Seven years of evaluation of ectoparasites and vector-borne pathogens among ring-tailed coatis in an urban park in southeastern Brazil.

Wild animals have been recognized as potential reservoirs of vector-borne pathogens. Proximity between these animals and urban areas increases the need to know which pathogens these are and whether they can infect domestic animals and humans. In Mangabeiras Municipal Park in Belo Horizonte, Brazil, coatis live near the urban area, which is mainly occupied by human residents and their domestic animals. Therefore, the objective of this study was to detect, through molecular and direct methods, the presence of ectoparasites and hemoparasites in coatis. A total of 216 samples were collected, of which 209 samples were from first-captures and seven were from recaptures. The following parasites were found: ticks of the genus Amblyomma, lice of the species Neotrichodectes pallidus and fleas of the species Rhopalopsyllus lutzi lutzi and Ctenocephalides felis felis. All the samples were negative for the family Anaplasmataceae and the species Leishmania sp. and Trypanosoma cruzi. The hemoparasites Trypanosoma evansi, Hepatozoon procyonis, Babesia sp. and Sarcocystis neurona were found. The area of the present study is not endemic for T. evansi, which therefore suggests that these coatis may be acting as reservoirs or sentinels of this parasite. This finding is of great epidemiological importance and should be investigated more closely. Thus, this study showed that there is a great variety of pathogens in the park that transit among coatis and, probably, among other animals that inhabit or live close to the park.

Molecular detection of bovine immunodeficiency virus (BIV) in bovines from the state of Minas Gerais, Brazil / Detecção molecular do vírus da imunodeficiência bovina (BIV) em bovinos do estado de Minas Gerais, Brasil

O vírus da imunodeficiência bovina é o agente causador da imunodeficiência viral bovina que é conhecido por infectar bovinos em todo o mundo. Como em outras infecções por retrovírus, os hospedeiros desenvolvem uma infecção de longo prazo e a maioria dos animais infectados permanece assintomática. O objetivo deste estudo foi detectar DNA proviral BIV em amostras de sangue de bovinos e estimar a ocorrência de infecção no estado de Minas Gerais, Brasil. Amostras de sangue de 391 bovinos foram coletadas de duas regiões do estado, Zona da Mata e Central. O DNA proviral foi detectado por reação em cadeia da polimerase semi-nested (SN-PCR). Os resultados de SN-PCR indicaram uma ocorrência de BIV de 12,5% no estado. Os produtos amplificados foram confirmados como BIV por sequenciamento e a similaridade da sequência de nucleotídeos com a estirpe de referência (R-29) foi de 99%. Este é o primeiro estudo que relata a presença do BIV em Minas Gerais, Brasil. Os resultados indicam a necessidade de realizar um estudo detalhado sobre a prevalência da infecção por BIV no Brasil.(AU)

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples.

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot-and-mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT-ddPCR) using one-step and two-step approaches. Analytical sensitivity and specificity were done in parallel with real-time PCR, RT-qPCR (one-step and two-step) for comparison of sensitivity and specificity of both methods. In the standardization of RT-ddPCR, the double-quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one-step techniques showed superior performance than two-step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT-ddPCR and RT-qPCR using the one-step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT-PCR had a better performance than RT-PCR when swine serum pools were tested. According to the results, the one-step RT-ddPCR and RT-qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT-ddPCR), being a useful tool for vesicular diseases control programs.

Genetic variability and phylogeny of the 5' long terminal repeat from Brazilian bovine leukemia virus.

We conducted a phylogenetic analysis of 22 strains of bovine leukemia virus obtained by polymerase chain reaction to amplify a 582-base pair fragment of the transcriptional regulatory region 5' long terminal repeat (LTR). Twenty-two samples of proviral DNA from peripheral blood mononuclear cells containing bovine leukemia virus from naturally infected bovine from 4 distinct geographic regions in Brazil were investigated. The products obtained by polymerase chain reaction were subjected to direct sequencing and sequence alignment. Fragments of 422 nucleotides were obtained, located between positions -118 and +303 base pairs of the 5'LTR. These fragments corresponded to 80% of the LTR region and included 56% of sub-region U3, 100% of R, and 82.5% of U5. Phylogenetic analysis of these sequences showed a high conservation degree in the 5'LTR region, with 5 well defined groups. However, a hotspot occurrence in the R-U5 region was also observed, which contained 40% of all nucleotide variability observed.

