Studies on the Biology of Trichinella Spiralis

Suggested short title

STUDIES ON THE BIOLOGY OF TRICHINELLA SPIRALIS

C. S. Shanta

STUDIES ON THE BIOLOGY OF TRICHINELLA SPIRALIS (OWEN, 1835), RAILLIET, 1895

by Charles Sumitra Shanta

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science

Institute of Parasitology McGill University Montreal June 1966 ACKNOWLEDGEMENTS

I would like to express my deep appreciation and thanks to my research superviser, Professer E. B. Meerovitch of the Institute of Parasitology, McGill University, for his continued guidance and useful criticisms during the course of this research project. My special thanks go to Professer K. G. Davey, Director of the Institute of Parasitology, McGill University, for instruction and advice in the use of the photomicroscope and other photographie equipment; to Miss J. 1. Smith, for obtaining reference material not available in the Institute; to Mrs. M. Couture, for the typing of this thesis; and last, but by no means least, to all members of the Institute of Parasitology, and to fellow graduate students, who, in numerous ways, by word and deed, have contributed to the completion of this research project.

I would also wish to place on record my gratitude and thanks to the Malaysian Government for financial assistance under a Federal scholarship during the first half of this project, and to the External Aid Office, Ottawa, Canada, for financial assistance under the Colombo Plan during the second half of this project. TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS • • • • • • • • • • • • • • • • • • i LIST OF TABLES ...... iv LIST OF FIGURES . . • • . . . • • • • • • • • • • v

Chapt er

I. INTRODUCTION • • • • • • • • • • • • • • • • 1 II. GENERAL MORPHOLOGY AND TAXONOMie POSITION OF TRICHINELLA SPIRALIS • • • • • • • • • • • • 3

A. General Morphology •• • • • • • • • • • • 3 1. The Adult Male 2. The Adult Female B. Taxonomie Position • • • • • • • • • • • • 5

III. HISTORICAL REVIEW--THE BIOLOGY OF TRICHINELLA SPIRALIS • • • • • • • • • • • • • • • • • • 7

A. Early History • • • • • • • • • • • • • • 7 B. Development and Moulting in the Intestinal Phase • • • • • • • • • • • • • • • • • • 8 c. In Vitro Studies of Moulting in the Intestinal Phase • • • • • • • • • • • • • 13 D. Migration of Larvae in the Muscle Phase • 14 1. Time of Appearance of Larvae in Tissues 2. Route of Migration of Larvae 3. Predilection for Striated Muscles E. Moulting of Larvae in Muscle Phase • • • • 19 F. Encystment of Larvae • • • • • • • • • 20 iii - Chapter Page IV. MATERIALS AND METHODS • • • • • • • • • • • • 23 A. Stock Animals • • • • • • • • • • • • • • 23 B. Strains of _r. s:Qiralis Used • • • • • • • 23 c. Preparation of Larvae for Infection • • • 24 D. Infection of Experimental Animals • • • • 25 E. Re co very and Study of Migrating Larvae in Blood • • • • • • • • • • • • • • • • • • 27 F. Recovery and Study of Migrating Larvae in Peritoneal Cavity • • • • • • • • • • • • 27 G. Recovery and Study of Developing Larvae in Muscle • • • • • • • • • • • • . • • • • • 28 1. The Maceration Method 2. The Digestion Method H. Preparation of Tissues for Histological Examination • • • • • • • • • • • . • • • 31 1. Fixation in Osmic Acid 2. Fixation in Zenker's Formol I. Recovery and Study of Intestinal Worms • • 32

v. OBSERVATIONS AND COMMENTS • • . • • • • • • • 36

A. Development in the Intestinal Phase • • • 36 B. Migration of Muscle Larvae • • • • • • • • 53 c. Development of Larvae in the Muscle Phase 58

VI. DISCUSSION • • • • • • • • • • • • • • • • • 70

VII. SUMMARY AND CONCLUSIONS • • • • • • • • • 81

LITERATURE CITED • • • • • • • • • • • • • • • • • • /V'

LIST OF TABLES

Table Page

1. Percentage moults occurring in males between 10-34 hours after infection • • • • • • • • • • 37

2. Percentage moults occurring in females between 10-34 hours after infection • • • • • • • • • • 38 3. Counts of larvae obtained in examinations of blood and peritoneal washings of mice • • • • • 54 v1

LIST OF FIGURES

Figure Page

1. Histogram showing percentages of males moulting between the periods 10-34 hours after infection • • • • • • • • • • • • • • • 37 2. Histogram showing percentages of females moulting between the periods 10-34 hours after infection • • • • • • • • • • • • • . . 38 3. Tail end of male going through first moult at 12 hours post infection. Note that no copulatory appendages are visible • • • • • • 42 Tail end of male going through first moult at 16 hours post infection. Note that copulatory appendages (arrow) are just visible 42 5. Tail end of moulting male going through first moult at 16 hours post infection. Note that copulatory appendages (arrow) are just visible 43 6. Tail end of female going through first moult at 12 hours post infection • • • • • • • • • • 43 Female at first moult (14 hours after infection) showing (arrow) very rudimentary vaginal opening • • • • • • • • • • • • • • • 45 8. Female at second moult (30 hours after infection) showing (arrow) well developed vaginal opening • • • • • • • • • • • • • • • 45 9. Tail end of male going through second moult (JO hours after infection) showing seminal vesicle full of spermatozoa • • • • • • • • • 46 10. Seminal receptacle of female going through second moult (32 hours after infection) showing no spermatozoa • • • • • ••••• • • 46 vi

Figure Page

11. Anterior end of muscle larva freed from its cyst. Note that the amphids are not developed • • • • • • • • • • • • • • • • • • 49 12. Anterior end of intestinal worm at 6 hours after infection. Note that the amphids are well developed • • • • • • • • • • • • • • • • 49 13. Anterior end of intestinal worm at 10 days after infection. Note that amphids are absent • • • • • • • • • • • • • • • • • • • • 50 14. Section of masseters of mouse, infected 24 days previously, showing portions of a coiléd up larva. Note the cuticle of the larva and the absence of cuticle-like material on the inside of the muscle tissue spaces. Stained with Verhoeff's Van Gieson stain • • • • • • • 50 15. Portion of 15-day-old muscle larva showing cuticle with ridges (arrow} on the dorsal aspect of the worm. Stained with iodine • • • 61 16. Portion of 15-day-old muscle larva showing cuticle with ridges (arrow) on the dorsal aspect of the worm. Stained with iodine • • • 61 17. Posterior end of a degenerating larva (ll days after infection) in whole mount preparation. Note artefact at posterior end resembling a moult ••••• • • • • • • • • • • • • • • • 64 18. Posterior end of degenerating larva (15 days after infection) in whole mount preparation. Note imprint in mounting medium resembling a moult • . . • . • . . . • . • ...... 64 I. INTRODUCTION

The literature on the biology of Trichinella spiralis is full of many confusing and conflicting reports. The reviews of Reinhard (1958} and Schwartz {1960) of the early work on T. spiralis show that the unique character of the biology of this worm confounded the early workers, who attempted to unravel its entire life cycle. Although many studies have since been made on the biology of this parasite, and much is now known about it, it will be seen from the literature review on the subject that many varied and contradictory reports have been made. Most of these reports have raised questions and doubts, which have not been resolved up to the present time.

The entire life cycle of T. spiralis is made up of 2 phases of development. One phase refers to the sequence of events that take place when infective larvae enter into the intestinal environment of a definitive host until they reach the adult stage. This phase is referred to here as the intestinal phase of the life cycle of T. spiralis. The other phase refers to the 2 sequence of events that take place when young emerging larvae, born of gravid females, enter into the extra intestinal environment of their hosts, migrate to the striated muscle fibres, and develop there to become the infective stage larvae. This phase is referred to here as the muscle phase. Many descriptions of the events that take place in these 2 phases of development have been given. Perhaps the most controversial aspect of the biology of this parasite has been the question of the times of moulting and numbers of moults that this parasite undergoes during its entire life cycle. The present studies were carried out chiefly to learn more of this aspect of the biology of the parasite. II. GENERAL MORPHOLOGY AND TAXONOMIC POSITION OF TRICHINELLA SPIRALIS

A. General Morphology

The general anatomical characters of both the adult male and female of T. spiralis have been described by Gould (1945) as follows.

1. The Adult Male The adult male usually measures 1.4 to 1.6 mm. in length and from 0.033 to 0.040 mm. in diameter, but may vary from 0.6 to 2.2 mm. in length. The oesophagus of the male worm runs posteriorly, slightly over half the length of the body. The male possesses two character istic, conical, winged copulatory appendages at the posterior extremity, which are believed to serve as holding organs during copulation. The cloacal opening in the male is surrounded by a row of 4 tubercles. The male has a single testis, which almost completely fills the posterior half of the body cavity. The testis is cylindrical in shape but wider posteriorly than anteriorly. At its posterior end the testis becomes narrower and curves backward to continue along the 4 ventral side as the narrow vas deferens, which leads to a dilated seminal vesicle that is filled with spermatic cells. The seminal vesicle terminates by joining the cloaca, which opens at the posterior end. The cloaca of the male is wider than that of the female. It opens between the copulatory appendages, and may become everted in the shape of a bell to serve as a copulatory organ. After copulation, the projected cloaca is withdrawn by the action of the musculi retractores cloacae, which are situated just anterior to the cloaca and between the muscular layer of the worm and the intestine.

2. The Adult Female The adult female usually measures from 2.2 to 3.6 mm. in length and from 0.060 to 0.072 mm. in width, but may vary from 1.1 to 4.8 mm. in length (Roth, 193Sa}. Growth of the female is more rapid than that of the male. The growth of the sexual apparatus of the female is particularly marked so that the oesophagus and "cell body" (stichosome) are relatively shorter than in the male, the oesophagus running posteriorly only one-third of the length of the body. The female is further characterized by the presence of a genital opening or vulva, which opens on the ventral aspect onto the cuticle at the posterior end of the first fourth or fifth of the body. The vulva leads posteriorly to a narrow vagina, 5 which in turn leads to the prominent uterus. In the gravid female, the anterior portion of the uterus contains numerous larvae in varying stages of development, while the posterior portion of the uterus is occupied by younger stages of developing ova. Behind the relatively wide uterus, there is a constriction which separates it from the narrow single ovary. The ovary reaches nearly ta the posterior end of the body. Ova are formed at one side of the ovary. When they mature, they become detached from the ovarian wall and eventually reach the posterior portion of the uterus, i.e., the seminal receptacle. Spermatozoa are stored in the seminal receptacle and fertilization of the ova takes place there. Towards the anterior portion of the uterus, the ova develop into little curved embryos or larvae, and as the latter approach the vagina they become freed from their envelopes. The larvae, having developed in the uterus, can pass only singly along the narrow vagina ta be expelled through the vulva.

