This dissertation has been 69-11,668 microfilmed exactly as received

MALIK, Prem Dutt, 1918- SOME IMMUNOLOGICAL AND OTHER STUDIES IN MICE ON INFECTION WITH EMBRYONATED EGGS OF (WERNER, 1782).

The Ohio State University, Ph.D., 1968 Agriculture, pathology Health Sciences, immunology

University Microfilms, Inc., Ann Arbor, Michigan SOME IMMUNOLOGICAL AND OTHER STUDIES IN MICE

ON INFECTION WITH EMBRYONATED EGGS OF

TOXOCARA CANIS (WERNER, 1782)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Prem Dutt Malik, L.V.P., B.V.Sc., M.Sc

******

The Ohio State University 1968

Approved by

Adviser / Department of Veterinary Parasitology ACKNOWLEDGMENTS

I wish to express my earnest thanks to my adviser, Dr. Fleetwood

R. Koutz, Professor and Chairman, Department of Veterinary Parasitology,

for planning a useful program of studies for me, and ably guiding my

research project to a successful conclusion. His wide and varied

experience in the field of Veterinary Parasitology came handy to me at

all times during the conduct of this study.

My grateful thanks are expressed to Dr. Harold F. Groves, for his

sustained interest in the progress of this work, and careful scrutiny of the manuscript. Thanks are extended to Dr. Walter G. Venzke, for making improvements in the manuscript. Dr. Marion W. Scothorn deserves my thanks for his wholehearted cooperation.

To Dr. Walter F. Loeb, I am really indebted for his valuable time

in taking pictures of the eggs, the larvae, and the spermatozoa of

Toxocara canis. The help of Mr. William A. Bruce, a graduate student

in acarology, in identifying and taking pictures of Myocoptes musculinus mites, is acknowledged. My thanks also go to my friend and fellow

student, Dr. Demetrice I. Lyles, for his valuable discussions and mutual help.

Lastly, I bow my head in solemn prayer for those cute, and lovely

little mice whom I layed to eternal rest to satisfy man's hunger for

the unknown. VITA

August 15, 1918 Born - Multan, India (now Pakistan)

1941 ...... L.V.P., Punjab Veterinary College, Lahore

1942-1957. . . . Veterinary Assistant Surgeon, Civil Veterinary Department, Punjab, India.

1952 ...... B.V.Sc., Punjab College of Veterinary Science and Animal Husbandry, Hissar, India.

1955 ...... Advanced training in rabies research, Central Research Institute, Kasuli, India.

1956 , ...... Post-graduate course in Veterinary Science, Indian Veterinary Research Institute, Mukteswar/Izatnagar, U.P., India.

1958-1960. . . . Research Assistant, College of Veterinary and Animal Science, Hissar, India.

1960-1965. . . . Assistant Disease Investigation Officer (Camels) Punjab, College of Veterinary and Animal Science, Hissar, India.

1966 ...... M.Sc., The Ohio State University, Columbus, Ohio.

PUBLICATIONS

"Amphistomiasis in Cattle in the Punjab." Journal of Animal Health, Calcutta, 1960.

"Incidence, Diagnosis, and Control of Immature Amphistomiasis in the Punjab." Conference on Parasites and Parasitic Diseases, Indian Council of Agricultural Research, 1961.

"Parasitic Gastro-enteritis in India with Particular Reference to the Main Pathogenic forms and Their Control." 11th All India Confer­ ence on Parasitic Diseases, Indian Council of Agricultural Research, 1964.

"Salmonella Serotypes from Camel in India." Journal of Research, P.A.U., Ludhiana, 1967.

iii VITA (continued)

Four Annual Technical Reports on "Normal Worm Burden, and Normal Fecal Egg Output Rate in Sheep, Goats, Cattle, and Buffaloes in the Punjab." Submitted to the Indian Council of Agricultural Research, New Delhi, 1957-1960.

Five Annual Technical Reports on "The Diseases of Camels in the Punjab. Submitted to the Indian Council of Agricultural Research, New Delhi 1960-1965.

FIELDS OF STUDY

Major Field: Veterinary Parasitology

Studies in Advanced Veterinary Parasitology. Professors Fleetwood R. Koutz and Harold F. Groves

Minor Problems in Veterinary Parasitology. Professors Fleetwood R. Koutz and Harold F. Groves

Studies in Principles of Infection and Resistance. Professor M. C. Dodd

Studies in Serology. Professor F. W. Chorpenning

Studies in Pathogenic Protozoology. Professor Julius P. Kreier

Studies in Veterinary Endocrinology. Professor Walter G. Venzke

Studies in Medical Entomology. Professor Carl E. Venard

Studies in the Prevention and Control of Diseases of Poultry. Professor Clyde A. Marsh

Minor Problem in Veterinary Clinical Laboratory Diagnosis. Professor Walter F. Loeb

Minor Problem in Zoology (Molluscs). Professor David H. Stansberry. TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iii

LIST OF T A B L E S ...... vii

LIST OF ILLUSTRATIONS ...... viii

INTRODUCTION ...... 1

Objectives...... 3

LITERATURE REVIEW ...... 5

Nomenclature ...... 5 Classification ...... 3 Historical review ...... 6 Life h i s t o r y ...... 8 Prenatal infection ...... 9 Migratory behavior ...... 9 Survival of larvae in host t i s s u e s ...... 11 Central nervous system involvement ...... 11 Morphology of second-stage larvae ...... 14

MATERIALS AND METH O D S ...... 16

Collection of e g g s ...... 16 Collection of eggs from fe c e s ...... 17 Collection of parasites from hosts ...... 17 Collection of eggs from parasites ...... 18 1. Maceration of the whole w o r m s ...... 18 2. Dissection of the u t e r i ...... 18 3. (a) Natural oviposition...... 20 (b) At incubator t e m p e r a t u r e ...... 21 (c) Maintaining female worms with male worms . . 21 (d) Maintaining worms in 1 per cent dextrose s o l u t i o n ...... 21 Experimental ...... 22 Preparation of inoculum ...... 32 Experimental procedure ...... 33 Plan of s t u d y ...... 41

v TABLE OF CONTENTS (Continued)

Page

RESULTS ...... 46

Longevity of larvae in tissues ...... 47 Lethal effects of Toxocara canis eggs ...... 56 Lethal doses of Toxocara canis eggs ...... 60 Challenge experiments ...... 65 Conclusions of challenge experiments ...... 70 Infectivity of 27-month-old Toxocara canis eggs .... 71 Lethal effects of 27-month-old Toxocara canis eggs . . . 73 Challenge experiments with 27-month-old Toxocara canis e g g s ...... 73 Treatment and control of murine mange ...... 76 Toxocara canis spermatozoa ...... 77

DISCUSSION...... 80

Criteria of immunity inhelminth i n f e c t i o n s ...... 80 Lethal doses ...... 82 Resistance to reinfection ...... 83 Prepatent period ...... 88 The effect of storage on viability of Toxocara canis e g g s ...... 90

SUMMARY ...... 91

LITERATURE CITED ...... 95

vi LIST OF TABLES

Table Page

1 The design of experiment to determine the lethal effect of different doses of 30-day-old embryonated eggs of Toxocara canis by oral intubation...... 42

2 The design of experiment for determining the minimum lethal dose (M.L.D.) and lethal dose 50 (L.D. 50) of Toxocara canis 30-day-old embryonated eggs for mice by oral intubation . . 43

3 The design of experiment to determine the minimum immuniz­ ing dose (M.I.D.) of Toxocara canis eggs for m i c e ...... 44

4 The design of experiment to test the infectivity of Toxocara canis eggs stored in a refrigerator for 27 m o n t h s ...... 44

5 The design of experiment to determine the lethal effects of ■27-month-old Toxocara canis eggs for m i c e ...... 45

6 Lethal effects of 30-day-old embryonated eggs of Toxocara canis for mice by oral i n t u b a t i o n ...... 57

7 Minimum lethal dose of 30-day-old embryonated eggs of Toxocara canis for mice by oral intubation...... 63

8 Lethal dose 50 (L.D. 50) of Toxocara canis eggs for mice by oral intubation...... 64

9 Results of challenge of surviving mice with 40,000 eggs of Toxocara canis four weeks after primary infection ...... 68

10 Results of challenging previously infected mice with 15,000 and 20,000 eggs of Toxocara canis three weeks after the primary infection ...... 69

11 Distribution of Toxocara canis larvae in white mice tissues infected orally with 27-month-old eggs stored in a refrigerator ...... 72

12 Lethal effects of 27-month-old embryonated eggs of Toxocara canis for mice by oral i n t u b a t i o n ...... 74

13 Results of challenging mice by oral intubation (Primary infection, 27-month-old eggs) with 30-day-old eggs of Toxocara canis. three weeks after the primary infection . . 75 vii LIST OF ILLUSTRATIONS

Figure Page

1 Toxocara canis eggs (X190), fertile, infertile, and abnormal e g g s ...... 19

2 tetraptera egg (X750)...... 24

3 Aspiculuris tetraptera egg (X500), showing a developing e m b r y o ...... 25

4 Myocoptes musculinus (X350, phase contrast), female ventral v i e w ...... 26

5 Myocoptes musculinus (X350, phase contrast), female dorsal v i e w ...... 27

6 Myocoptes musculinus (X350, phase contrast), male ventral v i e w ...... 28

7 Myocoptes musculinus (X350, phase contrast), male dorsal v i e w ...... 29

8 Myocoptes musculinus (X350, phase contrast), six-legged larva, ventral v i e w ...... 30

9 Myocoptes musculinus (X350, phase contrast), egg on hair showing polar c a p ...... 31

10 Toxocara canis larva (X190) ...... 37

11 Toxocara canis larva (X190), a freshly emerged larva alongside its empty s h e l l ...... 38

12 Toxocara canis larva (X500), anterior end ...... 39

13 Toxocara canis larva (X500), posterior end ...... 40

14 A ruptured Toxocara canis egg just before the emergence of larva ( X 7 5 0 ) ...... 48

15 A large immature Toxocara canis egg without the shell (X500)...... 49

16 Toxocara canis eggs and spermatozoa (X190)...... 50 viii Figure Page

17 Toxocara canis eggs and spermatozoa (X500) ...... 51

18 Toxocara canis spermatozoa (X190) ...... 52

19 Toxocara canis spermatozoa (X750) ...... 53

20 Toxocara canis spermatozoa (X750, phase contrast) . . 54

21 Toxocara canis sperm (X1900) ...... 55

22 Brain of a mouse showing petechial and ecchymotic hemorrhages, congestion of blood vessels and extra­ vasation of blood on the brain surface...... 61

23 A mouse showing petechial and ecchymotic hemorrhages on the subcutaneous surface of the s k i n ...... 62

24 Lethal dose 50 (L.D. 50) of 30-day-old embryonated eggs of Toxocara canis for mice by oral intubation . . 66

ix INTRODUCTION

Toxocara infections in dogs had received scant attention at the

hands of research workers until Beaver, et: ajL. (1952) determined its

larvae to be the etiological agents of a rather serious condition in

humans, which they called "". The condition had

been known for long before and attributed to various different causes

without a proper diagnosis and understanding. The infection in dogs

readily yields to treatment with any of the common , and

presents no serious menace to their health after they are six months

of age. Petrov (1941) and Nifontov (1949) reported that prenatal

infections of Toxocara canis in pups resulted in a large percentage of

still births and early mortality after birth. These and other findings

stimulated the interest of many workers in this parasite and its larval

phases. Sprent, Schacher, Nichols, Tiner, Webster, Oshima and several

other workers contributed a voluminous amount of information in regard

to the life history, migration pattern and distribution of larvae in

the tissues of normal and abnormal hosts, larval morphology and growth,

and standardization of technics.

Laboratory animals have been known to exhibit immunological

responses on infection with eggs. It has been generally observed that a previous infection induces resistance which influences

the migration and survival of the larvae. Stewart (1916) failed to

1 find any larvae in the lungs of a rat on a subsequent reinfection with heavier doses of eggs. Similarly, Yoshida (1919)

demonstrated the resistance of guinea pigs to reinfection with Ascaris

lumbricoides eggs after 27 and 42 days of initial infection. Wagner

(1933) cited by Taffs (1964) recovered fewer larvae of Ascaris lumbri­

coides from the liver and lungs of previously infected mice than from

those given an equal number of eggs for the first time. Kerr (1938a)

showed that single or multiple sublethal inoculations of eggs of pig ascaris rendered guinea pigs resistant to subsequently administered

lethal doses of infective eggs. Sprent and Chen (1949) observed the development of acquired immunity in mice on exposure to sublethal infection with the eggs of Ascaris lumbricoides. This was demonstrated in retarded growth and migration of the larvae which remained for longer periods in the liver before migrating to the lungs on a subsequent rein­ fection. Similar observations on acquired immunity in guinea pigs on infection with ascaris eggs have been recorded by Fallis (1942, 1944,

1948).

In order to study the immune reactions of a host, it is essential first to determine a lethal dose of the infection for the host. This enables one to work out a safe sublethal dose which will stimulate the defensive mechanism of the host without killing him. Lee (1960) found that a single dose of 2000 Toxocara canis eggs killed three mice out of ten, with typical symptoms of verminous pneumonia 7, 10, and 15 days after inoculation. Stewart (1916) also reported death of one mouse out of four on the day following infection with Ascaris marginata (Toxocara canis). and recovered larvae from the liver. On the other hand, Sprent (1952a and 1953a) infected mice with a dose of 2500 Toxocara canis eggs

each and observed them for one to six months without any one dying.

Again Sprent (1958) used a dose of 5000 Toxocara canis eggs to infect

14 mice which were killed 1-14 days after infection, without any dying

as a result of the infection. Nichols (1956a) infected mice with doses

of Toxocara canis eggs varying from 200 to 12,000 but reported no mor­

tality as such. Tiner (1951) observed that as many as 100 Toxocara canis larvae may remain alive in a mouse brain for 90 days without pro­ ducing any noticeable symptoms in the animal. Scothorn (1963) recovered

111 and 105 larvae of Toxocara canis from the brains of two mice on the

seventh and eighth days, respectively, after infection but no symptoms were exhibited by these animals. He, however, noticed central nervous

system involvement with resultant paralysis in three mice, which had been infected for 66 days.

In view of such conflicting claims by different workers, it became necessary to decide this issue first before proceeding further with other aspects of immunological studies in this direction. The follow­ ing objectives and experiments were designed for the purpose:

Objectives:

1. To study the lethal effect of embryonated eggs of

Toxocara canis in mice by oral administration direct

into the esophagus.

2. To determine the minimum lethal dose (M.L.D.) of

Toxocara canis eggs for mice. 3. To determine the lethal dose 50 (L.D. 50), that is, a dose

of Toxocara canis eggs which will kill 50 per cent of the

mice under experiment.

