examined 
in which unfertilized eggs occurred alone, 265 (91,7%) had 
light infections. This is the group in which diagnosis is 
most likely to be missed if unfertilized Ascaris eggs are not 
recognised.
During lung migration of Ascaris larvae, there may be 
exhibitioin of scattered moulting in the lungs, accompanied 
by dyspnoea of an asthmatic type, fever up to 40°C and 
spasm of coughing. Occasionally, blood-tinged spUtum and 
eosinophilia in the absence of tuberculosis is highly
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suggestive of Ascaris pneumonia. Later, when the
worms have matured in the small intestine, diagnosis 
is usually made on the recovery of the characteristic 
eggs (fertile or infertile) from the faecal samples.
A single female worm produces a sufficient brood of 
eggs each day to guarantee their recovery in one or two 
direct faecal films. In patients with suspected Asocris 
pneumonitis, a history of present or previous intestinal 
infection in the individual or in other members of the 
family, or of soil pollution, is suggestive, Keller 
et al (1932) stated that in children with negative 
tuberculin tests there may be evidence of a widening in 
the hilar areas of the lungs with increase in the broncho- 
vascular markings, presumably due to repeated migration of 
Ascaris larvae through the lungs.
The main materials that are examined for Ascaris 
infections are faeces, sputum, and bile samples. However, 
because of activities of houseflies, and other insects
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which assist in disseminating As carls eggs as well as in 
contaminating fruits, root and leaf vegetables, these food 
items are also examined for ova that are stuck to their 
surfaces. In many areas of the world, human faeces is still 
actively used as fertiliser without proper treatment to kill 
tho parasites in them The farmhand is therefore grossly 
polluted with eggs which are later passively end widely 
disseminated by rain, floods and strong winds., It is 
therefore customary to examine soil and air samples as well 
to determine the level of contamination of the environment 
with Ascaris eggs.
Amongst the various diagnostic methods, the direct smear 
method is the oldest and simplest method. It is however 
inefficient in light infections except one uses a large 
sample material. Kato's cellophane thick smear method is 
very efficient and is recommended for mass examination of 
many faecal samples. It is simple, rapid and cheap (Katof./Vbiurc! 
1954). It is however unreliable when the faecal material is
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fibrous and contains fermentation gas. It detects all 
the common helminth ova except the minute ones. Uhen 
the Koto's cellophane thick smear method is used, further 
examination of faecal sample by a concentration method is 
unnecessary.
The concentration methods are of two types. The 
floatation methods which make use of the differences in 
the specific gravity between the helminth ova and faecal 
materials in the medium. The objective is to float the 
ova on the medium surface. Commonly used salt solutions 
are those of sodium chloride and Magnesium sulphate. The 
sedimentation methods are better favoured and more widely 
used than floatation methods because they eliminate most 
of the faecal debris as well as increase yield of ova.
The morphology of the ova is often distorted in Acid-ether 
sedimentation but the yield in the Formalin-ether test is 
good and can be stored in formalin (Ridley & Hawgood, 1956).
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The quantitative methods that have been widely 
used include the followings
ti) Stoll dilution egg count method 
(Stoll, 1923, ■ );
(ii) Beaver egg count method 
(Beaver, 1949, 1950);
(iii) McMaster egg count method.
The value of quantitative methods have been fully 
appreciated in helminthic infections where egg production 
is fairly constant, and the approximate daily output p er 
female worm is known as in Ascaris, Trichuris and Hookworm 
infections. They, however, provide only estimates of the worm 
burden because of the many factors that are known to effect 
the total daily egg output. Some of these factors include 
the fluctuations that occur in the individual worn's egg 
output and the varying a«oounts of faeces passed. Honetheies 
the recorded egg counts enable us to classify infections as 
light, moderate and heavy and they are useful in detsrnining 
the success or failure of control programmes and mass
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chemotherapy. Since some intestinal helminths shed 
their ova ireegularly into faeces, modifications have 
been introduced to the commonly used quantitative methods 
to adjust for this peculiarity ( Martin &
Beaver, 1968).
The Stoll* s dilution egg count method (1923,
The stoll egg count method was first introduced in 
1923, but the small drop modification described by Stoll 
and Housheer in 1926 has been the most widely used quanti­
tative method. This is a dilution method and the eggs are 
counted in O,075rnl sample from 5mg of faeces which is an 
estimated equivalent of 1 in 200 of the original faecal 
sample. It is a reliable method which has been widely 
accepted.
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Beaver's Standard Smear Method (1949, 1950)
Beaver’s egg count method uses a small sample of 
faeces when compared with Stoll's method. It is simpler 
to perform anflfloss time consuming than the Stoll’s method. 
Beaver's method is however not suitable for routine work 
in hospitals and clinics especially in developing countries 
because of the use of the photometer and the precise measure' 
ments required for the faecal slide density. The photometer 
which is essential, is easily damaged and needs to be 
constantly recaiiberated. Although this method is known
give reliable counts with high reproducibility, Maldonado 
(1956) and MoIvin et al, (1956) have suggested that Stoll’s 
method shows no advantage over Beaver's despite tho rela­
tively small size used in Beaver's method.
McMaster’s Egg Count Method
This method, like the Stoll's is a dilution method 
and it uses saturated salt solution and sieve to freo 
helminth ova from tho faecal debris. The amount of faecal
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sample used is smaller than in Stoll's method and the 
helminth ovcj float on the saturated solution, being 
higher than the debris. These are counted in the 
counting chambers which have been calibrated. This 
method is unsuitable for trematode eggs which invariably 
collapse and refuse to float, nonetheless, it is a fairly 
reliable method. Other methods for quantitative estimation 
of eggs are Koto's Cellophane thick, smear method (Kato 
& Miura, 1954).
Ascaris infection can also be diagnosed by finding 
the adult worms in the stool, or vomit. When freshly 
passed out from the intestine the adult worm is light 
brown or pink in colour but it gradually changes to white. 
In shape, it is rounded and tapers at both ends, the 
anterior end being thinner than posterior. The mouth 
opens at the anterior end and possesses three finely 
toothed lips, one dorsal and two ventral. The posterior 
extremity is neither curved nor pointed but conical and
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X-Ray Diagnosis
The presence of Ascarls lumbricoides has been 
demonstrated by radiography with barium emulsion, which 
being ingested by the worm within 4 to 6 hours, casts 
an opaque string-like shadow*
Dermal Reaction (Allergic)
Scratch test with powdered Ascaris antigen has often 
been found to be positive but the results are variable.
This reaction has some value in the diagnosis of Asoaris
S
infection.
2.20 Control of Human Ascariasis
The control of ascariasis is very difficult especially 
in tropical countries where ascariasis is most common 
amongst young children, who habitually pollute the compounds 
with faeces and become infected while playing on the ground, 
as a result of infective eggs adhering to the fingers and
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being later transferred to the mouth. Whenever possible, 
all infected patients should be given specific treatment, 
but this in itself, does not control infection, since 
reinfection from contaminated sites may occur repeatedly. 
The benefits of single treatments for Ascaris lumbricoides 
have been shown to be of short duration in areas where 
infections are perpetuated by gross pollution of dooryard 
and environs (Cor't, Schapiro & Stoll, 1929; Otto, 1930; 
Otto & Cort, 1934 and Headlee, 1936).
Fayard (1949) treated 2,000 patients harbouring 
Ascaris lumbricoides using piperazine citrate and recorded 
70 to 95% success. Mouriguand, Roman & Coisnard (1951) 
also recorded good results when he used piperazine to 
treat some patients. Other investigators like Oliver- 
Gonzalez, Santiago-Stevenson & Hewitt (1949); Etteldorf 
& Crawford (1950); Gbanem (1954), White & Standen (1953) 
and Bumbaio et al (1954) recorded some degree of success
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and they condluded that if used over a period of time 
piperazine, a relatively non-toxic agent is useful in 
controlling As carls infection. Mykoff and Altmarcn (1956) 
had shown that some molluscicides that are often applied 
to soil have ovicidal activity on the embryonated eggs 
of Ascaris lumbricoides. It is of interest that all the 
4-thicyano-compounds they tested were toxic at high 
dilution to Ascaris eggs. Muazzam et_ gl̂  (1960) using 
pyrantel pamoate, another potent, yet safe drug with 
minimum toxicity reported that in two different reports, 
the therapeutic success of those drugs has been from 76% 
to 88$ (MHO., 1964) and the effect of other drugs such 
as thiobendazoie has been even less (MHO,, 1967). Ogata 
(1925) in a series of experiments on the destruction of 
Ascaris eggs concluded that Ascaris eggs were easily 
destroyed by heat at 10G°C. At this temperature, the eggs 
lost their power of development and that Ascaris eggs 
containing embryos, after hot water had been poured on
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them lost the ability to infect. Hoshi & Morizaki (1952) 
and Yamada (1952), dealing with some groups of people with 
large populations gradually lowered prevalence and density 
of Ascaris infection by repeated treatments, but complete 
elimination was not attained. While deliberate large- 
scale efforts to control ascariasis have been attempted in 
comparatively few countries, it can be assumed that its 
prevalence has gradually fallen in large part of Europe 
and America (WHO,, 1967). This has occurred as an unplanned 
by-product of a generally improved standard of living and 
of the popular acceptance of relatively high standards of 
personal hygiene. The experience of a few countries, notably 
Japan and the USSR, indicates cleawly, however, the results 
that may be expected from deliberate, planned efforts on a 
national scale. Adequate epidemiological data ate available 
to assess the efficacy of measures carried out, and this 
experience in ascariasis control is particularly instructive 
in planning control efforts in other countries. In general, 
the approaches to ascariasis control have proved valuable:
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(i) the treatment of infected populations;
(ii) measures to prevent environmental contamination 
with human faeces Gnd
(iii) education of the public in personal hygiene.
In the most successful programmes, all the three measures
have been undertaken simultaneously. Comparatively few
attempts have been made to demonstrate the effect of any
one of these approaches as the sole method of control,
although in Singapore, a sharp decline in ascariasis was
noted within a year of reCilltjcating families in modern
houses (WHO., 1967). Environmental measures, especially
the provision of latrines and of safe sources of water,
have not, in themselves eliminated ascariasis from certain
parts of Western Europe and North America (WHO., 1967).
In fact, high prevalences of infection still persist
locally in both these areas. In addition, various studies
provision
have shown that the ' of latrines in villages as
the sole preventive measure may have little or no effect 
upon prevalence. For example, in certain Egyptian villages
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without latrines, the prevalence was 73% while in 
similar villages in which latrines were present, it was 
70% (MHO., 1968), In such villages, less than one third 
of the latrines and the wells were in use after six to 
ten years. In area where Ascaris prevalence in the highest 
infected age group was 77% in 1926, in 1950-1951, it was 
72%, In the same period, the prevalence of hookwotm fell 
from 37% to 47%r thus showing that the methods usually 
employed to control hookworm infection have no effect in 
ascariasis control (Craig & Faust, 1970), In areas where 
human faeces is used as fertilizer fotr vegetables which 
are eaten raw, fermentation at 50°C or higher is needed 
to kill the eggs (Davey & Crewg, ,1973). Furthermore, if a 
small plot of ground is known to be seeded with Ascaris 
eggs, thus exposing children, it may be desirable to 
spade under the top two inches of soil and bury the eggs 
or to sterilise the soil with live steam (Craig & Faust,1970).
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Control of Pig Ascariasis
The chief problem in the control of ascariasis in 
pigs is the great survival capacity of the egg. In houses 
the use of caustic soda, applied concentrated to walls and 
floors and left for two or three days, to be hosed away 
before the pigs are re-introduced, is effective and, where 
it is available, live steam is also highly efficient. When 
the problem is a serious one in range pigs, the animals 
must be moved to fresh ground, and the old ground culti­
vated or grazed by other stock. The application of the 
new piperazine compounds or other effective anti-helminthics 
represents a possible means of controlling Ascaris sUum by 
planned periodic treatment (Kelley, Olson & Garwood, 1956), 
In order to prevent the spreading of the eggs, A.suum 
should not be permitted to mature within the host. About 
70 days are required for A.suum to reach maturity. Thus 
if the market pigs are treated at 2 and 4 months of age 
and the brood stock at bi-monthly intervals, the worms
would not mature and no new eggs would be deposited on the soil.
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This programme of planned treatment would lower th© 
number of infective Ascaris eggs available and eventually 
eliminate '-caris from the herd.
Vaccination with Whole b'ortn
Attempts to vaccinate against parasitic nematodes 
with dead whole organisms, homogenates or extracts began 
in the 1930s. Kerr (1938) could not detect any increased 
resistance to the dog hookworm Ancylostoma caninum in mice - 
or Ascaris suum in guinea-pigs following repeated vaccination 
with heat-killed larvae or extracts prepared in various ways, 
Sprent & Chen (1949) did not detect any resistanc£rigainst 
Ascaris lumbricoides in mice following vaccination with 
homogenates of worm tissues. The failure, or at most, 
marginal success, of the majority of these early attempts 
to vaccinate against parasitic nematodes gave rise to the 
view that the antigens needed to stimulate an effective 
immune response, the functional antigens, were not present 
in sufficient quantity in homogenates of dead worms, but
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DAN LIBRARY 
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might be actively secreted by the living parasites.
Taffs (1961) reported that adult worms in infections 
of pigs or laboratory hosts with Ascari? spym may be 
expelled by a self-cure mechanism; and that the larvae 
of a challenge infection in an immune host are affected 
when they approach the moulting period. In one of the 
first investigations of immune response to gastiro-intestinal 
nematodes in cattle and sheep, Stoll 0929) and Soulsby(1961), 
trmrte some observations which strongly suggested that an immune 
reaction capable of blocking egg production and causing 
expulsion of many adult worms was triggered by an early 
stage in the development- of a challenge infection. This 
so-called "self-cure" reaction has been most studied in 
the response of sheep to the stomach-worm, Haemonchus 
contortus. After sensitisation of sheep by several 
infections, a further challenge can result in a local 
hypersensitivity reaction in the abomasum which causes 
rejection of most of the adult worms from this site
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(Stewart, 1953). This reaction occurs at a time when 
the larvae of the challenge exposure are moulting from 
the third to th© fourth larval stage (Soulsby &Siewart, 
193D).
2*21 Identification of Bacterial Microflora 
in Human alimentary tract
(. are microscopic in size and 
relatively simplo, often unicellular in structure. The 
morphological features of importance are the size, shape, 
and grouping of the cells, and their possession of distinct 
tive structures such as endospores, flagella, capsules, 
and intracellular granules. Staining reactions are 
observed after treatment by special procedures such as 
Gram's stain (Gram, 1884). The Gram's stain divides 
bacteria into two categories in separate colours due to 
their permeability to certain decolourising agents.
Limanna & Mallette (1959) also discussed the principle 
of different staining procedures and concluded that
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staining helps in the grouping of bacteria. Furthermore, 
systematic position of microflora of the gut can be 
verified by other methods (Baltimore, 1957). Gram (1334) 
divided bacteria into Gram-negative bacteria and Gram-positive 
bacteria. These a*® many but those ones that ar© present 
in the alimentary tract of man include the following:
(a) Staphylococci:
These are Gram-positive cocci, usually arranged in 
clusters* Smears from liquid culture may show Gram-positive 
cocci in pairs, short chains or small clumps. Staphylococci 
are catalase positive, non-motile, non-spore forming. They 
grow on most laboratory media at 37°C in the presence of 
oxygen. Manito1-salt-agar is one of the selective media 
for the isolation of Staphylococci from contaminated 
specimens. Some strains show pigmentation which occurs 
better at room temperatures and some species are haemolytic. 
Staphylococci must be differentiated from Micrococci which 
are also Gram-positive cocci. This can be done by their
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attack on glucose. St^nhvlocqcci ferment glucose 
whereas.Micrococei attack glucose oxidatively or do not 
attack it at all. Coagulase testing separates the 
pathogenic strains of S-s-nohvlococci from the nan-pathogenic 
strains, Cowan (1939). Thus coagulase positive StaphvJ'■'<'ooa 
are referred to as Staphylococcus aureus (Staphylococcus 
pyogenes) and coagulase negative ones as Staphylococcus albus. 
Staphylococcus aureus produce a substance called coagulase 
which, in-vivo deposits fibrin on the surface of Staphylococci 
rendering them 1 ess susceptible to phagocytosis,
(b) Streptococci;
They are Gram-positive cocci arranged in pairs, short 
or long chains. They are non-sporing, generallypon-notile 
and catalaS^negative. Most species are aerobic, or 
facultative anaerobes, and a small group are obligatory 
anaerobes. Many species of Strentococci elaborate a substance 
called C-carbohydrate, which, when extracted by hydrochloric 
acid or formamide or lysed by enzymes, forms an antigen used
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• f S ,
for their serological grouping (Lancefield's groups, 
Lancefield \1933), The common Lcmcefield's groups ore 
A - G and Enterococci which are present in human alimentary 
tract belong to Lancefield* s grovp D# Ent@r5S.qcci may be
r
haemolytic Skt> Wj>̂ ! 1 p h a (o( ) or beta haemolysis on
Blood agar medium. On MacConkey’s agar medium, they 
form small pink colonies. They are heat-resistant - 60°C 
for 30 minutes - and will grow at 45 C, They will also 
hydrolise aesculin agar slope.
(c) Lactobacilli;
The organism in this group are aciduric, Gram-positive, 
non-motile, non-sparing rods. They are catalase negative, 
often long and slender and sometimes pleomorphic. They 
grow best on a glucose-containing medium or on Tomato 
juice medium having an acid pVi. The group is commonly
found in faeces of both adultSand infants
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(d) Clostridium perfringes\S(Clostridium welchii) s
The Clostridia are anaerobic or microaerophilie 
Gram-positive rods, forming spores. The position of the 
spore is an aid in identification. Some species decompose 
protein (proteolytic); others ferment sugars (saccharolytic), 
while some species are both proteolytic and saccharoiytic.
The majority are saprophytic organisms, their natural habitat 
being the soil and intestine of man and higher animals.
Many are pathogenic to man. Among the pathogens are the 
Clostridium tetani, the causative organism of tetanus, the 
Clostridium welchii (Clostridium perfringe^) and the allied 
ones are organisms associated with gas gangrene. The 
isolation and identification of the Clostridia is not a 
simple task, as materials for cultures will invariably 
contain other organisms including Proteus. Isolation 
media should therefore include blood agar media^ 
incubated aerobically and anaerobically; MacConkey's agar
. incubated aerobically and anaerobically and cooked
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meat medium and ThiogJLycoilate medium̂ , which _ after 
an overnight incubation,is later subcultured onto solid 
blood agar plates for both aerobic and anaerobic 
incubation.
2. Gram-negative Bacteria;
These are also many in genera and species but those 
found in human alimentary canal of man include;
(a) Escherichia coli - which is the commonest type of 
faecal coliform and a frequent cause of urinary tract 
infections and gasfro-enteritis in infants. It will grow 
readi iy on most laboratory media and ferments lactose and 
most sugars. It is nearly always indole positive and 
produces gas in MacConkey broth medium at 44°C (Eijkman's 
test). It does not utilise citrate medium. On account 
of its fermentation of lactose, it is pink in colour on 
MacConkey-Lactose-Bile-Salt medium (MCC).
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(b) Proteus .-.group of organisms are Gram-negative motile 
aerobic bacilli found in normal faeces, urinary tract 
infections, and in suppurating wounds, idost species 
tend to swarm over the surface of solid media such as 
Blood agar medium. There are four species of Proteus 
according to somo carbohydrates and biochemical reactions.
The species are P,vulgaris which ferments glucose, maltose 
and sucrose with acid and gas production. It is indole 
positive. It produces hydrogen sulphide; P.mirabilis 
which ferments glucose and sucrose with acid and gas 
production. It is the only species among the group that 
does not produce indole, P.morgani ferments glucose with 
acid and gas production. It is indole positive and it 
produces hydrogen sulphide. The fourth species is P.rettgeri 
which ferments glucose and mannitol with acid only. It 
does not produce gas in the sugars, It is indole positive, 
and hydrogen sulphide negative.
