Systematic monitoring of glanders-infected horses by complement fixation test, bacterial isolation, and PCR.

Glanders is an equine zoonosis caused by Burkholderia mallei that is responsible for considerable economic loss. Complement fixation testing (CFT) using warm or cold incubation are recommended by the OIE, but many routinely used detection tests may present misleading results. To increase accuracy of glanders diagnosis and establish an appropriate protocol in collaboration with the National Equine Health Program, seven horses positive for glanders kept in isolation in Brazil were examined fortnightly by CFT, microbiological screening, and molecular testing. Warm and cold serologies with USDA and c.c.Pro antigens, respectively, were performed on 132 samples using the US Department of Agriculture protocol. The warm and cold serologies showed, respectively,12.9% and 17.3% seroreactive, 85.7% and 65.2% non-reactive, 0.8% and 3% inconclusive, and 0% and 2.3% anticomplementary. The agreement of CFT protocols was moderate. Of 213 clinical samples submitted to selective culture (167 nasal swabs, 5 ocular swabs, 3 lymph node punctures, and 38 tissue samples from four horses that died), 1.9% tested positive for B. mallei. Fourteen samples and one nasal swab (7%) tested positive with PCR. Cold CFT with the USDA and c.c.Pro antigens, in combination with PCR to increase sensitivity, may be useful for diagnosis of chronic glanders.

Bovine papillomavirus type 1 in brains of cattle with a neurological syndrome: pathological and molecular study

Bovine papillomavirus (BPV) infection is endemic in Brazilian herds. Papillomaviruses are oncogenic, with a trophic response in squamous epithelial and mucosal tissues, and are associated with asymptomatic infections, proliferative benign skin lesions (papillomas), and malignant epithelial lesions (carcinomas). The presence and expression of BPV in the blood of healthy and papillomatosis-affected cattle has been demonstrated. Experimental inoculation of Bovine papillomavirus (BPV) into calf meninges can result in meningiomas and papillomatosis, but it´s not known if its natural infection causes neoplasia and neurological syndrome in cattle. We assessed the frequency of BPV in 300 Central Nervous System (CNS) samples from cattle with neurological syndrome from several Brazilian regions obtained from surveillance of neurological syndrome. Samples were negative for rabies, Neospora caninum, BoHV-1 and BoHV-5, bovine leukemia virus, and catarrhal malignant fever (PCR). Samples were fixed in 10% buffered formalin and submitted to macroscopic examination. For histological analysis, slides were submitted to a staining protocol using hematoxylin and eosin. PCR for BPV detection was applied in CNS frozen samples using generic primers FAP59 and FAP64 (L1 gene). Thirteen (4.3%) samples were positive for BPV by PCR, with 11 of these showing no pathological changes in microscopy, and two exhibiting nonspecific non-purulent meningoencephalitis. No CNS samples showed neoplasia. Nine of the 13 BPV positive samples (69.2%) came from females and four (30.8%) from males. The 13 positive animals were age 5 to 168 months with seven over 36 months (53.8%). Five were dairy cattle, four crossbred, and three beef cattle. Only one of the 13 positive samples provided sufficient BPV DNA for sequencing, which emonstrated 99% identity to samples of BPV-1 obtained from cutaneous papillomas in cattle in Brazil. The small quantity of BPV DNA in the CNS and the low number of PCR-positive samples may be associated with low neurotropism, unspecific inflammation, or BPV-infected lymphocytes in CNS tissues or bloodstream. Natural BPV-1 infection was not associated with cerebral neoplasia or neurological syndrome.(AU)

Low occurrence of equine herpesvirus 1 (EHV-1) as cause of abortion and perinatal mortality in Brazil / Baixa ocorrência de herpes-vírus equino 1 (HVE-1) como causa de abortamento e mortalidade perinatal no Brasil

Equine herpesvirus type 1 (EHV-1) is an important pathogen that causes abortion, neonatal disease, respiratory disorders, and neurological syndrome in equine populations worldwide. To evaluate EHV-1 as a cause of abortion and perinatal mortality in Brazil, tissue samples from 105 aborted equine fetuses, stillbirths, and foals up to one month of age were examined using virus isolation, immunohistochemistry (IHC), histopathology, and nested polymerase chain reaction (PCR). Two fetuses were positive for EHV-1 by PCR, one of which showed syncytia and eosinophilic intranuclear inclusion bodies in bronchial epithelia, but it was negative by virus isolation. The other showed no characteristic histological lesions, but it was positive by viral isolation. No sample was positive by IHC. The results presented low occurrence of EHV-1 in the studied population and suggested that the use of a combination of techniques increases the likelihood of an accurate diagnosis of EHV-1.

