I

CELLULAR REACTIONS IN GUINEA-PIGS AND JIRDS

DURING PARASCARIS EQUORUM INFECTION

A. Waheed Hashmi

A Dissertation

Submitted to the Graduate School of Bowling Green State University in_.partial fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY

August 1979 il

Abstract

Guinea-pigs and jirds were used as laboratory models

in order to assess comparatively the pathogenicity and

immunogenicity of Parascaris equorum infection. Each animal

was infected with embryonated eggs of the parasite by oral

route according to its body weight, and cellular responses

to the infection were observed in blood and lesions during

the 18 day period of infection.

Day to day hematological changes in both species

were monitored and analysed statistically. A significant

decrease in total erythrocyte count and concomitant decline

in packed cell volume and hemoglobin values were found in

jirds as compared to guinea-pigs. Generally, there was a

significant elevation in total leukocyte count in both animals

during the course of infection. But in the initial phase

of infection in guinea-pigs a noticeable decline in total

leukocyte population was accompanied by a significant shift

in differential leukocyte count, characterized by increased

neutrophilia. By contrast, in jirds an elevation in total

leukocyte population was accompanied by a similar trend in

the lymphocyte count. The level of neutrophils rose sig­ nificantly through the course of infection in both hosts, but their build up was delayed considerably in jirds. In both animals a rise in lymphocyte population was observed in the final phase of infection. A gradual increase in eosinophilia with peaks from 10 through 14 DOI (day of lilt

infection) were observed in both animals. Monocytes showed

an increase in the middle of the infection, and there was

significant rise in basophils towards the end of the infection.

Again, both these trends were more marked in guinea-pigs.

Extensive tissue damage and hemorrhage were observed

in lesions of the liver and lungs of jirds. This was accom­

panied by inadequate cellular response, which largely com­

prised of mononuclear cells. Massive cellular response

was observed in the lesions in guinea-pigs. This, however,

was local in the liver and generalized in the lungs. Among

other infiltrates, polymorphonuclear cells were abundant,

especially aggregates of eosinophils, which were indicative

of an immediate type hypersensitive response in guinea-pigs.

Data obtained on the yield of active larvae from the visceral organs of the two hosts revealed a greater recovery from the jirds. The rate of development of the larvae was faster in the guinea-pigs, but the range in size of the larvae recovered was greater in jirds. Consolidated evidence suggests a better host-parasite compatibility between guinea-pig and Parascaris equorum due to a more effective cellular response by the host to the immunogenic stimulus of the parasite. ACKNOWLEDGEMENTS

I wish to acknowledge my deep sense of appreciation for Dr. Francis C. Rabalais for his able guidance, enthusiasm and constant support throughout this investigation

My special thanks go to Mr. Richard Green, for extending all possible help at his disposal to facilitate the supply of Parascaris equorum at his horse abattoir.

Much gratitude is due to Dr. Ralph S. John for pro­ viding his counsel on statistical work involved in these studies.

My appreciation is also extended to Dr. Stephen H.

Vessey for his ideas and criticism for the improvement of the manuscript of this dissertation.

I also want to acknowledge my thanks to Dr. Richard E. Crang and Dr. Robert C. Graves for serving on my advisory committee. IV

TABLE OF CONTENTS

Page

INTRODUCTION...... 1

Statement of the Problem ...... 6

PLAN OF STUDIES...... 13

MATERIALS AND METHODS...... 14

Preparation of Inoculum...... 14

Selection of Size of the Inoculum for Guinea-pigs and Jirds...... 15

Bleeding Procedures...... 16

Hematological Methods...... 17

Histological Methods ...... 20

Statistical Methods...... 21

Experimental Designs...... 23

RESULTS...... 27

Erythrocyte Parameters in Jirds...... 27

Leukocyte Populations in Jirds...... 30 Erythrocyte Parameters in Guinea-pigs...... 44

Leukocyte Populations in Guinea-pigs...... 48

Histopathological Observations...... 65

Development of Parascaris equorum Larval Stages in Guinea-pigs and Jirds...... 75

DISCUSSION...... 79

SUMMARY...... 108

LITERATURE CITED...... 113

APPENDIX...... 121 V

LIST OF TABLES

Table page

1. Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Intervals in Infected and Controls (Group No. 1, Jirds)...... 59

2. Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 2, Jirds) ...... 60

3. Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 3, Jirds)...... 61

4. Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 1, Guinea-pigs) ...... 62

5. Multiple Comparisons of Various Hematological Va Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 2, Guinea-pigs) ...... 63

6. Multiple Comparisons of Various Hematological Variables Durig Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 3, Guinea-pigs) ...... 64

7. Rate of Growth of Parascaris equorum Larvel Stages During First Infection in Guinea-pigs and Jirds Over 18 Days Period...... 78

8. Comparison of the Number of Active Larvae Recovered from the Visceral Organs of Jirds and Guinea-pigs During Parascaris equorum Infection After Inoculum of 600 C+ 50) and 5,000 (+ 100) Embryonated Eggs Respectively at the Given Intervals...... 79 Vl

LIST OF FIGURES

Figure page

1. Experimental Design for Bleeding Infected and Uninfected Control Jirds During Parascaris equorum Infection...... I I I 7 ... 25

2. Experimental Design for Bleeding Infected and Uninfected Control Guinea-pigs During Parascaris equorum Infection...... ~~Z : I : 7 26

3. Absolute Erythrocyte Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls. ’ I I I I : . 28

4. Pack Cell Volume and Hemoglobin Values of Jirds Infected with Parascaris equorum Against Uninfected Controls. 7...... 31

5. Absolute Leukocyte Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls. 1 I I I I . “ I I . 32

6. Absolute and Percent Lymphocyte Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls . 34

7. Absolute and Percent Neutrophil Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls . 36

8. Absolute Eosinophil Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls...... 38

9. Percent Eosinophil Count in the Circulating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls...... 39

10. Absolute and Percent Monocyte Count in the Cir­ culating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls...... 41

11. Percent and Absolute Basophil Count in the Cir­ culating Blood of Jirds Infected with Parascaris equorum Against Uninfected Controls...... 4 3

12. Absolute Erythrocyte Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls...... 45 Vil

13. Pack Cell Volume and Hemoglobin Values of Guinea- pigs Infected with Parascaris equorum Against Uninfected Controls...... 47

14. Absolute Leukocyte Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Coritrols...... 4 9

15. Percent and Absolute Lymphocyte Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls . 50

16. Percent and Absolute Neutrophil Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls . 52

17. Percent and Absolute Eosinophil Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls . 54

18. Percent and Absolute Monocyte Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls . 56

19. Percent and Absolute Basophil Count in the Circulating Blood of Guinea-pigs Infected with Parascaris equorum Against Uninfected Controls . 59

20. (Jird, Liver 8 DOI x 400) Showing mechanical damage to hepatic parenchyma due.to migrating , larvae, hemorrhage and limited leukocytic response in lesions...... 68

21. (Jird, Liver 8 DOI x 400) 3rd stage larva of Parascaris equorum without surrounding cellular response, some cellular response could be seen along the tracks of larval migration...... 68

22. (Jird, Liver 8 DOI x 1,000) Accumulation of pre­ dominantly mononuclear cells in the damaged area...... 69

23. (Guinea-pig, Liver 8 DOI x 300) Massive Cellular response in the damaged area caused by larval migration, infiltrates include both mononuclear and polymorphonuclear cells...... 70

24. (Guinea-pig, Liver 8 DOI x 250) An older lesion an inner fibrotic zone (F), showing the lesion being resolved, a disintegrating larva (L) could be seen surrounded by a degenerating mass of eosinophils and other leukocytes ...... 70 vili

25. (Jird, Lung 14 DOI x 40) Hemorrhage caused by the migrating larvae in the peripheral area of the lung, cellular infiltration more marked around bronchioles and perivascular area .... 71

26. (Jird, Lung 14 DOI x 250) A close-up of the above figure, showing an area of hemorrhage, mono­ nuclear cells predominate in this area...... 71

27. (Jird, Lung 14 DOI x 250) Larvae in the alveolus are free of any surrounding cellular response. . 72

28. (Jird, Lung 14 DOI x 1,000) Showing predominantly mononuclear response in an area where hemorrhage has occured, greenish brown hemosiderin granules (h) are the digestive byproduct of effete ery­ throcytes after their phagocytosis ...... 72

29. (Guinea-pig, Lung 14 DOI x 40) Massive and generalized cellular response caused by the parasite ...... 73

30. (Guinea-pig, Lung 14 DOI x 1,000) Massive and generalized cellular response caused by the parasite...... 73

31. (Guinea-pig, Lung 14 DOI x 250) Disintegrating larva trapped in cellular response consisting of degenerated eosinophils and other leukocytes. 74 CHAPTER I

INTRODUCTION

Parasitism, like most other environmental stresses,

must be considered in ones concept of a biocenose. Most of

the parasitic infections which become patent in terrestrial vertebrates are known principally because they can be con­ veniently detected by their reproductive stages being passed in faces or their presence in some form in blood or external body tissue. Many of those parasitic infections which es­ tablish themselves in foreign hosts, without becoming patent, are known to occur in man and domesticated animals. Such infections are generally termed Zoonoses, although Pavlovsky

(1966) differentiates non-patent infections in man trans­ mitted from animals as anthropozoonoses. Where host response to such infections is severe, ultimately an investigation could result in the detection of the causative agent. A benign infection may go undetected for the whole life-time or may be discovered by surgery, biopsy or autopsy. For some zoonotic infections in man and domestic animals diagnosis could be established through blood or roentogenological examination in combination with serologic and intra-cutaneous tests.

Zoonoses occuring in other animal communities not associated with man are almost unknown. Even those zoonotic 2

infections which could be transmitted from man and his live­

stock to other terrestrial vertebrates, particularly mammals,

remain largely obscure. According to Lammler (1973) only

150 zoonotic infections have so far been recorded. A number

of animals, including rodents, lagomorphs, primates, and birds

have been employed as experimental laboratory models for

various parasitic infections. In some of the experimental

animals, which do not otherwise act as normal hosts, foreign

parasites have become established to the extent of becoming

patent in them. Some examples among nematodes include

(1) Brugia pahangi, which is a natural parasite of cats,

dogs and tigers, reached sexual maturity when experimentally

infected in jirds (Ash and Riley, 1970). (2) Necator americanus

is a natural parasite of man. Sen (1972) succeeded in es­

tablishing patent infection of Necator americanus in golden

hamsters. (3) Trichinella has been shown by many workers

to infect and reach sexual maturity in a wide variety of vertebrate hosts. The actual magnitude of zoonoses is pro­ bably much greater than the few examples listed.

The part which zoonotic relationships have played in the evolution of present day patent parasitic infections in natural hosts is not understood. Some very important parasitic infections of man such as trichinellosis, schisto­ somiasis, dracunculosis and some other strains of filarid worms are natural to other mannals as well. There seems no doubt that these parasitic maladies were either transmitted 3

from other mannals to man or vice versa. It seems unlikely

that these parasitic infections became patent immediately

in either kind of host, a process of gradual adaptation

seems more logical. Whether the zoonotic parasitic infections

which do not become patent in their abnormal, but frequently

encountered hosts, are on their way to greater degree of

establishment and complete adaptation remains to be seen.

It seems likely and should merit further investigation.

Ascarids (Ascaroidea) are the largest intestinal

nematode parasites of man and other land vertebrates and

have the most resistant infective egg stage among helminths.

These helminths infect a variety of hosts other than the

natural hosts causing a disease known as visceral larva

migrans. According to Beaver (1969) more than 200 cases

have been recorded since the first description of visceral

larva migrans in man caused by Toxocara canis, a natural

parasite of dogs. As in the natural host, the infective

larvae of the worm penetrate the intestinal wall and reach

the liver through the portal system. But unlike the natural

host, they do not develop beyond the second stage larva.

In the liver they cause hepatomegaly and eosinophilia (Chaudhri

& Saha, 1959). In adult sheep Schaeffler (1960) demonstrated

that larvae did not migrate beyond the liver, but in young lambs the larvae underwent erratic migrations and were found in the mesenteric nodes, pancreas, heart, lung, kidneys and brain from the fourth day onwards. Erratic migration 4

of Toxocara larvae has also been reported in young children.

Ocular lesions due to the migration of Toxocara larvae in

the eyes of children have been reported by Ashton (1960),

Duguid (1961, 1961a) from England, Beaver (1962) from eastern

United States, Hawaii, Mexico, Puerto Rico and Phillipines.

According to Duguid (1961, 1961a), who reported 28 cases of

Toxocara infection at the Institute of Opthalmology, University

of London, ocular lesions due to this parasite had in the

past been attributed mistakenly to other causes such as

organised hemorrhage or exudate, Coat’s disease or chorditis.

According to Levine (1968) cancer is often suspected in ocular

granulomatosis due to Toxocara larvae. Dent et al. (1956)

described and illustrated a case in New Orleans in which

granulomas and T. canis larvae were present throughout the

viscera and central nervous system of a child who died of

serum hepatitis. In mice, rats and guinea-pigs Toxocara

canis larvae remain in the liver for a few days and then

migrate to the brain and muscles, where they could be found

several days later (Galliard, 1974). Under natural conditions,

such small mammals can act as paratenic hosts, in which hatched larvae of Toxocara remain unchanged until eaten by the predatory natural host. Beaver (1966) points out from experimental evidence that Toxocara canis larvae can live in monkeys for eight to nine years. While man and other mammals are unsuitable hosts for the adult stage of this parasite, they are not abnormal hosts for the larval stage. 5

Many workers, including Robert (1934), Brown and Chan

(1955), Olsen and Kelley (1960), Boisvenue et al. (1968),

Jenkins (1968), Jeska (1969), Guerrero & Silverman (1971)

and Crandall and Crandall (1976) have used guinea-pigs, mice,

rats, and rabbits for experimental infection of Ascaris suum

and Ascaris lumbriocoides for studies on the larval migration,

pathology and the general immune response of the host. In

these and a number of other mannals, including cattle,

Ascaris suum does not remain as second stage larvae in the

liver like Toxocara spp., but moults to third stage larvae

and migratesto the lungs (Kennedy, 1954). The same is true

of Ascaris lumbricoides of man where experimental infection

has been used in the animals mentioned above. In rabbits

it has been reported by some workers that the third stage

larva undergoes another moult to become a fourth stage larva.

In this way, as compared to Toxocara, Ascaris goes further

to establish itself by undertaking third and fourth stage

development in an experimental host.

Most workers consider Ascaris suum of pigs and Ascaris lumbricoides of man as morphologically and biologically

indistinguishable. Ascaris suum produces patent infection in man and likewise human Ascaris develops to maturity in the pig. Takata (1951), Yajima (1955), and Akamatsu (1958,

1959), subjected human volunteers to both pig and human

Ascaris infections. The only notable difference they found was in the prepatent period, 4-6 weeks and 6-8 weeks for 6

Ascaris suum and Ascaris lumbricoides respectively.

The larval development in the lung and migration to

the intestine were faster in Ascaris suum than Ascaris lum­

bricoides during their prepatent period in the pig (Galvin,

1964). Robert (1934) suggested that, although infestation

of the pig has been of sufficient duration to evolve a dis­

tinct strain assuming man as the original and primary host,

the relationship between the pig and the parasite has not yet

reached perfect adjustment. On these bases many workers

still continue to designate Ascaris suum as Ascaris lumbricoides

var. suum.

Statement of the Problem

Much concern is currently being expressed by environ­

mentalists, health care agencies and the Food and Drug Admin­

istration about the various contaminants which find their

way into food and the human environment. Treated sludge and

sewage water which are used for land application are in­

criminated as main carriers of contaminants which are con­

sidered potential health hazards. Among other infective stages of helminths found mainly in the sludge, infective Ascaris eggs are of special importance because of their: (1) resistance to chemicals and adverse factors in the environment, (2) long­ evity as viable infective stage and (3) tremendous biotic potential of the female worm. In view of these characteristics suggestions have been made to use Ascaris eggs as an indicator 7

of the viability of other helminths as well (Akin et al. 1978).

Are not the eggs of other ascarids especially Para­

scaris equorum (of equines) and Neoascaris vitulorum (of

bovines) finding their way into municipal sludge being

utilized for land application? In the case of Ascaris suum,

the major source of eggs is refuse from hog abattoirs, which

is dumped into the city sewage. The same is true in the

case of Neoascaris vitulorum of cattle. Horse meat is

consumed primarily by pet and zoo animals and to some degree

by humans. The refuse (particularly, fecal contents) from

the horse abattoirs located within corporation limits is

discharged into city sewage. Some operators of the horse

abattoirs outside the corporation limits use or sell gastro­

intestinal contents of horses for land application (personal

observation) and, like sludge, again add to the risk of con-

timination. It may be true that Parascaris equorum is not

as prevalent as Ascaris suum, but it takes one fourth the time

to become infective (Clayton, 1978), and because of its larger

size and thicker protective egg shell, is seemingly more resistant.

A preliminary study in this laboratory was conducted to test the resistance of Parascaris equorum and Ascaris suum eggs against one common variable, desiccation. Eggs were kept without water in an incubator maintained at 30 °C and

45% relative humidity for ten days. At the end of this period each batch was orally inoculated into mice. On autopsies 8

performed at predetermined days of liver and lung migration

of ascarid larvae, only mice infected with Parascaris equorum

exhibited lesions in the lungs, and larvae were recovered

from the liver by use of the Baermann’s apparatus. A further

and precise evaluation of their comparative resistance needs

more elaborate experimentation, but it seems Parascaris equorum, besides developing more rapidly, has an added ad­ vantage of withstanding more adverse conditions of the environment.

