The demonstration of Toxoplasma in animals, with particular reference to members of the mustelidae

111

III.

THE

DEMONSTRATION

OF

TOXOPLASMA

IN

ANIMALS,

WITH

P A R T I C U L A R R E F E R E N C E T O M E M B E R S OF T H E M U S T E L I D A E BY

R. LAINSON, PH.D.*

London School of Hygiene and Tropical Medicine Toxoplasma gondii, generally regarded as a protozoan of doubtful affinities, has been described in a wide range of mammalian hosts, in some marsupials and among the birds. From experimental data and observations on natural infections there is evidence to suggest that no homothermous animal has complete natural immunity to the infection : poikilothermous animals have not yet been proved to harbour the parasite in nature. Interest in Toxoplasma was largely restricted to the zoologist until human infections were reported. CASTELLANI(1913) described organisms in spleen smears from a 14-year-old boy with protracted fever and splenomegaly in Ceylon, and considered them as Toxoplasma. Considerable doubt existed, however, as to the validity of this diagnosis, especially on the part of WENYON (1923, 1926) who regarded the organisms as fungal spore contaminants. A similar doubt was cast on the human infections claimed by FEDOROVITCH (1916) and CHALMERS and KAMAR (1920). JANK6 (1923) and TORRES (1927) both described what are now accepted as undoubted cases of human toxoplasmosis, but it was not until 1939 that WOLF et al. succeeded in actually isolating Toxoplasma from a human infant by the inoculation of tissues into mice and guinea-pigs. Since that time there have been numerous reports of the pre-natal human infection in many parts of the world, including this country. Until the comparatively recent use of serological techniques for the diagnosis of toxoplasmosis, infection in man was considered to be uncommon and largely restricted to the newborn ; only very rarely was the disease recorded in adults. It was soon shown, however, that antibodies against Toxoplasma could be demonstrated in a surprisingly high percentage of the normal adult population : figures of 50 per cent. or more were given for the higher age-groups. Medical interest being now aroused, intensive studies began to determine the source, or sources, from which infection might be acquired. The parasite was first described, in domestic rabbits, by SI'LENDORE (1908) in Brazil. Since that time infections have not only been repeatedly recorded in rabbits in many parts of the world, but also in dogs, cats, sheep, cattle, rats, various domestic fowls, and other animals. All these animals are at times closely associated with man, but until the mode (or modes) of transmission of Toxoplasma are fully known it is difficult to assess the significance of the incidence of the parasite in different host species. Knowledge of such incidence will be of great value when the life cycle of Toxoplasma is finally elucidated and may itself be instrumental in achieving this end. * I wish to thank all who have helped me in this work, in particular Professor P. C. C. Garnham. My thanks are also due to Mr. R. J. Clarke, Mr. R. S. Jailler and Miss B. B. Jones, all of the Ministry of Agriculture and Fisheries, Infestation Control Division, who trapped and sent me house mice. Finally, the examination of all such animals would have been impossible but for the technical assistance of Mr. R. Killick, at that time senior technician at our Field Station.

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DEMONSTRATION OF

Toxoplasma I N

ANIMALS

An attempt to deal with animal toxoplasmosis as a whole and as reported in other countries is beyond the scope of the present discussion ; rather is it intended to discuss some of the problems encountered in conducting surveys for Toxoplasma in selected animal populations by methods entailing the isolation of the parasite. Although some work has been carried out in this country to demonstrate the presence of antibodies against Toxoplasma in the human population, little has been done to study the prevalence of the organism in our wild and domestic animals. COLES (1914) found a single case of toxoplasmosis in a red squirrel ; FINDLAYand MIDDLETON(1934) described 19 infections among 63 Microtus sp. examined ; PERDRAt~and PtJGH (1930), MAclNTYRE et al. (1948) and BONSER (1950) have each described single cases of toxoplasmosis in dogs, while CAMPBELLet al. (1955) have reported 16 further cases in these animals. BEVERLEYet al. (•954) showed that 39 per cent. of wild rabbits from a locality in Wales possessed dye-test antibodies against Toxoplasma : LAINSON (1955a) reviewed the incidence of infection in rabbits as recorded in different parts of the world. By the inoculated of brain tissue into mice he found six of 113 domestic rabbits obtained from markets near Weybridge, Surrey, to be infected, but could not isolate Toxoplasma from 122 wild rabbits from Hertfordshire. The available literature on the prevalence of toxoplasmosis in dogs has been reviewed by the same author (LAINSON, 1956), who showed that 42 per cent. of 113 dogs from London possessed complement-fixing antibodies against Toxoplasma. The following work has been carried out in an attempt to extend our knowledge on the incidence of the parasite in some other animals in this country. MATERIALS

