Recent epidemiologic, clinical, subclinical and genetic diversity of Toxoplasma gondii infections in bats.

The protozoan Toxoplasma gondii infects virtually all warm-blooded animals, including bats. Depending on the diet, bats are classified as frugivorous, insectivorous, omnivorous, hematophagous, nectarivorous and carnivorous. The seroprevalence of T. gondii was higher in insectivores bats than fructivores bats. Owls, eagles, and cats can be predators of bats. Bats may be important in the epidemiology of T. gondii because they can be sentinels and can spread infection. Viable T. gondii has been isolated from brain, heart and pectoral muscle of bats. ToxoDB genotypes #9, #10, #6, #19, #69, #162 were identified from bats tissues. Genotypes #9 is prevalent in Asia. Genotypes #6 is widely distributed across Africa and Brazil. This result was matched with genotypes distribution from other hosts. The present review summarizes worldwide information on the seroprevalence, molecular epidemiology, isolation, genotypes and clinical cases of T. gondii infection in bats. Further studies are needed to verify the validity of serological and molecular tests, and the transmission routes of T. gondii infection in bats.

Recent aspects on epidemiology, clinical disease, and genetic diversity of Toxoplasma gondii infections in Australasian marsupials.

BACKGROUND: Toxoplasma gondii infections are common in humans and animals worldwide. Among all intermediate hosts of T. gondii, captive marsupials from Australia and New Zealand are highly susceptible to clinical toxoplasmosis. However, most free-range marsupials establish chronic T. gondii infection. Infected marsupial meat may serve as a source of T. gondii infection for humans. Differences in mortality patterns in different species of kangaroos and other marsupials are not fully understood. Lifestyle, habitat, and the genotype of T. gondii are predicted to be risk factors. For example, koalas are rarely exposed to T. gondii because they live on treetops whereas wallabies on land are frequently exposed to infection. METHODS: The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, and genetic diversity of T. gondii infecting Australasian marsupials in their native habitat and among exported animals over the past decade. The role of genetic types of T. gondii and clinical disease is discussed. RESULTS: Fatal toxoplasmosis has been diagnosed in captive Australasian marsupials in Argentina, Chile, China, Germany, Hungary, Japan, Spain, Turkey, and the USA. Most deaths occurred because of disseminated toxoplasmosis. Genetic characterization of T. gondii strains isolated from fatal marsupial infections identified Type III as well as atypical, nonclonal genotypes. Fatal toxoplasmosis was also diagnosed in free-ranging wombats (Vombatus ursinus) in Australia. Genetic characterization of DNA amplified directly from host tissues of subclinical culled kangaroos at slaughter identified many mixed-strain infections with both atypical and recombinant genotypes of T. gondii. CONCLUSIONS: Most Australasian marsupials in their native land, Australia and New Zealand, have high prevalence of T. gondii, and kangaroo meat can be a source of infection for humans if consumed uncooked/undercooked. The genotypes prevalent in kangaroos in Australia and New Zealand were genetically distinct from those isolated or genotyped from most macropods in the USA and other countries. Thus, clinical toxoplasmosis in marsupials imported from Australia is most likely to occur from infections acquired after importation.

Recent epidemiologic, clinical, and genetic diversity of Toxoplasma gondii infections in non-human primates.

Toxoplasma gondii infections are common in humans and animals worldwide. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, diagnosis, and genetic diversity of T. gondii in non-human primates (NHP) for the past decade. Seroprevalence estimates of T. gondii worldwide were tabulated for each host. Risk factors associated with T. gondii infections are evaluated. New World NHP in captivity are highly susceptible to T. gondii infection with high mortality associated with disseminated toxoplasmosis. T. gondii can be transmitted to NHP in contact with symptomatic NHP. Therefore, precautions should be taken to prevent transmission of T. gondii to humans while handling symptomatic NHP. There were no reports of clinical toxoplasmosis in Old World NHP. Among the different genera of New World NHP, susceptibility to clinical toxoplasmosis varies a great deal; however, factors affecting this susceptibility are not fully understood. Genetic characteristics of T. gondii strains from monkeys is summarized.

Epidemiological and Public Health Significance of Toxoplasma gondii Infection in Wild Rabbits and Hares: 2010-2020.

