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Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. are related Apicomplexan parasites that have 2 hosts in their life cycles. The definitive hosts excrete unsporulated (Neospora caninum, T gondii) or sporulated (Sarcocystis spp.) oocysts in their feces after ingesting tissue cysts from the tissues of ruminant intermediate hosts. These coccidians can cause abortion and neonatal mortality in ruminants. T gondii and Sarcocystis hominis (from cattle) are zoonotic. This article reviews information on the etiology, life cycle, diagnosis, control and prevention of these parasites and the diseases they cause in ruminants. ; Public domain authored by a U.S. government employee

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. ; Public domain authored by a U.S. government employee

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. ; Public domain authored by a U.S. government employee

The life cycle of Sarcocystis species is heteroxenous (2-host), with carnivores being the definitive host and herbivores serving as intermediate hosts in predator-prey relationships. Raptors (eagles, hawks, falcons, and owls) are apex predators and are not consumed routinely by other carnivores, making the occurrence of sarcocysts in their muscles unusual. Recent reports of sarcocysts in eagles and owls with Sarcocystis encephalitis suggests that this condition may be becoming more frequent, and Sarcocystis falcatula has been implicated as the agent of encephalitis in golden (Aquila chrysaetos) and bald eagles (Haliaeetus leucocephalus) as well as great horned owls (Bubo virginianus). The present study was done to determine the prevalence of sarcocysts of Sarcocystis species in the muscles of raptors from the southeastern United States. Pectoral and heart muscle from 204 raptor patients from the Carolina Raptor Center, Huntersville, North Carolina were tested for the presence of Sarcocystis species using histology. Only a few sarcocysts were seen in sections of pectoral muscle from 39 of 204 raptors (19.1%) and heart muscle from 9 that also had sarcocysts in their pectoral muscle. Two structural types of sarcocysts, thin-walled (1 mu m; 62%) or thick-walled (>2 mu m, 38%), were seen. Statistical analysis of raptor age and gender was done by Fisher's exact test on samples from raptors with 20 or more samples per group. The prevalence of sarcocysts by age (2 yr or more) was significant for red-shouldered hawks (Buteo lineatus) (P = 0.022) and Cooper's hawks (Accipiter cooperii) (P = 0.028). Sarcocyst prevalence in male raptors from these groups evaluated statistically were always less than in females. Prevalence in female red-tailed hawks (Buteo jamaicensis) (42.1%) was significantly greater than in males (6.7%) using Fisher's exact test (P = 0.047). Examination of case histories from the 39 sarcocyst-positive raptors did not reveal an association with sarcocysts in raptor pectoral or heart muscle and in a diagnosis of encephalitis. Additional studies are needed to determine the epidemiology and relationships of Sarcocystis spp. that use raptors as intermediate hosts and the importance of Sarcocystis spp. in the overall wellbeing of raptors in their natural environments. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; Intramural Research Competition (IRC) grant from the VirginiaMaryland College of Veterinary Medicine ; We would like to thank the staff and students at the Carolina Raptor Center in Huntersville, North Carolina for providing us with samples. This work was supported by grant 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to A.v.D. and an Intramural Research Competition (IRC) grant from the VirginiaMaryland College of Veterinary Medicine to D.S.L. We thank the numerous undergraduate students from Johnson C. Smith University and undergraduate and graduate students from Virginia Tech for assistance with this project. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture;; USDA is an equal opportunity provider and employer. ; Public domain authored by a U.S. government employee