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(c) Other Gram-negative organisms usually found in
faeces of man include:
Klebsiella species - They are Gram-negative, non-maiil®, 
indole negative organisms. Some mucoid strains produce 
capsule and the virulent strains are highly pathogenic 
for mice, thus, mice are used for their diagnosis. Citrate 
is utilised whidch helps to separate these lactose fermenting 
organisms from Escherichia coli - another lactose fermenting 
organism. Also found in the alimentary canal of man is 
Pseudomonas aeruginosa, which is a motile Gram-negative 
rod-shaped organism. It may produce a yellow-green pigment 
and may also produce a characteristic smell. It is catalase 
positive and oxidase positive. Other Gram-negativ© organisms 
occasionally encountered in the alimentary canal of man 
apart from the above mentioned ones a£e fully identified by
the methods of Baltimore (1957), Cruickshank (1965) and
Baker (7967).
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2.?2 Ascoris ULumbricoides and Bacteria:
In spite of tne fact that Ascaris infection is one 
of th© most cosmopolitan and most common helminth: infections 
in man and domestic animals and although our knowledge of 
these worms dates far back to antiquity, a review of the 
literature shows that very little is known about how these 
parasites feed.
Leuchart (18^6) observed in Enterobius vermicularis 
that th© worm intestine usually contained yellow fluid 
which microscopic examination proved to be identical with 
the liquid masses normally found in the large intestine 
of the host between the solid part of the faeces. In 
Oxyuris equi, he was able to demonstrate that th© intestinal 
contents contained small particles of vegetable material
identical with the contents of the intestine of the horse
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Weinberg (1907) stated that he could not find rod 
blood-corpuscles in the intestine of Ascaris but that; they 
were usually very numerous in the intestine of Strongylus 
of the horse; he expressed, therefore, the opinion that 
Ascaris feeds on the contents of the intestine of the host. 
Hoeppli (1927) recorded an experiment carried out by Dr.Voge 
in the Tropen-lnstitute at Hamburg. A patient, positive for 
Ascaris was fed with powdered animal charcoal throe times 
a day for three days. On tho fourth day, a female Ascaris 
was expelled with an ascaricide, but a thorough examination 
of the worm intestine showed no particles of charcoal; but 
the same author was later informed by Dr. Vogel that in a 
second experiment carried out in a similar way, numerous 
charcoal particles were found in the intestine of Ascaris 
lumbricoides.
Archer & Peterson (1929), in their experiment on 
"Roentgen Diagnosis of Ascariasis", made the interesting 
observation that soon after the ingestion of barium cereal
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- 70-
meal by the patients, a cylindrical filling defect
showing the displacement by the Ascaris specimens could
be seen in the jejunum. Later after the contrast meal had
entirely passed out of the jejunum, stringlike shadows,
representing the barium-filled enteric canal of the parasites
remained still for sometime in the jejunum. Furthermore, they
observed that the parasites would not ingest the barium, if
the patient had eaten prior to drinking the contrast meal,
and in that case, the enteric canals of the worms would not
be outlined. Their explanation of this observation is that
if the patient had eaten food prior to the administration
of barium, the enteric canal of the parasites would be
already filled so that no barium would be ingested. These
that
observations indicate Ascaris spp/^ lumbricoides) swallowed 
the barium meal and that they normally feed on the intestinal
contents of man.
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2.23 INTERRELATIONSHIP OF BACTERIA AND INTESTINAL 
NEMATODES (ASCARIS SPECIES)
Although the literature reports of the interrelationship 
of bacteria and intestinal nematodes are scanty, Newton £t al, 
(1959) and Wescott (1968) studied Nematospiroides dubius 
infection in germ-free mice and Przyjalkowski (1967) worked 
with Trichinella spiralis in germ-free and monocontaminafed 
mice.
Microbe-microbe interactions are numerous and have 
been reviewed by Donaldson (1968), Broitman & Gianneia 
(1971) and Bryant (1972). The antagonistic interactions 
include competition for nutrients, alteration of physical 
environment and elaboration of antagonistic substances or 
toxic metabolite, while the synergistic interactions involve 
production of growth factors, enzyme sharing and antibiotic 
resistance (Mettrick & Rodesta, 1974), Relationship between 
protozoa and bacteria of the gut and bacteria -nematode 
interactions" have been reveiwed by Hungate (1972) and 
Wescott (1970).
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Generally, bacteria, -protozoa, interactions are 
antagonistic, including competition for nutrient or, in 
some cases the protozoa ingesting the bacteria (Kumgat©,19/2). 
The relationship between nematodes and intestinal bacterial 
flora involve both synergistic and antagonistic component 
(Hettrick, 1973). The first is characterised by the 
microflora contributing to the survival and well-being 
of adult nematodes, increasing the percentage of larval 
nematodes developing to adultf and improving the survival 
times and fecundity of the adult worms. The second is 
characterised by alterations in the host's response to 
the tissue stages of the parasites. Hosts with normal 
flora were more successful in preventing the larval 
nematodes from completing their migrations and wore more 
successful in healing the lesions produced by migrating 
larvae (Wescott, 1970).
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Studies of the interaction of host microflora and 
metazoan or protozoan parasites became practical with 
the development of gnotobiotic technique (Reynier, 1959). 
These techniques allowed the study of parasitism in host 
devoid of bacteria or with a flora' of his choice. Philips 
& Holff (1959) working with Entamoeba histolytica in germ- 
free guinea-pigs were the first to show experimental 
example of parasite-microflora interaction using bacteria- 
free hosts and revealed the importance- of the application 
of gnotobiotics to the study of parasitism. Since that 
time, experimental infection of gnotobiotic hosts with 
metazoan and protozoan parasites has been reported by many 
investigators (Bradley & Reid, 1966; Doll & Franker, 1963, 
and 1964; Houser & Burns, 1968; Newtons, Weinstein & Jones, 
1959; Newtons, Beardon & Deleva, 1960; Prxyjalkowski, 1961, 
1966, 1969 and 1978; Weinstein, Newton, Sawyer & Sommerville, 
1.969), thus increasing our knowledge about the bacteria-
nematode interaction
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CHAPTER 1
3.1 DEVELOPMENT OF HUMAN AND PORCINE ASCARIS
EGGS TO INFECTIVE LARVAE IN DISTILLED HATER AND IN 
2% FORMALIN AT 25°C AND 27°C AND UNDER ACTUAL FIELD
Conditions
INTRODUCTION
Wharton (1915) reported that Ascaris eggs developed 
completely in 15 days to larval stage at 27°C, Schwartz 
(1922) observed that fresh unsegmented ascarid eggs 
cultured in a thin layer of 2% formalin solution in petri 
dishes developed to the first stage larvae in ten to twelve 
days at 25°Cf Refuerzo^ \1954), found that in distilled 
water culture at 27 C the eggs developed into the first 
stage larvae in six days. Tiner (1953 ) observed that in 
distilled water, ascarid eggs developed to the first stage 
larvae in 17 days at room temperature of 27°C* Further
experiments are necessary to ascertain whether the difference 
in temperatures is responsible for the differences in incu>- 
bation periods and also to find out the action of formalin
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on ths developing eggs. The study is also to find out 
the time it takes Ascaris eggs to develop to larval stages 
under actual field conditions in our tropical climate.
MATERIALS AND METHODS
Ascaris eggs used in this study were taken from 
the uteri of adult gravid female worms. Some of the 
eggs were harvested from faecal suspensions by sedimen­
tation and floatation on saturated sodium chloride solution. 
The Ascaris eggs were obtained from living worms collected 
from freshly passed faeces from dewormed children. Ascaris 
suum eggs were collected from living worms collected from 
thea abattoir. After the uteri were removed from the 
female worms and washed in several changes of sterile 
distilled water, they were placed in a petri-dish and 
the eggs were teased out of each uterus. Only the eggs . 
in the portion of the uterus proximal to the vagina were
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used because Ackert (1931) has shown that this part of 
the uterus contains a high percentage of fertile eggs.
The eggs were cultured using a modified method of Hansen 
et al (1954). This modification involved the preparation 
of an artificial digestive juice which would break down 
the uterine wall surrounding the eggs. This solution 
contains 0,5% hydrochloric acid and 1% pepsin. In 4-3 
minutes, contact with the^solution, the uterine walls were 
completely digested leaving only the eggs. The eggs were 
washed 4-6 times in distilled water and were cultured in 
different media thus:
(1) Some eggs were cultured in ordinary 
distilled water and incubated at 
25°C and 27°C,»
(2) Some eggs were cultured in ?.% formalin 
and were incubated at 25°C and 27°C.
Two incubators one of which was maintained at 25°C and the
other 27°C were used. The temperatures of 25°C incubator
and those of 27°€ incubator were recorded by means of
maximum and minimum thermometers kept close to the petridishes
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-85
The depth of the culture medium was about 2cm and the
actual temperature of the cultures was measured by
inserting a thermometer in the dishes. About half a
dozen petri-dishes were used for each medium and for a
particular temperature. The contents of the petri-dishes
were agitated twice a day to aerate them. The cultures
were observed under the light microscope everyday to see
the rate of development. At each observation, 3 separate
total differential egg counts were made from three of the
culture dishes selceted at random. Both the low and high
power objectives of the microscope were used in the process.
For each count, the culture was mixed well, and then taken
with a 50-dropper Pasteur pipette and delivered onto a clean 
microscope slide and covered with a coverslip. The eggs
on the slide were counted under the microscope with a
moving stage. Fresh pipette was used for each count and
three such counts were made and the average taken. The
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total counts per ml, was obtained by multiplying the 
number counted p@r slide by 50, Each stage of development 
was then counted and the percentage was calculated.
When the eggs appeared to have reached the first stage 
larvae at the end of the sixth day a quantity from each 
culture dish was fed to a white mouse. There was nothing 
that happened to the infected mice. Immediately the larvae 
moulted from the first stage larva® to the second stage 
larvae with their clearly discernible moulted cuticles on 
the thirteenth day of the experiment, a quantity from the 
culture dish was again fed to a white mouse. The infected 
animals did not show any sign of infection. When the 
cultures were seventeen days old, the feeding experiment 
of the white mice was repeated and all the infected animals 
died from Ascaris pneumonia. Ascaris larvae were recovered 
from the mice lungs thus showing that the eggs were then 
The e*£?eriment was then terminated at the end of 
the eighteenth day. The whole experiment was repeated
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•07-
using Ascaris suum oggs of pigs one! the sgitig results 
were obtained. The results recorded here are those of 
Ascaris lutnbriooides of mem For the field conditions,
Ascarid eggs-were cultured in both distilled water and in 
2% formalin and were incubated in pair thus:
(i) A pair consisting distilled water and
2% formalin cultures were incubated under 
direct sunlight;
(ii) Another pair under grasses and the last 
pair under big trees.
The cultures were examined daily and the temperatures 
measured with maximum and minimum thermometer^* The 
average temperature under direct sunlight was 42°C, that 
of under grasses was 30 C and the temperature under the 
trees was 22°C. The aqueous media were replenished, as %<L y 
dried up in cultures under direct sunlight^ under grasses
and under the tree
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RESULTS
Figs. 1 and 2 show Ascaris eggs with different 
stages of development. Fig. 2 particularly shows 
unfertilized egg showing vacuolated protoplasm with 
highly refractile granules; unsegmented fertile egg; 
advanced morula stage of development and early larval 
stage of development.
Fig. 3 shows fertile egg with advanced morula 
stage of development while Fig,4 shows tadpole-like 
larva inside the egg-shell and also first stage larva. 
Fig.5 shows second stage larva coming out of the egg­
shell while Fig.6 shows second stage larva expressed 
out of the egg-shell with its clearly discernible
sheath of the larva
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-89-
 ̂ - Shoeing different stages of
.\scaris development
2 days ole! x 200
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- 90-
Showing different stages of development*
Notice both fertile cmd infertile egs®
2 days old * 200
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— /
FIG 3s - ‘fertile egg with advanced
mof^ia stage of development 
3 days dd x 200
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- 92-
F1G.4; . Showing Tadpole-like larva andfirst stage larva rnside the egg - 
4 days old x 200
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- 9 3 -
FIG.5: Shô jû f second stage larva 
coming out of egg-shell 
IS days old x 200
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- 9 4 -
FIG.6: - Showing 2nd stage larva which has-been
expressed out of the egg-shell. Notice 
the sheath of the infective larv a and 
the egg-shell. 15 days old x 2.00
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UNIVERSIT 0Y 0 OF IBADAN LIBRARY 
0
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STAGES OF DEVccOPMENT EXPRESSED AS PERCENTAGE
UNIVERSITY OF IBADAN LIBRARY
irl  
Table 1 shows different stages of development of 
Ascaris lumbricoides eggs incubated at 27 C. The table is 
graphically illustrated in Fig.7.
Both the cultures in distilled water and in
formalin reached the prelarval stage on the third day,
but the cultures incubated at 25°C reached the pre-larval
stage three days later. This slow rate of development
at 25°C also happened to 1he rate of further development
at this temperature. The cultures at 25°C reached the
motile larval stage at the end of the eleventh day, while 
those at 27 oC reached the larval stq^e at the end or^sixth 
day. The same results were obtained in 7.% formalin cul­
tures. Under the field conditions, the eggs did not 
develop beyond the 4-3 cell stages at 42°C. Under the 
grasses, the Ascaris lumbricoides eggs developed to the 
first stage larvae in 15 days and under big tree, the 
eggs developed to the first stage larvae in 21 days.
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DISCUSSION
Fertilised eggs of Ascaris species require a 
period of incubation during which development and 
maturation takes place before they can be infective to 
the next host. This fact shows that immediate transfer 
of Ascaris eggs from one host to another does not happen 
in Ascaris infection. The eggs will have to undergo 
further development in the outer world before infection 
of the next host can take place, Ascaris eggs are 
complex organisms and before they can infect another 
animal must go through a period of development and 
adjustment. The percentage of fully embryonated eggs 
which develop and the rapidity of development depend on 
several environmental conditions. Commonest amongst 
these conditions are moisture, temperature, and oxygen.
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- 9 9 -
Under laboratory conditions, the three pre-requisite 
requirements are provided for by ciuturing the eggs in 
aqueous media and incubating them at favourable tempera­
ture. The dividing egg has a very high oxygen requirement 
and the development of the eggs incubated at 27 C (Table 1) 
reached the pre-larval stage on the third day. On the 
fifth day, the eggs developed into the tadpole-like 
larvae. Motile larvae clearly discernible inside the 
egg-shell were present at the end of the sixth day. This 
result does not agree with the findings of Tiner (19530 
who reported that the development of Ascaris eggs reached 
the first stage larvae in 17 days, but the result of the 
25°C cultures confirms the findings of Schwartz (1922) 
and Wharton (1915). The result of this study also
confirms the findings of Refuerzô 'j"'*'1' 1195/j-) and the 
result shows that the four-day difference between the two
investigations were due to the 2 OC difference in temperature
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-1 0 0 -
This study shows that the use of 2% formalin had no 
effect whatsoever on the development of the eggs because 
both the eggs cultured in formalin solution and in ordinary 
distilled water developed to the first and second stage 
larvae at the same time.
There was an interval of about seven days between 
the development of the first stage larva anc! the second 
stage larva, giving a total period of about thirteen days 
for the unsegmonted eggs to develop to the second stag© 
larvae (Table 1), This interval is necessary because the 
first stage larvae (L.j) needs to feed vigorously and grows 
within the period. Furthermore, within the interval, there 
is always a stage of lethargus at which time the larva does not 
feed before it moults to the second stage larva.
in nature, the eggs of Ascaris lumbricoides and 
Ascaris suumtake ten days before the first stage larvae 
are formed and another eight days before the first stage 
larvae develop into the second stage larva (Dunn, 1978).
UNIVERSITY OF IBADAN LIBRARY 
101
Therefore, the short incubation period of thirteen days 
from the unsegmented eggs to the formation of second stage 
larvae, in this study, is only possible under laboratory 
conditions. This observation is made plausible by the 
findings of Davey and CrewS-(1973) who reported that) the= 
eggs of Ascaris developed to the infective second stage 
larvae in a suitable environment in about a month. This 
long incubation period under the field condition where 
the two most important bionomic factors are temperature 
and moisture is probably due to the prevention of proper 
aeration by the faecal pad and also to unstable temperature 
both in the day and the night.
The time interval between the first stage larvae and 
the second stage larvae is n®ceessry because during this 
period the larvae must develop all necessary organs for 
their existence in the definitive host.
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Alicata (1934) had shown that in eggs of certain 
members of the family Ascaridae (Ascaris lumbricoides, 
Ascaris suum, Parascaris equoruw, Toxocara canis,
Toxocara leonina), moulting is a pre-requisite for 
infectivity. The results of this study confirm his 
findings because the first stage larvae failed to infect 
the white mice. The results show that the first stage 
larvae were not infective to the mice because tho larvae 
at this stage of development cannot survive in the animals 
and they died, for in the early stages of this nematode th 
larvae are adapted to free-living conditions (Dunn, 1973), 
Furthermore, the first stage larvae are incapable of 
secreting the hatching fluid which breaks down the egg 
shells and the sheaths. This observation is in agreement 
with Roger (1958) who showed that the larvae of ascarids 
secrete a moulting fluid which breaks down the egg shell 
to release itself. The results of this study also show 
that the second stage larvae failed to infect the mice
UNIVERSITY OF IBADAN LIBRARY 
immediately the larvae reached the second stage. This 
shows that the second stage larvae still require feu 
days post moulting period before they could become 
infective. The interval between the time the larvae 
moulted to the second stage larvae and the time they 
became infective seemed to be connected with the deve­
lopment of certain organs responsible for the secretion 
of the hatching fluid. The second stage larvae'which 
are parasitic can infect the mice by ingestion. Xt is 
when the second stage larvae arrive in the stomach, hatch 
and cast off their sheaths that they can bore their way 
through the stomach wall. There are two factors responsible 
for the exsheathing, namely, the extrinsic and the intrinsic 
factors. The extrinsic factor is the preliminary stimulus 
and the factor which induces it is present in the gastric 
and duodenal contents. The action of the extrinsic factor 
results in the release by the larva itself of the intrinsic 
factor and it is this factor which accomplishes the process
UNIVERSITY OF IBADAN LIBRARY 
104
of exsheathing. The secretory region for the intrinsic 
exsheathing fluid lies anteriorly in the larva between 
the excretory pore which is in itself about the mid 
oesophag£al area and the ba&e of the oesophagus. It is 
the infective stage larvae that possess this excretory 
pore thus the failure of newly formed second stcge larvae 
to infect the mice. Finally, the results of this investi­
gation show that the number of the infective larvae rose 
to a maximum by the close of the experiment, and the 
infective larvae (Figs 5 and 6) were typical sheathed 
filariform larvae (Fig.6). The study shows that the use 
of thermostatically controlled incubators for culturing the 
eggs of ascarids or other helminths will reduce the un­
necessary lengthy period of incubation especially when there 
is not much time at the disposal of one carrying out the 
experiment. Under the field conditions, ascarid eggs 
failed to develop to the larval stage in six days as com­
pared with the laboratory results. The eggs failed totally
UNIVERSITY OF IBADAN LIBRARY 
105
to develop into larval stage under the direct sunlight.
This probably shows that the sunlight had a direct lethal 
solar effect on the eggs. The average temperature recorded 
under direct sunlight was 42°C and this temperature was 
capable of destroying the eggs before larval formation. 
Wharton (1915) showed that at temperature around 37°C, ova 
of Ascaris lumbrisoides developed to the eight-cell stage 
and then died. The results of this study agree with his 
findings. The results also agree with the work of Uefuerzo 
& Albis-Jeminez (1954) who showed that Ascaris eggs were 
destroyed in five hours, thirty minutes when exposed to 
direct sunlight at temperature between 34°C and 42°C.
The findings in this study show that in the tropical 
areas where people defaecate on big stones directly exposed 
to sunlight, Ascariasis may be relatively low as most of the 
eggs would have been destroyed by the direct sunlight.
Again, the result here shows that direct solar effect 
could be used as a means of Ascaris control because once
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106
the Ascaris eggs have failed to develop to larvae 
infection of people would be impracticable.