Toxoplasmosis is a zoonosis of global distribution, and Toxoplasma gondii infections are common in humans and animals worldwide. Hares and rabbits are important small game species, and their meat is consumed by humans in many countries. Demand for rabbit meat for human consumption is increasing; therefore, toxoplasmosis in rabbits and hares is of epidemiological significance. Viable T. gondii has been isolated from rabbits. The present review summarizes worldwide information on the seroprevalence, parasitological investigations, clinical cases, isolation, and genetic diversity of T. gondii in wild rabbits, free domestic rabbits, hares, and other rabbits from 2010 to 2020. Differences in prevalence, susceptibility, genetic variants, and clinical implications of T. gondii infection in rabbits and hares are discussed. This review will be of interest to biologists, parasitologists, veterinarians, and public health workers. Additional studies are needed to increase our knowledge of genetic variants and the population structure of T. gondii in rabbits and hares and to understand the differences in susceptibility to T. gondii in hares in different areas.

Recent epidemiologic and clinical Toxoplasma gondii infections in wild canids and other carnivores: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, diagnosis, and genetic diversity of T. gondii in wild canids and other carnivores for the past decade. Seroprevalence estimates of T. gondii worldwide were tabulated for each host. Seroprevalence in wild foxes was very high compared with farmed Arctic foxes. Economic and public health aspects of some of the carnivore species raised for fur and meat (raccoon dogs, mink) are discussed. Diagnostic efficacies of different serological methods and PCR methods are discussed. Clinical toxoplasmosis was observed mainly in carnivores concurrently infected with immunosuppressive Canine Distemper Virus infection. Abortion and blindness were noted in mink. Genetic diversity of isolates using DNA derived from 162 (89 viable T. gondii isolates and 73 DNA extracted from tissues) of wild carnivores from several countries is discussed. However, 69 of the 162 T. gondii isolates were strains from USA and these were genetically diverse with predominance of ToxoDB genotypes #4 and #5 (haplogroup 12). Only limited information is available concerning genotyping of T. gondii isolates from other countries; none of the 93 T. gondii isolates from other countries (Brazil, China, France, Grenada) were haplogroup 12.

All about Toxoplasma gondii infections in pigs: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. Toxoplasma gondii infection in pigs continues to be of public health concern. Pigs are important for the economy of many countries, particularly, USA, China, and European countries. Among the many food animals, pigs are considered the most important for T. gondii transmission in USA and China because viable parasites have rarely been isolated from beef or indoor raised chickens. Besides public health issues, T. gondii causes outbreaks of clinical toxoplasmosis in pigs in China, associated with a unique genotype of T. gondii (ToxoDB genotype #9 or Chinese 1), rarely found in other countries. The safety of ready to eat pork products with respect to T. gondii infection is a matter of recent debate. Here, we review in detail seroprevalence, prevalence of viable and nonviable T. gondii, epidemiology, risk assessment, diagnosis, and curing of pork products containing T. gondii for the past decade. This review will be of interest to biologists, parasitologists, veterinarians, and public health workers.

Recent epidemiologic and clinical importance of Toxoplasma gondii infections in marine mammals: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. T. gondii causes mortality in several species of marine mammals, including threatened Southern sea otters (Enhydra lutris) and endangered Hawaiian monk seals (Monachus schauinslandi). Marine mammals are now considered sentinels for environmental exposure to protozoan agents contaminating marine waters, including T. gondii oocysts. Marine mammals also serve as food for humans and can result in foodborne T. gondii infections in humans. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, and genetic diversity of T. gondii infecting marine mammals in the past decade. The role of genetic types of T. gondii and clinical disease is discussed.

Toxoplasma gondii infections in dogs: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, diagnosis, and genetic diversity of T. gondii in dogs (Canis familiaris) from 2009-2020. Seroprevalence estimates of T. gondii worldwide were tabulated. Reports of high seroprevalence in canine population and high congenital transmission of T. gondii in dogs in Brazil are reviewed. Most reports from China were published in Chinese, and these reports are now summarized here. Dogs have an additional importance in some countries such as China, Vietnam, and Nigeria; whereas in many cities dog meat is sold commercially for human consumption and given to felids, and transmission of T. gondii could occur if meat is not cooked properly. Dogs can ingest T. gondii-infected cat feces and these oocysts remain viable after passage through the digestive tract of the dog; T. gondii DNA was found in feces of dogs from New York City parks in USA. Most clinical canine cases of toxoplasmosis were in immunosuppressed dogs, and ulcerative dermatitis was one of the main presentations. Genetic diversity based on PCR-RFLP markers using DNA derived from 133 viable T. gondii isolates from dogs from several countries is discussed. T. gondii strains from Asia and Americas were more genetically diverse than those from Africa. This review will be of interest to biologists, parasitologists, veterinarians, and public health workers.

Distribution of Toxoplasma gondii Tissue Cysts in Shoulder Muscles of Naturally Infected Goats and Lambs.

