Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis.

Researchers have learned much concerning the population biology of Toxoplasma gondii over the past 2 decades. It is now apparent that many atypical genotypes exist besides the typical 3 genotypes (type I, type II and type III) first described from samples from Europe and the United States. These genotypes can differ in pathogenicity and transmissibility from the typical genotypes that have been used in the majority of scientific research over the past 70 years. These differences impact much of what we used to believe as facts about congenital toxoplasmosis (CT) and will be important in developing new recommendations for prevention of CT and the monitoring of women at risk for developing CT. The present review highlights new information on T. gondii genotypes and how this information will change the way we convey information about CT to pregnant women, physicians and students.

Re-Evaluation of Asynchronous Asexual Development of Cystoisospora canis in Intestines of Dogs.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9-11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia ( Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called "cysts." However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term "types" as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species.

New Observations Allowing the Differentiation of Late Asexual Stages of Cystoisospora canis from Developing Microgamonts in the Intestines of Experimentally Infected Dogs.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generations of meronts are reported to occur in the small intestine of experimentally infected dogs before gametogony and oocyst formation. Oocyst excretion in the feces occurs at 9 to 11 days post-inoculation (PI). We examined the late asexual and sexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 sporulated C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria, throughout the small intestine in sections stained with hematoxylin and eosin from both dogs. In other studies of the C. canis life cycle, little attention has been given to distinguishing the last asexual generation of meronts and early microgamonts that can appear similar due to their stage of maturation and both having multiple nuclei. Here we report newly identified features of developing meronts and microgamonts and their distinction from each other by using sections processed using the periodic acid-Schiff (PAS) reaction. Using this method, we demonstrated that PAS-positive granules could be used to identify microgamonts and differentiate them from developing meront stages. These findings will aid pathologists and others in properly identifying coccidial parasites, in determining the cause of microscopic lesions in intestinal tissue, and in accurately identifying etiological agents.

Optimization of the use of C57BL/6 mice as a laboratory animal model for Neospora caninum vaccine studies.

The development and testing of vaccines for Neospora caninum in mice require challenge studies to demonstrate a reduction in clinical signs or prevention of vertical transmission of the parasite after vaccination. Genetic susceptibility to N. caninum varies with the strain of mice. In this study, C57BL/6 mice were evaluated as a model for Neospora vaccine studies. A lethal challenge model was developed and the LD(50) was determined to be 1.5 x 10(7)N. caninum tachyzoites/mouse, delivered intraperitoneally. Brain lesions encountered in sections from sub-lethally challenged mice were scored on the basis of severity and total number of lesions to develop a histopathological scoring system for vaccine efficacy. A vertical transmission model for N. caninum vaccine studies was developed by studying mice that were infected either 2 weeks prior to mating or between days 12 and 14 of pregnancy. It was found that infection prior to mating reduced the average number of pups per litter. DNA extracted from fetal tissue was examined by a N. caninum specific polymerase chain reaction (PCR). The rate of vertical transmission was 0, 100 and 90.5% for the uninfected controls, mice infected during pregnancy and mice infected before mating, respectively. This study demonstrates that the C57BL/6 strain of mice is a good model for N. caninum vaccine studies because it is possible to establish a clear-cut lethal challenge model in C57BL/6 mice and they transmit the disease to their offspring efficiently.

Serological survey of Leishmania infantum and Trypanosoma cruzi in dogs from urban areas of Brazil and Colombia.

Leishmania infantum and Trypanosoma cruzi are zoonotic parasites that are endemic throughout many parts of Latin America. Infected dogs play an important role in transmission of both parasites to humans. A serological survey of Leishmania and Trypanosoma infection was conducted on 365 dogs from São Paulo, Brazil and Bogatá, Colombia, South America. Serum samples were examined by the indirect immunofluorescent antibody test (IFAT). Anti-Leishmania IgG antibodies were detected in 5 of 107 from Brazil (4.7%) and in 4 of 258 dogs (1.6%) from Colombia. Titers ranged from 1:25 to 1:100. Anti-T. cruzi antibodies were not detected in any of the dogs from either Brazil or Colombia. The results show a low prevalence of anti-Leishmania antibodies and no antibodies against T. cruzi in these canine populations. Our study suggests that dogs play a limited role in the spread of L. infantum and T. cruzi in these urban areas of Brazil and Colombia.

Protozoal meningoencephalitis in sea otters (Enhydra lutris): a histopathological and immunohistochemical study of naturally occurring cases.