The results of the Ascaris eggs incubated under 
the grasses at an average temperature of 30°C shows 
that the eggs were capable of development to the larval 
stages in 15 days. This finding is not in agreement 
with the laboratory experiment of the egg cultures in 
which the eggs developed to the larval stage on tho 
sixth day at 27°C; a temperature which is lower than 
30°C. The explanation to this could probably be duo 
to the drop in temperature during the nights and the 
early mornings of the period of experiment; the tempe­
rature was not constant at 30°C unlike the one in tho 
laboratory with thermostatically controlled temperature. 
The finding, however, is in agreement with the result 
of the work of Brown (1927) who showed that eggs of 
Ascaris incubated under the field condition at 30°C,
developed to the motile embryo in 15 days. He considered
UNIVERSITY OF IBADAN LIBRARY 
107
this temperature to be the optimum. The result is 
also in accordance with the findings of the work of 
Fanret-Fremiet (1913) who showed that the ova of /.scciris 
suum are capable of development at varying temperatures 
below 35°C, but that the optimum temperature is about 30°C.
The results of the eggs culture incubated under the 
big tree at an average temperature of 22°C shows that 
the eggs developed into larval stage on the twentyfirst 
day of the experiment. This shows the slow rate of 
development at lower temperature. This slow rat© of 
development was not unexpected as it is natural that 
under lower temperature, the metabolic rate of any living 
animal is greatly reduced. The slow rate of development 
showed the ability of the tr$e to cut off the lethal solar 
effect, to reduce the temperature and to prevent evaporation 
of arueous media used. The tree also preventithe destruc­
tion of the eggs by the letKC$l solar effect. Those findings
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confirm $pindler*s (1940) view that under natural 
conditions, ascarid eggs are protected by the faecal 
mass during early stages of development. It is likoly 
that the embryomated eggs under this condition will 
survive for quite a long time. The observations here are 
made plausible by the findings of Refuerzo & Albis-Jeminez 
(1954) who showed that ascarid eggs under legume (Pueraria 
javanica) survived for 76 days while the eggs under grasses 
(Polling fulva) survived for 66 days. This survival period 
is very important in the epidemiology of aseariasis and this 
could probably account for the long survival of the eggs 
in shaded moist environments in tropical areas.
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7-
ACTION OF INTESTINAL BACTERIA ON THE 
DEVELOPMENT OF ASCARIS EGGS IN-VITRO
INTRODUCTION:
Although Grzyb & Szydlowska (1964) studied the 
effect of antibiotics on Ascaris suurn eggs in cultures 
of Escherichia coll and Proteus and found that the 
bacteriostatic doses of antibiotics acted ovostaticcilly, 
little or no work has been don© on the effect of mono­
cultures of intestinal bacteria on the development of 
ascarid eggs in-vitro. Therefore, in this study, the 
action of monocultures of intestinal bacteria on the 
cleavage and development of larval stages on Ascaris 
lumbricoides and Ascaris suurn in-vitro was investigated.
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110
MATERIALS AND METHODS
Experiment 1;
Fertile eggs of gravid female Ascaris lumbricoides 
were collected from uteri of gravid worms and were washed 
several times in sterile physiological saline (0,85/3 Macl). 
The eggs were deshelled and sterilised with equal volumes 
of 3/o sodium hydroxide and 3% sodium hypochlorite overnight 
using Hansen et a l (1954 & 1956) method. The eggs were 
subsequently washed 4-6 times with sterile saline to remove 
the deshelling solution. They were then checked bacteriolo- 
gically for their sterility by culturing them onto enriched 
and differential media and into Brewet’s thiogly4i©ilate 
medium for both aerobic and anaerobic organisms.
The washed sterile eggs were suspended in small volume 
of sterile normal saline and were counted using improved 
Neubauer counting chamber. Ten thousand of the prepared 
eggs were put in each of ten 6?xiinch long test tubes
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Ill
containing an overnight broth cultures of Escherichia coli; 
Proteus mirabilis, Staphylococcus aureus. Streptococcus 
faecalis (Enterococci), Bacillus subtills, Bacillus coreus, 
Pseudomonas aeruginosa and Clostridium welchii (in Brewer's 
medium). A control tube containing eggs in sterile nutrient 
broth without bacteria was put up. The tubes were covered 
back aseptically with their sterile cotton wool covers.
They were incubated at 30°-31°C for 14 days. One-day old 
cultures of bacteria were pipetted off using sterile 
Pasteur pipettes and a fresh sterile nutrient broth was 
added to a level of about one inch above the bottom of the 
tube. Brewer's medium was used for Clostridium welohii 
culture and the culture incubated anaerobically using 
Anaerobic jar. Each tube was examined everyday under 
the light microscope for its development. During this 
incubation and re-incubation periods, control egg cultures 
were tested for their sterility and the bacierial-egg- 
cultures for their monoculture state and their development.
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112
This process requires strict aseptic precaution under 
the microflow cabinet.
After the incubation period of 14 days all the 
bacterial-egg-cultures were disinfected using clorox 
solution. After an overnight incubation in this 
solution - which is the same solution as that used to 
sterilise the surface of the eggs at the beginning of 
this exercise, the eggs were washed several times with 
sterile saline. This treatment removed both the bacteria 
and their metabolic by-products. After the last washing, 
each tube was tested for sterility as previously described 
Fresh sterile nutrient brô fch was added to each -feube and 
fresh Brewer's medium added to Cl.welchii tube. All the 
tubes were incubated again at 30-31°C for a further period 
of 14 days.
>
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Experiment 2;
Quantitative Determination of the used Bacteria Species
Dilutions of the overnight broth cultures of the 
test bacteria were carried out. The surface viable count 
of Miles & Mizra (1933) was used. This method of utilising 
dropping-pipette is probably the most accurate of the 
viable count techniques (Baker, 1967).
Required?
_5Q-drop pipettes
fcyvbaried nutrient agar plates
Sterile 3 x \  inch tubes.
Method;
The nutrient agar plates were dried in drying oven 
on the tilt for two hours, to prevent a drop of fluid From 
running over the surface of the plates. With a grease 
pencil, the bottom of the plates was divided into 8 segments 
labelling them from 10" , 10 upwards. Using a dropping 
pipette 45 drops of sterile nutrient broth were addod to 
each of 10 sterile^Sx 1 inch tubes. The pipette was discarded
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114
with a fresh dropping pipette, 5 drops of culture were
added to tube 1, This pipette was also discarded. Uith
a fresh sterile pipette the content of tube 1 was mixed
ten times and 1 drop was added to segment labelled 10
and 5 drops were transferred to tube 2, The used pipette
was discarded. With a fresh pipette, the operation with
tube 2 was repeated upwards. The drops on the plates were
allowed to be absorbed into the medium before the plates
were incubated at] 37°C for 24 hours. For accurate viable
count, tubes 8,9 and 10 were used. The segment containing
discrete colonies was counted. Two of such counts were
made and the average found. The calculation of the number
of colonies of organisms per ml was done.
The organisms; used were:
Escherichiat_coli 2 t . Q  xIO*. orgs/ml,
Staphylococcus aureus 7 ’ S  X f # 13-  "  "
Streptococcus faecalis 5*oxnr,i- » •*
Pseudomonas .aeruginosa k  o x 10^ orgs/ml. 
Proteus mlrahilis x 10tO •«
Bacillus cereus j/S'x
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1 *5
Doubling dilutions of each of the used organism were made 
in 10-15 sterile cotton-wool-plugged 6 x t inch tubes, 
using fresh sterile pipette for each dilution and starting 
from the highest concentration to the lowest concentration 
of organism per ml. Four control tubes were set Up for 
each organism - on© control tube contained only nutrient 
broth plus eggs, the other contained distilled water plus 
eggs. One control tube contained nutrient broth only and 
the fourth control tube contained distilled water only.
Ten thousand eggs treated as in experiment 1 above 
was added to each tube including the controls except one 
tube containing nutrient broth only and one tube containing 
distilled water only. These two tubes were put up to test 
for the sterility of the broth, the distilled water and 
the egg. The tubes were incubated at 30-31°C for 14 days. 
The procedures used with the overnight broth cultures were 
also used in this series for checking for the development 
or otherwise of the eggs.
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Experiments 1 and 2 were repeated using the eggs
of Asearis suum, The same results as in Ascarls lumbricoides 
eggs were obtained, so the data recorded here are for 
Asearis lumbricoides of man.
RESULTS
The result of the bacteriological test on deshelled 
eggs after an overnight incubation period on different media 
was negative. The control tubes containing eggs plus 
nutrient broth/distilled water remained sterile throughout 
the period of the investigation; and the eggs in the con­
trol tubes developed into motile first and second stage 
larvae between the sixth and the fourteenth day of deve­
lopment (Table 1), Egg cultures in Gram-negative bacterial 
cultures with the exception of those in Pseudomonas . 
aeruginosa developed into two-cell stages. Those eggs in 
culture of Bacillus species also did not develop at all.
The use of Clorox (3% sodium hydroxide and 3$ sodium hypo­
chlorite) to sterilise the bacteria-egg-cultures die! not
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1i,
affect the protoplasm of the eggs as the eggs after 
washing in sterile saline and cultured in nutrient 
broth developed to larval stages.
Table 1, shows the effect of overnight broth cultures 
on the development of fertile Ascaris lumbricoides eggs.
In this concentration, the eggs were found to have multiplied 
only to 2-cells stages in some cultures and there was no 
development at all in others. Table 2 shows the develop­
ment of eggs into various developmental stages after killing 
off the bacteria with ClorOx. Table 3 shows the develop­
ment in various stages in dilutions of Escherichia coli, 
there was no development beyond 2-cell stage up to the 
concentration of 20 x 10^ organism/ml, but the eggs started 
to develop as the number of bacteria decreased in the tubes.
Effect of Stapylococcus cureus on the development 
of the Ascaris eggs «
In the cultures of this organism the eggs started to
3
develop beyond 2-cell stage at the concentration of 75x10 
organism/ml and the development reached the motile larval
stage at a concentration of 750 organisms per ml and to a
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118
second stage larva at a concentration of 0.75 
orgs/ml (Table 6).
The Effect of Streptococcus faecalis on the
development of the ascarid eggs
Table 5 shows this result. The higher concentra­
tions of the bacteria inhibited the development beyond 
the 2-cell stage. However, as the bacteria decreased in 
number, the eggs started to develop. The development 
reached the motile larval stage at a concentration of
50.5 orgs/ml thus the critical concentration was 505 xIO 3
organism per mi.
The Effect of different concentration of Pseudomonas 
aeruginosa on the development of Ascaris eggs
In the dilution of this organism, the eggs failed 
to develop until there were only 1000 organism per ml in 
the broth culture (Table 7). The eggs developed into larval 
stages where the tube contained almost no organism per ml 
of the broth, that is, when the concentration was 0,1 organ! 
sms per ml. The last dilution of this organism at which
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119
the asoarid eggs will not develop was 10,000 
organisms per ml.
The effect of different concentrations of Proteus 
mirabilis on the development of eggs is shown in Table 8.
In this dilution of the organism, the eggs did not 
devekop at all until the bacterial concentration was 
reduced to 8900 organisms per ml. The development 
reached the first stage larva at a concentration of 
8,9 organisms per ml. The critical concentration at which 
the eggs will not develop at all was the undiluted overnight
broth culture
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TABLE 1: SHOWING DEVELOPMENT OF ASCARIS JJJMBRTCOIDES*  'T  V  «’.U  *».« » > . j v . f j
EGGS IN OVERNIGHT BROTH CULiukES OF DIFFERENT 
BACTERIAL SPECIES
CONTROLS
E. Pro- Pseudo Strep, B• .sub 3 Cl,.yel NVt. Dis.
DAY coli teus monas Staph faoc, tills cereus chli ' '.bro, water
1 - - - - n mm mm >-~mm
2 2 2 - 2 2 - - - 2 2
3 2 2 mm 2 2 — - - - 4 4
4 2 2 mm 2 2 - - - M M
5 2 2 e ymm 2 - - - TP TP
6 2 2 mm 2 2 - - - LI LI
7 2 2 - 2 2 - - LI LI
8 2 2 & * 2 2 - - mm LI LI
9 2 2 - 2 2 - - - LI LI
10 2 2 - 2 2 - - - LI LI
11 2 2 - 2 2 - - mm LI LI
12 2 2 - 2 2 - - - LI LI
13 2 2 - 2 2 - - *•<* L2 , L2
14 2 2 M 2 2 - - mm L2 L2
KEYS; - = No Development H = Morula stage
2 = 2-cells stage TP = Tadpole larva 
4 = 4-cells stage LI s 1st stage larva 
8 = 8-cells stage L2 = 2nd stage larva
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TABLE 2: SHOWING THE DEVELOPMENT OF A5CARIS LUi-BRICOIDFS 
EGGS AFTER KILLING THE BACTERIAL SPtwitS WITH 
CLOROX
DAY E. Pro- Strep, B. B. Nut. Dis.coli teus Staph. faec. subtilis Cereus Broth water
1
2 2 2. 2 2 2 2 9 2
3 4 4 4 4 4 4 4 4
4 H M M M M M M M
5 M M M M M M M M
6 TP TP TP TP TP TP TP TP
7 LI LI LI LI LI LI LI LI
8 LI LI LI LI LI LI LI LI
9 LI LI LI LI LI LI LI LI
10 LI LI LI LI LI LI LI LI
n LI LI LI L» LI LI LI LI
12 LI LI LI LI LI LI LI LI
13 L2 L2 L2 L2 L2 L2 L2 L2
14 L2 L2 L2 L2 L2 L2 L2____ L2
- = No development
2 2-cells stage
4 = 4-cells stage
8 = 8-cells stage
M - Morula stage
TP s Tadpole larva
LI = 1st stage larva
L2 2nd stage larva
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TABLE 3 ^HOWIMr- TMF EFFECT OF DIFFERENT 
CONCENT R/FTT6n~ OF ESCHERICHIA r.CH I ON THE 
DEVELOPMENT OF ASCARI5 LUHBRICGIDFS EGGS
TUBE NQ„ L  Poj-Jv. Incubation RESULTS
Concentration Period in Days
In Broth
1 20 x 109 1
0 20 x 108 2
3 20 x 107 3 2
4 20 x !06 4 2-4
5 20 x 105 5 4-8-M
4
6 20 x 10 6 TP
7 20 x 103 7 LI
a 20 x TO2 8 LI
9 20 x 10 9 LI
10 20 10 LI
11 n LI
12 0.2 12 LI
13 0.02 13 L2
14 0.002 14 L2
15 0.0002 L2----
■KEYS 0•
—  = No Development M = Morula stage
2 = 2-cells stage TP = Tadpole larva
A 4-cells stage LI = 1st stage..larva
8 ~ 8-cells stage L2 = 2nd " I
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TABLE 4 SHOWING THE RESULTS OF THE FOUR CONTROLS 
SET UP FOR EACH SPECIES OF BACTERIA USED 
USING A3CARXS LUMBRlCOTr^S EGGS.
Nutrient Nutrient Disvillcd Distilled 
DAY broth only broth plus water only water plus 
oggs .egga
1 . RS - RS
2 RS 2 RS 2
3 RS 4 RS 4
4 RS 8 RS Vn
5 RS M RS M
6 RS TP RS T?
7 RS LI RS LI
S RS LI RS LI
9 RS LI RS LI
10 RS LI RS LI
11 RS LI RS L1
12 RS LI RS LI
13 RS L2 RS L2
14 RS L2 RS ..-L2
- - No Development
O = 2-colis stage
4 s 4-ce.lls stag©
8 = 8-cells stag©
M - Morula stage
TP = Tadpole iarvo
LI. = 1st stage larva
L2 = 2nd
RS S Remained Sterile
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I V
TABLE 5; SHOWING DEVELOPMENT OF A-SCARTC LUNBRXCOIDFS 
EGGS .IN MONOCULTURE DILUTIONS W  Z w T p ff"™  
FAPCALTS DURING 14 DAYS INCUBATION PERIOD
TUBE Street. faecalis Incubation 
NO. concentration in period in Days . .RESULTSbroth
1 505 x '10.A 1 w
2 505 x 10F 2 O
3 505 x ID6 3 n
4 505 x 105 4 o
5 505 x 104 5 2
6 505 x K)3 6 2
•7f 505 x IQ'3' 7 4
3 505 x 10 8 3-M
9 505 9 Tp
10 50.5 10 LI
11 5.05 11 L1
12 0.505 12 LI
13 0.0505 13 L2
14 0.00505 14 L2
15 0.000505 -----to-
KEYS
= No development M = Morula stage
2 " 2-sells stage TP = Tadpole larva
4 = 4-cells stage LI - 1st stage larva
3 = 0-cells stage L2 = 2nd "
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’25
TABLE 6: SHOWING DEVELOPMENT OF ASCARTS ILUMBRICOXDES
EGGS IN VARIOUS DILUTIONS OF AN OVERNluH f BROTH
CULTURE OF ST W Y l  "COCCUS AUREUS
" -.— -—
TUBE Staph, aureus Incubation 
NO. concentration .in period in Days RESULTS
broth
1 75 x l o " 1 -
2 75 x IQ10 2 2
3 75 x io9 3 2
4 75 x 108, 4 2
5 75 x 107 5 2
6 75 x io6 6 2
7 75 x io5 7 2
8 75 x 1Q4 8 4
9 75 x100° 9 8 and M
10 75 x 10 10 TP
11 75 x 10 11 LI
12 75 12 LI
13 7.5 IS L2
14 0.75 14 L2
15 0.075 L2~
KEYS:
- = No development M = Morula stage
2 = 2-cells stage TP = Tadpole larva
4 = 4-cells LI = 1st stage larva
8 = 8-cells II L2 = 2nd " II
U
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126
TABLE 7: SHOW IMG DEVELOPING ASCARIS LUMBRir.proFS _
EGGS m  VARIOUS DXLU1 IONS “Ô ' PSEUDOMONAS 
AERUGINOSA
TUBE Pseudomonas Incubation RESULTS
NO. concentration period in Daysin broth
1 10 x 109 1 —
2 10 x ID8 2 -
3 3 -
4 10 x 106 4
5 10 x IQ5 5 -
6 10 x 10‘ 6 -
7 7
8 10 X 102 8 2
9 10 x 10 9 2
10 10 10 4
11 1 11 8 and M
12 0.1 12 TP
13 0,01 13 LI
14 0.001 14 L2
15 0,0001 — L2
KEYS:
mm = No development M = Morula stage
2 = 2-cells stage TP = Tadpole larva
4 = 4-cells I I LI = 1st stage larva
8 = 8-cells II L2 = 2nd
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ITY OF IBADAN LIBRARY 
TABLE' 8: SHOWING THE EFFECT OF DIFFERENT 
CONCENTRATIONS OF PROTEUS JiTRABILIS 
ON THE DEVELOPMENT ‘OF‘*<CARI$ J ^ oRICOIDES
Proteus Incubation 
TUBE concentration period in Days RESULTS
NO. in Nutrient Froth
g
1 89 x 10 1 M
o 89 x 107 2 r>.f.
3 89 x 106 3 2
4 09 x 10° 4 2
5 09 x 10; 5 2
6 o89 x 10u 6 £
7 89 x 10"'" y 4
8 89 x 10 r3*>, 8 and M
9 89 9 TP
10 0.9 10 LI
11 0.89 11 LI
12 0.089 12 LI
13 0.0089 13 L2
14 0.00089 14 L2
15 . 0.000089 L2
KEYS:
- - No development M = Morula stage 
2 - 2-cells stage TP = Tadpole larva 
4 - 4-coils LI = 1st stage larva 
8 - 8-cells I L2 = 2nd
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128
TABLE 9: SHOWING THE EFFECT OF DIFFERENT
CONCENTRATIONS OF BACILLUS CEREUS ON 
THE DEVELOPMENT QP‘Xs£fe$ LUijpCOIDES 
EGGS *'
Bacillus Incubation 
TUBE concentration in period in Days RESULTS,
NO. Nutrient broth
1 75 x 109 1 -
2 75 x 10S 2 -
3 75 x IQ7 3 -
4 75 x TO6 4 m
5 75 x 105 5 m
6 475 x 10 6 m
7 75 x IQ3 7 -
na 75 x 103 8 2 and 4
9 75 x 10 9 8 and M
10 75 10 TP
11 7.5 11 LI
12 0,75 12 LI
13 0.075 13 L2
14 0.0075 14 L2
15 0.00075 - L2
KEYS:
= No development M = Morula stage
2 = 2-cells stage TP = Tadpole larva
4 = 4-cells LI = 1st stage larva
8 = 8-cells L2 = 2nd " II
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-129-
Tablo 9 shows th© result of the egg development 
in various concentrations of Bacillus cereus. Development 
did not begin until the number of organisms per ml dropped 
to 75,000 organisms per ml.' Thereafter, th© development 
got to the first larval stage at a concentration of 75 
organisms per ml, thus showing increased ovostatic action 
of this bacterium on the egg,
.... , . . DISCUSSION
Table 1 shows that almost all the species ofe bacteria 
used in this study were capable of unhibiting the cleavage 
and development of both the human and th® porcine Ascaris 
eggs. According to Stefanski & Przyjalkowski (1965 & 1966), 
some helminth eggs need intestinal bacterial flora for their 
growth and maturation. On the other hand Przyjalkouski & 
Jaekowski (1968) and Przyjalkowski 1973 a and b) found 
that eggs of Ascaris suum, Ascaridia galli and Aspicularis 
tetraptera developed well without a bacterial flora in their
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130
environment. The findings in this study arc thoroforc in total 
agreement with the observation of the above-named workers.