O herpes-vírus equino tipo 1 (HVE-1) é um importante agente patogênico causador de aborto, doença neonatal, distúrbios respiratórios e síndrome neurológica em populações de equinos em todo o mundo. Para avaliar a ocorrência do HVE-1 como agente causal de abortamento e mortalidade perinatal no Brasil, foram examinadas amostras de 105 fetos equinos abortados, natimortos e potros de até 1 mês de idade, utilizando as técnicas de isolamento viral, imuno-histoquímica (IHQ), histopatologia e reação em cadeia da polimerase aninhada (nested-PCR). Dois fetos foram positivos na análise de PCR, e um deles apresentou corpúsculos de inclusão viral eosinofílicos e sincícios no epitélio brônquico, porém foi negativo na análise de isolamento viral. O outro feto não apresentou lesões histológicas características de infecção herpética, mas foi positivo na análise de isolamento viral. Nenhuma amostra apresentou resultado positivo pela análise de IHQ. Os resultados demonstraram baixa ocorrência de HVE-1 na população estudada e que o uso de diferentes técnicas diagnósticas aumenta a probabilidade de um diagnóstico preciso para o HVE-1.

Bovine Vaccinia in dairy cattle and suspicion of vesicular disease on milkers in Brazil / Vaccinia bovina em gado leiteiro e suspeita de doença vesicular em ordenhadores no Brasil

ABSTRACT: Bovine vaccinia (BV) is a vesicular disease induced by the Vaccinia virus (VACV) that affects milk production and is an occupational zoonosis. This research had the following objectives: (i) detection of VACV by qPCR in cattle with clinical suspicion of vesicular disease; (ii) symptoms characterization in animals and milkers with clinical suspicion of the disease and virus detection in humans; and (iii) identification of risk factors for infections of VACV in herds from several Brazilian states. A total of 471 bovine epithelial samples from dairy farms, in 15 Brazilian states, were evaluated between 2007 and 2012. The samples were tested by quantitative PCR (qPCR) using SYBR Green® reagents, validated with a lower limit of detection of 100 TCID50/50µL (1.7x100 viral particles), and 45.1% of VACV positive samples were detected. Using official forms for epidemiological investigation (FORM-IN), the risk factors for VACV infections in cattle were determined to be farms with a lack of technological facilities (P=0.029) and the presence of rodents (P=0.001). There was an effect of seasonality in cattle with a higher occurrence of BV during the dry season. A total of 420 epidemiological questionnaires were applied at public health care centers, where 100% of the milkers had vesicular lesions on their hands (98.1%) and on their arms (6.9%). The most frequent clinical symptoms in humans were: local swelling (74.2%), headache (20.7%), fever (10.4%) and inguinal lymphadenopathy (74.2%). Only 19.98% of milkers aged between 39 and 58 years were seroreactive to VACV and were immunized with the human anti-smallpox vaccine. There was an increase in the frequency of BV in older individuals due to their natural decrease in specific immunity. It has been shown that the implementation of zootechnical management techniques and health planning are important for the prevention of BV in animals and humans.