Fitzgerald, Fox and Arther (1976, 1977a, 1977b) found high concentrations of viable Ascaris eggs in effluents from the five Chicago sewage plants. Larkin and his FDA co-workers (1978) were able to recover Ascaris eggs from seeded radishes and lettuce and surrounding soil in Cincinnati,

Ohio. In these studies it is questionable whether quality controls were stringent enough to differentiate the eggs of different species of ascarids. Particularly, Ascaris spp. from Neoascaris vitulorum and Parascaris equorum which have more or less equal chance of ending up in municipal sludge.

As indicated earlier, each species has a host specificity to become patent in a group of mammalian hosts and most of them have been proven to establish zoonotic infection as visceral larva migrans in so to speak "foreign hosts."

Parascaris equorum is an important parasite of equines, i.e., horses, asses, mules and zebras. In horses alone, according to the U.S. Department of Agriculture (1954), 9

the annual loss runs approximately $200 million in lost

work days. The loss in capital investment and treatment

expense could make the figure much higher. Evidence also

exists that this parasite can infect man (Hoeppli, 1949;

Sprent, 1965a), mice (Hoeppli, 1949), and accomplish lung

migration in these hosts. Despite its veterinary and zoonotic

importance, no well-documented studies have been carried out

to reveal aspects of its establishment, migration, immunity

and pathology in the experimental host, particularly in a

small mammal as laboratory model. Even in the natural host,

it is only recently that experimental infections have been

conducted in pony foals to study the clinical signs and pathology (Clayton and Duncan, 1977, 1978). The difficulty with using large domestic animals as experimental subjects is the inability to control the variables which effect the clinical and physiological conditions of the disease. The animals are usually genetically heterogeneous and the response to the inoculum varies greatly among individuals. Other problems include evaluating natural or "spontaneous" infections, exposure to elements in the environment, housing conditions as well as economics. In addition to using a small, more easily manageable mammal as a laboratory model to substitute for the large mammalian host, this study is mainly meant to explore the degree of establishment of the parasite in two laboratory models for a comparative assessment. Small mammals such as rodents share their habitat with large animals 10

like equines and are exposed to "nidi" or sources of trans­

mission more or less in the same manner by their food habits

as are the larger animals. In view of this, they are very

likely to be infected by Parascaris equorum under natural

conditions. As indicated earlier Parascaris eggs have a

great potential for survival, and probably like Ascaris

have the ability to infect a variety of mammalian hosts.

It could be surmised that other mammalian hosts having a

greater degree of association with the equine host would have

an adaptability to house the parasite somewhat similar to

the natural host. In order to explore this kind of host-

parasite relationship in the case of Parascaris equorum

two experimental models were used on the basis of their

divergent ecological and geographical distribution. The

guinea-pig (Cavia procellus) is an original inhabitant of

temperate to sub-tropical regions in Brazil, Peru and Argentina.

It favors clayey soil and somewhat humid climate. It cannot

live without vegetation such as grass because of its nutri­

tional requirement (Wagner & Manning, 1976). It shares to

a certain degree the habitat and niche with the larger her­

bivorous mannals such as bovines and equines. By virtue of

this association the guinea-pig may be exposed to foci of parasitic infections of larger mammals, which among other helminthic infections include ascarids. The Mongolian jird

(Merionus unguiculatus), on the other hand, is distributed in extremely dry and arid regions such as the Mongolian 11

desert. The habitat of the jird, therefore, is not indicative

of a close association with larger herbivorous mannals and

contact with the foci of transmission of their natural

helminth infections.

Ash and Riley (1970) and Ash (1973) reported the

susceptibility of the jird to Brugia pahangi, a natural para­

site of some canines. They found a "poor host-parasite relation­

ship" because of the migration of the parasite to such

unusual locations as the heart and pulmonary arteries.

Extensive nervous disturbances and absence of a cellular

inflammatory response at the site of infection in the nervous

system due to Dipetalonema viteae infection in jirds was

observed by Weinstein (1965). Boisvenue (1965) infected

jirds and mice with equal inocula of Ascaris suum. As a

result of this he found that jirds manifested more severe

clinical symptoms and greater consolidation of the lungs

as compared to mice.

In light of these observations, there is suggestive

evidence that jirds are immunologically more naive to those parasitic infections which happen to be natural in mammals distributed in relatively humid (temperate to tropical) areas of the world. Comparatively, guinea-pigs are extensively used as experimental animals for eliciting a potent immuno­ logical response, both at humoral and cellular levels against many parasitic infections.

In these studies guinea-pigs and jirds were infected 12

with Parascaris equorum in order to study the host-parasite

relationships at two levels. (1) General adaptation syndrome

(GAS) is caused by the release of metabolites of the parasite

which results in stress. According to Smirnov (1962), changes

occuring in the blood of animals subjected to stress constitute

a significant part of the concept of stress. Although a

decrease in red blood cells and hemoglobin levels may be

caused by homorrhage due to larval migration and direct

feeding of the larvae on the blood, changes occuring in differ­ ent populations of leukocytes represent successive phases of the syndrome. Information on such changes in leukocyte populations in circulating blood is sketchy even in the case of more thoroughly studied Ascaris suum infections. (2) Local adaptation syndrome (LAS) is caused by the migration and other activities of the larval stages of the parasite in the tissues.

The types of leukocytic response in the visceral organs involved in the Parascaris equorum infection in the two hosts was observed in order to assess the immunogenecity of the parasite.

Based on studies done with other parasites in both jirds and guinea-pigs, one would predict that the guinea-pig would react to Parascaris equorum in a more immune competent manner. Jirds, on the other hand, have been demonstrated to lack immunocompetence to a number of helminthic infections. 13

PLAN OF STUDY

Total erythrocyte cout, hematocrit and hemoglobin

levels were determined on a day to day basis during the course

of infection. Total leukocyte counts were determined along

with differential counts of basophils, eosinophils, lympho­

cytes and monocytes on a day to day basis during the course of infection. Visceral organs directly involved in the infection were prepared for observation of the extent of lesions and cellular response in the lesions corresponding with the changes in leukocyte populations observed in circulat­ ing blood. Some guinea-pigs and jirds were infected and later sacrificed to study the larval development and yield at different time intervals during the 18 day infection.

On the basis of findings based on GAS and LAS, larvel recovery and development, an assessment was made of the immune competence of the two hosts with respect to the immunogenecity of the parasite. CHAPTER II

MATERIALS AND METHODS

Six to eight-week-old laboratory bred male mongolian

jirds and guinea-pigs were used as subjects for the present

study. The weights of the jirds varied between 40-48 grams;

guinea-pigs ranged from 395-425 grams. The animals, both

control and infected, were kept in air-conditioned quarters

which had an automatically alternating 12 hour light-dark

(light period 0900-2100 hours) cycle, an ambient temperature

of 22°C + 0.5°C. Six to nine jirds were housed in bedded

polycarbonate cages size 19 x 10-3/2 x 6 with wire bar covers

(Cat. No. 10047, Lab. Products Inc.). Four to seven guinea-

pigs were housed in polyethylene guinea-pig bins (Cat.

No. 10099 Lab. Products Inc. size 38-3/4 x 36-3/4 x 10). Jirds were fed-on Lab-BloxR (Wayne Lab. Animal Diets Inc.)

ad-lib. Guinea-pigs were fed on Guinea-pig Diet (Wayne Lab.

Animal Diets) ad-lib supplemented with alfalfa, carrots

and lettuce from time to time. I Preparation of the Inoculum

The females of Parascaris equorum were obtained

from the small intestines of young ponies killed at a small abattoir located near Delta, Ohio. According to the method used by Clayton (1977) the female worms, two hours after 14 15

their recovery from the host, were placed in normal saline

kept at 37°C. The sedimented eggs were collected and washed

with 0.25% sodium hypochlorite (Clorox) to remove the outer

sticky uterine coat. As suggested by Jackson (1977) this

was done to keep the eggs from clumping together to yield

a uniform suspension of eggs. The eggs were washed several

times to remove the Clorox. After sedimentation in distilled

water, the eggs of each worm were transferred to 100 x 20 mm.

petri dishes with 2 ml. deep 0.5% formalin solution in water.

The eggs so maintained were incubated at 27°C for ten days.

At the end of this period motile second stage larvae could

be seen in the eggs with the help of a microscope, indicating

their having reached the infective stage. The infective

eggs in each petri dish were washed in water to remove the

formalin. After sedimentation they were transferred to test tubes containing 0.1 N sulphuric acid in distilled water.

The test tubes were then plugged with styrofoam and stored in a regrigerator at 5-7°C. In order to determine the size of the inoculum the eggs of Parascaris were counted in a counting slide (No. 3298 K 10, Arthur H. Thomas Co. Philadelphia).

The number of eggs desired to be given to the animals were carried in 1 ml. by concentrating or diluting the suspension of eggs.

Selection of the Size of the Inoculum for Guinea-Pigs and Jirds

Subjecting the animals to low challenge or mild 16

invasion of a parasite usually will produce a subclinical

syndrome. On the other hand administration of too high a

challenge dose can produce drastic physiological stress and

can possibly cause the animal to succumb to secondary infections.

According to Tarczynski (1962) the symptoms of ascaridosis

in animals invaded by a large challenge dose are of a general

nature, and not at all specific. In view of this and consider­

ing the relative difference in the average weight of animals

employed of each species, guinea-pigs were infected with

5,000 (+ 100) and jirds with 600 (+ 50) emtryonated eggs of

the parasite. In both animals the inoculum was administered

using the stomach tubing. The animals were kept without

food and water six hours before and after administration

of the infective dose.

Bleeding Procedures

Guinea-pigs

Blood was obtained by cardiac puncture from guinea-

pigs under light ether anesthesia. Normally, approximately

one half cc. of blood was drawn for performing various hemo-

tological analyses. The blood from syringe was immediately

transferred to screw-capped vials, each containing a droplet

of dipotassium ethylenediamine tetracetate (EDTA) to prevent

clotting of blood.

Jirds Light ether anesthesia was used during the bleeding 17

procedure. Approximately 0.3 ml. of blood was drawn from

retro-orbital plexus with the help of a 9 inch disposable

pasteur pipett coated with EDTA. The blood from pasteur

pipett was transferred to 2 ml. screw-capped vial for hemo-

tological analyses. All the control animals were bled first,

followed by the infected ones. Both guinea-pigs and jirds

were bled each day according to schedule at approximately

the same time (2100-2300).

Hematological Methods

Total White Blood Cell and Red Blood Cell Counts

Red and white blood cells were counted in a Coulter

Counter (Coulter Co., Model F, Hialiah, Florida). Twenty microliters of whole blood were placed in a clean sterile test tube with 10 ml. of isotonic saline specifically manu­ factured for Coulter Counters (Scientific Products, Inc.,

Cleveland, Ohio). This constituted 1:500 dilution used for white blood cell counts. After stirring the mixture to get the uniform dilution of blood cells, 0.1 ml. of solution was pipetted into another clean test tube containing 10 ml. of isotonic saline. This constitutes 1:50,000 dilution used for the red blood cell count. All of the red blood cell counts were conducted before the white counts using a pre­ determined aperture threshold setting on the Coulter Counter

(in accord with the Coulter Counter operational manual).

The infected and control blood samples were alternated as the tests were conducted. The Coulter Counter was designed 18

to draw in 0.5 ml. of the dilution and automatically determine

the number of red cells per cubic millimeter of whole blood.

All procedures were in accord with standard methods for this

laboratory technique.

The aperture threshold was increased to predetermined

setting for white blood cell counts. Two drops of Lysing

Agent Manual (Scientific Products McGraw Park, Illinois)

were added to 1:500 dilution sample to lyse selectively the

red blood cells while the white blood cells (all forms)

remained intact. The Coulter Counter drew in 0.5 ml. of the

solution and automatically determined the number of white

blood cells per cubic millimeter of whole blood.

Pack Cell Volume (PCV) and Hemoglobin Percentage Determination

A microtechnique using heparinized capillary tubes was employed for the determination of packed cell volume

(PCV). Each tube was filled with blood, sealed at one end with plastic clay, and centrifuged at 13,000 g for 4 minutes in Adams Microchemistry centrifuge (Clay Adams Inc., New York).

At the completion of centrifugation process, packed erythrocyte column measured as PCV % (hematocrit) for each sample were determined with the help of micro-hematocrit tube reader

"CRITOCAP" (Scientific Products, Evanston, Illinois). The buff zone or white blood cell portion of the hematocrit was not included in the PCV determination. Hemoglobin percentage was determined using method given in Hycel Manual for the 19

Quantitative Determination of Hemoglobin in Blood (undated).

Differential Leukocyte Counts

Modifications were made in the conventional method

of differential count to reduce the margin or error. 40-45

x 25 mm. thin blood films were prepared on 75 x 25 mm. clean

slides from a measured 2 lambdas (microliters) blood drop

placed in the center and near edge of the longitudinal axis

of each slide. Using a similar slide as a spreader from its

broad side, the edge of the spreader was placed on the narrow

side of the slide at 30° angle a little inside the blood drop

When the spreader made contact with the blood drop the blood

spread across the slide by capillary action, the spreader

was then pushed with a smooth and moderate motion of the

hand. After drying, the blood films were stained with

Wright's stain (Cameo Quick Stain, Scientific Products,

Cleveland, Ohio).

Only well-made blood films were used for enumeration

of the relative proportions (percentages) of various types

of leukocytes, neutrophils, eosinophils and basophils. Such

counts were carried out from edge to edge vertical to the

longitudinal plane of the blood film. A laboratory counter

(Clay Adams, Parsipany, New Jersey) was used for convenient

differential counts. The percentages of each type of leuko­

cytes were calculated from the average of 200 leukocytes counted from each blood film. Absolute counts of various types of leukocytes were calculated by multiplying the 20

percentage of each type of leukocyte from the differential

count by the Coulter Counter reading of the total leukocyte

population for each sample.

Histological Methods

The tissues were fixed in 10% formal saline. Two

methods were used for preparing the tissues for sectioning.

Conventional Paraffin Method

For dehydration, the tissues were passed through

grades of alcohol in increasing increments of 10% from 30%

onwards ending up in absolute alcohol. Among a number of intermediaries (used for their miscibility both with alcohol and paraffin) tried, methyl salicylate was found more acceptable. It did not harden the tissue like xylene did. For infilitration and embedding the tissues, the pro­ cedure given by Humason (1967) was followed.

Paraffin-dioxan Method

This method was found to be simpler and more rapid for two reasons: (1) a closely graded series of mixtures, for dehydration, is not necessary. In other words, the difference in density between water and dioxan is equal to the difference between densities of 70% and 80% alcohol,

(2) there is no need for an intermediary (as required in conventional paraffin method) because dioxan is miscible with paraffin. The procedure given by Galigher & Kozloff

(1964) was followed for preparing the tissues by the dioxan- 21

paraffin method for sectioning.

The blocks of tissues embedded in paraffin processed

through the two methods described above were sectioned on

a rotary microtome (No 820 American Optical Co, Buffalo,

New York). Sections of tissues were stained with Harris's

hematoxylene and eosin according to the method given by

Humason (1967). After dehydration in alcohol and clearing

in toluene the section of tissues were mounted in Permount

(Fisher Scientific Co, Fairlawn, New Jersey). Microphoto­

graphs of histopathology involved in Para scari s infection

were prepared with the help of a combination of Leitz Ortholux

research microscope and Leitz Orthomat 35 mm. camera attachment.

S tati st i cal Method s

Computer program spssh was used for procuring mean,

sum and standard deviation for all hematological variables

monitored in each group in single day comparisons between

infected and control animals.

A two way analysis for repeated measures was also performed on the data of all 14 hematological veriables monitored. In most cases where control animals exhibit more or less stable trend, and initial variance between the control and infected animals is not too high for all three groups of an animal, this method provided meaningful interpretation of the data.

A two way analysis of variance for repeated measures primarily yields the significance of two main effect i.e., 22

Parasite Effect and Days Effect. Parasite Effect remains

significant if there is no overall net effect (+ or -) in

infected animals as compared to their controls, on a given

variable, throughout the infection period. Days Effect

remains insignificant if there are no fluctuations in the

levels of a variable during the period of infection. Marked

fluctuations during the course of infection are indicative

of significant Days Effect. Very important, however, from

the standpoint of this study is the Days by Parasite inter

action. This remains insignificant if levels of response

in infected animals remain parallel with the control animals

during most of the infection period, no matter how high or

low. A significant Days by Parasite interaction shows marked

divergence in levels of response between infected and control

animals on certain day or combination of days, at one or more than one occasion, during the course of infection.

The analysis of variance on all variables was performed at p < .05 level of significance. Bonferroni's (1974) method of multiple comparisons was used for changes in hematological variables in infected animals on day to day basis in comparison with control animals.

For each variable of a given group, intervals were formed of the type: /V L + Bs (L)

L = Xc - Xi 23

where Xc = Mean value of a given variable for control group on a certain day.

Xi = Mean value of a given variable for infected group on a certain day.

If the interval contained zero [(0) either - or +] it was

concluded that there is no difference between control and

infected animals (for a given variable) i.e., L = yc - yi = 0.

At 95% confidence interval using the formula:

B = t(l - , 48) = 2.7

At p < .05 level of significance these 48 are the degrees

of freedom (see Appendix). Comparison includes 2 sub-groups

i.e., infected and controls with 7 trials (days) in each group.