The inhibition of development of Ascaris lumbricoides eggs 
by the cultures of bacteria added to sterile broth was 
found to be ovostatic which is anatogous to bacteriostatic 
action exhibited by some antibiotics on certain organisms.
In this case when cells which are inhibited by the presence 
of a bacteriostatic agent are removed by centrifugation, 
washed thoroughly in the centrifuge and resuspended in 
fresh growth medium, they will resume normal multiplication.
In this study, the eggs of Ascaris lumbricoides that were 
freed from bacteria and were resuspended in sterile butrient 
broth developed to motile larval stages after a period of 
re-incubation. This shows that the ovostatic action of 
bacteria occured only in the presence of living and r 
actively multiplying bacteria/cells. This finding agrees 
with the observation of Przyjalkowski (1973), who observed
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that the ovostatic action of bacteria was not effected
with the metabolic products of bacteria present in the
filtrate of bacterial cultures or with unfiltered culture
of bacteria killed after 48 hours of incubation. Furthermore,
the ovostatic action of bacteria might be due to respiration
processes.These processes are based on the oxidation-reduction
reactions and, in the case of bacteria are, connected with=
*
oxygen consumption. This assumption is made plausible by 
the observation in the study of egg development in cultures 
of strict aerobes and facultative anaerobes. The use of 
^^gt^idium welchii incubated anaerobically did not allow 
the development of Asccris lumbricoides eggs because ,under 
anaerobic conditions, oxygen content in the anaerobic jar 
was replaced with hydrogen gas thereby leaving no oxygen 
for the growth and development of the ascarid eggs.
Pseudomonas aeruginosa is a strict aerobe commonly used for 
the cultivation of facultative and obligatory anaerobes 
bocou»« its growth requires oxygen consumption from the
environment.
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132
The ovostatic action of Escherichia soli was much 
reduced (Table 3) because Escherichia coll can grow well 
in the presence of a trace of free oxygen. Therefor©,
motile larvae were formed in a concentration of 20 x TO 3
organisms per ml., the highest concentration to support 
the development of Ascaris lumbricoides eggs to larvae 
in this investigation. Tables (5 and 6) show that the 
organisms used were capable of growing both in the presence 
and absence of oxygen. Therefore, their ovostatic action 
was more than that of E.coli. There is a good reason to 
suggest that the marked inhibitory action exhibited by 
Pseudomonas aeruginosa and Bacillus cereus in Tables 7 and 9 
might be due to the production of certain enzymes and 
antibiotic substances by the two organisms. In addition., 
to the fact that Pseudomonas aeruginosa is a strict 
aerobe which requires all the available oxxgen for its 
growth, thus depriving the eggs of Ascaris lumbacicoidos
of necessary oxygen requirement for further development,
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this organism produces an antibiotic substance called 
bacteriocin© which acts ovostatically on Ascciris ova 
(Grzyb &SzydIowsfea, 1964). In the case of Bacillus 
species, there are n'.any antibiotic substances elaborated 
by this organism. Some of those antibiotics aro bacitracin, 
poiymyxinS and colistin, Ail these substances act ovosta- 
tically on the eggs of the ascarids. Furthermore Bacillus 
species produce certain enzymes especially proteases v/hich
* ' s Jr ‘
are capable of Iftfo^nMJAscaridae in-vitro (Emanuilov, 1956).
The lack of inhibitory action or phenomenon in the 
control tubes containing only nutrient broft) and distilled 
water was therefore due to the absence of organism which 
made the abundant oxygen present in the tubes to be availabl 
for the use of the • Ascaris egg development.
The ovostatic phenomenon is explained by the fact 
that the Ascarls eggs cannot start cleavage or continue 
further development■without oxygen. The oxygen present in 
a liquid medium is consumed rapidly by actively feeding and
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134
multiplying bacteria which have a shorter generation 
time than Ascaris eggs. Bacteria growth rate is 
expressed in terms of generation per hour (Jawetz et a1, 
1970). Since all the organisms used in this study reproduce 
by binary fission and their generation time is 40 minutes, 
the growth rate of the organisms is therefore 1.5 generation 
per hour. Table 4. shows that there was no development 
of the eggs during the first day and that the eggs 
developed only to 2-cells stage on the second day. Comparing 
this development with that of the bacteria used, the 
bacteria is at a considerable advantage over the eggs 
because of its enormous population available (1.5 x 43 
generations) by the commencement of egg cleavage. During 
this period almost all the available oxygen would have been 
used up by the large population oif organisms, leaving 
little or no oxygen for the egg development. The oxidation- 
reduction potential of Ascaris agg which is related to its 
oxygen consumption, therefore, becomes very low.
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The actively multiplying bacteria in a liquid medium 
therefore act ovostaticcilly by completely consuming the 
oxygen which is necessary for the development of Ascarls
eggs.
The observations in this study confirm the work of 
Grzyb & SzydXowska (196A), which showed that bacteriostatic 
doses of antibiotic on Ascaris suum eggs in cultures of 
E.coli and Proteus acted ovostaticoily. The results of 
the study also agree with the findings of Zwanczuk & 
Dozanska (1957) who showed that there was inhibition 
of cleavage of Ascaris lunbrlcoides eggs in a town sewage, 
but that after disinfection of the sewage with chlorine 
at a bacteriostatic concentration, the helminth ova 
started their cleavage and further development.
The use of Glorox has been shown in this study to be 
safe for sterilising the surface of Ascaris eggs because
it had no deleterious effect on the protoplasm of the eggs.
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13 6
This observation agrees with that of Hansen ©t ai(1954, 1956).
Finally, the results of this study show that the 
development of Ascaris eggs could be controlled in bacterial 
culture and this phenomenon could be used to advantage 
especially in storage and in transportation of Ascaris eggs.
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- 1 3 7 -
CHAPTER 3
3.3 BACTERIAL FLORA OF ASCART* SMJM (GOEZE, 1782)
AMD ITS RELATIONSHIP TO iHE HOST FLORA.
- - - -r-r— IN’ TRODUCTION
The earliest work on the bacterial flora of the 
intestine of a parasitic nematode was done by Weinberg 
in 1907. He found 33 .Sclerostomes positive for bacteria 
when he examined the intestine of 97 Sclerostomes 
(Stroncjylus) from 25 horses. McCoy (1929 a & b) found 
that living bacteria constituted the food of hookworm 
larvae, Ancylostoma caninum, developing to the infective 
stage. He did not attempt to isolate bacteria from the 
intestine of the worm. Li (1933a), studied the intestinal 
flora of the following nematodess Spiroeerca lupi, 
Physaloptera clausa, Cheilospirura hamulosa, Bunostomum 
trigonocephalum, and Enterobius vermlcularis-. Plain agar
was used as the isolation medium and cultures were incubated
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138
under aerobic conditions. The dominant bacteria in the 
intestine of about half of the nematode belonged to the 
"coli group".
A series of feeding experiments was conducted by Li 
(1933b) on certain roundworms belonging to the Ascaroidea 
and Oxyuroidea, He concluded that Ascaris lusnbricoldes. 
Toxocara canis. Toxoscaris leonina, Ascaridia galll, and 
Heterakis gallinarum fed on the intestinal contents of 
their host. According to Ackert and Whitlock (1941) 
ascarids and oxyurids normally feed on mucus, desquamated 
mucosal cells, and blood elements that are free in the 
lumen of the intestine. The intestinal flora of chicken 
has been under investigation for a long time. Kern (1397) 
studied the intestinal flora of 24 birds and isolated 88 
species of bacteria. He concluded from direct preparations 
of the stomach and intestinal contents that a large number 
of species did not grow when submitted to ordinary culture 
methods. Kern reported that the following species were
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139
obligate intestinal forms: Bacterium coli. Bacillus 
vegatus, Pseudomonas ejrcmulo-fca. Bacillus clefessus. and 
Bacterium verruscosum. The last four species are not 
listed in the seventh edition of Bergey's Mammal of 
Determinative Bacteriology (1957).
Rahner (1901), King (1905), Gage (1911), and 
Emmel (1930) reported that Escherichia coli was the 
predominating type of bacterium in the chicken intestine.
It was observed that the microflora was most abundant in 
the caecum and colon and gradually decreased in numbers 
towards the duodenum. Thoy concluded that diet and extrinsic 
factors associated with management practices could influence 
intestinal flora.
Shapiro and Sarles (1949), quantitatively studied 
the microfiora of the intestine of chicks of different 
ages and found that newly hatched chicks had few micro­
organisms in the intestinal tract. They nctod that normal
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140
s
flora became established in the contents of the duodenum, 
ileum, eaecal pouches, and colon after th© chicks were fed 
for 16 hours within 40 hours after hatching. Total numbers 
of bacteria were highest in the contents of the colon, ileum, 
and duodenum. Their results indicated that Lactobacillus 
species composed th© most numerous group of bacteria in all 
the regions of the intestinal tract, Escherichia coll was 
found to be the predominant coiiform organism and the pre­
dominant enterococci, (Streptococcus faecalis), was present 
in large numbers only in the caeca! pouches and colon. The 
principal obligate anaerobe present in the intestinal tract 
of th© chicken studied was Clostridium perfringeihC’ ,
A quantitative study of the intestinal flora of 
chickens parasitised with Ascaridia galli and of uninfected 
control chickens was carried out by Shear (1957). Ho reported 
that the numbers of bacteria increase from th© gizzard toward 
th© cloaca. The lactic acid bacteria wer© the most numerous 
group and the predominating bacteria in this group v/ere
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Lactobacillus species. The probable predominating
coliform organism was Escherichia coll and the pre­
dominating enterococcus form was Streptococcus faecclls.
The probable predominating obligate anaerobe was 
Clostridium perfringoflfi •
Przyjalkowski (1961) studied the bacteria flora of 
the intestine of Parassaris ©quorum and the small intestine 
of a horse and reported that mostly Sinobvlococcus and 
Escherichia coli v/ere isolated both from the contents of 
the parasite and that of their host. He did not isolate 
any anaerobic organism.
The present study was carried out in view of tho 
paucity of information on the microflora of Ascaris suum, 
Goocfce, 1782 of pigs and their relation to the flora of 
the hosts. Because of the problems of relationship between 
bacteria and parasites, the study also attempted to verify 
and eventually define the group of the bacteria on the body
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surface, inside the eggs, and in the internal organs of
the adult worms. Furthermore, the experiment attempted to
establish the type of 'relationship that exists between the
/
parasite and the bacterial flora with the hope that such 
information could be useful in culturing parasites in-vitro 
and in understanding the potential role of nematodes in 
transmitting pathogenic bacteria in human and animal gut,
- .-MATERIALS AND METHODS
Experiment 1;
Adult worms were collected from abattoir. The worms 
were washed several times in sterile distilled water until 
clean, liith the aid of a pair of sterile forceps, each 
worm, after identifying the sex, was rubbed onto selective 
and differential agar plates and also rinsed in Nutrient 
broth medium, in Brewer’s thioglycollate broth and in Robert 
cooked meat broth to ensure the recovery of all bacteria 
adhering firmly on the body surface of the worms. The 
specimens of faeces were cultured onto and into selective 
media for the recovery of the bacteria present in them.
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143
The adult worms were killed by dipping soraee o'? 
them in hot water which also helped to sterilise the body 
surface of the worms as was done by Prsyjalkowski (1961). 
Some of the worms were dipped ini : dilution of mercuric
chloride. The worms were kept in the mecuric chloridQ 
disinfectant for 5 minutes as was done by Li (1933a), The 
worms were dissected to extract the peritoneal fluid, abdo­
minal fluid, the intestine and the internal organs. The 
eggs were also recovered from the uteri.
Experiment 2:
Dissection of Adult Worms;
Dissection of female Ascaris suum:
Female Ascaris suum was laid on a sterile dissecting 
tray. From the mid-region of the lateral line, the worm 
was opened towards the anterior end using sterile dissecting 
instruments. Using a sterile pasteur pipette with rubber 
teat, the peritone<3cL fluid was taken and cultured onto 
enriched, differential and selcetive media and also into
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144
selective liquid media; the abdominal fluid was taken and 
cultured accordingly. The intestine was removed and washed 
several times to remove the adhering fluid and it was then 
cut into fore-gut, mid-gut and hind-gut. Each part was then 
washed and opened and the contents cultured. The ovary, the 
uterus, the oviduct and the vagina were extracted separately, 
washed in sterile water, crushed and then cultured separately 
on various media. The cultures were incubated at S7°C for 
24-72 hours aerobically, anaerobically and under carbon 
dioxide (C0f).
Dissection of Male Ascaris suums
The worm was opened from the posterior end. The 
peritoneC^l fluid and the abdominal fluid were taken and 
cultured. The intestine was removed and treated as in the 
case of the female worms. The testes, the vas deferens and 
the seminal vesicle were extracted separately, v/ashed, 
crushed and cultured separately. All the cultures were 
treated as in the case of female worms.
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145
Experiment 3:
Eggs were obtained from the uteri of adult worms. 
After the uteri were removed from the female worms and 
washed in several changes of tap water, they were placed 
in petridishes and the eggs teased out of each uterus.
The eggs were washed by centrifugation in several changes 
of water. The surface of the eggs were then sterilised 
using equal parts of 3% NaOH and 3% solution of commercial 
sodium hypochlorite (5,25% NaOCl according to the formular 
of Elliot, 1954), The eggs in this solution wore incu­
bated at 30-33°C for 24 hours. The eggs were then washed 
free of clorox solution by repeated centrifugation using 
sterile distilled water. The eggs were cultured onto 
different agar and into different media to find out the 
sterility of their surface. The plates and liquid cultures 
were incubated at different atmosphere^for 24-72 hours at 
37°C.
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146
Using sterile Griffith grinding tubes, the eggs 
were grounded in batches to release the internal contents 
which were cultured as before.
To determine whether the solution used to sterilise 
the eggs surface would kill the eggs materials, some of 
the clorox solution and some of the sterilised eggs were 
cultured together in sterile tubes according to the method 
of Hansen _et _al (1954)and (1956), The cultures wore 
placed in an incubator with a temperature of 3G°-33°C for 
14 days. At the end of the 14 days period, the eggs were 
examined under the light microscope for the development 
of motile larvae inside them.
Experiment 4;
Faeces cultures
Samples of the intestinal contents of the pigs 
harbouring the adult worms were collected into sterile 
screw-capped containers at the abattoir. . The samples 
were cultured onto different media and into various liquid
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147
media. The cultures were incubated at 37°C in different 
atmosphereSfor 24-72 hours. Cultures were examined 
macxSbscopicaliy and microscopically to determine the 
species of bacteria present.
Biochemical tests were performed and each species 
of bacteria identified.
u> -
Bacteriological Examination
Bacteria on the body surface, in the contents of 
the intestine and other parts of the worms and those from 
different parts of the intestine of the pigs were classi­
fied into groups according to the normal flora of the [Digs 
and also the normal flora obtained from the stool specimens 
of man. The following groups were used;
(1) Coiiform under aerobic condition
(2) Staphylococci under " "
(3) Enterococci " " "
(4) Proteus
(5) Pseudomonas " " "
(6) Bacillus
(7) Lactobacilli " tticroae^ophilic condition.
(8) Clostridium welchii (Cl.perfringeasunder anaerobic
condition.
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148
The Clostridium welchli serves as a representative of the 
anaerobes of the intestine.
This method of group classification was based on the 
example of Baron, Hansen 8-. Lord (1960). The workers discerned 
in their qualitative studies only coliforms. Enterococci 
(Streptococcus faecalis) and a genus of Lactobacilli,The 
choice of the proper selective media was based on preli­
minary tests#
(1) Coliforms group, Proteus and other Gram-negative bacilli
of the gut - an agar medium after NacConkey (1900) was used#
(?) Staphylococcus - 4P|amptnan*s (1946) medium with 7,5% and 
10% contents of sodium chloride and mannitol salt agar was used,
(3) Enterococci - Agar medium after MacConkey was used 
because on this medium the colonies of Enterococci (Stroptoco 
ecus faecalis) are small usually magenta-coloured.
Studies of the anaerobic flora were conducted separately 
and both the worm body surface and the contents of the 
intestine and internal organs, tho contents of the eggs and
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the specimen of faeces from the intestine of pigs and 
man were examined. Oaspak method of anaerobiosis, using 
sterile blood agar plates and necessary controls was 
employed. Similarly, bacteriological method for isolating 
Clostridium perfrin,ge^£. Sacteriodes fragilis using 
Robertson's cooked meat medium, Brewer's anaerobic 
thiogiycollate medium and Hagler's egg-yolk modittot were 
employed.
Controlled Investigations;
Controlled investigations were set up for the 
determination of the degree of selectivity of the media.
At first, the quality control of the batch of media used 
v/as carried out by inoculating the determined bacteria 
on separate selective media. After necessary incubation 
period, at the right atmosphere and temperature, the test 
were successful and the corresponding bacteria showed 
good growth only on the appropriate selective media.
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150
Present Investigation:
After the incubation of the cultures overnight, at 
37°C, the culture plates were examined both macroscopically 
study the colonial appearance and microscopically to 
study the morphology of the bacteria. Microscopical pre­
parations stained with Gram's method were made from the 
colonies characteristic of coliforms, Proteus, Pseudomonas 
and other groups under investigation. Spore staining methods 
were done on all characteristic colonies on anaerobic 
plates. So also was Nagler's reaction for rapid detection 
of Cl.welchii was carried out. Biochemical and carbohydrate 
fermentation tests were carried out by inoculating various 
carbohydrate sugars such as glucose, lactose, maltose, 
mannitole, dulcitol, urea slope, sucrose and citrate 
medium. All the sugars were incubated at 37°C for 24 hours. 
Glucos^ mannitol and lactose were fermented with the pro­
duction of acid plus gas; indole was produced, Escherichia 
coli and Proteus organisms were motile. Proteus mirabllis
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did not produce indole and citrate was not utilised by 
E.coli, Pseudomonas aeruginosa was oxidase positive. The 
colonies on MacConkey’s medium that resembled Enterococci 
(Streptococcus faecalis) were further tested using aeoulin 
test, and heat-resistant test, Stqphvlococci pathogenicity 
test was determined by using fresh human citrateu/plasma for 
both the slide and the tube techniques. Using the methods 
of Baltimore (1957), Christensen (1946), Kovacs (1956),
Baker (1967), Cruickshank (1965) and Cowan & Steel (1974), 
the systematic positions of the other groups of the organisms 
were verified,
RESULTS
Table 1 shows the results of the bacteriological 
examination of the body surface of adult worms of Ascaris 
suum. From the body surface of the worms were isolated, 
Escherichia coli, Streptococcus faecalis (Enterococci), 
Staphylococcus aureus. Staphylococcus albus, Proteus vulgaris 
Proteus mirabilis. Pseudomonas aeruginosa, Lactobacillus
acidophilus
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Candida albicans was also isolated. There was no
anaerobic organism and aerobic spore bearer isolated.
Tables 2 and 3 show that both the male and the 
female Ascaris suum carry in their gut and internal organs 
different general of bacteria in different percentage^ - 
The microscopic preparations from the materials stained 
for the presence of spores to demonstrate anaerobic 
spore-forming bacilli were negative.