AND

METHODS

The isolation of the parasite constitutes the only real proof of infection and this immediately raises several problems on the techniques to be employed. All attempts to cultivate Toxoplasma on media free from living cells have so far failed ; this means that, in order to determine whether or not a particular animal is infected, and to obtain the living parasites for further study, tissues must be passaged into " clean " laboratory animals.

a) Choice of laboratory animals Before such animals can be used it is essential to be sure that spontaneous infections do not exist among the stock from which they are drawn. So many reports of natural infections among domestic and laboratory rabbits have been recorded in the past (see above), that these animals can at once be excluded. Similarly, spontaneous infections have frequently been found in guinea-pigs (CARINI and MmLIANO, 1916 ; NICOLAU, 1932 ; MARKHAM, 1937 ; MARIANI, 1941 ; and many others). There have been few reports of spontaneous toxoplasmosis in laboratory mice and rats : NICOLAUand BALMUS(1933) described Toxoplasma-like cysts in the brain of one laboratory mouse but pointed out that a wild house mouse caught in the same building was also infected and that the infection may have originated from this source. MOOSER(1950) found infections in a number of his white mice which, however, were obtained from a dealer who had introduced wild mice into his colony to invigorate the stock (SABIN, in a personal note to WOKE et al., 1953). Among laboratory rats, the only reports of natural infections appear to be isolated cases described by SANCIORGI(1914a, 1914b). In the above respect it would appear that rats and mice are both suitable animals to

R. LAINSON

113

use for the isolation of Toxoplasma : it is very difficult, however, to infect adult rats experimentally and when an infection is established it is of the chronic type in which the parasites are not easily demonstrated. The mouse, on the other hand, is highly susceptible to the infection and has the further attribute of being easily bred, housed and handled. There has been no record of Toxoplasma occurring in our colony of laboratory mice at the London School of Hygiene and Tropical Medicine, and these animals were selected for use in the following studies.

b) Choice of inoculum and inoculation route Natural, inapparent infections by Toxoplasma are invariably associated with the presence of the resistant aggregations of the parasites (" pseudocysts ") in the central nervous system, particularly the brain : this, therefore, was the tissue selected for passage into mice. For the examination of large numbers of animals a system of pooling the tissues had to be adopted ; the number of brains in a single pool varied but was never more than five. After fixing small portions of the brains, the remaining tissues were ground together in a suitable quantity of sterile normal saline in an M.S.E. homogenizer ; from 1.0 to 1.5 ml. of the resulting suspension was inoculated into each of two mice. Different routes of inoculation, while sometimes affecting the course of the infection in mice, are as a rule equally efficient in producing infection. It was convenient, therefore, to use the easiest method, namely the intraperitoneal inoculation. There are added advantages in selecting this route ; the parasites multiply most readily in the cells of the lymphoidmacrophage system and the peritoneal fluid is an immediate and rich source of such cells ; this fluid is easily removed early in the infection for examination or sub-inoculation into further animals ; finally, smears of this material are among the easiest to examine for the organisms.

c) Demonstration of Toxoplasma in the mice All examples of Toxoplasma studied so far appear to be morphologically identical, and serologically and immunologically very similar : they are now generally regarded as belonging to a single species, Toxoplasma gondii. Many of the parasites isolated from different host species, however, and even from different animals of the same species, may differ greatly in the degree of virulence they exhibit in experimentally infected animals. Toxoplasma from an animal which has died from a spontaneous, acute infection may not necessarily prove pathogenic in a wide range of laboratory animals ; on the other hand, parasites from an apparently healthy host may produce rapidly fatal infections in these same laboratory animals. As Toxoplasma, in nature, is generally a benign parasite producing only chronic infections, the great majority of animals to be studied in parasitological surveys will be apparently healthy ones ; after inoculation of tissues from such animals into mice the following may take place : Firstly, toxoplasms from the inoculum may proliferate rapidly and produce fulminating and fatal infections, the mice usually dying in about a week. In such cases the sickness of the mice is a useful guide and large numbers of the parasites are to be found in the peritoneal exudate (which is usually abundant) and in liver, spleen and lung smears. Secondly, there may be no visible signs of infection in the inoculated mice (which develop chronic infections) but sub-inoculation of their tissues into further mice produces the acute disease.

