Sarcocystis spp. in sheep and goats: frequency of infection and species identification by morphological, ultrastructural, and molecular tests in Bahia, Brazil.

Sarcocystis spp. are cyst-forming coccidia that infect numerous animals species, including several livestock species. Despite the importance of sheep and goat production in Brazil, little it is known about the Sarcocystis species that infect small ruminants in the country and their potential impact on meat condemnation due to the presence of macroscopic cysts of the parasite. The aims of the present study were to determine the frequency of infection by Sarcocystis spp. in goats and sheep intended for human consumption in Bahia State, Brazil, as well as to identify the parasite species in selected samples. The entire tongue, esophagus, and heart were collected from 120 goats and 120 sheep. Tissues were examined for Sarcocystis spp. by macroscopic evaluation, light microscopy, electron microscopy, and molecular tests. Microscopic cysts of Sarcocystis spp. were detected in 95.8 % of sheep and 91.6 % of goats. Using either transmission electron microscopy or partial sequencing of the 18S region of the ribosomal DNA (rDNA) for species identification, Sarcocystis tenella and Sarcocystis arieticanis were observed in sheep and Sarcocystis capracanis in goats. Macroscopic cysts were not detected in the analyzed samples. We concluded that goats and sheep destined for human consumption in Bahia possess high frequencies of Sarcocystis infection. Carcass condemnation due to Sarcocystis macrocysts seems to be rare in the studied region. S. arieticanis and S. capracanis were confirmed for the first time by electron microscopy or by molecular tests in small ruminants from Brazil.

Sarcocystis sp. shed by the common boa snake (Boa constrictor) in Brazil.

The genus Sarcocystis contains around 200 species and 25 of these infect snakes. Two Sarcocystis spp. shed by snakes have called special attention of the scientific community. S. nesbitti, which is shed by scrub pythons (Simalia amethistina), causes myopathy in humans that consume water or food contaminated with the parasite. Sporocysts of S. singaporensis, excreted by reticulated pythons (Malayopython reticulatus), is letal for rats and was successfully tested in the biological control of these rodents. A high biodiversity of snakes is found in Brazil, however, scarce information is available about Sarcocystis spp. in Brazilian snakes. Herein, we investigated Sarcocystis sp. in feces of the common boa (Boa constrictor) from Salvador, as it is widely distributed in Brazil and it is also bred in other countries. Feces of 65 boas were examined, and Sarcocystis sp. was found in 1/65 (1.53%) snakes. All snakes were alive, and for this reason, intestinal scrapping, which is the most sensitive method to detect the parasite, was not performed. Morphometric evaluation of sporocysts showed significant differences in their sizes. PCR and multilocus sequencing of four genetic markers (cox1, 18S, ITS1, and 28S) revealed that sporocysts corresponded to a new Sarcocystis species. Sequences of cox1 and 18S had identities of 100% and higher than 98%, respectively, with sequences obtained from the rodent Lagostomus maximus in Argentina. ITS1 and 28S sequences did not match with any known Sarcocystis sp. No ITS1 and 28S sequences were available for the Sarcocystis sp. found in the Argentinian L.maximus. Bioassay using the boa sporocysts was conducted in three mouse lineages and in Rattus norvegicus, but no parasitic stages were detected in these rodents. We concluded that the common boa is probably the definitive host of a new species of Sarcocystis sp. that has L. maximus or related rodents as intermediate hosts.

Translation, cross-cultural adaptation, and validation of the Behavioral Activity Rating Scale (BARS) for the Brazilian population.

