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Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

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Neospora caninum is an important abortifacient agent affecting mainly cattle worldwide. The aim of the present work was to describe the histopathological findings in a naturally infected beef cow and its midterm fetus caused by a genetically defined N. caninum isolate in Argentina. A N. caninum seropositive multiparous Aberdeen Angus pregnant cow and its fetus in the sixth month of gestation were submitted for histopathological, immunohistochemical, serological, and molecular studies and parasite isolation. The cow belonged to a beef herd under extensive management, with a N. caninum seroprevalence of 11%, and low level of annual abortion rate (≤ 5%). The dam had mild lymphocytic infiltrate in CNS, heart and uterus and no parasites were detected by Immunohistochemistry (IHC). No parasitic DNA was detected in the dam's brain, and gamma interferon knockout mice inoculated with brain material did not become infected. Clusters of tachyzoites and parasitic DNA were detected in the placenta by IHC and PCR, respectively. However, isolation from the placenta was unsuccessful. The fetus developed specific antibodies and an inflammatory response was detected in multiple organs. Furthermore, in vitro and in vivo isolation was achieved from gamma interferon knockout mice inoculated with CNS from the fetus. Multilocus-microsatellite typing revealed a genetically defined N. caninum isolate similar to the previously reported as MLG 72. We report the first N. caninum isolate from beef cattle in Argentina.

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Neospora caninum is a coccidian parasite originally reported in dogs and widely prevalent in numerous species of wild and domestic animals and has as definitive hosts some species of canids. The white-lipped peccary (WLP) ( Tayassu pecari) is a Tayassuidae mammal, found from Mexico to south of Brazil and north of Argentina. It is a game species with great economic importance in the Peruvian Amazon. Blood samples from 101 WLPs were collected from near or within three different conservation reserves located in the southeastern region of the Peruvian Amazon. For the detection of antibodies against N. caninum, indirect fluorescent antibody tests (IFAT) were performed using collared peccary ( Pecari tajacu) and swine ( Sus scrofa domesticus) heterologous secondary antibodies. For both IFAT tests, the cutoff was 1:50. Positive samples were titrated by a two fold serial dilution. In addition to IFAT, samples were also analyzed using an immunoblotting test (IB) with anti-swine conjugate. To confirm the viability of the anti-swine conjugate, the results of these samples previously tested by a modified agglutination test (MAT) for Toxoplasma gondii were used as reference. From the total of 101 samples tested, 5 (4.9%) were N. caninum positive by the three tests and an extra sample was positive by both IFATs and negative in the IB. Comparing both IFATs and considering IB as the gold standard, the relative sensitivity of IFATs was 100%, the specificity was 98.9%, the positive predictive value was 83.3%, and the negative predictive value was 100%. The agreement between tests was characterized by a κ value of 0.904 (95% confidence interval, 0.717 to 1.0) and an SE of 0.095. This is the first report of N. caninum antibodies in free-ranging T. pecari, and swine and collared peccary conjugate can be used as a secondary antibody for detection of antibodies in Tayassu species.

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A majority of emerging infectious diseases in humans are zoonoses. Understanding factors that influence the emergence and transmission of zoonoses is pivotal for their prevention and control. Toxoplasma gondii is one of the most widespread zoonotic pathogens known today. Whereas only a few genotypes of T. gondii dominate in the Northern Hemisphere, many genotypes coexist in South America. Furthermore, T. gondii strains from South America are more likely to be virulent than those from the Northern Hemisphere. However, it is not clear what factor(s) shaped modern-day genetic diversity and virulence of T. gondii Here, our analysis suggests that the rise and expansion of farming in the past 11,000 years established the domestic cat/mouse transmission cycle for T. gondii, which has undoubtedly played a significant role in the selection of certain linages of T. gondii Our mathematical simulations showed that within the domestic transmission cycle, intermediately mouse-virulent T. gondii genotypes have an adaptive advantage and eventually become dominant due to a balance between lower host mortality and the ability to superinfect mice previously infected with a less virulent T. gondii strain. Our analysis of the global type II lineage of T. gondii suggests its Old World origin but recent expansion in North America, which is likely the consequence of global human migration and trading. These results have significant implications concerning transmission and evolution of zoonotic pathogens in the rapidly expanding anthropized environment demanded by rapid growth of the human population and intensive international trading at present and in the future.

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The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.

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Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and Besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.