Because the experimental designs for both guinea-pigs and

jirds were similar the value of B = 2.7 is the same for both.

s^ (L) = y (6 infected and 4 control animals in a group)

In ANOVAR table MSE (mean square of error) represents MSb x swg

Hence s(£) = / MSE + 5^ b 4

and Bs(L> = 2.7/ 0 4

Thus, B s(L) of each group was calculated and compared with the difference between the means of infected and control 23a

animals (i.e., Xi - Xc) for each day as given in Tables

1, 2, and 3 for jirds and Tables 4, 5, and 6 for guinea-pigs.

Experimental Design for Infecting Guinea-pigs and Jirds for Undertaking Studies in Larval Yield and Larval Development

Eighteen guinea-pigs and 18 jirds were infected

with embryonated eggs of Parascaris equorum with challenge

doses by oral as established earlier. Eight guinea-pigs

and 8 jirds were set aside for monitoring larval development.

The remaining 10 guinea-pigs and jirds were used for observing

larval yeilds.

Larval Development

One of each experimental host was sacrificed in

intervals of 6 hours, 24 hours, days 4, 7, 9, 14, and 18

during the infection period (Table 1) for studies on larval

development. After being sacrificed, the animals were

immediately eviscerated. Either lung or liver, depending on the developmental phase of the parasite, was teased with needles and then lightly masticated with fingers to make into a kind of pulp. This method produced only active larvae which were separated and harvested by using Baermann's apparatus.

Larval Yields

On late 6th DOI, 5 jirds and 5 guinea-pigs were sacrificed and the larvae from the livers of animals were 24

retrieved by the method outlined above. On 14th DOI the

remaining 5 guinea-pigs and 5 jirds were killed, their

lungs removed, teased with needles, and the active larvae

harvested by using Baermann’s apparatus.

Experimental Design for Bleeding Guinea-pigs and Jirds for Hematological Evaluation (Pigs» 1 & 2) Thirty jirds and 30 guinea-pigs were selected after

screening them for similar baseline WBC count for each species.

The animals in each batch of a species were randomly sub­

divided into 3 sub-groups of 10 animals each. Of the group

of 10 animals 6 were infected with Parascaris equorum and

4 were kept as controls. The animals in each group were first bled once before day 0 to establish a baseline for all the blood parameters (i.e., on days -8 and -6). Group No. 1 of both guinea-pigs and jirds was bled on every third day beginning from day 1 followed by day 4, 7, 10, 13 and 16.

After -7 day Group No. 2 was bled on day 2, 5, 8, 11, 14 and 17. Similarly, Group No. 3 of each animal after day —6 was bled on day 3, 6, 9, 12, 15 and 18. Using this design it was possible to bleed the infected and control animals each day over the span of 18 days, the duration of the

Parascaris equorum infection. Hematological observations were conducted according to the methods described earlier. 30 GUINEA PIGS

BLED ON DAYS BLED ON DAYS BLED ON DAYS -8,1,4,7,10,13,16 -7,2,5,8,11,14,17 -6,3,6,9,12,15,18

INFECTED CONTROL INFECTED CONTROL NFECTED CONTROL

FIG. 2. EXPERIMENTAL DESIGN FOR BLEEDING INFECTED AND UNINFECTED CONTROL GUINEA-PIGS DURING PARASCARIS EQUORUM INFECTION. NUMBERS ABOVE INFECTED AND CONTROL ARE SAMPLE SIZE. ------

NJ Ln 3 0 JIRDS

BLED ON DAYS BLED ON DAYS BLED ON DAYS -8,1,4,7,10,13,16 -7,2,5,8,11,14,17 -6,3,6,9,12,15,18

6 4 6 4 6 4 INFECTED CONTROL INFECTED CONTROL INFECTED CONTROL

FIG. 1. EXPERIMENTAL DESIGN l-UR BLEEDING INFECTED AND UNINFECTED CONTROL JIRDS DURING PARASCART'; EQUORUM INFECTION. NUMBERS ABOVE INFECTED AND CONTROL ARE SAMPLE SIZE. f^ASCARIS

to o> CHAPTER III

RESULTS

Erythrocyte Parameters in Jirds (Fig. 3, Tables 1, 2, & 3)

Total preinfection erythrocyte counts in infected

jirds (group 1 and 2) were singificantly higher than their

controls i.e., on -8 and -7 days. Group 3 showed a lower

erythrocyte count than the controls at day -7. The lower

count, however, was not statistically significant. Although

the trend in total erythrocyte count showed a noticeable

decline on day 3, the count did not become statistically

singificant until day 6. A further decline on days 8, 9,

10, 11 and 12 in all the three groups of infected animals

represented the minimum level attained with a mean of

5.6 million/cu. mm. By comparison the mean was 6.26 million/

cu. mm. for- the controls for the same days. Erythrocyte levels

in group 1 and 2 returned to near the levels of the controls

for these groups. Group 3, however, did not show this •S recuperation and levels remained below the levels for its control groups. The insignificant degree of recuperation in group 3 ,may have been due to the inidial difference in the infected and control groups. The erythrocyte counts in control jirds at all the three groups during the course of infection fluctuated on

27 INFECTED CONTROL

T3 ©o >o E E si © cn -4 •L»UJ U bu O

HAYS FIG. 12. ABSOLUTE ERYTHROCYTE COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS INFECTED WITH PARASCARIS EQUORUM AGAINST UNINFECTED CONTROLS'.

hJ 00 29

both negative and positive sides of the mean (6.33 million/

cu. mm.) with a standard deviation +0.25. Comparatively,

infected jirds in all three groups had a mean erythrocyte

count of 6.0 million/cu. mm. with a standard deviation

+0.644.

Two way analysis of variance was performed on the

erythrocyte data on each group. Groups 1 and 2 showed highly

singificant "days effect" (p < 0.001) and "parasite by days

interaction" (p < 0.001). Whereas "parasite effect" in both these groups was insignificant (p >_ 0.61). This probably was due to a considerable degree of recuperation in erythro­ cyte count during the final phase of infection which reduced the net effect.

However, in group 3 the situation is different.

It showed an insignificant "days effect" (p >_ 0.5) and "days by parasite interaction" (p 0.27). This seems to be due to lesser variability on a day to day basis in group 3 as compared to groups 1 and 2. On the other hand "parasite effect" in this group was quite significant (p < 0.01) because of higher net effect.

From the trends based on the quantitative analyses of erythrocytes in all three groups, it is apparent that with the inoculum of the parasite administered, enough hemorrhage was caused to significantly depress the erythrocyte count in the peripheral blood.

The mean packed cell volume (PCV) of the blood or 30

hematocrit and hemoglobin % (Fig. 4) were also monitored

throughout the 18 day period in all three groups. Both of

these variables corresponded very closely with the changes

in total erythrocyte count. Multiple comparisons (Tables 1,

2 & 3) and two way analyses of variance (Appendix) of these

variables further corroborate this statement. Also, the

correlation of hematocrit and hemoglobin % with total red

cell count further substantiates the observation that con­

siderable hemorrhage occured during the course of Parascar1s

equorum infection in jirds.

Leukocyte Population in Jirds

Total Leukocyte Counts (Fig. 5, Tables 1, 2, & 3)

Total leukocyte counts monitored over 18 days showed

contrasting fluctuations between the 3 groups in the initial phase of infection. Because each group was bled at different intervals from day 0 (day of infection), it is very likely that the total leukocyte population which is comprised of

5 main sub-populations of WBCs could have undergone frequent fluctuations.

In the first 24 hours after infection, there was no significant elevation. A significant elevation in the total leukocyte count was observed on day 2. This was followed by a fall on day 3, rise on day 4 and fall on day 5. In all three groups an elevation in levels of total leukocyte counts was noticed after day 5. Except for group 1 this F MEAN PACKED RED CELL VOL(%) MEAN HEMOGLOBIN (grams I G .

4 .

P A C C K O E N D T

R C O E L L S L .

V O L U M E

A N O

H E M O G L O B I N

V A L U E S

O F

J I R D S

I N F E C T E D

W I T H

P A R A S C A R I S

E O U O R U M

A G A I N S T

U N I N F E C T E D t o DAYS u> FIG. 5. ABSOLUTE LEUKOCYTE COUNT IN THE CIRCULATING BLOOD OF JIRDS INFECTED WITH PARASCARIS EQUORUM AGAINST NJ UNINFECTED CONTROLS. 33

elevation progressed until day 12. An insignificant but

noticeable decline in WBC levels was noticed during the

terminal phase of infection.

Two way analysis of variance for repeated measures

was performed on each group. Statistical analyses showed

highly significant "parasite effect" (p £ 0.013), "days

effect" (p £ 0.002) and parasite x days interaction (p < 0.002).

Lymphocytes (Fig. 6, Tables T, 2 & 3)

The relative and absolute peripheral blood lymphocyte

levels of control jirds in all of the three groups were

fairly stable. An increase in adsolute lymphocyte level in infected jirds was observed on day 1 and 2 in groups 1 A and 2 respectively. Although, by contrast to Bs(L) values,

Xc - Xi values for total lymphocyte count does not seem to be significant. Due to the corresponding increase in the leukocyte count, even though the percent lymphocytes were found significantly reduced, the total lymphocyte count remained, more or less at control levels from day 5 through day 12. Near the terminal phase of Parascaris equorum infection in jirds a significant rise in total lymphocyte count was observed. This rise occured in all three groups from day 13 through day 18 in single day comparisons with controls.

Due to significant changes in the total leukocyte population and relatively dominant population of lymphocytes, a two-way analysis of variance on each group for total F I

G THOUSANDS OF CELLS/cujnm blood PER CENT .

6

A E B Q S U O O L R U U T M E

A A G A N I D N

S P T E

R C U E N N I T N

F L E Y C M T P E H D O

C C Y O T N E T

R O C L O S U . N T

I N

T H E

C I R C U L A T I N G

B L O O D

O F

J I R D S

I N F E C T E D

W I T H

P A R A S C A R I S G 4 ^ J 35

lymphocyte counts showed more meaningful results (Appendix).

All three groups showed singificant "days effect"

Ip (group 1 & 3) £ 0.001 & p (group 3) £ 0.05] significant

"parasite by days interaction" (p < 0.01). But only group 3

exhibited a significant "parasite effect" (p < 0.01), "Para­

site effect" in groups 1 and 2 was insignificant (p > 0.1).

This seems more likely due to the longer duration of the

final phase of Parascaris infection of this group (18 days

in group 3, as opposed to 16 days in group 1 and 17 days in

group 2), when, in general, total lymphocyte levels were

highest during the course of infection.

Neutrophils (Fig. 7, Tables i, 2 & 3)

Total neutrophil counts like total lymphocyte counts

provided more meaningful results than the relative neutrophil

counts because of two reasons: (1) Among other leukocytes

neutrophils are the second most, ominant population, and

(2) fluctuations in total neutrophil counts account in part for the changes in total leukocyte population.

A statistically significant rise in total neutrophil

count in infected jirds according to multiple comparison

(Table 2) occured on day 4 in group 1. This trend continued today 5 in group 2. An elevation in neutrophil level through day 11 and 12 in group 2 and 3 was observed. However after day 7 group 1 showed no further rise. But according to Bonferroni's multiple comparison total neutrophil counts F I G

. THOUSANDS OF CELLS / PERCENT cu.mm blood 7 .

A E B Q S U O O L R U U T M E

A G A A N I O N

S P T E

R C U E N N I T N

F E N C E T U T E R D O

P C H O I N L T

R O C L O S U . N T

I N

T H E

C I R C U L A T I N G

B L O O D

O F

J I R D S

I N F E C T E D

W I T H

P A R A S C A R I S U C T > t 37

in 3 groups remained significantly higher than their controls

up to 15 DOI. The decline in all three groups from 14-18

DOI is in contrast with steady rise of lymphocyte population

to significant levels during the terminal phase of Parascaris

infection in jirds.

Two way analyses of variance performed on both the

relative and total neutrophil counts of all groups revealed

significant "parasite effect" (p <0.02), highly significant

"days effect" (p <_ 0.002) and "days by parasite interaction"

(P < 0.002) .

Levels of percent neutrophils in control jirds in

the three groups remained within + 5% during 18 days period.

A majority of neutrophils were younger forms with ring shaped,

lobulated or twisted nuclei.

Eosinophils (Figs. 8 & 9, Tables i, 2, & 3)

In some individual cases a noticeable increase in

the eosinophil population was observed on the second DOI.

But in general, eosinophilia became statistically significant

in infected jirds on day 4 followed by a steady rise in both percent and total eosinophil counts. Peaks of eosinophillia showed up on day 10, 11-14 and 12 in groups 1, 2 and 3 respectively.

In all three groups statistical analyses manifested very significant "parasite effect" (p <_ 0.02), highly significant "days effect" (p < 0.002) and "days by parasite OJ FIG. 9. ABSOLUTE EOSINOPHIL COUNT IN THE CIRCULATING BLOOD OF JIRDS INFECTED WITH PARASCARIS EQUORUM AGAINST LO UNINFECTED CONTROLS. ----- 40 interaction" (p < 0.002) in both relative (differental) and

absolute counts of eosinophils (see Appendix).

Monocytes (Fig. TO, Tables 1, 2 & 3)

According to Naegil (1931) the monocytosis is transi­

tory and not as constant as neutrophilia or subsequent lym­

phocytosis. This may well be due to a comparatively smaller

population of monocytes which could increase the experimental

error (+) due to the counting method used. With this in

view both relative (%) and absolute count of monocytes were taken into consideration at the same time. According to the multiple comparison in 3 groups of both relative and absolute monocyte count, a significantly higher monocyte count over a longer period was observed in the later.

Group 1

Significantly higher absolute monocyte count on day 7, 10, 13 and 16

Significantly higher relative (_%) monocyte count on day 10

Group 2

Significantly higher absolute monocyte count on day 8 and 17 Significantly higher relative # (%) monocyte count on day 8

Group 3

Significantly higher absolute monocyte count on day 9 and 15 INFECTED CONTROL ▲ A GROUP 1

• O GROUP 2 T N E C

R E P / S L L E C

F d O o

o S l D b E

R m D m N . U u H c

FIG. 10. ABSOLUTE AND PERCENT MONOCYTE COUNT IN THE CIRCULATING BLOOD OF JIRDS INFECTED WITH PARASCARIS EQUORUM AGAINST UNINFECTED CONTROLS. 42 Significantly higher relative (%) monocyte count on day 15

On the basis of these observations, coupled with the

graph of both percent and absolute monocyte count, a more

or less general trend of monocytosis could be visualized.

Overall levels of monocytosis in the three groups were sig­

nificantly higher during the middle of the infection period.

Two way analyses of variance of relative monocyte

counts (%) showed significant "parasite effect" (p £ 0.05)

in group 1 and 2 and insignificant "parasite effect" (p < 0.05)

in group 3. "Days effect" for relative monocyte counts was significant (p £ 0.05) in all three groups. "Parasite by days interaction" was only significant (p < 0.05) in group 1 and insignificant in group 2 and 3 (p < 0.05).

Two way analyses of variance of absolute (total) monocyte counts revealed significant "parasite effect"

(p £ 0.016) and "days effect" (p £ 0.039) in all three groups.

"Days by parasite interaction" for total monocyte counts was significant for group 1 (p £ 0.001) and not significant in groups 2 and 3 (p ?■ 0.05).

Basophils (Fig. 11, Tables 1, 2 & 3)

Basophils constituted the smallest population of leukocytes. Both relative and absolute counts were considered for a meaningful interpretation of basophil levels during the infection period. Accordingly, multiple comparison DAYS FIG. 11. PERCENT AND ABSOLUTE BASOPHIL COUNT IN THE CIRCULATING BLOOD OF JIRDS INFECTED WITH PARASCARIS EQUORUH AGAINST UNINFECTED CONTROLS. u> 44

conducted on both relative (%) and absolute counts in group 1

revealed that only on day 16 was the basophil population ele­

vated to significant levels. In group 2 an elevation in

basophil level towards the end of the infection was visible,

but did not approach a level of significance. However, in

group 3 a multiple comparison of basophils at both levels

shows a significant increase of these cells on day 15 and 18.

The data on basophils was subjected to a two way

analysis of variance for repeated measures (see Appendix).

Significant "parasite effect" (p < 0.05) at both relative

and absolute levels was manifest in all the three groups.

"Days effect" was insignificant (p 0.80) at both levels

in group 1 and 2; whereas, "days by parasite interaction"

was significant (p _< 0. 038) at both levels in group 3 only.

Erythrocyte Parameters in Guinea-pigs (Fig. 12, Tables 4, 5 & 6)

According to the multiple comparisons, there appeared

to be no significant drop in erythrocyte levels in guinea- pigs of group 1 and 2 in day to day comparisons with their

controls. To some degree in group 2 this could be due to an initial (day -7) higher RBC count than the controls.

But this difference hardly seems significant enough to com­ pensate for a significant loss of blood during the infection period.

In group 3, as shown in Table 8, the erythrocyte counts recorded on days 3, 6, 9, 12, 15 and 18 approach INFECTED CONTROL

E E si o

CZÏ

O

CZÏ

o

DAYS FIG. 12. ABSOLUTE ERYTHROCYTE COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS INFECTED WITH PARASCARIS EQUORUM AGAINST UNINFECTED CONTROLS.

<_n 46 borderline significance. Some consideration should be given

to the lower baseline (day -6) counts of the infected group

as opposed to the control group.

