Marjorie L. Melton

Toxoplasma gondii: The Oocyst, Sporozoite, and Infection of Cultured Cells

The infective form of Toxoplasma gondii fouind in cat feces is an oocyst wihich, when sporutlated, resembles that of the genus Isospora in havinig two sporocysts. Sporozoites obtained by artificial excystation of the oocyst are infective for monkey kidney cell cultures. Ultrastructural characteristics of sporozoites resemble those seen previously in proliferative stages of Toxoplasma gondii.

Toxoplasmosis in chickens.

Pools of tissues from the ovaries and oviducts of apparently healthy hens, obtained at a poultry processing plant, were examined for Toxoplasma gondii cysts by the digestion-inoculation technique. Each pool contained organs from ten birds. Twelve pools of a total of 62 were found positive. A subsequent survey of 108 individual hens revealed four with chronic toxoplasma infection; none of 108 shelled eggs taken from these birds was positive for toxoplasma. Three hundred and twenty-seven eggs laid by 16 chickens during the chronic stage of infection produced experimentally with chicken strains of T. gondii were also tested and one positive egg was found. Parasitemia was demonstrated in all experimental birds during the acute stage of infection. All birds had toxoplasma cysts in one or more of the following organs or tissues, when killed 3 to 10 months postinoculation: brain, muscle, ovary, oviduct, kidney, gizzard, and intestine. A recent review by Siim, Biering-S0rensen, and M0ller (1963) on toxoplasmosis in domestic animals summarizes most of the reports on this infection in chickens. To our knowledge, the only additional citations needed to complete their list is a report of a spontaneous case in Argentina (Mayer, 1961), one by Kulasiri (1965) on experimental infection of chickens with an avirulent strain of toxoplasma, and another report of experimental infection by Kinjo (1961) that we have not seen. (One paper cited by Siim et al., by Pande, Shukla, and Sekariah, 1961, on toxoplasma from the eggs of domestic fowl, was subsequently questioned, Science 134: 945, 29 September 1961.) In earlier work done in two laboratories of this Institute and reported by Jones et al. (1959), it was found that the chicken is relatively tolerant to toxoplasmosis. Only very large inocula produced disease in mature birds, or even in young chickens. This was true despite hematogenous spread of the organisms early in the course of infection. Furthermore, when tissues of experimentally infected chickens were examined for residual parasites 4 weeks or longer after inoculation, the brain, liver, lung, or muscles were only occasionally found positive. However, these experimental results were in contrast to the observations of others on disease due to toxoplasmosis in naturally infected chickens (Hepding, 1939; Ericksen and Harboe, 1953; Fankhauser, 1951; Nobrega et al., Received for publication 14 May 1966. * Present address: Division of Biologics Standards, NIH, Bethesda, Maryland 20014. 1955). Also, while Ericksen and Harboe had not been able experimentally to reproduce the disease they had observed in epidemic form, Geissler (1955) did produce the clinical picture in experimentally infected chickens. The question of the importance of birds in the epidemiology of toxoplasmosis also requires more study. Gibson and Eyles (1957) found infected chickens among other infected animals in an area adjacent to the home of a case of human toxoplasmosis. Kimball and her associates (1959), in a survey of human beings in Minnesota, reported a correlation of dye test positivity and contact with chickens and other birds, although not with the ingestion of raw eggs. Sparapani (1950) found toxoplasma cysts in developing eggs from the ovaries of hens dead of toxoplasmosis. Biering-S0rensen (1956) also found toxoplasma cysts in the ovaries of chickens with disease due to toxoplasma. Geissler demonstrated, by inoculation into mice, toxoplasma in five eggs from experimentally infected hens and also found complement fixing antibodies in the sera of eight chicks hatched from the eggs of such hens. [He claims much more usefulness for the complement fixation test than do Harboe and his co-workers (Ericksen and Harboe, 1953; Harboe and Ericksen, 1954)]. The reports just cited all have dealt with chickens showing overt disease. It seemed worthwhile to us to reexamine the apparently healthy chicken as a natural host of toxoplasma, and to attempt to appraise the possible importance of eggs as a source of infection for other animals.