RESUMO: Vaccinia bovina (VB) é uma doença vesicular induzida pelo Vaccinia virus (VACV) que afeta a produção de leite e é uma zoonose ocupacional. Este trabalho teve os seguintes objetivos: (i) detecção de VACV por qPCR em bovinos com suspeita clínica de doença vesicular; (ii) caracterização dos sintomas apresentados por animais e ordenhadores com suspeita clínica da doença e detecção do vírus em humanos; e (iii) identificação de fatores de risco para infecção por VACV em rebanhos de vários estados brasileiros. Um total de 471 amostras de epitélio bovino de fazendas leiteiras, em 15 estados brasileiros, foram avaliados entre 2007 e 2012. As amostras foram testadas por PCR quantitativa (qPCR) usando reagentes SYBR Green®, validados com um limite inferior de detecção de 100TCID50/50μL (1,7x100 partículas virais) e 45,1% das amostras positivas de VACV foram detectadas. Usando formulários oficiais de investigação epidemiológica (FORM-IN), os fatores de risco para infecções por VACV em bovinos foram determinados como fazendas com falta de instalações tecnológicas (P=0,029) e presença de roedores (P=0,001). Houve um efeito da sazonalidade no gado com maior ocorrência de VB durante a estação seca. Um total de 420 questionários epidemiológicos foram aplicados nos centros públicos de saúde, onde 100% dos ordenhadores apresentaram lesões vesiculares nas mãos (98,1%) e nos braços (6,9%). Os sintomas clínicos mais frequentes em humanos foram: inchaço local (74,2%), cefaleia (20,7%), febre (10,4%) e linfadenopatia inguinal (74,2%). Apenas 19,98% dos produtores de leite com idade entre 39 e 58 anos foram sororreagentes ao VACV e foram imunizados com a vacina contra a varíola humana. Houve um aumento na frequência de BV em indivíduos mais velhos devido à sua diminuição natural na imunidade específica. Demonstrou-se que a implementação de técnicas de gestão zootécnica e planejamento sanitário são importantes para a prevenção da VB em animais e seres humanos.

Malignant Catarrhal Fever in Brazilian cattle presenting with neurological syndrome

Abstract Malignant Catarrhal Fever (MCF) was investigated in the central nervous system of cattle with neurological syndrome. Two-hundred-ninety samples were analyzed by histology, and molecular methods to detect ovine herpesvirus type 2 (OvHV-2) were optimized and validated. The qualitative polymerase chain reaction (qualitative PCR) analytical sensitivity was 101 DNA copies/µL and found 4.8% (14/290) positive for OvHV-2. The quantitative polymerase chain reaction (qPCR) analytical sensitivity was 100 DNA copy/µL and 5.9% (17/290) positivity, with 47.1% (8/17) of the positive samples presenting histological evidence of non-purulent meningo-encephalitis. The qualitative PCR products (422 bp of the ORF75 region) were sequenced and submitted to phylogenetic analysis. Identity matrices showed 100% similarity in OvHV-2 samples obtained in this study and those recovered from GenBank, corroborating other studies.

Malignant Catarrhal Fever in Brazilian cattle presenting with neurological syndrome.

Malignant Catarrhal Fever (MCF) was investigated in the central nervous system of cattle with neurological syndrome. Two-hundred-ninety samples were analyzed by histology, and molecular methods to detect ovine herpesvirus type 2 (OvHV-2) were optimized and validated. The qualitative polymerase chain reaction (qualitative PCR) analytical sensitivity was 101DNA copies/µL and found 4.8% (14/290) positive for OvHV-2. The quantitative polymerase chain reaction (qPCR) analytical sensitivity was 100DNA copy/µL and 5.9% (17/290) positivity, with 47.1% (8/17) of the positive samples presenting histological evidence of non-purulent meningo-encephalitis. The qualitative PCR products (422bp of the ORF75 region) were sequenced and submitted to phylogenetic analysis. Identity matrices showed 100% similarity in OvHV-2 samples obtained in this study and those recovered from GenBank, corroborating other studies.

Neospora caninum as causative agent of bovine encephalitis in Brazil / Neospora caninum como agente causal de encefalite bovina no Brasil

For supporting the Brazilian bovine encephalitis surveillance program this study examined the differential diagnosis of Neospora caninum in central nervous system (CNS) by histological analysis (HE staining), immunohistochemistry (IHC), and nested-PCR using a set of primers from the Nc5 region of the genomic DNA and ITS1 region of the ribosomal DNA. A sample of 302 cattle presenting neurological syndrome and negative for rabies, aged 0 to 18 years, from herds in 10 Brazilian states was evaluated for N. caninum from January 2007 to April 2010. All specimens tested negative with IHC and nested-PCR using primers from the ITS1 region of ribosomal DNA, while two positive cases (0.66%) were found using primers from the Nc5 region of genomic DNA: a 20 month-old male and a 72 month-old female, both from São Paulo State. Only the male presented severe multifocal necrotizing encephalitis associated with mononuclear cell infiltration, a pathognomonic lesion caused by parasites of the family Sarcocystidae, and only this case was associated with N. caninum thus representing 0.33% positivity. Future studies should explore the association of IHC and nested-PCR with real-time PCR, a quantitative method that could be standardized for improving the detection of N. caninum in bovine CNS specimens.

