Macroscopic, histological, and molecular aspects of Sarcocystis spp. infection in tissues of cattle and sheep.

The macroscopic, histological, and molecular aspects of Sarcocystis spp. were examined in the tissues of two cattle and four sheep, 16 and eight fragments analyzed respectively, condemned in the slaughterhouse. All 24 samples were collected and analyzed for detecting macrocysts and macroscopic lesions. Subsequently, subdivided for direct examination, polymerase chain reaction and histopathological examination. All sheep tissues samples had grossly white round to oval tissue cysts, ranging from 0.3 to 1 cm in diameter. In contrast, cattle tissues did not present grossly visible cysts but had randomly distributed white-yellow foci with irregular contours. All samples from cattle and sheep had microscopic cysts. In the histological examination of sheep tissues, circular to elongated, encapsulated, basophilic structures ranging from 30 to 3,000 µm in length and 20 to 1,000 µm in width were observed within the skeletal muscle fibers. In cattle tissues, all cardiac muscle four fragments analyzed contained circular to elongated basophilic structures inside cardiomyocytes and in some Purkinje fibers. PCR were performed using the primers: 2L and 3H. In conclusion, all 24 tissues were infected with Sarcocystis spp., and S. gigantea (in sheep) and S. cruzi (in cattle). were the identified species by sequencing.

Contaminated water confirmed as source of infection by bioassay in an outbreak of toxoplasmosis in South Brazil.

The protozoan Toxoplasma gondii is a causative agent of toxoplasmosis, an important and widespread zoonotic disease. The transmission of this disease in humans includes ingestion of sporulated oocysts present in contaminated water or food. T. gondii oocysts are widely distributed and toxoplasmosis is considered a major food- and waterborne pathogen worldwide, making drinking water containing sporulated T. gondii oocysts a major source of contamination for people. In the first half of 2018, an unprecedented outbreak of toxoplasmosis was reported in the city of Santa Maria, southern Brazil. The temporal and spatial distribution of the cases strongly suggested a waterborne infection. Thus, the aim of this study was to investigate a possible involvement of treated water as a source of the outbreak. For this, piglets received potentially contaminated water ad libitum for 21 days and the infection was monitored by serology through IFAT and investigation of T. gondii DNA in tissues by PCR amplification of a 529 bp followed by mouse bioassays. All piglets receiving test water ad libitum for 21 days as well as positive controls seroconverted to T. gondii. T. gondii DNA was detected in 62.5% of the piglets that received test water. All mice inoculated with tissues from each positive piglet were PCR-positive. These results strongly indicated the presence of viable oocysts in the test water administered to the animals during the study.

Molecular Detection and Characterization of Sarcocystis Infection in Naturally Infected Buffaloes, Brazil.

ABSTRACT: Sarcocystosis is a disease caused by various Sarcocystis species, a coccidian protozoan parasite that infects humans and animals and is commonly found in ruminants. Although Sarcocystis occurs all over the world, the species responsible for infecting buffaloes in Brazil have not been identified. In this study, we used molecular methods to estimate the prevalence of natural Sarcocystis infection in buffaloes. Phylogenetic analyses were conducted for the first time to identify the species of this protozoan that are responsible for infecting buffalos in southern Brazil, Rio Grande do Sul state. Heart samples from 80 buffaloes were subjected to microscopic examination followed by molecular analysis. Microcysts were present in 19 (23.75%) of 80 samples. The genomic DNA from the 19 cyst samples was extracted and amplified, and six sequences were obtained. The analysis was performed with the StandenPackage software, and the nucleotide sequences generated were analyzed by comparison with sequences in GenBank. All the sequenced samples were identified as Sarcocystis levinei.

Isolation, genetic and immunohistochemical identification of Toxoplasma gondii from human placenta in a large toxoplasmosis outbreak in southern Brazil, 2018.

The present study aimed to describe a molecular analysis of environmental and pork samples, the isolation, genetic identification and immunohistochemistry (IHC) of Toxoplama gondii from placenta and amniotic fluid from five pregnant women that miscarried during a toxoplasmosis outbreak in 2018, Santa Maria, Rio Grande do Sul. Environmental and pork samples were submitted to polymerase chain reaction (PCR); placenta and amniotic fluid samples to histopathology, IHC, mouse bioassay and PCR. All samples were genotyped by PCR-RFLP with 11 loci. Histopathologic and IHC were compatibles with toxoplasmosis. All pregnants were positive in PCR and bioassay, the genotypes were compared, and all were equal suggesting a same source of infection. Among the environmental and food samples, a sludge sample from a water tank and two porks samples were positive in PCR, and the genotypes were different from the pregnant women isolates. It is concluded that obtain and compare isolates is essential to elucidate outbreak source.

