Seroepidemiology Survey of Bovine Alphaherpesvirus 1 (BoHV-1) in Unvaccinated Beef Bubalines (Bubalus bubalis) from Southern Brazil

Background: The bovine alphaherpesvirus type 1 (BoHV-1) belonging to the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae and genus Varicellovirus.Bovine alphaherpesvirus 1 (BoHV-1) infections have a great importance due to the high rates of dissemination in cattles worldwide. Although, the BoHV-1 was largely related in cattle, buffaloes were also classified as host for the virus. However, studies that determine seroepidemiological data in this species are scarce and necessary. The aim of the present study was to determine the presence of antibodies against BoHV-1 in healthy beef buffaloes using the virus neutralization (VN) technique.Cases: This work portrays an epidemiological survey, based on a sectional study characterized by blood samples collected from 54 Murrah buffalo, aged 6 to 24 months, from the municipalities of Guaraqueçaba, Ponta Grossa, Antonina and Doutor Ulysses, located in Paraná state, being 20, 14, 10 and 10 samples from each location, respectively. Thirty-seven percent (20/54) of the samples were collected at slaughterhouse with registration at the Federal Inspection Service (SIF) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), and 63% (34/54) at the buffalo's farms. The serum samples were collected in sterile tubes without anticoagulant in stored isothermal boxes, with serum separation and stored at -20ºC. The samples were sent to the Animal Virology Laboratory of Universidade Estadual de Londrina, UEL for serological analysis. Serological diagnosis using the virus neutralization (VN) technique was performed according the OIE manual of diagnostic tests and vaccines for terrestrial animals, using the BoHV-1 Los Angeles strain. Forth-five of the 54 samples (83.3%) evaluated generated titers of antibodies against BoHV-1 present in all evaluated herds, and the samples collected in herd from Antonina city were 100% positive, following by 80% in Guaraqueçaba and Doutor Ulysses city, and 78.6% in Ponta Grossa city. In relationship to the titration of anti-bovine herpesvirus 1 antibodies (BoHV-1), it was found that 23/45 (51.12%) of the samples had titers ≤ 16, 13/45 (28.88 %) with indexes between 32-64, and 9/45 (20%) with levels above 128.Discussion: The confirmation of the presence of antibodies against BoHV-1 in the buffalo samples tested in the present study proves the circulation of the agent in the studied species, with a homogeneous distribution of bovine alphaherpesvirus 1 in all the evaluated herds. There was high variability in antibody titers against BoHV-1 from all herds, and the occurrence of a high number of buffaloes reactive to BoHV-1 and with high antibody titers suggests a course of active or reactive infections in these animals. In only one of the herds, the age of the animals studied varied between 6 to 8 months. The fact that these animals are lactating raises the possibility of anti-BoHV-1 antibodies being acquired by breastfeeding. The results obtained allow to conclude, considering the fact that the animals are not vaccinated against BoHV-1, the presence of circulating antibodies is from a natural infection, and the positivity in all farms tested denotes the endemicity of the BoHV-1 infection in herds. To the best knowledge of the authors, this is the first study on the detection and measurement of antibodies against BoHV-1 in healthy and unvaccinated buffaloes in the state of Paraná, southern Brazil, showing a high frequency of seroreagent animals.

Serological and molecular investigation of viral agents in free-living jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência – RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onças-pintadas nesta região.This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest – SFIMT and Rapid Fluorescent Focus Inhibition Test – RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region

Seroepidemiology Survey of Bovine Alphaherpesvirus 1 (BoHV-1) in Unvaccinated Beef Bubalines (Bubalus bubalis) from Southern Brazil

