Frances E. Jones

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.

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.

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.

Fluorescent Antibody Reactions against Six Species of Simian Malaria in Monkeys from India and Malaysia.

Sera from 45 Malaysian and 51 Indian monkeys were tested for fluorescent antibody response to antigens of Plasmodium fieldi, P. gonderi, P. inui, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii. A total of 17 Malaysian and 33 Indian monkey sera had positive antibody responses with the larger number reacting with P. fieldi and P. gonderi. The results suggest the possibility of prior malaria infection or of nonspecific antibody to other parasites. The intensity of this response to heterologous or common antigens may be sufficient to mask a species-specific response. The use of monkeys having no initial fluorescent antibody titer for Plasmoclium is suggested for studies involving antibody in monkey malaria. The staining of monkey malaria parasites using the fluorescent antibody (FA) technique was first reported by Ingram et al. (1961) using Plasmodium cynomolgi bastianellii. Additional studies by Tobie and Coatney (1961) and Tobie et al. (1962) showed cross-reactions between P. vivax and P. cynomolgi bastianellii. Reported here are the fluorescent antibody reactions of monkey sera from Malaysia and India against six different species of monkey malaria. MATERIALS AND METHODS Thin blood films, containing the parasites of P. fieldi, P. gonderi, P. inzi, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii, were prepared and stored without fixation at -70 C in a mechanical freezer. Prior to use, they were raised to room temperature in a desiccator and then fixed for 30 min in acetone at -20 C. The slides were dried and then dehemoglobinized by immersion for 5 min in 0.1% HC1, followed by 1-min rinses each in distilled water and phosphate-buffered saline (PBS), pH 7.0. The antigen slides were then ready for immediate use. Rabbit antiserum (globulin fraction) to monkey globulin conjugated with fluorescein isothiocyanate was obtained commercially (Sylvana Chemical Company) and prepared by absorption once with rabbit liver powder and once with rabbit bone marrow powder, and stored at 1: 40 in PBS at -20 C. The 1: 40 dilution was used throughout the studies. Received for publication 28 July 1964. 81 A Leitz SM fluorescence microscope with an Osram HBO-200 light source was used with BG-12 and UG-2 exciter filters and a Wratten 2A barrier filter. For each test, several drops of the serum dilution to be tested were applied to the antigen slide and allowed to react for 20 min at room temperature in a moist chamber. The slide was then given 3 5-min rinses in PBS. Several drops of the labeled rabbit antimonkey globulin were applied and allowed to react for 20 min. The slide was again given three 5-min rinses in PBS and then mounted under a cover slip in buffered glycerine. Fluorescence was graded 1+ to 4+. A reading of 2+ was considered positive and reproducible. Each serum sample was titrated simultaneously against all six antigens. The test sera were from several species of Macaca and Presbytis monkeys collected in the Selangor, Pahang, and Perak states of Malaysia in 1961, and from Macaca mulatta monkeys arriving in our laboratory from New Delhi, India, in 1963. Of the Malaysian monkeys, only one (C-22) was infected with Plasmodium (species undetermined) at the time of collection. Since these were field-collected sera, slides were made only the 1 day. It is therefore impossible to determine if other of these monkeys were suffering from chronic infections. None of the Indian monkeys had malarial infections as determined by the microscopic examination of blood films taken on two separate days. Previous laboratory studies had shown that monkeys having chronic infection with Plasmodium sp. had FA end points of 1: 40 or greater. Even though all the sera were tested starting at the 1: 10 dilution, it was decided to report only those FA reactions of 1 : 40 or greater as being positive. This content downloaded from 157.55.39.249 on Wed, 03 Aug 2016 05:47:52 UTC All use subject to http://about.jstor.org/terms 82 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 1, FEBRUARY 1965 TABLE I. Fluorescent antibody test reactions (expressed as reciprocal of titer) of 17 positive sera from Presbytis sp. and Macaca sp. monkeys collected in Malaysia in 1961. Monkey Monkey Species of Plasmodium species no. species no. P. fieldi P. gonderi P. inui P. coatneyi P. obscura 0-61 40 40 P. cristatus C-18 40 C-22 40 40 -25 40 160 C-27 40 C-28 40 40 -29 40 40 C-31 40 C-32 160 C-38 40 M. irus M-312 40 -313 80 M-314 40 M-315 40 M-316 40 M. nemestrina N-93 80 N-94 40 Positive responses 13 4 1 4 Control sera from monkeys known to be free of previous malarial infection gave negative responses. In addition, the labeled antimonkey globulin alone failed to give any response. Monkeys having previous malaria reacted strongly both to homologous and heterologous antigens.

Amebio Infections in Dogs.

Fecal specimens from a series of 143 dogs were examined for amebae by this laboratory as a part of a larger epidemiologic study of intestinal parasites in the Memphis, Tennessee area. The purpose of this study was to determine whether or not the dog might serve as a reservoir from which man could acquire infections, particularly of Endamoeba histolytica, in this region. Secondarily, the study was designed to provide additional information on the amebic parasites of the dog. A review of the available literature shows that the dog has been found infected naturally with E. histolytica in a number of areas. Isolated instances of canine infection have been reported from India, Manchuria, Egypt, Panama, China, IndoChina, and the United States; however, studies which provide prevalence data are less frequent. In this country Swartzwelder and Avant (1952) reported 10 per cent of 70 dogs examined in New Orleans to be naturally infected. Frye and Meleney (1932) examined 22 dogs at New Hope in Jackson County, Tennessee but found no infections. No reports of other systematic investigations of dog populations were found.