Table 4 shows that the contents of the eggs of Ascaris 
suum harbour different genera of bacteria in various per­
centages. No anaerobic organism nor Lactobacillus species
was isolated from the protoplasm of the eggs
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- 1 5 3 - TABLE 1
*0 I i- - i:l AL FLORA OF THE BODY SURFACE OF ADULT ASCARIS SUUFi
...' " 1 ' " T"_l ' ..' X:
MONGER AND PERCENTAGE OF POSITIVE RESULTS
<,> i ,̂\s 0.1 iuu SPECIMEN SPECIMEN SPECIMEN specimen SPECIMEN SPECIMEN
NUMBER OF POSr* Ir>T: • IVE POSITIVE POSITIVE POSITIVE POSITjve POSITIVE POSITIVE
ADULT l*UU. FOR FOR FOR FOR FOR _FQR
•fORMS L. » Wlu. IS ENTEROCOCCI STAPHYLO- PROTEUS PSEUpOMnwAS LACTOBACILLI AEROB'Cp ■ / ? i rj“\ 1 r- p. COCCI - SPGREBEARER
J LNiO. No. Pef No. >f?3 No. /c/i > ° NO. p No. . No. %
MALE MALE 
ASCARIS . 
SUUM. 26" -|30- 13 50 5 19 7 27 A4  ̂5 3 12 ,... .... .
FEMALE
ASCARIS
SUUM f‘L o  [• ISP 14 56 4A 16 7 28 3 12 1 4 . . ~ -
rl.iN?i  • nLO i r  fNDIDA ALBICANS WAS ISOLATED FROM THE BODY 0F 0NE MALE /.SCARED (](4S)
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-154- TABLE 2
EXPERIMENT 2:
2B
'BACTERIAL FLORA OF THE GUT AMD INTERNAL ORGANS OF MALE A5CARIS SUUM
TYPE OF NUMBER OF SECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN
SPECIMEN SPECIMEN POSITIVE POSITIVE POSITIVE POSITfVE POSITIVE POSITIVE POSITIVE
EXAMS; ;ED . FOR FOR FOR FOR FOR FOR FOR
E.COLI :eSTEROCOCCI STAPHYLOCOCCI PROTEUS p<SFUDOMOMAS LACTOBACILLI AEROBIC
SPORE
BEARER
" "Mo. P No. yr*? Mo. No. i'-3 No. y• f»* Mo. .,S No o'.
Peritoneum
fluid 26 20 76 8 31 4 15 4 15 2 8 2 ■J 1 4
Abdominal
fluid 26 16 62 8 31 4 15 10 35 2 O«*> 2 6 1 4
lore-gut 26 .2 . 0 TOO 9 34 8 31 8 31 3 12 2 Of 1 4
Mid-gut 26 r .* 100 9 34 8 31 3 31 3 12 0 1 4
llind-gut p2. 6 2.6 TOO 9 34 8 31 8 31 3 12 O 1 4
T ostes oMl2 6 31 7 27 4 15 4 15 1 4 4 15 - -
Vas deferens 2.6 TO 38 10 38 6 23 4 15 2 8 2 On - -
Seminal p
vosicle 26 31 10 38 6 23 2 8 1 4 2 o
n> - —
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-155- TABLE 3
BACTERIAL FLORA OF THE GUT AND INTERNAL ORGANS OF FEMALE ASCARIS 5UUM
TYPE OF NUMBER, X7 SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN
SPECIMEN SPECIMEN POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE
EXAMINED FOR FOR FOR FOR FOR FOR . FOR
E.COLI ENTEROCOCCI. STAPHYLOCOCCI PROTEUS PSEUDOMONAS /"iRRQSiC
3ACELLI SPORE
BEARER
—  • . , io.
rPt9 No. P No. * No. cA>f No. * 1 >a0 ft... ic?f 4̂ 0 • c?fi
Peritoneum
fluid 10 75 8 33 4A 17 4 17 KoJ 13 /O- . .
Abdominal
fluid r £ 14 53 6 25 4 17 5 21 3 18  OO r„
!ore-gut ' r, i 100 8 33 5 ?1 6 ■ 25 4 17 6 .25 2 Or??
i iid-gut 24 r ’ 100 dO 33 5 21 6 25 4 17 6 25 2 frj>
Hind-gut ' 24 100 e 33 5 21 6 25 4 17 6 25 0 oodm
Ovary 24 ID 42 6 25 4 17 7 29 4 17 3 13 1 Or
Oviduct O A 12 50 6 25 4 17 6 25 3 13 4 17 - -
Uterus 12 50 8 33 5 21 6 25 1 4 O 13 - -
Vagina 24 «ipl  -’/»1• 58 10 42 8 33 8 33 2 3 0* 13 2 8
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!
o»
UH
-J
-156- TABLE 4s
BACTERIAL FLORA OF THE CRUSHED EGGS OF A3CARIS SUUM
TYPE OF i; I.J.'JlJ L*.« » Ot* SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN SPECIMEN
SPECIMEN POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE
EXAMINED ■~J */. Ji V u >. L—O JLl >1 FOR FOR FOR FOR FOR FOR FOR
" T' ,* * “r̂ V̂tU if tT-Qo E.COLI ENTEROCOCCI STAPHYLOCOCCI PROTEUS PSEUDOMONAS LACTOBACILL.Z AEROBIC
— — ..... • - - “ | SPORE I BEARER
I
........— S■ M1O . p No. i\!G « /o No. % No. No. if-  -1p \jQ • C?p
CATCHES OF fo ,, ,?
CRUSHED n r 30 94 Fi 66 13 56 13 41 9
EGGS.
) '.J._________ _____ _______ ________ ______ : rz zz z:  :z vzL . . » l n;:snazz L-
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The study on the bacterial flora cf the intestinal 
contents of the pigs showed that all the organisms identified 
from the worm materials were also isolated and identified 
from the pEg hosts. Hence from the intestine of pigs in 
Experiment 4 were isolated Clostridium welchii, Lactobacilli, 
Escherichia coli, Streptococcus faecalis (Enterococci) 
Staphylococcus aureus. Staphylococcus albus, Proteus vulgaris, 
Proteus mirabilis. Pseudomonas aeruginosa,Bacillus subtilis 
and Candida albicanVThis shows that the specific flora of 
Ascaris .suum and its host were similar, but unlike in the 
case of Ascaris suum, Clostridum welcbii was isolated from 
the host.
Before the microflora of the eggs of Ascaris suum 
could be investigated, it was necessary to determine whetb^6T 
the clorox solution used to sterilise the egg surface would 
kill or injure the eggs protoplasm. Table 4 shows that the 
disinfectant had no deleterous effect on the contents of
the eggs
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158
The result of the egg development at 30 O C - 33 oC 
after the use of the disinfectant (Clorox) to sterilise 
the egg surface showed that the solution had no lethal 
effect on the protoplasm as many of the eggs developed 
into larvae after 14 days incubation period.
DISCUSSION
The use of hot water to kill the adult Ascaris suum 
worms had no damaging effect on the contents of the worms.
The use of j: S O D  dilution of mercuric chloride to sterilise 
the body surface of the adult worms also did not have any 
dexterous effect on the bacteria contained in the internal 
organs of the worms. From the results obtained, it was 
concluded that mercuric chloride at the concentration at 
which it was used and for the period of 5 minutes had a 
germicidal effect on the bacteriatflord of the cuticle of 
Ascaris suum because no bacteria was isolated afterwards
from the cuticle after sterilisation. Both the male and the
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female As carls suum carry on their cuticle many 
genera of bacteria at various percentages' Aerobic 
spore bearers were, however, not isolated from the 
surface cuticle although this organism was isolated 
from the gut. The finding of different genera of 
bacteria on the surface cuticle of the worms is in 
accordance with the work of Przyijalkowski (1961) who 
isolated from the nutrient broth cultures different 
genera of bacteria from the body surface of Ascaris spp. 
with E.coli having the highest incidence of recovery.
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160
The results of the dissection of adult worms are 
shown in Tables 3 and 4. The results show that the rut 
and the internal tissues of the worms harbour many bacterial 
species at various percentage^' These results^compared with 
the results of the intestinal contents of the pigs, show 
that both the adult worms and the pigs harbour almost the 
same species of bacteria. The only slight difference was 
the isolation of anaerobic organism in the pigs but not 
in the ascarids. Although the flora of the intestine of 
the ascarids and their hosts were similar, there were 
increased percentage yields of the isolates especially the 
Enterococci and the Staphylococci from the pigs than from 
the ascaridA This type of results were not unexpected 
since Ackert & Whitlock (1941) had shown that ascarids and 
oxyuridj normally feed on mucus, desquamated mucosal cells, 
and blood olemests that are free in the lumen of the intes­
tine of the host. Furthermore, the relative size of the 
ascarids to the pigs could probably explain this because the
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bacterial flora have more space and more nutrients in the
pigs than in aecarids. Therefore, the results strongly 
suggest that Aswaris suum feeds on the intestinal contents
3
including the microflora of their hast. The results of 
this study therefore confirms the findings of Hoeppli 
(1927) who recorded an experiment carried out by Dr.Vogel 
in the Tropen-lnstitute at Hamburg in which a patient, 
positive for Ascaris was fed with powdered animal 
charcoal three times a day for three days. On the 
fourth day, a female Ascaris was expelled by an ascaricide 
and numerous charcoal particles were found in the intestine 
of the worm. The results also agree with the findings 
of Archer & Peterson (1929), who made cm intej 
observation that soon after the ingestion of a barium 
cereal meal by some patients, a cylindrical filling defect 
showing the displacement by the Ascaris specimen could be 
seen in the jejunum. Later, after the contrast meal had
entirely passed out of the jejunum, stringlike shadows,
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162
representing the borium-filled enteric canal of the 
parasite remained still for some time in the jejunum. 
Furthermore, the results show that the parasites possess 
their own bacterial flora usually similar to that; of the 
host intestine. The results of the study are in accordance 
with the findings of McCoy (1929) and Li (1933a) who 
isolated many Escherichia coli from the intestine of some 
nematodes, and Li (1933b) twho in a series of feeding 
experiments concluded that Ascarioidea and Oxyuroidea 
feed on the intestinal contents of their hosts. The 
results of this study are made plausible by the work of 
Tolslova (1951) who isolated from the intestine of Ascaris 
species 76 bacterial strains completely analogous to the 
microflora of the host in which these parasites lived, 
and Jettmar (195?) who isolated certain Gram-negative 
bacteria from the intestine of Parascaris equorum and 
Baron ejt al .(I960) who, comparing the bacterial flora of 
the small intestine of hens with that of the intestine of 
Ascaridia qalli isolated Escherichia coli and Lactobacilli
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163
species from both the host and the parasites.
From the results of this study it is clear that 
there are Very good relationships between the parasite 
and the environment containing bacteria ancPis confirmed 
by the observation of Liebmann (1953), that Lamblia 
iptestinalis occurs in human intestine in large number 
when Escherichia coli is present in the small intestine.
The most interesting finding in this study is the 
presence of some species of bacteria in both the host and 
the parasite. This is interesting because bacteria(jspecies 
are highly pathogenic when once they leave their normal 
habitat, and Ascaris larvae and adults alike, are noted for 
wondering up and down the intestine of their host and into 
other organs off their usual route. For instance, Sprent 
(1955) & Mochizuki^jC 1954) hafcfeshown in mice experimentally 
infected with Toxocara canis that the larvae of Ascaris 
migrated to the brain of the mice. Beautyman & Woolf(1951) 
had demonstrated Ascaris larva in the human brain,. Woodruff
& Thacker (1964). Woodruff et al(1966)&Woodruff(1968) showed tha
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larvae .in their migration may be capable of carrying 
virus and bacteria from the intestine to the brain and 
other tissues of their host. The wondering Ascaris larvae 
and adults carry with them the bacteria on their cuticle, 
in their gut and other tissues and they could be excreting 
these bacteria from time to time as long as they are feeding 
and developing. One good explanation for this type of 
association may be that both the larvae and the adult 
worms produce certain antibiotic substances in their 
intestine and other organs which render the bacter^inside 
them non-pathogenic to them and probably to their hosts too. 
It is therefore necessary to confirm conclusively the type 
of the antibiotic substances present inside the Ascaris 
suum because data recorded strongly suggest that there are 
some inhibitory substances inside the worms which render 
the harboured bacteria non-pathogenic.
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165
Studies on the effect of Clorox disinfectant 
solution on Ascaris eggs revealed that the solution did 
not penetrate the vitelline membrane of the eggs in 
sufficient quantity to kill or injure the protoplasm of 
the egg cell. It can be assumed that the bacteria present 
on the protoplasm of the eggs were likewise not injured 
since different species of bacteria were isolated from 
the eggs, thus Table 4 shows that the eggs of Ascaris suum 
harbour different species of bacteria withE. coli on the 
lead; followed by Fnterococci, Stt^pl^t^^cci, Proteus and 
Pseudomonas in that order. Lactobacilli and Aerobic spore
* srs*-■ . _ --r«* r~  "  - •
bearers were noij however, isolated from the contents of 
the eggs. The finding of bacteria in egg contents, in the 
uterus, and in the ovary strongly suggests that transovarian 
transmission of some bacteria with Ascaris ova can not be 
ruled out. Although the finding of bacteria inside the 
Ascaris eggs is not in total agreement with the report of 
Baron et ed (1960) who recorded very scanty growth of bacteria
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w»n«; nf As<ggfl|ĵ <| gcilXi which he examined, it 
supports the work of Hutchinson (1965) who established a 
relationship between NeVascaris eqq and Tox^nlasmci trans- 
mission in which Toxoplasma organism were incorporated 
into the eggs of Mepascaris worm inhabiting the intestinol 
lumen of infected individuals. The finding is also in 
accordance with the work o f  Adede-ji -who isolated
different species of bacteria from inside the eggs of 
Ascaris lumbricoides of man which he studied.
With all th ese findings, it is, therefore necessary 
to confirm conclusively, whether or not there is trans- 
ovarian transmission of some of the bacterial species 
recorded because data both in this study and in previous 
work recorded strongly suggest that this may be the case.
Finally, the findings in this study indicate that the 
flora of the worm is similar to that of the host in kind ■- 
and in relative frequency. These data confirm those of 
Li (T933b) and Ackert & Whitlock (1941), who reported trhat 
ascarid parasites normally feed on the host's intestinal 
contents. >' *
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The data therefore demonstrate that there are mutual 
relationships which exist between the flora of the parasite 
on one hand and the host on the other. These relationships 
start from the early development of the parasite. The rela*? 
tionships that exist between the parasite and the host seem 
to be a mutual one as long as the worms are not many in 
number and as long as the worms do not wander off their
usual route
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168
CHAPTER 4
3.4 THE EFFECT OF INTESTINAL FLORA ON THE DEVELOPMENT,
XNFECTXVXTY AND PATHOGENICITY OF ASCARXS S'lUM LARVAE 
IN PIGLETS
INTRODUCTION
Stefonski & Przyjalkowski (1965 & 1966) reported that 
normal intestinal flora favours the development of Trichinella 
spiralis in mice, as do also some Gram-negative bacteria: 
Escherichia coil. Pseudomonas aeruginosa and Proteus species 
given to conventional mice orally in monocultures. In 
subsequent studies, Stefonski & Przyjalkowski (1967), carried 
out seme experiments with chickens infected with Asoaridia 
galii, mice infected with Hymenofepis nano and Aspicuiaris 
tet rapt era, and rats infected with H. nana. In all these 
experiments, the authors observed a favourable influence 
of the total intestinal flora, as well as of all used 
Gram-gegative monocultures of bacteria, viz: E.coli.
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P.aeruginosa, and Proteus species. Gram-positive 
Bacillus subtilis also had an action like all the above 
mentioned Gram-negative bacteria.
In this study, apart from finding out the effect of 
monocultures of some intestinal bacteria on the development, 
infectivity and pathogenicity of Ascaris suum larvae in 
piglet, the study also attempted to determine if Ascaris 
suum larvae are capable of disseminating bacteria that 
adhere firmly on the surface cuticle during their migration, 
thus transmitting bactgria mechanically to the host. The 
study also found out the synergistic effect of Ascaris suum 
migration and various different species of bacteria.
MATERIALS AMD METHODS
Piglets, mature* eggs of Ascaris suum and monocultures 
of different species of intestinal flora were used.
The piglets used were supplied by the University of 
Ibadan Teaching and Research Farm. The animals were kept
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on a good standard ration throughout th© period of the 
experiment. The pigs were kept in clean pens first for
one week to find out whether or not they were harbouring 
helminth ova• Examination of their faeces during this 
period showed that they were free of any helminth infection.
The twenty piglets, weaned at 6-8 weeks of age were divided 
into 4 groups and each group was placed in concrete pens that
were cleaned and washed with water daily. The test animals 
were fed with ascarid larva© in overnight broth culture of 
a particular species of intestinal flora, the second group 
were fed with only ascarid larvae, the third group received 
overnight broth culture of the bacteria in use; while th© 
last group had nothing other than their normal ration.
This last group served as the control animals. Each pig 
was weighed at the beginning of the experiment and at" the 
end of the experiment. Ascarid eggs were removed from th© 
uteri of gravid female. As car is suut% collected from abattoir.
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17
The eggs were washed in normal saline (0.85/1 NaCl) and 
were kept in this solution for three days to remove from 
them the sticky material which causes clumping of the 
eggs in aqueous cultures. Eggs were cultured in petridishes 
and the tap water environment was replaced with fresh tap 
water every two days; some eggs were cultured in tap water 
to which a few drops of 5:S formalin was added and others 
were cultured in 2% formalin. The cultures were kept at 
room temperature and agitated once or twice daily for 21 days. 
At the end of this period, g quantity of the matures Ascarls 
3uum eggs from each culture dish was fed to a white mouse.
All the mice died of ascarid pneumonia and ascarid larvae 
were recovered from their lungs thus showing that the eggs 
were viable. The various cultures were combined, the eggs 
were concentrated and washed 4-6 times in sterile distilled 
water. They were then deshelled and sterilised in equal 
volumes of 2% sodium hydroxide and 3% sodim hypochlorite 
overnight according to the method of Hansen jet jal (1954 & 
1956). The eggs were subsequently washed 4-6 times with
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sterile saline to remove the deshelling solution; they 
were checked hacteriologically for their sterility by 
culturing them into and onto enriched, differential and 
selective media. The cultures were incubated aerobically 
and anaerobically at 37 C for 48 hours. The eggs wore 
later concentrated and stored in sterile tap water for 
feeding to the experimental piglets.
The species of bacteria used were Escherichia coli 
type 0128, Pseudomonas aeruginosa, Proteus mirabilis, 
Staphylococcus aureus. Streptococcus fmecclis (Enterococci) 
Bacillus subtilis and Bacillus cereus. Approximately
6,000 embryonated eggs were added to an overnight broth 
culture of a particular species of bacteria. Some sterile 
sand was added. The mixture was shaken gently to release 
the larvae from the shell into the broth. The infective 
larvae were kept in contact with the bacteria broth 
culture for thirty minutes to enable the bacteria to 
adhere firmly onto the cuticle of the larvae as was done
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by Taylor & Purchase (1931). The pigs were fF&cS by 
mouth ©ach day before their breakfast of breeder's 
grain ration. Three other piglets were fed by mouth 
v/ith ascarid larvae only. Seven piglets were fed each 
with overnight broth culture of bacteria only. Three 
piglets were not given anything other than their normal 
ration. The first set of seven piglets served as the 
test animals, the second set of three piglets served as 
the Ascaris control, the next set of seven piglets served 
as bacterial control while the last set of three piglets 
with only normal ration served as the negative controls. 
The experimental infections were carried out for eleven 
days. Towards the end of the first week of the beginning 
of the experiment, the infected pigs with Ascaris larvae 
only and with Ascaris larvae plus bacterial species were 
thumping badly. Some of them became very uneasy and they 
showed no desire for food. These symptoms are indicative 
of the passage of ascarid larvae through the lungs and
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174
the symptoms were not observed in the pigs that received 
bacteria only and those that served as negative controls.