INTRODUCTION: Few instruments are available in Brazil to evaluate psychomotor activity in psychiatric emergency, clinical, and research settings. This study aimed to perform a cross-cultural adaptation of the Behavioral Activity Rating Scale (BARS) into Brazilian Portuguese and assess the adapted scale's psychometric properties. METHOD: An expert consensus committee conducted a translation and back-translation of the original scale, resulting in the BARS-BR. Four pairs of physicians administered the BARS-BR and the Sedation-Agitation Scale (SAS) to patients in a hospital psychiatry emergency room and patients in the hospital's psychiatric wards. The BARS-BR was compared to the SAS to assess concurrent validity and internal consistency was evaluated with the Bland-Altman technique. RESULTS: In the emergency room, the correlation coefficients between the first and second assessments were rho = 0.997 and rho = 1.0, respectively. In the hospital wards, the correlation coefficient between the pair of evaluators was rho = 0.951. There were strong correlations between the BARS-BR score of the first examiner and the SAS score of the second examiner (rho = 0.903) and between the SAS score of the first examiner and the BARS-BR score of the second examiner (rho = 0.893). CONCLUSION: The BARS-BR showed good psychometric properties, and we recommend its use because it constitutes an easy method for assessment of changes in psychomotor activity. Further studies are suggested to evaluate adoption and comprehension of the BARS-BR scale by all classes of healthcare professionals.

Cryopreservation of jaguar (Panthera onca) sperm cells using different cryoprotectants and different thawing temperatures.

The cryopreservation of jaguar semen must be improved to produce high-quality biobanking doses. Until now, the rare studies of semen freezing in the species have only evaluated glycerol, always with a significant reduction in sperm quality in thawed semen. The purpose of this study was to assess the efficacy of three cryoprotectants, dimethylsulfoxide (DMSO), glycerol (GLY), and methanol (MET), in the cryopreservation of jaguar semen in an LDL-based extender, as well as the effect of thawing temperature on dosage quality. Five mature males with a history of reproduction were used. On the males, an infrared thermal image (IRT) was captured, the spicules and testes were analyzed, and the CASA system was used to evaluate the quality of fresh and thawed sperm. The superficial IRT was 4.6 ± 1.2 °C cooler than the anal sphincter, and the semen measured between 27.3 and 28.7 °C shortly after exiting the urethra. The total motility of fresh sperm was 55.3 ± 22.6%, and progressive motility was 36.3 ± 18%. The total motility of thawed sperm was 5.28 ± 2.51%, 4.49 ± %2.49, and 0.51 ± 0.62% for DMSO, GLY, and MET, respectively. DMSO and GLY performed better than MET, and there was no difference in thawing temperature (37°C 30 s vs. 50°C 12 s). All animals exhibit a considerable level of morphological changes in sperm. Low amounts of total and progressive motility were found in the thawed sperm. Males with a high level of sperm morphological changes were found to be fertile, but the lone male with normospermia was infertile. Thus, we contest the applicability of the commonly used morphological classification for bovines to felid species.

Reactivity of Horse Sera to Antigens Derived From Sarcocystis falcatula-Like and Sarcocystis neurona.

Sarcocystis neurona and Sarcocystis falcatula are protozoan parasites endemic to the Americas. The former is the major cause of equine protozoal myeloencephalitis, and the latter is associated with pulmonary sarcocystosis in birds. The opossum Didelphis virginiana is the definitive host of these parasites in North America. Four Didelphis species are found in Brazil, and in most reports in this country, Sarcocystis species shed by opossums have been classified as S. falcatula-like. It is unknown whether reports on S. neurona-seropositive horses in Brazil are also derived from exposure of horses to S. falcatula-like. The aim of this study was to test the sera reactivity of 409 horses in Brazil using antigens derived from a Brazilian strain of S. falcatula-like (Sarco-BA1) and from a North American strain of S. neurona (SN138). Samples were examined by immunofluorescent antibody tests (IFATs) at start dilutions of 1:20, and a selected number of samples was tested by Western blot (WB). Sera from 43/409 (10.5%) horses were reactive to S. falcatula-like and 70 of 409 (17.1%) were reactive to S. neurona antigen; sera from 25 animals (6.1%) were positive for both parasites by IFAT. A poor agreement was observed between the two employed IFATs (κ = 0.364), indicating that horses were exposed to more than one Sarcocystis species. Horse sera evaluated by WB consisted of four sera reactive to S. falcatula-like by IFAT, six sera positive to S. neurona by IFAT, two sera that tested negative to both parasites by IFAT, and a negative control horse serum from New Zealand. Proteins in the range of 16 and 30 kDa were recognized by part of IFAT-positive sera using both antigen preparations. We concluded that Brazilian horses are exposed to distinct Sarcocystis species that generate different serological responses in exposed animals. Antigens in the range of 16 and 30 kDa are probably homologous in the two parasites. Exposure of the tested horses to other Sarcocystis species, such as Sarcocystis lindsayi, Sarcocystis speeri, and Sarcocystis fayeri, or Sarcocystis bertrami cannot be excluded in the current study.