4. To determine the prepatent (incubation) period, that is,

the period between administration of infection and mani­

festation of clinical signs, and to study the factors

influencing this period.

5. To study the clinical signs and macroscopic lesions produced

in mice as a result of infection with varying doses of

Toxocara canis eggs.

6. To study if the sublethal infection or infections of Toxocara

canis in mice resulted in the development of resistance to

subsequent infection and the nature of such a resistance.

7. To determine the minimum immunizing dose (M.I.D.), that is,

a minimum dose of Toxocara canis eggs which will confer

protective immunity on mice.

8. To determine the immunizing dose 50 (I.D. 50), that is, a

dose of Toxocara canis eggs which will confer protective

immunity on 50 per cent of the mice under experiment. LITERATURE REVIEW

Nomenclature:

Name: Toxocara canis (Werner, 1782) Johnston, 1916.

First described by Werner in 1782 as Lumbricus canis. it has

since been identified and named differently by different workers.

Many synonyms of it are, therefore, known which are enumerated below.

Johnston in 1916 classified it as Toxocara canis (Werner, 1782), which name is current today.

Synonyms: Lumbricus canis Werner, 1782; Ascaris canis (Werner,

1782) Gmelin, 1790; Ascaris werneri Rudolphi. 1793; Ascaris marginata

Rudolphi, 1802; Belascaris marginata (Rudolphi, 1802); Ascaris mystax canis (Werner, 1782) Blanchard, 1888, Railliet, 1893; Toxascaris

limbata Railliet and Henry, 1901; Toxascaris marginata Leiper. 1907;

Toxascaris canis (Werner, 1782) Castellani and Chalmers, 1913; Belas­ caris canis (Werner, 1782) Garin, 1913.

Classification:

Several classifications of the nematoda have been made from time to time by various authorities including Baylis and Daubney (1926),

Stiles and Hassal (1926), Yorke and Maplestone (1926), Chitwood and

Chitwood (1950), Morgan and Hawkins (1953), Mozgovoi (1953), Whitlock

(1960), and Yamaguti (1961). All of these authorities based their classification of the Toxocara canis on the structure of the esophagus, the anterior end of the intestines, and a few other salient features.

Hartwich (1957), however, formulated his classification on the basis of the excretory system. The classification enunciated by Chitwood and Chitwood (1950), and Morgan and Hawkins (1953) has been adopted in this paper:

P h y l u m ...... Nemathe lminthe s

C l a s s ...... Nematoda

Subclass ...... Phasmidia

O r d e r ......

Suborder ...... Ascaridina

Superfamily...... Ascaridoidea

F a m i l y ......

Subfamily ...... Anisakinae

G e n u s ...... Toxocara

Species ...... Toxocara canis (Werner, 1782)

Historical Review

A survey of literature has revealed that relatively little work has been done so far with regard to the study of immune reactions of either the normal or the abnormal host to an invasion by Toxocara canis eggs, except that the lesser incidence in adult dogs has been surmised as an evidence of immunity. In a survey of 1,465 dogs, Ehrenford (1956) reported an overall incidence of 21 per cent for Toxocara canis. Ana­ lyzing his data of 1,324 dogs on the basis of sex, he reported an incidence of 32.8 per cent for males and 9.4 per cent for females.

Further analysis of his data for 1,294 dogs on age and sex basis, revealed that puppies of both sexes had a high incidence of infection; male dogs had a significantly higher incidence than the females. The males showed no evidence of immunity up to 36 months of age, while female dogs exhibited a marked and increasing immunity from 6-36 months of age. However, a vast amount of literature is available on the immu­ nity studies against other and cestode infections of man and animals, such as, Ascaris lumbricoides, , Ancylo- stoma caninum, Trichuris vulpis, , viviparus. , hydatigena, Taenia pisiformis,

Taenia ovis. and several other parasites. The subject of immunity and immune responses of hosts to metazoan parasites is both a fascinating and a challenging one. Some attempts have met with a promising success while others are being pursued and some are yet in exploratory stages.

The subject still remains open and attractive in view of the present- day concepts of disease control on a mass prophylactic scale.

As early as 1916 Kolmer, et al. demonstrated, on the basis of a series of complement fixation tests, the presence of antibodies in the sera of dogs infected with Toxocara canis. Taenia serrata, Diplydium caninum, and Trichuris vulpis. They also stated that the production of antibodies was especially in evidence in with tapeworms

Taenia serrata and Diplydium caninum; to a less degree with ascarids,

Toxocara canis; and to a still lesser degree with the whipworms,

Trichuris vulpis. They also observed group reactions between the two species of tapeworms and heterologous reactions to a lesser extent between widely divergent species. Sadun, et al. (1957) in their efforts to develop a serological test for the laboratory diagnosis of visceral larva migrans, found evidence of the production of specific antibodies following active infection or artificial immunization of rabbits with Toxocara canis. Again Ivey (1965), and Ivey and Slanga

(1965) reported existence of specific and heterologous antibody-antigen

systems between Toxocara canis. Ascaris lumbricoides. and Trichine11a

spiralis.

Life History

Yokogawa (1923), and Wright (1935), cited by Webster (1958b), carried out early investigations into the life history of Toxocara canis. The similarity between the migration of Toxocara canis and

Ascaris lumbricoides through the liver and lungs was first reported by

Ransom and Foster (1917), and Stewart (1918). The migration of ascaris larvae through the somatic tissues of laboratory animals, mice, guinea pigs, and rabbits was studied by Ransom and Cram (1921). They reported no apparent difference between the migration of these larvae through the laboratory animals and the definitive hosts, except that in the laboratory animals the larvae were unable to complete their life cycles after returning to the intestine, and passed out with the feces. Later

Sprent (1952a,b; 1957, 1958), Tiner (1953), Nichols (1956a,b), Schacher

(1957), Oshima (1961), and several other workers investigated the dif­ ferent phases of the life cycle of Toxocara canis. Prenatal Infection

A large majority of stillbirths, and early mortality in young dogs and fox pups was determined by Petrov (1941) and Nifontov (1949) to be the result of their prenatal infection with Toxocara canis larvae. Shillinger and Cram, earlier in 1923, thought prenatal infec­ tion to be a probable contributing factor in such cases but considered' weather and other physical conditions to be more unfavorable to young life at that age.

Adult bitches free from intestinal infections and isolated from any contact with infective eggs during gestation, still produced infected litters, Yutuc (1949), Scothorn, et al. (1965). The encapsu­ lated larvae in the somatic tissues of the bitches were presumably responsible for the prenatal infections, Yutuc (loc. cit.), Koutz, et al. (1966). But the actual mechanism of the release of encapsulated larvae from the host tissues still remains a matter of speculation.

Yutuc (loc. cit.) thought the debilitating effect of pregnancy and lowered resistance to be responsible for this phenomena, while Webster

(1958b) attributed it to hormonal changes during pregnancy.

Migratory Behavior

Three types of migratory behavior - the tracheal, the somatic, and the nonmigratory, by ascarid larvae in mice tissues have been recognized and studied by Fulleborn (1929), Hoeppli, et a_l. (1949),

Sprent (1952a,b; 1953a,b; 1954, 1955a,b; 1958), Beaver, et al. (1952),

Smith and Beaver (1953), Tiner (1953), Noda (1961), Oshima (1961), and

Olson (1962). Ascaris lumbricoides. and larvae follow the tracheal route of migration. Here the larvae hatch in the

intestine, pass through the liver, and lungs, and return to the intes­

tines via trachea, pharynx, and esophagus. They eventually disappear

from the intestines with the feces or otherwise. In the somatic type

of migration, the larvae having passed the liver and lungs, are carried

by the blood stream to various somatic tissues, where they encapsulate

and survive for long periods. Toxocara canis, Ascaris columnaris and

Ascaris mustelarum (now Ascaris devosi) belong to the somatic type of

migration. Oshima (1961), however, observed that although most of the

larvae of Toxocara canis followed the somatic route, some of them fol­

lowed the tracheal route, returned to the intestine and re-entered the

tissues. A second wave of larval invasion of the tissues was observed

by him. Malik (1966) also reported seeing larvae in the intestinal

contents a second time after their passage through liver and lungs,

but he did not observe a second invasion of the tissues by these larvae.

Sprent (1951) reported that in the dog - the definitive host,

Toxocara canis larvae, followed both tracheal and somatic routes, pre­

dominantly tracheal in young pups below three to six weeks of age, and mostly somatic in older dogs above six months old.

Toxascaris leonina, and Toxascaris transfuga follow the third

type of nonmigratory behavior, in which the larvae are encapsulated mostly in the intestinal wall without any further migration. 11

Survival of Larvae in Host Tissues

Live and active larvae of the dog ascarid were recovered, from the liver of white mice for at least six weeks after infection, by

Hoeppli, et al. (1949). The encapsulated larvae were present after two months. They observed that a majority of the larvae were encapsulated in three to four weeks but some were still free in tissues for six weeks or more. Webster (1958b) noticed that Toxocara canis larvae sur­ vived for at least six months in the somatic tissues of old dogs as second stage larvae. Beaver (1956) remarked that a high percentage of larvae persisted and retained infectivity for, at least, two years in mice, rats, guinea pigs, and rabbits. The larvae were eventually encap­ sulated in organs other than the brain. Sprent (1953a) recovered living larvae of Toxocara canis from mouse brain up to six months after experi­ mental infection. The present writer recovered living and active larvae from the brain of a mouse that died about 14 months after infection, while a mixed population of living and dead larvae were recovered from the muscle tissues of the same mouse.

Central Nervous System Involvement

Ascaris larvae have been observed by many workers in the central nervous system with or without any manifestation of nervous symptoms.

Beaver, et al. (1952) found active larvae of Toxocara canis in the pressed preparations of cerebellum as well as entire mouse brain up to six months after infection. Smith and Beaver (1953) observed a higher concentration of Toxocara canis larvae in the brain and muscles of mice after one to six months of infection. They recovered 152 larvae; 120 from the brain, 17 from the liver, and 15 from the carcass of a mouse

killed 10 months after infection. No gross lesions were seen by them

in the brain and spinal cord of another mouse killed one year after

infection, although it had been paralyzed for two weeks before death.

Microscopically, no striking lesions were noted except for a few

scattered foci of recent and earlier hemorrhage manifested by hemosid­

erosis and perivascular infiltration. Beautyman and Wolf (1951) found

a nematode larva, apparently a toxocara, from a macroscopic lesion in

a child's brain, Beaver (1954) attributed the death of a child to an

overwhelming invasion of many organs, including heart and brain. He

further observed that the individual larvae may produce extraordinary

damage if specific tissues, such as nerve centers of sense organs, were

invaded. He also remarked that ascarid larvae, probably Toxocara canis

had been reported twice from human brain. Blindness and chronic endoph

thalmitis in human beings, as a result of Toxocara canis larvae, have

also been reported by many workers, including Ashton (1960), Duguid

(1961a,b), Rey (1962), and Unsworth, et al. (1965). Rubin and Saunders

(1965) reported four cases of intraocular larva migrans due to Toxocara

canis larvae in dogs — the definitive host.

Tiner (1949, 1951) reported central nervous system damage in

rodents, invariably followed by death due to Ascaris columnaris larvae.

He, however, corroborated the earlier work of Fulleborn in affirming

that no brain damage was produced by Toxocara canis infections in mice

despite a larger number of larvae being present there. He observed,

"... as many as 100 Toxocara canis larvae may remain alive in a mouse brain 90 days without producing any noticeable symptoms in the animal. 13

Growth rate and size maxima correspond to the damage produced in the brain." Beaver (1956) observed "... whether these damaging effects are due to trauma produced by the greater size of the larvae, to toxic­ ity of their metabolic products or to other factors has not been satis­ factorily answered, so far."

Tiner (1953) described the symptoms of brain impairment in rodents infected with Ascaris columnaris larvae as frequently slight unsteadi­ ness, trembling, slowness or lameness. They were followed by more pronounced symptoms of circling, spinning, rolling, paralysis, blindness and coma. The death resulted from starvation and dehydration. Similar symptoms in mice were described by Sprent (1955b) in 12 out of 32 infec­ tions with Ascaris columnaris; in two out of 20 infections with Ascaris devosi; in one out of 23 infections with Toxocara canis. He, however, stated that the symptoms slightly varied in different mice. Scothorn

(1963) observed nervous symptoms, indicative of central nervous system derangement, in three mice after 66 days of infection with Toxocara canis larvae. Malik (1966) did not observe any paralytic or nervous signs up to 63 days after infection of mice with 1,000 Toxocara canis eggs, although about 60 to 80 larvae were found in the brain at various times.

Sprent (1955a) was in accord with Tiner (1951) when he remarked that probably the traumatic effect of in the central nervous system was directly proportional to the size and activity of the para­ site. The relatively small larvae of Toxocara canis may wander in the brain without causing sufficient damage to provoke symptoms. He also observed that the larvae reaching the brain produced characteristic 14 hemorrhages on the surface of the cerebral hemispheres in the early stages of infection. He further concluded that the larvae reached the brain via the arterial blood stream, left the arteries at the point where their diameter approximated that of the artery. This was mostly on the surface of the brain. Thereafter the larvae penetrated the brain tissue from the subarachnoid space. Larvae were recovered from the brain of only two out of 12 dogs fed 3000-5000 Toxocara canis eggs while no larvae were observed by him in the brains of naturally infected dogs.

Morphology of Second-Stage Larvae

Alicata (1934) first observed a molt of the larva within the egg of Toxocara canis and correlated it with its infectivity. The morphol­ ogy and diagnostic features of second-stage larvae of Toxocara canis which distinguish it from other larvae of Toxocara cati, Ascaris lumbricoides. americanus, stercoralis. and

Ancylostoma caninum were studied at length by Nichols (1956a,b). He determined that the width at mid-gut level, rather than the length of

Toxocara canis larvae, was the characteristic feature distinguishing it from Toxocara cati larvae. The larvae of the other species men­ tioned above could be readily distinguished, according to him, from those of Toxocara canis by specific features of the presence or absence, and size of lateral alae and posterior excretory columns, the type of the intestine, and the relative diameter of the body. 15

Schacher (1957) described the morphology of five developmental stages of Toxocara canis larvae. He found no evidence of another molt or any further development except for a slight increase in the size of the secretory cell in the sections of encapsulated larvae in the older dogs' tissues. Sprent (1958), in similar studies, confirmed the above findings of Nichols (1956a,b), and Schacher (loc. cit.). MATERIALS AND METHODS

Collection of Eggs

Before embarking on any research project requiring the use of

large numbers of Toxocara canis eggs, one has first to ensure a regu­

lar supply of clean, viable and sufficient numbers of eggs for his

experiments and enough to spare for an emergency. The steps detailed

below are involved in arranging adequate numbers of these eggs:

1. To locate a source or place where large numbers of

young pups are regularly housed.