Toxoplasma gondii: transmission through feces in absence of Toxocara cati eggs.

When incubated at room temperature (23�C) for 3 to 14 days, feces from cats infected 4 to 8 days with Toxoplasma gondii, and free of Toxocara cati eggs, produced toxoplasmosis in mice. Results indicate that the nematode egg is not necessary for transmission of the parasite.

Effect of pyrimethamine and sulfadiazine on the fine structure and multiplication of Toxoplasma gondii in cell cultures.

Rhesus monkey kidney cell cultures were inoculated with Toxoplasma gondii organisms obtained from peritoneal fluid of mice infected with the RH strain. Pyrimethamine and sulfadiazine were added either singly or in combination to the cultures 4 hr after inoculation. Twenty-four hours later the effect of the drugs on the parasites were studied by light and electron microscopy. Pyrimethamine (1.0 mug/ml) inhibited multiplication of the parasites and caused striking morphological changes. Organisms were rounded and often had a fragmented nucleus. Division was inhibited as indicated by abnormal daughter membrane formation during endodyogeny. No effect was evident in sulfadiazine-treated parasites when concentrations up to 50 mug/ml were used. However, combination of ineffective levels of pyrimethamine (0.1 mug/ml) and sulfadiazine (0.5 mug/ml) produced effects similar to those seen at a higher concentration of pyrimethamine indicating a synergistic action of the 2 drugs.

The prevalence of toxoplasmosis in wild pigeons.

The basis for such conjectures consists largely of reports on the occurrence of cases of toxoplasmosis in dogs, cats, and other animals (for review, see Callahan, Russell and Smith, 1946) and on several surveys on the prevalence of the infection in murine hosts. In the first of these surveys, Perrin, Brigham, and Pickens (1943) found 8.7 per cent of wild Norway rats in Savannah, Georgia infected. Hiilphers, et al. (1947, fide Laven and Westphal, 1950) reported finding Toxoplasma in 27 of 840 hares in Sweden. Laven and Westphal (1950) tested serologically a total of 81 rats from 3 sections of Germany and found 10 positive. Eyles (1952) examined 90 Norway rats by inoculating 18 pairs of guinea pigs each with pooled brain tissue from 5 rats and found Toxoplasma infections in 5 of the pairs. Christiansen and Siim (1951) in Denmark examined histologically a large number of hares shot in a sick condition or found dead in the field. Of 2,812 animals, they found 264 or 9.4 per cent positive for toxoplasmosis. As to birds, Toxoplasma has been reported from about 45 species, mostly on morphological evidence which is not always conclusive. It has been well established, however, by Carini (1911) and by Reis and Nobrega (1936) that the pigeon is a natural host of Toxoplasma. It was also shown by Nobrega and Reis (1942) that toxoplasmas of pigeons are capable of infecting rabbits, guinea pigs, and mice. Also, dogs and cats were infected with the same strain obtained from Reis and Nobrega by Guimaraes and Meyer (1942). In the United States, T. gondii was isolated in mice from the tissues of a healthy pigeon in Cincinnati, Ohio by Feldman and Sabin (1949). This pigeon had a dye test titer of 1 : 1024; two other pigeons, of 20 tested, showed titers of 1: 64. Manwell and Drobeck (1951) found 1 of 60 pigeons from the Syracuse, New York area positive in the dye test, and on this basis postulated a 2 per cent rate for naturally occurring toxoplasmosis in these birds. Epizootics of toxoplasmosis in pigeons have been reported from Panama (Johnson, 1943), Brazil (Springer, 1942), and the Belgian Congo (Wiktor, 1950). Our interest in pigeons as a possible reservoir of infection was aroused as a result of studies on the parasitemia in experimentally infected birds (Jacobs and Jones, 1950). Pigeons infected with the RH strain of T. gondii showed a high parasitemia even in the absence of acute disease. It was considered possible that pigeons with asymptomatic toxoplasmosis might serve for infecting bloodsucking arthropods in nature. Since these birds are ubiquitous and have considerable contact with man, it was deemed worthwhile to obtain information on the occurrence among them of natural infection with T. gondii.