ABSTRACT: Toxoplasmosis has been recognized as a major public health problem worldwide. The consumption of uncooked or undercooked meat infected with Toxoplasma gondii tissue cysts is one of the main means of transmission of this parasite. Although sheep, goats, and pigs are commonly infected with T. gondii, little information is available on the distribution of T. gondii tissue cysts in naturally infected meat. In this study, we investigated the distribution of viable T. gondii tissue cysts in shoulder muscles of naturally infected lambs and goats. Hearts and shoulders of 46 lambs and 39 goats from a local grocery store were tested for T. gondii infection. Animals were evaluated for the presence of anti-T. gondii antibodies in heart blood and clots by the modified agglutination test. Fourteen of the 85 animals (seven lambs and seven goats) were seropositive. Six to 12 samples weighing 5, 10, and 50 g were obtained from shoulder muscles of each seropositive animal and used for bioassay in mice. The distribution of viable T. gondii differed according to the size of the sample analyzed, but in general larger sample sizes resulted in higher isolation rates (P < 0.05). Results of the study revealed an uneven distribution of T. gondii in muscle samples of lambs and goats and that T. gondii can be transmitted by consumption of very small servings (5 and 10 g) of meat when it is consumed raw or is undercooked.

Low prevalence of viable Toxoplasma gondii in fresh, unfrozen American pasture-raised pork and lamb from retail meat stores in the United States.

In a national survey of fresh, unfrozen, American pasture-raised lamb and pork, the prevalence of viable Toxoplasma gondii was determined in 1500 samples selected by random multistage sampling (750 pork, 750 lamb) obtained from 250 retail meat stores from 10 major geographic areas in the USA. Each sample consisted of a minimum of 500g of meat purchased from the retail meat case. To detect viable T. gondii, 50g meat samples of each of 1500 samples were bioassayed in mice. Viable T. gondii was isolated from 2 of 750 lamb samples (unweighted: 0.19%, 0.00-0.46%; weighted: 0.04%, 0.00-0.11%) and 1 of 750 pork samples (unweighted: 0.12%, 0.00-0.37%; weighted: 0.18%, 0.00-0.53%) samples. Overall, the prevalence of viable T. gondii in these retail meats was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat. Cooking meat to an internal temperature of 66°C kills T. gondii.

Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus.

Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti-Neospora caninum rabbit antibodies. Transmission electron microscopic observation on lung tissue revealed tachyzoites consistent with Neospora, including many rhoptries. Polymerase-chain reaction (PCR) using primers designed to amplify the rRNA gene internal transcribed spacer 1 (ITS1) of the Sarcocystidae was performed on DNA extracted from fetal tissues. Comparison of the ITS1 amplified from the foal tissue to sequences available in GenBank revealed 100% sequence identity to the ITS1 from three isolates of Neospora hughesi.

Isolation and Genetic Characterization of Toxoplasma gondii from Tissues of Wild Turkeys (Meleagris gallopavo) in Pennsylvania.

Toxoplasmosis in wild turkeys (Meleagris gallopavo) is of epidemiological interest because turkeys feed from the ground, and detection of infection in turkeys indicates contamination by oocysts in the environment. During the 2018 spring hunting season in Pennsylvania, fresh (unfixed, not frozen) samples were obtained from 20 harvested wild turkeys and tested for Toxoplasma gondii infection. Hearts from all wild turkeys and skeletal muscle from 1 were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Antibodies to T. gondii were detected in 1:5 dilution of neat serum from 5 of 15 wild turkeys and in fluid from the heart of 1 of 4 wild turkeys with the modified agglutination test (MAT); neat serum was not available from 4 wild turkeys. Viable T. gondii was isolated from hearts of 5 wild turkeys, 1 with MAT of 1:10, 1 with MAT of 1:5, and 3 seronegative (MAT < 1:5). Toxoplasma gondii was isolated from both heart and skeletal muscle in the 1 wild turkey that had skeletal muscle submitted. The KO mice inoculated with tissue from all 5 infected wild turkeys died or were euthanatized when ill, 7-21 days post-inoculation (PI). Tachyzoites were detected in lungs of all KO mice, and the T. gondii strains were successfully propagated in cell culture. The SW mice inoculated with tissues of wild turkeys remained asymptomatic, and tissue cysts were seen in the brains of infected mice when euthanatized in good health at 46 days PI; 1 of the 2 SW mice inoculated with the heart of 1 turkey died on day 26, and tachyzoites were detected in its lung. Genetic typing on DNA extracted from culture-derived tachyzoites using the PCR restriction fragment length polymorphism with 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed that 4 isolates belonged to ToxoDB PCR-RFLP genotype #5 and 1 was genotype #216.