2. The collection of eggs from the feces of the

naturally-infected pups.

3. The manner of collection of parasites from the pups.

4. The mode of obtaining eggs from the parasites.

Toxocara canis is generally a parasite of young pups below the

age of six months, with an incidence' decreasing with age in older dogs

(Webster, 1958a,b). The best places to find young pups in large num­ bers are either the clinic of a veterinary college or a dog dealer's premises. The latter is by far the most suitable place, particularly

if the dealer is contracting with dog pounds in the area for their

regular clearance. Another good source may be the kennels of humani­

tarian societies, but it is rather difficult, nay impossible, to deal with those good and kind-natured people, with whom any form of research on animals tantamounts to cruelty. 16 17

Collection of Eggs From Feces

The feces of the naturally-infected dogs with pure Toxocara canis

infections were processed for collection of eggs. Various methods have been employed for the purpose by different workers. Tiner (1953) harvested Ascarid lumbricoides eggs from the fecal suspensions by sedi­ mentation and flotation on saturated sodium chloride solution. Nichols

(1956a) used a combined sedimentation-zinc sulfate flotation technic to " concentrate Toxocara canis eggs from the feces of naturally-infected dogs. In the present study sodium nitrate solution as described by

Koutz (1941) was employed to obtain Toxocara canis eggs from the feces of naturally-infected dogs. It was found that the recovery of eggs from the feces of dogs entailed a very cumbersome and time-consuming process. In spite of the best efforts in cleaning the eggs several times, the fecal debris could not be completely eliminated.

Collection of Parasites From Hosts

The parasites were collected from pups either by deworming them while alive or collecting them at post-mortem soon after death. The dogs were killed either by intravenous injection of nembutal in our laboratory, or by euthanising them in a gas chamber at the dealer's premises. Where deworming had to be done in live dogs, as in the veterinary clinic, one of the various piperazine formulations was used.

The worms were washed several times in tepid water soon after collec­ tion to free them of any fecal matter or other debris, and then placed in physiological saline solution. Fully mature, young, female worms were found to be good layers of eggs, while immature and senile worms 18 were either poor layers or layed a majority of infertile, deformed and

abnormal eggs (Fig. 1). The ideal age of pups for getting young mature worms was found to be six to eight weeks and even up to twelve weeks. Pups older than this usually harbored a majority of old worms.

Collection of Eggs From Parasites

The collection of ascarid eggs from the uteri of worms has been

reported by Sprent (1952a), Tiner (1953), Nichols (1956b), Noda (1961),

Oshima (1961) and numerous other workers. In order to find a conve­ nient and more suitable method for obtaining clean, and viable Toxocara

canis eggs, different methods, described below, were tried for this

purpose.

1. Maceration of the whole worms.- The worms were cut into small pieces about an inch long and ground in a Waring blendor for 30 seconds at slow speed. The suspension was at first strained through a double

layer of cheesecloth to remove heavy, coarse debris. It was then

strained through a series of sieves of various gradations, and finally collected in beakers. Three or four washings with physiological saline

solution were given daily and the eggs were incubated at room tempera­ ture for 30 days. A large amount of debris still remained at the end which made the egg suspension unsuitable to use when large doses of eggs were to be administered to small laboratory animals.

2. Dissection of the uteri.- The worms were split open along their long axis. The uteri were freed and removed from the intestinal coils, and macerated into a beaker over a fine sieve of 100 meshes to an inch. The eggs thus obtained were reasonably clean but a fair Fig. 1. Toxocara canis eggs (X190).

a. A fertile egg with larva

b. An infertile egg

c. An abnormal egg 20 proportion of infertile and immature eggs was present in such cultures, because the uteri naturally contain eggs in various stages of develop­ ment. The dissection of the worms and separation of the uteri from the intestines was a difficult task. Several times either the uteri or the intestines broke while handling. The rate of embryonation in such cultures was found to be about 70 to 80 per cent.

3. Natural oviposition.- (a) The worms, after thorough cleaning in tepid water were finally washed in physiological saline solution before being distributed in petri dishes. Nonwettable plastic petri dishes were used because of the minimal sticking of eggs to their bottoms, and ease in daily collection of eggs. The female worms were placed two or three to a petri dish containing physiological saline and kept at room temperature for a period of 10-15 days. The eggs layed naturally by the worms in each petri dish were collected daily and fresh saline solution was replaced after every collection. The worms were found to lay viable eggs for five to six days, after which infertile and improperly formed eggs (Fig. 1) began to appear along with some debris which increased gradually thereafter. The worms remained alive for 10-14 days usually, although one worm lived for 20 days and continued to lay eggs up to the 18th day. The first week's collection was maintained separately from the second week's collection.

The cultures were incubated for 30 days at room temperature in physio­ logical saline solution containing 1 per cent formalin to check any bacterial or mycotic infection. The cultures were washed weekly with physiological saline containing 1 per cent formalin. The embryonation 21

rate of the first week's cultures was 90-93 per cent, while that of

the second week's cultures was anywhere from 60-70 per cent.

(b) At incubator temperature.- Six petri dishes with three female

Toxocara canis worms in each in physiological saline solution were kept

in the incubator. The worms soon became loose and flaccid, lost their

tenacity and disintegrated in three to four days without laying many

eggs. This method of maintaining worms was not found suitable and was

discarded.

(c) Maintaining female worms with male worms.- One male worm to

two female worms each were kept in six petri dishes, while other petri dishes contained only three female worms to each as usual. The petri dishes were covered with a double layer of thick brown paper all the time to simulate the darkness in the intestines. This was done in the

supposition that the presence of the male with the females may provide a stimulus to the females to lay more eggs or copulation may take place between them in the petri dishes, with a consequent greater percentage of fertile eggs in the resultant cultures. But no such effect was observed and the male worms rather added their debris to the cultures instead of increasing egg numbers. Hence, this practice was not adopted for general use.

(d) Maintaining worms in 1 per cent dextrose solution.- Dextrose

1 per cent was added to six petri dishes containing three female worms each in physiological salt solution. It was believed that it might provide a source of energy to the worms, and enhance their longevity and laying capacity. It was found, on the other hand, that the dextrose 22

provided a favorable medium for extraneous bacterial infections which

killed the worms early. The internal organs of some of the worms were

all devoured and dissolved in three to four days leaving just the

transparent skeleton sheath of the worms. This process was thus not

found suitable for maintaining worms.

The 27-month-old eggs used in Experiments 5 and 6 were collected

on June 18, 1966, and used on September 26, 1968. They were collected

and processed for embryonation as described above under natural ovipo-

sition (a). They were then stored in a refrigerator all this time,

and physiological saline solution with 1 per cent formalin was changed

every month.

Experimental Animals

White Swiss mice, seven to eight weeks old, and weighing 25-35

gms., were employed for experimental purposes throughout this study.

The mice were raised in our laboratory because of the previous experi­

ence of difficulty in procuring parasite-free mice from laboratory animal breeding establishments, as and when required. Initially 24 mice, eight males and 16 females, four to five weeks old, were obtained on July 20, 1967, from Harlan Industries, Inc., Cumberland, Indiana, for starting our mouse colony. The mice were distributed three to a cage usually; the males being housed separately from the females. The fecal examination of the mice on arrival in our laboratory revealed infection with mouse pinworms (oxyurids) Aspiculuris tetraptera and

Syphacia obvelata. The infection was treated with thiabendazole 23

administration. Thereafter weekly fecal examination of all experi­

mental mice during the course of this study was regularly carried out.

The pictures of the eggs of Aspiculuris tetraptera are exhibited in

Figures 2 and 3.

One of the male mice accidentally died in a cage during the night

between July 28 and July 29, 1967, just eight days after its receipt in

our laboratory. Large numbers of mites could be seen with the naked

eye all over its hair coat in the morning. The microscopic examination

revealed the mites as Myocoptes musculinus. and the diagnosis was con­

firmed in the Acarology Laboratory at The Ohio State University. The

pictures of the mites taken at the time are presented in Figures 4-9.

The treatment of all mice was undertaken with various acaricidal formu­

lations, which results are presented in the results section.

For breeding purposes two females and one male mouse, age seven

to eight weeks, were confined in a cage for 10-14 days, after which the

male was removed. The females were separated from each other just a

few days before parturition. Baby mice were weaned at three weeks of

age, sexed and placed three to a cage in separate cages.

The mice were fed Purina mouse breeder chow, which was partially

sterilized by exposing it to live steam in an autoclave for one minute

just before feeding, to kill any parasite eggs or larvae. Clean, fresh water in sterile bottles was provided. Sterile litter of corn cobs

formed the bedding for the mice in the cages and was changed every week. %

26

Fig. 4. Myocoptes musculinus (X350, Phase contrast),

female ventral view, showing an egg inside. Fig. 5. Myocoptes musculinus (X350, phase contrast),

female dorsal view, showing an egg inside. Fig. 6. Myocoptes musculinus (X350, phase contrast),

male ventral view. Fig. 7. Myocoptes musculinus (X350, phase contrast),

male dorsal view. Fig. 8. Myocoptes musculinus (X350, phase contrast),

six legged larva, ventral view. 31

■ RH 1■

Fig. 9. Myocoptes musculinus egg on hair showing

polar cap (X350,phase contrast). 32

The mouse cages with ventilation slits in the lids at the top

were found unsuitable. The wild mice in search of food would run over

the tops, dropping their feces and urine into the cages, which can be

a source of extraneous infections to the experimental animals. Some­

times a whole experiment may be ruined because of the stress produced

by extraneous concurrent infections in the experimental animals. It

is believed that the second attack of mites infection in our mice was

brought in this way, because once a wild mouse was trapped which was

covered with the mites of Myocoptes musculinus species. Thereafter,

the feed pellets were always kept inside the cages to minimize the

chances of wild mice getting on the cages. It is thought that cages

with ventilation slits in the side walls near the top may prove better

than those with ventilation slits on the top.

Preparation of Inoculum

The eggs stored in the refrigerator in 1 per cent formal saline

were given six washings with physiological saline to remove all traces

of formalin two days before the scheduled infection day for an experi­ ment. The washings were given in the refrigerator itself with refrig­

erated saline to avoid frequent variations in temperature of the larvae within the eggs. On the day before infection day, the washed eggs were

transferred to a number of 50 ml. centrifuge tubes depending upon the

estimated number of eggs in each beaker. The suspension was allowed to

settle for 15 to 20 minutes before centrifugation to avoid a heavy

sticking of eggs to the tube walls, which usually happened if centri­

fuged immediately. In the latter stages plastic centrifuge tubes with 33

nonwettable surface were used for the purpose because of a very minimum

sticking of eggs to their sides. After centrifugation the supernatant

in excess of 10 ml. was withdrawn. The count of eggs in the remaining

10 ml. of the fluid in each tube was made by gently stirring and simul­

taneously withdrawing 0.02 ml. of an even suspension of eggs at one

time. Hamilton microliter syringes were used for the purpose. An

average of six such counts of viable eggs only, containing actively

motile larvae, was taken and multiplied by 500 to give the total number

of eggs in each tube. The suspension was centrifuged again and stored

in a refrigerator for use the next day.

The tubes containing counted eggs were withdrawn at the time of

inoculation. The concentration of the eggs in the suspension was so

balanced as to contain the required number of eggs in not more than 1.0 ml. of the inoculum, when the eggs had to be administered by intubation,

and in 0.5 ml. when subcutaneous inoculations were to be given. The mice were intubated by means of 2 1/2-inch long, 18-guage, hypodermic needles, the sharp points and bevelled edges of which had previously been removed.

Experimental Procedure

All mice used in this study were invariably infected between

0800 and 0900 hours, and likewise sacrificed at the same time on the

scheduled day. A careful antemortem examination of all the mice was carried out before necropsy. An accurate record of any abnormal signs was maintained for annotation and check later. The mice were killed by decapitation and bled thoroughly before being laid on the necropsy table. The internal organs, including liver, spleen, kidneys, genitalia, alimentary tract (esophagus, stomach, intestines, caecum and rectum), heart, lungs, and brain were removed and stored separately in 25 ml. of physiological saline solution, but the carcass was stored in 50 ml. of the solution. The brain was removed intact along with the meninges, being severed from the spinal cord immediately behind the medulla. The eyes were removed and placed in a petri dish containing a little physio­ logical saline solution. They were cut by means of a small fine scis­ sors for a larval search inside their chambers. The omentum, mesentry, and peritoneum were stripped off the abdominal organs, and discarded along with the skin, feet and tail. The anterior portion of the head to a level behind the eyes was cut off and the rest with the masseter muscles was included in the carcass. The tissues not required for examination were disposed of by incineration.

The stomach and caecum were cut off from the alimentary canal and emptied of their contents. The intestines were flushed out with physi­ ological saline solution. The parietal surface of these viscera was lightly scraped with the blunt end of a scalpel and rinsed. The con­ tents of the stomach, caecum and intestines, and their washings were all collected in a beaker, marked as "i.C." (intestinal contents).

This was examined after several washings for the presence of any eggs, larvae, or adult parasites. The cleaned tissues of the alimentary canal were cut into small portions and ground in a Waring blendor at slow speed for 45 seconds with 25 ml. of the saline solution in which they were stored. The resultant emulsion was divided into three aliquots and poured into three 50 ml. conical centrifuge tubes, 35 containing 5 ml. of 1 per cent trypsin solution freshly prepared on the same day. More saline solution was added to make approximately

35 ml. of the solution in each. The pH of the medium in each tube was adjusted to 7.0 with 0.1 N sodium hydroxide, using hydrion papers as indicators. The tubes were incubated at 37°C, for four hours, being stirred every hour to ensure even digestion. These were then centri­ fuged at 1000 rpm for five minutes, after which all but 10 ml. of the sediment was removed by suction. The supernatant solution was dis­ carded because the previous experience had shown that it contained no larvae.

The liver, spleen, kidneys, gentialia, heart, lungs and brain, before being ground were examined for any gross pathological lesions.

The liver, spleen, kidneys, genitalia, and heart were ground separately in a Waring blendor at slow speed for 30 seconds each. The resultant emulsion from each was digested and processed for final examination as described earlier for the intestinal wall. The liver emulsion had to be divided into three or four aliquots, depending upon the size of the individual liver, for ease of larval search. All tubes were properly labelled for differentiation and always arranged in a definite serial order in the tube rack to avoid an inadvertent mixing error. The lungs and brain were also processed similarly, except that these were ground for a lesser time, for about 20 to 25 seconds. The brain emulsion had to be divided into four aliquots, and the lungs into two.