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roxoplasma I N

ANIMALS

Thirdly, Toxoplasma may only become pathogenic after a long period of frequent sub-inoculation, when the parasites become sufficiently abundant to be ea.sily detected. Finally, many strains persist at the lowest threshold of pathogenicity for mice and do not change this characteristic even after a year or more of continual passage from mouse to mouse (LAINSON, 1955b). Parasites of such strains are the most difficult to detect ; in such cases the toxoplasms undergo a low grade proliferation in the cells of the peritoneal fluid and then, within a week or two, disappear from this site to form the " pseudocysts " in the viscera, musculature and central nervous system (the chronic infection). ~here is no sickness at all on the part of the recipient mice ; hence, although some authors consider that one or two sub-inoculations are sufficient to register the presence or absence of Toxoplasma in a given tissue, it is my own experience that this may result in overlooking infections of this nature. The following procedure was adopted in the present work : One week after the inoculation with the brain suspension, the mice were killed. Impression smears of the peritoneal fluid were made by gently pressing glass slides on to the exposed viscera ; the smears were rapidly dried, fixed with methyl alcohol, and stained for one hour in Giemsa stain (2 drops of stain per 1 ml. of distilled water, p H 7.2 to 7.4). They were examined for 15 minutes under the 16 mm. objective and then, if Toxoplasma could not be seen, for a further 15 minutes under the oil immersion lens. Serial passage of tissues was continued, using a pooled suspension of the exudate (or saline washings from the peritoneal cavities), spleen, liver and brain from each mouse. Six such weekly subinoculations were made before rejecting the run as negative.

d) Maintenance of experimental mice and Toxoplasma strains Precautions must be taken to ensure that infections are not introduced from outside sources : in view of the possible transfer of infection by wild rodents, traps were set and all bedding and food-stuff kept in closed bins. Care was taken to see that water-bottles were returned to the same cages when refilled ; soiled cages, trays and water-bottles were steeped in dilute " lysol " and scrubbed in hot water. The pairs of inoculated mice were kept in well separated cages, so that there was no chance of contamination by faeces or urine.

e) Animal species studied The world-wide distribution of Toxoplasma in so many different animals makes it very difficult to select any particular species as definite reservoirs of infection for man : this difficulty is added to by our ignorance of the parasite's life cycle and by the evidence suggesting that there may be several methods of transmission. Until these problems are solved it must be assumed, therefore, that any infected animal may serve as a source of human infection, while certain hosts may be of greater importance due to their closer association with man. The following formed the main subjects for the present investigation : rats (Rattus norvegicus) ; house mice (Mus musculus) ; and birds, largely jackdaws (Corvus monedula spermologus), pigeons (Columba palumbus palumbus) and rooks (Corvus .frugilegusfrugilegus). Details of the numbers and sources of these animals are included in the Table together with the results. RESULTS

Toxoplasma was isolated from one out of 99 Norway rats ; two out of two weasels (Mustela nivalis) ; one out of one ferret (Mustela (Putorius)furo) ; two out of two ferret/polecats (hybrid animals). Toxoplasmacould not be isolated from 399 house mice, 65 jackdaws, 14 pigeons, six rooks.

R. LAINSON

115

Only in one instance was a highly pathogenic strain of Toxoplasma isolated :: this was from a R. norvegicus and appeared in the second passage mice which died from acute toxoplasmosis 6 days after inoculation. The five strains from the carnivores produced no sickness in the mice and were variably detected in the first or fourth mouse passage. It has been possible to compare these toxoplasms with two strains of human origin : morphologically and serologically they appear identical. After a long search, "pseudocysts" of Toxoplasma were demonstrated in sections prepared from the brains of the five carnivores. TABLE. Results of inoculation of brain 'tissue from some animals into mice for the isolation of Toxo-

plasma gondii.

Animals examined

Number examined

Toxoplasma Locality

isolated

Mouse passages required

Mammals Weasels Ferrets Polecat/ferrets Norway rats 7'

House mice

St. Albans, Herts.

1,4 1

1,4 85 10 4

North Finchley, London Cole Green, Herts. St. Albans, Herts.