Overall, in all of the three groups day 6 through

12 showed a noticeable difference between control and infected

guinea-pigs. The difference between mean reticulocyte count

from days 6-12 for control groups (He = 5.383 million/

cu. mm. blood) and mean for infected guinea-pigs (Ui = 5.208

million/cu.mm. blood) is 0.178 million/cu. mm. blood. Thus,

an inoculum of 5,000 infective eggs of Parascaris equorum

did not induce sufficient hemorrhage to show a tangible

reduction in the total erythrocyte counts in any phase

of infection in guinea-pitjs.

A two-way analysis of variance for repeated measures

was performed on the total erythrocyte data of all the three

groups of guinea-pigs. An insignificant "parasite effect"

(p > 0.05) was noted in all groups. However, this was

accompanied by a significant (p < 0.05) "days effect" and

"days by parasite interaction" in each group.

The mean packed cell volume (PCV) of the blood or hematocrit and hemoglobin percentage (Fig. 13, Tables 4, 5 & 6) was also monitored throughout the 18 days period in each of the three groups. Both of these variables corresponded very closely with the changes within total erythrocyte counts.

Multiple comparisons (Tables 4, 5 & 6) and two way analysis of these variables (Appendix) further support these findings. F MEAN PACKED RED CELL(^) MEAN HEMOGLOBIN (grams/IOO ml) I G .

1 3 . U P N A I C N K E F D E

C T C E E L D L

C O V N O T L R U O M L E S

. A N D

H E M O G L O B I N

V A L U E S

O F

G U I N E A - P I G S

I N F E C T E D

W I T H

P A R A S C A R I S

E Q U O R U M

A

G

A

’ •

I O N S T 4 ^ 48 . Leukocyte Populations in Guinea-pigs

Total Leukocyte Counts (Fig. 14, Tables 4, 5, & 6)

A ¿Light depression in total WBC count was noticed

on day 2 in group 2. Total leukocyte count became signifi­

cantly higher in infected guinea-pigs as compared to their

controls on the 3rd DOI in group 3. This was followed by

a gradual rise of WBC levels in all three groups.

WBC level in group 2 exhibited a more significant *

elevation up to day 11 when its peak was reached. Total

leukocyte count in group 1 and 3 reached their peak on day

11 and 13 respectively. WBC counts in each group after reaching

a peak at day 14 manifested a noticeable decline.

The data on total WBC count of each group was subjected

to a two way analysis of variance for repeated measures

separately. This showed a highly significant "parasite effect"

(p < 0.001), "days effect" (p < 0.001), "days by parasite

interaction" (p < 0.001) in all groups.

Lymphocytes (Fig. 15, Tables 4,5, & 6)

Lymphocytes in guinea-pigs as in jirds constituted a dominant population amongst leukocytes. Relative lymphocyte count only accounted for the percentage of lymphocytes in comparison with the populations of other leukocytes.

The changes taking place in the total lymphocytes during the course of Parascaris infection were derived from the DAYS

FIG. 14. ABSOLUTE LEUKOCYTE COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS INFECTED WITH PARASCARIS EQUORUM >b. AGAINST UNINFECTED CONTROLS. VO F I

G THOUSANDS OF CELLS/cu.mm blood .

1 5 .

E P E Q R U C O E R N U T M

A A N G D A

I N A S B T S

O L U U N T I E N

F E L C Y T M E P D H O

C C Y O T N E T

R O C L O S U . N

T

I N

T H E

C I R C U L A T I N G

B L O O D

O F

G U I N E A - P I G S

I N F E C T E D

W I T H

- P - A - R - A - S - C - A - R - I - S 51 corresponding changes occuring in the total leukocyte popu­

lation. The total lymphocyte counts thus were more useful

in interpreting the changes in lymphocyte population.

A depression in total lymphocyte count was observed

on day 1 and 2 in group 1 and 2 respectively. A significant

rise in the lymphoctye count was observed on day 6 almost

concomitant with the decline in relative (%) neutrophil count.

This was also accompanied by rising WBC levels. The total

lymphocyte count remained significantly higher from day 6

onwards in all three groups. During the final phase of the

infection (day 14-18) a cumulative rise in lymphocytes both

at relative (%) and absolute scales were monitored.

Two way analyses of variance for repeated measures

of both relative and absolute lymphocyte counts revealed

singificant (p < 0.05) "parasite effect," "days effect" and

"days by parasite interaction."

Neutrophils (Fig. 16, Tables 4, 5 & 6) An early and significant rise in relative and total neutrophils was observed on the first day of infection (DOI)

(i.e., 24 hours after infecting the animals). Relative neutrophil count (%) of infected guinea-pigs on day 2 showed a highly significant difference of 16% from the controls

S'. as compared to Bs (L) = 6.3%. Though the total neutrophil count on day 2 showed a noticeable rise, it remained short of becoming significant when compared with the corresponding

Bs(L) value. The lack of significance was probably due to 50

40

U

20

d 4 o o l b

m m . u c / S L L

E 2 - C

F O

S D N A S U O H T -J---- 1------L//-I-----1___ I____L I I J------1------1------1------1------1------1____ I_____I I I I -8 -7 -6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DAYS Ul to FIG. 16. PERCENT ANO ABSOLUTE NEUTROPHIL COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS EQUORUM AGAINST UNINFECTED CONTROLS. INFECTED WITH PARASCARIS 53

a depression in the total WBC counts which were recorded as

lower than the controls on day 2. In this situation, it

appeared that a multiple comparison of percent neutrophil

count offered a more meaningful information.

Total neutrophil counts in all the three groups re­

mained almost significantly higher than the controls from

day 3 onwards. A noticeable decline in the total neutrophils

was observed on day 16-18. This decline was significant

on day 17 in group 2.

A two way analysis of variance was performed on the

data of both relative and absolute neutrophil counts of all

three groups (Appendix). Relative neutrophil counts of

group 1 showed a highly significant "parasite effect"

(p < 0.001), "days effect" (p < 0.001) and "days by parasite

interaction" (p < 0.001). Relative neutrophil counts of group

2 exhibited a highly significant "days effect" and "days

by parasite interaction" (in both p < 0.001), but an insig­

nificant "parasite effect" (p > 0.170). Relative neutrophil counts in group 3 demonstrated a highly significant "days

effect," "parasite by days interaction" (p < 0.001) and an insignificant "parasite effect" (p > 0.300). All three effects were highly significant (p < 0.001) for total neutrophil counts in the three groups.

Eosinophils (Fig. 17, Tables 6, 7 & 8)

Both relative and absolute eosinophil counts in 25

20

.i..... i_____J-y/-1__ -i____ I____ i____ i____ «____i____ •____ t. . .i. i « i i i i i i -8 -7 -6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DAYS <_n 4^ FIG. 17. PERCENT AND ABSOLUTE EOSINOPHIL COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS INFECTED WITH PARASCARIS EQUORUM AGAINST UNINFECTED CONTROLS. 55

guinea-pigs followed a similar pattern of changes due to

Parascaris equorum infection. An increase bordering on

significance in eosinophilia as compared to corresponding

Bs (L) value showed up on day 5 in group 2. On day 6 in group 3

the eosinophilia as compared to corresponding Bs (L) value

is insignificant, but very near to singificance. More sig­ nificant and regular increases began on day 7 and continued until peaks were observed on days 10, 11 and 12 in group 1,

2 and 3 respectively. A considerable decline in eosinophil population was noticed in all three groups in the following days. Despite this, eosinophil levels remained singificantly higher than controls from day 13-18.

Statistical analyses performed on the relative and absolute eosinophil counts showed highly significant (p <0.0011

"parasite effect," "days effect" and "days by parasite inter­ action ."

Monocytes (Fig. 18, Tables 6, 7 & 8)

According to Braunsteiner (1962) the monocytic stage is the least constant and less striking as compared to neutrophilic and lymphocytic stages. If absolute counts of monocytes are statistically analysed, their significance may be entirely lost.

The foregoing statement by Braunsteiner is probably based on the fact that the population of monocytes is quite small as well. Being so, it runs a higher risk of experimental F I G

. HUNDREDS OF CELLS/cu.mm blood PER CENT

1 8 .

P E E Q R U C O E R N U T M

A A G N A D I

N A S B T S

O L U U N T I E N

F M E O C N T O E C D Y

T C E O N

T C R O O U L N S T .

I N

T H E

C I D R C A U Y L S A T I N G

B L O O D

O F

G U I N E A - P I G S

I N F E C T E D

W I T H

P A R A S C A R I S 57

error in counting. Therefore, it seems preferable to rely

more on changes in relative counts during the infection.

But at the same time it would not be desirable to ignore

the corresponding changes in absolute monocyte counts especially

on days when both relative and absolute counts were signi­

ficantly higher than the controls.

In group 1 both relative and total monocyte counts were highly significant on day 7. On day 13 and 16 absolute counts were observed to be of border line significance as compared to Bs(L) = 18.13. In group 2 both relative and absolute monocyte counts were significantly elevated on day 8. Total monocyte counts were found significant on days 5,

11, 14 and 17. In group 3 both relative and absolute monocyte counts were significant on day 8. Total monocyte counts were found significant on days 3, 9 and 12. Total monocyte counts were significant from day 3 through 18. On the whole, days 7, 8, 9 and 12 seem to be more consistently significant considering both relative and absolute parameters at the same time.

Analysis of variance performed on the data of absolute monocyte counts revealed a very significant "parasite effect"

(p £ 0.006) in all three groups. "Days effect" was highly significant in group 3 (p < 0.001), significant in group 2

(p < 0.022) but insignificant in group 1 (p > 0.090).

"Days by parasite interaction" was again highly significant in group 3 (p < 0.001), very significant in group 2 (p < 0.006)., 58 and insignificant in group 1 (p > 0.124). Relative monocyte counts revealed a significant "parasite effect" (p < 0.045) in all the three groups. "Days effect” was highly signifi­ cant in group 3 (p < 0.001), but insignificant (p _> 0.239) in group 1 and 2. "Days by parasite interaction" was highly significant in group 3 (p < 0.001), but insignificant in group 1 and 2 (p >_ 0.174).

Basophils (Fig. 19, Tables 4, 5 & 6)

Basophils occur infrequently as compared to other leukocytes such as lymphocytes, neutrophils and eosinophils.

Their relative count in uninfected or control guinea-pigs ranged from 0 to 1%. Even in infected guinea-pigs the baso­ phil population was very small relative to other leukocyte populations cited above. This could induce greater experimental error, which may become highly misleading when percent basophil counts are converted to absolute basophil counts for a day or number of days. With this in mind it may be safer to disregard the counts of borderline significance. On this basis day 12-18 in the three groups could more safely be evaluated relative to the infection when the basophil popu­ lation rose to substantial proportions.

A two way analysis of vairance was performed on the data of relative basophil counts (Appendix). In group 1 there was a significant "parasite effect" (p < 0.00), "days effect" (p < 0.0201 and "days by parasite interaction to oo v\

DAYS

FIG. 18. PERCENT AND ABSOLUTE MONOCYTE COUNT IN THE CIRCULATING BLOOD OF GUINEA-PIGS INFECTED WITH PARASCARIS EQUORUM AGAINST UNINFECTED CONTROLS. Table 1: Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Intervals in Infected and Controls (Group 1 —Jirds)

VARIABLES Bs(li) Xi - Xc on days

1 6 7 10 13 16

RBCs (total) 28*1,000.0 492,000.0* *192,000.0* -170,000.0 -530,000.0* -564,000.0* - •560,000.0* -202,000.0 . « » PCV (%) - 2.02 3-59* 2.83* -O.92 -3.62 -6.00* -3.75 -1.67* . » * Hemoglobin (%) 0.6733 1.19* 0.94* -O.3O -1.16 -1.33 -1.25 -O.56 WBCs (total) 1,6?8.6 -85O.O 52O.O 3,260.0* 3,670.0* 3,210.0* 3,610.0* 3,280.0* Lymphocytes » 9.82 -»1.08 -O.67 -17-75* -32.75* -31.70* -21.75* -9.0 (%) Lymphocyte 930.0 -80*1.5 998.6* 1,758.2* (total) 366.0 298.0 102.9 680.8 Neutrophils 6.00 -1.00 14.17* 27.25* 17.92* 13.58* 0.62 (%) 9.54 Neutrophils 1,930.0* 3,100.0* 2,160.0* 1,900.0* (total) 1,235-4 66.1 60.0 760.0 Eosinophils # . * , * 3.24 0.25 7.00* (%) 2.29 3-37 6.31 8.67 6.50 Eosinophils 280.0* 396.8* 723.6* 665.I* 688.7* (total) 316.5 82.0 138.0 « Monocytes (%) 2.25 -1.0*1 -0.71 -0.12 1.75 3.21 2.13 1.87 Monocytes 195.7* 298.1* 222.0* x* (total) 177.0 -60.7 -*13.2 18.5 179.6 « Basophils (%) 1.085 -O.38 -0.18 0.33 0.70 0.75 0.06 1.17 Basophils » (total) 82.8 -23.O -6*1.0 31.6 61.9 77.7 26.0 85.2

significant Ln Table 2: Multiple Comariaons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 2—jirds) ~

VARIABLES Bs(L) XI - Xo on days

-7 2 5 8 11 14 17

RBCs (total) 264,000.0 420,000.0* 260,000.0* 0 380,000.0* 930,000.0* 130,000.0 100,000.0 x * PCV (%) 1.759 2.92* 2.25* O.83* -2 <67 -3-33* -O.25 0.67 Hemoglobin (%) 0.5863 0.97* 0.75* 0.28 -0.89* -1.11 -0.09 0.22 WBCs (total) 2,241.0 -539.0 3,380.0 943.0 3.030.0* 4,380.0* 4,524.0* 3,723.0* Lymphocytes 10.0 1.67 -22.62* -29.67* -34.33* -15.83* -8.08 (%) -2.25 Lymphocyte (total) 1,791.2 -5I8.I 2,?3O.O -640.8 -29.9 200.2 1,731-3 2,135.9 Neutrophils -2.1? 4.62 23.75* 19.55* 20.63* (%) 9.99 5.37 2.08 Neutrophils 1,350.9* 2,196.3* 2,850.0* 1,650.0* (total) 949.4 -2I.7 410.8 532.0 Eosinophils „ * * n # -0.8 2.54 8.2* 8.37* (5«) 3-85 3.87 5.33 8.17 Eosinophils 463.1 817.0* 8?2.9* 769.1* (total) -3.5 I87.8 238.1 439.9 Monocytes (%) 2.91 0.12 0.67 0.62 3.1?* 1.13 1.13 2.20 Monocytes 286.5* 214.4* (total) 195.8 7.1 53-5 43.4 149.6 39.6 Basophils 2,241.0 -539.0 3,388.0 (%) 943.0 3,030.0 4,380.0 4,524.0 3,723.0 Basophils -11.2 (total) 178.7 -6.3 105.5 85.2 65.9 102.0 147-3

X significant

OA o Table 3: Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 3—jirds)

VARIABI£S Bs(L) XI - Xc on days

-6 3 6 9 12 15 17

RBCs (total) 497,000.0 -282,000.0 -470,000.0 -810,000.0* - 1174,000.0* - 1200,000.0* -83Q000.0* -750,000.0* „ * PCV (%) 3.42 -1.83 -3.17 -6.17 -8.08* -8.75* -5.03* -5.0* x* Hemoglobin (%) 1.1*1 -0.61 -I.06 -2.06 -2.69* -2.92* -1.94* -1.67* WBCs (total) 1,719.0 -28*t.O 715-0 2,930.0* 3,765.0* 5,540.0* 5,660.0* 3,378.0* Lymphocytes 16. *»7 -II.92 -27.04* -31.17* -26.87* -14.5 (%) 1.67 -7.25 Lymphocytes 1,020.1 1,309.0* 2,923.0* 1.987.0* (total) -IO5.9 -125.9 123.3 227.0 Neutrophils x * -O.58 23.13* 17-83* (%) 15.3** 10.08 21.67 5.25 1.58 Neutrophils 1,5*12.0 2,440.0* 2,670.0* 3,100.0* 1,710.0* (total) -111.0 736.8 590.0 . . * » « Eosinophils (%) 2.32 0.5 1.67 1.87 4.42 6.54* 5.50 5-37 Eosinophils 395-0* 405.4* 600.6* 459.9* (total) 29*1.0 29-0 89.0 161.2 * Monocytes (%) 1.76 -0.50 -0.20 1-59 1.67 0.08 1.92 1.2 Monocytes 19.0* 23.03* (total) 16.35 -2.68 -O.77 15.54 9.06 12.68 * Basophils (%) 1.06 -O.25 0.04 0.08 0.33 0.87 1.83 1.67* Basophils 185.4* 140.8* (total) 97.5 -1*1.3 3.1 12.8 37.2 92.4

significant

cn H* Table 4: Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 1—Guinea-pigs)

VARIABLES Bs(L) XI - Xc on days

-8 1 4 7 10 13 16

RBCs (total) 200,000.0 138,000.0 178,000.0 77,000.0 -U8,000.0 -190,000.0 -2,000.0 -130,000.0 PCV (%) 1.70 1.08 1.0 0.17 -1.33 -2.1?* -O.33 1.08 Hemoglobin (%) 0.54 0.36 0.33 0.06 0.44 -0.73* -0.11 O.36 WBCs (total) 145.0 440.0 61.0 2,750.0 4,004.0 4,770.0 5.350.0 5,130.0 lymphocytes « 6.12 -23.58* -20.0* -12.45* -24.0* -14.67* (%) 0.3 -19.5 Lymphocyte 847.6 1,493.0* 968.4* 1,523.7* 1,858.7* (total) -3I6.7 -715.2 462.0 Heutrophils . * , * 0.21 21.0 14.42* 7.25* M 5.65 4.25 9.U2 6.50 Neutrophils 732.0 1,171.4 1,768.6* 1,625.8* 2,153.5* 2,225.0* 2,180.0* (total) Eosinophils « 4.0 0.11 12.88* 10.95* 6.25* (%) 0.25 2.67 5.75 Eosinophils 619.0* 1,373.8* 1,273.1* 798.0* (total) 389.0 269.0 223.0 306.8 x * Monocytes (%) 2.45 -O.45 2.42 0.25 6.05 O.67 I.50 1.33 Monocytes 646.0* 222.0* (total) 181.3 16.0 123.0 68.0 157.0 152.0

Basophils 1.83* (%) 1.17 -0.3 -0.17 0.50 0.33 0.13 0.8?