The carcass tissue was cut into small portions and ground in a

Waring blendor for 45 seconds at slow speed with 50 ml. of the saline solution, in which it was stored. The resultant emulsion was mixed 36

with 50 ml. of the pepsin solution (1 gm pepsin/50 ml saline solution)

in a 150 ml. beaker. The pH of the suspension was adjusted to 1.0

with hydrochloric acid using hydrion indicator papers. This was then

incubated at 37° C and stirred every hour. After two hours incubation

the suspension was strained through a sieve (20 meshes per square inch)

into a clean 150 ml. beaker and returned to the incubator for another

two hours digestion. The undigested muscle particles on the sieve were

transferred to a petri dish, mixed with some saline solution and exam­

ined under the dissection scope for larvae. A few larvae were occa­

sionally found which were added to the carcass count. After four hours

digestion, all but 30 ml. of the fluid at the bottom was carefully

removed by suction so as not to disturb the sediment. The fat layer

in the.supernatant fluid contained some amount of sediment adherent to

the fat globules. This portion, too, was examined under the dissection

scope for larvae but was invariably negative. In cases where the

carcass was badly emaciated due to heavy infection, no fat layer was

found on the top of the supernatant fluid. All of the sediment in such cases settled down to the bottom and needed several washings with phys­ iological saline before any larvae could be seen.

Usually the lower power of a dissection scope was sufficient for identification of larvae. In case of doubt, however, higher magnifi­ cations helped. The larvae (Figures 10-13) appeared silvery grey in a reflected low blue light from under the scope. The sediment in each tube was well stirred before being transferred to a petri dish, which had been marked on the under-surface with a central straight line and three concentric rings, dividing its surface area into eight portions. 37

Fig. 10. Toxocara canis larva (X190). Fig. 11. Toxocara canis larva (X190), a freshly emerged

larva alongside its empty egg shell. iivaraaisswsittuwJK*’ ^ 40

Fig. 13. Toxocara canis larva (X500), posterior end. 41 This was done to facilitate ease of area of search. The actively-

motile larvae were easy to identify but the coiled, semi-paralyzed or

dead ones were difficult to identify but could not escape an experi­

enced eye.

The number of larvae counted cannot be considered a total count;

first, because the blood was not included in the search for larvae and

secondly, because of inevitable losses during the handling processes.

The count can be termed as fairly accurate and representative as far

as possible.

Plan of Study

A number of experiments, as detailed below, were designed in this

study to achieve the results as contemplated in the objectives outlined

earlier.

Experiment No. 1.- The first experiment consisted of infecting

three mice each with different doses of 30-day-old embryonated eggs of

Toxocara canis by oral intubation. This experiment was meant to demon­

strate the lethal effect of Toxocara canis eggs for mice. This also determined the prepatent (incubation) period of Toxocara canis infection in mice, and the effect of varying doses of these eggs on this period, the clinical signs, lesions, and the course of infection. The design of this experiment is shown in the following table. 42

TABLE 1

THE DESIGN OF EXPERIMENT TO DETERMINE THE LETHAL EFFECT OF DIFFERENT DOSES OF 30-DAY-OLD EMBRYONATED EGGS OF TOXOCARA CANIS BY ORAL INTUBATION

Group Number of Number Eggs Group Number of Number Eggs Number Mice per Mouse Number Mice per Mouse

1 3 500 8 3 20,000

2 3 1,000 9 3 30,000

3 3 2,000 10 3 50,000

4 3 5,000 11 3 100,000

5 3 7,500 12 3 200,000

6 3 10,000 13 3 500,000

7 3 15,000 14 3 Controls

Experiment No. 2. As there was no death in mice administered a dose of 10,000 Toxocara canis eggs and all of the mice died with the next higher dose of 15,000 eggs in the first experiment, hence this second experiment was designed to locate exactly the minimum lethal dose (M.L.D.) and lethal dose 50 (L.D. 50) between these two doses by oral intubation. The design for this experiment is shown in Table 2. 43

TABLE 2

THE DESIGN OF EXPERIMENT FOR DETERMINING THE MINIMUM LETHAL DOSE (M.L.D.) AND LETHAL DOSE 50 (L.D. 50) OF TOXOCARA CANIS 30-DAY-OLD EMBRYONATED EGGS FOR MICE BY ORAL INTUBATION

Group Number of Number Eggs Group Number of Number Eggs Number Mice per Mouse Number Mice per Mouse

1 6 7,500 6 6 14,000

2 6 10,000 7 6 15,000

3 6 11,000 8 6 20,000

4 6 12,000 9 3 Controls

5 6 13,000

Experiment No . 3 .- The surviving mice of experiments 1 and 2 were challenged with 40,000 eggs (double the lethal dose 100) of

Toxocara canis per mouse by oral intubation after four weeks of the

original infection.

Experiment No. 4 .- As all of the mice in experiment No. 3 suc­ cumbed to the challenge dose, an abridged form of experiments 1 and 2 was repeated and the survivals were challenged with 15,000 and 20,000 embryonated eggs of Toxocara canis per mouse by oral intubation, after three weeks of the original infection. The design of this experiment is presented in Table 3. 44

TABLE 3

DESIGN OF EXPERIMENT TO DETERMINE THE MINIMUM IMMUNIZING DOSE (M.I.D.) OF TOXOCARA CANIS EGGS FOR MICE

Group Number of Number Eggs Group Number of Number Eggs Number Mice per Mouse Number Mice per Mouse

1 3 5,000 5 3 15,000

2 3 7,500 6 3 20,000

3 3 10,000 7 2 Controls

4 3 13,000

Experiment No. 5 .- This experiment was designed to test the infectivity of Toxocara canis eggs stored in a refrigerator for 27 months. The eggs were collected on June 18, 1966, and fed to mice by oral intubation on September 26, 1968. The mice were killed on days

1, 2, 3 and 7 post-infection day. The tissues were processed as described earlier and examined for the presence of larvae in various tissues.

TABLE 4

DESIGN OF EXPERIMENT TO TEST THE INFECTIVITY OF TOXOCARA CANIS EGGS STORED IN A REFRIGERATOR FOR 27 MONTHS

Group Number of Number Killed days Number Mice of Eggs after infection Remarks

1 1 1,000 1 Examined tissues emulsion for larvae 2 1 1,000 2 if 3 1 1,000 3 It 4 1 1,000 7 It 45

Experiment No. 6.- This experiment was designed to determine whether the Toxocara canis eggs retained their virulence and the capacity to kill mice even after a long storage of 27 months in a refrigerator. The mice were administered the infection by direct intubation into the esophagus.

TABLE 5

DESIGN OF EXPERIMENT TO DETERMINE THE LETHAL EFFECT OF 27-MONTH-OLD TOXOCARA CANIS EGGS FOR MICE

Group Number of Number Eggs Number Mice per Mouse Remarks

1 3 10,000 Eggs stored in refrigerator, and fed by oral intubation

II 2 2 15,000

3 2 Controls No infection given

Experiment No. 7.- The three survivals and two controls of experiment No. 6 were challenged by oral intubation with 20,000 freshly embryonated (30-day-old) eggs of Toxocara canis after three weeks of their initial infection. RESULTS

The results of this investigation to study the immunogenic

responses of mice on infection with the embryonated eggs of Toxocara

canis are presented in the following pages. Seven experiments were

carried out during the course of this study. The eggs used in experi­

ments No. 1 to 4 were embryonated for 30 days at room temperature and

had, sometimes, to be stored in the refrigerator for just a few weeks

before being commissioned for use. The eggs used in experiments No. 5

to 6 had been embryonated similarly for 40 days, and then stored in a

refrigerator for a little over 27 months before use. However, freshly

embryonated 30-day-old Toxocara canis eggs were used to challenge the mice in experiment No. 7.

One hundred and twenty-four mice were used in these experiments,

of which 10 mice were employed as controls. All of the mice used in

these experiments, except for the basic stock of 24 mice, were raised in our laboratory. Only six mice, two males and four females, from the basic stock were used for breeding and the remainder were used in experiments. The infection of the basic stock with the mites of

species Myocoptes musculinus was noticed within a week of their re­ ceipt in our laboratory. It is believed that they harbored the infection before being imported which may possibly have been picked up during transit. Therefore, the steps taken towards the treatment and eradication of these mites are also presented in these pages.

46 47

Other incidental observations, likewise, which happened to be noticed during the course of this study, such as the actual observa­

tion of the spermatozoa being voided by the two male Toxocara canis worms, on two different occasions, are also recorded. The pictures

of the spermatozoa and eggs taken on the occasion are exhibited here

(Figures 14-21).

Longevity of Larvae in Tissues

The data on larval count and clinical signs observed in one mouse infected with 1,000 (40-day-old) embryonated eggs on 7/25/66 are also recorded here. This mouse was infected by oral intubation with the above-mentioned dose of Toxocara canis eggs, along with

other mice, but was held over for further observations (Malik, 1966).

After about two weeks of infection the mouse exhibited signs of

reduced activity, dullness, standing hair, harsh coat, drowsy appear­ ance, and trembling movements of the head. These signs persisted for one week and then receded gradually; thereafter, he began to improve and gain in condition. No untoward signs or loss of condition was noticed for about a year after the first indisposition. The mouse died on 9/16/67 after about 14 months of infection. About one month before his death, the mouse began to lose in condition, became list­ less, with hair coat rough and staring, and exhibited general cachexia.

The cachectic signs increased progressively until death, which followed as a result of extreme emaciation and debility. At no time did the mouse show any nervous or paralytic signs as reported by Beaver, et al. (1952) and Scothorn (1963). The present observations confirm, Fig. 14. A ruptured Toxocara canis egg just before the

emergence of larva (X750). Fig. 15. A large immature Toxocara canis egg without

the shell (X500). This is the stage when

the sperms gain entrance into the eggs.

The hyaline egg membrane in this stage is normally smooth when laid. The markings and lines visible on the membrane in the above picture, appear on longer standing in physiological saline solution as it becomes more concentrated by evaporation. 50

Fig. 16. Toxocara canis, eggs and spermatozoa (X190).

a. A ruptured egg.

b. Spermatozoa.

c. An egg with two-cell division stage.

52

I

Fig. 18. Toxocara canis spermatozoa (X190). 53

Fig. 19. Toxocara canis spermatozoa (X750).

The periphery is smooth when laid but becomes rough on longer standing in 0.85 per cent saline solution. 54

Fig. 20. Toxocara canis spermatozoa (Phase contrast, X750),

showing morphological details in various stages

of development.

a. Undivided central mass.

b. Divided central mass arranged along the periphery.

c. A developing break in the ringed mass. ia?vH!;X>»i\K ir. vC:-.r»w /'>*-iv:^r'«a>«'.v»tKaj^rfi>areaw'i.;i:

55

iTf I 1* * • h

Fig. 21. Toxocara canis sperm (X1900), showing

a pore at one end. 56 rather further extend, the findings of Tiner (1951). He observed "as many as 100 Toxocara canis larvae may remain alive in a mouse brain for 90 days without producing any noticeable signs in the animal."

Sprent (1955, a,b) also observed many larvae of Toxocara canis. and lesions caused by them in the brain of mice but rarely any signs.

At post-mortem examination of this mouse, no gross macroscopic lesions were observed in any other organ except in the lungs, which had a dirty brick-colored appearance with evidence of healed lesions.

The brain, too, did not show any gross macroscopic lesions.

The larval count revealed the presence of 187 actively motile larvae in the brain, and 132 larvae were recovered from the skeletal muscles. All other organs and tissues were negative for the presence of any larvae.

Lethal Effects of Toxocara Canis Eggs

The first experiment was designed to study the lethal effects of

30-day-old embryonated eggs of Toxocara canis for mice by oral intuba­ tion. Forty-two mice, seven weeks old and weighing about 30 gms. each were used in this experiment. Thirteen groups of three mice each were infected with varying doses of 500 to 500,000 eggs per mouse. The fourteenth group of three mice served as controls. All of the mice were maintained under the same physical conditions in separate cages.

The results of this study are presented in Table 6.

The fecal droppings were examined from all cages on the days following infection. A large number of unhatched eggs with active larvae within them were found on fecal examination of mice which had TABLE 6

LETHAL EFFECTS OF 30-DAY-OLD EMBRYONATED EGGS OF TOXOCARA CANIS FOR MICE BY ORAL INTUBATION

Mouse Egg Dose Degree of Larval Invasion of Different Organs No. per Mouse Results Sp Li Ki Lu Ht Br Gn Cr Ey I.W. i.e.

1 500 S Challenged with 40,000 eggs four weeks after infection. 2 500 S Do 3 500 S Do 4 1,000 S Do 5 1,000 S Do 6 1,000 S Do 7 2,000 S Do 8 2,000 S Do 9 2,000 S Do 10 5,000 S Do 11 5,000 S Do 12 5,000 S Do 13 7,500 S Do 14 7,500 S Do 15 7,500 S Do 16 10,000 S Do 17 10,000 S Do 18 10,000 S Do 19 15,000 D6 4 3+ 2+ 4+ 3+ 5+ 6 5+ 4 15 27 20 15,000 D6 7 4+ 2+ 5+ 2+ 4+ 10 5+ 9 — 5 21 15,000 D8 2 3+ 3+ 4+ 2+ 5+ 7 4+ 7 11 — 22 20,000 D6 5 5+ 2+ 5+ 2+ 5+ 4 5+ 11 17 8 23 20,000 D6 10 5+ 2+ 4+ 2+ 5+ 12 5+ 8 9 2 24 20,000 D7 7 4+ 3+ 5+ 2+ 5+ 9 5+ 14 8 4 — continued on next page TABLE 6 (Continued)

Mouse Egg Dose Degree of Larval Invasion of Different Organs No. per Mouse Results Sp Li Ki Lu Ht Br Gn Cr Ey I.W. I.C.