Isolation and molecular characterization of Toxoplasma gondii from placental tissues of pregnant women who received toxoplasmosis treatment during an outbreak in southern Brazil.

Toxoplasma gondii is a protozoan that has great genetic diversity and is prevalent worldwide. In 2018, an outbreak of toxoplasmosis occurred in Santa Maria, Brazil, which was considered the largest outbreak ever described in the world. This paper describes the isolation and molecular characterization of Toxoplasma gondii from the placenta of two pregnant women with acute toxoplasmosis who had live births and were receiving treatment for toxoplasmosis during the outbreak. For this, placental tissue samples from two patients underwent isolation by mice bioassay, conventional PCR and genotyping using PCR-RFLP with twelve markers. Both samples were positive in isolation in mice. The isolate was lethal to mice, suggesting high virulence. In addition, the samples were positive in conventional PCR and isolates submitted to PCR-RFLP genotyping presented an atypical genotype, which had never been described before. This research contributes to the elucidation of this great outbreak in Brazil.

Anti-Sarcocystis Antibodies in Lambs Deprived of Colostrum.

INTRODUCTION: The objective of this study was to evaluate the presence of anti-Sarcocystis spp. specific IgG antibodies in serum samples from precolostral lambs to determine the occurrence of transplacental transmission of Sarcocystis spp. in sheep. METHODS: Blood samples were collected from 80 ewes and their respective lambs, immediately after lambing and before colostrum ingestion, respectively. The presence of anti-Sarcocystis spp. IgG was evaluated in serum samples using the indirect fluorescent antibody test (IFAT). Positive samples of the lambs were submitted to titration and IFAT to detect anti-T. gondii and anti-N. caninum specific IgG. RESULTS: Anti-Sarcocystis spp. IgG was detected in 62.5% of the ewes (50/80) and in 4% of the lambs of the seropositive ewes (2/50). None of the lambs from seronegative ewes were positive. The final titers of the positive lambs were 80. No cross reaction was detected among the positive samples to anti-Sarcocystis spp., anti-N. caninum, and anti-T. gondii IgG. The detection of anti-Sarcocystis spp. antibodies in serum samples of lambs deprived of colostrum suggests transplacental transmission of infection. Thus, the vertical transmission may be an alternative route of infection of Sarcocystis spp. also in sheep. Further studies are warranted to confirm transplacental transmission in sheep and to explain the importance of this infection pathway.

Sarcocystis species identification in cattle hearts destined to human consumption in southern Brazil.

Sarcocystis spp. are ubiquitous protozoan parasites that can form cysts in striated muscle and CNS of cattle. Cattle hearts are commonly infected by microscopic sarcocysts. Humans can get infected by consuming cattle meat containing the zoonotic parasites Sarcocystis hominis and Sarcocystis heydorni. However, bovine myocardium is generally infected by Sarcocystis cruzi. The aims of this study were to investigate the occurrence of sarcocysts and the identity of Sarcocystis species present in cattle hearts destined to human consumption in the Central region of the Rio Grande do Sul state, southern Brazil. A total of 314 cattle myocardium samples collected from a local abattoir were microscopically examined for the presence of sarcocysts. The sarcocysts isolated from 134 of these samples (ten sarcocysts per sample) were subjected to DNA extraction and PCR amplification. The PCR-amplified DNA fragments were digested with the restriction enzymes BclI and RsaI (PCR-RFLP) for differentiation among S. cruzi, S. hirsuta, and S. hominis. Sarcocystis species identification was confirmed using DNA sequencing of the cox1 mitochondrial DNA. Sarcocysts were detected in all the bovine myocardium samples. PCR-RFLP analysis resulted in successful amplification of 78 of the 134 samples tested. Only the S. cruzi DNA restriction pattern was identified from all of the 78 amplified samples. DNA sequencing also confirmed the presence of S. cruzi DNA. In conclusion, all myocardium samples evaluated were infected with microscopic sarcocysts. S. cruzi was the only species detected infecting the cattle hearts.

Antibodies against Apicomplexa protozoa and absence sarcocysts in heart tissues from horses in southern Brazil.