Protozoal meningoencephalitis is considered to be an important cause of mortality in the California sea otter (Enhydra lutris). Thirty nine of 344 (11.3%) California (CA) and Washington state (WA) sea otters examined from 1985 to 2004 had histopathological evidence of significant protozoal meningoencephalitis. The aetiological agents and histopathological changes associated with these protozoal infections are described. The morphology of the actively multiplicative life stages of the organisms (tachyzoites for Toxoplasma gondii and merozoites for Sarcocystis neurona) and immunohistochemical labelling were used to identify infection with S. neurona (n=22, 56.4%), T. gondii (n=5, 12.8%) or dual infection with both organisms (n=12, 30.8%). Active S. neurona was present in all dual infections, while most had only the latent form of T. gondii. In S. neurona meningoencephalitis, multifocal to diffuse gliosis was widespread in grey matter and consistently present in the molecular layer of the cerebellum. In T. gondii meningoencephalitis, discrete foci of gliosis and malacia were more widely separated, sometimes incorporated pigment-laden macrophages and mineral, and were found predominantly in the cerebral cortex. Quiescent tissue cysts of T. gondii were considered to be incidental and not a cause of clinical disease and mortality. Protozoal meningoencephalitis was diagnosed more frequently in the expanding population of WA sea otters (10 of 31, 32.3%) than in the declining CA population (29 of 313, 9.3%). Among sea otters with protozoal meningoencephalitis, those that had displayed neurological signs prior to death had active S. neurona encephalitis, supporting the conclusion that S. neurona is the most significant protozoal pathogen in the central nervous system of sea otters.

Isolation, Culture and Cryopreservation of Sarcocystis species.

More than 200 valid Sarcocystis species have been described in the parasitological literature. The developmental life cycle in the intermediate host and definitive host has only been described for a few species. Sarcocystis parasites are common pathogens infecting a wide range of animals, including humans, and this unit reviews the methods used for isolating infective stages of the parasite from both definitive and intermediate host(s), as well as methods used to initiate cultures from sporocysts and merozoites and for cryopreservation of various Sarcocystis spp. These methods are based on published reports and our experience with Sarcocystis species in cell culture over many years. The information presented is suitable for the efficient culture of many Sarcocystis species; however, some minor modifications may be needed based on the unique developmental patterns of some species. © 2017 by John Wiley & Sons, Inc.

Sarcocystis pantherophisi n. sp., from Eastern Rat Snakes (Pantherophis alleghaniensis) as Definitive Hosts and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Hosts.

Here, we report a new species, Sarcocystis pantherophisi n. sp., with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n = 15) from intestinal contents of the snake were 10.8 × 8.9 µm. Sporocysts were orally infective to KO mice but not to laboratory-raised albino outbred house mice (Mus musculus). The interferon gamma KO mice developed schizont-associated neurological signs, and schizonts were cultivated in vitro from the brain. Mature sarcocysts were found in skeletal muscles of KO mice examined 41 days postinoculation (PI). Sarcocysts were slender, up to 70 µm wide and up to 3.5 mm long. By light microscopy, sarcocysts appeared thin-walled (<1 µm) without projections. By transmission electron microscopy, the sarcocyst wall was a variant of "type 1" (type 1i, new designation). The parasitophorous vacuolar membrane (pvm) had approximately 100-nm-wide × 100-nm-long bleb-like evaginations interspersed with 100-nm-wide × 650-nm-long elongated protrusions at irregular distances, and invaginations into the ground substance layer (gs) for a very short distance (6 nm). The gs was smooth, up to 500 nm thick, without tubules, and contained a few vesicles. Longitudinally cut bradyzoites at 54 days PI were banana-shaped, 7.8 × 2.2 µm (n = 5). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes indicated a close relationship with other Sarcocystis parasites that have snake-rodent life cycles. The parasite in the present study was molecularly and biologically similar to a previously reported isolate (designated earlier as Sarcocystis sp. ex Pantherophis alleghaniensis) from P. alleghaniensis, and it was structurally different from other Sarcocystis species so far described.

Sarcocystis strixi n. sp. from a Barred Owl ( Strix varia) Definitive Host and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Host.