Development of Cystoisospora felis in Cell Culture and in vitro Formation of Monozoic Tissue Cysts.

Cystoisospora felis is a coccidian parasite commonly found in feces of domestic cats. Infection in cats occurs by ingestion of sporulated oocysts or consumption of rodents infected by the parasite. Scarce information is available about extraintestinal stages of C. felis in naturally infected intermediate hosts, as well as in cell culture. The aim of the current work was to investigate the development of C. felis in Vero cells (African green monkey kidney) and MDCK cells (Madin-Darby canine kidney). Cell monolayers were inoculated with mechanically released sporozoites of C. felis, and parasite growth was daily examined using light microscopy. After cell invasion, only parasitophorous vacuoles containing a single zoite were observed. Five days post-inoculation with sporozoites, unstained cell monolayers were evaluated by differential interference contrast (DIC), and also by Romanovsky stain using conventional light microscopy. Single zoites, each surrounded by a cyst wall, were observed by both methods. Multiplication by endodyogeny did not occur in any cell monolayer. Treatment of encysted parasites with HCl-pepsin for 15 min led to dissolution of the cyst wall and release of intact and motile zoites. To our knowledge, this is the first demonstration of in vitro production of monozoic tissue cysts of C. felis. As kittens commonly shed C. felis in their feces, oocysts are easily available for in vitro production of monozoic tissue cysts of the parasite. Development of C. felis in cell culture may be employed as a model on tissue cyst formation of Cystoisospora spp. and closely related coccidia.

Serologic cross-reactivity between Sarcocystis neurona and Sarcocystis falcatula-like in experimentally infected Mongolian gerbils.

Sarcocystis neurona is the major cause of the equine protozoal myeloencephalitis (EPM) in the Americas and has opossums of the genus Didelphis as definitive hosts. Most isolates of Sarcocystis sp. shed by opossums in Brazil differ genetically from the known species of Sarcocystis. These Brazilian isolates behave similarly as Sarcocystis falcatula, which causes sarcocystosis in birds, and for this reason, have been classified as Sarcocystis falcatula-like. Genes coding for the immunodominant surface antigens SAG2, SAG3 and SAG4 of S. falcatula-like are similar to those from S. neurona. It is unknown the Sarcocystis species that causes EPM in Brazil, as S. neurona has never been genetically confirmed in Brazilian horses. All cases associated with EPM in Brazil were diagnosed by immunological tests, which are not specific for S. neurona infection. It is possible that S. falcatula-like may infect horses in Brazil. The aims of the current study were to test the susceptibility of gerbils (Meriones unguiculatus) to experimental infections with S. neurona and S. falcatula-like, and to investigate potential serologic cross-reactivity to these parasites by immunofluorescent antibody test (IFAT) and Western blot (WB). A total of 27 gerbils, distributed in five experimental groups (G1-G5), were employed in this work (G1: 4 negative controls; G2: 6 infected with S. neurona merozoites, G3: 6 infected with S. falcatula-like merozoites; G4 and G5 (5 and 6, respectively, infected with different doses of sporocysts). None of the 17 animals that seroconverted for the parasites in IFAT presented any visualized organism or Sarcocystis DNA in the examined tissues. No serologic cross-reactivity was observed using IFAT. However, sera from animals infected with S. falcatula-like and S. neurona presented the same pattern of antigenic recognition when S. neurona merozoites were used as antigen in WB, including reactivity to proteins of 30 and 16 kDa, regarded as specific markers for S. neurona-infected animals. Gerbils did not sustain infection by these parasites, although produced antibodies after inoculation. These results are suggestive that other animal species that are exposed to S. falcatula-like, including horses, may present serologic cross-reactivity to S. neurona in WB. IFAT was demonstrated to be more specific that WB for the detection of antibodies to S. falcatula-like and S. neurona in the experimental conditions of this study.