25 30,000 D4 28 5+ 3+ 5+ 2+ 5+ 19 5+ 11 32 15 26 30,000 D5 21 5+ 2+ 5+ 2+ H.I. 14 5+ 8 17 9 27 30,000 D5 13 H.I. 3+ 5+ 2+ H.I. 21 5+ 16 24 10 28 50,000 D4 25 H.I. 3+ H.I. 3+ H.I. 18 H.I. 14 38 27 29 50,000 D4 41 5+ 2+ H.I. 2+ H.I. 12 H.I. 6 21 55 30 50,000 D4 17 H.I. 2+ H.I. 2+ H.I. 9 H.I. 9 43 61 31 100,000 D2 45 H.I. 3+ H.I. 3+ 283 27 5+ 2 169 98 32 100,000 D3 29 H.I. 2+ H.I. 3+ 246 13 4+ 3 218 63 33 100,000 D3 36 H.I. 2+ H.I. 2+ 152 5 3+ -- 133 58 34 200,000 D1 53 H.I. 2+ 3+ 3+ — 5+ -- 4+ 2+ 35 200,000 D2 71 H.I. 3+ 5+ 3+ — 8 H.I. 1 3+ 2+ 36 200,000 D2 113 H.I. 2+ 5+ 4+ —— 4+ -- 2+ 2+ 37 500,000 D1 25 H.I. 155 89 104 —— 3+ -- H.I. 4+ 38 500,000 Dl -- H.I. 2+ 124 117 — — 4+ — H.I. 3+ 39 500,000 D1 — H.I. 74 96 130 -- — 3+ — H.I. 3+ 40 Control -- Challenged with 40,000 eggs four weeks after infection 41 Control — Do 42 Control “ •• Do

S *s surviving mice Ht - heart 1+ - 1-5 larvae per field D * death on day shown by numeral Gn - genitalia 2+ = 6-10 " " Sp = spleen Ey - eyes 3+ = 11-15 ” Li = Liver I.W. = intestinal wall 4+ = 16-20 " " " Ki = kidneys I.C. = intestinal contents 5+ = 21-25 " " " Lu = lungs Cr = carcass H.I. = heavy , more than 5+ Br = brain A total count was made in organs where number of larvae was low. 59

received higher doses, 100,000 eggs and above. The number of eggs

found in the feces decreased with the decrease in dose of eggs

administered.

No signs were exhibited by mice infected with higher doses of

100,000 eggs and above, except for a slight dullness, and general

inactivity on the day following infection. Most of them died in one

or two days, and only two in three days. The mice infected with

doses of 15,000 to 50,000 eggs per mouse died in four to eight days.

Two days after infection these mice began to exhibit signs of vermin-,

ous pneumonia with shallow, rapid and labored breathing. The mice walked with a stiff gait. The hair coat was harsh looking and stand­

ing, with general debility and emaciation. The eyes were congested

and sunken in their sockets with complete or partial opacity of one

or both of the eyes.

They died with a characteristic posture of hunched back, the

front feet being held in close apposition. The mouth was always found

open, indicative of mouth breathing. Some bloody discharge was invari­

ably seen at the nasal orifices after death.

The post-mortem examination revealed severe congestion, petechial

and ecchymotic hemorrhages in all of the organs. There was evidence of

free blood in the body cavities which received heavier doses of eggs

100,000, and above. The intestines were highly congested and exhibited

small pin-point hemorrhages throughout their length. The abdominal membranes were congested and thickened. The liver and kidneys were also congested with petechial markings. The urinary bladder was greatly distended and full of urine mixed with blood in two mice that 60 had received doses of 15,000 and 30,000 eggs; in one there was more blood than urine. The testes also showed petechial markings in a few

cases. The lungs were the most severely affected of all organs, with

entire lung surfaces being highly cyanotic and dark blue in appearance.

The heart showed petechial hemorrhages. The pleurae were congested,

thickened, dull in appearance and adherent. Petechial, ecchymotic and diffuse hemorrhagic areas were seen on the brain surface, Figure 22.

The brain substance was generally softened and malacic. The meninges were congested, thick and adherent. The cranial blood vessels were greatly congested. Ecchymotic and petechial hemorrhages were seen on the subcutaneous surface of the skin, Figure 23.

Lethal Doses of Toxocara Canis Eggs

Since there was no death in mice administered 10,000 Toxocara canis eggs and all of the mice died with the next higher dose of

15,000 eggs in the previous experiment, hence the second experiment was designed to find out the minimum lethal dose, and lethal dose

50 — a dose that will kill 50 per cent of mice by oral intubation.

Eight groups of six mice each were administered varying doses of

Toxocara canis eggs, ranging between 7,500 to 20,000 eggs per mouse.

The ninth group of three mice served as controls. The results of mice that died and those that survived at different doses are given in Table 7. 61

Fig. 22. Brain of a mouse showing patechial and ecchymotic

hemorrhages, congestion of blood vessels and

extravasation of blood on the brain surface. The

mouse died on the 7th day after infection with

15,000 Toxocara canis eggs. 62

Fig. 23. A mouse showing petechial and ecchymotic

hemorrhages on the subcutaneous surface

of the skin. The mouse died after 7 days

of infection with 15,000 eggs of Toxocara

canis. 63

TABLE 7

MINIMUM LETHAL DOSE OF 30-DAY-OLD EMBRYONATED EGGS OF TOXOCARA CANIS FOR MICE BY ORAL INTUBATION

Group Number Egg Dose Number Group Number Egg Dose Number No. of Mice per Mouse Died No. of Mice per Mouse Died

1 6 7,500 0 6 6 14,000 4

2 6 10,000 1 ' 7 6 15,000 5

3 6 11,000 1 8 6 20,000 6

4 6 12,000 2 9 3 Controls

5 6 13,000 4

There was no death in the group administered a dose of 7,500

eggs per mouse, and all of the mice died with a dose of 20,000 eggs

per mouse. One mouse died on the eighth day from the group of six mice administered a dose of 10,000 Toxocara canis eggs. Thus, the minimum lethal dose of Toxocara canis eggs was determined to be

10,000 eggs for mice by oral intubation.

The lethal dose 50 (L.D. 50) for mice has been calculated from

the above data in accordance with the method described by Reed and

Muench (1938), Table 8.

In this method it is assumed that a mouse which has survived at

a given dose, would have survived at a lower dose. Column "c" is,

therefore, added from the bottom and the cumulative total of survivals

for each dose is entered in Column "e". Conversely, a mouse which died at a given dose of eggs, would have died at a higher dose. Column "d" is, therefore, added from the top and the cumulative total

of mice dying at each dose is entered in column "f". Percentage of

mortality is calculated from the total of columns "e" and "f".

TABLE 8

LETHAL DOSE 50 (L.D. 50) OF TOXOCARA CANIS EGGS FOR MICE BY ORAL INTUBATION

Dose Number Mice Mice Cumulative Total Per cent eggs of mice alive dead Alive Dead mortality a b c d e f

7,500 6 6 0 25 0 0.0

10,000 6 5 1 19 1 5.0

11,000 6 5 1 14 2 12.5

12,000 6 4 2 9 4 30.8

13,000 6 2 4 5 8 61.5

14,000 6 2 4 3 12 80.0

15,000 6 1 5 1 17 94.4

20,000 6 0 6 0 23 100.0

It is evident from the above table that 50 per cent end point

lies between 12,000 and 13,000 eggs. The formula for calculation of

the proportional distance of 50 per cent end point from the dose next below is:

50 per cent - per cent mortality at next Proportional distance ------lower d o s e ------per cent mortality above 50 - per cent mortality below 50 65

Substituting the proper values in the above equation we get:

Proportional distance = 50-30.8 61.5-30.8

19.2 30.7

= 0.622.

The lethal dose 50 is now obtained by adding 622 to the dose next

below 50 per cent, that is, 12,000 in this case. Thus, the lethal dose

50 (L.D. 50) of Toxocara canis eggs for mice by oral intubation is

12,622 eggs, or 12,600 in round figures.

The same results have been obtained graphically as shown in

Figure 24, which conform with the results obtained by calculations.

Challenge Experiments

The mice surviving in experiments Nos. 1 and 2, which had received a dose of 10,000 eggs or more had exhibited signs of general dullness, lassitude, standing hair, off-feed, labored breathing,

crouched appearance, and loss of condition for about a week after

infection. The mice administered smaller doses of eggs, exhibited

less severe symptoms than these. The clinical signs disappeared gradually, and the mice regained their sprightly appearance.

All surviving mice were in apparently normal condition and eat­ ing well when they were challenged with 40,000 eggs (double the lethal dose 100) after four weeks of infection. No discomfort was exhibited by any mice for a day or two after the challenge dose. Thereafter they began to show symptoms of general dullness, off-feed, labored breathing, and emaciation, as described earlier. All of the mice died iue 4 Lta Ds 5(..5) f a-l Ebyntd Eggs Embryonated Day-Old 0 3 of 50) 50(L.D. Dose Lethal 24. Figure

NUMBERS OF MICE 30-1 0 2 500 IPO 100 2P00 1,0 1,0 I 0 0 p 0 2 0 0 P I5 14,000 13,000 0 0 P I2 11,000 IOPOO 0 0 ,5 7 - UBR F GS E DOSE PER EGGS OF NUMBER MICE SURVIVING f ooao a s o Mc y Intubation by Mice for is can Toxocaro of c l e f al 10) table(col. of “e" clV'f al 10) table 'of (col.V MICE DYING— 66 within four to eight days after the challenge dose. In general, the mice that received smaller doses of eggs on the first infection suc­ cumbed earlier to the challenge infection than those which received

larger doses. One mouse, which received 500 eggs on the first infec­ tion, died on the seventh day after challenge while one with an

infective dose of 12,000 eggs died after four days of challenge, and one with 15,000 infective dose died on the fifth day after challenge.

Therefore, no specific conclusions emerge from the death pattern in this experiment because there was no uniformity in the death pattern of controls. The results of this experiment are presented in Table 9.

As all of the surviving mice died on challenge in the previous experiment, an abridged form of the first two experiments was repeated to determine the immunizing effect of Toxocara canis eggs in mice challenged with 15,000 and 20,000 embryonated eggs after Jthree weeks of primary infection. Six groups of three mice each were infected with different doses of Toxocara canis eggs. Two mice were employed as controls, Table 3.

Seven mice, one which received 13,000 eggs, and three each which received 15,000 and 20,000 eggs, died within six to eight days after the primary infection. They exhibited similar clinical signs before death, and identical lesions were seen on post-mortem as described earlier under the first experiment. The remainder of the 11 infected mice and two controls were challenged with 15,000 and 20,000 embryo­ nated eggs of Toxocara canis after three weeks of the primary infec­ tion. The results are presented in Table 10. 68

TABLE 9

RESULTS OF CHALLENGE OF SURVIVING MICE WITH 40,000 EGGS OF TOXOCARA CANIS FOUR WEEKS AFTER PRIMARY INFECTION

Egg dose Death Egg dose Death Egg dose Death Mouse first on Mouse first on Mouse first on No. infect. day No. infect. day No. infect. day

1 500 4 18 7,500 6 35 12,000 4

2 500 4 19 7,500 7 36 12,000 6

3 500 7 20 7,500 7 37 12,000 6

4 1,000 4 21 7,500 8 38 12,000 7

5 1,000 4 22 10,000 5 39 13,000 6

6 ,1,000 4 23 10,000 6 40 13,000 6

7 2,000 5 24 10,000 6 41 14,000 6

8 2,000 5 25 10,000 6 42 14,000 7

9 2,000 6 26 10,000 6 43 15,000 5

10 5,000 5 27 10,000 7 44 Control 4

11 5,000 5 28 10,000 7 45 Control 4

12 5,000 8 29 10,000 8 46 Control 6-

13 7,500 5 30 11,000 5 47 Control 6

14 7,500 5 31 11,000 6 48 Control 6

15 7,500 5 32 11,000 6 49 Control 7

16 7,500 6 33 11,000 7

17 7,500 6 34 11,000 8 TABLE 10

RESULTS OF CHALLENGING PREVIOUSLY INFECTED MICE WITH 15,000 AND 20,000 EGGS OF TOXOCARA CANIS, THREE WEEKS AFTER THE PRIMARY INFECTION

Egg dose Egg dose Day of Mouse primary challenge death after ______Degree of Larval Invasion of Different Organs No. infection infection challenge Sp Li Ki Lu Ht Br Gn Cr Ey I.W. I.<

1 5,000 15,000 5 17 4+ 3+ 5+ 2+ 5+ 12 4+ 9 15 ^ , 2 5,000 15,000 6 18 5+ 2+ 5+ 1+ 5+ 7 5+ 2 1+ 4 3 5,000 20,000 4 2 5+ 3+ 5+ 2+ H.I. 13 4+ 5 9 -■ 4 7,500 15,000 5 10 3+ 2+ 4+ 1+ 4+ — 3+ 3 — 5 7,500 15,000 6 — 5+ 1+ 5+ 1+ 5+ 8 5+ 7 — -■ 6 7,500 20,000 5 5 5+ 3+ 5+ 1+ 5+ 3 3+ 11 1+ 1 7 10,000 15,000 10 2 1+ 1+ 2+ 1+ 4+ 11 4+ 6 5 -• 8 10,000 20,000 8 1+ 3+ 2+ 3+ 1+ 5+ 7 3+ 4 1+ 2 9 10,000 20,000 7 4 3+ 1+ 3+ 2+ H.I. — 5+ 10 -- 3 10 13,000 15,000 18 -- 1+ 1+ 1+ 8 5+ 2 3+ 8 6 - 11 13,000 20,000 37 -- 1+ ' 4 1+ — 4+ 1 3+ 14 — - 12 Control 15,000 6 8 4+ 2+ 5+ 1+ 5+ 14 4+ 12 8 5 13 Control 20,000 6 15 3+ 2+ 5+ 2+ H.I. 10 5+ 8 11 1

Sp = spleen Gn = genitalia 1+ = 1-5 larvae per field Li = liver Cr = carcass 2+ - 6-10 " 11 " Ki = kidneys Ey = eyes 3+ = 11-15 " " " Lu = lungs I.W. - intestinal wall 4+ = 16-20 " 11 11 Ht = heart I.C. = intestinal contents 5+ = 21-25 11 11 " Br = brain H.I. = heavy infestation, more than 5+ A total count was made in organs where number of larvae was low. 70

From Table 10, it may be seen that all surviving mice from a

previous infection succumbed to the challenge dose on different days.

The usual clinical signs and lesions in all organs, as described

earlier, were also seen in these mice. In addition, they exhibited

paralytic signs of falling, circling, inability to maintain balance,

general stupor and prostration for some days before death. Some of

the significant observations which resulted from this study are enumer­

ated below.

Conclusions of Challenge Experiments

1. The mice that received a lower primary infection of 5,000

and 7,500 Toxocara canis eggs succumbed to a challenge dose of either

15.000 or 20,000 eggs earlier than those which received a larger dose

of 10,000 or 13,000 eggs on primary infection.

2. The order of recovery of Toxocara canis larvae from the mice with lower primary infection and dying on challenge with either 15,000

or 20,000 eggs was not very much different from that of the mice which

received 15,000 or 20,000 eggs on primary infection, Table 6. The

challenge or super infection rather advanced the death date of such

mice, over those that received the same dose on first infection.

3. The mice that received a primary infection of 10,000 or

13.000 eggs withstood the challenge infection for a longer time than

the others. One mouse which received a primary infection of 13,000

eggs and was subsequently challenged with 15,000 eggs survived the

challenge infection for 18 days, while another that received an equal 71

primary infection of 13,000 eggs and was challenged with 20,000 eggs

died after 37 days of the challenge. The order of recovery of larvae,

too, from such mice was distinctly lower than that of the mice which

received lower doses of eggs primarily.