Basophils 130.0* 197.2* (total) 83.6 -11.4 -1.8 54.3 61.8 63.7

significant

Ch to Table 5: Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 2—Guinea-pigs)

VARIABUSS Bs(L) XI - Xc on days

-7 2 5 8 11 16 17

RBCs (total) 177,000.0 319,000.0* 216,000.0* -66,000.0 -139,000.0 21,000.0 78,000.0 71,000.0 2.33* PCV (%) 1.66 1.17 -O.33 -O.92 0.0 0.62 0.33 Hemoglobin (%) 0.606 0.78 -O.39 -0.11 -0.31 0.0 0.16 -O.I3 WBCs (total) 1,670.0 -50.0 -76O.O 2,700.0 4,880.0 6,120.0 . 5,880.0 5,060.0 Lymphocytes -18.0* 19.75* 27.75* 38.08* -12.67* 27.76* (*) 7.134 2-33

Lymphocyte -1,202.0* 2,413.5* 2,798.1* 1,826.6* 2,662.2* (total) 1,099.9 78.6 361.18 Neutrophils (%) 6.30 1.83 16-33 13.63 0.25 -1.5 -I.92 -8.0

Neutrophils 1,794.8* 1,472.7* 1,692.5* 1,567.5* (total) 811.0 -106.7 407.3 801.6 Eosinophils 3.12 6.83* 10.42* 11.17* 4.3* (%) 0.33 0.8 3-33

Eosinophils 307.2* 630.0* 1,330.0* 1,395-0* 619.8* (total) 323.0 13-4 5-2 « Monocytes (%) 2.16 -0.83 1.42 1.96 2.3 1.0 1.38 0.55 Monocytes I83.O* 266.0* 236.O* 233.0* (total) 162.0 -38.0 61.0 119.0 * Basophils (%) 1.17 0.0 0.05 1.17 1.12 1.0 1.79* 1.75* Basophils 12.15* 13-57* 21.12* 17.01* (total) 9.94 0.04 -O.53 8.19

significant

cn GJ Table 6: Multiple Comparisons of Various Hematological Variables During Parascaris equorum Infection at Given Time Intervals in Infected and Controls (Group 3—Guinea-pigs J

VARIABLES Bs(L) XI - Xc on days

-6 3 6 9 12 15 18

RBCs (total) 116,000.0 -66,000.0 -120,000.0* -294,000.0* -279,000.0* -392,000.0* -285,000.0 * -219,000.0* PCV (%) 1.03 -0.67 -0.92 -0.35 -2.17 -3.O8 -2.25 -I.75 . * Hemoglobin (%) 0.385 -0.22 -0.31 -0.41 -0.73* -I.03* -0.75* -0.58* WBCs (total) 775-0 -64.2 1,840.0* 2,600.0* 3,880.0* 4,840.0* 6,010.0* 4,790.0* Lymphocytes » 8.50 2.0 16.83* 9.42* IO.58* 27.42* 11.83* (%) -19.5 Lymphocytes 825.2 68.7* 973-*** 1,484.2* 1,491.1* 2,527.0* 2,430.0* (total) 43.7 Neutrophils II.58* 4.8* (%) 5.05 0.67 0.75 0.25 2.42 2.92 Neutrophils 1,335.3* 1,146.7* 1,291.1* 1,473-0* 1,567.9* 1,144.0* (total) 440.0 41.1

Eosinophils 4.05* „ * 12.2* 11.21* 8.04* (%) 4.7 0.25 2.12 8.71

Eosinophils 923.7* 1,356.5* 1,426.3* 904.9* (total) 433.8 -12.6 193-7 361.9 Ä * • Monocytes (%) 1.47 O.58 2.92 1-31 1-55 2.42* 1.58 0.83 Monocytes (total) 181.3 -30.2 229.2* 139.9 127.O 327.8* 210.0* 162.2 Basophils (%) 1.14 -O.33 -0.70 -O.33 1.08 2.08 2.48 1.37 Basophils 199.4* 276.2* 137.1* (total) 95-8 -I5.5 50.3 31.0 86.2

« significant 65 (p _< 0.022). In group 2 there was a significant "parasite

effect" (p _< 0.003), "days effect" (p < 0.030) and "days by

parasite interaction" (p _< 0.006). Group 3 showed highly

significant "parasite effect" (p < 0.001), "days effect"

(p < 0.030) and "days by parasite interaction" (p < 0.001).

Total basophil counts were subjected to a similar

statistical analysis (Appendix). In groups 1 and 2 a signi­

ficant (p < 0.001) "parasite effect," "days effect" and

"days by parasite interaction" was observed. In group 3

a significant "parasite effect" (p < 0.002) along with a

highly significant (p < 0.001) "days effect" and "days by

parasite interaction" was observed.

Histopathological Observation

Lesions and Cellular Response in the Liver of Jirds

Little cellular response could be observed in liver

tissue at 4th DOI. This evidently may be due to less damage

to hepatic parenchyma involved and limited degree of larval

migration at that time.

Macroscopic examination of the liver at 8th DOI

showed no white or "milk spots." However, small dark red

areas differentiating in contrast from the general color of the liver were observed. These probably indicated the areas of homorrhage. An examination of microscopic prepara­ tions at this stage showed the following pathological changes

(1) considerable mechanical damage to hepatic parenchyma 66

(Plate 1, Fig. 20) due to migrating larvae was observed, with

red blood cells accumulated in a number of damaged areas.

(2) There was some cellular infiltration, specially along

the tracks of migrating larvae (Plate 1, Fig. 21). The

infiltrates consisted of predominantly macrophages, lymphocytes,

some neutrophils, but no eosinophils, (3) Larvae found in

sections showed no cellular response around them (Plate 1,

Fig. 21). They appeared viable and lacked a precipitate,

indicating the absence of an atigen-antibody reaction to

inhibit their development.

Lesions and Cellular Response in the Liver of Guinea-pigs .....

Virtually no cellular response or hepatic damage

could be observed at 4th DOI. This may be due to a limited

degree of larval migration, which seemed to accelerate after

the first moult in the host.

At the 8th DOI in infected guinea-pigs a macroscopic examination of liver revealed "milk spots" on the surface which was described by White (1941) as "focal interstitial hepatitis." Histopathology of the liver in general is characterized by: (1) large clusters of infiltrating cells

(Plate 3, Fig. 23), consisting of lymphocytes, neutrophils, macrophages and eosinophils. At a little later stage i.e., in older lesions these clusters differentiated into a central area of fibroblasts, encircled by macrophages, lymphocytes, and eosinophils (Plate 3, Fig. 24), (2) larvae encircled by 67

leukocytes and on their way to disintegration (Plate 3, Fig. 24),

(3) less damage to the hepatic parenchyma and markedly less

hemorrhage than jirds.

Lesions and Cellular Response in the Lungs of Jirds '

At 14th DOI microscopic examination of the lungs of

jirds showed a consolidation of the peripheral areas in the

lower half of lungs. Petechial hemorrhage spots were generally

distributed throughout the other areas of the lungs. Micro­

scopic examination of the infected lungs revealed: (1) peri­

pheral areas of the lungs (Plate 4, Fig. 25) with extensive

hemorrhage and collapse of intra-alveolar septa, (2) limited

cellular infiltration relative to the damage caused by

migrating larvae, (.3) a dominant population of mononuclear

cells, (4) greenish brown pigment granules, most likely

hemosiderin in areas of extensive hemorrhage, (.5) a number

of larvae (Plate 5, Fig. 27) in alveoli, without any cellular

involvement or antigen-antibody precipitate around them.

Lesions and Cellular Response in the Lungs of Guinea-pigs

Macroscopic examination of infected guinea-pigs at

the 14th DOI showed petechial hemorrhage spots and small consolidated areas generally scattered over the surface of lungs. The hemorrhage spots and areas seemed cloudy or whitish as compared to the intense reddish color in jirds.

Histopathology of lungs at this stage was characterized by: PLATE 1 68

Pig. 20 (JTird, Liver 8 DOI X 4-00) Showing mechani­ cal damage to hepatic parenchyma due to migrating larvae, hemorrhage and limited leukocytic response in lesions.

Pig. 21. (Jirdr Liver 8 DOI X 4-00) Third stage larva of Parascaris equorum without surrounding cellular response; some response could be seen along the track of larval migration. PLATE 2

69

Pig. 22 (Jird, Liver 8 DOI X 1000) Accumulation of predominantly monomialsar cells in the damaged area. PLATE 3 70

Pig. 23 (Guinea-pig, Liver 8 DOI X 300) Massive cellular response in the damaged area caused by larval migration, infiltrates include both mono­ nuclear and polymorphonuclear cells.

Pig. 24 (Guinea-pig, Liver 8 DOI X 250) An older lesion showing an inner fibrotic zone(P), showing the lesion being resolved, a disintegrating larva (L.) could be seen surrounded by a degenerating mass of eosinophils and other leukocytes. PLATE 4 71

Pig. 25 (Jird., Lung 14- DOI X 40) Hemorrhage caused by the migrating larvae in the peripheral area of the lung, cellular infiltration more marked around bronchioles and perivascular area.

Pig. 26 (Jird, Lung 14 DOI X 250) A close up fo the above figure, showing an area of hemorrhage, mono­ nuclear cells predominate in this area. PLATE 5

72

Pig. 27 (Jird, Lung lA DOI X 250) Larvae in the alveolus are free of any surrounding cellular response.

Pig. 28 (Jird, Lung 14- DOI X 1,000) Showing predominantly mononuclear response in an area where hemorrhage has occured; hemosiderin granules (h) are the digestive byproduct of effete erythrocytes after their phagocytosis. PLATE 6

Pig. 29 (Guinea-pig, Lung 14 DOI X 40) massive and generalized cellular response caused by the parasite. ■

Pig. 30 (Guinea-pig, Lung 14 DOI X 1,000) Accumula­ tion of eosinophils (reddish granulaes) and degranulated basophils (bluish granules) indicative of immediate type hypersensitive response. PLATE 7 74

Pig. 31 (Guinea-pig, Lung 3.4 DOI X 250) Disintegrat­ ing larva trapped in cellular response consisting of degenerated eosinophils and other leukocytes. 75

(1) general hypersensitive reaction indicated by considerable

cellular infiltration (Plate 6, Fig. 24), the cellular in­

filtration was dense around perivascular and peribronchial

areas, (2) little noticeable hemorrhage and collapse of

intra-alveolar septa, (3) the presence of leukocytes such

as lymphocytes, neutrophils and macrophages, eosinophils as

dominant cell types in some areas in the lungs showing a

hypersensitive response, among the aggregates of eosinophils

could be detected dark blue basophil granules (Plate 6,

Fig. 30), (4) the cytoplasm of some macrophages contained

hemosiderin granules, (5) many larvae found in sections were

surrounded by a dense ring composed of degenerating eosino­

phils, macrophages and lymphocytes (Plate 7, Fig. 31).

DEVELOPMENT OF PARASCARIS EQUORUM LARVAL STAGES IN GUINEA-PIGS AND JIRDS (TABLE 7)

The difference in larval growth of P ara s cari s equorum

in the two experimental hosts became evident after the moult

from the 2nd stage larva to 3rd stage. In Parascaris equorum

the moult from the 2nd stage larve to the 3rd stage seems

to occur between the 5th and 6th day. This is followed by

enhanced growth of the 3rd stage larvae in both hosts coupled with their migration to the lungs where they undergo further growth. However, as indicated above, a significant difference between growth rates of larvae in guinea-pigs and jirds could be observed after the 4th DOI. The maximum growth levels attained by the 4rd stage larvae in the lungs of the two 76 hosts showed that on the 18th day average larval size in jirds lagged behind guinea-pigs by a margin of 550 u x 16 u.

This difference was accentuated because of the wide range between the sizes of larvae recovered from jirds. Table 7: Rate of Growth* of Parascaris equorum Larval Stages During First Infection in Guinea-pigs and Jirds over 18 Days Period.

Time lapse in Larval** Total length and max. diameter*** infection stage Location Guinea-pigs Jirds

6-24 hours 2nd lumen of intestine 305-320 (312.5) 310-320 (315) & liver x 20-25 (22.5) x 22-24 (23) 4 days 2nd liver 450-500 (475) 390-460 (418) x 28-32 (30) x 25-29 (27)

7 days 3rd liver 1155-1200 (1180 810-1050 (950) x 45-52 (48) x 32-50 (40)

9 days 3rd lung 1350-1500 (1440 1020-1220 (1115) x 48-60 (54.8) x 35-50 (44)

10 days 3rd liver None recovered 800-1065 (930) x 30-55 (40)

14 days 3rd lung 2040-2060 (2052) 1050-1500 (1255 x 55-65 x 38-60 (48)

18 days. 3rd lung 2035-2075 (2050 1300-1800 (1490) x 60-75 (68) x 45-60 (52)

* Ranges and (averages) in microns for 5 larvae randomly selected. ** Identification of 2nd stage and early 3rd stage according to Robert (1934): late 3rd stage according to Douvres et. al (1969) in Ascaris suum. *** Diameter at level of base of oesophagus of larvae. -j Table 8: Comparison of the Number of Active Larvae Recovered from the Visceral Organs of Jirds and Guinea-pigs During the Parascaris equorum Infection After Inoculum of 600 (+50) and 5,000 (+ 100) Embryonated Eggs Respectively at the Given Intervals

Visceral No. of Animals Range of Standard Animal Organ Day used counts Deviation

Jird

Liver 6 5 87-138 21.25

Lungs 14 5 33-68 12.08

Guinea-pig

Liver 6 5 20-41 8.4

Lungs 14 5 14-25 2.9 CHAPTER IV

DISCUSSION

Hematological changes during the migration of the larval stages of Parascaris equorum were documented in two laboratory models representing two different ecotypes. An important consideration in determining the inoculum was not to overburden the experimental host with an excessive infective dose. Such an approach would result in exaggerated symptoms of the disease, and may cause the animal to succumb to secondary infections. Shope (1957) reported increased severity of cholera in hogs due to already existing heavy Ascaris suum infection.

An excessive challenge dose could induce abnormal larval growth and influence the migration of the larvae, i.e., migration into organs not normally utilized by the parasite. According to Olsen and Kelley (1960) a significantly reduced growth rate of Ascaris suum larvae undergoing visceral migration in mice resulted from a large egg does (10,000 to 100,000). This result may have been produced by the crowding effect or by lowering the fitness of the environment provided for the larvae.

The massive larval migration could cause adverse physiological effects on the host, thus lowering the potential for proper larval growth.

With an infective dose of 600 (+ 50) infective eggs

79 80 given to jirds, a significant decrease in erythrocyte count

and corresponding packed cell volume and hemoglobin values

were observed during the course of infection. Guinea-pigs,

on the other hand, subjected to an infective dose of 5,000

(+ 100) eggs showed little or no significant changes in

erythrocyte parameters and corresponding PCV and hemoglobin

values in three groups in a day to day comparison with their

controls.

In this regard a comparative assessment of the number

of actively migrating larvae retrieved by using Baermann’s

apparatus (Table 1) in the two hosts provides some interesting

information. Fewer larvae were recovered from the liver and

lungs of guinea-pigs on 6 DOI despite approximately a nine­

fold higher infective dose as compared to jirds. It seems

some inhibitory factor was operating on the parasite in guinea-pigs during the course of larval migration. This inhibitory factor may have been in the form of a non-specific resistance on the part of the host, particularly in view of its first experience with the parasite.

Histopathological findings in the liver and lungs of infected guinea-pigs and jirds further corroborate this assumption. Important features observed in the liver of jirds at 8 DOI were: (1) extensive damage to hepatic parenchyma,

(2) hemorrhage, where considerable damage to hepatic tissue has taken place, (3) very little to moderate cellular infiltration where there is damage to tissue, (4) almost no cellular involvement 81 around the larvae to inhibit their migration and growth.

Comparatively, in guinea-pigs, the histopathology revealed:

(1) moderate to little damage to hepatic parenchyma, (2) scant hemorrhage, (3) heavy cellular infiltration indicating a general hypersensitive response, (4) cellular involvement around larvae arresting their growth and migration and final disintegration.

A similar comparison of the lung histopathology in jirds at 14th DOI was characterized by: (1) migrating larvae causing extensive hemorrhage, (2) breakdown of intra-alveolar septa at many places, (3) little cellular infiltration in general and no cellular involvement around larvae found in alveolar spaces. By contrast, the histopathology of the guinea- pig lung on 14th DOI showed that: (1) cellular infiltration was extremely heavy as a result of hypersensitive response and no damage to alveolar septa was observed, (2) no signifi­ cant hemorrhage was visible, (3) a number of larvae were found trapped in a cellular response, with larvae at various stages of disintegration.

On the basis of larval yield and related cellular response in both animals, convincing evidence exists that jirds offered an inadequate immune cellular response to effectively resist hemorrhage during the first infection of

Parascaris equorum.