At the end of the eleventh day of the experiment, the 
infected animals showed signs of pneumonia, cough with 
respiratory difficulties. Later, the symptoms began 
to moderate and the infected pigs ceased thumping and 
regained their appetites. Both the infected and uninfected 
pigs were then left on their normal ration for about 70 
days. The faeces of the infected animals were examined 
at the end of the sixtieth day and they were found to 
contain Ascaris ova. At this time the piglet that received 
Escherichia coli type 0128 and one piglet that received 
only Ascaris larvae were given fresh larvae plus .E. coli 
type 0128 and fresh larvae respectively. During the 
first 48 hours that followed this dose, the two piglets 
became very uneasy and they showed no desire for food. 
Within a week, the two piglets became moribund. Both 
the two piglets, the pig earlier infected with only Ascaris
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and a negative control pig were killed for Postmortem 
examination. The pigs were weighed before they were 
killed. Ail the other pigs including the controls were 
also weighed. Smears v/ere taken from the samples of 
lungs, spleen, liver, intestine and kidney and were 
stained by Gram's stain method. Samples of the liver, 
lungs, intestine, spleen and kidney were collected and 
preserved in Unbuffered neutral formalin ,(BHF) for 
histological examinations. Sections were cut at 6 microns 
(yu) and stained with Haematoxylin and Eosin (H & E) stain 
and also with Gram's stain. Samples of liver, lungs, 
spleen and kidney v/ere cultured for bacterial growth.
The isolates were identified both macroscopically and 
microscopically. Crush preparations of the lungs and 
the liver were made and examined for larvae under the 
microscope. The packed cell volume )(PCV), total white 
blood cells and differential leucocytes counts were 
performed on all the animals including those that were killed.
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All the remaining piglets were not killed for post-mortem 
examination for economic reasons. The University of Ibadan 
Teaching and Research Farm supplied the piglets on the 
understanding that the experiments were not terminal ; and 
that the animals were to be returned to the farm after the 
end of the experiments, thus the use of microscopic method 
to show whether or not the pigs were harbouring Ascaris ova.
RESULTS
The infected animals became very unthrifty compared 
with uninfected ones as shown in Figs 1 & 2. The infected 
animals weighed less than the uninfected animals. Table 1 
shows the PCV, WBC, and differential leucocytes counts of 
the pigs infected with Ascaris larvae and bacteria while Tabl 
2 shows the PCV,VJBC, and differential leycocytes counts of 
the pigs infected with Ascaris larvae only. Table 3 shows th 
PCV,h'BC and differential leucocyte counts of control animals. 
The summary of data on weight gains of pigs experimentally 
infected with Ascaris larvae plus bacteria, Ascaris larvae on
and of uninfected control animals is shown in Table 4
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TABLE 1l MEAN PACKED CELL VOLUMES, TOTAL WHITE 
BLOOD CELLS COUNTS AND DIFFERENTIAL 
LEUCOCYTE COUNTS OF PIGLETS INFECTED WITH
ASCARIS LARVAE AND BACTERIAL SPECIES
Animal Packed Total Eosino Lympho- Neutro­ Monocytes
infec­ cell W3C phils cvtes phils %
ted with volume % % %
(PCV)
Ascaris
plus 36 21,800 9 51 38 2
Proteus
Ascaris
plus 42 17,200 8 54 34 4...
Pseudo.
Ascaris
plus 36 18,100 11 50 38 1___
B.cereus
Ascaris
plus 36 24,600 8 49 42 _ 1____
Staph.
Ascaris
plus 37 26,000 10 40 38 2
Faecal • - .... —
strept.
Ascaris
plus 40 22,300 8 46 42 ..4___
B.subtil is
Ascaris
plus 36 27,500 9 40 46 5
E.coli 
tyge 0128
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TABLE 2°. ; J,' /•••. v ' •
MEAN PACKED CELL VOLUMES, TOTAL
WHITE BLOOD CELL COUNTS AND DIFFERENTIAL
LEUCOCYTE COUNTS OF PIGLETS, INFECTED
WITH ASCARIS ONLY
..— —
Animal Packed Total Eosino Lympho- Neutro- Monocytes
infected cell WBC phils cytes phils
with volume % % % %
(PCV)
Ascaris
only 42 22,600 2 80 18 ____=
Ascaris
only 37 23,100 2 82 16 2
.■* .■. . .
Ascaris
onlv 44 21,300 1 81 16 2
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TABLE 3; MEAN PACKED CELL VOLUMES, TOTAL WHITE 
BLOOD CELLS COUNTS AND DIFFERENTIAL 
LEUCOCYTE COUNTS OF NEGATIVE CONTROL 
PIGLETS
Packed Total Eosino Lympho Neutro Mono •_ 
Control cell WBC. phils cytes phils cytes
Animals volume % % % __ cl____
(PCV)
1 38 15,300 9 67 28 ..____ 9
2 44 13,900 2 69 26 __ ,3_.
3 40 14,550 2 70 24 4
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TABLE 4s SUMMARY OF DATA ON WEIGHT GAINS OF
PIGLETS EXPERIMENTALLY INFECTED WITH
A^ARTS AND BACTERIA
Piglet infected Weight Weight Weight Percentage 
with Ascaris at at end of Gained Weight 
and Bacteria beginning experiment Gained “
Proteus 261bs 341bs 81bs 24
Pseudomonas 24 " 35 " 11 " 31
B. cereus 21 " 31 " 10 " 32
Staphylococci 23 " 26 " 3 " 12
B.subtilis 20 " 28 " 8 " 28
Enterococci 20 " 30 " 10 " 33
E.coli 0128 25 " 33 " 8 “ 24
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TABLE 5: SUMMARY OF DATA OH WEIGHT GAINS OF 
PIGLETS EXPERIMENTALLY INFECTED WITH 
ASCARJS ONLY
Piglet infected Weight Weight Weight Percentage
with ;Ascaris at at end of Gained Weight
only beginning experiment " 'Gained'
1 241 bs 31 lbs 71bs ____23__
2 23 " 35 " 12 11 34
3 9 " 12 " 3 " 25
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TABLETS. SUMMARY OF DATA ON WEIGHT GAINS
OF CONTROL PIGLETS
Control Weight Weight at Weight Percentage
Pig.lote at end of Gained Weightbeginning experiment Gained_____
1 131bs 46 lbs 28 lbs 61%
2 13 :I 34 21 " 62%
3 20 " 46 " 57%
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The control pigs wore worm-free. In the infected 
pigs no helminth parasite other than ascarids were found.
As can be seen in Tables 4 ,5  & 6, the total amount of 
weight gained by the infected animals was lower than the 
weight gained by the uninfected pigs. The Gram*s stained 
slides of the piglet infected with Ascoris plus E,coll 
0128 showed Gram-negative bacilli. There was no bacteria 
seen in the other slides. The crush preparation showed 
larvae in the lungs.
Gross Post-mortem findings
Four piglets (P149/83, PI50/83, P161/83 and P162/83) 
that were slaughtered had the following necropsy findings: 
Piglet PI49/83 which was infected with only Ascaris larvae 
initially had generalised serous atrophy of body fat. There 
was also a patch of congestion at a point between the apical 
(anterior) and cardiac lobes of the right lung. The stomach 
contained normal ingesta and the small intestine was 
virtually empty but there were some adult ascarid worms in
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u vJ'Ti*
the intestine. There were multifocal areas of milky- 
white slightly depressed spots measuring !-2cm in 
diameter in the liver, this is the so-called milk-spots. 
There was no significant gross findings in other organs.
Piglet PI50/83 which was infected with Ascaris larvae 
plus Escherichia coli 0128 had a generalised serous atrophy 
of body fat. The pericardial and perirenal fats were 
gelatinous. There was a slight firm and nodular area of 
grey hepatisation with abscess pocket in the intermediate 
lob© and another one on the anterior i of the diaphragmatic 
lobe of the right lung. There was also a patch of red hepa­
tisation on the antero-ventral part of the left apical lobe. 
The greyish area contained a cream-coloured purulent exudate 
in a fibrous tissue capsule which on culture yielded E.coli 
serotype 0128. The liver contained l-2cm greyish-white 
and depressed focus immediately dorsal to the area of 
attachment to the gall bladder. There were multifocal 
areas of 2-3cm sized pale necrotic, slightly firm (Fibrotia) 
spots on the parietal and visceral surfaces of the liver.
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\
t
Thor© were two 2 x3cm sized circumscribed firm whitish 
foci on the liver. When incised, they contained fibrous 
connective tissue surrounding some diaiated hyperaemic 
tunnels. There was enlargement of the hepatic lymph node 
(Fig.3), The small intestine contained a lot of greyish 
watery material. There were some adult worms in the intestine. 
There was no significant gross lesion in other organs.
Piglet PI61/03, the control piglet tbct was uninfectod 
showed no gross lesion. The stomach and intestinal contents 
appeared normal and there was no significant gross lesion in 
the organs.
Piglet PI62/83, that was infected with Ascaris and 
later on reinfected with fresh dose of Ascaris larvae 
towards the end of the experiment had multifocal areas 
of petechial to ecchymotic haemorrhages on the parietal 
and visceral surfaces of the cardiac and anterior half of 
the diaphragmatic lobe of the right lung. These areas 
were slightly firm and rubbery. The intestinal contents
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186
appeared normal but there were some adu It ascarid worms 
in the intestine, /.scaris larvae were found in the crush 
preparation of the lungs. There were numerous milk-spots 
in the liver and a few nodular foci which consisted of 
fibrous connective tissue surrounding some hyperaemia 
tunnels. There were no significant gross lessions in 
other organs.
Histological Examination
Microscopical examinations of the organs of the 
infected and the uninfected control animals revealed the 
following findings; In Piglet P149/83 there were a few 
haemorrhagic foci in the lung. There were also a few 
atelectatic areas with congested alveolar capillaries 
and haemosiderosis (Fig.4). There was no parasite section 
seen. The 1 iver showed some degree of fatty degeneration 
and a few haemorrhagic tracts in some lobules. There 
were also a few foci of fibrous connective tissue prolife­
ration in some lobules (Fig.5). There was no significant 
finding in the spleen and intestine. There was no bactoritl̂ vj 
seen in the Gram's stained section.
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187
In Piglet P150/J3, the lungs sections showed areas 
of diffuse neutrophilic infiltration into the alveoli 
and some bronchioles end marked fibroplasia of the inter­
lobular connective tissue (Pig.6). There were a few largo 
focal areas of caseous necrosis with: numerous bacterial 
colonies within the necrotic debris. There was also a 
fibrous capsule around the necrotic mass and layer of 
inflammatory cells. The necrotic centre was surrounded 
by a zone of marked cellular infiltration consisting of 
neutrophils and eosinophils (Figs 7 E 0). There was 
hyperplasia of the bronchial epithelial cells in areas 
of the lung adjoining the necrotic mass as well as diffuse 
alveoli and peribronchiolar infiltration marked by neutro­
phils (Fig.9)0 Some areas of the alveolar tissue had been 
replaced by fibrous connective tissue. The liver showed 
several foci of granulomatous nodules with central hae­
morrhagic areas with dying and dead cells. The central 
area of each nodule was surrounded by large areas of 
mononuclear ceil infiltration predominantly lymphocytes
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and eosinophils (Fig.10). There were also a few foci 
of lymphoid follicle formation within the area of cellular 
infiltration which surrounded the necrotic centre. The 
area was surrounded by a fibrous tissue capsule. There 
are a few areas with bile-duct hyperplasia and fibrous 
tissue proliferation in other parts of the liver. In the 
hepatic lymph node, the germioal centres of the lymphoid 
follicles were very prominent and there was also increased 
cellularity in the paracortical and medullary areas. Most 
of the cells were lymphocytes. There was no significant 
finding in the kidneys, spleen, and intestine. The Gram- 
stained section of the lungs and liver showed Gram-negative 
bacilli within the necrotic debris.
In piglet PI61/G3, most areas of the lungs were 
normal (Fig.11) but there were a few focal areass of 
peribronchial lymphoid hyperplasia and alveolar haemor­
rhages. There were no significant findings in the spleen, 
and kidneys. No bacterial colonies were seen in the 
section stained by Gram*s method.
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In Piglet P169/83 many bronchial and bronchioles,*
lamina contained sections of helminth parasite which 
are Asearls species (Pigs IP, 13 & 14). There was 
usually an accompanying peribronchial and peribronchiolar 
infiltration by mononuclear cells which were predominantly 
lymphocytes and eosinophils(Figs. 15,16 & 17). There was 
also fibrous connective tissue infiitretan in the area of 
peribronchial cellular infiltration (Fig.16) and hyper­
plasia of the bronchial epithelium (Fig.13). Some 
portions of the bronchi had been destroyed and replaced 
by infiltrating colls and sections of the parasite (Figs*
14 & 17). The peribronchial areas contained normal alveole 
inspite of parasite sections within the lumen of bronchi 
in such areas (Fig.19), The histological picture of the 
liver revealed a feu large-sized granulomatous nodules 
with central areas containing sections of a parasite 
(Ascaris spa); and necrotic cells and surrounded by a 
zone of marked mononuclear cell infiltration, mainly 
lymphocytes and eosinophils bounded by a fibrous tissue
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190
capsule (Figs. 20 & 21). There were a few areas of 
bile-duct hyperplasia emc! pseudolobulation in the 
liver tissue adjoining the parasitic granuiomata 
(Fig, 2? ). There was marked reactive hyperplasia 
in the hepatic lymph node. There were no significant 
findings in the kidneys, spleen and intestine.
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191
FIG.1: Showing unthriftiness and retarded
growth of the infected piglets at the 
end of the esperiment.f(k — | ?
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192
FIG#?J Showing control piglets at the end
of the experiment. Hot©(  t(hte — Id1?z .freronoe3.
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193
FIG.3; Enlarged hepatic lymph node (arrowed) 
due to synergistic effect of Ascarl6 
plus -E--.--c-o—li  infection. 'n  /,  V ,3 /
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194
FIG, 4: Lung showing area of slight alveolar
haemorrhage and congestion of alveolar 
capillaries. Many alveoli are normal. 
(H & E) x 160
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195
FIG,5: Liver showing a r e a s of fibrous tissue
proliferation (repair) of the liver 
sequel to parasitic infection.
(H & E) x 160.
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196
FIG. 6; Lung showing diffuse infiltration by
some mononuclear cells indicating tsiat 
the animal is progressing towards 
pneumonia (H & E) x 62
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197
FIG.7: Lung showing area of necrosis with
bacterio colonies end surrounding 
thickened wall. Notice the bacteria 
Granuloma formed following initio.-, 
parasitic infection (H & e) x 6 .̂»
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- 198-
FIGt S;
Lung showing area of necrosis containing 
bacteria on the right with surrounding areas 
of neutrophilic infiltration and an outer 
zone of mononuclear cells infiltration which 
are predominantly eosinophils and macrophages 
- evidence of parasitic infection.
(H & E) x 400
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199
FIG.9: Lung showing peribronciolar and alveolar
neutrophilic infiltration due to secondary 
bacterial infection. Notice the bronchiole 
which is intact and no infection itkside it 
except around it (H & E) x 250.
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FIG,10; Liver showing central area of necrosis 
and mononuclear infiltration around the 
necrotic areas - evidence of bacterial 
infection, (H & E) x 62,
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FIG, 11i Normal Liver of PI61/83
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202
FIG. 12: Lung showing cross-section of
parasite in a bronchiole. There is 
slight peri-bronchiolar mononuclear 
cells infiltration around the cross-section 
of the parasite predominantly eosinophils 
- indicating parasitic infection.
(H & E) x 160.
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203
FIG. 13: Lung showing parasite sections in
bronchi with peribronchial mononuclear 
ceils infiltration.
The intervening alveoli are normal.
(H & E) x 62
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? Q4
FIG.14:
Lung showing lungitudinol section of 
parasite in the bronchus.^
(H & E) x 62
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?05
FIB,15: Liver showing area surrounding
parasitic granuloma; Hepatocellular 
necrosis, infiltration by eosinophils 
and few neutrophils (H & E) x 160,
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906
FIG,16; Lung showing area of marked mononuclear 
cell infiltration in an area where normal 
bronchial tissue has been lost.
Notice the formation of fibrous connective 
tissue, (H & E) x 160,
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207
FIG, 17: Lung showing u .  of parasite in a
bronchus. Note loss of bronchial 
epithelium (arrowed), haemorrhages 
(arrowed) and marked mononuclear cell 
infiltration into the bronchial wall 
and around the parasite section,
(H & E) x 62,
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•it  
FIG, 18: Lung showing hyperplastic bronchial
epithelium with parasite section in the 
lumen* This is an evidence of respiratory 
embarrassment caused by the parasite. The 
area will secrete more mucus to wash out 
the irritant (parasite), (H & E) x 160,
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209
FIG. 19: Lung showing cross-section of the
parasite in bronchus with normal 
surrounding alveoli - indicating that 
the parasite was just passing through 
the bronchtlS and has not destroyed 
the wall of the branch
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210
FIG. 20: Liver showing parasitic granuloma
with fibrous tissue capsule surround 
and mononuclear cell (eosinophils) 
infiltration around section of parasite 
in centre of the lesion (H & E) x 62,
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- J
-210A ,
FIGx 21.
Liver showing parasitic granuloma 
surrounded by a zone of cellular 
infiltration and a fibrous connective 
tissue capsule. (H & E ) x 6.c:
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mam  
21 OB
Liver tissue in vicinity of parasitic 
granuloma showing pseudolobulation.
(H & E) x 62
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211
DISCUSSION
/
Although the literature on the interrelationship 
of bacteria and intestinal helminths in animals is scanty 
Newton _et _al,( 1959^, W.fescott & Todd (1964), and Wescottt 
i * (j960) h ave studied Nematospiroides dubius 
infection in germfree mice and Przyjalkowski (1967) has 
worked with Trichinella spiralis in fre© and mono- 
contaminated mice.
In the present study, Asccris suum lcrvae and 
monocultures of intestinal bacteria were used. The data 
presented in this study provide clearly data on 
the extent to which infections of ascarid only and 
ascarid plus bacteria can affect adversely the growth 
rate of pigs in an environment of heavy bacteriafconta­
mination and in an environment with little or no bacteriat 
contamination. Infections with large intestinal round- 
worms (As Claris suum) constitute a problem in swine 
production that is of considerable economic importance.
; • '-..2 >:■
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212
Theso worms are among the most widespread and injurious 
of the intestinal helminth parasite that infect pigs 
(Ransom, 1927, Burch, 1930 and Roberts, 1934), Light 
infections may produce some degree of unthriftiness
and retarded growth where severe infections may result
*
in death of the affected animal (Schwatz, 1937), Fig,1 
shows the effect of Ascaris suum infection in piglets. 
The infected animals were very unthrifty and showed 
retarded growth. These animals can be compared with 
the control animals in Fig.2 which appeared healthy and 
robust. It is reasonable to assume that apart from the 
damage caused by the migrating larvae of ascarids to the 
pigs* internal organs, sensitisation is caused during 
larvae migration and is intensified by the repeated 
infection (Jung, 1953). Furthermore, the presence of 
worms in the small intestine, their growth, metabolism, 
and secretions might escort an adverse effect on the 
nutrition of the host. Many worms in th© small bowel of 
the host undoubtedly rob the pigs of nourishment by
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utilising the semi-digested food. Tables 1,2 and 3
show that the packed cell volumes of the pigs, infected
*
with Ascarids only, those infected with ascarids plus
(!
bacteria and the control pigs are
within the normal range (normal packed cell volume 
(PCV) of pig 32-50, >), but the differential leucocyte 
counts are quit© interesting. Inthe pigs infected with
j
ascarids plus bacteria, the neutrophils are somehow higher- 
in count than those of animals with ascarid only. This 
high count of neutrophils is an evidence of bacterial 
infection whereas the animals infected with ascarids only 
gave very high count of lymphocytes in response to ascarids 
infection. This is characteristic of jĵ eiminth infections 
and the results obtained in this study agree with the 
findings of Przyjalkowefci and liescott (1969), who recorded 
very high percentage (71.1?? - 82,4/b) of lymphocytes in 
animals infected with Trichinellg spiralis. There is 
also a big difference in the percentage counts of eosino­
phils of the parasitised and unparasitised piglets. The
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214
high eosinophil counts recorded in the parasitised pigs 
is not surprising as Strafuss and Zimmerman (1967) found 
that peaks of eosinophils appeared in pigs infected with 
nematodes. Tables 4,5 & 6 summarised the data on weight- 
gains of both infected and control animals. The results 
show that all the infected animals harbouring adult worms 
had their growth retarded. For example, the presence of 
adult worms was sufficient to destroy the health of the 
pigs to the extent that progressive loss of weight occurred
vbfecW
so that the ̂ animals weighed less at the termination of the 
experiment than they did at the beginning. The maximum 
weig ht gains in infected animals was 34% whereas in the 
uninfected control animals it was 62%. These results agreo 
with the findings of Spindler (1947) who found that pigs 
infected with 2,0 or more -worms at 8 weeks of age failed 
in gain of weight in proportion to the number of worms 
they harboured. He also reported that one pig with 109 
worms gained no weight at all, whereas uninfected pigs 
gained an average weight of IQOlbs. Because of the b.
