High titers of anti-Leishmania spp. antibodies in apparently healthy dogs in the North Pioneer Mesoregion of the state of Paraná, Brazil.

Leishmaniasis is an anthropozoonosis with vector transmission, and knowledge regarding the occurrence of this parasitosis in sentinels can contribute to infection and disease control measures in humans. The objectives of this study were to evaluate the occurrence of Leishmania exposure and infection in dogs from urban and rural areas in the North Pioneer Mesoregion of the state of Paraná, to evaluate possible risk factors, and to analyze the statistical agreement between the serological techniques that were used. Using a convenience sampling, serum and whole blood samples were collected to perform serological and molecular assays, respectively. The enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) identified 29/204 (14.2%) and 20/204 (9.8%) seropositive dogs, respectively. Five dogs (2.4%) were seropositive for both serological tests, and four dogs presented high titers in the IFAT. None of the samples tested positive for Leishmania spp. DNA according to polymerase chain reaction analysis. No factors were significantly associated with infection. Leishmania parasites circulate in urban and rural dogs in the North Pioneer Mesoregion of the state of Paraná. Despite the absence of clinical cases, seropositive animals with high antibody titers should serve as a warning to the local population that should be properly informed regarding the prevention.

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.

Genetic characterization of bovine viral diarrhoea virus detected in persistently infected cattle in Southern Minas Gerais, Brazil / Caracterização genética do vírus da diarreia bovina detectada em bovinos do Sul de Minas Gerais, Brasil, persistentemente infectados

Isolates of bovine viral diarrhoea virus (BVDV) detected in serum samples of two persistently infected animals (PI) identified in a herd located in the southern state of Minas Gerais, Brazil, underwent genetic characterization trough partial nucleotide sequencing and analysis of the 5’ Untranslated Region (5’UTR) of the viral genome. The isolates were characterized as belonging to genotype BVDV-1, subgenotype BVDV-1b. The results of this study suggest BVDV-1b as an agent of importance in the occurrence of bovine viral diarrhoea (BVD) in the herds of the region. Moreover, the genotypic characterization of isolates of BVDV helps to better understand the epidemiology of the disease, as the genetic variability of BVDV interferes in the serological tests and has implications for the use of vaccines, whose majority is produced only with reference strains of BVDV. Therefore, the investigation on the genetic diversity of BVDV existing in Brazil is required for the improvement of the disease prevention and control measures.

Isolados do vírus da diarreia viral bovina (BVDV) detectados em amostras de soro sanguíneo de dois animais persistentemente infectados (PI), identificados num rebanho bovino localizado na região sul do Estado de Minas Gerais, Brasil, foram submetidos à caracterização genética através do sequenciamento parcial de nucleotídeos da região 5’UTR do genoma viral. Os isolados foram caracterizados como pertencentes ao genótipo BVDV-1, subgenótipo BVDV-1b. Os resultados do presente estudo sugerem o BVDV-1b como um agente de importância na ocorrência da diarreia viral bovina (BVD) nos rebanhos da região. Ademais, a caracterização genotípica dos isolados do BVDV contribui para a melhor compreensão da epidemiologia da enfermidade, pois a variabilidade genética do BVDV interfere nos testes sorológicos e também possui implicações na utilização de vacinas, cuja maioria é produzida apenas com estirpes de referência do BVDV, requerendo, portanto, investigações sobre a diversidade genética do BVDV existente no Brasil para o aprimoramento das medidas de prevenção e controle da enfermidade no país.

Asymptomatic encephalitis in calves experimentally infected with bovine herpesvirus-5.

This study demonstrated that bovine herpesvirus 5 (BoHV)-5 infected calves can develop encephalitis and remain asymptomatic. Seven calves were infected intranasally and monitored for 30 days. Cerebrospinal fluid (CSF) analysis was performed from the onset of neurological signs. Multiple sections of brain and the trigeminal ganglion were submitted to histopathology. Virus detection (PCR and isolation) was performed on CSF and tissues. Four calves developed signs of neurologic disease and died. Three calves remained asymptomatic and were euthanized 30 days post-infection. Cerebrospinal fluid mononuclear pleocytosis occurred in symptomatic and asymptomatic calves. BoHV-5 was isolated and viral DNA was detected in multiple areas of the encephalon of all calves. The viral DNA was detected in the CSF of 2 calves showing neurological signs. Histologically, inflammation was noted in the brain of all calves and confirmed that the encephalitis caused by BoHV-5 may be mild and asymptomatic.