Sarcocystis spp., Neospora spp., and Toxoplasma gondii are Apicomplexa protozoa that can infect horses. This study aimed to investigate the occurrence of antibodies against Sarcocystis spp., Neospora spp., and T. gondii in horses slaughtered in southern Brazil. The presence of histological lesions, tissue cysts, and Sarcocystis spp. DNA in the hearts of these horses was also investigated. A total of 197 paired serum and heart samples were evaluated by serology and direct microscopic examination; 50 of these samples were subjected to histopathological and PCR analyses. Antibodies against at least one of the protozoa were detected in 146 (74.1%) of the serum samples. The frequencies of positive serology were: 36% (71/197) against Sarcocystis spp., 39.1% (77/197) against Neospora spp., and 47.2% (93/197) against T. gondii. No cysts, Sarcocystis spp. DNA, or histopathological lesions were observed in myocardial tissue samples. The frequencies of antibody seropositivity against Sarcocystis spp., Neospora spp., and T. gondii showed that horses are frequently infected by these parasites in southern Brazil. The absence of sarcocysts in horse tissues is compatible with their role as aberrant/accidental hosts in the life cycle of Sarcocystis spp..

Antibodies against Apicomplexa protozoa and absence sarcocysts in heart tissues from horses in southern Brazil / Anticorpos contra protozoários do filo Apicomplexa e ausência de sarcocistos no miocárdio de equinos no sul do Brasil

Abstract Sarcocystis spp., Neospora spp., and Toxoplasma gondii are Apicomplexa protozoa that can infect horses. This study aimed to investigate the occurrence of antibodies against Sarcocystis spp., Neospora spp., and T. gondii in horses slaughtered in southern Brazil. The presence of histological lesions, tissue cysts, and Sarcocystis spp. DNA in the hearts of these horses was also investigated. A total of 197 paired serum and heart samples were evaluated by serology and direct microscopic examination; 50 of these samples were subjected to histopathological and PCR analyses. Antibodies against at least one of the protozoa were detected in 146 (74.1%) of the serum samples. The frequencies of positive serology were: 36% (71/197) against Sarcocystis spp., 39.1% (77/197) against Neospora spp., and 47.2% (93/197) against T. gondii. No cysts, Sarcocystis spp. DNA, or histopathological lesions were observed in myocardial tissue samples. The frequencies of antibody seropositivity against Sarcocystis spp., Neospora spp., and T. gondii showed that horses are frequently infected by these parasites in southern Brazil. The absence of sarcocysts in horse tissues is compatible with their role as aberrant/accidental hosts in the life cycle of Sarcocystis spp..

Resumo Sarcocystis spp., Neospora spp. e Toxoplasma gondii são protozoários que pertencem ao filo Apicomplexa e que podem afetar equinos. O objetivo deste estudo foi investigar a ocorrência de anticorpos contra Sarcocystis spp., Neospora spp. e T. gondii. A presença de lesões histológicas, cistos teciduais e DNA de Sarcocystis spp. no miocárdio de equinos abatidos no sul do Brasil também foi investigado. Um total de 197 amostras de soro juntamente com as respectivas amostras de coração, foram avaliadas por sorologia e exame microscópico direto. Destas amostras, 50 foram selecionadas e submetidas a análise histopatológica e PCR. Anticorpos contra pelo menos um dos protozoários foi detectado em 146 (74,1%) das amostras de soro. As frequências de sorologia positiva foram: 36% (71/197) para Sarcocystis spp., 39,1% (77/197) para Neospora spp. e 47,2% (93/197) para T. gondii. Não foram encontradas lesões histopatológicas, cistos e DNA de Sarcocystis spp. nas amostras de miocárdio dos equinos. As frequências de soropositividade para Sarcocystis spp., Neospora spp. e T. gondii mostra que os equinos podem ser frequentemente infectados por estes parasitas no sul do Brasil. A ausência de sarcocistose no coração dos equinos é compatível com seu papel como hospedeiro errático/acidental no ciclo de vida deste protozoário.

Molecular detection of protozoa of the Sarcocystidae family in sheep from the State of Rio Grande do Sul, Brazil / Detecção molecular de protozoários da família Sarcocystidae em ovinos no Estado do Rio Grande do Sul, Brasil