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215
presence of adult warns in the pigs, there is bound to be 
loss results from condemnation of carcases for,jaundice 
owing to blockage of bile-ducts. In this study, the 
number of adult worms recorded from each pig killed was 
relatively small compared with the number of larvae
administered. This is difficult to explain^ Lj A aasi fl
O i V W  *
The results of the study also show that Ascaris larvae 
and Ascaris larva© plus bacteria species were infectiv4 
for the pigs. The Ascaris larvae developed to maturity 
both in the presence of bacteria species as shown by the 
presence of Ascaris eggs in the faeces of all the infected 
animals. The findings in this study are in accordance 
with the works of liescott & Todd (1964), Stefanski &
Przyjalkowski (1965 & 1966), Przyjalkowski (1967) and 
Stefanski (196y) who found that intestinal microflora of 
the host favoured the establishment of intestinal nematodes.
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In the animal infected with Ascarls larvae only, 
the histological findings show patchial haemorrhages whic 
is an evidence of traumatic damage probably due to the 
larval migration of the parasite. There was no major . ' 
lesions observed on this animal possibly because of 
tissue repair sequel to initial parasite damage. Further 
more, there was no parasitic section in the tissue 
probably because the parasite had already left the lungs. 
The liver shows fibrous tissie repair sequel to previous 
damage. In Piglet PI49/83, it did appear that the animal 
had recovered from the infection. The parasite had 
completed its developmental and migratory phases in the 
liver and lungs with minimal damage. Such damage was 
not serious enough to leave any major lesions in the 
organs. However, the areas of fibrous tissue prolifera­
tion in the liver will suggest a repair process sequel 
to a previous parenchymal damage. This damage was severe
enough to have caused extensive tissue damage which could
%
not be replaced by regenerated hepatocytes but by fibrous
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o
connective tissue.
The lung lesion in Piglet P150/03 suggests a 
significant secondary bacterial infection of a pre-existing 
wound. The pneumonia is suppurative and of a long duration 
in view of connective tissue proliferation which led to 
a fibrous adhesive pleuritis in the lung. The inflammatory 
reaction which seemed to be mainly alveolar, peribronchial, 
and peribronchiolar will suggest that the bacteria most 
probably reached the lung by other routes other than the 
aerogenous route. This finding suggests that the bacteria 
wore carried to the lung by the migrating Ascaris larvao 
since the animals were infected with both bacteria and 
larvae together. The results of the study -show that 
Ascaris larvae are capable of transmitting bacteria mecha­
nically, because, after infection during the necropsy, 
bacteria was isolated from the organs both in direct 
smears and in cultures. These findings agree with the 
work of Weinberg, (1907) who concluded that by biting and
boring into the intestinal wall, or by wounding it with
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218
hooks and suckers, the majority of helminths favour the 
penetration of microbes, these being carried on the 
exterior and in the intestine of the worms. The 
increased pathogenicity caused by both the Ascaris 
larvae and L’scherichla coli shows that the two agents 
work together to produce a disease condition more severe 
than the sum total of effects produced by each indepen­
dently. The observation in .this study is in accordance 
with the findings of A’ayak & Kelley (l965) who showed 
that Ascaris suum increased the severity of swine 
influenza in pig. There are several ways that migrating 
Ascarls larvae may contribute to this synergism. Nayak 
& Kelly (1964) have shown that there is a greater amount 
of haemogglutination antigen produced in compounded 
Ascaris-influenza-bacferia infection and this material 
may be toxic to the lung tissue. Furthermore, the trauma 
produced by larvae rupturing alveoli racy induce sufficient 
stress to lower the animals resistance to very severe 
infection.. Capillary damage produced by penetrating
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219
larvae may allow more viral, or bacterial antigens to 
gain access to the systemic circulation and thus produce 
vireamia, bactereamia or toxaemia.
The presence of a few eosinophils will suggest an 
underlying hypersensitivity or parasitic infection which 
was superimposed by the bacteria pneumonia. The liver 
lesion which showed some inflammatory response most 
probably suggests an initial market! damage to the liver 
and secondary bacterid infection. The cellular response 
suggests a long-standing lesion which is most probably 
due to both parasitic, bacterial andkWpersensitivity 
reaction. The areas of nodule formation indicate areas 
of massive tissue damage with replacement by infiltrating 
colls reacting to an antigenic stimulation and presence 
of fibrous connective tissue to repair some of the damaged 
tissues. The bile-duct hyperplasia is also suggestive 
of reaction of the bile-duct system to an irritant or 
foreign body. The hepatic lymph node shows a remarkable
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reactive hyperplasia probably as a result of inflammatory
r
reaction occuri/lf) 1° the liver. The absence of any inflam­
matory reaction in the intestine of the infected animal 
suggests that the worms which sere found did not attach
"to the mucosa.
Piglet P161/83 sections of the liver and lungs 
appear normal. This is an animal used as control with 
neither parasitic nor bacterial infection, and these 
sections could therefore be compared with those from 
infected animals for proper assessment of the damage 
done to the organs by both the parasite and the bacteria 
either individually or together.
In Piglet P162/83, the lung lesion indicates the 
presence of developmental stages of the Ascaris suum as 
they pass through the bronchioles and bronchi. There 
is little or no damage to the bronchiolar epithelium but 
the bronchial epithelial damage suggests some pressure 
atrophy induced by the parasites which occupied the 
bronchial lumen. The marked cellular reaction around the
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bronchi and bronchioles and the cell types suggest an 
antigen-antibody reaction probably induced by the para­
sites, their metabolic by-products or the antigenic coat 
released into the lung tissue during moulting. The 
presence of fibrous connective tissue capsule indicates 
that the lesion is a parasitic nodule being walled off 
from the normal lung tissue. Similar parasitic nodules 
are present in the liver and perhaps this suggests that 
the ages of the liver and lung lesions may be about the 
same. The pathogenesis of the liver lesion is possibly 
similar to the lung lesion. Reaction is mainly mononucloar 
cells. Enlargement of goblet cells shows increased mucus 
production. There is diffuse neutrophilic infiltration 
like in PI50/83 which suggests that the major lesion is 
due to parasitic demage only.
In summary, the findings in this study concerning 
pathogenicity and pathogenesis of Ascaris suum in pigs aro 
in total agreement with the works of Spindler (1947),
Roberts (1934), and Soulsby (1967), The action of the given
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222
monocultures of intestinal bacteria observed in this 
study therefore offers a promising model system for 
fWflfher investigation of the interaction of bacteria 
and intestinal helminths.
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223
CHAPTER 5
S5SSS5SSS
3.5 SOURCES OF HUNAN I! FECTION OF ASCARIASIS 
Xt'j ISADAN
INTRODUCTION
In areas where human faeces is used as manure or where 
promiscuous defaecation is practised, these factors contri­
bute significantly to the pollution of soil, and helminth 
eggs and larvae may be readily demonstrated on fresh 
vegetables purchased from the market (Chang & Chin, 1943).
It is therefore reasonable to assume that in those areas, 
the prevalence of intestinal helminths is in part due to 
the ingestion of infective stages adherent to the uncooked 
food; plants and water. Where refrigeration and commercial 
conning are little used as in Nigeria, vegetables are freely 
bought in the market or are taken from gardens and eaten 
raw in order to satisfy the consumers' need and desire for
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224
balanced diets. In such foods, the development and 
survival of the infective stages of helminths exert 
an undetermined influence on the prevalence of these 
parasites. Reported observations by Chang & Chin (1949) 
indicate that none of the common preservatives such as 
salt, alcohol, and vinegar in the usual concentration 
can be relied upon to kill the eggs of Ascaris lumbricoides. 
It has been noted by Fueki (1952) that vegetables, soil, 
dust and fingers constitute the principal sources of 
Ascaris infection.
Since there has been no published record in Nigeria 
on the importance of roof and leaf vegetables and other 
food items consumed rat; in the transmission of Ascariasis, 
this study was designed to find out this possible role 
in Ibadan.
MATERIALS AND METHODS
Different food items that are usually eaten rat; or 
uncooked such as Gari, leaf vegetables, root vegetables 
and fruits were brought from different shops and open
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225
markets in Ibadan. The food items were examined for 
Ascaris eggs. Each food item was washed in sterile 
distilled water several times. They were then 
centrifuged at a top speed of an international centrifuge 
at 2,500rpm, for 2. minutes using sterile universal con­
tainers already covered. The deposit was examined, the 
number of Ascaris eggs present was counted and the number 
of embryonated eggs noted, using light microscope.
The supernatant and the sediments of the centrifuged 
foods were cultured on different culture media for bacte­
rial growth. Palm win a was centrifuged at] a top speed 
of an international centrifuge for 2 minutes at 25Q0rpm. 
Both the deposit and the supernatant were treated as 
above. Gari was suspended in sterile distilled water. It 
was mixed very well and then allowed to sediment. The 
supernatant was collected. More water was added and 
most of the grains of gari was removed by staining through
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226
sterilo four-piygause, The whole supernatant was then 
centrifuged at 35Q0rpm for 2 minutes. The deposit was 
examined for Ascarls ova. Doth the deposit and super­
natant were cultured.
In addition, the deposits and the supernatants of 
all the food items were separately inoculated into nutrient 
broth containing 5'% and 10$ acetic acid and into 70$
Ethyl alcohol. Both the acid and alcohol cultures wore 
left for 15 minutes, 30 minutes before subculturing them 
onto nutrient media and the eggs present cultured for 
larval development. This egg culture was made after 
washing off the acid and alcohol from the eggs 4-6 times 
with sterile distilled water. The sediments and the 
supernatants wore also boiled at 100°C for 5 minutes,
TO minutes and 15 minutes. They were cooled down and then 
subcultured onto culture media and the eggs present were 
cultured for larval development. Identification of the 
isolated bacteria was based on the Gram's stain, motility 
and biochemical reactions as given in Sergey's Manual of 
Determinative Bacteriology, Baltimore (1957).
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R E S U L T S
sssss& ssaasss
TABLE 1; FREQUENCY OF ASCARIS EGGS
ON EDIBLE FOODS
Kinds of Number Positive Percentage Total Emforyonated
Specimen Examined Specimen Positive Egg Eggs
Counts
Lettuce 43 11 25.6 27 10
Cucumber 55 9 16.4 21 6
Carrots
(roots) 45 11 24.4 38 12
Mango 35 8 22.9 20 6
Tomato 52 5 9.6 13 3
Gtfwrcta'v 63 10 15.9 15 2
Sweet pepper 32 7 21.9 10 3
Onion (bulb) 29 4 13.8 7 1
Gari 36 7 19.4 18 7
Palm-Nine 29 5 17.2 11 5
NR; Th© sp re a d  i s  shown in  A p p en d ix  1 — 10
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TABLE 2: EFFECT OF ACETIC ACID AND
ETHYL ALCOHOL ON BACTERIA
Type of “ 5jT~ ' 10$ 70$ Time of 
Organism Acetic Acetic Ethyl Exposure 
Acid “ Acid Alcohol in minutes
Staph.aureus NG ng G 15
Staph.aureus NG NG NG 30
Esch.coli NG NG G 15
Esch.coli NG NG NG 30
Proteus spp. NG NG G 15
Proteus spp. NG NG G 30
Aerobic
sparebearer 0 G G 15
Aerobic
sporebearer G G G 30
KEYS;
G a Growth on subculture 
MG = No growth on subculture.
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TABLE 3; EFFECT OF ACETIC ACID AMD ALCOHOL
ON DEVELOPMENT OF BOTH EMBRYONATED 
AND NON-ENBRYONATED ASCARIS LUMRRTCGXDES
^ « 8  V  ■ « i M .  ■ *  —  .-is**.As>v.-
EGGS
•
5% 10# ‘ 70% Incubation
Type of Acetic Acetic Ethyl period in
Specimen Acid Acid Alcohol Days
Ascaris
lumbricoides ++ +-S- ++ 21
eggs plus 
Bacteria
Ascaris
lumbricoides ++ ++ ++ 21
eggs only
KEYS;
++ = Development of eggs to infective larval
stage
NOTE; Acetic acid and ethyl alcohol had no effect 
on either the embryonated or developmental 
stages.
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Table 1 shows the recovery rate of Ascaris eggs 
on the food items examined. The bacteriological exami­
nation of the food items yielded some bacteria/ species.
The species recovered from the cultures of the water 
used to wash the food items were Staphylococcus aureus, 
Staphylococcus albus, Escherichia coli, Proteus mirabilis 
Proteus morgani and Proteus vulgaris. No anaerobic 
organism was isolated.
Tables 2 and 3 show the effect of acetic acid 
and ethyl alcohol on bacteria and Ascaris lumbricoides 
eggs. The 5% acetic acid killed all the bacteria isolated 
from the food items but hac! no effect on the eggs of 
Ascaris lumbricoides. The acetic acid and ethyl alcohol 
appeared to be essentially without effect on either the 
embryoncted (infective) or developmental stages (Table 3).
Boiling the eggs and bacteria at 100°C is effective 
in killing them and this is confirmed by the failure of 
eggs to develop in culture.
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DISCUSSION
In Nigeria, intestinal nematodes abound both in 
the rural and urban environments because our tropical 
climate offers excellent opportunities for easy and 
rapid development of the different stages of these 
parasites which are rapidly disseminated in our soil 
through our gross and indiscriminate defaecation habits 
The prevalence of Ascaris infection among the people 
therefore has been at a level of 5.81,? (Ramsay, (1934) 
in the North and 73*4$ (Okpala, 1956) in the South.
Table 1 confirms that various food items that are 
eaten raw con serve as vehicles of Ascaris infection to 
man. Fueki (1952) found Ascaris eggs on many vegetable 
specimens. The finding of Ascaris eggs on edible vegeta 
fruits therefore emphasises the need for strict obser­
vation of simple hygiene methods aimed at eliminating 
the Ascaris eggs before consumption of the uncooked
f r a n d  vegetables
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There ere many ways by which these food items can
be contaminated with Ascaris lumhricoides eggs. The
eggs of Ascaris could be transferred from objects to
fingers and from fingers to the mouth or directly from
the objects to the mouth. In countris where Ascaris
eggs are abundant in the environment they have been
recovered from any object including paper money (Dolt
& Themme, (1949) and Gonzalez-Castro (1951).
*
Infection with Ascaris eggs can be soil-borne 
in the case of vegetable and roots, water-borne in 
the case of palm-wine. The contamination of food 
items is very common in materially less favoured 
countries like Nigeria where the absence of community 
services result in low sanitary standards and greater 
opportunities for the common spread of infection in 
water supplies or foods contaminated by cases and 
carriers. This contamination is also common in areas 
where there are overcrowding and careless droppings of 
small children in the gardens and the open ditches aroun
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the farms anc) houses. In areas where human faeces are 
used as fertilizer of garden crops, all the raw vegetables, 
including roots, stem, leaves and fruits which ripen in 
or near the ground are especially exposed and can be 
easily contaminated and be sources of infection when 
improperly washed or cooked before consumption. Further­
more, Ascaris eggs remain viable for a long time. Davains 
(1863) found that the ova of Ascaris lumbricoides remained 
infective after storage for five years and Bailliet 
(1866) observed that the eggs of Ascaris remained viable 
after twelve months exposure to heat of summer and to the
cold of winter. Other studies (Brown, 1927a, Cort, 1934,port &
& Otto„1933, Otto & Cort, 1?34, and Headlee,1936 have $hown that 
Ascariasis is essentially a household infection and the infectivity
o f the soil is maintained for the most part by promis­
cuous defaecation. For the recjsons given above, all 
fruits getting in touch with contaminated soil would 
likely carry Ascaris eggs on their surface. Ascaris eggs
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are adhesive, therefore vegetables and other food 
items that are not properly washed or cooked serve as 
sources of infection. The results of this study confirm 
the work of Fueki (1952), Kobayashi (1954), Nishimura 
(1957) and Fuji! (1957) who carried out a survey on the 
soil of farming land for Ascaris eggs and found that the 
soil of green leavy vegetable fields was heavily conta­
minated with Ascaris eggs.
Dust is also known to play an important part in the 
dispersal of Ascaris eggs Bbgojawlenski & Demidowa (1928). 
Nakayama (1956) hafe&shown that Ascaris eggs provide a 
physical characteristic of aerosol. This fact indicates 
that the eggs can be regarded as the dust in nature and 
therefore can be air-borne. Ascaris eggs adhering to 
small particles may be carried away by the wind from the 
surface of cultivated land (Kobayaski, 1955 and Morishita 
et al, 1959) and adhere to vegetable, enter into house 
and contaminate food items like gari and others. Ascaris
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eggs in the faeces used as fertilizer in agricultural 
farms or in faecal deposit in farms and gardens are 
scattered on the soil and occasionally onto vegetable. 
Attachment of viable eggs onto vertical surfaces up to 
30cm above soil surface has been observed by Beaver (1902). 
The eggs on the surface of the soil may be splattered onto 
vegetables when it rains. The eggs also flow out of farming^ 
lend during heavy rainfall and may be carried away to other 
places by the running water.
Although acetic acid has not been studied as an Ascaris 
ovicidal agent^ as the principal active constituent of 
natural vinegars, it is a well known food preservative, 
and its toxicity for bacteria, yeasts and moulds has been 
the subject of investigations which have demonstrated that 
at room temperature, 15 minutes contact (or less) with 5y 
concentration (or less) kills several strains of food 
poisoning Staphylococci, Salmonella typhi, Eschericia ooli 
and others (McCulloch, 1945). In this study, the use of 5%
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acdtic acid failed to kill Ascaris lumbricoides eggs.
These findings agree with the results of Soh (1960) and 
Faust (1970) who showed that chemical sterilisation of 
Ascaris Ijumbricoides eggs is impractical, since the eggs 
thrived and maturedwhen immersed in strong chemical solutions. 
The use of 5% acetic acid was, however, capable of killing 
all the species of bacteria isolated from both the super­
natant and the sediment of water used to wash the vegetables. 
The results of this study confirm the work of Beaver & 
Deschamps (1949) who showed that 5% acetic acid kills 
several strains of food poisoning - Stanhylococci, Salmonella 
typhl and E.coii. This finding clearly shows that the use 
of acetic acid would not be effective as a prophylactic 
measure against Ascaris infection.
The use of 70% Ethyl alcohol cannot b© ralifid upon as
a propby1actic measure as it failed to kill the eggs of 
As earns._ The use of boiling water to destroy both the 
bacteria arid the Ascaris eggs present seems to be the only 
way to got cut of Ascaris infection in edible fruiis. This
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finding is in accordance with the observation of
Ogata (1925) who studied the effect of heat in the
destruction of the ova of Ascaris lumbricojdes anc!
found that eggs of Ascaris exposed to 70 o C-100 oC 
for one minute were killed. Boiling treatment could 
however, not be used on fruits and vegetables that are 
normally eaten raw.