According to Baker and Douglas (1966) blood loss through hemorrhage in helminthic infections results in loss 82 of iron from circulating erythrocyte and plasma. As a result of this, avoidance of anemia would require an equally great increase in the amount of iron supplied to bone marrow for haemopoiesis. In general, such rapid compensation cannot occur and a normocytic anemia in jirds appeared during the active phase of larval migration and development in the liver and lungs where blood was a major larval food source. With the larvae ending their visceral migration near the final phase of infection a prompt recuperation of erythrocyte count and corresponding packed cell volume and hemoglobin concentration followed.

According to Cline (1975) leukocytes may be regarded as the major defense against invasion of foreign organisms.

During the course of infection leukocytes complement each other, yet they are independent in their functions. In the very beginning of an infection mononuclear phagocytes and perhaps other leukocytes may release materials that stimulate the bone marrow to produce phagocytic cells. In this way mature circulating cells may be involved in feed back control of leukopoiesis. Thus, monocytes (Robinson,

1975), by releasing a colony stimulating factor, can influence immediate granulopoiesis in response to a foreign invader.

Later, lymphoid, cells are called into play.

B lymphocytes elaborate immunoglobulin which together with the complement "osponize" the invader. Later, the organism is engulfed or attacked by phagocytes i.e., neutrophils, 83 monocytes and macrophages. T and B lymphocytes elaborate a

variety of effector substances that help to activate and

direct the distribution of mononuclear phagocytes. The

phagocytes may localize anitgen and present it to lymphocytes

in a manner that facilitates their immunological response.

Changes in the leukocyte picture in the blood, there­

fore, are to a great degree indicative of the phase of

infection and related immune state of the host.

Total leukocyte counts in guinea-pigs (Fig. 14),

as compared to jirds (Fig. 5) showed a more gradual, less

transient build up. In the later phase of the infection

the total counts in guinea-pigs attained higher levels than

jirds.

Among leukocyte sub-populations lymphocytes were the most dominant followed by neutrophils in both experimental hosts. Comparative analyses of the changes in these two sub­ populations showed that they had reciprocal relationship during the course of 18 days infection. At the onset of infection a sharp increase in percent neutrophils in infected guinea-pigs demonstrated the animal's ability and preparedness to mount a sudden immune response. The rise in absolute count was not so spectacular for two reasons: (1) neutrophils are not the most dominant population among leukobytes, and

(2) build up in total leukocyte count in the initial phase of infection did not achieve significant levels. After a levelling off in absolute neutrophilia a decline was observed 84 in both percent and absolute neutrophil count near the final

phase of infection. The rise in both absolute and percent

neutrophils in jirds was late, becoming significant from

4th day onwards. However, a decline in neutrophil counts

in jirds was also noticed during the terminal phase of in­

fection. As indicated earlier, lymphocyte populations had

a more or less reciprocal relationship with neutrophil populations in both experimental animals. Thus, a sharp rise in percent neutrophils in guinea-pigs was accompanied by a sharp decline in lymphocytes, and a decline in neutrophil count was concomitant with increase in lymphocyte count.

Similarly, in jirds a late build up in neutrophil count was concomitant with increase in lymphocyte count, and in the end a rise in lymphocyte count was coupled with a decline in neutrophils.

The effectiveness of the immune mechanism of the host depends on the early mobilization of neutrophils to the inflammatory site in the event of an infection. This, however, is preceded by stimuli such as colony stimulating factor of mononuclear phagocytes which increases granulopoietic activity at the bone marrow level. As a result, greater than normal quantities of neutrophils enter the circulation. The stimuli, which later are responsible for the recruitment of neutrophils to infection sites, could be more specific. This recruitment is carried out by a process known as chemotaxis. Most of the materials which are considered to carry out this process 85 are identified as immune complexes. Senn (1972) considers

the inability of neutrophils to migrate in a normal and timely

manner from the microcirculation to participate actively in

an inflammatory reaction may lead to a significant risk

of infection.

A significantly delayed response in the build up of neutrophilia was observed in jirds as compared to guinea- pigs. This delayed response in jirds could have impaired

the host’s ability to resist Parascaris equorum infection.

The lack of a sufficient circulating neutrophilia could greatly influence the migration of neutrophils to the site of infection since the route taken by these leukocytes to the infection site is by necessity the circulatory system. However, as indicated, this migration is dependent upon the efficiency of the chemotactic factors, or in other words the immunogenicity of the parasite at the infection sites. The degree of cellular response at the infection sites, therefore, determines the immune competence of the host. A comparative assessment of histopathological findings showing the cellular response in visceral organs of both experimental hosts is given in the later part of this discussion.

In guinea-pigs, after an initial drop in absolute lymphocyte count on day 1 and 2, a gradual rise was observed.

This rise became statistically significant from day 6 onwards.

Highest levels in absolute lymphocyte count in all three groups were observed from day 13-18 DOI. Comparatively, 86 in jirds after a sudden and significant increase in absolute

lymphocyte count on 2nd DOI its level declined to almost control

until day 12, a rise in absolute lymphocyte count became

significant again on day 13 which lasted through day 18.

An early and sudden decline in lymphocyte count

(lymphopenia) in Ascaris infection has been reported by

Purcherea (1970) and by Smirnov (1935) in guinea-pigs.

De la Blaze (1946) observed depression in lymphocytes in

humans immediately after the onset of acute illness and

poisoning. In guinea-pigs infected with Parascaris equorum,

it seems, early lymphopenia was an alarm reaction initiated

by the parasite with increase in neutrophils at the same time.

However, in jirds an early increase in absolute lymphocyte

count on day 2 was not in accord with the reaction in guinea- pigs. Braunsteiner (1962) has documented certain cases where

a dramatic increase in lymphocytes was observed immediately

following a bacterial or viral infection as well as injection of a foreign protein.

The disease associated with parasitic infections is often the sure outcome of a complex but harmonious earlier association (Davies, 1977). In view of this, the immune response may differ between two different hosts, especially in divergent ecotypes, on the basis of their earlier experience with the parasite. It could be suggested that may be Parascaris equorum is too "foreign" for the jird host, so much so that it has not evolved enough ability to interact immunologically 87

with the parasite despite its undbouted antigenicity.

Generally, the lymphoid cells are believed to be the

carriers of delayed-type hypersensitivity or cell-mediated

immunity. Soulsby (1972) reported that the lymphoid cell

response to Ascaris suum infection in guinea-pigs was local.

According to him, peripheral lymphocyte responsiveness to

an antigen (as assessed by the uptake of tritiated thymidine)

required 8-10 days. In the present study with Parascaris

equorum infection in guinea-pigs, a shift towards a signifi­

cant build up in lymphocyte count was observed on 6th DOI

which became highly significant on 8th DOI. In jirds, the

significant rise in lymphocyte count was delayed until 13th DOI.

This lapse could have contributed to the impaired ability

of the jird to mount an effective immune response to the first

Parascaris infection.

Eosinophilia

In both guinea-pigs and jirds, eosinophilia became

significant after 7th DOI and remained significantly higher

than their controls until the last or 18th DOI. However,

in guinea-pigs, eosinophilia attained levels twice as high

as jirds. Eosinophilia is more common in atopic diseases

including helminthic infections, particularly involving

the tissue phase in the mammalian host. The most striking

attribute of eosinophils is their interaction with immune complexes. Bastsen and Beeson (1970) provided evidence 88

that eosinophilia is dependent on a product of thymus dependent

lymphocytes. They also provided the evidence that these

cells are also responsible for mediating eosinophilotaxis.

In addition to being important inducers of eosino­

philia, helminths are also known for their ability to evoke

reaginic antibody (IgE) response in a number of hosts. In

such hosts both eosinophilia and IgE levels in the blood could

go hand in hand. But in the case of first infection, it occurs

as an immediate type hypersensitive response in which lym­ phocytes do not play a principal role.

Lymphocytes, as indicated earlier, play a principal role in delayed type hypersensitivity or cell-mediated immunity

Administration of an antigen in experimental animals initiates a series of cell divisions and cellular differentiation within the lymphoid system (Cline, 1975). Eventually this leads to a rise in lymphocyte levels in the circulating blood.

It is not a mere coincidence that the rise in eosinophils and lymphocytes, especially in guinea-pigs during Parascaris equorum infection, occured after the second stage larvae in the liver had undergone a moult to become third stage larvae on the 6th DOI. SoulSby (1957 ), working with Ascaris lumbricoides in guinea-pigs has shown that immunity to a challenge infection is directed principally against the moulting period between the 2nd and 3rd larval stages.

According to him in reinfected immune animals most of the larval population does not enter this transition stage. 89 Soulsby (1958 ) has further shown that the first marked

release of antigen occured after the third moult.

Basophils

A significant rise in basophils in both guinea-pigs

and jirds occured during the final phase of Parascaris equorum

infection. In infected jirds, basophils counts became signifi­

cantly higher than their controls on 15th, 16th and 18th DOI, whereas in guinea-pigs the basophil count became significant

earlier i.e., on 12th DOI and remained consistently significant

up to the last or 18th DOI. The levels of basophil counts in

guinea-pigs during their significant build up were Considerably higher than jirds during the same period.

The blood basophils and tissue mast cells are similar in structure and chemical characteristics and tend to respond to various stimuli in a similar manner (Barbero, 1972). One of the most important roles of basophils and mast cells i.e., synthesis and release of histamines, has been well recognized in literature. According to Ishizaka (1970) the human blood basophil fixes IgE antibody with a release of histamine when

Sadun (1972) in his review has given a number of examples showing the ability of Parascaris equorum and Ascaris spp. when injected or infected in animals, to induce anaphylactic or acute allergic reaction. The most outstanding features of allergy are a build up in eosinophilia, IgE titre and histamine level. Rothwell and Dineen (1972) made observations 90

on the cellular reaction in immune guinea-pigs during Tricho-

strongylus colubriformis infection. They found that after

initial proliferation in bone marrow, the eosinophils and

basophils enter the circulation and show a significant increase.

Later, both eosinophils and basophils migrate to the site of

infection. Considerable evidence exists that eosinophils

antagonise the role of basophils. Broom and Archer (.1962)

found that eosinophils inhibited edema caused by histamine.

They also showed that eosinophil extract counters the changes

induced by histamines, like constriction of smooth muscles

and capillary permeability.

Basophil appearance in the circulating blood was

delayed up to 15 days after infection with Trichostrongylus

colubriformis in non-immune (previously uninfected) hosts

(Rothwell and Dineen, 1972). This is of interest because present studies on Parascaris equorum infection also involve previously uninfected guinea-pigs and jirds. In both of these experimental hosts a significant build up in basophil count was also noticed in the last phase of infection. It may be misleading to draw any kind of parallel between two different nematode infections, especially when the two parasites are developing in two different locations, Parascaris being parenteral and Trichostrongylus being enteral. As a rule parenteral or more particularly visceral migration and develop­ ment of the parasite accompanied by its interaction with local tissues mobilize, among other responses, greater cellular and 91 allergic response. Pruzansky (1970) observed that the percentage

of basophils decrease in allergic individuals when inoculated

with the antigen. Apparently, this happens due to the degranu­

lation of basophils subsequent to their interaction with the

antigen. It is hypothesized that during Parascaris equorum

infection basophils were mobilized at the same time as the

eosinophils as a result of granulopoiesis in the bone marrow.

But due to the release of antigenic elements produced by the

developing and migrating parasites in the blood, they degranu­

lated. Their significant rise in the blood could be correlated

with the counter shock phase when most of the parasitic larvae

were being eliminated from the host. Studies undertaken on

the level of histamines during Parascaris equorum infection

could yield more definite information on the quantity of

basophils entering the blood.

Monocytes

More consistent rise of monocyte counts in the circu­

lating blood were noticed in the middle of the infection,

covering the period from day 6 through 13 in both experi­ mental animals. In guinea-pigs, however, the peaks in both

absolute and relative monocyte counts (%) were higher than

in jirds. The blood monocyte is an intermediate form in the cell

line between the younger monoblast or promyelocyte and most mature form, macrophage. The younger forms originate in bone 92 marrow, whereas mature forms are found in tissues (van Furth et al., 1972). The most recognized function of monocyte- macrophage system is the removal of damaged or dying cells and cell debris by phagocytosis. Schilling (1955) has described the "monocytic phase" as a "defense stage" which follows the earlier "neutrophilic phase" which he describes as a "fighting stage." During the present studies, in two experimental hosts, the significant increase in monocytes also followed the initial neutrophilia. However, the changes in monocyte popu­ lation were less marked and consistent as compared to other leukocyte populations. Naegli (1931) observed similar trends in monocytosis on the basis of his findings in other infections.

Among other functions of the monocyte-macrophage system is the interaction of mononuclear phagocytes with lymphoid cells in certain phases of the immune response. This view is supported by several observations in recent years :

(1) Human monocyte and macrophages exposed to the antigen in vitro and then washed free of extracellular antigen can induce transformation of autologous lymphocytes (Cline & Summer, 1972; Hanifin & Cline, 1970).

(2) Normal, non-immune lymphoid cells can induce antibody synthesis in X-irradiated recipient animals when living macrophages containing antigen are also transferred (Kunin, S. et al., 1972 & Unanue, E. R. et al., 1968).

(3) Pure populations of lymphocytes containing no phagocytes show a reduced blastogénie and antibody response to specific antigens in vitro; addition of macrophages restores this response to normal values (Hersh & Harris, 1968 & Pierce, 1969).

(5) Addition of macrophages to lymphocytes augment the pro­ duction of certain effector substances, such as interferon, in response to specific antigen and non-specific mitogen (Epstein, L. G. et al., 1971). 93

These observations have led to the concept that macro­

phage function may be divided into two phases during the

immune response; in the "afferent" limb during the induction

of immunity by antigen, and in the "efferent" limb during

the expression of cellular immunity. The role of the macro­

phage in the reacting with antigen during recognition phase

of the immune response appears to be quite separate from

its role in cell-mediated immune reactions.

To briefly recapitulate the importance and changes

in leukocytic syndrome during Parascaris equorum infection

in guinea-pigs and jirds: Neutrophils are the front line

fighting cells which are mobilized at once in the event of an

infection in a competent host to phagocytize or lyse the

invading organism. As compared to guinea-pigs, jirds exhibited

a delayed neutrophil increase in circulating blood. This

could mean an inability in jirds to meet the challenge of

infection at its initial stage.

Lymphocytes become involved in delayed type hyper­

sensitivity or cell mediated immunity after interaction with phagocytes and antigen. Their significant rise in peripheral blood is indicative of the proliferation after this reaction, which means a maturation of immune response to increase the antigen binding affinity. In guinea-pigs, this maturation of the immune response at the cell-mediated level seemed to begin earlier than jirds.

Circulating eosinophils in helminthic infections 94 involving visceral migration like Parascaris are indicators of hypersensitive or allergic reaction of the host. They are also indicative of reaginic antibody (IgE) levels in the blood.

There is evidence that they neutralize the effects of histamines.

Guinea-pigs demonstrated a higher circulating eosinophilia than jirds.

Basophils, like eosinophils, are mobilized as a part of the hypersensitive response of the host to an infection.

As mast cells in the tissues, they release histamines at the inflammatory sites and in the blood as a reaction to the antigen.

In addition, they fix reaginic antibody (IgE). In both hosts, the basophil counts achieved significant levels during the final phase of infection, but basophil levels in guinea-pigs were higher than jirds.

An increase in circulating monocytes suggested their migration to infected sites in greater than normal numbers.

In tissues, they exist as migrating and fixed cells. They are directly involved in phagocytosis of cell debris, but indirectly involved in the complex intereactions with lymphoid cells for stimulating immune responses. A significant build up in monocytosis in the two hosts occured during the middle of the infection, again in guinea-pigs the peaks of monocyte counts were higher than jirds.

There is ample evidence that many of the leukocytic changes are controlled by hormones (Braunsteiner, 1962,

Hampton, 1972) both in health and under the stress of a disease. 95

But it is difficult to evaluate the influence of hormones

on these changes because of conflicting results.

Schilling (1955) designed a general scheme which was

applicable to all qualitative changes in the blood picture

seen in a great variety of conditions, particularly infections.

Following a prodromal phase, Schilling recognized three stages:

neutrophilic "fighting stage," the monocytic "defense stage"

and lymphocytic-eosinophilic "convalescent phase." These phases

could be outlined more clearly in local infections. The leuko­

cyte response curve has been demonstrated to be more rapid

and circumscribed in studies by William and Johnson (1976)

using turpentine and dextran injections. Spector, W. G. et al.

(1965) demonstrated the same response by injecting fibrinogen.

Comparatively, Parascaris equorum infection is more

complex. It is a migratory type of infection involving

three visceral organs. The infection begins with the pene-

teration of Parascaris larvae through the gut wall, followed

by a period of migration, moulting and growth in the liver.

Later, the larvae infect the lungs, where they undergo further period of growth. A pattern similar to Schilling’s seemed to have emerged, though less delineated because of overlapping leukocytic rhythms in each phase of the infection. The phases may be characterized by an initial "neutrophilic stage," an inter­ mediate "monocytic stage" followed by a final "lymphocytic- eosinophilic stage" (particularly in guinea-pigs). 96 Lesions and Cellular Response in the Two Experimental Hosts: A Comparative Assessment

The 8th DOI in the liver for histopathological observa­ tions was selected because of two reasons: (1) the larvae, after undergoing a moult between the 5th and 6th DOI, show an accelerated growth. After moulting, they double their size by the 8th DOI. This is coupled with their active migration within the liver and outside to the lungs; (2) as mentioned before in the discussion, the moulting process carries with it an antigenic potential for the host. The extent of immune response to this stimulus from the host at the site of infection, as a rule, is also expressed in the form of increased cellular response.