ABSTRACT: Infections caused by protozoa belonging to the Sarcocystidae family have worldwide distribution and are common in ruminants, leading to considerable economic losses. This study evaluates Sarcocystis spp., Toxoplasma gondii and Neospora caninum infections in sheep from Southwest region of Rio Grande do Sul, Brazil. Myocardium samples of 80 sheep raised on extensive system were collected. Tissue cysts were detected by direct examination and presence of infective agents was confirmed by PCR. Macroscopic evaluation did not reveal changes, but direct microscopic examination showed cysts in 76.2% (61/80, 95% CI: 66.9 - 85.9) samples, and all cysts were morphologically similar to those caused by Sarcocystis tenella or Sarcocystis arieticanis. PCR detected Sarcocystis spp. DNA in 21.2% (17/80, CI: 12.3-30.2) of the tested samples and T. gondii DNA in 15% (12/80, CI: 7.2-22.8). Moreover, 6.2% (5/80, CI: 2.1-13.9) samples contained DNA of both protozoan. The presence of N. caninum nucleic acids was not observed in tested samples. However, all PCR-positive samples (23.7%-19/80, CI: 14.4-33.1) were also positive by direct examination (microscopic cysts). Thus, a high occurrence of microscopic tissue cysts was detected in sheep from southwest region of Rio Grande do Sul State. Although PCR did not show good sensitivity to identify the causative agents of these cysts, it revealed the presence of Sarcocystis spp. and T. gondii in ovine cardiac muscle samples. This may predispose the contamination of animals and humans by these protozoa.

RESUMO: Infecções causadas por protozoários da família Sarcocystidae apresentam distribuição mundial, sendo comuns em ruminantes, responsáveis por causar importantes perdas econômicas. Este estudo avaliou infecções de Sarcocystis spp., Toxoplasma gondii e Neospora caninum em ovinos da região sudoeste do Rio Grande do Sul, Brasil. Foram coletadas amostras de miocárdio de 80 ovinos criados em sistema extensivo. Cistos teciduais foram detectados por exame direto, com a presença dos agentes confirmada por PCR. A avaliação macroscópica não revelou alterações, porém, no exame microscópico direto, foram verificados cistos em 76,2% (61/80, 95% IC: 66,9-85,9) das amostras, sendo todos morfologicamente semelhantes ao Sarcocystis tenella ou Sarcocystis arieticanis. A PCR detectou DNA de Sarcocystis spp. em 21,2% (17/80, IC: 12,3-30,2) das amostras testadas e DNA de T. gondii em 15% (12/80, IC: 7,2-22,8). Em 6,2% (5/80, IC: 2,1-13,9), foram detectados DNA de ambos os protozoários. Todas as amostras positivas no PCR (23,7%-19/80, IC: 14,4-33,1) também foram positivas no exame direto (cistos microscópicos). Assim, uma alta ocorrência de cistos teciduais microscópicos em ovinos da região sudoeste do Rio Grande do Sul foi detectada. Apesar de a PCR não ter mostrado uma boa sensibilidade na identificação dos agentes causadores desses cistos, foi possível verificar a presença de Sarcocystis spp. e T. gondii em amostras do músculo cardíaco de ovinos. Dessa maneira, a presença destes protozoários pode predispor a contaminação de humanos e animais.

DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

Anthelmintic resistance in gastrointestinal nematodes of beef cattle in the state of Rio Grande do Sul, Brazil.

Gastrointestinal nematodes resistant to anthelmintics have been reported in several regions of Brazil, and they may be associated with economic losses for the cattle industry. This study aimed to evaluate the resistance status of gastrointestinal nematodes from naturally infected beef cattle to several commercially available anthelmintics, as well as to test the efficacy of combinations of anthelmintics against multi-resistant gastrointestinal nematodes. Ten farms located in Rio Grande do Sul state were selected by: farmers' consent; extensive raising system; availability of calves aged from 7 to 9 months naturally infected by gastrointestinal nematodes; absence of anthelmintic treatment for 60 days before the study; and presence of 70-100 calves or more of both genders with ≥ 200 eggs per gram of feces (EPG) (sensitivity of 50 EPG). These calves were distributed into 10 groups (of 7-10 animals) per farm and treated with ivermectin, doramectin, eprinomectin, fenbendazole, closantel, nitroxynil, disophenol, levamisole, albendazole, or moxidectin. Feces were collected 2 days before treatment and 14 days after treatment. Additional groups of 7-10 calves were used to test six different two-drug combinations at four of the studied farms. In general terms, fenbendazole was the most effective drug, followed by levamisole, disophenol, and moxidectin. However, parasite resistance to multiple drugs was found in all herds, especially in the genera Cooperia spp., Trichostrongylus spp., and Haemonchus spp.. Some of the two-drug combinations were effective against nematode populations identified as resistant to the same compounds when used as single drugs. The most effective combinations were moxidectin + levamisole, doramectin + fenbendazole, and levamisole + closantel. In this study, parasites resistant to the main commercially available anthelmintics were found in all herds, and some combinations of two active components belonging to different chemical groups were effective against multi-drug resistant gastrointestinal nematodes.