Observations on toxoplasmosis in dogs.

There is no question that under some circumstances Toxoplasma gondii is the occasional cause of a severe disease of dogs. Since Mello described the first spontaneous case of canine toxoplasmosis in 1910, over 50 cases of the infection in dogs have been reported from all parts of the world (for review, see Habegger, 1953). Some of these reports have described a fatal disease, manifested by such symptoms as severe respiratory distress, diarrhea, convulsions, and partial paralysis. On the other hand, some of the reports merely record the finding of toxoplasmas in histological sections of dog tissues, indicating that Toxoplasma may exist in dogs without producing symptoms. There is also serological evidence that dogs are frequently found infected in nature. For example, Miller and Feldman (1953) have reported the finding of anti-Toxoplasma antibodies in 59 percent of 51 dogs investigated. This naturally has led to the conjecture expressed-by various authors (Chamberlain et al., 1953; Fankhauser, 1951; Cole et al., 1953; Westphal and Finke, 1950) that the dog may serve as a reservoir of human infection. Some surveys have indicated a correlation between antibodies in dogs and in their owners and handlers (Otten, Westphal, and Kajahn, 1951) although the serological procedures used give results different from those reported by other investigators. The widespread occurrence of anti-Toxoplasma antibodies among humans and dogs makes it extremely difficult to decide in any particular case that the dog infection manifested by such antibodies was related even in time to human infection. Moreover, the histories of mothers of cases of congenital toxoplasmosis sometimes have contained denials of any even casual contacts with dogs. For example, the mother of one congenital case that we have studied serologically claims that dogs are not allowed in the large apartment development in which she lives and that she hardly ever saw dogs, let alone came in close contact with them, during the time of her pregnancy. The question, therefore, of the epidemiological importance of dogs in regard to human toxoplasmosis has remained obscure. In order to judge the role of the dog in the spread of Toxoplasma, information in regard to the manner by which the parasite might be transmitted from the dog to other animals and man must be accumulated. Some statements can be found on this point in the literature. For example, the pneumonitis described in the acute disease and the finding of toxoplasmas in the alveoli of the lungs (MacHattie, 1938; Olafson and Monlux, 1942) suggest that the organism might be present in sputum from the animals. Also, the parasite has been found in the urine and feces of animals with the acute disease (see Jacobs, 1953 for a review of these observations). Consequently, even though toxoplasmas in the only stage in which we know them

Experimental results on possible arthropod transmission of toxoplasmosis.

The most commonly recognized manifestation of toxoplasmosis in humans is a severe disease of the central nervous system in newborn infants. Neonatal toxoplasmosis is definitely of congenital origin; yet the mother shows no history of illness preceding delivery. Thus, in adults, Toxoplasma infection may be entirely asymptomatic, and the occurrence of the disease in infants is due to the unfortunate circumstance that the mother acquires the infection during the gestation period. On the basis of survey data, it appears that Toxoplasma infection is widespread in man (Feldman and Sabin, 1949) and animals (Jacobs, Melton, and Jones, 1952). The mode of transmission, however, is still a matter of conjecture. The organism, in the stage in which we know it, is not resistant to environmental conditions outside the host, and there is little evidence that the contaminative method could serve for its spread. Transmission by bloodsucking arthropods has been postulated by several investigators, and a few experimental attempts have been made to test various arthropods as vectors. References to the pertinent literature are included in the bibliographies of Weyer (1951), Piekarski (1950), Blanc, Bruneau, and Chabaud (1950), and Jacobs (1953). This article summarizes the results of an extensive series of experiments which were made to test a number of arthropods (17 species) as possible vectors of Toxoplasma gondii. As donors in attempts to infect arthropods, rabbits, guinea pigs, chicks, and pigeons were used. These animals were infected by intraperitoneal or intradermal inoculation with the RH strain of Toxoplasma. This strain of the parasite was isolated by Sabin (1941) from a fatal human case and has since been carried in mice; it is highly virulent for all of the animals used, except chicks. Assurance that the donors actually carried the infection is given by the fact that all rabbits and guinea pigs died within the expected period following inoculation; acute infections in rabbits have been demonstrated always to result in a high parasitemia. The same has been found true of acute infections in birds (Jacobs and Jones, 1950). The chicks and pigeons that did not die were shown to have an infection by inoculation of their blood into mice.