Here we report a new species of Sarcocystis with a barred owl ( Strix varia) as the natural definitive host and interferon gamma gene knockout (KO) mice as an experimental intermediate host. A barred owl submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because of paralysis. Fully sporulated 12.5 × 9.9 µm sporocysts were found in intestinal scrapings from the owl. Sporocysts from the barred owl were orally fed to 4 laboratory-reared outbred Swiss Webster (SW) ( Mus musculus) and 8 KO mice. All mice remained asymptomatic. Microscopic sarcocysts were found in all 5 KO mice euthanized on day 32, 59, 120, 154, and 206 post-inoculation (PI), not in KO mice euthanized on day 4, 8, and 14 PI. Sarcocysts were not found in any SW mice euthanized on day 72, 120, 206, and 210 PI. Sarcocysts were microscopic, up to 70 µm wide. By light microscopy, the sarcocyst wall < 2 µm thick had undulating, flat to conical, protrusions of varying dimensions. Numerous sarcocysts were seen in the histological sections of tongue and skeletal muscles from the abdomen, limbs, and eye but not in the heart. By transmission electron microscopy, the sarcocyst wall was "type 1j." The ground substance layer (gs) was homogenous, up to 2 µm thick, with very fine granules, and a few vesicles concentrated toward the villar projections. No microtubules were seen in the gs. Longitudinally cut bradyzoites at 206 days PI were 7.8 × 2.2 µm. Based on molecular characterization using 18S rRNA, 28S rRNA, and cox1 genes and morphology of sarcocysts, the parasite in the present study was biologically and structurally different from species so far described, and we therefore propose a new species name, Sarcocystis strixi n. sp.

Sarcocystis jamaicensis n. sp., from Red-Tailed Hawks (Buteo jamaicensis) Definitive Host and IFN-γ Gene Knockout Mice as Experimental Intermediate Host.

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-γ gene knockout (KO) mice as an experimental intermediate host in which sarcocysts form in muscle. Two RTHs submitted to the Carolina Raptor Center, Huntersville, North Carolina, were euthanized because they could not be rehabilitated and released. Fully sporulated 12.5 × 9.9-µm sized sporocysts were found in intestinal scrapings of both hawks. Sporocysts were orally fed to laboratory-reared outbred Swiss Webster mice (SW, Mus musculus) and also to KO mice. The sporocysts were infective for KO mice but not for SW mice. All SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in any SW mice euthanized on days 54, 77, 103 (n = 2) or 137 post-inoculation (PI). The KO mice developed neurological signs and were necropsied between 52 to 68 days PI. Schizonts/merozoites were found in all KO mice euthanized on days 52, 55 (n = 3), 59, 61 (n = 2), 66, and 68 PI and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neural tissue. The schizonts/merozoites were located in neural tissue and were apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice by subcutaneous inoculation and when seeded on to CV-1 cells. Microscopic sarcocysts were found in skeletal muscles of 5 of 8 KO mice euthanized between 55-61 days PI. Only a few sarcocysts were detected. Sarcocysts were microscopic, up to 3.5 mm long. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. By transmission electron microscopy, the sarcocyst wall classified as "type 1j" (new designation). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes revealed a close relationship with Sarcocystis microti and Sarcocystis glareoli; both species infect birds as definitive hosts. The parasite in the present study was biologically and molecularly different from species so far described in RTHs and we therefore propose a new species name, Sarcocystis jamaicensis n. sp.

Antibody Prevalence and Risk Factors for Toxoplasma gondii Infection in Women from Multan, Pakistan.

Toxoplasma gondii infections are prevalent in humans and warm-blooded animals. Maternal infections during pregnancy may have devastating consequences for transplacentally infected neonates. This study was conducted to examine the seroprevalence of antibodies to T. gondii in pregnant women of childbearing age and determine risk factors associated with pregnancy history, pet ownership, social and cultural factors at Nishtar Hospital, Multan. Samples were collected from 403 women and examined using a commercially available enzyme-linked immunosorbent assay (ELISA). The overall prevalence of antibodies to T. gondii was 17.6% (71) in the 403 samples collected from women. Antibodies to T. gondii were present in 19.4% (45) of 232 pregnant women and 15.2% (26) of the samples from 171 non-pregnant women. This study identified miscarriage history, pet ownership, type of residence, marital status, source of drinking water and eating habits as significant (P < 0.05) risk factors associated with the presence of antibodies to T. gondii infection. Seroprevalence was not significantly different (P > 0.05) in women from different ethnic groups based upon lifestyle and culture.

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue.

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.

Variation in Eimeria oocyst count and species composition in weanling beef heifers.

Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from 11 weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent samples were collected from the same animals on 15 April 2005 and 9 June 2005. Oocyst numbers were determined by the modified McMaster's test, and species were identified by examination of oocysts recovered with the Wisconsin sugar flotation technique. Soil samples were collected from the heifer pasture on 8 June 2005, and oocysts were quantified and identified to species. Mean fecal oocyst counts varied little at all sampling dates ranging from 134-377 oocysts/g. Ten Eimeria spp. were identified in fecal samples collected in November and April and 11 in June. Eimeria bovis was the most common species identified at all samplings. Mean species composition showed little variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. The results indicate that clinically normal weanling beef heifers are likely to be infected with a diverse, but relatively stable, community of Eimeria spp.

Comparison of Legionella longbeachae and Legionella pneumophila cases in Scotland; implications for diagnosis, treatment and public health response.

The reported incidence of Legionnaires' disease caused by Legionella longbeachae has increased since 2008 in Scotland. While microbiological and epidemiological studies have identified exposure to growing media as a risk factor for infection, little is known about the differences regarding disease risk factors, clinical features and outcomes of infection with L. longbeachae when compared with L. pneumophila. A nested case-case study was performed comparing 12 L. longbeachae cases with 25 confirmed L. pneumophila cases. Fewer L. longbeachae infected patients reported being smokers [27% (95% CI 2-52%) vs. 68% (95% CI 50-86%), P = 0.034] but more L. longbeachae patients experienced breathlessness [67% (95% CI 40-94%) vs. 28% (95% CI 10-46%), P = 0.036]. Significantly more L. longbeachae-infected patients received treatment in intensive care [50% (95% CI 22-78%) vs. 12% (95% CI 0-25%), P = 0.036]. However, the differences in diagnostic methods between the two groups may have led to only the most severe cases of L. longbeachae being captured by the surveillance system. No differences were observed in any of the other pre-hospital symptoms assessed. Our results highlight the similarity of Legionnaires' disease caused by L. pneumophila and L. longbeachae, and reinforce the importance of diagnostic tools other than the urinary antigen assays for the detection of non-L. pneumophila species. Unfortunately, cases of community-acquired pneumonia caused by Legionella species will continue to be underdiagnosed unless routine testing criteria changes.

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum.

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.

Prevalence of IgG Antibodies to Toxoplasma gondii in Veterinary and Undergraduate Students at Virginia Tech, Blacksburg, Virginia.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects humans and other warm-blooded vertebrates. Felids are the only definitive host for T. gondii, and they excrete oocysts in their faeces. The national prevalence in humans is declining in the United States. This zoonotic organism is of particular interest due to its importance in pregnant women, in individuals with altered immune systems, and in reactivated ocular infections. Exposure to the parasite in humans is usually associated with consumption of raw or undercooked meat or by accidental ingestion of oocysts. It was hypothesized that veterinary students would have a greater chance at exposure to the parasite than an average population of undergraduate students due to increased contact with cats who are infected. A commercially available ELISA was used to examine serum samples from 336 students (252 veterinary students and 84 undergraduate students) at Virginia Polytechnic Institute and State University and the Virginia-Maryland Regional College of Veterinary Medicine for serum IgG antibodies to T. gondii antigen. The prevalence of T. gondii in these subjects was 5.6% in veterinary school students (n = 252) and 2.4% in undergraduates (n = 84). There was no significant difference (P > 0.05) in the prevalence of T. gondii antibodies in veterinary versus undergraduate students. The overall prevalence of 4.8% in all students in this study reflects the continuing decline of antibodies to T. gondii in humans in the United States.

Experimental transmission of Cystoisospora felis-like coccidium from bobcat (Lynx rufus) to the domestic cat (Felis catus).