Background: The bovine alphaherpesvirus type 1 (BoHV-1) belonging to the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae and genus Varicellovirus.Bovine alphaherpesvirus 1 (BoHV-1) infections have a great importance due to the high rates of dissemination in cattles worldwide. Although, the BoHV-1 was largely related in cattle, buffaloes were also classified as host for the virus. However, studies that determine seroepidemiological data in this species are scarce and necessary. The aim of the present study was to determine the presence of antibodies against BoHV-1 in healthy beef buffaloes using the virus neutralization (VN) technique.Cases: This work portrays an epidemiological survey, based on a sectional study characterized by blood samples collected from 54 Murrah buffalo, aged 6 to 24 months, from the municipalities of Guaraqueçaba, Ponta Grossa, Antonina and Doutor Ulysses, located in Paraná state, being 20, 14, 10 and 10 samples from each location, respectively. Thirty-seven percent (20/54) of the samples were collected at slaughterhouse with registration at the Federal Inspection Service (SIF) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), and 63% (34/54) at the buffalo's farms. The serum samples were collected in sterile tubes without anticoagulant in stored isothermal boxes, with serum separation and stored at -20ºC. The samples were sent to the Animal Virology Laboratory of Universidade Estadual de Londrina, UEL for serological analysis. Serological diagnosis using the virus neutralization (VN) technique was performed according the OIE manual of diagnostic tests and vaccines for terrestrial animals, using the BoHV-1 Los Angeles strain. Forth-five of the 54 samples (83.3%) evaluated generated titers of antibodies against BoHV-1 present in all evaluated herds, and the samples collected in herd from Antonina city were 100% positive, following by 80% in Guaraqueçaba and Doutor Ulysses city, and 78.6% in Ponta Grossa city. In relationship to the titration of anti-bovine herpesvirus 1 antibodies (BoHV-1), it was found that 23/45 (51.12%) of the samples had titers ≤ 16, 13/45 (28.88 %) with indexes between 32-64, and 9/45 (20%) with levels above 128.Discussion: The confirmation of the presence of antibodies against BoHV-1 in the buffalo samples tested in the present study proves the circulation of the agent in the studied species, with a homogeneous distribution of bovine alphaherpesvirus 1 in all the evaluated herds. There was high variability in antibody titers against BoHV-1 from all herds, and the occurrence of a high number of buffaloes reactive to BoHV-1 and with high antibody titers suggests a course of active or reactive infections in these animals. In only one of the herds, the age of the animals studied varied between 6 to 8 months. The fact that these animals are lactating raises the possibility of anti-BoHV-1 antibodies being acquired by breastfeeding. The results obtained allow to conclude, considering the fact that the animals are not vaccinated against BoHV-1, the presence of circulating antibodies is from a natural infection, and the positivity in all farms tested denotes the endemicity of the BoHV-1 infection in herds. To the best knowledge of the authors, this is the first study on the detection and measurement of antibodies against BoHV-1 in healthy and unvaccinated buffaloes in the state of Paraná, southern Brazil, showing a high frequency of seroreagent animals.

Concomitant bovine viral diarrhea, mycotoxicosis, and seneciosis in cattle from northern Paraná, Brazil

O objetivo desse estudo foi avaliar a causa de mortalidade observada em um rebanho bovino constituído por vacas da raça Nelore criadas no norte do Estado do Paraná, região sul do Brasil, no qual 9,5% (23/242) das vacas morreram após manifestação clínica de andar cambaleante, febre, diarreia transitória com sangue, dispneia e decúbito lateral. O rebanho era mantido em pastagens degradadas e ingeriram silagem de milho mofada e broto de Senecio brasiliensis. Amostras de soro (n=17) foram coletadas para a detecção de anticorpos neutralizantes para o vírus da diarreia viral bovina (BVDV) e herpesvírus bovino 1 (BoHV-1). Três vacas foram necropsiadas e os fragmentos de tecidos foram avaliados por RT-PCR e PCR para a identificação de BVDV e BoHV-1, respectivamente. Os achados macroscópicos incluíram aumento da consistência hepática, sangramento intestinal e hemorragia pulmonar. A histopatologia revelou fibrose hepática, enterite hemorrágica, necrose tubular renal e vasculite generalizada. Todas as amostras de soro apresentaram anticorpos neutralizantes para o BoHV-1 e seis amostras foram positivas para o BVDV. A RT-PCR amplificou produtos da região não-traduzida do genoma do BVDV a partir de tecidos de ambos os animais. Análises filogenéticas dos produtos amplificados demonstraram que as sequências agruparam com o BVDV subgenotipo 1d. Todas as amostras analisadas por PCR para a identificação do DNA do BoHV-1 foram negativas. Análises toxicológicas nas amostras de silagem milho mofada apresentaram altas concentrações de ocratoxina (75,94 ?g/kg). Esses resultados confirmam que as ações combinadas da infecção com o BVDV-1d, a micotoxicose e a intoxicação por Senecio contribuíram com a mortalidade de vacas no rebanho avaliado.This study investigated the causes of mortality in cattle from northern Paraná, southern Brazil, during which 9.5% (23/242) of Nellore cows died after clinical manifestations of uncoordinated gait, fever, transient bloody diarrhea, dyspnea, and lateral decumbency. All cattle were maintained on poor pastures, and ingested moldy silage and sprouts of Senecio brasiliensis. Serum samples (n=17) were collected for virus neutralizing (VN) antibodies assays against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1). Three cows were necropsied; fragment of tissue samples collected during necropsy from two cows were used in polymerase chain reaction (PCR) and reverse transcription- PCR assays to identify BoHV-1 and BVDV; blood samples (n=6) were obtained for hematological and biochemical analyses. Significant gross findings of all cows included increased liver consistency, intestinal bleeding, and pulmonary hemorrhage. Histopathology revealed hepatic fibrosis, hemorrhagic enteritis, renal tubular necrosis, pulmonary hemorrhage, and widespread vasculitis. All serum samples contained VN antibodies against BoHV-1, but only four of these reacted positively to BVDV. The RTPCR assays amplified specific amplicons of the untranslated region of BVDV from tissues samples of both animals; direct sequencing and sequence analyses demonstrated that these sequences clustered within the BVDV subgenotype 1d; all PCRs were negative for BoHV-1. Toxicological analyses of the moldy silage demonstrated elevated concentrations of ochratoxin (75.94 ?g/kg). Laboratory evaluations revealed renal and hepatic dysfunctions. These findings confirmed the combined actions of BVDV-1d infection and toxicosis by mycotoxin and S. brasiliensis in cattle mortality at this farm