4. An interesting observation of some significance was that a

vast majority of larvae, recovered from all the other somatic tissues

and organs, was dead while the larvae recovered from the brain were mostly active and motile.

5. The results of this and previous experiments indicate con­

clusively that no protective immunity is conferred by an exposure to

Toxocara canis infection to a subsequent superinfection with similar

eggs in mice.

Infectivity of 27-Month-Old Toxocara Canis Eggs

Four mice, seven weeks of age, were infected with 1,000 eggs

each of Toxocara canis stored in a refrigerator for 27 months after

embryonation. The mice were killed 1, 2, 3, and 7 days after infec­

tion day. The usual lesions expected on the day of the kill were

seen in the respective organs. The results of larval counts are

presented in Table 11, which show that these eggs retained their

infectivity even after 27 months. Their rate of larval recovery,

24.5 per cent on the first day, 31 per cent on the second day, 35.5 per cent on the third day, and 39.7 per cent on the seventh day fol­

lowing infection, compares favorably with the corresponding rates of infectivity reported by Malik (1966) for 40-day-old, one year-old, and 18-month-old embryonated eggs of Toxocara canis. TABLE 11

DISTRIBUTION OF TOXOCARA CANIS LARVAE IN WHITE MICE TISSUES INFECTED ORALLY WITH 27-MONTH-OLD EGGS STORED IN A REFRIGERATOR

Killing day after Degree of Larval Invasion of Different Organs Jo. per mouse infection Sp Li Ki Lu Ht Br Gn Cr Ey I.W. I.C. Total

1 1 1,000 J. — 108 133 4 245

2 1,000 2 — 285 — 9 — -- 12 — 4 310

3 1,000 3 — 79 — 27 92 — 157 —— 355

4 1,000 7 6 1 — 181 209 -- 397

Sp - spleen Gn = genitalia Li = liver Cr = carcass Ki = kidneys Ey = eyes Lu = lungs I.W. = intestinal wall Ht ~ heart I.C. = intestinal contents Br = brain 73

Lethal Effects of 27-Month-Old Toxocara Canis Eggs

Five mice were infected by oral intubation, three with 10,000 eggs and two with 15,000 eggs each of Toxocara canis stored in a refrigerator for 27 months. Two mice were employed as controls. One mouse infected with 10,000 eggs, and the other with 15,000 eggs died on the seventh and sixth day, respectively, following infection. The clinical signs exhibited and lesions observed on post-mortem were similar to those described earlier. The results of the larval count are presented in Table 12.

The larval yield from mice dying in this experiment, as seen from Table 12, is of about the same order as seen in mice dying with corresponding doses of freshly embryonated 30-day-old Toxocara canis eggs, Table 6. It has, thus, been confirmed that the Toxocara canis eggs stored in a refrigerator for 27 months retained their capacity to fatally infect mice as well as the freshly embryonated eggs.

Challenge Experiment With 27-Month-Old Toxocara Canis Eggs

The three surviving mice and the two control mice of the pre­ vious experiment were challenged with 20,000 freshly embryonated 30- day-old eggs of Toxocara canis three weeks after the primary infection.

All mice died within six to ten days following challenge. There was no significant difference between the death pattern and the larval yield of the challenged and the control mice, Table 13. Therefore, no specific conclusions may be drawn from this experiment, except that TABLE 12

LETHAL EFFECTS OF 27-MONTH-OLD EMBRYONATED EGGS OF TOXOCARA CANIS FOR MICE BY ORAL INTUBATION

Mouse Egg Dose Degree of Larval Invasion of Different Organs No. per Mouse Results Sp Li Ki Lu Ht Br Gn Cr Ey I.W. I.C.

1 10,000 S Challenged with 20,000 eggs three weeks after infection.

2 10,000 S — Do —

3 10,000 D7 1 3+ 2+ 2+ 1+ 4+ 37 5+ 8 12

4 15,000 D6 5 H.I. 2+ 4+ 1+ 3+ 22 3+ 3 9 8

5 15,000 S Challenged with 20,000 eggs three weeks after infection.

6 Control — — Do --

7 Control — — Do --

S s surviving mice Ht = heart 1+ = 1-5 larvae per field D = death on day shown by numeral Gn = genitalia 2+ » 6-10 " tl II VI llll Sp spleen Cr = carcass 3+ » 11-15 " II II Li = liver Ey = eyes 4+ = 16-20 " II II Ki = kidneys I.W. rr intestinal wall 5+ ** 21-25 " Lu s lungs I.C. = intestinal contents H,.1. = heavy infestation, more Br = brain than 5+ A total count was made in organs where number of larvae was low. TABLE 13

RESULTS ON CHALLENGING MICE BY ORAL INTUBATION (PRIMARY INFECTION, 27-MONTH-OLD EGGS) WITH 30-DAY-OLD EGGS OF TOXOCARA CANIS, THREE WEEKS AFTER THE PRIMARY INFECTION

Egg Dose Egg Dose Day of Death Mouse Primary Challenge After Degree of Larval Invasion of Different Organs No. Infection Infection Challenge Sp Li Ki Lu Ht Br Gn Cr Ey I.W. I.C.

1 10,000 20,000 6 16 3+ 1+ 4+ 1+ 5+ 23 4+ 5 12 3

2 10,000 20,000 7 9 2+ 29 3+ 1+ 3+ 7 2+ 3 — --

3 15,000 20,000 10 15 2+ 1+ 2+ 1+ 4+ 14 3+ 10 35 8

4 Control 20,000 7 7 5+ 2+ 4+ 2+. 5+ 19 H.I. 14 1+ 4

5 Control 20,000 8 11 5+ 3+ 5+ 2+ H.I. 13 H.I. 11 1+ 13

Sp = spleen Gn = genitalia 1+ = 1-5 larvae per field VI ii ii Li = liver Cr ® carcass 2 + = 6-10 Ki - kidneys Ey = eyes 3+ = 11-15 If ii it Lu = lungs I.W. = intestinal wall 4+ = 16-20 II if ii Ht = heart I.C, - intestinal contents 5+ = 21-25 " 11 " Br = brain H.I. = heavy infestation, more than 5+ A total count was made in organs where the number of larvae was low. 76 I the primary infection with 27-month-old eggs, did not confer any pro­

tective immunity on mice to subsequent challenge with freshly embryo­ nated Toxocara canis eggs.

Treatment and Control of Murine Mange

The mice received from Harlan Industries, Inc., Cumberland, Ind., were found to be infested with the mites, Myocoptes musculinus. The

following treatments were employed to control the infection.

1. The mice were dusted with an' insecticidal powder (Ectocide,

Nordon Laboratories, Lincoln, Neb.) containing the active ingredients

of rotenone 0.375 per cent; pyrethrins 0.075 per cent; piperonyl butoxide 0.75 per cent; other cube resins 0.75 per cent; and inert

ingredients 98.05 per cent. The powder was also mixed with the bedding

of corn cobs in the cages. The treatment was repeated three times at weekly intervals but without complete success. The mice were seen

scratching and restless between treatments. The denuded areas per­

sisted on the head and behind the ears.

2. Next, an insecticidal spray (Kemic pet spray, Vet. Kem.

Laboratories, Dallas, Tex.) with the active ingredients as carboxyl

0,5 per cent; pyrethrins 0.05 per cent; piperonyl butoxide 0.1 per

cent; N-octyl bicycloheptene dicarboximide 0.166 per cent; 2,2- methylene bis (4-chlorophenol) dichlorophene 0.1 per cent; inert

ingredients 99.084 per cent, was sprayed under pressure on the entire body surface of the mice and the ’'ectocide" powder was mixed with the bedding in the cages. The treatment was repeated after one week but without any significant improvement, in the general condition of the mice. 77

3. Lastly, the condition was successfully treated and controlled with the use of Aramite-15W, containing 2-(P-tert-butylphenoxy) isopropyl-2-chloroethyl sulfite 15 per cent and inert ingredients 85 per cent, in a 2 per cent solution with 0.1 per cent detergent. The mice were completely dipped, head and tail, in the solution for one

second and then partially submerged at swimming depth for 15 seconds.

They were completely dipped once again for a second before being taken out of the solution. The mice looked good following the treatment.

The general condition of the mice and their hair-coats improved in a

few days. A second treatment was given after two weeks.

Two months following the last treatment with Aramite-15W, the mice were found to be scratching again and small denuded areas were

seen behind the ears and near the inner canthus of the eyes. The

treatment with Aramite-15W was used again. During this time a wild mouse was trapped in the laboratory which was heavily infested with

the same Myocoptes musculinus mites. As it was not possible to com­ pletely stop wild mice from visiting the laboratory for food, the practice of a monthly treatment with Aramite-15W to all the mice in the colony was instituted. This practice worked well to keep the mouse colony free from this infestation, as the condition was not

seen at any time after that and the mice remained perfectly healthy.

Toxocara canis spermatozoa

At one time while collecting eggs by natural oviposition method

it was decided to keep one male worm with two female worms in each petri dish to see its effects on the egg laying by the females. 78

During the routine daily microscopic check of all the petri dishes for collection of eggs, one male worm was seen to ejaculate. The ejaculate gushed out in a jet stream fashion from the distal end of the two spicules which were joined together to form a groove.

A small drop of the ejaculate was immediately taken on a slide and examined under the microscope. The spermatozoa were seen as small, round, circular and sluggishly motile bodies. The motility noticed was of a gliding progressive type, unlike a pseudopodic amoeba type or rotary type. None of the spermatozoa formed pseudo­ podia during their movement. One of the sperms was seen to enter a large sized immature egg (Fig. 15), which had the vitelline membrane only without the thick shell membrane around it. The sperm made its entry into the hyaloid vitelline membrane by means of a small round pin-like structure, which perhaps it could extend in and out, because this little projection was not seen during its movements.

A few days later another male worm was seen in an agitated state with extended spicules. It was immediately removed to a clean petri dish, containing physiological saline solution. After a while this worm was also seen to ejaculate and similar sperms were seen. The measurements of the sperms were taken which ranged from 15 to 18p< in diameter on the average, with only one sperm measuring 20M* .

The pictures of the spermatozoa were taken on both occasions.

The sperms in the latter pictures are somewhat crenated because of the increasing concentration of salt in the medium due to evaporation.

The sperm shells were perfectly smooth when freshly layed. The phase contrast picture of the spermatozoa (Fig. 20) shows the outer layers 79 and an internal granular mass. The different sperms in this picture show this internal mass in different stages of division. In some, this mass is undivided and compact, while in others it is divided and arranged along the periphery in a ringed form somewhat like the multi- lobed nucleus of a polymorphonuclear leukocyte, with a developing cavity in the center. One sperm (Fig. 20c) shows a developing break in this ringed mass, forming a pore. This pore is seen more complete in another picture of a sperm taken under the oil immersion lens (Fig.

21). This picture shows the ringed granular mass to have disintegrated into small granules scattered throughout the internal cavity. The internal most layer and the middle, layer have broken at one end in the shape of a pore. Whether these different phases represent stages in the development and maturation of a sperm cannot be answered at this time.

The large sized eggs without the thick shell membrane, Fig. 15, were usually seen in increasing numbers five to six days after the worms had been laying the normal eggs. Their size varied from 140p, to 170(0, in diameter, while the normal eggs measured about 80p, to 90|j, in diameter. Other eggs of various shapes and intermediate sizes, with partially developed shell membranes were also seen. It is, thus, inferred that the Toxocara canis eggs are large sized when layed by the ovaries, but shrink in size as the thick shell membrane gets secreted around them on passage through the uterus. DISCUSSION

Nematode parasites and their larvae have been known to induce immune reactions in their normal and abnormal hosts. The production of antibodies by hosts against parasite invasions has been demon­ strated by the development of various serological tests as aids in the diagnosis of these conditions. Kolmer, et al_, (1916) on the basis of several complement fixation tests, reported the presence of antibodies in dog's sera against Toxocara canis, and frequently observed group reactions between closely related species and infrequently between widely divergent species. The evidence of production of specific anti­ bodies following an infection of rabbits with Toxocara canis was demon­ strated by Sadun, et al. (1957). Sharp and Olson (1962) observed cross reactions between Toxocara canis. Ascaris lumbricoides. and Trichine11a spiralis. Recently Ivey (1965), and Ivey and Slanga (1965), likewise, reported specific and heterologous antigen-antibody systems between these same parasites. The present studies were undertaken to determine if the antibodies produced by sublethal infections of Toxocara canis eggs in mice had any protective value for the mice against a subsequent challenge with lethal doses of the eggs.

Criteria of Immunity in Helminth Infections

Various criteria have been devised by different workers to test the development of immunity or resistance by the hosts on exposure to

80 81

a sublethal infection. Some of the more common criteria employed are:

1. Reduction in the number of larvae recovered from the

liver and more specifically from the lungs.

2. The delayed and stunted growth of the larvae.

3. The ability to withstand a lethal dose of the eggs.

4. An increased cellular response or encapsulation of

the larvae.

While a majority of the workers, Kerr (1936, 1938a,b,c), Lee

(1960), Olson (1962), Taffs (1964), Crandall and Arean (1965), and

several others employed one or more of the above-mentioned criteria in

their studies, Sprent and Chen (1949) based their criteria of immunity mainly on the liver ratio, besides some of the other more usual points.

They defined the liver ratio as the number of larvae in the liver

divided by the total number in the liver and the lungs. This ratio

obviously is variable in accordance with the duration of the infection.

The criteria of immunity employed in the present studies were:

1. The survival or the death of mice on challenge with a

lethal dose of the eggs after recovery from a primary

exposure.

2. The delay in the day of death on challenge.

3. Reduction in the number of larvae recovered from dif­

ferent mice after a challenge infection.

Kerr (1936) observed that the comparison of the number of larvae

recovered from lungs of resistant and nonresistant animals was not

only tedious but also subject to considerable experimental error. The

present study indicates that although the survival of a host on 82 challenge was a very significant point in the consideration of immunity development, other criteria, such as the reduction in the number of

larvae and their state of physiological development were of value in deciding this issue.

Lethal Doses

In the study of immunity the first step to be decided is the

lethal dose of the parasite eggs or larvae for the particular host

species. This is because5 the resistance can be measured by challenging with a dose that will surely kill the animal. Some have used a multi­ ple of the minimum lethal dose, a dose that can kill even one animal under experimental conditions. Taffs (1964) used 5 to 10 times the minimum lethal dose in different experiments while working with Ascaris

lumbricoides infections in guinea pigs. Kerr (1936) working with

Ancvlostoma caninum infections in mice used a dose greater than, what - he called, the average lethal dose, which was virtually a dose that killed all of the animals. In some other experiments he used a test dose of more than five times the average lethal dose. At another time working with Ascaris lumbricoides infections in guinea pigs, Kerr

(1938a) used a test dose of twice the average lethal dose.