B. Taxonomie Position

Trichinel1a spiralis has been placed in the arder Trichuroidea by Yorke and Maplestone (1926), Chitwood and Chitwood (1941} and Yamaguti (1961}. The basis for this 6 classification is best described by Chitwood and Chitwood (1941) as follows: a. muscular tissue of posterior part of oesophagus somewhat reduced; b. oesophageal glands outside contour of oesophagus in the form of a single or double row of cells (stichocytes); c. intestine not extending anterior to base of oesophagus; d. male with one or no spicules; e. female reproductive system highly developed, with vagina greatly elongated and tubular.

T. spiralis has been further placed in the family Trichinellidae by Yorke and Maplestone (1926), Chitwood and Chitwood (1941) and Yamaguti (1961). Again, the basis for this classification is best described by Chitwood and Chitwood (1941) as follows: a. females are viviparous; b. males are without spicules; c. stichosome is made up of a single row of cells.

The family Trichinellidae is monogenic and con tains the single species, T. spiralis. III. HISTORICAL REVIEW--THE BIOLOGY OF TRICHINELLA SPIRALIS

A. Ear1y History

The review of Reinhard (1958) gives an interesting account of the early history of T. spira1is. This account is briefly described as fol1ows. The 1arvae of T. spira1is were first discovered in the muscles of man by James Paget in 1835. In that same year, Owen described the parasite and named it Trichina spira1is. Rai11iet, in 1895, changed the name to Trichinella spiralis because the generic name Trichina had previously been used to designate a genus of flies.

Leuckart (1860) was the first to attempt to unravel the life cycle of T. spiralis. Both he and Virchow (1860a, 1860b), working independently with laboratory animals, proved that the entire life cycle of the worm took place in one host. This fact made T. spiralis unique among parasitic nematodes.

Leidy, in 1846, was the first to discover the larvae of T. spiralis in the extensor muscles of the thigh of a hog (Gou1d, 1945). Herbst, who in 1850 encountered Trichinella cysts in a cat, was the first to establish that an animal which eats trichinous meat may develop cysts in its muscles (Reinhard, 1958). Since then, natural infections have been found in mouse, rabbit, beaver, pole cat, domestic cat, palm civet, dog, wolf, coyote, fox,marten, ferret, European and American badgers, raccoon, polar bear, common bear, and mongoose (Gram, 1941). It has also been found in wild boar, rat, coypu, hedgehog, marmot, mole, hippopotamus, and porcu pine (Gould, 1945). Thus "Trichinella spiralis is perhaps the single species of parasitic nematode least restricted in its range of hosts" (Bachman and Rodriguez Molina, 1933).

B. Development and Moulting in the Intestinal Phase

Infection with T. spiralis begins when infective larvae are ingested in a viable state as in meat that is raw or insufficiently cooked. The muscle fibres and the capsules which enclose the parasites are digested within a few hours and the larvae are liberated. These larvae pass into the small intestine, where they anchor themselves to the mucosa, from which they obtain tissue juice for nutrition and oxygen for their metabolism {Heller, 1933). In experimental work with mice and hamsters, Boyd and 9

Huston (1952) found that penetration of the small intestine by the larvae occurred within an hour after ingestion of the encysted larvae. Gursch (1949), who did a detailed study on the intestinal phase of the life cycle of T. spiralis, found that, in rats, penetration of the small intestine by the larvae began at about 2 hours, with the majority of the larvae embedded in the mucosa by about the 4th hour post infection. After 20 hours of infection, most worms were located in the lumen of the intestine. He thought that this emergence into the lumen was for copulation. However, Doerr and Menzi {1933) thought that copulation must take place in the mucosa because they never observed mature females and males in the lumen. Gursch (1949) found that, after 2 days, the worms again penetrated deep into the mucosa with only a few adults remaining in the lumen. He also found that males were embedded close to the muscular layer. This finding did not agree with the observations of Kreis (1937), who believed that only sexually mature females bore into the intestinal villi. Gould (1945) states that, after copulation, the male dies while the gravid female, whose anterior portion is embedded in the mucosa, continues to grow in size. The female is viviparous, and gives birth to her motile, minute larvae, which, according to Gould {1945), are deposited within lü

the mucosa or directly into the central lacteals of the villi or other lymphatics.

From the time of ingestion of infective larvae to the attainment of sexual maturity, moulting of the Trichinella worm takes place. However, many confusing reports on the number of moults, the times of moulting and of copulation by the worms have been made. Hemmert Halswick and Bugge {1934), working with rats, found that the first moult occurred at about the 5th hour and the second moult between the lSth and 26th hour after ingestion of infective larvae. They determined that copulation of intestinal worms occurred about 40 hours after infection. Further development was rapid and completed within 4 days. Kreis (1937), also working with rats, stated that the female worm undergoes 4 moults, the first moult between the 2nd and Sth hour, the second between the l2th and l6th hour, the third at the 4Sth hour, and the fourth and final moult at about the 72nd hour post infection. Insemination of the female was said to occur between the second and third moults. According to Kreis (1937), in the male, 2 moults occurred before the sexual stage (about the lSth hour) and only l more moult after this stage at about the 20th hour after infection. Thus, in his view, the male underwent only 3 moults. The development of copulatory appendages in 11 the male was said to occur at about the lSth hour after infection. Lapage {1956) also stated that there are 4 intestinal moults. Hyman (1951), on the other hand, states that there is only 1 intestinal moult. Berntzen {1965), working with rats, concurs with Hyman (1951) and states that the 1 moult can occur any time between the 8th and the 72nd hour post infection. Richels (1955), working with mice, found the first occurrence of a moult at the 16th hour post infection. She also found moults occurring at the 24th, 36th and 54th hours post infection. She did not mention, however, that these corresponded to the first, second, third and fourth moults respectively, and it is to be inferred that they referred to only one and the same moult. Wu and Kingscote (1957} described only the final moult as observed in rats. This occurred between the 27th and 32nd hour after infection. While it is to be inferred that no further moult took place after the 32nd hour, it did not preclude the possibility of one or more moults occurring before the final moult. Villella (1958), working with rats, stated that the pre adult undergoes 4 moults to reach the adult stage. Most of the larvae developing into females undergo successive moults at the 6th, 12th, làth and 24th hours after infection, while those destined to become males moult 6 hours later, i.e., the 12th, lSth, 24th and 30th hours after infection. Podhajecky (1964b), in his experiments 12 with mice, found that 2 moults occurred, the first between the 14th and 18th hour and the second between the 22nd and 37th hour after infection. He observed copulation of the intestinal worms to have occurred after the second moult, i.e., 37 hours after infection. Thomas (1965), also working with mice, found slightly different resulta. The first moult in both males and females occurred between the 12th and 15th hour after infection. In the female, the second moult occurred between the 26th and 29th hour, while in the male, it occurred between the 20th and 26th hour after infection.

Young emerging larvae are deposited singly through the vulva of the mother worm, and according to McCoy's (1931) studies on rats, it was estimated that a single female worm released from 200 to 400 larvae. Roth (1939) estimated an average of 1,000 to 1,500 young larvae. Nolf (1937), also working with rats, found an average of just over 1,000 larvae, whilst Edney, Arbogast and Stepp (1953) calculated that each female worm produced about 345 larvae. The young 1arvae are said to be released,on the average, at the rate of 1 every half hour (Roth, 1938b, 1939}. Podhajecky (1964a} observed, in his in vitro studies, that larvae were released at intervals of less than 15 minutes. 13

c. In Vitro Studies of Moulting in the Intestinal Phase

Many investigators have attempted to grow decapsu lated larvae of T. spiralis and have had varying degrees of success. The earliest worker to attempt culture of this worm was Keilty (1914). He was able to maintain decapsulated muscle larvae in a balanced salt solution, to which nutrient material had been added. He reported an increase in length but there was no tissue differenti ation or organogenesis. Weller (1943) cultivated decapsulated muscle larvae axenically in roller tube tissue cultures, and he reported sorne growth and organo genesis leading to sexual differentiation, along with the first reports of moulting in vitro. Most of his worms went through 2 moults, but he also obtained sorne super numerary moults, in which the external genitalia appeared after the 3rd incomplete moult. Supernumerary moults were also observed by Kim (1961, 1962), but these as well as those of Weller (1943) were ascribed to abnormal conditions in vitro (Meerovitch, 1965). Berntzen (1962) was able to culture T. spiralis decapsulated larvae axenically to the adult stage, in which the males con tained spermatozoa and the females contained developing embryos. During this growth, he observed only l moult. He states that worms could be induced to produce more than l moult; however, under these conditions, worms 14 would differentiate, reduce in size, and produce multiple sheaths. None of the worms were able to free themselves of their multiple sheaths and no females produced embryos. Tarakanov (1964) obtained 4 moults in his culture system and he observed the development of sex organs and appendages at about the 16th to 18th hour after culture. Berntzen, in 1965, again reported on the culture of T. spiralis decapsulated larvae. In this report, he mentioned that the females became gravid, but that during the period of cultivation, he observed again only 1 moult. Meerovitch (1965) obtained 2 moults with his culture system and he observed the appearance of the caudal appendages in the male and the vulvae in the female at the 2nd moult.

D. Migration of Larvae in the Muscle Phase

1. Time of Appearance of Larvae in Tissues New-born Trichinella larvae are cylindrical in shape, measure 80 to 120~ in length and 5 to 6)U in diameter {Gould, 1945). They are thus slightly narrower than the diameter of a red blood cell. Their size remains apparently unchanged during their successive transit from the intestine into the tissues of the host. Phillipson and Kershaw (1960) found that in mice, larvae 15 recovered from the muscle at the earliest stage of infection were of the same size as those expelled from the adult female worms, and as those found in the blood.