Cystoisospora felis is an ubiquitous coccidian of cats. The domestic cat (Felis catus) is its definitive host and several mammalian and avian species are its optional intermediate/transport hosts. Nothing is known if it is transmissible to wild felids. In the present study C. felis-like oocysts were found in two naturally infected bobcats (Lynx rufus) from Pennsylvania. To study transmission of C. felis-like parasite from bobcats to domestic cats, sporulated oocysts of C. felis-like from one bobcat were orally inoculated into interferon gamma gene knockout (KO) mice, and 56 days later tissues of KO mice were fed to two coccidian-free cats; two littermate cats were uninoculated controls. The inoculated cats and controls were euthanized five and seven days later, and their small intestines were studied histologically. One inoculated cat excreted C. felis-like oocysts seven days post inoculation (p.i.) and was immediately euthanized. Mature schizonts, mature male and female gamonts, and unsporulated oocysts were found in the lamina propria of small intestine; these stages were morphologically similar to C. felis of domestic cats. No parasites were seen in histological sections of small intestines of the remaining three cats. The experiment was terminated at seven days p.i. (minimum prepatent period for C. felis) to minimize spread of this highly infectious parasite to other cats. Although oocysts of the parasite in bobcats were morphologically similar to C. felis of domestic cats, the endogenous stages differed in their location of development. The bobcat derived parasite was located in the lamina propria of ileum whereas all endogenous stages of C. felis of domestic cats are always located in enterocytes of intestinal epithelium. Characterization of DNA isolated from C. felis-like oocysts from the donor bobcat revealed that sequences of the ITS1 region was only 87% similar to the ITS1 region of C. felis from domestic cats. These results indicate that the parasite in bobcat is likely different than C. felis of cats.

Source monitoring.

A framework for understanding source monitoring and relevant empirical evidence is described, and several related phenomena are discussed: old-new recognition, indirect tests, eyewitness testimony, misattributed familiarity, cryptomnesia, and incorporation of fiction into fact. Disruptions in source monitoring (e.g., from confabulation, amnesia, and aging) and the brain regions that are involved are also considered, and source monitoring within a general memory architecture is discussed. It is argued that source monitoring is based on qualities of experience resulting from combinations of perceptual and reflective processes, usually requires relatively differentiated phenomenal experience, and involves attributions varying in deliberateness. These judgments evaluate information according to flexible criteria and are subject to error and disruption. Furthermore, diencephalic and temporal regions may play different roles in source monitoring than do frontal regions of the brain.

Development and ultrastructure of Besnoitia oryctofelisi tachyzoites, tissue cysts, bradyzoites, schizonts and merozoites.

Development and structure of different life cycle stages of Besnoitia oryctofelisi which has a rabbit-cat life cycle was studied by light and transmission electron microscopy. For light microscopy, Besnoitia oryctofelisi-infected tissues were stained with haematoxylin-eosin, periodic acid Schiff (PAS) reagent, and immunohistochemically with rabbit anti-B. oryctofelisi polyclonal antibodies and anti-BAG-1 antibodies. In vitro and in vivo-derived tachyzoites were 5-6 microm long and they were found to divide by endodyogeny. In tachyzoites, the nucleus was often central, and micronemes were few and located anterior to the nucleus. Earliest tissue cysts were seen in gerbils starting 12 days p.i. Early tissue cysts had an outer PAS-positive cyst wall, a middle PAS-negative host cell layer, and an inner PAS-negative parasitophorous vacuolar membrane. Organisms in early tissue cysts were PAS-negative, did not stain with anti-BAG-1 antibodies, and amylopectin granules and enigmatic bodies were absent. Tissue cysts beginning 17 days p.i. contained organisms that became PAS-positive and reacted with anti-BAG-1 antibodies, indicating they were bradyzoites. Immunoreactivity with polyclonal anti-B. oryctofelisi antibodies suggested that Besnoitia species bradyzoites are encapsulated by the host cell. Bradyzoites (10 microm) were about twice the length of tachyzoites and contained enigmatic bodies characteristic of Besnoitia bradyzoites. Unlike tachyzoites and tissue cysts, schizonts were located intravascularly in the lamina propria of the small intestine of cats. Merozoites were 5-6 microm long, had few rhoptries and amylopectin granules, had numerous micronemes and had a terminal nucleus.

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula.

Sarcocystis neurona was isolated in nude mice and gamma-interferon knockout mice fed sporocysts from faeces of naturally infected opossums (Didelphis virginiana). Mice fed sporocysts became lethargic and developed encephalitis. Protozoa were first found in the brain starting 21 days post-inoculation. Sarcocystis neurona was recovered in cell culture from the homogenate of liver, spleen and brain of a nude mouse 11 days after feeding sporocysts. The protozoa in mouse brain and in cell culture multiplied by schizogony and mature schizonts often had a residual body. Sarcocystis falcatula, which has an avian-opossum cycle, was not infective to nude or knockout mice. Protozoa were not found in tissues of nude mice or knockout mice after subcutaneous injection with culture-derived S. falcatula merozoites and sporocysts from the faeces of opossums presumed to contain only S. falcatula. Results demonstrate that S. neurona is distinct from S. falcatula, and that opossums are hosts for both species.