Diagnóstico etiológico de enfermidades do sistema nervoso central de equinos no Estado de Minas Gerais, Brasil / Etiologic diagnosis of diseases of the central nervous system of horses in Minas Gerais State, Brazil

O Brasil possui o quarto maior rebanho equino do mundo, e o Estado de Minas Gerais detém o maior número de equinos do país. Portanto, um diagnóstico preciso das doenças neurológicas dos equinos é prioridade no estado. Sendo assim, o objetivo deste estudo foi identificar, utilizando a Reação em Cadeia pela Polimerase (PCR), os agentes infecciosos responsáveis por enfermidades que afetam o sistema nervoso central (SNC) de equinos. De janeiro de 2009 a janeiro de 2011, foi realizado um levantamento dos casos de encefalites e encefalomielites em equinos no Estado de Minas Gerais, utilizando-se amostras de SNC de equinos que morreram com sinais neurológicos. Das 217 amostras de SNC, 47 (21,7%) foram positivas para o vírus da raiva pelo método de imunofluorescência indireta e inoculação em camundongos. Nas 170 amostras negativas para o vírus da raiva, o herpes-vírus equino-1 (EHV-1) foi diagnosticado em 20 (11,8%) e o herpes-vírus suíno-1 (SHV-1), em uma amostra por meio de PCR, e o vírus encefalite de Saint Louis (SLEV), em outra amostra, através de transcrição reversa (RT) e PCR (RT-PCR). Constatou-se que o vírus da raiva é o principal agente causador de encefalite em equinos, apesar do crescente número de casos de encefalomielite associados ao EHV-1 no Estado de Minas Gerais.(AU)

Brazil has the fourth largest equine herd in the world and the State of Minas Gerais has the largest equine population in the country. Therefore, an accurate diagnosis of cases of neurologic diseases is a priority in Minas Gerais. The aim of this study was to identify by Polymerase Chain Reaction (PCR) infectious agents associated with neurological disease in the central nervous system (CNS) of horses. A survey of encephalitis and encephalomyelitis in horses in Minas Gerais State was performed on samples of CNS from horses that died with neurological signs from January 2009 to January 2011. Forty seven CNS samples from 217 (21.7%) horses were positive for rabies virus by the indirect immunofluorescence assay and mouse inoculation. Among the 170 samples that were negative for rabies, EHV-1 was detected in 20 (11.8%) and the swine herpesvirus-1 (SHV-1) was detected in one sample by PCR, and the Saint Louis encephalitis virus (SLEV) was identified in another sample by reverse transcription (RT) and PCR (RT-PCR). Rabies virus is the most common causative agent of encephalitis in horses, despite the increasing number of cases of encephalitis associated with EHV-1 in the State of Minas Gerais.(AU)

PCR em tempo real para detecção do vírus da doença de Aujeszky / Real time PCR for detection of Aujeszky's disease virus

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.

The aim of this study was to validate a low-cost real-time PCR for a quick and sensitive diagnosis of the disease. The fluorofore used was a DNA intercalating agent, one of the cheaper detection systems. Primers amplified a 123 base pairs fragment of the gene coding for glycoprotein D. PCR was tested on 25 samples of pig brain positive for AD and 85 samples negative in viral isolation and serum neutralization. The detection limit was calculated on samples of pig brain contaminated with a Brazilian isolate of SuHV-1. The technique had a detection limit of 10-0,5 TCID50/50µL. PCR was able to distinguish nonspecific reactions due to primer dimers (thus avoiding false positives) and get faster results.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.

Confirmação de infecção por Brucella abortus em um rebanho bovino certificado livre em Minas Gerais: relato de caso / Brucella abortus infection in an officially brucellosis-free bovine herd in Minas Gerais state - Brazil: a case report

Relata-se a ocorrência de um surto de brucelose em um rebanho de aproximadamente 1000 animais, livre da doença há 18 anos, certificado pelo Ministério da Agricultura, Pecuária e Abastecimento desde 2006. Dois animais reagiram aos testes sorológicos de diagnóstico por ocasião dos procedimentos de recertificação em 2008. Após o sacrifício deles, Brucella abortus, biovariedade 1, amostra não vacinal, foi isolada e identificada por meio de provas bioquímicas e de biologia molecular (PCR AMOS). A origem do agente no rebanho é de difícil determinação. No entanto, a adoção de procedimentos preconizados pelo Programa Nacional de Controle e Erradicação da Brucelose permitiu evitar a disseminação da enfermidade. Ocorrências como essas, em que rebanhos livres foram infectados após anos sem a ocorrência de brucelose, nunca haviam sido relatadas no Brasil.

A Brucellosis outbreak is reported in a bovine herd free from the disease for over 18 years, officially free since 2006. One heifer and one cow tested positive in serological tests for the 2008 annual recertification in a herd of almost 1000 animals. Isolation and identification by biochemical tests and molecular biology (AMOS PCR) confirmed the infection by a Brucella abortus biovar 1 field strain. It wasn't possible to find the source of the infection. However, adoption of standard procedures prescribed by the Brazilian National Brucellosis and Tuberculosis Control and Eradication Program hindered the spread of the disease. This is the first report of a Brucella infection in an officially Brucellosis-free bovine herd in Brazil.