The first occurrence of young 1arvae in the b1ood and tissues of infected hosts have been reported differ ent1y. Hel1er (1933) stated that he observed young 1arvae in rats and cats at 80 to 90 hours after infection. Dun1ap and Wel1er (1933) found 1arvae in the myocardium of white rats 5 days after infection. Hemmert-Halswick and Bugge (1934} stated that they observed the release of 1arvae 5 days after infection of rats, following which they found larvae in the striated muscles. Spink and Augustin (1935), working with rats, observed the first young 1arvae being deposited in the lymph spaces on the 4th day. Roth (l938a) found them on the 6th day and Lehmensick and Senadisaya (1941) observed them on the 5th day after infection. In man, larviposition is said to begin at 7 days after infection (Gould, 1945). Ketz {1952) observed larvae in hamsters at 7 days post infection; in rats,they were observed at 7 days by Szeky and Nemeseri (1956); in white mice, at 6 days by Phi11ip son and Kershaw (1960), and at 5 days by Podhajecky (1964a). Berntzen, working with rats, found larvae in the body fluids at 5 days post infection. 16

2. Route of Migration of Larvae The emerging young larvae have been reported to be deposited directly into the lymphatics or the lacteals of the intestinal villi (Askanazy, 1895; Graham, 1897; Staubli, 1905). However, Leuckart (1860) maintained at that time, that larvae reached the various parts of the host body by migration from the intestine through the connective tissue. Staubli, in 1909, reported finding migrating larvae in body fluids of rabbits, but he found them in larger numbers in the blood. Heller (1935) considered that the migration of larvae was mainly a passive carriage through the bloodstream by way of lymph vessels and that direct penetration of capillaries did occur. Nolf and Crum {1937) found very few larvae in blood obtained from the left ventricle, the right ventricle, and the portal vein of infected rats, but none in the lymph cistern, liver and lungs. Matoff (1943}, working with guinea pigs and rabbits, felt that the lymph vessels were the chief distribution route for the young larvae. Gould {1945) states that the young larvae pass through the intestinal lymphatics, thoracic duct, right side of the heart and pulmonary circulation, until they reach the striated muscles of the body and penetrate the sarcolemmal sheaths of the muscle fibres. Phillipson and Kershaw (1961), working with mice, found very few larvae 17

in b1ood during the peak of 1arval production, and con sidered that if the 1arvae were carried by the blood, they must leave the circulation very quickly.

Only recently, Berntzen (1965) made a detailed study of the migration of larvae from the intestine of white rats to their final location in the striated muscles. He examined body fluids, mesenteric lymph nodes, mesentery, blood, thymus, 1iver and lungs. He was unable to find larvae in the thymus, liver and lungs, but he found a few larvae in blood obtained by total exsanguination in only sorne of his animals. He found however, large numbers of larvae in the body fluids of both the abdominal and thoracic cavities, and direct microscopie examination of fresh mesentery showed larvae migrating through the connective tissues. He concluded, therefore, that larvae migrate through the connective tissues but that carriage by the bloodstream can and does occur. He suggests the possibility that the routes of migration of larvae may differ from one species of host to another.

3. Predilection for Striated Muscles Whatever their mode of travel, the young larval worms penetrate the sarcolemma of the skeletal muscle fibre (Virchow, 1860b; Hertwig and Graham, 1895; Jensen and Roth, 1938}, with the aid of a minute spear and 18 perhaps, with the help of a histolytic enzyme (Jensen and Roth, 1938). Lewert and Lee (1954}, however, feel that no such enzyme is produced to facilitate penetration of muscle fibres.

Various reasons have been advanced as to why Trichinella larvae will develop only within striated muscle fibres. Flury (1913) believed that it was because of the parasite's need for glycogen, but Trichinella larvae will not develop in the liver, which is abundantly rich in glycogen. Berger and Stahelin {1928), working with guinea pigs, showed that the degree of trichinous invasion was not proportional to the amount of work done by a muscle, and therefore, to the amount of blood supplied to it. Doerr and Schmidt (1929) injected gravid female Trichinella adults directly into the circulation of the guinea pig and determined the intensity of local ization of muscle larvae, which developed in various muscles. They found that the distribution of larvae in various striated muscles, whether by natural enteric infection or by parental infection, was not determined merely by the mechanical factor of transportation of the larvae in the bloodstream. Hemmert-Halswick {1934) referred to the affinity of the larvae for striated muscle as "organotaxis" or chemical affinity. Heller (1935) suggested that oxygen may be the decisive factor 19 influencing the intensity of infection within the muscles. Scheifley (1937), working with dogs, showed that intensity of infection was not dependent upon the blood supply to given muscles, whereas Britov (1960), also working with dogs, stated that the distribution of Trichinella larvae in the muscles depended on the physical activity as well as the blood supply of the given muscles. The true reason, therefore, for the localization of Trichinella larvae in striated muscle is not clearly understood (Gould, 1945}.

E. Moulting of Larvae in Muscle Phase

One of the most controversial aspects of the life cycle of T. spiralis has been the question of moulting during the muscle phase of its development. Chandler, Alicata and Chitwood (1941) postulated, on analogy with other nematodes, that there were possibly 2 moults within the muscle phase. Hyman (1951) states that the encysted muscle larva is the 4th stage larva, inferring therefore, that there are 3 moults before encystment. Lapage (1956) states that the infective larva goes through 4 moults in the intestines and therefore, the infective larva must be the lst stage larva. Meerovitch (1965) reported to have observed moulting in the muscle phase and felt that the infective larva was probably the Jrd stage larva. 20

Berntzen (1965) claims to have observed the first moult occurring in utero, and that there are 2 further moults before encystment. He concurs with the statements made by Hyman (1951). Finally, Thomas (1965), on the basis of indirect circumstantial evidence, believes that there is probably 1 moult in the muscle phase prior to encystment.

E. Encystment of Larvae

In white mice, penetration of the muscle fibres can be observed as early as the 7th day post infection (Zarzycki, 1951). Themann (1960) found that larvae penetrated into the muscle fibres of rats on the lOth to 12th day post infection. Phillipson and Kershaw (1960) found that, in mice, the growth of the first deposited larvae during the first few days appeared to be slow; it accelerated between the 12th and 20th day, of infection, and then slowed down again until the maximum length was reached about the 24th day. At about this time, the worms had attained a length of about 1 mm. and a diameter of 0.035 mm. At this stage they became sexually differ entiated, and in general, the females were longer than the males. They also found that on the 17th day, the larvae were resistant to acid-pepsin digestion and were infective to new hosts. Coiling of larvae appeared on • about the 17th day • 21

In guinea pigs, Amelung (1952) found that larvae • were infective from the 15th day after infection and the beginning of coiling of the larvae was observed at 18 days after infection. In hamsters, Schaaf and Lampe (1958) found invasive forms on the 19th day after infection. Nolf and Edney {1935) found them on the 17th day after infection in rats.

Wantland, Bardes and Levine (1945) have given a comprehensive description of the nature of the mechanism of encapsulation in trichinosis which is as follows:

Following entrance of trichina larvae into the striated musculature of the host, inflammation is set up by the movements and waste products of the rapidly growing larvae, the fiber becomes granu lar and swells, giving rise to pressure necrosis and degeneration of the surrounding fibers. Considerable white cell infiltration occurs and the fibers of the interfascicular connective tissue undergo hypertrophy. A delicate parietal layer is then formed around each larva, which is apparently the beginning of a ~ibrosis resulting from the alteration of the normal condition of the tissues. The infected tissue undergoes rapid histological changes and an intercellular exudate is formed. The delicate parietal layer surrounding the invading parasite arises by a direct and gradual transformation of the minute elements of the exudate. This transformation is effected by the pressure and tension which is applied to the fibrillations that appear in the exudate as a result of inflammation and swelling of the invaded fiber. The enveloping sheath becomes thicker as more fibrillated layers are added. The wall becomes homogenous and hyaline in nature and its deeper layers unite at the poles of the cyst. In sorne instances complete atrophy of the muscle fiber results; at other times the striations appear normal above and below the cyst and the sarcolemma is continuous with the most external layer of the cyst wall. 22

They conclude by saying that the fibrous nature of the cyst can be readily seen in both fresh and fixed preparations of infected rat and rabbit diaphragm muscle when viewed directly with the microscope.

Cyst formation in man, laboratory animals and naturally infected domestic animals begins at about 19 to 20 days after infection and calcification starts in the 14th to 16th month, the first fully calcified cysts appearing after 2 years (Berezantsev, 1961, 1962). In hamsters, Schaaf and Lampe (1958) found that all muscle larvae were coiled up by the 30th day and encapsulated by the 60th day, but even 14 months after infection, no calcification could be detected. Berezantsev {1957) states that the rate of calcification varies not only with the host species but also with the host's physio logical condition, but that the mechanism of migration and cyst formation is the same in all hosts (Berezantsev, 1963). IV. MATERIALS AND METHODS

A. Stock Animals

In the present work, only Swiss albino mice were used. They weighed approximately 15-20 gm. and were in good physical condition. Every attempt was made to ensure that in each experiment, only animals of the same sex and weight were used.

B. Strains of T. spiralis Used

Two strains of T. spiralis were used for all the experimental studies. One was the "Joliette" strain, originally obtained from infected pork, which caused an outbreak of trichinosis in Joliette, Quebec Province, and maintained in this laboratory for over 2 years by serial passage in rats.

The other strain used was the 110ld" strain, main tained in this laboratory for over 11 years by serial passage in rats, and originally received from the Animal Diseases Research Institute in Hull, Quebec Province. 24

c. Preparation of Larvae for Infection

Ground infected muscle tissue was digested in the proportion of approximately 4 gm. of tissue to 100 ml. of acid-pepsin digesting fluid, which was made up of 0.5% pepsin powder N.F. (Fisher Chemical Company) and 0.7% hydrochloric acid, according to the formula of Avery (1941). Digestion was facilitated by the use of a magnetic stirrer, set at low speed and carried out for about 5 hours in an incubator maintained at a constant temperature of J7.5 C. At the end of digestion, the freed larvae were allowed to settle for about JO minutes. Sorne of the supernatant was siphoned off and the remain ing fluid containing the larvae was filtered through a double layer of cheese cloth into sedimentation glasses. The larvae were again allowed to settle for another JO minutes and the supernatant discarded. Lastly, the larvae were washed J times with 0.$5% saline and then allowed to settle for 15 minutes. After discarding the supernatant, the washed larvae were collected into conical centrifuge tubes and an estimate of the total number of larvae was made by use of the McMaster nematode egg counting slide. A total of J counts was made to obtain an average, from which the total number of larvae was calculated.

