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Burkholderia mallei is the causative agent of glanders, a zoonosis listed by the World Organization for Animal Health as of mandatory notification. In this work, a comparison of three qPCR protocols was made, two of them based on articles by other authors and one standardized in house, this last one aiming at a genomic region that does not exist in other species of the Burkholderia genus. All qPCRs showed high efficiency and good repeatability. However, reactions with Cq between 36 and 40 were considered suspicious and unreliable, requiring greater clinical criteria to analyze the results.

Bovine leukemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus . BLV infects cattle worldwide and is responsible for significant economic losses. The objective of this study was to validate real-time quantitative PCR (qPCR) for the detection of BLV. After identification of the most efficient qPCR, the limits of detection, repeatability, and reproducibility were determined. The results indicate that qPCR can be easily reproduced between laboratories with high sensitivity. The test variation was low in samples from lesions suggestive of bovine leukosis or whole blood.

The vesicular stomatitis virus belongs to the Rhabdoviridae family, genus Vesiculovirus . Four species (New Jersey, Indiana, Cocal, and Alagoas) are responsible for disease outbreaks in Western Hemisphere countries. In Brazil, the Alagoas virus is responsible for the main outbreaks of the disease, mainly in the states of the Northeast, Midwest, and Southeast regions of the country. The present study aimed to perform the genetic characterization of 41 vesicular stomatitis virus samples. RNA was extracted using Trizol and used to amplify part of gene P. Amplicons were sequenced using the Sanger method. The phylogenetic trees generated showed that Alagoas vesiculoviruses were positioned into three groups: group A formed by the first virus isolate; group B by isolates from states in the Northeast region; and group C by isolates from the states of Bahia, Goiás, and Tocantins. Their divergence to date has generated the formation of two genotypes evolving independently in regions that until the present study had little geographic overlap.

The classical swine fever virus is the etiologic agent of one of the diseases with the greatest impact on swine farming worldwide. An extensive area of Brazil is considered free of the disease, but some states in Northeast Brazil have registered outbreaks since 2001. The objective of this study was to analyze the genetic variations of the virus and its spread over time and space. Partial sequences of the viral E2 protein obtained from samples collected during the Brazilian outbreaks were compared with sequences from the GenBank database (NCBI). The results demonstrated the continuous presence of the virus in the state of Ceará, with diffusion to at least two other states. The Brazilian Northeast virus presents specific polymorphisms that separate it from viruses isolated in other countries.

The pseudocowpox virus (PCPV) is recognized for causing exanthematic lesions in cattle and humans. The diagnosis is important because it is a zoonosis and its clinical signs can be confused with foot-and-mouth disease, a high-impact bovine disease in livestock. The objective of this work is to validate a SYBR Green qPCR and a conventional PCR for virus detection in bovine samples. Detection limit tests, repeatability, reproducibility, sensitivity, and specificity were compared. When two analysts were compared, results demonstrated that training and pipetting influence the repeatability. The qPCR was more sensitive than conventional PCR but showed nonspecific reactions distinguishable by the melting curve. Both showed high repeatability and reproducibility.

Bluetongue virus (BTV) is an RNA virus that infects cattle and sheep. The objective of this study was to compare two real-time PCRs for the detection of BTV and to monitor Orbivirus viremia in sheep and cattle for 6 months. The PCR results showed the occurrence of infected animals throughout the experiment without records of clinical signs. The number of positive animals reduced during the experiment, but some animals were positive for BTV RNA during the entire experiment. The performance of the two RT-qPCRs for BTV detection techniques used in this work revealed a kappa index of 0.71 for cattle and 0.75 for sheep.

This article describes the recurrence of outbreaks of Vesicular Stomatitis in the State of Maranhão, Brazil. The procedures for treating the outbreak of vesicular disease, sample collection, laboratory tests performed, and the results obtained were described. The clinical signs and observed injuries have been described. The sera showed antibodies that cross-react between the Vesiculovirus Indiana, Cocal, and Alagoas. The serological profile shows the presence of high antibody titers for Alagoas vesiculovirus in cattle, swine, and horses. Higher antibody titers indicate the viral serotype present in the outbreak. The genetic sequencing of the isolates confirmed the presence of Alagoas vesiculovirus, which grouped with the virus isolated in 2013 from cattle from the State of Maranhão.

For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants – 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media – 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.

Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country. RESUMO: O objetivo do presente estudo foi caracterizar (fenotipicamente e genotipicamente) duas cepas de Brucella abortus identificadas como pertencentes ao biovar 4 isolada de bovinos no Brasil. As cepas foram isoladas de bursite cervical de bovinos dos estados do Pará e Rio Grande do Sul, respectivamente. Na identificação fenotípica, os isolados foram positivos na exigência de CO2, produziram H2S, foram resistentes à fucsina básica (20 µg / mL) e sensíveis à tionina (20 µg / mL e 40 µg / mL) e apresentaram antígeno de superfície M, mas o antígeno de superfície A foi ausente. Os isolados foram positivos na PCR para o gene bcsp31 (gênero específico) e na PCR - AMOS, ambos os isolados apresentaram perfil de banda consistente com B. abortus biovar 1, 2 ou 4. Além disso, ambos os isolados também apresentaram padrões de restrição idêntica à cepa de referência quando testada pelo omp2b PCR-RFLP. Na genotipagem usando Multiple Locus Variable Number of Tandem Repeat (VNTR) - MLVA (MLVA16), os isolados apresentaram diferenças em vários loci (Bruce42, Bruce19, Bruce04, Bruce16 e Bruce30); no Multiple Locus Sequence Typing (MLST), os isolados também exibiram diferenças na sequência tipo (ST), amostra 16/02 ST1 (2-1-1-2-1-3-1-1-1) e amostra 128/11 ST (22-1-1-8-9-3-1-1-1). A extensa tipagem de cepas de B. abortus isoladas de bovinos no Brasil por diferentes abordagens confirmou a rara ocorrência de B. abortus biovar 4 no país.