Sarcocystis falcatula-like derived from opossum in Northeastern Brazil: In vitro propagation in avian cells, molecular characterization and bioassay in birds.

Most reported isolates of Sarcocystis spp. derived from Brazilian opossums (Didelphis sp.) have genetic characteristics distinct from the known species of Sarcocystis, but behave similarly as Sarcocystis falcatula, as they are infective to budgerigars. In previous studies, these Brazilian isolates, classified as Sarcocystis falcatula-like, were originated from South and Southeast regions of Brazil. In the current work, we aimed to culture and to perform multilocus sequence analysis of Sarcocystis sp. derived from a Brazilian opossum (D. aurita/D. marsupialis) that inhabited the city of Salvador, Bahia, in the Northeast of Brazil. The parasite was isolated in Vero cells, referred here as Sarco-BA1, and propagated in avian cells (DF-1). Molecular analysis of Sarco-BA1 revealed that the nucleotide sequence of the internal transcribed spacer 1 (ITS1) of the rDNA was identical to all isolates (n = 19) of Sarcocystis spp. reported in two studies from South and Southeast regions of the country. Two budgerigars were inoculated with 10 and 1000 sporocysts of Sarco-BA1, respectively, and developed acute sarcocystosis, showing that the parasite behaves like S. falcatula. It was interesting to observe that Sarco-BA1 had almost identical ITS1 and SAG sequences to all 16 isolates of S. falcatula-like recently described in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Espírito Santo state, Brazil. Our results suggest that Sarco-BA1 and S. falcatula-like may represent a single species of Sarcocystis. Propagation of the parasite in a permanent avian cell line significantly improved the yield of merozoites in cell culture. To our knowledge, this is the first molecular study and in vitro isolation of S. falcatula-like derived from Northeastern Brazil. Studies are under way to determine the infectivity of Sarco-BA1 to other animal species, as well as to investigate serological cross-reactivity among Sarco-BA1, S. neurona and related species.

Translation, cross-cultural adaptation, and validation of the Behavioral Activity Rating Scale (BARS) for the Brazilian population

Abstract Introduction Few instruments are available in Brazil to evaluate psychomotor activity in psychiatric emergency, clinical, and research settings. This study aimed to perform a cross-cultural adaptation of the Behavioral Activity Rating Scale (BARS) into Brazilian Portuguese and assess the adapted scale's psychometric properties. Method An expert consensus committee conducted a translation and back-translation of the original scale, resulting in the BARS-BR. Four pairs of physicians administered the BARS-BR and the Sedation-Agitation Scale (SAS) to patients in a hospital psychiatry emergency room and patients in the hospital's psychiatric wards. The BARS-BR was compared to the SAS to assess concurrent validity and internal consistency was evaluated with the Bland-Altman technique. Results In the emergency room, the correlation coefficients between the first and second assessments were rho = 0.997 and rho = 1.0, respectively. In the hospital wards, the correlation coefficient between the pair of evaluators was rho = 0.951. There were strong correlations between the BARS-BR score of the first examiner and the SAS score of the second examiner (rho = 0.903) and between the SAS score of the first examiner and the BARS-BR score of the second examiner (rho = 0.893). Conclusion The BARS-BR showed good psychometric properties, and we recommend its use because it constitutes an easy method for assessment of changes in psychomotor activity. Further studies are suggested to evaluate adoption and comprehension of the BARS-BR scale by all classes of healthcare professionals.

Toxoplasmatinae Parasites in Bats from Bahia State, Brazil.