After the dose rate of each experimental animal was determined, suspensions of infective larvae were made 25

in such a manner that 0.25 ml. of the suspension contained 1 infective dose.

D. Infection of Experimental Animals

All experimental animals were deprived of solid food for a period of 6-8 hours prior to infection. The animals were anaesthetized by ether and the larvae were introduced directly into the stomach, by use of a tuberculin syringe fitted with an 18-gauge hypodermic needle which was slightly bent and blunted at the tip. Different batches of experimental animals were infected at different dose rates according to the following schedule:

a. Experimental animals that were used for the study of migration of larvae in blood, peritoneum, muscles and for the study of histological specimens

fixed in Zenker1 s formol, were infected at the dose rate of 30 larvae per gm. of bodyweight. The "Old" strain of T. spiralis larvae was used in these experiments.

b. Experimental animals that were used for studies of the development of intestinal worms were infected at the dose rate of 40 larvae per gm. of body weight. The "Joliette" strain of T. spiralis wa.s used • in these experiments • 26

c. Experimental animals ~rom which histological specimens ~ixed in osmic acid were prepared, were i~ected at the dose rate o~ 60 larvae per gm. o~ bodyweight. The reason ~or this high dose rate was to obtain a greater in~iltration o~ muscle larvae during the early phase o~ larviposition by the gravid ~emales. Each of these animals was treated 8 days after infection with the anthelmintic, methyridine (trade name npromintic" o~ Imperial Chemical Industries, Ltd.}, courteously provided by Dr. D. Campbell o~ Ayerst, McKenna and Harrison, Ltd. The dose rate given was 500 mgm. per Kgm. bodyweight.

The drug was, there~ore, made into a 5% solution in sterile 0.85% saline and each animal was injected sub cutaneously at the dose rate of 0.1 ml. of the diluted drug per 10 gm. bodyweight. The reason for treatment with methyridine was to eliminate all adults present at the 8th day of infection, so that the age of migrating larvae studied in histological sections, could be more accurately established. The drug is believed to have very little or no action on the migrating larvae, whereas it is excellent in its action on the intestinal worms (Schanzel and Hegerova, 1963}. My preliminary experiments, conducted to test the efficacy o~ the drug, showed that methyridine had lOO% efficiency against the intestinal worms. 27

E. Recovery and Study or Migrating Larvae in Blood

Blood ror study was obtained by exsanguination because, in earlier experiments, recovery or blood by cardiac puncture was round to be unsatisractory. The animals were anaesthetized with ether, an incision or the skin made on one side or the neck and with a sharp pair or scissors, the blood vessels were severed. The blood was allowed to drip into a centriruge tube containing about 1 ml. of a 1% solution of sodium citrate. After as much blood as could be obtained was collected, the blood was mixed with the citrate solution by gentle shaking to prevent clotting.

Enough de-ionized water was then added to the citrated blood to cause lysis or the red blood cells. The contents were then centriruged ror about 2 minutes at 1000 r.p.m. The supernatant was discarded and enumeration of the larvae in the sediment was made under the low-power objective (X 100 magnification) or the light microscope.

F. Recovery and Study or Migrating Larvae in Peritoneal Cavity

Arter exsanguination, the animals were stretched out on their backs on a dissecting board and held down firmly. The skin on the ventral part of the abdomen was 28 first dissected out, a puncture made through the abdominal muscles with the end of a pasteur pipette and about 3 ml. of 0.85% saline was introduced into the peritoneal cavity. The abdominal cavity was washed by repeated pipetting in and out, and the washings were collected into a conical centrifuge tube. The contents were centrifuged at 1000 r.p.m. for about 2 minutes and the larvae in the sediment were counted under the lower power objective (X lOO magnification) of the light microscope.

The mesentery was then dissected out and released from its intestinal and abdominal attachments. It was spread out, pressed between two glass slides and examined for migrating larvae under the law-power objective (X lOO magnification) of the microscope.

G. Recovery and Study of Developing Larvae in Muscle

Two methods were used in the recovery of muscle

larvae ~-

l. The Maceration Method The method used was essentially that of Levin (1941). The animals were skinned and eviscerated and the carcasses were finely minced with scissors. Only half of the minced tissue was used in the maceration method; the 29 other half was subjected to the process of digestion described below. For the maceration method, the tissue was placed in a 250 ml. Erlenmeyer flask and about lOO ml. of 0.85% saline was added. A few glass beads were introduced into the suspension and the whole mixture gently shaken by hand for about 3 minutes. The whole suspension was filtered through a 60-mesh sieve and col lected into a sedimentation glass. The filtrate was a1lowed to stand for about 30 minutes, the supernatant then siphoned off and the sediment containing 1arvae collected for further processing.

2. The Digestion Method The method followed was essentially the same as that used by Berntzen (1965). One hour before autopsy, a solution of 2 gm. of trypsin 1:250 (Difco) in lOO ml. of 0.85% saline and 0.5 ml. of a 5% NaHco solution was 3 warmed in an incubator at 37.5° c. and continually stirred with a magnetic stirrer at 1ow speed. The remaining half of the minced tissue was added to this solution, and digestion was allowed to proceed for 2 hours at )7.5 C. The digested material was filtered through one layer of cheesecloth and collected into two 50 ml. conical centrifuge tubes. These were centrifuged for 10 minutes at about 1000 r.p.m. The supernatant was siphoned off and the sediment containing larvae was 30 collected for further processing.

Larvae obtained following maceration and digestion of muscle were processed and examined in the following ways:

a. Fresh material containing live larvae was examined under both the light and phase contrast micro scopes at X 100 and X 400 magnifications.

b. Sorne of the fresh material was lightly stained with 10% aqueous iodine solution and examined under both the light and phase contrast microscopes at X 100 and X 400 magnifications.

c. A few drops of pig serum were added to the balance of the sediments and mixed. Portions of the mixed suspension were spread out on albuminized slides as thin films, and allowed to evaporate until almost dry. The slides were then immersed in Schaudinn's fixative for 24 hours and after treating in the usual manner for mercuric chloride fixed specimens, stained with Harris' haematoxylin, mounted and examined. Examinations were carried out under the light and phase contrast micro scopes at X 100, X 400, and X 1000 magnifications. 31

H. Preparation of Tissues for Histological Examination

Preliminary examination of pieces of muscles from different parts of the animal body showed that the con- centration of larvae was high in the masseters. For this reason only the masseters were used for histological studies.

1. Fixation in Osmic Acid The method used was patterned on that described by Davey (1965}. Since preliminary studies showed that 1% osmic acid did not penetrate satisfactori1y into the masseter muscles, the tissues were fixed for 24 hours in ice-cold 1% osmic acid in 0.9% NaCl, rinsed in distilled water, and transferred to the ethyl gallate solution of Wigglesworth (1957) for at least 24 hours. The tissues were washed, dehydrated through ethyl alcohol, embedded in nEster wax 1960n {British Drug Houses, Ltd.), and sectioned on a rocking microtome at a thickness of 2AI, After the wax had been removed from the sections, they were mounted, without further staining, in Farrant's medium containing a trace of ethyl gallate, and these were examined by both light and phase contrast microscopy at magnifications of X 100, X 400, and X 1000.

In these studies, masseter muscles were examined at 3-day interva1s beginning from the 12th day until the 36th day post infection. 32

2. Fixation in Zenker's Formol Masseter muscles were fixed in Zenker's formol for a period of 6 to 7 hours. They were then washed over night in running water, processed in the usual manner, and were embedded in "Tissuemat" (Fisher Scientific Company). Sections were eut on a rocking microtome at 7)U, mounted on albuminized slides and stained with Verhoeff's Van Gieson elastic stain.

In these studies, masseter muscles were examined every day beginning from the 9th day until the 35th day post infection.

I. Recovery and Study of Intestinal Worms

Killed animais were stretched out on their backs and held down firmly. The skin covering the abdomen was dissected out and the abdominal muscles eut open. The duodenum was severed at the pyloric end and the entire small intestine was released from its mesenteric attach ments by means of gentle traction. The small intestine was severed again at the ileo-caecal junction.

The small intestine was placed in a petri dish containing sufficient warmed Tyrode's solution with glucose, and it was eut into convenient lengths of about 10 cm. These short lengths of intestine were eut open 33 with scissors, very gently shaken in the Tyrode's solu tion to loosen and remove gross particles of food. The washed lengths of intestines were placed in a 250 ml. Florence flask containing approxirnately lOO ml. of warmed Tyrode's solution with glucose. Carbon dioxide was then gently bubbled through the suspension for approximately 2 minutes. The reason for this procedure was to simulate as far as possible an anaerobie environment within the Florence flask, similar to that prevailing in the small intestine. It was felt that prolonged exposure of the intestinal worms to atmospheric oxygen might have an adverse effect on the worms. Such adverse effects could alter the interpretation of findings. It was also felt that the bubbling effect of the gas might help mechanically in the release of worms from the mucosa. After the bubbling, the outlet and the inlet of the flask were tightly clamped and the flask was incubated for 45 minutes at 37.5 0 C. After incubation, when the worms had detached themselves from the mucosa, the suspension of intestines was filtered through a coarse mesh tea strainer into a sedimentation glass. The filtrate was allowed to stand for approximately 15 minutes and the supernatant was siphoned off. The remaining suspension containing larvae and adults was mixed with an equal volume of 10% buffered formol saline. 34

Enumeration of moulting larvae was carried out in the following manner. All of the formalinised larvae were resuspended as evenly as possible and half of the suspension was pipetted off into a conical centrifuge tube. The tube was centrifuged for about 2 minutes at approximately 1000 r.p.m. The supernatant was then siphoned off and all of the sediment was wet mounted on a slide under a 22 x 40 mm. coverslip. The numbers of both male and female moulting and non-moulting worms were counted under the low-power objective (X 100 magnification) of the light microscope. The results obtained were converted into percentages and represented an enumeration based on 50% of the total number of worms.

As mentioned earlier, Bern~zen (1965), in his studies of moulting in T. spiralis, observed the first moult of the embryo occurring while it was still within the uterus of the gravid female. In an attempt to observe this, embryonic larvae in utero were examined in the following manner. Gravid females, which were larvipositing, were picked out with the aid of a needle and collected in a small quantity of 0.85% saline in a cavity block. The uteri, containing developing eggs and larvae, were teased out of the female worms by means of fine needles. Further teasing of the uteri by needles caused the eggs and larvae to spill out into the saline. 35

A drop of this fluid containing eggs and larvae was pipetted onto a glass slide, covered with a cover slip, and examined under the light and phase contrast micro scopes at X 100, X 400 and X 800 magnifications.