Detecção dos genes de Staphylococcus aureus, enterotoxinas e de resistência à meticilina em leite / Detection of genes of Staphylococcus aureus, enterotoxins and methicillin resistance in milk

Realizou-se a detecção do gene de Staphylococcus aureus, de enterotoxinas e de resistência à meticilina com extração de DNA feita diretamente de amostras de leite. Das 200 amostras estudadas, 145 (72,5%) amplificaram o gene femA, e estas foram analisadas quanto à presença dos genes sea, seb, sec e mecA. Os genes das enterotoxinas mais prevalentes foram: sea (60%), seb (37,9%) e sec (6,9%). Foram encontradas 18 amostras de leite (11,0 %) com S. aureus portadores do gene mecA. A detecção de S. aureus diretamente do leite, sem a necessidade de isolamento bacteriano e a caracterização do potencial enterotoxigênico, demonstra que a técnica de PCR é muito útil para estudos epidemiológicos das infecções estafilocócicas da glândula mamária. O alto percentual (72,5%) de amostras de leite positivas para a presença do gene femA sugere que S. aureus constitui um dos principais agentes causadores de infecções intramamárias na microrregião de Sete Lagoas-MG e que seu potencial enterotoxigênico e presença do gene mecA, que identifica o S. aureus resistente à meticlina, representa um risco potencial à saúde pública.

This work was performed to detect the Staphylococcus aureus gene, enterotoxins resistance to methicillin with the extraction of DNA directly from milk samples. Of the 200 samples studied 145 (72.5%) amplified the femA gene, which were analyzed regarding the presence of sea, seb, sec and mecA genes. The most prevalent enterotoxins genes were: sea (60%), seb (37.9%) and sec (6.9%). 18 milk samples (11 %) had S. aureus carrying the mecA gene. The detection of S. aureus directly from the milk, with no need for bacterial isolation and the characterization of the enterotoxigenic potential demonstrate that the PCR technique is very useful for epidemiological studies of staphylococcal infections of the mammary gland. The high percentage (72.5%) of positive milk samples for the presence of the femA gene suggests that S. aureus constitutes of the main agents which cause intramammary infections in the micro region of Sete Lagoas-MG and that its enterotoxigenic potential and the presence of the mecA gene, which identifies the S. aureus resistant to methicillin, represent a potential risk to public health.

PCR Multiplex para detecção dos principais herpesvírus neurológicos de ruminantes / Multiplex PCR for detection of Suid herpesvirus 1, Bovine herpesvirus 1, Bovine herpesvirus 5, Ovine herpesvirus 2

Desenvolveu-se uma PCR multiplex (mPCR) para diagnóstico diferencial de encefalite bovina causada por herpesvírus suíno 1 (SuHV-1), herpesvírus bovino 1 (BoHV-1), herpesvírus bovino 5 (BoHV-5) e herpesvírus ovino 2 (OvHV-2). Os iniciadores foram projetados após alinhamento de sequências disponíveis no banco de genomas (GenBank) e a reação foi padronizada levando-se em consideração a concentração dos reagentes e os tipos diferentes de DNA polimerase. Após determinação da especificidade e sensibilidade, 65 amostras de encéfalo de bovinos com síndrome neurológica foram submetidas à análise. A sensibilidade analítica para detecção de BoHV-1, BoHV-5 e SuHV-1 foi, respectivamente, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL na reação multiplex. Das 65 amostras analisadas, 10 foram positivas para BoHV-5, uma para BoHV-1 e cinco para OvHV-2. A mPCR descrita neste trabalho mostrou-se uma técnica útil para o diagnóstico diferencial de enfermidades relacionadas ao sistema nervoso central de bovinos.

The aim of this study was to develop a multiplex PCR (mPCR) for the differential diagnosis of bovine encephalitis caused by the suid herpesvirus 1 (SuHV-1), bovine herpesvirus 1 (BoHV-1), bovine herpesvirus 5 (BoHV-5) and ovine herpesvirus 2 (OvHV -2). The primers were designed after alignment of sequences available in GenBank and the reaction was developed by taking into account the concentration of reagents and different types of DNA polymerase. After determining the specificity and sensitivity to PCR, 65 brain samples from cattle with neurological syndrome were submitted to the reaction. The analytical sensitivity for detection of BoHV-1, BoHV-5 and SuHV-1 was, respectively, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL. Ten samples were positive for BoHV-5, one for BoHV-1, one for SuHV-1 and five for OvHV-2. The mPCR described here is a useful technique for the differential diagnosis of diseases related to the central nervous system of cattle.

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.