There is an emerging concern that snakes are definitive hosts of certain species of Sarcocystis that cause muscular sarcocystosis in human and non-human primates. Other species of Sarcocystis are known to cycle among snakes and rodents, but have been poorly characterized in the USA and elsewhere. Although neurological sequalae are known for certain species of Sarcocystis, no such neurological symptoms are known to typify parasites that naturally cycle in rodents. Here, sporocysts of a species of Sarcocystis were found in the intestinal contents of a rat snake (Pantherophis alleghaniensis) from Maryland, USA. The sporocysts were orally infective for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred mice. The KO mice developed neurological signs, and were necropsied between 33 and 52 days post-inoculation. Only schizonts/merozoites were found, and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neuropil. The schizonts and merozoites were located in neuropil, and apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice and CV-1 cell line. DNA extracted from the infected mouse brain, and infected cell cultures revealed the highest identity with Sarcocystis species that employ snakes as definitive hosts. This is the first report of Sarcocystis infection in the endangered rat snake (P. alleghaniensis) and the first report of neurological sarcocystosis in mice induced by feeding sporocysts from a snake. These data underscore the likelihood that parasites in this genus that employ snakes as their definitive hosts constitute an ancient, globally distributed monophyletic group. These data also raise the possibility that neurological sequalae may be more common in intermediate hosts of Sarcocystis spp. than has previously been appreciated. ; Public domain authored by a U.S. government employee

We are interested in the disease ecology of Sarcocystis species that infect birds of prey as definitive and intermediate hosts. The present study was done to test our hypothesis that a laboratory model can be developed for sarcocystis infection in mammals using gamma interferon gene knockout (KO) mice as a source of Sarcocystis strixi bradyzoites and mammalian cell cultures as a source of sporulated S. strixi oocysts. Sporocysts of S. strixi from a naturally infected barred owl (Strix varia) were fed to KO mice to produce sarcocysts, and the enclosed bradyzoites were obtained by acid-pepsin digestion of abdominal and thigh muscles. Bradyzoites, metrocytes, and an unusual spherical stage were seen in digest before the inoculation of host cells. The spherical stages stained dark with Giemsa stain, but no nucleus was observed, and they were seen free and associated with the concave portion of some bradyzoites. Examination of infected cell cultures demonstrated that macrogamonts and microgamonts were present at 24 hr post-inoculation. Since sporulated oocysts were not observed, we had to reject our current hypothesis. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; Virginia-Maryland College of Veterinary Medicine; DOEUnited States Department of Energy (DOE) [DE-SC0014664] ; Published version ; This work was supported in part by grant number 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to A.R.V.D. and an Internal Research Competition (IRC) grant from the Virginia-Maryland College of Veterinary Medicine to D.S.L. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by Oak Ridge Associated Universities (ORAU) under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. The USDA is an equal opportunity provider and employer. ; Public domain authored by a U.S. government employee

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 10.838.9 lm. 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 lm wide and up to 3.5 mm long. By light microscopy, sarcocysts appeared thin-walled (, 1 lm) 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-wide3100-nm-long bleb-like evaginations interspersed with 100-nm-wide 3 650-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 bananashaped, 7.832.2 lm (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. ; Public domain authored by a U.S. government employee

Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host. (C) 2016 Elsevier Ireland Ltd. All rights reserved. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; Virginia Maryland College of Veterinary Medicine ; This work was supported by grant # 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to ACR and an IRC grant from the Virginia Maryland College of Veterinary Medicine to DSL. ; Public domain authored by a U.S. government employee

Felis catus, the domestic cat, is the definitive host for parasites that may result in adverse health outcomes in humans. Prevalence data of zoonotic parasites in feral cats, which are free-roaming domestic cats that are born and live in the wild, are limited. The objective of this study was to assess seroprevalence of Toxoplasma gondii antibodies and copro-prevalence of potentially zoonotic parasites in feral cats and to evaluate risk factors for seropositivity and faecal excretion of parasites. In this cross-sectional survey, 275 feral cats at Trap-Neuter-Release clinics in Central Virginia were tested for parasites via faecal flotation, direct immunofluorescence assay (faeces) and modified agglutination testing (serum). Toxoplasma gondii seroprevalence was 22.35% (95% CI: 17.47-27.86). Faecal prevalence of T.gondii-like oocysts was 1.04% (95% CI: 0.13-3.71), Toxocara cati 58.85% (95% CI: 51.54-65.89), Ancylostoma spp. 18.75% (95% CI: 13.49-25.00), Giardia duodenalis 5.73% (95% CI: 2.89-10.02) and Cryptosporidium spp. 3.33% (95% CI: 1.37-7.24). Female cats were more likely than males to excrete faecal Ancylostoma spp. eggs (OR 2.88; 95% CI 1.34-6.17). Adults were more likely than immature cats to be seropositive (OR 2.10; 95% CI: 1.11-3.97) and to excrete faecal Ancylostoma spp. eggs (OR 2.57; 95% CI: 1.10-5.99). However, immature cats were more likely than adults to excrete T.cati eggs (OR 6.79; 95% CI: 3.31-13.90) and to excrete one or more potentially zoonotic species (OR 4.67; 95% CI: 2.28-9.55) in faeces. Results of this study have implications for human and animal health and highlight the importance of collaboration between public health, medical and veterinary communities in preventive efforts. ; Public domain authored by a U.S. government employee