A drop containing eggs and larvae was also stained lightly with aqueous iodine and preparations of these were examined in the same manner as the living material. V. OBSERVATIONS AND COMMENTS

A. Development in the Intestinal Phase

Examinations of mice for intestinal worms were carried out at 2-hourly intervals beginning from the 2nd until the 82nd hour after infection. Three mice were examined at each interval until the 34th hour after infection, and from then on, only 2 mice were examined at each interval. Counts of moulting worms were made from each mouse separately, and the averages are presented in Tables 1 and 2.

Examinations of mice for worms showed that at 2 hours after infection, the infective larvae were embedded in the intestinal mucosa and that when the intestines were opened along their entire length, no worms were seen in the lumen contents. This finding concurs with the observations of Gursch (1949}, who found that infective larvae, soon after they are ingested, become embedded in the intestinal mucosa.

The first occurrence of moulting in intestinal worms was observed at the 12th hour after infection and significant numbers of moulting worms were seen to 37

TABLE 1.--Percentage moults occurring in males between 10-34 hours after infection

Hours after 16 lS infection 10 12 14 20 22 24 26 2S 30 32 34

Percentages 0 14 13 14 0 3 1 6 12 25 9 4 0

~~ undifferentiated worms

males

25 til 20 Q) bO CIS .J.) 5:::: Q) 15 CJ J..t Q) p.. 10

: ,,

10 12 14 16 lS 20 22 24 26 2S JO 32 34 Hours after infection

Figure 1.--Histogram showing percentages of males moulting between the periods 10-34 hours after infection. TABLE 2.--Percentage moults occurring in females between 10-34 hours after infection

Hours after infection 10 12 14 16 là 20 22 24 26 28 30 32 34

Percentages 0 14 13 14 2 2 4 17 4 1 5 0 0

...;:: undifferentiated worms 25 fema1es 20 11.1 (1) b.O ~ 15 s::: (1) 0 ~ 10 Il..

10 12 14 16 18 20 22 24 26 28 30 3 2 3 4 Hours after infection

Figure 2.--Histogram showing percentages of females moulting between the periods 10-34 hours after infection. 39 persist until the 16th hour post infection. Thus, the first moulting period appeared to occur between the 12th and 16th hour after infection (see Tables 1 and 2, Figures 1 and 2). During this time, because of the experimental method used for the enumeration of per centages of moulting worms, it was difficult to differentiate between males and females. The sexes, during this period of time, could not be differentiated easily by visible external genitalia. From then on, the sexes were better differentiated, and male and female moulting worms were enumerated separately. From Table 1 and Figure 1, it will be seen that in males, a second significant rise in the numbers of moulting worms too~ place between the 24th and 32nd hour post infection. After this period, no more moulting males were seen. In females (see Table 2 and Figure 2}, the second rise occurred between the 22nd and 30th hour post infection. After this period, no more moulting females were seen.

As seen in Tables 1 and 2 and Figures 1 and 2, the moulting periods can be considered as typical, since the 2 "peaks" were taken to represent two moulting periods. In several preliminary experiments conducted in order to develop an accurate method for determination of moulting periods, it was observed that shifting of the 2 moulting periods along the timescale often occurred. This appeared 40 to depend upon the different batches of mice used, although the strain of T. spiralis larvae was the same, and the infective material was subjected to the same preparatory procedures. This might explain one of the reasons why contradictory reports have been made with regards to the times of moulting in those cases where 2 intestinal moults have been observed. Podhajecky (1964b), in his experiments with mice, found that the first moult occurred between the 14th and lSth hour after infection, whereas Thomas (1965) observed the first moult between the 12th and l5th hour after infection. The second moult in Podhajecky's {1964b) experiments occurred between the 22nd and 37th hour after infection, while Thomas (1965} observed it between the 26th and 29th hour after infection. Many other factors may cause wide dis crepancies to occur with regards to times of moulting. They may be the species of experimental animals, the strains of T. spiralis, the form in which infective larvae were fed (as infective meat or as freed larvae after digestion), and the time of infection related to environmental conditions or to any circadian rhythms that may occur within the host animals or the parasites.

As mentioned earlier, the procedure used for enumerating the moulting worms did not take into account the detailed internal anatomy of either the males or the 41 females. However, when at a later period a more careful study of individual worms was made, certain structural characters became more apparent. It was seen that whereas males moulting at the earliest period {i.e., 12 hours post infection) did not show a development of copulatory appendages (see Figure 3), those that were moulting at the 16th hour were beginning to show slight protuberances at the hind end (see Figures 4 and 5). Kreis (1937) found development of copulatory appendages in males at the làth hour post infection. This time is very nearly in keeping with the findings in the present experimental work. Figure 6 represents a female worm going through the first moult at the l2th hour after infection (compare with Figure 3). At the time of the second moult, between the 22nd and 32nd hour post infection, the males showed well developed copulatory appendages. At this time it was observed that there were sorne moulting males with seminal vesicles devoid of spermatozoa, and sorne with seminal vesicles containing stored spermatozoa (see Figure 9). It must be emphasized that since females were not seen to be fertilized until the 36th hour after infection, it was inferred that moulting males, which did not show stored spermatozoa in the seminal vesicles, were immature worms. This inference is being mentioned because it could have been interpreted that males, whose seminal vesicles were devoid of spermatozoa, were possibly males 42

Figure 3.--Tail end of male going through first moult at 12 hours post infection. Note that no copulatory appendages are visible. Phase contrast, X 1000.

Figure 4.--Tail end of male going through first moult at 16 hours post infection. Note that copulatory appendages (arrow) are just visible. Phase contrast, X 1000. 43

Figure 5.--Tail end of moulting male going through first moult at 16 hours post infection. Note that copulatory appendages (arrow) are just visible. Phase contrast, X 1000.

Figure 6.--Tail end of female going through first moult at 12 hours post infection. Phase contrast, X 1000. 44 that had already copulated and, therefore, were fully mature adults.

Examination of the internal structures of the female worms at the time of the first moult showed that the site of development of the vaginal opening was in the form of an indentation (see Figure 7). Such an indenta tion was not observed in the encysted muscle larvae. At the time of the second moult, the vaginal opening was seen to be much more developed (see Figure 8}. Seminal receptacles of moulting females were never seen to con tain spermatozoa (see Figure 10). Inseminated females were first seen at the 36th hour post infection. There fore, insemination occurred only after the second and final moult. These findings are at variance with those of Kreis (1937) who stated that the female intestinal worms undergo 4 moults, 2 before and 2 after copulation, and that the males undergo 3 moults, 2 before and 1 after copulation. It is difficult to comprehend the reasons why males and females should moult at all after they have reached sexual maturity and have mated.

The many in vitro studies made by several workers in attempts to grow larvae of T. spiralis to the adult stage resulted in reports which also disagreed as to the number of moults that the larvae underwent. Weller {1943) obtained worms that mostly went through 2 moults, 45

Figure 7.--Female at first moult (14 hours after infection) showing (arrow) very rudimentary vaginal opening. Phase contrast, X 1600.

Figure 8.--Female at second moult (30 hours after infection) showing {arrow) well developed vaginal opening. Phase contrast, X 1000. 46

Figure 9.--Tail end of male going through second moult (30 hours after infection) showing seminal vesicle full of spermatozoa. Phase contrast, X 1000.

Figure 10.--Seminal receptacle of female going through second moult (32 hours after infection) showing no spermatozoa. Phase contrast, X 1600. 47 although sorne of his worms showed "supernumerary moults." Meerovitch (1965) also obtained 2 moults in his culture system, whereas Tarakanov (1964} observed 4 moults. Kim (1961, 1962) also observed 4 moults, but he also obtained as many as 6 incomplete moults. Berntzen (1962, 1965) obtained only 1 moult in his culture system. It has been suggested that one of the chief reasons why decapsulated larvae of T. SEiralis go through so many different moults has been that the in vitro conditions were abnormal to the developing worms. It is difficult, therefore, to conclude anything from these varied reports, and it must be further remembered that what may or may not occur in vitro need not necessarily apply to the in vivo conditions.

Berntzen {1965), in his studies on the life history of T. SEiralis, observed the first moult to occur within the uterus of the gravid female. However, studies made on embryonating eggs and larvae, obtained by dis section of gravid female worms, did not show the occurrence of a moult in utero. Since there are no other reports available regarding the occurrence of an in utero moult, it is not possible to comment on the discrepancies between the findings of Berntzen (1965) and those obtained in the present studies.

During the course of studies on the intestinal worms, a pair of sac-like structures at the anterior end were observed to develop into prominence at certain times during the course of infection. These structures are similar to those described by Richels (1955) and are believed to be the amphids. Richels (1955) describes them as ellipsoïdal organs, situated at the anterior end of the worm between the mouth opening and the nerve ring. She did not find any amphidial pores or glands and, therefore, concluded that these were atrophied structures. In the present experimental studies, these amphids were never observed at any time during the muscle phase of growth of the larvae and they were also not observed in the encysted worms. Figure 11 shows the anterior end of a decapsulated muscle larva, in which no amphids are visible. As early as 2 hours after entry of the larvae into the intestine, these amphids became visible and they increased in size until about the 6th hour, when they appeared to reach their maximum size. Figure 12 shows the anterior end of a worm at the 6th hour post infection, in which the amphids are greatly enlarged. Subsequently, as infection in the intestine progressed, these amphids became reduced in size, so that at 10 hours post infection they were only about two-thirds their maximum size. In a few days they had become very much reduced and were hardly visible. Figure 13 shows the anterior end of a worm at 10 days, in which the amphids have become so reduced that they are not visible. 49

Figure 11.--Anterior end of muscle larva freed from its cyst. Note that the amphids are not developed. Phase contrast, X 1500.

Figure 12.--Anterior end of intestinal worm at 6 hours after infection. Note that the amphids are well developed. Phase contrast, X 1500. 50

Figure 13.--Anterior end of intestinal worm at 10 days after infection. Note that amphids are absent. Phase contrast, X 1500.