25

Weekday Cross, Nottingham Woodthorne, Wolverhampton Quarry Dene, Leeds Different areas in Herts.

40 10 324

2

.=--_

Birds Jackdaws Wood pigeons Rooks

65 14 6

Different areas in Herts.

DISCUSSION The possibility that dogs and cats may become infected by Toxoplasma, after being fed raw rabbit flesh, has previously been considered in discussing the significance of canine and rabbit toxoplasmosis (LAINSON, 1955a, 1955b). In the present work the morphology, biology and serology of all the strains of Toxoplasma from five carnivorous animals studied was indistinguishable from that of six strains previously isolated from domestic rabbits. It is tempting, therefore, to suggest that the mustelid toxoplasms may have originated from rabbits, especially as the stoat family commonly preys on these animals. Infection after ingesting the flesh of domestic rabbits could well apply to the three caged carnivores, the ferret and the two polecat/ferrets (hybrids resulting from a ferret and polecat cross). These were housed at our Field Station and maintained largely on the flesh of domestic and wild rabbits together with laboratory rats and mice. In the case of the weasels, however, such a source of infection is more difficult to explain, for these animals presumably do not have ready access to domestic rabbits, and Toxoplasma could not be isolated from the wild rabbits examined. It is possible that the distribution of Toxoplasma is very patchy. If the findings

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DEMONSTRATION OF

Toxoplasma IN

ANIMALS

of BEVERLEY et al. (1954), for example, are taken to indicate present or past infections it would indicate that Toxoplasma exists in wild rabbits in the form of isolated zoonoses. Unfortunately, it was not possible to obtain rabbits from the same locality in which the two weasels were shot as they had been virtually eliminated by myxomatosis at that time. I have been unable to trace any previous reference to Toxoplasma infection in the weasel, but mention must be made of the finding of this parasite in caged ferrets at Mill Hill laboratories, London (CouTELEN, 1932). Twelve of 150 examined were infected and, although no suggestion was made as to the possible source of the infections, it was mentioned that all the ferrets were strictly isolated from one another. This, again, suggests that infection may have been acquired per os after the ingestion of infected flesh. It would be very interesting to examine a much larger number of purely carnivorous animals to see whether or not Toxoplasma infection is more prevalent in these animals than in herbivores and omnivores. Study of the Toxoplasma strains previously isolated from rabbits and here from the carnivores emphasizes the fact that the parasite is normally a well-adapted one and that disease directly attributable to the organism is the exception rather than the rule ; this is, of course, to be expected if the parasite is to maintain itself successfully in nature. It is perhaps a pity that so much attention has been devoted to the study of what I would prefer to term the atypical or altered strains of Toxoplasma rather than to the avirulent forms described above : most experiments on the transmission of the organism, for example, have been carried out using such highly virulent toxoplasms as provided by the well-known " RH " strain. That it is important to consider the apparently avirulent toxoplasms in relation to human infection and disease has already been stressed (LAINSON, 1955b). The rabbit strains were passaged in mice for a year with no attending increase in pathogenicity for mice and guineapigs ; similarly, 14 rapid, weekly passages in mice did not alter the virulence of the parasites. On introduction of the strains into multimammate rats, 31astomys coucha (Rattus coucha), however, the toxoplasms became highly pathogenic and always produced fatal infections in these animals ; in canaries the pathogenicity was less marked but nevertheless considerable. This suddenly increased virulence in the new hosts was fully maintained on the first passage of the organisms back into laboratory mice and guinea-pigs, which died from acute infections. The toxoplasms isolated from the mustelids have since been shown to behave in a similar manner. M. coucha has, up to now, been little used as an experimental animal in the laboratory. The great susceptibility of this animal to Toxoplasma infection in the above cases (greater than that of the laboratory mouse) indicates that it might be considered as the experimental animal of choice in work on this parasite ; they are easily bred all the year round, and have large litters. GUNDERS (1956) has shown M. coucha also to be susceptible to Plasmodium berghei, Babesia rodhaini, Trypanosoma congolense and Leishmania donovani. There is little doubt that the animal would also be useful for the study of many other parasitic infections. SUMMARY

(1) Until further information is available on the life cycle of Toxoplasma, it is important to gain some knowledge of the incidence of the parasite in animal species which may form reservoirs of infection for man. Little work of this nature has been done in England. (2) Isolation of the parasite is the sole proof of infection and such isolation is so far dependent on the inoculation of suspect tissues into clean laboratory animals ; up to now