Ovine herpesvirus type 2-induced malignant catarrhal fever in a heifer

Os achados clínicos, patológicos e moleculares associadas às lesões da febre catarral maligna induzidas por herpesvírus ovino 2 (OvHV-2) são descritos em uma novilha, 12 meses de idade, raça mista, da Região Norte do Estado Paraná, Brasil. O animal demonstrou comportamento agressivo, nistagmo, incoordenação motora, decúbito, movimentos de pedalagem e morte dentro de 48 horas, após a qual foi submetido à necropsia de rotina. Fragmentos do cérebro foram utilizados em ensaios de PCR para a identificação de agentes infecciosos (OvHV-2; herpesvírusbovino-1 e -5 e Histophilus somni). A raiva foi descartada por exame realizado em laboratório oficial de diagnóstico do estado. Alterações patológicas significativas incluíram vasculite disseminada com infiltrado inflamatório linfoplasmocitário no cérebro, rins, fígado e miocárdio. A PCR amplificou o amplicon específico do gene da proteína do tegumento do OvHV-2 a partir de amostras do cérebro; todas as outras avaliações foram negativas. Estes resultados confirmam a participação de OvHV-2 na etiopatogenia da doença observada nesta novilha.The clinical, pathological, and molecular findings due to malignant catarrhal fever lesions induced by ovine herpesvirus type 2 (OvHV-2) are described in a 12-month-old, mixed-breed, heifer from northern Paraná, Brazil. The animal demonstrated clinical signs including manifestations of aggressive behavior, nystagmus, incoordination, decumbency, paddling movements, and death within 48 hours, after which was submitted for routine necropsy. Brain fragments were used in PCR assays for identification of infectious agents (OvHV-2; bovine herpesvirus-1 and -5; and Histophilus somni). Rabies was discarded in test done at official state diagnostic laboratory. Significant pathological alterations include disseminated vasculitis with lymphoplasmacytic inflammatory infiltrate within the brain, kidneys, liver, and myocardium. PCR assay amplified the specific422base pair fragment of the OvHV-2 tegument protein gene from the brain; all other evaluations were negative. These findings confirm the participation of OvHV-2 in the etiopathogenesis of the disease observed in this mixed-breed heifer

Infectious Disease Agents Associated with Pulmonary Alterations in Aborted Bovine Fetuses

This study investigated the occurrence of selected pathogens of bovine respiratory disease in fetal pulmonary tissue of cattle and associated these with patterns of disease. Fetal pulmonary (n = 37) tissues were evaluated by histopathology; immunohistochemical assays identified intralesional antigens of bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and Mycoplasma bovis. Molecular assays were performed to amplify reproductive disease pathogens and bovine gammaherpesvirus 6 (BoGHV6) from 12 lungs. The 2 patterns of pulmonary diseases were interstitial pneumonia (12/37) and suppurative bronchopneumonia (1/37). The frequency of the intralesional antigens identified was BRSV (16.2%; 6/37), BVDV (13.5%; 5/37), BoAHV1 (8.1%; 3/37), M. bovis (5.4%; 2/37), and BPIV-3 (2.7%; 1/37). Interstitial pneumonia was associated with BRSV (n = 3), BoAHV1 (n = 3), and BVDV (n = 2); suppurative bronchopneumonia contained a Gram-positive bacterium and BVDV and BRSV. Reproductive pathogens detected included Leptospira spp., (n = 3), BVDV, Neospora caninum, and Brucella abortus (n = 2). BoGHV6 DNA was identified in the lungs of two fetuses with interstitial pneumonia. These findings suggest that these fetuses were infected transplacentally by several pathogens. The role of some of these pathogens herein identified must be further elucidated in the possible participation of fetal disease