Figure 14.--Section of masseters of mouse, infected 24 days previously, showing portions of a coiled up larva. Note the cuticle of the larva and the absence of cuticle-like material on the inside of the muscle tissue spaces. Stained with Verhoeff's Van Gieson stain. Phase contrast, X 2000. 51

Lee {1965} describes the amphids as paired structures found on the head end of a great number of nematodes. They are cuticular pits which vary in shape in different nematodes. In sorne free living forms, these structures are elaborate spirals, whereas in most para sitic nematodes they are simple slit-like openings. Internally, each amphid consists of a pouch supplied with several nerve endings from the amphidial nerve, which originates in the lateral ganglia of the nerve ring. The amphidial gland, which runs alongside the pharynx, also opens into this pouch. The amphids are well developed in free living nematodes, but are greatly reduced in animal and in sorne plant parasitic nematodes.

Various physiological functions have been ascribed to the amphids. Lee {1965) states they may function as chemoreceptors, but no one has verified this assumption. Thorson {1956} found that the amphidial glands of Ancylostoma caninum contain a substance which inhibits coagulation of host blood and is thought to be secreted by the nematode during feeding. Stephenson (1942), in his studies on the free-living soil nematode, Rhabditis terrestris, found that when he immersed the worms in concentrated saline solutions, there was an increase in the internal osmotic pressure of the nematodes. This was proved by the shrinkage of the body, which also showed 52

that this increase in the osmotic pressure was largely due to the removal of water. At the same time, he found that the amphids and the phasmids became greatly enlarged when the nematodes were placed in these solutions of high osmotic pressure. He also observed that vacuoles were present over the general body surface, suggesting that some penetration of osmotically active substances occurred from the outside through the cuticle and also possibly through the hypodermis. During recovery in these con centrated solutions, there was slow removal of osmotically active material from the body. He suggests that the hypertrophy of the amphids and the phasmids may be related to this removal of osmotically active material from the body.

It is conceivable that the amphids of • spiralis act very similarly to those of Rhabditis terrestris. There is evidence that Parascaris eguorum, Ascaris lumbricoides and severa! related species normally live in a slightly hypertonie medium within the host (Rogers, 1962). The larva of T. spiralis, during its migration and growth within the muscle tissues, and its sojourn within the cyst, probably feeds on body and tissue fluids of the host. It grows and lives in an environment that is probably isotonie with its own body fluids. However, as soon as the larva enters into the intestinal 53 environment, it finds itself in a hypertonie medium. It is possible, then, that the amphids begin to hypertrophy and function as osmoregulatory organs. After the larva has become well adjusted to the change in osmotic pressure in the intestinal environment, the amphids cease to function and become reduced in size. Richelé' (1955) description of the amphids of T. spiralis might be taken to infer that they were not functional in this worm, but there is reason to believe, on the basis of these observations and comparison with other nematodes, that they are functional, especially during the early stages of infection in the intestines.

B. MigratiQn of Muscle Larvae

Two mice were examined daily, between the 5th and the 15th day after infection, to determine the presence and the numbers of migrating larvae. From the 16th day on, until the 32nd day after infection, only 1 mouse per day was examined. The results of these examinations are summarized in Table J.

It will be seen from Table 3 that only very occasionally were larvae recovered from the bloodstream, and also that they were recovered in extremely small numbers. Although the method of bleeding the mice did

V1 V1

32 32

e e

of of

31 31

muscles muscles

30 30

washings washings

29 29

-·-

y·-

28 28

abdominal abdominal

27 27

the the

peritoneal peritoneal

2 2

4 4

26 26

25 25

15 15

and and

from from

25 25

55 55

74 74

14 14

14* 14*

2'!:: 2'!::

blood blood

24 24

13 13

18 18

450 450

escaped escaped

of of

2* 2*

25 25

23 23

12 12

413 413

have have

10* 10*

12 12

11 11

195 195

ce ce

21 21 22

50* 50*

mi mi

10 10

might might

125 125

165 165

examinations examinations

9 9

3* 3*

8 8

3 3

20 20

16 16

in in

2 2 2

8 8

9 9

7* 7*

which which

1 1

19 19

106 106

7* 7*

7 7

50 50

14 14

18 18

4* 4*

larvae, larvae,

l l

6 6

obtained obtained

procedures. procedures.

17 17

11 11

13 13

5 5

- -

31 31

16 16

larvae larvae

larger larger

of of

operative operative

with with

washings washings

Infection Infection

during during

Mixed Mixed

* *

2 2

3.--Counts 3.--Counts

1 1

after after

• •

Blood Blood

Peritoneal Peritoneal

Blood Blood

Peritoneal Peritoneal

Days Days

TABLE TABLE

Days Days

Mouse Mouse

-- -- 55 not yield constantly the same volumes of blood each time, the fact remains that no larvae, or only extremely few, were obtained from blood.

Larvae were first recovered from peritoneal washings on the 6th day after infection and subsequently they were constantly recovered in considerable numbers until about the 16th day after infection. From the 17th day onwards, until the 2Sth day after infection, larvae in decreasing numbers were recovered and they were very often mixed with larger larvae. These were obviously older larvae which were residing in the abdominal muscles and which were inadvertantly freed into the abdominal cavity during operative procedures. From the 29th up to the 32nd day after infection, no larvae were recovered from the peritoneal cavity.

Examinations of mesentery pressed between slides showed very often the presence of migrating larvae between the folds of the peritoneum. They were never seen to occur in any of the blood or lymph vessels that lead from the intestinal wall towards the larger blood or lymph vessels.

Varying reports as to the routes of migration of the larvae of T. spiralis have been made by different workers. According to sorne workers, emerging young larvae from gravid females have been reported to be 56 deposited directly into the lymphatics or the lacteals of the intestinal villi (Askanazy, 1895; Graham, 1897; Staubli, 1905). Matoff (1943), working with guinea pigs and rabbits, also stated that the lymph vessels were probably the chief distribution route for the young larvae. Heller (1935) and Gould (1945) felt that the larvae, after first getting into the lymphatics, were distributed throughout the body by passive carriage through the bloodstream. Nolf and Crum (1937), Phillip son and Kershaw (1961}, and Berntzen (1965) found very few larvae in the blood of mice and rats infected with T. spiralis.

Using the criterion of the high recovery of larvae obtained in the peritoneal washings as an index of the peak of deposition of larvae, it would appear that this peak period occurred between the 8th and the 13th day after infection. This finding follows quite closely the observations of Phillipson and Kershaw (1961) who found in their experiments with mice that the peak period was between the 7th and the llth day post infection. The results as outlined in Table 3 show that in spite of heavy deposition of larvae during the peak period, extremely few or no larvae were obtained from the blood. Moreover, examinations of fresh mesentery consistently showed larvae migrating outside of blood and lymph 57 vessels, and were never seen inside these vessels. These observations and findings are significant as they indicate that the migration of freshly deposited larvae occurs through the peritoneal cavity, and thence onwards by way of connective tissue to the far reaches of the host body. Few larvae are carried by way of the blood stream, but this is not the rule. It is also felt that if the larvae regularly emerged from the blood capillaries in order to reach the various striated muscles, there would be extensive occurrence of petechiae. Because such petechiae were not observed at autopsy, it is believed that such a method of migration does not, as a rule, take place. These results and observations concur with the findings of Berntzen (1965), who found that, in rats, large numbers of migrating larvae were obtained from body fluids and very few from blood and from other internal organs. He believes that the main route of migration is through connective tissues. He suggests the possibility that, in different species of host animals, the route of migration may vary, but it is difficult to comprehend why this should be so. It is interesting to note that even as far back as 1866, Leuckart, who was one of the first to unravel the life history of T. spiralis, maintained that migration of larvae from the intestines occurred through the con nective tissues. 58

C. Development of Larvae in the Muscle Phase

Larvae in small numbers were first observed in teased diaphragm muscle on the 6th day after infection. The finding of larvae in muscle tissues occurred simultaneously with the first recovery of larvae from peritoneal washings. This suggests that larvae, after traversing through the peritoneal cavity and connective tissues, very quickly enter into muscle tissues. Examina tions of "teased-muscle" preparations and of histological sections of muscle, showed that, as infection progressed, there was a graduai increase in the infiltration of muscle tissues with Trichinella larvae.

After penetration into the muscle fibres and during the period of their graduai growth within the muscle fibres, the larvae were seen to lie extended. On about the 17th day after infection, sorne of the larvae were seen to coil. On about the 2lst day after infection, the beginnings of cyst formation were visible a:round sorne of the coiling worms.

~mile Heller {1933) stated that he observed young larvae in rats and cats as early as 80 to 90 hours after infection, the finding of the larvae in muscles at 6 days post infection in the present experimental studies is generally in keeping with the findings of other 59 workers (Roth, 1938a; Phillipson and Kershaw, 1960}.

Phillipson and Kershaw (1960) found that the rate of growth of the first deposited larvae during the first few days appeared to be slow; it accelerated between the 12th and 20th day of infection and then slowed down again until the maximum length was reached on about the 24th day. They found that on the 17th day, the larvae were coiled in the muscle, encased in a cyst and were resistant to acid-pepsin digestion. At that time they were also infective to new hosts. Schaaf and Lampe (1958}, working with hamsters, found invasive forms on about the 19th day after infection, and that by the 30th day, almost all the larvae were coiled. The findings in the present studies come quite close to the results obtained by all these workers.

The cuticle of the early migrating live larvae and those in histological sections of muscles obtained during the earlier phases of infection, was seen to be very fine and delicate. Certain simple experiments were carried out in order to test the permeability and resistance of the cuticle. Larvae, obtained from animals by the maceration technique at different stages of infection, were immersed in distilled water for periods of 1 to several hours. It was observed that very young live larvae, which were allowed to remain in water for 1 hour, 60 showed degeneration of the internal structures and were immobilized. In older larvae, recovered up to about 18 days after infection, degeneration of internal structures continued to occur, although the process took longer. Thus, the time taken for the degeneration of internal structures of larvae was directly proportional to the age of the growing larvae. From the 19th day onward, the larger of the muscle larvae were not affected by prolonged immersion in distilled water. They were also resistant to acid-pepsin digestion, and were infective when fed to mice. The larger larvae were resistant to the penetration of histochemical stains used in the studies. Examinations of whole mounts and of histological sections showed a considerable thickening of the cuticles of these larger forms, with wrinkling on the concave side.

