In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

The objective of this work is to describe the distribution of outbreaks of vaccinia virus (VACV), pseudocowpox virus (PCPV), and bovine papular stomatitis virus (BSPV) in Brazil. The Official Laboratory of the Brazilian Ministry of Agriculture received 89 samples from different locations in Brazil in 2015 and 2016 for diagnosis of vesicular and exanthematous disease. Poxvirus coinfections occurred in 11 out of 33 outbreaks, including the first reported triple infection by BPSV, PCPV, and VACV. This occurrence may be associated with the circulation of these viruses in Brazilian cattle.

No abstract

Abstract. Vesicular diseases are of high importance for livestock, primarily because of foot-and-mouth disease (FMD), which is a high-morbidity disease that generates direct losses caused by low milk production, weight loss, and indirect losses because of the need for sanitary barriers. Other vesicular diseases are also of importance for livestock because of direct impacts or because their clinical signs may be confused with those of FMD. We report herein the detection of multiple infections in cattle with suspected vesicular disease in the Brazilian states of Amazonas (AM), Mato Grosso (MT), and Roraima. Thirtyseven epithelial samples from cattle and 1 sample from a buffalo were sent to the laboratory for testing for FMDV and similar disease agents. All samples from MT were positive for parapoxvirus (Pseudocowpox virus and Bovine papular stomatitis virus). In addition, 3 samples were positive for Bluetongue virus, and 5 samples were positive for Bovine herpesvirus 1. Among these samples, 1 was positive for all of these 3 agents. Only 2 samples from AM were negative for parapoxvirus. The molecular tests conducted in this study detected multiple infections, with a high prevalence of parapoxvirus.

Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents

A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing

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.