Detection of pseudocowpox virus in water buffalo (Bubalus bubalis) with vesicular disease in the state of São Paulo, Brazil, in 2016

Background: Parapoxviruses are zoonotic viruses that infect cattle, goats and sheep; there have also been reports of infections in camels, domestic cats and seals. Objective: The objective of this report was to describe a case of vesicular disease caused by pseudocowpox virus (PCPV) in water buffalo (Bubalus bubalis) in Brazil. Animals: Sixty buffalo less than 6 months old exhibited ulcers and widespread peeling of the tongue epithelium. There were no cases of vesicular disease in pigs or horses on the same property. Methods: Samples were analysed by PCR and sequencing. Phylogenetic analysis in MEGA 7.01 was reconstructed using major envelope protein (B2L) by the Tamura three-parameter nucleotide substitution model and the maximum likelihood and neighbor joining models, both with 1000 bootstrap replicates. The genetic distance between the groups was analysed in MEGA using the maximum composite likelihood model. The rate variation among sites was modeled using gamma distribution. Results: The presence of PCPV in the buffalo herd could be demonstrated in epithelium and serum. The minimum genetic distance between the isolated PCPV strain (262-2016) and orf virus and bovine papular stomatitis virus was 6.7% and 18.4%, respectively. The maximum genetic distance calculated was 4.6% when compared with a PCPV detected in a camel. Conclusions/Clinical Importance: The peculiar position of the isolated strain in the phylogenetic trees does not necessarily indicate a different kind of PCPV that infects buffalo. More samples from cattle and buffalo in Brazil must be sequenced and compared to verify if PCPV from buffalo are genetically different from samples derived from cattle

Estudo comparativo e validação de três técnicas de PCR em tempo real (qPCR) para diagnóstico de Peste Suína Africana

Resumo: Este estudo verificou o desempenho de três técnicas de PCR quantitativa (Real-Time) para o diagnóstico de Peste Suína Africana, uma doença exótica no Brasil, a partir de amostras de tecidos. As três técnicas escolhidas baseiam-se na amplificação de sequências do gene da proteína viral VP72 e são preconizadas, cada uma, por laboratórios oficiais da OIE (PSA-OIE), dos Estados Unidos (PSA-USDA) e da União Europeia (PSA-EU), respectivamente. Oligonucleotídeos iniciadores e sondas de hidrólise marcadas com fluoróforos foram sintetizados conforme a literatura de referência consultada. Sequências-alvo do DNA viral foram inseridos em plasmídeo sintético, os quais serviram de controle positivo para a padronização das técnicas e otimização de reagentes, determinação dos limites de detecção e testes de verificação de desempenho. Para aferição de repetibilidade e reprodutibilidade das técnicas, as técnicas padronizadas foram repetidas em dias diferentes, por um segundo analista, com alteração no mix comercial de reagentes utilizado e em um equipamento diferente, e também por outro laboratório. Realizaram-se, ainda, provas de sensibilidade analítica com amostras de DNA viral de referência e especificidade analítica e diagnóstica, com amostras negativas. As técnicas de PSA-EU e PSA-USDA apresentaram-se mais vantajosas quanto ao consumo de iniciadores. Não houve diferenças significativas nos resultados quantitativos variando-se os dias dos ensaios, os analistas, os equipamentos e o mix de reagentes. As três técnicas apresentaram alta especificidade analítica e diagnóstica e sensibilidade diagnóstica. As três técnicas de qPCR mostraram-se eficazes para serem adotadas por um mesmo laboratório para emissão de diagnósticos oficiais de Peste Suína Africana

Vaginal Microbiome Characterization of Nellore Cattle Using Metagenomic Analysis

<div><p>Understanding of microbial communities inhabiting cattle vaginal tract may lead to a better comprehension of bovine physiology and reproductive health being of great economic interest. Up to date, studies involving cattle microbiota are focused on the gastrointestinal tract, and little is known about the vaginal microbiota. This study aimed to investigate the vaginal microbiome in Nellore cattle, heifers and cows, pregnant and non-pregnant, using a culture independent approach. The main bacterial phyla found were <i>Firmicutes</i> (~40–50%), <i>Bacteroidetes</i> (~15–25%) and <i>Proteobacteria</i> (~5–25%), in addition to ~10–20% of non-classified bacteria. 45–55% of the samples were represented by only ten OTUs: <i>Aeribacillus</i>, <i>Bacteroides</i>, <i>Clostridium</i>, <i>Ruminococcus</i>, <i>Rikenella</i>, <i>Alistipes</i>, <i>Bacillus</i>, <i>Eubacterium</i>, <i>Prevotella</i> and non-classified bacteria. Interestingly, microbiota from all 20 animals could be grouped according to the respiratory metabolism of the main OTUs found, creating three groups of vaginal microbiota in cattle. Archaeal samples were dominated by the <i>Methanobrevibacter</i> genus (<i>Euryarchaeota</i>, ~55–70%). <i>Ascomycota</i> was the main fungal phylum (~80–95%) and <i>Mycosphaerella</i> the most abundant genus (~70–85%). Hormonal influence was not clear, but a tendency for the reduction of bacterial and increase of archaeal populations in pregnant animals was observed. Eukaryotes did not vary significantly between pregnant and non-pregnant animals, but tended to be more abundant on cows than on heifers. The present work describes a great microbial variability in the vaginal community among the evaluated animals and groups (heifers and cows, pregnant and non-pregnant), which is significantly different from the findings previously reported using culture dependent methods, pointing out the need for further studies on this issue. The microbiome found also indicates that the vaginal colonization appears to be influenced by the gastrointestinal community.</p></div

Major eukaryotic OTUs at the genus-level in each group.

<p>Average relative abundance is presented. (<i>a-d</i>) Comparison within each group. (e) Comparison between groups. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).</p

Universal primers used in the PCR reaction for <i>Bacteria</i>, <i>Archaea</i> and fungi.

<p>Universal primers used in the PCR reaction for <i>Bacteria</i>, <i>Archaea</i> and fungi.</p

Average alpha diversity in the analyzed groups, expressed as antilog of Shannon’s index.

<p>NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).</p

Bacterial phylogenetic tree, including all bacterial families encountered in the samples.

<p>The seven major families, comprising nine of the ten most abundant OTUs at the genus level are indicated by the red dots. Red circles indicate three important orders, namely <i>Clostridiales</i> (a), <i>Bacillales</i> (b) and <i>Bacteroidales</i> (c).</p

Major eukaryotic phyla found in the vaginal tract.

<p>Relative abundance comparisons within each group are shown in graphics <i>a</i> to <i>d</i>, and phyla comparison between groups are shown in graphic <i>e</i>. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).</p

Major archaeal OTUs at the genus-level in each group.

<p>Average relative abundance is presented. (<i>a-d</i>) Comparison within each group. (e) Comparison between groups. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).</p

Bacterial phyla found in the vaginal tract.

<p>Relative abundance comparisons within each group are shown in graphics <i>a</i> to <i>d</i>, and phyla comparison between groups are shown in graphic <i>e</i>. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).</p