Toxoplasma gondii and Neospora caninum are widespread cyst-forming coccidian parasites of the subfamily Toxoplasmatinae that infect a wide range of wild and domestic animals. Whereas T. gondii is a zoonotic disease, N. caninum is restricted to nonhuman animals. Some chiropteran species can be infected by T. gondii and present fatal toxoplasmosis. In most cases, T. gondii -infected bats are believed to remain asymptomatic and to act as an infection source to other animals. It is not known whether N. caninum can infect bats. We determined infection rates of T. gondii and N. caninum in free-living bats in the state of Bahia, Brazil. Brain samples from 97 bats of seven species, captured in 2008-15, were analyzed by PCRs for T. gondii and N. caninum . Two of the 97 samples were positive for T. gondii DNA. None of the samples were positive for N. caninum DNA, suggesting that the bats were not susceptible to N. caninum infection or that its prevalence was very low.

Diagnostic performance of anti-beta2 glycoprotein I and anticardiolipin assays for clinical manifestations of the antiphospholipid syndrome.

The objective of the present study was to analyse the performance of the tests for detection of anti-beta2 glycoprotein I (beta2 GP I) and anticardiolipin (aCL) antibodies for identification of clinical manifestations of the antiphospholipid syndrome (APS). Patients with systemic lupus erythematosus (SLE) as well as carriers of infectious diseases such as Kala-azar, syphilis and leptospirosis were studied. Particular interest was given to the presence of clinical complications related to APS. Anticardiolipin and anti-beta2 GP I antibodies were searched using an enzyme-linked immunosorbent assay (ELISA) assay. Clinical manifestations of APS were observed in 34 of the 152 patients (22.3%) with SLE and no patient with infectious disease had such manifestations. Antibodies to cardiolipin in moderate or high levels and anti-beta2 GP I were detected in 55 of 152 (36.1%) and 36 of 152 (23.6%) patients with SLE, respectively, and in 2 of 30 (6.6%) and 16 of 30 (53.3%) patients with Kala-azar, in 9 of 39 (23%) and 6 of 34 (17.6%) patients with leptospirosis, and 14 of 74 (18.9%) and 8 of 70 (11.4%) cases of syphilis, respectively. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratio (LR) of the anti-beta2 GP I test for the identification of the clinical manifestation of APS were, respectively, 29% [95% confidence interval (CI) = 24%-34%], 78% (95% CI = 73-83%), 15% (95% CI = 11-19%), 89% (95% CI = 85-93%) and 1.38. Regarding the aCL assay, the figure was 29% (95% CI = 24-34%), 76% (95% CI = 71-81%), 14% (95% CI = 10-18%), 89% (95% CI = 86-92%) and 1.26. As the validity and performance of the anti-beta2 GP I assay were similar to the aCL in demonstrating the presence of clinical phenomena associated with APS and due to the difficulties in performing as well as the lack of standardisation of the anti-beta2 GP I test, we suggest that the test for aCL should continue to be the first one performed when the presence of APS is suspected.

[Frequency and implications of autoantibodies in acute viral hepatitis]. / Freqüência e implicações dos auto-anticorpos em hepatites agudas virais.

There are interactions between hepatotropic viruses and the host immune system, which could contribute to liver damage in viral hepatitis. The aim of this study was to investigate the frequency of autoantibodies in patients with acute viral hepatitis and their relationship with biochemical activity, severity of acute illness and chronicity rate. From 1992 to 2000, 156 patients with acute viral hepatitis were enrolled in a prospective study. Among these, hepatitis A was detected in 32%, hepatitis B in 31%, hepatitis C in 8%, hepatitis E in 3% and 24% were considered non A-E hepatitis. During the acute phase, 20.5% of patients presented ANA and 14.8% anti-smooth muscle antibody positive. During convalescence, 6.4% of patients showed ANA and 3.9% anti-smooth muscle positive. Comparison between autoantibodies-positive and negative groups showed no differences regarding ALT and bilirubin levels. In conclusion, autoantibodies can occur in acute viral hepatitis but there are no prognostic consequences.