Frequency of group a rotavirus in piglet stool samples from non-vaccinated Brazilian pig herds

Neonatal diarrhea is one of the main causes of morbidity and mortality in piglets, and it leads to significant economic losses for pig farmers worldwide. The aim of this study was to determine the frequency of diagnosis, age group, and association of group A rotavirus (GARV) infection with diarrhea in piglets from pig herds in two (south and center-west)Brazilian geographical regions. The frequency of GARV diagnosis was evaluated between 2004 and 2007, using SS-PAGE on 681 fecal samples (428 diarrheic and 253 with normal consistency) from 1-4 week-old piglets. The animals were selected from 130 pig herds and 80 counties in the states of Rio Grande do Sul, Santa Catarina, Paraná, Mato Grosso do Sul, and Mato Grosso, Brazil. None of the herds were vaccinated against porcine GARV. Rotaviruses with the typical GARV electrophoretic pattern was identified in 193 (28.3 percent) fecal samples, and of these, 157 (81.3 percent) were diarrheic (p=0.001).Porcine GARV infection was identified in animals from all age groups evaluated, and the highest infection rate (54.7 percent; p=0.001) occurred in diarrheic piglets between 21 and 28 days of age. Diarrheic feces from 1-7 day-old piglets also had a high rate of rotavirus presence (32.3 percent), suggesting a failure in passive immunity. The high rate of porcine GARV infection in all geographical regions studied demonstrates the involvement of rotavirus in the etiology of neonatal diarrhea in Brazilian pig herds. This study highlights the importance of GARV infection for pig raising and the need of control and prophylactic measures for porcine rotavirus infection, including vaccination in the main areas of pork production in Brazil.

A diarréia neonatal é uma das principais causas de morbidade e mortalidade em leitões, ocasionando consideráveis prejuízos econômicos à suinocultura em todo o mundo. O objetivo desse estudo foi determinar a frequência de diagnóstico, distribuição etária e a associação com diarreia da infecção por rotavírus grupo A (GARV) em leitões de granjas situadas em duas (sul e centro-oeste) regiões geográficas brasileiras. A frequência de diagnóstico do GARV foi avaliada no período de 2004 a 2007, pela técnica de SS-PAGE, em 681 amostras fecais (428 diarréicas e 253 com consistência normal) de leitões de 1 a 4 semanas de idade. Os animais eram provenientes de 130 granjas, localizadas em 80 municípios dos estados do Rio Grande do Sul, Santa Catarina, Paraná, Mato Grosso do Sul e Mato Grosso. Nenhum dos rebanhos realizava vacinação contra o GARV suíno. Rotavírus suíno com perfil eletroforético característico do grupo A foi detectado em 193 (28,3 por cento) amostras, das quais 157 (81,3 por cento) diarreicas (p=0,001). A infecção foi identificada em animais de todas as faixas etárias avaliadas e a maior frequência (54,7 por cento; p=0,001) de diagnóstico ocorreu em leitões diarréicos com 21 a 28 dias de idade. A alta taxa (32,3 por cento) de diagnóstico do rotavírus em fezes diarreicas de leitões com 1 a 7 dias de vida sugere a necessidade da adoção de condutas que objetivem o incremento da imunidade passiva. A frequência de diagnóstico e a ampla distribuição das infecções pelo GARV, nos rebanhos das regiões geográficas avaliadas nesse estudo, ratificam a importância da rotavirose na etiologia dos episódios de diarreia de leitões no Brasil. Este resultado destaca também a importância do controle e medidas profiláticas na infecção por rotavírus suíno, incluindo a vacinação nas principais áreas de produção suína no Brasil.

Bovine herpesvirus 5 detection by virus isolation in cell culture and multiplex-PCR in central nervous system from cattle with neurological disease in Brazilian herds

Bovine herpesvirus 5 (BoHV-5) is an important cause of meningoencephalitis in young and adult cattle. The multiple etiology of neurological disturbances in cattle makes the quick and conclusive diagnosis of BoHV-5 infection important for animal and public health, mainly because of herbivore rabies that is endemic in Brazilian cattle herds. The objective of this retrospective study was to use a multiplex-polymerase chain reaction (multiplex-PCR) for BoHV-5 and BoHV-1 glycoprotein C gene detection from stored central nervous system (CNS) tissue fragments of cattle with neurological clinical signs. Forty-seven frozen CNS samples of young and adult cattle from 31 herds in three Brazilian geographical regions (South, Southeast, and Center-west) were evaluated. Eighteen (38.3 percent) of these CNS samples were BoHV-positive by virus isolation in cell culture. By multiplex-PCR 30 (63.8 percent) CNS samples were BoHV-5 positive. All 18 positive samples by virus isolation were confirmed as BoHV-5 by the multiplex-PCR, that provided a increase of 25.5 percent (12/47) in the BoHV-5 diagnosis rate. BoHV-1 was not detected in any CNS sample. This retrospective study demonstrated the wide regional distribution of BoHV-5 infection in Brazilian cattle herds since positive results were obtained in CNS samples of cattle with neurological disease from Paraná, São Paulo, Minas Gerais, Mato Grosso, and Mato Grosso do Sul States.