Este estudo contribuiu para o programa de vigilância epidemiológica de encefalite bovina no Brasil realizando o diagnóstico diferencial de Neospora caninum no sistema nervoso central (SNC) por análise histológica (coloração HE), imunohistoquímica (IHC) e nested-PCR utilizando-se primers da região Nc5 do DNA genômico e da região ITS1 do DNA ribossomal. Um total de 302 amostras de bovinos com síndrome neurológica, negativos para raiva, na faixa etária de zero a 18 anos, provenientes de rebanhos de 10 estados brasileiros foi avaliada para N. caninum no período de janeiro/2007 a abril/2010. Todas as amostras foram negativas na IHC e na nested-PCR usando-se primers da região ITS1 do DNA ribossomal, enquanto dois casos (0,66%) foram positivos à nested PCR, usando-se primers da região Nc5 do DNA genômico: um macho de 20 meses de idade e uma fêmea de 72 meses de idade, ambos do Estado de São Paulo. Apenas o macho apresentou severa encefalite multifocal necrotizante associada com infiltrado inflamatório mononuclear, lesão patognomônica causada por parasitas da família Sarcocystidae, mostrando que apenas este caso de encefalite foi associado à infecção por N. caninum, representando 0,33% de positividade. Sugere-se em estudos futuros utilizar também a PCR em tempo real para detecção do parasito.

Diagnosis of Neospora caninum in bovine fetuses by histology, immunohistochemistry, and nested-PCR.

Neospora caninum, a cause of abortion and stillbirth in cattle, was studied by histology, immunohistochemistry, and nested-PCR, using primers from the Nc5 region of the genomic DNA (PCR PLUS) and primers from the ITS1 region of the ribosomal DNA (PCR JB). A total of 105 fetal samples sent to the Centro de Pesquisa e Desenvolvimento de Sanidade Animal do Instituto Biológico from January 2006 to May 2008 were examined for evidence of N. caninum. Histological examination revealed 71.4% with non-suppurative inflammation in the heart, lung, liver, kidney, placenta, and brain. Immunohistochemistry detected infections in 8.6% of the samples, mainly in the brain, placenta, and heart. Nested-PCR JB revealed 6.7% with infections, while nested-PCR PLUS returned 20.9% positive results, mainly in brain and placenta, and in the pooled liver and heart. Kappa statistics demonstrated little agreement among the three techniques. The three methods are complementary, since they have distinct diagnostic characteristics and were combined to give a positivity rate of 24.8%.

Erradicação do herpesvírus bovino - 1 (BHV-1) de um rebanho bovino leiteiro em manejo semi-intensivo / Eradication of Herpesvirus Bovine-1 (BHV-1) from a dairy cattle herd with a semi-intensive management

Com o objetivo de erradicar o BHV-1 de um rebanho bovino leiteiro de alto valor genético sem a utilização de vacina, foi realizado um exame sorológico prévio em 154 animais, onde constatou-se 15,6% de reagentes ao BHV-1. A técnica utilizada foi a soroneutralização em microplacas. Dentre os animais soropositivos, as vacas vazias foram descartadas imediatamente e as prenhes isoladas e descartadas após o parto. Os bezerros apresentaram anticorpos colostrais até os seis meses de idade, motivo pelo qual não foram descartados; nos bezerros de 6 a 12 meses de idade e nas novilhas não foram diagnosticados animais soropositivos. Os animais foram examinados trimestralmente, por 21 meses, seguido de mais duas coletas semestrais. As vacas secas, prenhes e em lactação, soropositivas, revelaram ser a fonte de infecção do BHV-1. A manutenção de rebanho livre é possível, desde que sejam adotadas medidas como a utilização de sêmen livre de BHV-1, realização de quarentena no ingresso de animais e exames sorológicos anuais visando impedir a reintrodução do vírus. Com o conjunto destas medidas adotadas, a fazenda encontra-se há 18 meses livre do BHV-1