The first two experiments in this study were devoted to determin­ ing the minimum lethal dose (M.L.D.), lethal dose 50 (L.D. 50), lethal dose 100 (L.D. 100) of Toxocara canis eggs for mice, which were found to be 10,000 eggs, 12,600 eggs, and 20,000 eggs, respectively. These results are quite at variance with those of Lee (1960), who reported

2,000 eggs of Toxocara canis as an average lethal dose for mice, but 83 are substantially in agreement with the reports of Nichols (1956a),

Tiner (1951), Sprent (1952a, 1953a, 1958) and several other workers.

At no time in the several experiments undertaken in this study, was a mouse found to die with an infection of less than 10,000 eggs.

Resistance to Reinfection

Ducas (1921), cited by Taliaferro (1929), reported an acquired immunity in rats against reinfection with Trichinella spiralis as evidenced by the fact that the larval parasites were arrested in their development and swept out of intestines before reaching sexual matur­ ity. Sandground (1928) experimenting with Strongyloides stercoralis infections in dogs showed that dogs having overcome a first infection, or in some cases even before that, were refractory to a second infec­ tion. Stoll (1929) observed the self cure phenomenon in sheep infected with Haemonchus contortus and stated that it was accompanied by a high and apparently enduring protection against further by the same worm species. Kerr (1938a) working with Ascaris lumbricoides infections in guinea pigs found greater encapsulation and destruction of larvae in the liver of reinfected guinea pigs. He concluded that liver formed an important barrier to the migration of the larvae to the lungs. Sprent and Chen (1949) working with Ascaris lumbricoides infections in mice found a high liver ratio (the number of larvae recovered from the liver, divided by the total number found in the liver and the lungs) for five days after primary infection, which decreased to a low level by the 8th day due to the migration of the larvae from the liver to the lungs. On the other hand, he found that the liver ratio remained high for 8 days in immunized mice, because 84 the larvae did not migrate to the lungs. They also noticed that the larvae in the immunized mice were significantly small. Later this phenomenon of resistance or immunity by hosts to reinfection with the parasites of the same species has been demonstrated by numerous workers in case of different parasites. While a sizeable amount of literature on this subject is available regarding Ascaris lumbricoides. Trichi- nella spiralis. Trichuris vulpis and several other helminth infections, relatively very little literature, except for Lee (1960), and Olson

(1962), is available with regard to Toxocaris canis infections in mice.

Lee (1960) using repeated inoculations of mice with smaller doses of Toxocara canis eggs, to a total of 2,000, observed the development of resistance in test mice as compared to the control mice who were given a single dose of 2,000 eggs. He recovered fewer total larvae from the test animals as compared with the control groups. He also found relatively few larvae in the liver and the brain of superinfected mice. No larvae were found by him in the intestinal wall either after single or repeated inoculations. Olson (1962) reported that a previous infection of mice with Toxocara canis or Ascaris lumbricoides appeared to increase the resistance of mice to a challenge infection with

Toxocara canis resulting in less survival of larvae and delayed migra­ tion through the liver.

In the present study 43 mice out of 87 which had been given dif­ ferent doses of Toxocara canis eggs, varying from 500 to 15,000, survived in the first two experiments meant for the determination of lethal doses, Tables 6, 7, and 8. They were challenged four weeks later with 40,000 eggs, twice the lethal dose 100, Table 9. All of 85 the mice, along with six controls, died within four to eight days of the challenge infection, with a majority of them dying on the sixth day. Two of the control mice died on the fourth day, three on the sixth day, and one on the seventh day, Table 9. No uniformity or regularity of death pattern was seen between the previously infected mice and controls, either among themselves or in comparison to each other. The one tangible conclusion that can be deduced from this study is that, but for a few exceptions either way, the mice that received smaller doses of eggs initially, died earlier on challenge infection than those that received heavier doses of eggs on the first infection.

It is again evident from the results of the next experiment that none of the mice which received 5,000, 7,500, and 10,000 eggs each, died because of the primary infection, while one out of three which received 13,000 eggs each, died on the eighth day; the remaining two mice from this group survived on first infection. All of the mice which received 15,000 and 20,000 eggs died within six to eight days of the primary infection. The 11 surviving mice and two controls died within 4 to 37 days on challenge by oral intubation with either 15,000 or 20,000 eggs per mouse, Table 10. The mice which got lower initial doses of 5,000 and 7,500 eggs each succumbed to challenge between the fourth and sixth day after challenge. The death day was, however, prolonged in the case of mice getting higher initial doses of 10,000 and 13,000 eggs each. One mouse which received 10,000 eggs on initial infection died on the 10th day after challenge with 15,000 eggs, while one which had 13,000 eggs as primary infection and 15,000 eggs on 86

challenge died on the 18th day after challenge. Another mouse which

received 13,000 eggs on primary infection died after 37 days of chal­

lenge with 20,000 eggs. The last two results are highly important.

The order of larval recovery from different organs of mice with

lower initial infections and dying on challenge, was not significantly

different from the control mice which received no primary infection.

On the other hand, larval recovery from various organs of mice with

higher initial doses of 10,000 and 13,000 eggs each was quite evidently

lower than that of the control mice. The lower rate of larval recovery

and prolonged day of death in mice infected with higher initial doses

means that as the size of the egg dose increased on primary infection

there was evidence of increased resistance on the part of the host to

a subsequent challenge infection. These observations are in conformity

with those of Kerr (1938b) when he remarked, "It seems that it is a

matter of introducing enough antigen to stimulate a resistance, for

the experiments show that as the amount of antigen was increased more

of the injected guinea pigs survived the test infection."

The recovery of larvae from the brain and the carcass (muscle

tissues) was always higher as compared to other organs in all cases,

although it was somewhat lower in mice having higher initial doses

than in those with lower primary infections. This also shows that

the liver does not provide a very effective barrier to the invasion

of Toxocara canis larvae in mice to other organs and tissues of the body when larger infection or challenge doses are concerned. The size o

87 of the challenge dose between 15,000 eggs and 20,000 eggs did not materially affect either the day of death or the recovery of the larvae from the tissues.

The larvae were quite often recovered from the intestinal wall of mice dying either on primary infection or on challenge, which observation is at variance with that of Lee (1960), who did not find larvae in the intestinal wall either after single infection or repeated infections. Further, no diminution in the size of the larvae recovered from any of the organs of the challenged mice was ever noticed, which is contrary to the observation of Sprent and Chen (1949), who found lower size larvae in challenged mice in the case of Ascaris lumbri­ coides infections.

Another interesting observation was that a vast majority of the larvae recovered from tissues other than brain in challenged mice was invariably dead, some were sluggish, and only a few were actively motile. The larvae recovered from the brain of mice either dying of primary infection or on challenge infection were always actively motile, with rarely a few dead or sluggish larvae. A satisfactory explanation for this phenomenon cannot be offered at this stage.

Whether the larvae find a safe sanctuary in the brain away from the onslaught of host's defensive mechanism is just a conjecture at the moment.

None of the challenged mice in both experiments survived either a single or double the lethal dose 100. This shows that no protective 88 immunity is conferred by a primary infection of sublethal doses of

Toxocara canis eggs to a subsequent exposure to lethal doses on chal­ lenge in mice.

The evidence of open blood in the body cavities when heavier doses of eggs were given, the extravasation of blood on the brain surface, and recovery of the larvae from the vitreous humour, which was frequently streaked and tinged with blood, indicate that the larvae were able to injure and break out of the blood vessels. Buske and Engelbrecht (1968) working with Ascaris lumbricoides observed that the larvae frequently strike on the blood vessels during their active migration.

Some of the characteristic lesions observed in challenged mice were the dirty grey nodules in the liver and the lungs, evidence of healed lesions of the primary infection. Lesions to a lesser extent were seen in the kidneys, testes, and occasionally in the urinary bladder. The pleurae were extremely dirty and studded with areas of pustular foci. The lungs were highly cyanotic and dark purple-blue all over. The brain showed petechial and ecchymotic hemorrhages, congestion of blood vessels and extravasation of blood on the brain surface. This agrees with the statement of Sprent (1955a) that the larvae left the arteries on the surface of the brain where their diameter approximated that of the arteries.

Prepatant Period

From the various experiments undertaken during the course of this study, it was seen that the prepatant period of Toxocara canis eggs in mice was subject to the size of the dose of eggs administered. 89

When large doses, varying from 100,000 to 500,000 of eggs were given the mice exhibited signs of dullness, and depression in less than 24 hours after infection. They crouched morose and drowsy in a corner all the time, without an inclination to move even when disturbed.

The intake of food and water was totally suspended. The death fol­ lowed within one to three days.

In other cases, when 10,000 to 50,000 eggs were administered, the mice began to show signs of dullness, and general discomfort within two to three days. The well pronounced clinical signs of labored and rapid breathing were evident from fourth to fifth day.

The intake of food and water was diminished and finally suspended.

The death followed in six to eight days in a majority of such mice.

Similar signs were seen in mice which survived the doses of 10,000 to 15,000 eggs. After about eight to ten days they began to improve in condition with a gradual abatement in the signs of verminous pneumonia. The intake of food and water was gradually resumed and they seemed to be completely recovered after about two weeks of the infection.

The mice that got doses of less than 10,000 eggs did not show much discomfort or uneasiness. They never gave up feeding and water­ ing. The signs of verminous pneumonia were also seen in them after about four to five days of infection but these were milder in inten­ sity and of lesser duration.

The challenged mice that succumbed early and had received smaller doses of eggs initially, exhibited greater discomfort, dull­ ness and depression. They exhibited exaggerated signs of verminous 90

pneumonia, hard breathing, and general debility. The intake of food

and water was suspended. The mice were extremely emaciated before

death. The mice that survived the challenge dose longer for 10, 18,

and 37 days did not show much discomfort for about a week but began

to lose in condition gradually. They were similarly emaciated and

exhibited paralytic signs before death.

The Effect of Storage on Viability of Toxocara canis Eggs

Four mice were i . tubated orally with 1,000 eggs each which had

been stored in a refrigerator for 27 months after embryonation. The

results presented in Table 11 show that the eggs still retained their

infectivity even after such a long storage. The recovery of 24.5 per

cent to 39.7 per cent larvae from various tissues of mice on days 1,

2, 3, and 7 compared favorably with the results reported earlier by

Malik (1966) in this regard.

The death of two mice on days 6 and 7, after being infected with

10,000, and 15,000 eggs of Toxocara canis, similarly stored for 27

months, further confirmed that the eggs had retained their viability

during all of this period, Table 12.

Similarly, three mice which had been infected with 10,000 and

15,000 eggs of Toxocara canis, stored in a refrigerator for 27 months,

died on challenge with 20,000 freshly embryonated eggs, Table 13.

This showed that these stored eggs were immunogenically no better than

the freshly embryonated eggs, Tables 9 and 10. SUMMARY

The immunological responses of mice to infection with 30-day- old embryonated eggs of Toxocara canis by oral intubation have been studied. Enough information on the lethal effects of these eggs in mice was not available in the litera-ture, and widely divergent opinions had been expressed by different workers in this regard.

Therefore, as a preliminary to the study of immune responses by mice to this infection, minimum lethal dose (M.L.D.), lethal dose 50 (L.D.

50), and lethal dose 100 (L.D. 100) of these eggs for mice were first determined, which were found to be 10,000 eggs, 12,600 eggs, and 20,000 eggs, respectively. The mice that got heavier doses of the eggs, rang­ ing from 100,000 to 500,000 succumbed to such infections within one to two days without manifesting any noticeable clinical signs.

Forth-three surviving mice, out of 85 from experiments for the determination of lethal doses, were challenged with 40,000 eggs, double the lethal dose 100, after four weeks of the primary infection which ranged from 500 to 15,000 eggs per mouse. All of the challenged mice succumbed within four to eight days of the challenge infection, which death pattern was no different from that of the six controls employed in this experiment. Thus, it was demonstrated that the primary infec­ tions with varying doses of Toxocara canis eggs had failed to stimulate the production of sufficient protective immunity in mice to enable them to withstand a challenge infection with twice the lethal dose 100. 91 92

In a repeated experiment 18 mice in six groups of three each were infected with varying doses of Toxocara canis eggs ranging from

5.000 eggs to 20,000 eggs per mouse. Two mice were employed as con­ trols and were not given any infection. All of the mice that received

15.000 eggs and 20,000 eggs each and one out of three from the 13,000 group died within six to eight days of the primary infection. The remainder of the 11 surviving mice along with two noninfected controls were challenged with 15,000 eggs, and 20,000 eggs each after three weeks of the primary infection. All of them, including the controls, died but with a variation in the length of the survival period after challenge, and in the recovery of larvae from their tissues after the death of these mice. Some significant observations which resulted from this study were:

1. The mice that received heavier infections, ranging from 100,000

to 500,000 eggs of Toxocara canis, seemed to be overwhelmed with

such infections, and died within one to two days generally, with­

out manifesting any marked clinical signs.

2. The mice that received lower primary infections with Toxocara canis

eggs succumbed to a challenge infection earlier than those which

received higher primary infections.

3. The order of recovery of larvae from the mice with lower primary

infections and dying on challenge was not very much different from

that of the control mice. On the other hand, fewer larvae were

recovered from the mice with higher primary infections, dying on

challenge, than from the control mice. 93

4. A vast majority of larvae recovered from all the other somatic

tissues and organs of challenged mice was dead, while the larvae

recovered from the brain were mostly active and motile.

5. No protective immunity to lethal doses of Toxocara canis eggs was

seen to result from a previous exposure to sublethal doses.

Larvae were recovered from almost all of the organs, including

liver, spleen, kidneys, lungs, heart, brain, genitalia, intestinal walls, skeletal muscles, and eyes of mice receiving higher doses of

eggs. Paralytic signs were seen, more often, in challenged mice or

in mice receiving higher doses than 10,000 eggs. The evidence of

free blood in the body cavities of mice receiving doses of 100,000

eggs or more was quite visible. The liver, kidneys, genitalia, and

the urinary bladder showed petechial and ecchymotic hemorrhages. The

lungs were highly cyanotic and purplish blue. The pleurae were

thickened, dull, and studded with areas of pustular foci. The brain

showed petechial and ecchymotic hemorrhages, and extra vasation of

blood. The cranial blood vessels were congested. The meninges were

thickened. The brain tissue was soft and malacic. The mice receiving

lower doses of eggs showed correspondingly less severe lesions.

Experiments with 27-month-old Toxocara canis eggs, stored in a

refrigerator after embryonation, revealed that the eggs had retained

their viability and the lethal capacity to fatally infect mice. The

larval yield from mice tissues infected with these eggs was 24.5 per cent on the first day, 31 per cent on the second day, 35.5 per cent on the third day, and 39.7 per cent on the seventh day. 94

A mouse that died 14 months after infection with 1,000 embryo- nated eggs of Toxocara canis yielded a larval count of 187 actively motile larvae from the brain and 132 larvae from the skeletal muscles.