Toxoplasma gondii tissue cyst formation and density of tissue cysts in shoulders of pigs 7 and 14 days after feeding infected mice tissues.

Among the meat sources of Toxoplasma gondii, pork is considered important in the epidemiology of toxoplasmosis in the USA. How soon after infection T. gondii forms tissue cysts in pork is unknown. In the present study, eight serologically negative ˜3 months old pigs were fed mouse tissues infected with VEG (Type III) strain of T. gondii and euthanized 7 (4 pigs) and 14 days (4 pigs) post-inoculation (p.i.). Meat from the right shoulder of each pig was bioassayed in mice for T. gondii tissue cysts by peptic digestion. From each pig, the shoulder muscle was cut at random spots into 5 g, 10 g and 50 g portions. Extreme care was taken to use different scalpels and forceps to minimize cross contamination among 17 samples (6 replicates of each 5 g and 10 g portions and 5 replicates of 50 g). From the four pigs euthanized at 7 days p.i., a composite of ˜200 g of leftover meat from each shoulder was bioassayed in cats and their feces were tested for oocyst excretion. All eight pigs developed T. gondii antibodies (modified agglutination test, MAT, 1: 80 or higher) and viable T. gondii was isolated from shoulder meat of each pig. All four cats fed pork from excreted T. gondii oocysts. The density of T. gondii, based on mouse infectivity, varied within 5-50 g samples each pig, and between pigs within the same group, day 7 versus day 14 p.i. There were no significant differences in mouse bioassay results obtained with day 7 versus day 14 infected pigs. Overall, the rate of isolation of T. gondii increased with sample size of meat bioassayed. Results demonstrate that tissue cysts are formed early in infection and they are unevenly distributed.

Confirmation of Sarcocystis jamaicensis Sarcocysts in IFN-γ Gene Knockout Mice Orally Inoculated with Sporocysts from a Red-Tailed Hawk ( Buteo jamaicensis).

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-γ 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 µm thick) and smooth. Ultrastructural details of sarcocysts are described.

Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA.

Sarcocystis sarcocysts are common in many species of domestic and wild animals. Here, we report sarcocystosis in muscles from 91 free range elk (Cervus elaphus) from Pennsylvania, USA, tested by histopathology, transmission electron microscopy (TEM), and DNA sequencing. Sarcocysts were detected in hematoxylin and eosin (HE)-stained sections from 83 of 91 (91.2%) elk, including 83/91 (91.2%) tongues and 15/17 (88.2%) hearts. With respect to age, sarcocysts were found in 0/5 calves, 8/9 (88.8%) yearlings, and 75/77 (97.4%) adults. Sarcocysts were identified in 62/69 (89.4%) females and 21/22 (91.2%) males. Associated lesions were mild and consisted of inflammatory foci around degenerate sarcocysts. There were two morphologically distinct sarcocysts based on wall thickness, thin (< 0.5 µm) and thick-walled (> 4.0 µm). Thin-walled sarcocysts had a TEM "type 2" and villar protrusions (vps), identical to Sarcocystis wapiti previously described from elk in western USA. This species was present both in tongue and heart samples and was detected in all infected elk. Thick-walled sarcocysts consisted of three morphologic variants, referred to herein as subkinds A, B, C. Subkind A sarcocysts were rare; only four sarcocysts were found in three elk. Histologically, they had a 5-8-µm thick wall with tufted vp. By TEM, the sarcocyst wall was "type 12" and appeared similar to Sarcocystis sybillensis, previously described from elk in USA. Subkind B, Sarcocystis sp.1 sarcocysts were also rare, found in only 1 elk. These sarcocysts had 6.7-7.3-µm-thick wall with TEM "type 15b" vp. Subkind C Sarcocystis sp.2 sarcocysts were more common (22/91). Morphologically, the sarcocyst wall was 6.1-6.8 µm thick and contained "type 10b" vp. Comparisons of ribosomal DNA loci with published sequences indicated all sarcocysts were similar to what has previously been isolated from cervid hosts across the northern hemisphere. Phylogenetic analysis placed the thin-walled S. wapiti within a strongly supported clade with S. linearis and S. taeniata, while the thick-walled cysts were very closely related to S. truncata, S. elongata, S. silva, and S. tarandi. Further sequencing is needed to produce molecular diagnostics to distinguish among these species. North American elk are hosts to multiple Sarcocystis species with diverse morphology, deriving from two separate evolutionary lineages.

Role of Rats ( Rattus norvegicus) in the Epidemiology of Toxoplasma gondii Infection in Grenada, West Indies.