Of interest is the finding of Ascaris eggs in 
palm win© which is on© of the country's local wines and 
which is usually taken uncooked or unfiltered. Palmwine 
is tapped from palmtree and naturally one would expect 
such wine coming out of tall trees of palmwin© to be free 
from both bacteria and parasites contamination. The finding 
of Ascaris eggs in palmwine therefore, suggests that the 
contamination could be due to the use of contaminated 
water for the dilution of the wine. It could also tor® 
due to the use of contaminated containers and the
activities of flies and dust
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One of the most interesting findings of this 
study is the presence of Ascaris eggs in gari which is 
one of the staple foods in Nigeria. The preparation of 
gari involves frying the gari at such a high temperature 
that is capablo of destroying both bacteria, viruses, 
protorca cysts and helminth ova that may be present in 
it during fermentation period. The presence of Ascaris 
eggs in gari could probably be due to the use of dirty 
or contaminated containers and utensils for preparation, 
selling, buying or for drinking the food. Alternatively, 
it could be due to careless handling of gari by the gari 
sellers who probably are excreting Ascaris eggs in their 
faeces and thereby contaminate the food with the eggs in 
their fingers. Most importantly is the role of flies in 
contaminating gari in open markets. The gari sellers sell 
their gari in containers and not in polythelene bags or 
glass cased containers. The sellers stay in open markets 
where flies are always abundant and feeding freely. Flies
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like Musea domestica and other species of Musca possess 
both ex|"&</hal and internal structures particularly well 
adapted for picking up and carrying of living bacteria 
and parasites including Ascaris eggs from contaminated 
faeces to gari in the open markets, especially since 
markets are noar refuse dumps where people defaecate 
freely because there are no public toilets in our markets.
In the direct feeding position of the fly, cestode, tresnatode 
and helminth eggs including Ascaris eggs, can enter the gut. 
In this way, Ascaris eggs may be transmitted to gari while 
the fly is feeding. Ascaris eggs may be conveyed onto gari 
through the agency of the vomit, the so-called vomit-drop 
which is often used by the fly when feeding on solid food 
items. The fly may also carry Ascaris eggs on any part 
of the body particularly the proboscis, or on the glandular 
hairs of the pulvilli. These flies are for ever in search 
of food and because of their high intake of nourishment, 
they defaecate very often. This constant discharge of
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excreta is one of the many factors which make 
housefly a very dangerous carrier of Ascaris infection. 
Finally, the role of carriers engaged in gari handling 
either in preparation or distribution of the food stuff 
can b© incriminated as sources of infection. Vegetable 
salad, fruits, roots, palmwine and gari contaminated 
with human excreta are vehicles of infection and as long 
as our food items are sold in open containers in open 
markets, the food items will continue to be some of the 
sources of Ascaris infection to individuals who eat these
food items raw.
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4.0 CONCLUS IONSAND RECOMMENDATIONS
4.1 CONCLUSIONS
Arising from the observations and data, obtained during 
this study on Ascaris-bacterra relationship, the following 
conclusions have therefore been made:
(1) The time required for culturing Ascaris eggs to the 
infective larval stages could be shortened to the barest 
minimum by using thermostatically controlled incubators in 
the Laboratory;
(2) The use of formalin has no effect whatsoever on the 
development of Ascaris eggs, but formalin can be used to 
prevent bacterial contamination of the eggs;
(3) When Ascaris eggs are inOcul*3*®^ in broth culture of 
intestinal bacteria, the bacteria species used up all the 
available oxygen and thereby inhibited the cleavage and 
development of the eggs;
(4) The gut of both man and piglets^inhabited^both Ascaris 
and bacteria which probably live happily as individuals 
independently of the other. However, the larvae of Ascaris 
have been found to act as vehicles of bacterial transportation 
from the intestine;
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(5) The adult worms of Ascarls harbour bacterial species 
and could therefore be important agents of transportation of 
bacteria into the tissues. 3ucb an association was found 
to increase the changes of invasion of tissues by the 
harboured intestinal bacteria which turned out to be even 
□ore virulent than their like in the intestine. This close 
association was found to increase the pathogenicity of Ascaris 
in piglets;
(a) The results confirmed the work of Bradley _et £l (1966), 
Philips et ai (1959), llescott (1970), Brown & Perna (1958) 
and Woodruff (1968), who observed that close association of 
protozoa and microflora increased the pathogenicity of the
protozoa;
(7) The results of this study have confirmed that a close 
attention is required to prevent an indirect cause , of ill- 
health in man and piglets. From the results of this study, 
it is now very definite that some of the bacterial species 
especially E.coli, which are regularly associated with 
Ascaris worm can be easily transported by the worms to 
places outside the intestinal lumen;
( a )  The results have also established that certain food 
items like carrots, vegetables and fruits that are eaten 
raw by many people in Ibadan population serve as sources 
of Ascaris infection to man.
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243
4.2 RECOMMENDATIONS
The examination of faecal samples for Ascarls 
infection should bo mandatory to further elucidate 
possible causes of ill-health which may not be unconnected 
with normally commensal bacteria of intestine being trans­
ported to the tissues where they become invasive and 
pathogenic. The microscopic examination of stool from 
man and animal who show retarded growth (especially in 
children end young animals) and with pyrexia of unknown 
origin, no doubt, will eliminate late discovery of Ascarls 
infections. The result^ of this late discovery may be 
fatal, because when parasitic infection is discovered late 
and antibiotic discontinued, the bacteria carried by 
migrating Ascaris larvae and adults would have been 
permitted to overwhelm the patient and spread via the 
blood to the meninges, causing fatal septicaemia and
meningitis Periodic deworming of children and piglets
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244
are strongly recommended.
Finally, the observance of the simple rules of 
personal hygiene and the sanitary disposal of faecal 
wastes should be taught to all levels in the population 
in order to prevent this constant contamination of food 
items with Ascari3 eggs with a view to reducing the exposure
level of the population to Ascaris infection
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Parametric: evaluation of the prevalence 
of Entcsmoeba histolytica infection in Ibadan,
Trop. Geog, Med. 24; 370,
ACKERT, J.E, (1931);
The morphology and life history of the fowl 
nematode Ascaridia lineata (Schneider)
Parasitology, 23: 360-379.
ACKERT, J.E. & WHITLOCK, J.H. (1941);
Feeding habits of nematode parasite of vertebrates 
Introduction to Nematology (Chitwood & Chitwood) 
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ADEDEJX, S.O. (1981);
Studies on the Microflora occuring simultaneously 
with nematodes in the human gut in Ibadan,
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Pi.Sc. Thesis; University of Ibadan.
ALICATA, J.E. (1934);
Observations on the period required for 
Ascaris eggs to reach infectivity.
Proc. Helminthol. Soc. Wash.1(1); 12 (Helm. Abstr.)
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ARCHER, E & PETERSON, M (1929):
Roentgen Diagnosis of Ascariasis 
Quoted by Li (1933),
BAXLLET, C.C. (1866):
Nouveau dictionaire pratique de medicine, 
de chiurgie et d'hygisne veterinaries 
Article on Helminths Paris 8: 619-687
BAKER, F.J*
Handbook of Bacteriological Technique 
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Butterv'orths, 1967; pp,335-347,
BANESTIVA, J.E.
Current Problems in clinical Virology (1971)
1st Edition pp, 523-582,
New York Appleton-Century, Inc,
BARON, R.R.: HANSEid, H.F. & LORD, T.H. (1960):
Bacterial flora of the round worm
Ascaridia galli (Schrank) and its relationship
to the host flora.
Exptl, Parasitol. 9: 281 - 291,
BEAUTYMAN. W & WOOLF, A.L, (1951):
An Ascarls larva in the brain in association 
with acute anterior poliomyelitis,
J, Path. Sact, <63: 635-647.
BEAVER, P.C. (1949):
Quantitative hookworm diagnosis of direct smear* 
Jour. Parasitol, 35: 125 - 135.
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APPENDIX 1 8, ’ " THE SPREAD OF
A *5r A R T S  LUMBRIOD
t - m r r s k v -v -v ' w -  -:3IDES EGGS ONeraKra**B»,.c!Wi
..LETTUCE LEAVES
Total No. Number Total egg Emforyonated
Batch of : Positive Counts Eggs.
number specimens
examined.
1 - 4 1 2 —
2 2 - - -
3 8 2 3 1
4 4 2 7 3
5 2 1 2 -
6 10 2 6 4
7 3 1 3 1
8 2 - - -
9 4 1 2 2
10 4 ----  1 2 -
TOTAL 43 11 27 11
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APPENDIX 2 THE SPREAD OF 
AS.C/ nM IjPRICOIDES EGGS ON
CUCUMBER rRun~*
Total No.
Botch of Number total Embryonatef
Number specimens Positive Eggsexaminecl. Counts
1 7 1 OQ 1
2 2 - - -
3 5 1 3 1
4 9 - - -
0
6 .-1 - - -
7 6 2 5 2
0 6 1 —
9 7 2 3 2
10 S 1 O -
11 3 - - -
12 5- 1 3 -
TOTAL 55 9 21 6
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APPENDIX 3 THE SPREAD OF 
ASCARXS LUMBRICQIDES EGGS ON 
CARROTS f i
s O £ c
Batch of Number Total Embryonatod
Number specimens Positive ®ss Eggs—
examinee] __ Counts
v x g x m * ■< ae-«» -vm xsarM m sKZ«m xz*zBm am r*m
1 uo - -
A n 3 1
3 o - - ~
4 3 1 4 1
5 r.A\. 1 4 1
6 >5 2 6 2
y 7 2 8 2
u<=*> OO *-> rr-« *"
O
✓ ~ * * ■ * «
10 5 2 2
11 o 8 3
TOTAL 45 11 12
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APPENDIX 4s .L , THE SPREAD OF
ASCAPTS I URSRICO^DES eggs on
’ LaNgo-f r u i t’
Total No.
Batch of Number Total Emforyanated
Number specimens Positive egg Eggs
examined Counts
1 4 1 3 1
2 3 - - -
3 5 2 7 2
4 4 1 2 -
5 5 3 5 2
6 4 - - -
7 3 - - -
8 4 1 3 1
9 3 - - mm
TOTAL 35 20 6
sssss:ss:sssssss== =3s=:=s:=SSSSSSBSSSSS
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APPENDIX 5 ; i ' ..w ' THE SPREAD OF
a^CARTS UJH3R* .»Tr.PS EGGS m  
TOrfATO PkdlT
Total No.
Batch oF Number Total Embryonated
Number specimens Positive ©gg Eggs
examined. Counts
1 5 mm
9 6 - - -
3 10 1 2 -
4 9 1 4 1
5 12 2 5 2
6 10 1 o mm
TOTAL 52 5 13 3
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APPENDIX 6s \ 3 THE SPREAD OF
ASCARXS LUHBRIC^TDFS EGGS OH
Garden EGGS
—
Total No, 
Batch of Number Total Embryonated
Number specimens Positive egg Eggs
examined Counts
1 12 2 3 mm
2 10 2 4 1
3 10 1 2 -
4 11 2 2 -
5 10 2 3 1
6 10 1 1 mm
TOTAL 63 15 2
S 2 3  = S  = 2=5S=SSJ5!5
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APPENDIX 7: - —  _? THE SPREAD OF
*A ' SCAR IS l'«MF?{?TroTDFS EGGS OH # fc- ..iV X> a w  - - w
SWEE'I PEPPtR
Total No. 
Batch of Number Total Embryonated 
Number specimens Positive ©gg Eggs.!___
examined Count*..
1 2 - - -
2 2 - - -
3 4 1 2 -
4 10 3 4 1
5 6 1 1 1
6 8 2 3 1 ____
TOTAL 32 7 10 3
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APPENDIX 8: ■< •.« * ’ THE SPREAD OF
ASCARIS LUMBRICHTHps EGGS ON 
ONiONS (BULB)
Total No# 
Batch of Number Total Embryonated
Number specimens Positive egg Eggs
examined Counts
1 5 1 1 -
2 3 - - —
3 5 1 1 -
4 6 1 7 -
5 3 - -
6 7 1 3 1 ----
TOTAL 29 7 1
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APPENDIX 9: ■ ?r as ' the spread of
ASCAPTS LUMBRICOIDES EGGS ON GARI
Total No.
Batch of Number Total Embryooated
Humber specimens Positive egg Eggs
examined ~~ Counts
1 7 p 5 2
2 5 1 2 1
O 6 1 2 1
4 8 2 7 2
5 6 1 2 1
6 4 - -
TOTAL 36 7 18 7
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APPENDIX 10: ' ff*i$ ~ THE SPREAD OF 
ASCARIS LUfiB RICO TOES EGGS ON 
PALN V aNE
Total No* 
Batch of - Number Total Embryonated 
Number specimens Positive Egg Eggs „
examined. Counts
1 5 1 2 mm
7 1 3 2
3 6 1 3 2
4 4 1 1 -
5 5 1 2 1
6 2 - mm »n> -
TOTAL 29 5 11 5
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APPENDIX 11:
Direct Smear Method
Procedure - Place a drop of saline on a clean slide and 
take as much or slightly less than a match head of faecal 
material by means of a disposable applicator such as a 
swab stick or toothpick. Mix with saline thoroughly and 
examine microscopically after placing a coverslip on it. 
Note (1) Smear on the slide should be thin enough to 
read newspaper print through. Properly prepared faecal 
film is grey in colour,
(?) Saline may be replaced with glycerine-water 
(1:2) solution to prevent drying.
(3) The amount of saline used for mounting 
faecal material should be enough to fill all space beneath 
the coverslip without any saline flowing beyond theedges 
of the coverslip,
(4) Ascaris infection with a female worm may be 
theoretically detected by observation of three faecal 
films prepared by this method. But this is not true in
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the case of human infections with other species of
helminths because of much lower oviposition as compared
with that of Asearis.
APPENDIX 12;
Kato Cellophane Thick Smear Method
Procedure;
(1) Cellophane coverslips, 40^um in thickness and 
26 x 28mm in size.
(2) Cellophane staining medium containing 100 parts of 
distilled water (6% phenol), 100 parts of glycerine 
and one part of 3$ malachite green solution
(3) Soak the coversliras individually in the medium for 
more than 24 hours.
Procedure;
(1) Place 6Q-?Qmg (as much as a red bean) of faecal 
sample on a slide.
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(2) Cover it with the cellophane coverslip after 
removing the excess of th© medium by shaking.
(3) Press the coverslip by means of rubber stopper or 
finger to spread faecal material evenly to the edge 
of the slip. Examine it microscopically.
Note -
(i) In slides standing for 60 minutes at atmospheric 
temperature of 25°C and relative humidity of 75% 
deformation of eggs on these slides may occur, presumably 
due to overdrying.
(ii) Some eggs on the slides prepared by this method 
are observed as somewhat different from those prepared 
by the direct smear method. The thin-shelled eggs 
such as those of hookworms are seen flattened and 
rather roundish in shape in the slides used in this 
method. Both in thick-shelled eggs such as those of 
Ascaris and whipworm, no morphological deformation 
occurs in the direct film and cellophane thick-smear
method
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APPENDIX 13:
Brine Floatation Method 
Preparation:
Saturated Nacl medium with a specific gravity of 
1, 200, which is prepared by adding more than 400gm of 
NaCl into looml of water and dissolving well by slight 
heating and thorough stirring. In practice the supernatant 
fluid from the saturated NaCl solution with NaCl undissolvod 
at the bottom of a vessel after standing for a while is 
preferably used as a floatation medium.
Procedure;
(a) Place 0,5g of faecal sample taken from various parts 
of a faecal specimen into a small sized test tube 
containing a previously prepared small amount of
the floatation medium
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(b) Stir vigorously with on appropriate applicator,
(c) Place the test tube virtically and add the medium 
up to th© brim of the tube so as to form a convex 
surface of the medium at the month of the tube.
Then let it stand for 30-45 minutes.
(d) Carefully superimpose a grease-free coversiip on the 
convex surface in contcct with the medium without 
overflow and carefully lift the coversiip by a straight 
upward pull and then place it on the slide for micros­
copic observation,
h!ote (a) Incomplete stirring may result in no egg 
floatation to the surface of the fluid due to incomplete 
separation of eggs from the faecal debris.
(b) Egg floatation may be incomplete when the 
floatation period is too short (less than 30 minutes) 
or too long (more than 60 minutes). In the latter 
case, the eggs floating at the surface may descend again.
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(c) This method is suitable for detection of eggs 
with low specific gravity such as those of hookworm 
but not for heavy eggs such as trematode, whipworm 
eggs and Ascaris infertile eggs.
APPENDIX 14;
Hagnesium Sulphate Method
Preparation - Prepare -NaCl floatation medium by
adding 290gm of NaCl Gnd !35gm of HgC12 into 1,000ml of 
previously warmed water. Then stir thoroughly.
Procedure; - Follow the same procedure co in brine 
floatation method using MgS04 - NaCl medium instead of 
saturaded NaCl as in the former method.
APPENDIX 15s 
Formalin-Ether Hethod;
Procedure; (a) Place 0.5g of faecal sample with 2-3ml-
of water in a small-sized test tube and stir thoroughly
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(b) Filter the faecal suspension through gauze into a 
centrifuge tube after increasing its volume to 15ml 
with water.
(c) Centrifuge the tuba at 2#000rpm. for 1-2 minutes 
and decant the supernatant.
(d) Add 7-10ml of 10$ formalin and fix the sediment 
for 10 minutes. Add 3ml Ether.
(e) 5hake the stoppered tube vigorously for 20-30 seconds 
by hand.
(f) Centrifuge the tube at 2,000r*p.m. for a minute after 
removal of the stopper from the tube. Decant the 
supernatant with the faecal scum which is separated 
previously from the tube wall by means of an applicator
(g) Place the sediment on a clean slide by tilting the 
tube or by sucking up the sediment by a long capillary 
pipette and discharging onto the slide. After placing 
a coverslip examine microscopically.
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Note;
(a) Formalin-fixed sediment may be stored for long 
periods of time and sent anywhere for microscopic 
examination.
(b) Egg recovery rate by this method is somewhat lower 
than that by AH3 111 and Tween 80-Citrate buffer 
method and the amount of the final sediment obtained
by this method is somewhat larger than those previously 
mentioned.
APPENDIX 16;
HcNaster Egg Count Method 
Procedure;
(1) VJeigh out 2gm, faeces from sample.
(2) Thoroughly mix sample with 60ml of saturated salt 
solution, and pass through sieve into evaporating
dish or glass jar,
(3) Hash remaining debris in sieve with further SOmi 
saturated salt solution.
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(4) Agitate, fill both counting chambers by means of 
pipette, count all eggs in both chambers. In 
practice, faeces can be diluted at once through 
sieve with 60ml saturated salt.
Note; Eggs of Ascaris, bookworm, stronqyloides and 
trichostrongyles being higher than the salt solution float 
to upper surface of fluid and are easily seen, free from debris 
which is on lower plane. Trematode eggs colbpsed and do not floa' 
Each chamber Isq cm., divided by a grid into 6 areas each 
equal to diameter of microscope field with x 6 eyepiece, irds 
objective. Depth 1.5mm. Volume of each chamber therefore,
15ml. 2gm. faeces in 60ml = 1;3Q dilution. Volume counted 0.3ml 
- 1/lQ0th of 30ml. Therefore eggs counted x 100 = Eggs/per gm 
of faeces.
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APPENDIX 17;
Stoll's Egg Count Method 
Procedure;
(1) Place faeces until the surface of aqueous fluid 
reaches the 60ml mark in Stoll's graduated Erlenneyer 
flask containing previously prepared 0.IN NaOH up
to the 56ml mark.
(2) Add 10-20 glass beads (3-4mm in diameter) to . the 
flask and stand it with a stopper overnight.
(3) Shake the flask vigorously, pipette out an aliquot 
of 0.15ml onto a clean slide and place a coverslip 
on it.
(4) Count the total number of eggs concerned microscopically. 
Then multiply the number by 100 to obtain number of
eggs per gram (E.P.G.).
Note;
(1) Two ml of faecal sample are added to 58ml od. 0,1 El 
NaOH. E.P.G. may be obtained by multiplying the total 
number of eggs in 0.15ml aliquot of the faecal suspension
by 200.
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(2) looter content of faeces may vary with the nature of
faeces. To convert to the normally formed faeces basis,
the following factors should be used according to the nature
of faeces used: normally formed, 1;
*
mushyformed, 1.5; mushy 2.0; mushy diarrhoeic 3.0; 
obviously diarrhoeic 4.0 and watery 5.0. But no 
correction is made on values obtained from many 
people in the field-survey.
APPENDIX 10 s
Beaver's Egg Count Method.
This method involves the use of a photometer in the 
preparation of a faecal smear of a known standard density* 
This photometer is difficult to standardise and the facility 
is difficult to maintain in a place like Nigeria.
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