A feature of the cutieles of developing larvae was the presence of cuticular ridges which were apparent only on the dorsal aspect of the worms (see Figures 15 and 16). Young larvae, stained with aqueous iodine, showed these ridges quite clearly even up to the stage when they were about to coil and encyst. At this later stage, probably due to increased deposition of cuticular material, these ridges became less distinct, until in the fully encysted larvae, they were hardly visible. 61

Figure 15.--Portion of 15-day-old muscle larva showing cuticle with ridges (arrow) on the dorsal aspect of the worm. Stained with iodine. Phase contrast, X 880.

Figure 16.--Portion of 15-day-old muscle larva showing cuticle with ridges (arrow) on the dorsal aspect of the worm. Stained with iodine. Phase contrast, X 880. 62

Mention of these cuticular ridges has been made by Kalwaryjski (1938) but only recently, Richels {1955) has made further studies on these structures. According to her, iodine-silver impregnation staining techniques show annular or rib-like structures, which do not increase in number as larvae grow; the average number is 96. They are real morphological structures, which disappear when the larvae have been in the host intestines for a few hours. In the pv.esent studies, these ridges were not observed in worms developing in the intestines. Meerovitch (personal communication) round that, when he used an unsuitable culture medium for his in vitro work with T. spiralis larvae, no moulting of worms took place but the cuticular ridges were prominent. He observed that in the region of the ridges, there appeared visible precipitates, very similar to those observed in micro precipitation reactions. This suggests the presence of pores on these cuticular ridges, but the precise function of these pores is not known. It is not possible to explain why these pores (if they are present in the cuticular ridges} should persist during the muscle phase of the lire cycle of the worm, and then disappear soon after entry into the intestinal environment.

Examinations of fresh live larvae obtained from "teased-muscle" preparations and by the maceration method 63 showed no evidence of moults occurring at any time from the first appearance of the larvae in tissues (6th day} up to the 35th day after infection. Examination of larvae obtained by the trypsin-digest method, however, showed numerous larvae in varying stages of degeneration. The numbers of larvae showing degeneration of the internai structures were greatest in mice killed at earlier stages of infection and progressively decreased in those killed at later stages. Whole mounts of larvae prepared from macerated material and stained with Harris' haematoxylin showed no evidence of moulting at any stage during the course of this investigation. However, whole mounts prepared from trypsin-digested material and stained with Harris' haematoxylin showed varying numbers of degenerating larvae, which produced artefacts, that could easily be mistaken for moults. Figure 17 representa a larva, obtained from digested material, undergoing degeneration of the internai structures at the posterior end. The degeneration of the internai structures has caused the withdrawal of the tissues from the posterior end of the cuticle, leaving a small space. This could be mistaken for the separation of the shed cuticle in a moulting worm. Figure là represents another form of an artefact, very often observed in stained mounted specimens. Shrinkage of the worm took place during fixation in Schaudinn's fluid and this caused an imprint Figure 17.--Posterior end of a degenerating larva (11 days after infection) in whole mount preparation. Note artefact at posterior end resembling a moult. Phase contrast, X 1000.

Figure 18.--Posterior end of degenerating larva (15 days after infection) in whole mount preparation. Note imprint in mounting medium resembling a moult. Phase contrast, X 1000. 65 in the surrounding mounting medium (containing pig serum), which looked like the posterior end of a moulting worm.

Chandler et al. (1941) postulated on analogy with other nematodes that there were possibly 2 moults during the muscle phase of development of Trichinella larvae. Hyman (1951) states that the encysted muscle larva is the fourth stage implying thereby that the larva has undergone 3 moults; however, she makes no reference to any experimental work to support this statement. Lapage (1956), by stating that the parasite goes through 4 moults in the intestine, implies that the encysted muscle larva is the first stage larva. Meerovitch (1965) claimed to have seen moulting in the muscle stages, but gave no details of his observations. Berntzen (1965) claimed to have observed 2 moults during the muscle phase of development, following the first moult in utero. The second moult is said to take place from the llth day onwards and the third moult from the l5th day onwards. He described cell types as anatomical features to dis tinguish between second stage juveniles, third stage juveniles and fourth stage juveniles.

Berntzen (1965) made his studies and observations on larvae recovered by digestion with trypsin. In the present experimental studies, the methods used were as 66

close as possible to those employed by him, and in addition, for comparison, larvae were recovered by the maceration method. In the present experimental studies, no moults were observed at any time in the larvae recovered by the maceration method, but artefacts, resembling moults, were observed in larvae recovered by , the digestion technique. These results are significant. It is probable that Berntzen (1965} based his conclusions on these artefacts and therefore his claims must be viewed with sorne reservation.

Studies made on histological specimens stained with Verhoeff's elastic stain did not show any red staining of the lining of spaces in which larvae were deposited. Figure 14 represents portions of a larva embedded in muscle tissue. There is very often an inter vening space between the larva and the surrounding muscle tissue, probably due to the effects of fixation. Berntzen (1965) reported finding these red staining linings of spaces and concluded they were cast off sheaths of moulting larvae. It is felt, however, that even though the lining of spaces was stained as a thin red line, it only meant that sorne collagenous material was stained and that this did not necessarily imply that the material was shed cuticle. Furthermore, this is substantiated by the results of the present experimental 67 studies in which no moults at all were observed either in fresh material or in whole mount preparations. It is felt that the significance of cell types, as described by Berntzen (1965), has no relevance to moulting within the muscle phase of development of the larvae.

Thomas (1965) found that increase in length of the growing larvae was slow up to the llth or 12th day post infection. Following this, there was a rapid growth in length coming to a standstill on the 25th day post infection. Growth in diameter followed the same pattern with the exception of a comparatively rapid increase between the lOth or llth and 12th or 13th days post infection. He concluded, on the basis of this circumstantial evidence, that 1 moult took place during this period. Richels (1955) also found at first a slow increase in length of the growing larvae up to the llth day, and then there was a rapid increase in length up to the 25th day. But in her studies of the cuticles of the growing larvae (which are described below in detail), there was no evidence of a suggestion that moulting occurred during the muscle phase of growth. In view of this finding, together with the results of the present experimental studies, and in the absence of more con clusive evidence on the part of Thomas {1965), it is difficult to accept his hypothesis that 1 moult occurred 68 during the muscle phase of development of the larvae.

Richels (1955), in her detailed study of the cuticles of the growing larvae, found that the blood larva had a cuticle about 0.43;u in thickness, and that it was delicate and transparent. When the larva was about 200/U long, the thickness of the cuticle measured about 0.6/U and finally when the larva had reached maximum development, the thickness of the cuticle measured 1.17/U. She described the cuticle of the mature infective larva as being made up of 3 layers almost equal in thickness. The outermost layers, using silver-staining techniques, stained much more darkly than the inner one. Electron-microscope studies by Beckett and Boothroyd (1961) showed that the cuticle of the mature infective larva measured 0.6-l.l;u in total thickness and that it was composed of 2 definite layers, separated by an electron-dense line or membrane. The 2 layers did not have a constant relative thickness. The external border of the outer layer, which undulated to correspond with the annulations of the worm's surface, was covered by a triple electron-dense membrane, and the remainder of the thickness of this layer seemed to be composed of granular material. The inner layer showed evidence of an array of very fine fibrils about 60 A0 in thickness, mainly orientated parallel to the circumference 69 of the larva.

In the absence of any visible moults of T. spiralis larvae during their period of migration and growth within the muscle phase of the life cycle, a hypothesis suggest ing that new cuticles were produced, but not shed, could have been acceptable. However, the histological studies of Richels (1955}, and Beckett and Boothroyd (1961}, show beyond any reasonable doubt, that there could not possibly be any retention of unshed cuticles as such. Their findings do show, on the other hand, that the muscle larvae, during their period of migration and growth, produce a single, but very thick and resistant cuticle. VI. DISCUSSION

There is no doubt that T. spiralis is a most unusual parasite. This opinion is based on a number of facts. It is, perhaps, the single species of parasitic nematode least restricted in its range of hasts (Bachman and Rodriguez-Molina, 1933). Ordinarily the parasite is found only in carnivorous mammals including a wide range of domestic and wild animals. Because of its ability to infect a wide range of animals, T. spiralis can be con sidered ta be of recent evolutionary origin. It is also an unusual parasite because, according to the results of the present experimental studies, the larva does not undergo moulting between the time when it is expelled from the uterus of the gravid female and the time it encapsulates and reaches the infective stage within the muscle fibres. It is, perhaps, the only species of parasitic nematode in which the infective larvae are sexually differentiated. It is able to use the same species of animal for its intermediate and its final host, and finally, and perhaps most significantly, the evidence obtained in the present studies has shawn that 71 it undergoes only 2 moults during its entire life cycle.

On analogy with other nematodes, Chandler et al. (1941) postulated that T. spiralis undergoes 2 moults before reaching the infective muscle stage. Meerovitch (1965), because he obtained 2 moults in his culture system leading up to the adult stage, considered that the encysted muscle larvae were the 3rd stage larvae. In the light of the results of the present studies, the question can be raised as to why moulting does not occur during the muscle phase of development up to the time of encystment within the muscle fibres.

Lee (1965) describes moulting as occurring in 3 main steps: (1} the formation of the new cuticle; (2) the loosening of the old cuticle; and (3) the rupture and shedding of the old cuticle with the ensuing escape of the larva. However, the significance of moulting in the life cycle of nematodes is not clear. Rogers (1962} believes that in unspecialized species, moulting may be primarily a mechanism of growth related to the properties of the cuticle. In 19l~Looss stated that this non cellular outer covering may limit the size of each larval stage, and its periodic removal at each moult may be necessary for increases in the size of the worm (Rogers, 1962). Harris and Crofton (1957) found that the elastic properties of the cuticle were important in determining 72 the movements and structure of Ascaris lumbricoides. Rogers (1962) believes that this probably applies to many nematodes and suggests that "moulting is related to growth and is necessary, so that, among other things, the elastic properties of the cuticle are retained as the organism increases in size." On the other hand, it is also known that the cuticles of sorne worms are highly extensible and considerable growth is known to take place without the occurrence of intervening moults. In contrast to this, there are cases in which moulting takes place without any increase in size, as seen in Meloidogyne. Thus, it would appear that moulting may take place with out growth and growth can take place without moulting, and it is difficult to define in precise terms the relationship between moulting and growth.