Rodents are known to be reservoir hosts of Toxoplasma gondii infection for other animals, such as cats and pigs. From February to July 2017, 167 rats ( Rattus norvegicus) were trapped in Grenada, and serum, heart, skeletal muscle, and brain were examined for T. gondii infection by serological examination (modified agglutination test, 1:25) for T. gondii antibodies and for viable parasites by bioassay in mice. Samples of heart, skeletal muscle, and brain of all rats were bioassayed in Swiss Webster (SW) outbred albino mice and interferon gamma gene knockout (KO) mice. Toxoplasma gondii was isolated from heart and brain from 1 rat; this was the only seropositive rat. The T. gondii strain was avirulent for SW mice but killed KO mice. Tissue cysts were detected in the brains of SW mice, and tachyzoites were detected in the lungs of KO mice that died of acute toxoplasmosis. The strain was propagated in cell culture, and DNA derived from cell-cultured tachyzoites was genotyped using the 10 PCR restriction fragment length polymorphisms (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The strain was a clonal Type III (ToxoDB genotype no. 2) strain. Although the prevalence of T. gondii in humans and animals in Grenada is high, rats seem to have little importance in the transmission of T. gondii on this island.

CCR5 chemokine receptor gene polymorphisms in ocular toxoplasmosis.

CC chemokine receptor type 5 (CCR5) is a chemokine receptor that influences the immune response to infectious and parasitic diseases. This study aimed to determine whether the CCR5Δ32 and CCR5 59029 A/G polymorphisms are associated with the development of ocular toxoplasmosis in humans. Patients with positive serology for Toxoplasma gondii were analyzed and grouped as 'with ocular toxoplasmosis' (G1: n=160) or 'without ocular toxoplasmosis' (G2: n=160). A control group (G3) consisted of 160 individuals with negative serology. The characterization of the CCR5Δ32 and CCR5 59029 A/G polymorphisms was by PCR and by PCR-RFLP, respectively. The difference between the groups with respect to the mean age (G1: mean age: 47.3, SD±19.3, median: 46 [range: 18-95]; G2: mean age: 61.3, SD±13.7, median: 61 [range: 21-87]; G3: mean age: 38.8, SD±17.9, median: 34 [range: 18-80]) was statistically significant (G1 vs.G2: p-value <0.0001; t=7.21; DF=318; G1 vs.G3: p-value <0.0001; t=4.32; DF=318; G2 vs. G3: p-value <0.0001; t=9.62; DF=318). The Nagelkerke r2 value was 0.040. There were statistically significant differences for the CCR5/CCR5 (p-value=0.008; OR=0.261), AA (p-value=0.007; OR=2.974) and AG genotypes (p-value=0.018; OR=2.447) between G1 and G2. Individuals with the CCR5/CCR5 genotype and simultaneously the CCR5-59029 AA or AG genotypes have a greater risk of developing ocular toxoplasmosis (4% greater), which may be associated with a strong and persistent inflammatory response in ocular tissue.

Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is an economically important disease of cattle in many countries but its transmission remains a mystery. Wild felids are suspected to be its definitive hosts. The domestic cat (Felis catus) is known experimental definitive host for Besnoitia species of rodents. Here, we report for Besnoitia darlingi the first identification of a natural definitive host, the bobcat (Lynx rufus). Oocysts resembling Toxoplasma gondii (unsporulated; 10.9±0.8×12.1±0.2µm; n=5) were detected microscopically in the feces of two of 25 free ranging wild bobcats from Mississippi, USA. After detailed investigation, we identified these oocysts as B. darlingi and not T. gondii. The IFN-γ gene knockout (KO) mice fed oocysts from bobcats died of acute besnoitiosis and tachyzoites were found in their tissues. Oocysts were also mildly pathogenic to outbred Swiss Webster mice (SW) (Mus musculus). The SW mice fed oocysts became ill but generally survived and developed characteristic thick-walled Besnoitia tissue cysts in their tongue and heart muscles and brains. Two laboratory-raised domestic cats (Felis catus) excreted B. darlingi oocysts after ingesting murine tissues infected with bobcat-derived oocysts. The parasite was successfully cultivated in African green monkey kidney fibroblast cells (CV-1 cell line) seeded with infected murine tissue homogenate. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, and ITS-1) from culture-derived tachyzoites confirmed the parasite as B. darlingi. Our results suggest that bobcats may be an important link in the sylvatic cycle of Besnoitia species and bioassay or molecular tests are needed to differentiate Toxoplasma gondii-like oocysts in feces of felids, both domestic and wild cats.