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117

the mouse is still the experimental animal of choice as rabbits and guinea-pigs are frequently found to be spontaneously infected and rats are generally resistant to infection by Toxoplasma. (3) Brain tissue is the best material for passage into mice, the intraperitoneal route is to be preferred, and sub-inoculations should be carried out at weekly intervals to detect avirulent strains of Toxoplasma. (4) Toxoplasma was isolated from one of 99 Norway rats and all of five members of the Mustelidae examined. No infections were found in 399 house mice or 85 birds, including jackdaws, rooks and pigeons. (5) All strains except that from the rat were of very low pathogenicity to mice ; morphologically, biologically and serologically the mustelid toxoplasms were identical, and they were indistinguishable from parasites previously isolated from rabbits in this country. (6) It is suggested that the five carnivores found infected may have become so by the ingestion of infected flesh, possibly that of rabbits, and that further examination of habitually carnivorous animals may yield interesting results. (7) Toxoplasma, as indicated by the present findings, is a benign and well-adapted parasite. Disease caused by the organism is considered due to aberrant or altered strains : such alteration may be speedily effected by passage of the parasites into certain species of animals. (8) T h e multimammate rat (Mastomys coucha) was found to be more highly susceptible to Toxoplasma isolated from rabbits and some mustelid animals than the laboratory mouse. It is suggested that the multimammate rat might prove more suitable in studies on this parasite. REFERENCES BEVERLEY,J. K. A., BEATTIE,C. P. & ROSEMAN,C. (1954). ft. Hyg. Camb., 52, 37. BONSER, G. M. (1950). ft. Path. Bact., 62, 650. CAMPBELL, R. S. F., NIARTIN,W. B. & GORDON, E. D. (1955). Vet. Rec., 67, 708. CARINL A. & MIGLIANO, L. (1916). Bull. Soc. Path. exot., 9, 435. CASTELLANI,A. (1913). ft. Ceylon Branch, Brit. reed. Ass., 10, 20. CHALMERS,A. J. & KAMAR,A. (1920). ft. trop. Med. Hyg., 23, 45. COLES, A. C. (1914). Parasitology, 7, 17. COUTELEN,F. (1932). C. R. Soc. Biol. Paris, 111,284. FEDOBOVITeH,A. I. (1916). Ann. Inst. Pasteur, 30, 249. FINDLA¥, G. M. & MIODLETON,A. D. (1934). ft. Anita. Ecol., 3, 150. GUNDEBS, A. E. (1956). Trans. R. Soc. trop. ,~1ed. Hyg., 50, 300. JANKr0, J. (1923). Cas. L~h. Ces., 62, 1021, 1054, 1081, 1111, 1138. LAINSON, R. (1955a). Ann. trop. Med. Parasit., 49, 384. - (1955b). Ibid., 49, 397. - (1956). Ibid., 50, 172. MACINTYItE, A. B., TREVAN,D. J. & ~IONTGOMERIE,R. F. (1948). Vet. Rec., 60, 635. MAmANI, G. (1941). Riv. Biol. colon., 4, 47. MARKHAM, F. S. (1937). Amer. ]. It~,g., 26, 193. MOOSER, H. (1950). Sehweiz. reed. Wschr., 80, 117. NICOLAU, S. (1932). C . R . Soc. Biol., Paris, 110, 676. - & BALMI'S, G. (1933). Ibid., 113, 1002. PEltDRAU, J. R. & PUGH, L.P. (1930). ft. Path. Baet., 33, 79. SANCmRGI, G. (1914a). G. Accad. Med. Torino, 78, 383. (1914b). Pathologica, 7, 345. SPLENDORE,A. (1908). Rev. Soc. Sci. S. Paulo, 3, 109. TORRES, C. M. (1927). C . R . Soc. Biol., Paris, 97, 1778. WENYON, C. M. (1923). Trop. Dis. Bull., 20, 527. (1926). Protozoology, 2, 1041. London : Baillifire, Tindal & Cox. WOKE, P. A., JACOBS,L., Jo>~es, F. E. & MELTON, M. L. (1953). ft. Parasit., 39, 523. WOLF, A., COWEN, D. & PAIOE, B. H. (1939). Science, 89, 226. -

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