This showed that the Toxocara canis larvae could survive in the host tissues for 14 months without producing any noticeable clinical signs or lesions. LITERATURE CITED

Alicata9 J. E. 1934. Observations on the period required for Ascaris eggs to reach infectivity. Proc. Helm. Soc. Wash. 1:12.

Ashton, N. 1960. Larval granulomatosis of the retina due to Toxocara. Brit. J. Ophthal. 44:129-148.

Baylis, H. A., and Daubney, R. 1926. A synopsis of the families and genera of nematoda. British Museum, London.

Beautyman, W., and Woolf, A. L. 1951. An ascaris larva in the brain in association with acute anterior poliomyelitis. J. Path. & Bact. 63:635-647.

Beaver, P. C., Snyder, C. H., Carrera, G. M, , Dent. J. H., and Lafferty J. W. 1952. Chronic eosinophilia due to visceral larva migrans Pediatrics 9:7-19.

Beaver, P. C. 1954. Parasitic diseases of animals and their relation to public health. Vet. Med. 49:199-202,205.

Beaver, P. C. 1956. Parasitological Reviews. Larva Migrans. Expl. Parasit. 5:587-621.

Buske, M., and Engelbrecht, H. 1968. Uber die larva migrans viscer- alis von Ascaris lumbricoides in experimentalwirt (Maus.). Zeitschrift fur Parasitenkunde 30:337-346.

Chitwood, B. G., and Chitwood, M. B. 1950. An introduction to nematology. 2nd Ed. Sect. I. Baltimore pp. 12-19.

Crandall, C. A., and Arean, V. M. 1965. The protective effect of viable and nonviable Ascaris suum larvae and egg preparations in mice. Am. J. Trop. Med. Hyg. 14:765-769.

Ducas, R. 1921. L'immunite dans la trichinose. These. Paris (Jouve et Cie). p. 47.

Duguid, I. M. 1961a. Chronic endophthalmitis due to Toxocara. Brit. J. Ophthal. 45:705-717.

Duguid, I. M. 1961b. Features of ocular infestation by Toxocara. Brit. J. Ophthal. 45:789-796.

Ehrenford, F. A. 1956. Canine -a potential zoonosis. J. Parasitol. 42 (Suppl.):12-13. 95 96

Fallis, A. M. 1942. Resistance to Ascaris lumbricoides. L. in guinae pigs and the eosinophilia associated with infection. J. Parasit. 28 (Suppl.) :21.

Fallis, A. M. 1944. Resistance to Ascaris lumbricoides L. infection as demonstrated experimentally in guinae pigs. Canad. J. Publ. Hlth., 35:90.

Fallis, A. M. 1948. Ascaris lumbricoides infection in guinae pigs with special reference to eosinophilia and resistance. Canad. J. Res. 26:307-327.

Fulleborn, F. 1929. On the larval migration of some parasitic nematodes in the body of the host and its biological signifi­ cance. J. Helminth 7:15-26.

Hartwich, G. 1957. "Zur Systematik der Nematoden Superfamilie Ascaridoidea. " Zoo. Jahrb. Abt. Syst. 85:211-252. Helminth. Abst'r. 26: Aostr. No. 618, 332.

Hoeppli, R., Feng, L. C., and Li, F. 1949. "Histological reactions in the liver of mice due to larvae of different ascaris species. Peking Natural Hist. Bull. 18(2), 119-131. Helminth. Abstr. 18: No. 726d, 238.

Ivey, M. H. 1965. Immediate hypersensitivity and serological responses in guinae pigs infected with Toxocara canis or Trichi- nella spiralis. Am. j. trop. Med. Hyg. 14:1044-1051.

Ivey, M. H., and Slanga, R. 1965. An evaluation of passive cutaneous anaphylactic reactions with Trichinella and Toxocara antibody- antigen systems. Am. J. Trop. Med. Hyg. 14:1052-1056.

Kerr, K. B. 1936. Studies on acquired immunity to the dog , caninum. Am. J. Hyg. 24:381-406.

Kerr, K. B. 1938a. The cellular response in acquired resistance in guinae pigs to an infection with pig ascaris. Am. J. Hyg. 27: 28-51.

Kerr, K. B. 1938b. Attempts to induce an artificial immunity against the dog hookworm, . and the pig ascaris, Ascaris lumbricoides suum. Am. J. Hyg. 27:52-59.

Kerr, K. B. 1938c. Studies on the passive transference of acquired resistance to the dog hookworm and pig ascaris. Am. J. Hyg. 27:60-66.

Kolmer, J. A., Trist, M. E., and Heist, G. D. 1916. Complement fixation in intestinal parasitism of dogs. J. Infect. Dis. 18:88-105. 97

Koutz, F. R. 1941. A comparison of flotation solutions in the detec­ tion of parasite ova in feces. Am. J. Vet. Res. 2:95-100.

Koutz, F. R., Groves, H. F., and Scothorn, M. W. 1966. The prenatal migration of Toxocara canis larvae and their relationship to infection in pregnant bitches and in pups. Am. J. Vet. Res. 27 (No. 118):789-795.

Lee, H. F. 1960. Effects of super-infection on the behavior of Toxocara canis larvae in mice. J. Parasit. 46:583-588.

Malik, P. D. 1966. Studies on the migration pattern of Toxocara canis (Werner, 1782) larvae in white mice by enteral and parenteral administration. M.Sc. Thesis, The Ohio State University, Columbus.

Morgan, B. B., and Hawkins, P. A. 1953. Veterinary Helminthology. 3rd Ed. Burgess Publishing Co., Minneapolis:33-34.

Mozgovoi, A. A. 1953. On the study of phylogenetic relationships and lines of evolution of the Ascaridata, viewed from the biology and phylogeny of the Ascaridae. (In Russian). Izdatelstovo Akakemii Nauk USSR. Moscow; pp. 422-431; Helminth. Abstr. (1953), 22: Abstract No. 996 cn., 390.

Nichols, R. L. 1956a. The etiology of visceral larva migrans. I. Diagnostic morphology of infective second-stage Toxocara larvae. J. Parasit. 42:349-362.

Nichols, R. L. 1956b. The etiology of visceral larva migrans. II. Comparative larval morphology of Ascaris lumbricoides, , Strongyloides stercoralis. and Ancylostoma caninum. J. Parasit. 42:363-399.

Nifontov, S. N. 1949. Importance of intra-uterine invasion in the epizoology of Toxocara infections in dogs. Veterinariia 26 (10):32-34 (In Russian). Helminth Abstr. 18: No. 506i, pp. 190, 192.

Noda, R. 1961. Studies on the development of eggs of the dog Ascarid, Toxocara canis (Werner, 1782), with an observation on its infec­ tion in mice. Bull. Univ. Osaka Pref., Ser. B., 11:65-75.

Olson, L. J. 1962. Organ distribution of T^ canis larvae in normal mice and in mice previously infected with Toxocara, Ascaris, or Trichinella. Tex. Rep. Biol. Med. 20:651-657.

Oshima, T. 1961. Standarization of techniques for infecting mice with Toxocara canis and observations on the normal migration routes of the larvae. J. Parasitol. 47:652-660. 98

Petrov, A. M. 1941. Importance de l'invasion intrauterine dans 1 1-epizootologie de la toxocarose des renards noirs-argentes. Vestnik Selskokhozyaistvennoi Nauki. Veterinariia 3:84-92 (in Russian: French summary, p. 92). Helminth Abstr. 10, No. 518f, pp. 138-139.

Ransom, B. H., and Cram, E. B. 1921. The course of migration of Ascaris larvae. Am. J. Trop. Med. 1:129-159.

Ransom, B. H., and Foster, W. D. 1917. Life history of Ascaris lumbricoides and related forms. J. Agr. Res., U.S. Dept. Agri., 11:395-398.

Reed, L. J., and Muench, H. 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27:493-497.

Rey, A. 1962. Nematode endophthalmitis due to Toxocara. Brit. J. Ophthal. 46:616-618.

Rubin, L. F., and Saunders, L. Z. 1965. Intraocular larva migrans in dogs. Path. Vet. 2:556-573.

Sadun, E. H. , Norman, L., and Allain, D. 1957. The detection of antibodies to infections with the nematode, Toxocara canis, a causative agent of visceral larva migrans. Am. J, Trop. Med. Hyg. 6:562-568.

Sandground, J. H. 1928. Some studies on susceptibility, resistance, and acquired immunity to infection with Strongyloides stercor- alis (Nematoda) in dogs and cats. Am. J. Hyg. 8:507-538.

Schacher, J. F. 1957. A contribution to the life history and larval morphology of Toxocara canis. J. Parasit. 43:599-612.

Scothorn, M. W. 1963. Observations on the migratory behavior of larvae of the Toxocara canis species in white mice. M.S. thesis, The Ohio State University, Columbus.

Scothorn, M. W. , Koutz, F. R., and Groves, H. F. 1965. Prenatal Toxocara canis infection in pups. J. Am. Vet. Med. Assn. 146:45-48.

Sharp, A. D., and Olson, L. J. 1962. Hypersensitivity responses in Toxocara, Ascaris and Trichinella infected guinae pigs to homologous, and heterologous challenge. J. Parasit. 48:362- 367.

Shillinger, J. E., and Cram, E. B. 1923. Parasitic infestations of dogs before birth. J. Am. Vet. Med. Assn. N.S.V. 16,63:200-203. 99

Smith, M.H.D., and Beaver, P. C. 1953. Persistence and distribution of Toxocara canis larvae in the tissues of children and mice. Pediatrics 12:491-497.

Sprent, J.F.A., and Chen, H. H. 1949. Immunological studies in mice infected with the larvae of Ascaris lumbricoides. I. Criteria of immunity and immunizing effect of isolated worm tissues. J. Infect. Dis. 84:111-124.

* Sprent, J.F.A. 1951. On the migratory behavior of the larvae of various ascaris species in mice. J. Parasit. 37 (Suppl.):21.

Sprent, J.F.A. 1952a. On the migratory behavior of the larvae of various ascaris species in white mice. J. Infect. Dis. 90: 165-176.

Sprent, J.F.A. 1952b. (1) Migratory behavior of ascaris larvae in mice; (2) The dentigerous ridges of the human and pig ascaris. Trans. R. Soc. Trop. Med. Hyg. 46:378.

Sprent, J.F.A. 1953a. On the migratory behavior of the larvae of various ascaris species in white mice. II. Longevity of encap­ sulated larvae and their resistance to freezing and putrefac­ tion. J. Inf. Dis. 92:114-117.

Sprent, J.F.A. 1953b. On the life history of Ascaris devosi and its development in the white mouse and domestic ferret. Parasitology 42:244-258.

Sprent, J.F.A. 1954. The life cycle of nematodes in the family Ascarididae (Blanchard, 1896). J. Parasit. 40:608-617.

Sprent, J.F.A. 1955A. On the invasion of the central nervous system. I. The incidence and pathological significance of nematodes in the central nervous system. Parasitology 45:31-40.

Sprent, J.F.A. 1955b. On the invasion of the central nervous system by nematodes. II. Invasion of the nervous system in ascariasis. Parasitology 45:41-55.

Sprent, J.F.A. 15 7. The development of Toxocara canis (Werner, 1782) in the dog. J. Parasit. 43 (Suppl.):45.

Sprent, J.F.A. 1958. Observations on the development of Toxocara canis (Werner, 1782) in the dog. Parasitology 48:184-209.

Stewart, F. H. 1916. Further experiments on Ascaris infection. Brit. Med. J. 2:486-488. 100

Stewart, F. H. 1918. On the development of Ascaris lumbricoides and Ascaris mystax in the mouse. Parasitology 10:189-196.

Stiles, C. W., and Hassall, A. 1926. Key catalogue of the worms reported for man. Hyg. Lab. Bull. 142:152-156. (U.S.P.H.S.)

Stoll, N. R. 1929. Studies with the strongyloid nematode, Haemonchus contortus. I. Acquired resistance of hosts under natural reinfection conditions cut-of-doors. Am. J. Hyg. 10:384-418.

Taffs, L. F. 1964. Immunological studies on experimental infection of guinae pigs and rabbits with Ascaris suum Goeze, 1782. I. The minimum lethal dose (M.L.D.) and the minimum immunizing dose of Ascaris eggs in guinae pigs. J. Helminth. 38:303-314.

Taliaferro, W. H. 1929. The immunology of parasitic infections. The Century Co., New York and London, pp. 98,270-271.

Tiner, J. D. 1949. Preliminary observations on the life history of Ascaris columnaris. J. Parasit. 35:(Suppl.) No. 7:13.

Tiner, J. D. 1951. Observations on larval carnivore ascarids in rodents. J. Parasit. 37:No. 42 (Suppl.), p. 21.

Tiner, J. D. 1953. The migration, distribution in the brain, and growth of ascarid larvae in rodents. J. Infect. Bis. 92:105- 113.

Unsworth, A. C., Fox, J. C., Rosenthal, E., and Shelton, P. A. 1965. Larval granulomatosis of the retina due to nematodes. Amer. J. Ophthal. 60:127-134.

Wagner, 0. 1933. Immunisierungsvetsuche bie experimenteller Askaris infektion der maus. Z. Immun Forsch. 78:372-382.

Webster, G. A. 1958a. A report on Toxocara canis (Werner, 1782). Can. J. Comp. Med. 22:272-279.

Webster, G. A. 1958b. On prenatal infection and the migration of Toxocara canis (Werner, 1782) in dogs. Can. J. Zool. 36:435- 440.

Whitlock, J. H. 1960. Diagnosis of veterinary parasitisms. Lea & Febiger, Philadelphia:189-198.

Wright, W. H. 1935. Observations on the life history of Toxocara canis and , and the influence of environ­ mental factors on their development. Ph.D. Thesis, George Washington University. 101

Yamaguti, S. 1961, Systema Helminthum; The Nematodes of Vertebrates, Vol. Ill, Part I. Interscience Publishers, Inc. New York & London:573-578.

Yokogawa, S. 1923. On ascariasis and the life history of Ascaris. Taiwan Igakkai Zasshi, Taihoku. 229:241-301 (in Japanese). (English Summary 1-18).

Yorke, W., and Maplestone, P. A. 1926. The nematode parasites of vertebrates. J., and A. Churchill, London.

Yoshida, S. 1919. On the development of Ascaris lumbricoides L. J. Parasit. 5:105-115.

Yutuc, L. M. 1949. Prenatal infection of dogs with ascarids, Toxocara canis and , Ancylostoma caninum. J. Parasit. 35:358-360.