Macroscopic "milk spots" in guinea-pigs were focal lesions in which destroyed liver cells were replaced by fibrous tissue after infiltration with leukocytes (Plate 3, Fig. 24).

In the earlier stages of the lesions, neutrophils, lymphocytes, macrophages and eosinophils could be seen in clusters of infiltrates accumulated in the areas eroded by migrating larvae

(Plate 3, Fig. 23). This constitutes a very early response to combat the infection and help repair the damaged tissue.

From the density of the cellular elements in each cluster, it appears that cellular response was intense. Some of the larvae were found in a disintegrated condition surrounded by a degenerating mass of eosinophils and other leukocytes.

By contrast, in jirds "milk spots" were absent at this 97

stage. Microscopically, a number of foci with eroded hepatic parenchyma were apparent with limited or absent cellular in­ volvement. Considerable hemorrhage in the eroded areas was visible. Another important feature of contrast with the guinea-pig was the absence of cellular involvement around the larvae (Plate 1, Fig. 24), which appeared uninhibited in the liver tissue. Among the dominants of the infiltrates, mono­ nuclear cells were found involved around aggregates of spilled and effete erythrocytes. Eosinophils could not be detected in the lesions, suggesting a very low grade antibody-antigen reaction at the site of infection.

On day 14, Parascaris infection in the lungs of both experimental hosts was almost at its peak. The lesions in guinea-pigs were predominantly characterized by intense cellular infiltration exhibiting hypersensitive reaction to the infection.

The participants in the cellular reaction could be differentiated into neutrophils, macrophages, lymphocytes and eosinophils.

Many cellular reaction foci were dominated by eosinophilic or mononuclear cell infiltrates. Relatively, few larvae were found in sections, all were trapped in cellular reaction.

Comparatively, the larval invasion in the lungs of jirds caused extensive tissue damage and hemorrhage. There was little in the way of cellular response as compared to guinea-pigs to resist the offending parasite. Most of the infiltrates participating in the reaction were mononuclear cells. Very few eosinophils could be detected among other infiltrates in jirds, suggesting lack of a hypersensitive response. 98

Synthesis

A discussion of the correlation between the findings already presented based on the cellular response in blood and sites of infection in tissues, larval yield and develop­ ment is important. In addition, the factors related to the manifestations of these findings should also be considered for an overall comparative assessment of the immune state of the host and degree of establishment of the parasite.

A delay in neutrophil increase in jirds as compared to guinea-pigs subsequent to Parascaris infection may indicate a defective response to a potent antigenic stimulus. Delayed neutrophilia has been reported by a number of workers in man, more recently by Miller (1975), who named it "Lazy Leukocyte

Syndrome.”

There seems to be a cause and effect relationship between neutrophil increase and their later recruitment into lesions. However, the two processes are operated by more or less different factors. Neutrophils are regarded by a number of workers to be mobilized in the granulopoietic tissue by colony stimulating factor, but their chemotaxis to the site of infection is performed most commonly by the complement.

William and Johnson (1976) undertook studies on leukocyte dis­ tribution, particularly of neutrophils in response to the local inflammatory stimuli in rats. On the basis of these studies, they concluded that the increase in neutrophil counts corresponded with the time of their migration to inflammatory 99

sites.

As mentioned earlier, neutrophils are the first among

leukocytes to arrive at the site of infection after an endo­

thelial injury. Their inability to emigrate in a normal and timely manner from the microcirculation and to participate actively in inflammatory reaction may lead to considerable risk of infection, even if all neutrophil functions appear to be intact (Senn, 1972).

During the course of larval migration of Parascaris equorum, new lesions are formed. In view of this neutrophils are expected to be found at least in fresh lesions. In micro­ scopic examination of lesions caused by the parasite in the liver of jirds, on both the 4th and 8th DOI the infiltrates were predominantly mononuclear cells consisting of macrophages and lymphocytes, and the cellular response in these lesions was not commensurate with the trauma and damage to hepatic parenchyma.

A lack of neutrophils in lesions caused by the parasite in the liver of jirds could be due to a very limited chemotaxis for neutrophils. The principal candidate for their chemotaxis is the complement, which is formed of a number of components.

According to Ward et al., (1965) chemotaxis could be limited in the absence of limited interaction between the components of the complement. Keller and Sorkin (1966) rpovided experi­ mental evidence that cytotaxins are cell-type specific. In the case of neutrophils, complement has been found to be a 100

more specific cytotaxin than other materials. It appears that

chemotaxis of mononuclear cells at the site of the lesion

was governed by cytotaxins other than the complement.

Monocytes and macrophages may substitute for granu­

locytes (neutrophils, eosinophils and basophils) in some

functions such as phagocytosis (Braunsteiner, 1962). Hence,

the behavior of monocytes and macrophages in the case of

agranulocytosis or a weak and inadequate chemotactic response

for neutrophils could be of importance. Ghani (1969), among

others claimed that both monocytes and lymphocytes became

transformed into macrophages, fibroblasts and even endothelial

cells. Taliafero (1970) demonstrated the development of

lymphocytes and monocytes into macrophages.

The rise in lymphocyte population in jirds on 2nd DOI

could have been a step to increase the migration of lymphocytes

to inflammatory sites. Lymphocytes, by transforming into macrophages, could possibly have carried out phagocytosis to

substitute for granulocytes.

Microscopic observations of the lesions in the lungs of jirds revealed a number of hemosiderin granules (Fig. 28,

Plate 5) in those areas where accumulation of erythrocytes had occured due to the hemorrhage. Hemosiderin is an iron containing substance resulting from the splitting up of hematin during the phagocytic digestion of effete erythrocytes.

In comparatively older lesions, especially in the lungs of jirds, an acute cellular response could be observed. It 101

has been suggested by Page and Good (1958) that the absence

of polymorphonuclear (PMN) cells (neutrophils, eosinophils and

basophils) reduce the intensity of non-allergic acute response.

But Willoughby and Giroud (1969) provided experimental evidence

based on histological studies that acute inflammatory reaction

can continue in PMN cell-depleted rats, and this acute response

occurs mostly by infiltration with mononuclear leukocytes.

As mentioned earlier, the cellular response was not adequate

or reciprocal to the magnitude of infection and damage caused

by the parasite in the.liver of infected jirds. In fresh

lesions in the lungs of jirds there also seemed to be inadequate

resaponse as compared to the tissue damage. In older lesions,

sufficient response could be observed to be considered an acute

cellular response.

In infected guinea-pigs the cellular response in the

liver and especially in the lungs was massive. The cellular

elements participating in the reactions were PMN cells, phago­

cytes and other mononuclear cells.

Since this response was exhibited after the first

exposure of the guinea-pig host to the parasite, it was essen­

tially an immediate type hypersensitive immune response.

According to Barbaro (.1972) the immediate type hypersensitive response includes all those reactions that are the result of various kinds of antibodies found in the circulation and body

fluid. After challenge with specific antigen, mast cells or basophils degranulate releasing histamines at the site of 102

infection. At the same time mast cells fix IgE antibody.

Both histamines and antibody-antigen complexes are a powerful

chemotactic influence on eosinophils. In the present studies the eosinophilic aggregates and associated basophilic granules

(Fig. 30, Plate 6) in the lungs of guinea-pigs should be viewed as a hypersensitive response to Parascar1s equorum infection.

This immediate type of immune response initiated by antibodies already present strongly suggests a considerable degree of innate immunity present in guinea-pigs for the parasite.

Despite the fact that significant changes in eosinophilia were observed in the peripheral blood, accumulation of eosino­ phils at the site of infection was almost absent in jirds.

Higashi and Chowdhury (1970) were unable to demonstrate significant in vitro adhesion of eosinophils to Wuchereria bancrofti larvae and the reaction was shown to be complement dependent. The eosinophilia in the peripheral blood of jirds could be considered a hypersensitive response. It is also possible that a delayed type of hypersensitive response

(cell-mediated immune response) mediated by T - lymphocytes was developing in jirds. This response, according to Herbert

(1975), takes a number of days to develop. In view of this, in a short-lived infection by Parascaris equorum in the two experimental hosts, this type of response was not observed at the site of infection. However, a repeated infection could induce such a type of delayed type hypersensitive response. 103

A delayed type of hypersensitive response might

have developed in guinea-pigs along with the immediate hyper­

sensitive response. The accentuation of cellular response

in guinea-pigs was demonstrated by Taffs (1965) when subjected

to repeated infections with Ascaris suum. According to a

number of workers this increased cellular response was mediated

by lymphocytes. Delayed hypersensitivity or cell-mediated

immunity is short lived. The presence of demonstrable anti­

bodies against Ascaris suum infection in the serum of rabbits

were prolonged up to 114 days by repeated infections. It

remained to be seen whether cell-mediated immunity plays a

significant role in conferring any degree of protection to

jirds after repeated infection with Parascaris equorum.

As mentioned earlier, the number of active larvae

recovered from the liver and lungs of jirds were much higher

than recovered from these visceral organs of guinea-pigs

(Table 8). Table 7 illustrates a comparison of the larval

growth rate in the two experimental hosts during the larval

development and migration in the liver and lungs. With the

advancing development and increase in size of the larvae in

the two animals, it became apparent that the range in size of

the larvae recovered at different intervals from jirds became

greater. Comparatively, in guinea-pigs the range in size of the larvae was much smaller, and remained more or less

stable throughout their development.

From the evidence gathered and discussed, it has 104

become obvious that guinea-pigs mounted a much higher cellular

response especially at the sites of infection. One of the

important components of the hypersensitive response observed

was eosinophil accumulation in the lesions and particularly

around the parasite (Fig. 31, Plate 7). This response, as

it appeared, was responsible for trapping, immobilizing and

finally eliminating the parasite. In jirds, as mentioned

earlier, the parasites found in tissue sections of both liver

and lungs (Fig. 21, Plate 1; Fig. 27, Plate 5) initiated no

cellular response to inhibit their development. It has been

demonstrated by Grove et al. (1977) that larval success of

Trichinella spiralis in muscles was doubled in eosinophil

depleted mice. Similarly, Mahmoud et al (1975) showed that eosinophils are an essnetial factor in the resistance to a challenge infection of Schistosoma mansoni in mice.

It may be possible that larvae which developed faster and elicited lower antigenic response were able to survive the immune response in guinea-pigs. On the other hand, it could have been to a certain degree a process of selection and elimination carried out by the host through a negative feedback. An immune response is part of an immunologically competent host’s negative feedback control to limit the over­ crowding of parasitic organisms in the body of the host.

This is important for the physiological well-being of the host in order to provide a more stable and balanced host- paraside relationship. This negative feedback may be beneficial 105

to the parasite in that through selection it is provided with

a gene pool which expresses itself in a more compatible host-

parasite relationship.

In general, higher suiceptibility and related low-

grade immune response for Parascaris equorum infection in

jirds as compared to guinea-pigs was evident from the observa­

tions made on the cellular response particularly at the sites

of infection. A significant rise in eosinophilia concomitant

with the development of infection indicates an immune response

to an antigenic stimulus. But microscopic observations made

on the lesions caused by the parasite showed an almost lack

of eosinophilic participation. At the same time, a much

greater degree of damage in the visceral organs indicated a

poor host-parasite relationship. This also indicated a greater

accessibility of the parasite to the host tissues, which

might be the reason for greater yield of the larvae from vis­

ceral organs of jirds (Table 8).

An analysis of the data obtained on the growth rates

(Table 7) among larvae at different stages of development

revealed the increasing variability towards the later stages

of development. A plausible explanation of this could be found

in the physiological state of the host under increasing para­

sitic stress. Those larvae possessing better growth potential

after showing a faster pace of development during the initial phase of the infection could have continued their growth to some degree even during the later stages of development. 106

But the increasing stress caused by the parasite in the host

could have significantly reduced or stopped the growth of

larvae already showing a lower growth potential. This explana­

tion further supports the earlier indication of a poor host-

parasite relationship between jird and Parascaris equorum.

In view of the foregoing considerations, it may be

postulated that the jird, as an ecotype of arid region,

has had no intereaction with ascarids in general and Parascaris

in particular. Dobson (1972) believes that the hypersensitive

response occurs in animals infected by "foreign" parasites as

contrasted to natural parasites. But, at the same time, he

also admits that any hypothesis concerning the evolutionary

adaptation of parasites to the antagonistic responses of the host must account for both elimination of the debilitatory effects of these reactions and the possible synergic effect they may also have on maintaining a balanced host-parasite relationship. His former statement could be contested on the grounds that a hypersensitive response to an immunogenic stimulus is prompted by the presence of antibodies against the invading organism. The presence of these antibodies strongly suggests a prior experience of the host in an evolu­ tionary sense with a particular parasite or a related one with which it shares antigenic cross-reactivity.

Sprent (1959) proposed that the host-parasite inter­ action evolves by a process of reciprocal adaptation where invading organisms survive because of a series of adaptations 107

to the host, which in turn responds by modifying its defense mechanisms. In the light of observations made on immune responses at the cellular level, guinea-pigs seem to show a better reciprocal adaptation for Parascaris in the sense that:

(1) A hypersensitive response is beneficial for the host for

limiting the infection of more extensive and damaging pro­ portions and (2) for the parasite the hypersensitive response in the visceral organs provides a source of natural selection.

Larvae showing a better genetic make-up for withstanding the immune response from the host are selected for and are pro­ pagated at a greater rate in subsequent generations. CHAPTER V

SUMMARY

Despite the importance of Parascaris equorum as visceral

larva migrans in man and other mammals, no well documented studies have been carried out to reveal aspects of its estab­

lishment, migration, immunity and pathology in the zoonotic hosts. In an effort to explore these aspects, two lab models, the guinea-pig (Cavia procellus) and the jird (Merionus un­ guiculatus ) were selected because of their divergent ecological and geographical distribution for a comparative assessment.

The two experimental hosts were infected once with embryonated eggs of Parascaris equorum according to their weight. The infective dose for both hosts was maintained at a level which would not subject the animal to excessive trauma and exaggerated symptoms of the disease. At the same time, the inocula were predetermined to be potent enough not to produce a sub-clinical syndrome either. In view of this, guinea-pigs were inoculated with 5,000 (+ 100) and jirds with

600 (+50) embryonated eggs.

Parascaris equorum infection in both experimental hosts lasted approximately 18 days. During this period ob­ servations on the yield of active larvae from the visceral organs (liver and lungs) of both hosts were determined.

108 109

The yield of active larvae from jirds was much more than

guinea-pigs. Studies were also undertaken to monitor the

comparative development of the larvae in the two hosts. It

was found that although the larvae in their final stages

achieved a greater increase in size in the guinea-pig host,

the range in size of the larvae recovered was greater in jirds.

An experimental design was followed for undertaking

hematological studies in order to observe: (1) changes in

total erythrocyte populations and related hematocrit (PCV)

and hemoglobin values in the two experimental hosts, (2) changes

in the total leukocytes and both total and relative (%)

leukocyte sub-populations, such as lymphocytes, neutrophils, eosinophils, monocytes and basophils. The data obtained on all of the hematological variables recorded were statistically analysed using two way analysis of variance for repeated measures and Bonferonni’s method of multiple comparisons.

A significant decrease in the erythrocyte count was observed in jirds, which was followed closely by a similar decrease in values obtained for the hematocrit (PCV) and hemoglobin percentage during the course of the infection.

In both experimental hosts, a rise in the total leukocyte count was observed during the course of infection.

However, in guinea-pigs, a noticeable decline (statistically insignificant) during the initial phase of the infection was accompanied by a significant shift in the differential re­ sulting in an increased neutrophilia. 110

By contrast, in jirds an elevation in the total leuko­

cyte population was accompanied by a rise in lymphocyte cells.

The build up in neutrophilia in jirds was late and achieved

significant proportions after 4th DOI. Comparatively, relative

neutrophilia in guinea-pigs became highly significant on 1st

DOI. There seemed to be a deficiency in some immune function

of the jird which inhibited its ability to mount an initial

and effective immune response to the endothelial injury caused

by the parasite.

In both animals, a rise in lymphocyte levels, was ob­

served in the final phase of the infection. There was a gradual

increase in eosinophils starting at 7th DOI in both experimental

hosts with peaks from 10th through 14th DOI. Eosinophilia

in guinea-pigs, however, achieved levels twice as high as compare«

to jirds. In helminthic infections, particularly involving

visceral migrations, eosinophilia is a characteristic and

considered as an indicator of allergic or hypersensitive

response.

The changes in monocyte population in both hosts were

transient and less delineated as compared to other leukocytes.

But in general, some significant build ups were observed during

the middle of the infection, especially in guinea-pigs.

After the initial fighting stage or "neutrophilic stage"

an increase in monocyte indicated the defense stage accompanied by migration of monocytes in to tissues to act as macrophages, the role of which has been discussed. Ill

A significant increase in basophil population in the

two hosts was observed during the final phase of infection.

This could have been due to decrease in antigenic byproducts

secreted or excreted by the developing parasite (during the

decline of infection) which act to degranulate the basophils.