Whatever the physiological functions of moulting are, it appears that moulting, in sorne ways, facilitates entry into a new environment (Rogers, 1962). When the parasite is about to moult, it goes into a period of quiescence, during which it reorganizes itself structur ally, apparently in preparation for the next stage in its life cycle. This next stage may be the new environment of an intermediate or the final host, or it may be just the change of site during migration within a host. Thus, it would appear that moulting may be a prerequisite to 73 the reaching of the infective stage by the parasite.

In the life cycles of most parasitic nematodes, the formation of the infective stage is usually preceded by a moult or partial moult. Thus, it is found that in many of the Ascaroidea, the first moult precedes the infective stage, whereas in many of the Strongyloidea it is the second moult. In a number of other species of parasitic nematodes, the life cycle is not known in detail and it is uncertain how many moults precede the development of the infective stage. However, there is sufficient evidence to suggest that, in different species, almost any stage in the life cycle, except the adult stage, may be the infective stage for the final host (Rogers, 1962).

The larva of T. SEiralis, when it is seen in the tissues of the host at 7 days post infection, is about 107)U in length and it grows to a length of l225)U by the time it is encysted (Richels, 1955). It might be expected that the migrating and growing larvae would go through 1 or more moults before they finally encyst, but the studies of Richels (1955), and Beckett and Boothroyd (1962), and those reported here show that such a moult (or moults) does not take place. Within the muscle fibres, the larvae probably only feed and grow in size. During this period of growth in the muscle fibres, there is a gradual increase in the thickness of the cuticles of 74 the growing 1arvae as indicated in the studies of Riche1s (1955), and Beckett and Boothroyd (1962}. The cutic1e of the encysted muscle 1arva deve1ops to such a degree that it becomes resistant to a great variety of physica1 and chemical factors. For example, in sorne of the experiments described here, it was seen that the cuticles of the larger non-encysted and the encysted larvae were resistant to changes in osmotic pressure. Secondly, it is common knowledge that undercooked infected pork or its products have often been the cause of outbreaks of trichinosis in man. This means that infective larvae are resistant to considerable amount of prolonged heat; Ransom and Schwartz (1919) have stated that the thermal death point of T. spira1is 1arvae is about 131° F. Third1y, in the laboratory, it was frequently observed that infective trichinous meat, which was beginning to decompose, almost always contained viable Trichinella larvae. This suggests that the cutic1es of the larvae are resistant to bacterial action and other putrefactive agents. This resistant property of the cuticle allows the infective larvae to withstand the action of digestive juices during their entry into the intestinal environment.

When the infective larvae enter into the alimentary tract of a host, they begin development to the adult stage and in so doing, go through 2 moults in much the 75 same manner as many of the Strongyloidea. This phase of development is very rapid; as early as 32 hours post infection, while the second moult is still in progress, sorne of the worms are already sexually mature. Copulation takes place at about 36 hours post infection and about 6 days post infection, larviposition occurs. It would appear, then, that the parasite is in great haste to deposit its young. It is generally believed that follow ing copulation, the male intestinal worms die and are digested, and that following the birth of the brood of young larvae over a period of a few days, the females likewise die and are digested (Gould, 1945). There have been reports, however, that insemination of females may take place more than once (Gould et al., 1955; Thomas, 1965). Nevertheless, it has generally been observed that at about 2 weeks after infection, the adults begin to be eliminated from the intestines. These facts set T. spiralis apart from most gastro-intestinal nematodes, which have longer prepatant periods and are known to persist for considerable periods of time within the definitive host.

It is commonly assumed that nematodes normally undergo 4 moults before they complete their entire life cycle. However, the evidence obtained in the present studies shows that T. spiralis undergoes only 2 moults during its entire life cycle. This finding has led to 76

the postulation of 2 possible hypotheses. The first hypothesis presupposes that at sorne time in the past evolutionary history of T. spiralis,there were probably 2 moults during development of the larva to the infective stage. This presupposition is based on the consideration that if the infective larva undergoes only 2 moults to reach the adult stage, then, on the basis of the 4-moult nematode life cycle, the infective larva should be in the third developmental stage. This, in turn, implies that the parasite must go through 2 moults to reach the infective stage.

It is conceivable that in the past evolutionary history of the parasite, there did exist an intermediate host in the life cycle of T. spiralis, in which the 2 moults preceding the infective stage took place. This intermediate host could have been an arthropod or even an annelid. It is possible that, during the course of evolution the parasite had adapted itself to utilizing the final host as the intermediate host, thereby eliminating the need for an intermediate host. It is also conceivable that in the earlier stages of its adaptation to the new life cycle, the first and the second moults preceding the infective stage continued to take place, but that the newly emerging larva, on finding the muscle environment to be constant and unchanging 77 during the course of its evolutionary development, did not need to go through any moult. Instead of moulting, the larva had converted its inherent capacity of growing cuticles to developing a single thick and resistant cuticle. This thick and resistant cuticle has enabled the larva to become infective when it encysts and also to survive through many adverse conditions. In the circum stances, one may consider that the encysted muscle larva is a modified third stage larva and the 2 intestinal moults are actually the third and fourth moults, which the parasite has retained up to the present time.

The second hypothesis is based on the assumption that the newly emerging larva never did undergo any moult before reaching the infective stage, either in a hypo thetical intermediate host or in any of its present-day hosts. In this circumstance, one has to consider that the encysted muscle larva is in the first developmental stage. The fact that the parasite undergoes only 2 moults in the intestines to reach the adult stage, would suggest that the so-called adult T. spiralis is actually a third stage neotenic larva. It can be postulated further that the parasite, in its haste to produce the next generation of larvae, had done away with 2 further moults in the intestines after reaching sexual maturity.

The significance and the physiological functions 78

of moulting have already been discussed earlier. Briefly, moulting may be primarily a mechanism of growth related to the properties of the cuticle. Looss, in 1911, has stated that this noncellular outer covering may limit the size of each larval stage, and its periodic removal at each moult may be necessary for increases in the size of the worm (Rogers, 1962). Harris and Crofton (1957} have stated that the elastic properties of the cuticle determine the movements and structure of Ascaris lumbricoides. Rogers (1962) thinks that this property probably applies to many nematodes and suggests that moulting is related to growth and is necessary so that the elastic properties of the cuticle are retained as the organism increases in size. Moulting is also known to be an event in the life of a nematode, during which the organism reorganizes itself structurally, presumably in preparation for the next stage of its life cycle. If one considers these points as sorne of the factors related to moulting in nematodes with a typical 4-moult life cycle, one, perhaps, may be led to consider more favourably the hypothesis in which 2 moults were assumed to have existed in the past before the larvae reached the infective stage. It is possible that transmission experiments, carried out in various arthropod or other possible vectors to study the role they might have played in the life cycle of 79

T. spiralis, could shed more light either in supporting or discrediting this hypothesis. However, at the present stage of our knowledge of the biology of T. spiralis, and in the absence of more precise evidence to support this or the other hypothesis, it is not possible to say with any certainty which is the more probable.

T. spiralis, for want of a better taxonomie classification, has been placed in the Family, Trichinellidae within the Order, Trichuroidea. The basis for this classification has been described earlier. There are, however, many characters in this species of worm which bear resemblance to the nematodes belonging to the Order, Filarioidea. Sorne of these characters are the viviparity of the females of T. spiralis and of many of the filarial group of worms, and the ability of the larvae of T. spiralis to live and grow in the body tissues of its host, similarly to the microfilariae of many of the Filarioidea. Indeed, these very biological characters set • spiralis apart from the other members of the Trichuroidea. But the anatomical characters which have been described earlier and which have been largely used as a basis for classifying T. spiralis within the Order Trichuroidea, far outweigh the biological characters of T. spiralis which bear resemblance to the Order Filarioidea. For these reasons, it is considered that T. spiralis 80 should continue to remain in its present taxonomie position. The very many biological characters of T. spiralis described and discussed here, no doubt make this worm a most unusual and unique parasitic nematode. VII. SUMMARY AND CONCLUSIONS

The present experimental studies were carried out in order to elucidate more fully certain aspects of the life cycle of T. spiralis. The results obtained showed that when encysted larvae of T. spiralis were freed from infected muscle tissue by artificial digestion, and when fed to young mice, they moulted twice within the intestines to reach the adult stage. In the male worms, the first moult occurred between the 12th and the 16th hour post infection and the second moult occurred between the 24th and the 32nd hour post infection. In the females, the corresponding moulting periods occurred between the 12th and the 16th, and the 22nd and the 30th hour post infection. Fertilization of the females took place at about the 36th hour post infection. The first appearance of larvae in the extraintestinal tissues of the host was observed on the 6th day post infection. No moult of the second generation larvae was observed in utero.

After their expulsion from the uterus of the gravid female, the larvae were found to migrate through the peritoneal cavity and connective tissues to reach the striated muscle fibres. It is believed that this is the usual method of migration of the larvae and that they are not distributed through the blood and lymph vessels, as reported by several other workers. After entry into the striated muscle fibres, the larvae were found to lie extended and to grow in size without moulting. Coiling of the larvae was observed on about the 17th day after infection. On about the 19th day after infection, the larvae were resistant to acid pepsin digestion, and were infective to mice. The beginnings of cyst formation were observed on about the 2lst day after infection.

The evidence that only 2 moults occurred in the entire life cycle of T. spiralis has led to the postula tion of 2 possible hypotheses with regards to the parasite's life cycle. In the first hypothesis, it has been considered that, in the past evolutionary history of the parasite, there might have occurred 2 moults before the larva reached the infective stage. In the present stage of its evolution, it has, instead of moulting twice, developed a capacity to grow a very thick and resistant cuticle, which it has utilized to full advantage for self-preservation and for the perpetuation of the species. This implies that the encysted muscle larva is actually a modified third stage larva, and that, on entry into the intestinal environment it moults twice to reach the adult or the fifth stage. In the second hypothesis, it bas been considered that the encysted muscle larva is the first stage larva, and, because there are only 2 moults in the intestinal phase of development of the worm, the so-called adult Trichinella worm is actually a third stage neotenic larva. In the absence of more precise evidence to support one or the other of the 2 hypotheses, it bas not been possible to say with any certainty which of the 2 hypotheses is the more probable.

In view of the fact that the anatomical characters of T. spiralis, which bear resemblance to the other members of the Orde~Trichuroidea, far outweigh its biological characters which bear resemblance to the members of the Order, Filarioidea, it is felt that T. spiralis should continue to remain in its present taxonomie position. LITERATURE CITED

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