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-gamma 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 x 9.9-mu m 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 mu m 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. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; Internal Research Competition (IRC) grant from the Virginia-Maryland College of Veterinary Medicine ; This work was supported in part by grant no. 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to A.R.V.D. and an Internal Research Competition (IRC) grant from the Virginia-Maryland College of Veterinary Medicine to D.S.L. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA); USDA is an equal opportunity provider and employer. ; Public domain authored by a U.S. government employee

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-gamma gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk (Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 mu m thick) and smooth. Ultrastructural details of sarcocysts are described. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; Virginia-Maryland College of Veterinary Medicine; U.S. Department of Energy (DOE)United States Department of Energy (DOE); U.S. Department of Agriculture (USDA)United States Department of Agriculture (USDA); DOEUnited States Department of Energy (DOE) [DE-SC0014664] ; This work was supported in part by grant number 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to A.R.V.D. and an Internal Research Competition (IRC) grant from the Virginia-Maryland College of Veterinary Medicine to D.S.L. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by Oak Ridge Associated Universities (ORAU) under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. The USDA is an equal opportunity provider and employer. ; Public domain authored by a U.S. government employee

Toxoplasma gondii is a protozoan parasite that is responsible for approximately 24% of deaths attributed to foodborne pathogens in the United States. It is thought that a substantial portion of human T. gondii infections is acquired through the consumption of meats. The dose‐response relationship for human exposures to T. gondii‐infected meat is unknown because no human data are available. The goal of this study was to develop and validate dose‐response models based on animal studies, and to compute scaling factors so that animal‐derived models can predict T. gondii infection in humans. Relevant studies in literature were collected and appropriate studies were selected based on animal species, stage, genotype of T. gondii, and route of infection. Data were pooled and fitted to four sigmoidal‐shaped mathematical models, and model parameters were estimated using maximum likelihood estimation. Data from a mouse study were selected to develop the dose‐response relationship. Exponential and beta‐Poisson models, which predicted similar responses, were selected as reasonable dose‐response models based on their simplicity, biological plausibility, and goodness fit. A confidence interval of the parameter was determined by constructing 10,000 bootstrap samples. Scaling factors were computed by matching the predicted infection cases with the epidemiological data. Mouse‐derived models were validated against data for the dose‐infection relationship in rats. A human dose‐response model was developed as P (d) = 1–exp (–0.0015 × 0.005 × d) or P (d) = 1–(1 + d × 0.003 / 582.414)−1.479. Both models predict the human response after consuming T. gondii‐infected meats, and provide an enhanced risk characterization in a quantitative microbial risk assessment model for this pathogen.

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 12.539.9 lm 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 lm wide. By light microscopy, the sarcocyst wall, 2 lm 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 lm 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 3 2.2 lm. 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. ; National Science Foundation Historically Black Colleges and Universities Undergraduate Program [1505407]; IRC grant from the Virginia-Maryland College of Veterinary Medicine ; This work was supported by grant 1505407 from the National Science Foundation Historically Black Colleges and Universities Undergraduate Program to A.R. von D. and an IRC grant from the Virginia-Maryland College of Veterinary Medicine to D.S.L. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; USDA is an equal opportunity provider and employer. ; Public domain authored by a U.S. government employee