Although significant changes were observed in the popu­

lations of both polymorphonuclear cells (PMN) i.e., neutrophils,

eosinophils and basophils, and mononuclear cells (MNC) i.e.,

lymphocytes and monocytes, in the circulating blood of the jird,

the cellular response at the sites of the lesions was limited,

mostly comprised of MNCs. Although there is a cause and effect

relationship between leukocyte mobilisation and the later

recruitment at the sites of infection, the two processes are

governed by different factors, some of which have been mentioned

In lesions caused by the parasite in the liver and lungs

of jirds MNCs were predominant, which may have substituted

for the phagocytic role of PMNs. Comparatively, in the guinea- pig liver both PMN and MNCs were present. Microscopic examina­ tion of guinea-pig lungs revealed a general hypersensitive response, accompanied by aggregates of eosinophils and degran­ ulated basophils indicating an immediate type of hypersensitive response.

Larvae found in histological preparations of both liver and lungs of jirds were devoid of any surrounding cellular response. Whereas in guinea-pigs larvae found in sections were trapped in a cellular response with the participation 112 of eosinophils among other infiltrates.

Extensive damage to hepatic parenchyma and hemorrhage were observed in jirds. Some lesions were also observed in the guinea-pig liver. A massive cellular response was noticed in the area eroded by migrating larvae in the guinea-pigs.

A limited cellular response was seen in the jirds. Some older lesions in guinea-pig liver showed the initiation of the repair process in the form of fibrotic infiltration.

Similarly, the lesions in the lungs of jirds were characterized by a rupture of alveolar septa and extensive hemorrhage. Comparatively in guinea-pigs less damage and more hypersensitive response was observed.

Consolidated evidence based on the larval yield and development in the visceral organs, cellular response in the circulating blood and especially at the sites of infection suggest a better host-parasite compatibility between guinea- pig and Parascaris equorum. From this both host and parasite benefit, the host by limiting the infection from more extensive proportions, and the parasite by a process of selection by the host. On the other hand, it seems that jird is too

"foreign" for the parasite, so much so that it has not evolved enough compatibility to effectively interact immunologically with the parasite despite its undoubted antigenicity. 113

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Result of 2 Way Analysis of Variance for Repeated Measures of Total Erythrocyte Count in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 0.27 0.617

Days Effect 6 23.87 0.000

Parasite X Days 6 19.11 0.000 Effect

Group No. 2 Parasite Effect 1 0.02 0.883

Days Effect 6 13.92 0,000

Parasite X Days 6 11.86 0.000 Effect

Group No . 3 Parasite Effect 1 7.95 0.023

Days Effect 6 0.86 0.528

Parasite X Days 6 1.30 0.275 Effect

Significant at p / 0.05 122 Result of 2 Way Analysis of Variance for Repeated Measures of Hematocrit (PCV) in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 0.33 0 . 581

Days Effect 6 20.98 0.000

Parasite X Days 6 17.43 0.000 Effect

Group No . 2 Parasite Effect 1 0 .00 0.977

Days Effect 6 18.05 0.000

Parasite X Days 6 12.88 0.000 Effect

Group No . 3 Parasite Effect 1 8.30 0.021

Days Effect 6 1.00 0.437

Parasite X Days 6 1.65 0.155 Effect

Significant at p <, 0.05 123

Result of 2 Way Analysis of Variance for Repeated Measures on Hemoglobin Values (%) in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No . 1 Parasite Effect 1 0.33 0.581

Days Effect 6 20.98 0.000

Parasite X Days 6 17.43 0.000 Effect

Group No . 2 Parasite Effect 1 0.00 0.977

Days Effect 6 18.05 0.000

Parasite X Days 6 12.88 0.000 Effect

Group No . 3 Parasite Effect 1 8.30 0.021

Days Effect 6 1.00 0.437

Parasite X Days 6 1.65 0.155 Effect

Significant at p ( 0.05 124 Result of 2 Way Analysis of Variance for Repeated Measures of Total Leukocyte Counts in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 10.25 0.013

Days Effect 6 8.21 0.000

Parasite X Days 6 7.94 0.000 Effect

Group No. 2 Parasite Effect 1 15.04 0.005

Days Effect 6 4.17 0.002

Parasite X Days 6 4.04 0.002 Effect

Group No. 3 Parasite Effect 1 50.58 0.000

Days Effect 6 8.21 0.000

Parasite X Days 6 10.95 0.000 Effect

Significant at p < 0.05 125 Result of 2 Way Analysis of Variance for Repeated Measures of Relative Lymphocyte Counts (%) in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 57.98 0.000

Days Effect 6 9.38 0.000

Parasite X Days 6 12.40 0.000 Effect

Group No. 2 Parasite Effect 1 3.74 0.089

Days Effect 6 2.26 0.053

Parasite X Days 6 3.12 0.011 Effect

Group No . 3 Parasite Effect 1 113.96 0.000

Days Effect 6 2 .16 0.000

Parasite X Days 6 1.69 0.144 Effect

Significant at p < 0.05 126 Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Lymphocyte Counts in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 11.63 0.009

Days Effect 6 3.68 0.004

Parasite X Days 6 4.69 0.001 Effect

Group No . 2 Parasite Effect 1 61.31 0.000

Days Effect 6 10.79 0.000

Parasite X Days 6 13.70 0.000 Effect

Group No . 3 Parasite Effect 1 11 .57 0.000

Days Effect 1 5 . 76 0.000

Parasite X Days 6 9.89 0.000 Effect

Significant at p < 0.05 12?

Result of 2 Way Analysis of Variance for Repeated Measures on Relative Neutrophil Counts (%) in Jirds

•Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 17.24 0.003

Days Effect 6 7.69 0.000

Parasite X Days 6 8.14 0.000 Effect

Group No. 2 Parasite Effect 1 11.37 0.010

Days Effect 6 5.12 0.000

Parasite X Days 6 8.84 0.000 Effect

Group No. 3 Parasite Effect 1 83.21 0.000

Days Effect 6 1.60 0.168

Parasite X Days 6 1.49 0.202 Effect

Significant at p < 0.05 128

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Neutrophil Count in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 17.52 0.003

Days Effect 6 6.16 0 . 000

Parasite X Days 6 6.58 0 .000 Effect

Group No . 2 Parasite Effect 1 18.28 0.003

Days Effect 6 7.70 0.000

Parasite X Days 6 8.59 0.000 Effect

Group No . 3 Parasite Effect 1 61.47 0.000

Days Effect 6 2.31 0.049

Parasite X Days 6 2.34 0.046 Effect

Significant at p < 0.05 129

Results of 2 Way Analysis of Variance for Repeated Measures of Relative Eosinophil Count (%) in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 31.61 0.000

Days Effect 6 5.48 0.000

Parasite X Days 6 5.62 0.000 Effect

Group No. 2 Paresite Effect 1 10.40 0.012

Days Effect 6 5. 26 0.000

Paresite X Days 6 5.10 0.000 Effect

Group No. 3 Parasite Effect 1 13.56 0.006

Days Effect 6 8.99 0.000

Parasite X Days 6 8 .32 0.000 Effect

Significant at p < 0.05 130

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Eosinophil Count in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 26 . 79 0.001

Days Effect 6 4.87 0.001

Parasite X Days 6 4.94 0.001 Effect

Group No. 2 Parasite Effect 1 7.92 0.023

Days Effect 6 4.09 0.002

Parasite X Days 6 4.05 0.002 Effect

Group No . 3 Parasite Effect 1 15.14 0 . 005

Days Effect 6 9.45 0.000

Parasite X Days 6 9.61 0.000 Effect

Significant at p < 0.05 131

Result of 2 Way Analysis of Variance for Repeated Measures of Relative Basophil Count (%) in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 7.02 0.029

Days Effect 6 0.98 0.449

Parasite X Days 6 1.90 0.100 Effect

Group No . 2 Parasite Effect 1 11.83 0.009

Days Effect 6 0.48 0.82

Parasite X Days 6 0.66 0.685 Effect

Group No. 3 Parasite Effect 1 4.89 0.58

Days Effect 6 2.47 0.036

Parasite X Days 6 4.03 0.002 Effect

Significant at p < 0.05 132

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Basophil Count in Jirds

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 14.46 0.005

Days Effect 6 1.04 0.413

Parasite X Days 6 1. 77 0.125 Effect

Group No. 2 Parasite Effect 1 9.32 0.016

Days Effect 6 0.69 0.656

Parasite X Days 6 0.80 0.578 Effect

Group No. 3 Parasite Effect 1 6.15 0.038

Days Effect 6 3.36 0.008

Parasite X Days 6 4.31 0.001 Effect

Significant at p 0.05 133

Result of 2 Way Analysis of Variance for Repeated Measures on Relative Monocyte Count (%) in Jirds

Source of Degtees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 5.20 0.052

Days Effect 1 2.80 0.020

Parasite X Days 6 3.78 0 . 004 Effect

Group No. 2 Parasite Effect 1 5.57 0 . 046

Days Effect 1 • 2.29 0.051

Parasite X Days 6 1.67 0.148 Effect

Group No . 3 Parasite Effect 1 4.57 0.065

Days Effect 1 2.43 0.040

Parasite X Days 6 1.81 0.118 Effect

Significant at p < 0.05 134 Result of 2 Way Analysis of Variance for Repeated Measures on Absolute Monocyte Count in Jirds

Source of Degrees of Sample Group Variât ion Freedom F P

Group No. 1 Parasite Effect 1 5.20 0.052

Days Effect 6 2.80 0.020

Parasite X Days 6 3.78 0.004 Effect

Group No. 2 Parasite Effect 1 5.57 0.046

Days Effect 6 2.29 0.051

Parasite X Days 6 1.67 0.148 Effect

Group No. 3 Parasite Effect 1 9.99 0.013

Days Effect 6 2.44 0.039

Parasite X Days 6 2.07 0.075 Effect

Significant at p < 0.05 135

Result of 2 Way Analysis of Variance for Repeated. Measures on Total Erythrocyte Count in Guinea-Pigs

Sample Group Source of Degrees of Variation Freedom F P

Group No. 1 Parasite Effect 1 0.04 0.838 Days Effect 6 3.28 0.009 Parasite X Days 6 3.53 0.006 Effect Group No. 2 Parasite Effect 1 0.43 0.528 Days Effect 6 7.73 0.000 Parasite X Days 6 5.67 0.000 Effect Group No. 3 Parasite Effect 1 2.60 0.145 Day Effect 6 6.16 0.000 Parasite X Days 6 6.54 0.000 Effect

Significant at p<0.05 136

Result of 2 Way Analysis of Variance for Repeated Measures of Hematocrit (PCV) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No . 1 Parasite Effect 1 0.00 0.968

Days Effect 6 3.11 0.012

Parasite X Days 6 4.29 0.002 Effect

Group No . 2 Parasite Effect 1 0.29 0.607

Days Effect 6 6.60 0.000

Parasite X Days 6 3.99 0.003 Effect

Group No. 3 Parasite Effect 1 2 . 73 0.137

Days Effect 6 7.00 0.000

Parasite X Days 6 4.85 0.001

Significant at p 4 0.05 137 Result of 2 Way Analysis of Variance for Repeated Measures of Relative Hemoglobin Values (%) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No . 1 Parasite Effect 1 0.00 0.968

Days Effect 6 3.11 0.012

Parasite X Days 6 4.29 0.002 Effect

Group No. 2 Parasite Effect 1 0.29 0.607

Days Effect 6 6.66 0.000

Parasite X Days 6 3.99 0.003 Effect

Group No. 3 Parasite Effect 1 2.73 0.173

Days Effect 6 7.00 0.000

Parasite X Days 6 4.85 0.001 Effect

Significant at p < 0.05 138 Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Leukocyte Count in Guinea-pigs

Source of Degrees of Sample Group Variat ion Freedom F P

Group No . 1 Parasite Effect 1 116.17 0.000

Days Effect 6 15.44 0.000

Parasite X Days 6 14.82 0.000 Effect

Group No . 2 Parasite Effect 1 112.86 0.000

Days Effect 6 26.99 0.000

Parasite X Days 6 26.87 0.000 Effect

Group No . 3 Parasite Effect 1 184.16 0.000

Days Effect 6 51. 78 0.000

Parasite X Days 6 52.60 0.000 Effect

Significant at p 4 0.05 139

Result of 2 Way Analysis of Variance for Repeated Measures of Relative Lymphocyte Count (%) in Guinea-pigs

Source of Degrees of Sample Group Variât ion Freedom F P

Group No . 1 Parasite Effect 1 65.89 0.000

Days Effect 6 18.75 0.000

Parasite X Days 6 13.87 0.000 Effect

Group No. 2 Parasite Effect 1 13.81 0.006

Days Effect 6 9.60 0.000

Parasite X Days 6 8 .05 0.000 Effect

Group No . 3 Parasite Effect 1 36.16 0.000

Days Effect 6 5.09 0.000

Parasite X Days 6 4.18 0.002 Effect

Significant at p < 0.005 140

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Lymphocyte Count in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 16.02 0.004

Days Effect 6 7 . 21 0.000

Parasite X Days 6 8.09 0.000 Effect

Group No. 2 Parasite Effect 1 20.77 0.002

Days Effect 6 15.33 0.000

Parasite X Days 6 16.72 0.000 Effect 6

Group No. 3 Parasite Effect 1 47.62 0.000

Days Effect 6 10.90 0.000

Parasite X Days 6 10.78 0.000 Effect

Significant at p 4 0.05 141

Result of 2 Way Analysis of Variance for Repeated- Measures of Absolute Neutrophil Count in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 65.51 0.000

Days Effect 6 9.12 0 .000

Parasite X Days 6 7 .75 0.000 Effect

Group No. 2 Parasite Effect 1 67.75 0.000

Days Effect 6 6.82 0.000

Parasite X Days 6 5.78 0.000 Effect

Group No . 3 Parasite Effect 1 47.35 0.000

Days Effect 6 12.05 0.000

Parasite X Days 6 10.98 0.000 Effect

Significant at p < 0.05 Result of 2 Way Analysis of Variance for Repeated Measures of Relative Neutrophil Count (%) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 28.96 0.000

Days Effect 6 12.31 0.000

Parasite X Days 6 10.71 0.000 Effect

Group No . 2 Parasite Effect 1 2.27 0.171

Days Effect 6 10.99 0.000

Parasite X Days 6 12 . 29 0.000 Effect

Group No . 3 Parasite Effect 1 1.19 0.307

Days Effect 6 8.91 0.000

Parasite X Days 6 7.32 0.000 Effect

Significant at p < 0.05 143

Result of 2 Way Analysis of Variance for Repeated Measures of Relative Eosinophil Count (%) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 14.90 0.005

Days Effect 6 13.83 0.000

Parasite X Days 6 11.04 0.000 Effect

Group No . 2 Parasite Effect 1 88.05 0.000

Days Effect 6 17.03 0.000

Parasite X Days 6 13.73 0.000 Effect

Group No. 3 Parasite Effect 1 34.23 0.000

Days Effect 6 9.41 0.000

Parasite X Days 6 7.36 0.000 Effect

Significant at p <0.05 144

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Eosinophil Count in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 32.89 0.000

Days Effect 6 16.13 0.000

Parasite X Days 6 14.71 0.000 Effect

Group No. 2 Parasite Effect 1 105.27 0.000

Days Effect 6 28.21 0.000

Parasite X Days 6 26.24 0.000 Effect

Group No . 3 Parasite Effect 1 46.68 0.000

Days Effect 6 13.67 0.000

Parasite X Days 6 12.38 0.000 Effect

Significant at p 0.05 145

Result of 2 Way Analysis of Variance for Repeated Measures of Relative Monocyte Count (%) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 5.62 0.045

Days Effect 6 1.39 0.239

Parasite X Days 6 1.35 0.255 Effect

Group No . 2 Parasite Effect 1 5.82 0 . 042

Days Effect 6 0 .57 0.752

Parasite X Days 6 1.58 0.174 Effect

Group No. 3 Parasite Effect 1 11 . 21 0.010

Days Effect 6 4.79 0.001

Parasite X Days 6 4.61 0.001 Effect

Significant at p 4 0.05 146

Result of 2 Way Analysis of Variance for Repeated- Measures of Absolute Monocyte Count in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No . 1 Parasite Effect 1 13.75 0.006

Days Effect 6 1.96 0.090

Parasite X Days 6 1.78 0.124 Effect

Group No. 2 Parasite Effect 1 14.32 0.005

Days Effect 6 2.76 0.022

Parasite X Days 6 3.52 0.006 Effect

Group No . 3 Parasite Effect 1 26.79 0.001

Days Effect 6 6.75 0.000

Parasite X Days 6 6.30 0.000 Effect

Significant at p A 0.05 147

Result of 2 Way Analysis of Variance for Repeated Measures of Absolute Basophil Count in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 73.28 0.000

Days Effect 6 5.51 0.000

Parasite X Days 6 5.51 0.000 Effect

Group No . 2 Parasite Effect 1 20.63 0.002

Days Effect 6 5.13 0.000

Parasite X Days 6 5.57 0.000 Effect

Group No . 3 Parasite Effect 1 277.94 0 . 000

Days Effect 6 6.09 0.000

Parasite X Days 6 8.28 0.000 Effect

Significant at p Z 0.05 148

Result of 2 Way Analysis of Variance for Repeated Measures of Relative Basophil Count (%) in Guinea-pigs

Source of Degrees of Sample Group Variation Freedom F P

Group No. 1 Parasite Effect 1 13.73 0.006

Days Effect 6 2.83 0.019

Parasite X Days 6 2.75 0.022 Effect

Group No. 2 Parasite Effect 1 17.23 0.003

Days Effect 6 2.57 0.031

Parasite X Days 6 3.48 0.006 Effect

Group No. 3 Parasite Effect 1 103.78 0.000

Days Effect 6 2.48 0.036

Parasite X Days 6 5.38 0 . 000 Effect

Significant at p 0.05