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Neospora caninum and Toxoplasma gondii are coccidian parasites that infect a wide range of mammalian and avian species. While viable T. gondii has been in vitro isolated in natural infections from wild and domestic birds, attempts to isolate N. caninum from naturally-infected birds were unsuccessful. We speculate that body temperatures of birds, which are usually higher than those of mammals, may impair the multiplication of N. caninum. In contrast to N. caninum, T. gondii can grow in vitro at temperatures higher than 37°C. To test the hypothesis that N. caninum tachyzoites are impaired to grow in vitro at high temperatures, three strains of N. caninum (NC-1, NC-Liverpool, and NC-Bahia) and three of T. gondii (RH, ME-49 and NED) were cultivated at gradually increasing temperatures starting at 37°C up to 41.5°C. A permanent chicken cell line was chosen for the study. Parasites were observed microscopically and their presence in culture was evaluated by species-specific conventional PCRs. In a second experiment, growth rates of T. gondii (RH strain) and N. caninum (NC-1 strain) were evaluated after direct passage of tachyzoites from 37°C to 41.5°C, and quantified by real-time PCR. In addition to comparisons between N. caninum and T. gondii, growth rates of three T. gondii strains were compared at high temperatures. Neospora caninum tachyzoites could not sustain multiplication at temperatures between 39°C and 41.5°C. Toxoplasma gondii tachyzoites continued to multiply at the same experimental conditions. Direct passage of N. caninum tachyzoites from 37°C to 41.5°C caused a significant decrease in the number of parasites during 96h of observation, while T. gondii had a significant increase in the number of stages after the same period of time. T. gondii RH strain (clonal type I) presented a different growth rate at 41.5°C when compared with type II and type III strains. In conclusion, multiplication of N. caninum tachyzoites in vitro was inhibited at temperatures similar to those of chickens, what may be one of the reasons that isolation of the parasite is difficult in avian species. In contrast to N. caninum, T. gondii continued to grow at 41.5°C.

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Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis-like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5-µm-thick wall with slender villar protrusions (Vp); the Vp were up to 5 µm long, up to 0.5 µm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 µm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10-12 µm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 µm long, and up to 1.8 µm wide; the Gs was up to 2 µm thick and without vesicles. Its sarcocyst wall was up to 5.6 µm thick, the vp were bent at an angle, up to 5.8 µm long, the Gs was up to 2 µm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.

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Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38 kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered "Relative Standards of Comparison" (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8 % and 99.5 %, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the "majority of tests". All tests displayed high sensitivity and specificity values (greater than 95 %); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N. caninum antibodies in cattle from Argentina.

Neospora caninum es un parásito protozoo responsable de abortos y pérdidas económicas en bovinos. La realización de un diagnóstico serológico preciso y con resultados comparables obtenidos por diferentes pruebas contribuye al manejo de este problema y a encarar medidas de control. Los objetivos del presente trabajo fueron los siguientes: 1) evaluar en Argentina una prueba de enzimoinmunoensayo in-house con el antígeno nativo de 38 kDa de N. caninum (ELISA-p38) para el diagnóstico de la neosporosis bovina, utilizando un panel de sueros locales bien caracterizados, procedentes de bovinos infectados de modo experimental o naturalmente expuestos; 2) comparar el desempeño y establecer el nivel de concordancia de tres pruebas serológicas para la detección de N. caninum, ELISA-p38, inmunofluorescencia indirecta (IFI) e inmunoblot (IB), con el mismo panel de sueros. Los sueros que resultaron positivos o negativos a IFI e IB fueron considerados como estándares relativos de comparación (ERC) para evaluar la prueba de ELISA-p38. El análisis de característica operativa del receptor determinó que la prueba de ELISA-p38 fue altamente precisa (área bajo la curva= 0,982) usando el punto de corte 0,0905. La sensibilidad y especificidad relativa del ELISA-p38 fue 97,8 % y 99,5 %, respectivamente, con una concordancia casi perfecta (k= 0,97) respecto del ERC. La comparación del desempeño de las pruebas se realizó usando como gold standard el criterio de la decisión de la "mayoría de las pruebas". Las pruebas exhibieron altos valores de sensibilidad y especificidad (mayores del 95 %) y excelente concordancia. Este trabajo describe un buen desempeño de la prueba de ELISA-p38 evaluada localmente y adecuada performance diagnóstica de las pruebas serológicas analizadas para la detección de anticuerpos anti N. caninum en bovinos de Argentina.

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Donkeys (Equus asinus) are closely related to horses and are known to be infected by several equine pathogens. Neospora caninum and Neospora hughesi are protozoan parasites that infect horses, but they were not confirmed in donkeys up to this date. The aim of this study was to evaluate the exposure of donkeys (Equus asinus) to Neospora spp. using tachyzoites of N. caninum as antigen and employing two common serologic methods, IFAT and immunoblot. Sera from 500 donkeys were obtained from 30 municipalities in Bahia state and tested by IFAT. Two of 500 sera were positive for Neospora spp. by IFAT with antibody titers of 100, and recognized a 37kDa antigen in immunoblot. Approximately 22% of the samples showed strong apical reactions and/or incomplete fluorescence, what may cause confusion in the interpretation of IFAT. We concluded that Neospora spp. are possibly of minor importance for Brazilian donkeys. Future studies are necessary to prove that Neospora spp. can naturally infect donkeys.

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