Experimental toxoplasmosis in pigeons

Abstract Data are given on the parasitemia and antibody response in pigeons infected with 3 strains of Toxoplasma , of varying virulence. With the virulent RH strain, which killed about 45% of the birds infected with it, a high parasitemia was obtained regardless of the route of inoculation. The time of appearance or persistence of parasitemia was not markedly different in birds infected by different routes. The parasitemia remained high for about 2 weeks or until death; dilutions up to 1:10,000 of the blood of these birds were found infectious for mice. With the less virulent strains lower parasitemias were obtained; there appears to be a correlation between the pathogenicity of the parasite, in any host, and its spread via the blood stream. Parasitemia does not continue after the antibody titer becomes high. Antibody titer in pigeons as measured in the dye test, drops fairly rapidly and antibody may be undetectable less than a year and a half after infection. Persistence of parasites in the brain of infected birds for as long as 33 months has been demonstrated. Toxoplasmas may remain in brain pseudocysts for long periods without stimulating antibody production. The significance of this observation in relation to the value of serological tests in determining the possible toxoplasmic etiology of human chorioretinitis is briefly discussed.

The Survival of Toxoplasma gondii in Various Suspending Media.

In the handling of Toxoplasma gondii outside its host organisms, for such purposes as the preparation of standard inocula for test animals, some assurance is necessary that the toxoplasmas remain viable and infective in the media used. If the medium is not adequate, no eal standardization of inocula is possible, especially when the time which elapses following removal of the parasite from the host is extended. Because of these considerations, it has been our practice (Jacobs and Jones, 1950) to limit the time of handling to one hour. However, it is possible that even within such a short period deleterious effects on the toxoplasmas might result from an inadequate medium. Consequently it was considered necessary to assess various fluids for their ability to sustain toxoplasmas without harm and to ascertain reasonably safe time limits for the handling of the parasites in vitro. Some data have been reported in regard to the length of time that Toxoplasmainfected tissue may be stored without loss of the parasite. Sabin and Olitzky (1937) stated that the parasites could be preserved by storing an infected mouse brain in Tyrodes solution in the refrigerator without loss of infectivity for 14 days. Wolf, Cowen, and Paige (1940) kept pieces of brain and spinal cord of a fatal neonatal case of toxoplasmosis in sterile physiological saline for 11 hours at 7 degrees C. without noting any essential difference from fresh material in subsequent inoculations into mice. Manwell and his collaborators (1945) stated that infected tissues kept in the refrigerator seldom showed living toxoplasmas after 48 hours. More recently, however, Manwell and Drobeck (1951) report survival of toxoplasmas in infected mouse brains for as long as 18 days when the tissues were suspended in Difco skim milk and kept in the refrigerator; they noted no differences in the time of development of infections in mice inoculated with stored material. To our knowledge, beyond statements on the susceptibility of toxoplasmas to freezing and drying, there has been no report on the survival of small numbers of the organisms in various fluids which might be used for handling Toxoplasma in the laboratory. In this paper will be presented data on the effects of tissue storage on the toxoplasmas and information on the survival of small numbers of the parasites in saline, broth, and other fluids.

Activity of the anticoccidial compound, lasalocid, against Toxoplasma gondii in cultured cells.

The activity of the anticoccidial drug, lasalocid, was tested against Toxoplasma gondii in cell cultures. Multiplication of parasites was inhibited by 0.05 mug/ml of lasalocid added to the cultures prior to adding the parasite inoculum, with the parasite inoculum, or after the parasites had penetrated the culture cells. Penetration of culture cells was inhibited when 0.05 mug/ml lasalocid was added with the parasite inoculum. Incubation of extracellular parasites in 0.5 mug/ml lasalocid had no effect on penetration or multiplication. Ormetoprim, sulfadimethoxine, and a combination of the 2 were less effective than lasalocid. Monensin exhibited an inhibitory effect in all experiments.