O herpesvírus bovino 5 (BoHV-5) é um importante agente etiológico de meningoencefalite em bovinos jovens e adultos. A etiologia múltipla dos distúrbios neurológicos em bovinos torna o diagnóstico conclusivo do BoHV-5 importante tanto em termos de sanidade animal quanto de saúde pública, principalmente pela característica endêmica da raiva dos herbívoros nos rebanhos bovinos brasileiros. O objetivo desse estudo retrospectivo foi utilizar a reação em cadeia da polimerase (multiplex-PCR) para a detecção do gene da glicoproteína C do BoHV-5 e do BoHV-1 em fragmentos estocados de sistema nervoso central (SNC) de bovinos com sinais clínicos neurológicos. Foram avaliadas 47 amostras congeladas de fragmentos de SNC de bovinos jovens e adultos pertencentes a 31 rebanhos de três regiões geográficas brasileiras (Sul, Sudeste e Centro-oeste). Por meio do isolamento viral em cultivo celular foi possível o isolamento do BoHV em 18 (38,3 por cento) amostras. Pela técnica de multiplex-PCR 30 (63,8 por cento) amostras de SNC foram positivas para o BoHV-5. Todas as 18 amostras positivas no isolamento viral foram confirmadas como BoHV-5 pela multiplex-PCR, proporcionando um incremento na taxa de diagnóstico do BoHV-5 de 25,5 por cento (12/47). Em nenhuma das amostras avaliadas foi possível a identificação do BoHV-1 pela multiplex-PCR. Esse estudo retrospectivo demonstrou a ampla distribuição da infecção pelo BoHV-5 nos rebanhos bovinos brasileiros uma vez que resultados positivos foram obtidos em amostras de SNC colhidas de bovinos com doença neurológica, provenientes dos estados do Paraná, São Paulo, Minas Gerais, Mato Grosso e Mato Grosso do Sul.

Rapid detection and differentiation of bovine herpesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR.

A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.

Avaliação da capacidade adjuvante do cloreto de dimetildioctadecilamônio associado ao hidróxido de alumínio na indução da resposta imune humoral de bovinos vacinados com o vírus da diarréia viral bovina / Evaluation of the adjuvant activity of dimethyl dioctadecyl ammonium chloride associated with the ammonium hydroxide to induction of humoral immune response of cattle vaccinated with bovine viral diarrhea virus

A resposta imunológica humoral de bovinos vacinados com o vírus da diarréia viral bovina (BVDV) inativado, tendo como adjuvante o cloreto de dimetildioctadecilamônio (DDA cloreto) associado ao hidróxido de alumínio (vacina B), foi comparada com uma vacina contendo o mesmo antígeno adsorvido apenas com hidróxido de alumínio (vacina A). Duas semanas após a segunda dose foi avaliado o título de anticorpos neutralizantes dos animais que receberam as duas preparações de antígenos. Os animais que receberam a vacina B apresentaram melhor resposta imune humoral quando comparados com os animais vacinados com a vacina A. O título médio de anticorpos neutralizantes, expresso em Log2, dos animais que receberam a vacina B foi superior (P<0,05) ao observado no grupo vacinado com a vacina A. Esse resultado demonstra que, em bovinos vacinados com o BVDV inativado, a inclusão do DDA cloreto em formulações de vacinas adsorvidas com hidróxido de alumínio potencializa a resposta imune humoral.

Ensaio imunoenzimático comercial no diagnóstico sorológico das infecçöes por herpesvírus bovino 1 / A commercial enzyme immune assay in serodiagnosis of bovine herpesvirus 1 infections

Avaliou-se o desempenho de um ensaio imunoenzimático, obtido de fonte comercial, na identificaçäo de anticorpos contra herpesvírus bovino tipo 1 (BHV-1), induzidos tanto por infecçäo natural quanto por vacinaçäo, em 1000 amostras de soros sanguíneos de bovinos. A análise comparativa dos resultados obtidos no sistema avaliado e na técnica padräo de soroneutralizaçäo mostrou uma concordância de 97,05 por cento (K=0,94) entre as duas metodologias de diagnóstico sorológico.

Prevalência de anticorpos neutralizantes contra o herpesvírus bovino tipo 1, decorrente de infecçäo natural, em rebanhos com distúrbios reprodutivos / Prevalence of neutralizing antibodies against bovine herpesvirus type 1, due natural infection, in herds with reproductive problems

A detecçäo de anticorpos anti-Herpesvírus Bovino tipo 1 (BHV-1) foi realizada, através da técnica de soroneutralizaçäo, em 1235 amostras de soro de bovinos adultos, näo vacinados contra Rinotraqueíte Infecciosa Bovina. As amostras de soro analisadas foram colhidas em 81 rebanhos, com histórico de problemas reprodutivos, incluindo animais com aptidäo para carne e leite, provenientes de 30 municípios do Estado do Paraná. Na amostragem proveniente de rebanhos leiteiros, 41,9 por cento (409/977) das amostras de soro e 90,5 por cento (57/63) dos rebanhos foram considerados positivos. Em bovinos de corte, o índice de soropositividade foi de 50,8 por cento (131/258) e 100 por cento (18/18) para amostras de soro e rebanhos, respectivamente. As frequências de 43,7 por cento (540/1235) de animais e 92,6 por cento (75/81) de rebanhos soropositivos demonstram que as infecçöes por BHV-1 apresentam-se amplamente disseminadas nas regiöes estudadas.