RESUMO
The molecular typing of Mycobacterium bovis, which causes bovine tuberculosis, can be accomplished by combining different polymorphic markers, contributing to its epidemiological investigation. Multispacer sequence typing (MST) is a sequencing-based method that employs intergenic regions susceptible to higher mutation rates given the low selection pressure. It has been applied to M. tuberculosis, but not to M. bovis. The aim of this study was to evaluate a MST for M. bovis. A total of 58 strains isolated from tissues with lesions suggestive of bovine tuberculosis, coming from cattle herds in six Brazilian states and four standard samples of M. bovis were typified employing the MST technique. Fourteen intergenic regions were used, and four types of genetic events were reported: single nucleotide mutation (SNP), insertion, deletion, and tandem repeat (TR). Seven loci were chosen for typing. Twenty-eight type sequences (ST) were identified, indicating type sequences (ST) were identified, indicating a 92.9% HGDI (Hunter Gaston Discriminatory Index). The data were used to analyze the evolutionary patterns of these isolates and correlate them to phylogeographic lineages based on the formation of clonal complexes generated from eBURST software. Later, we associated the MST with spoligotyping technique, currently considered the gold standard for classification of M. bovis. The results support the MST as an alternative method for genotyping of M. bovis. The method has the advantage of sequencing and the availability of sequences analyzed in public databases, which can be used by professionals around the world as a tool for further analysis. This was the first study to identify the variability of isolates of M. bovis by the MST method.
RESUMO
The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.
RESUMO
O objetivo deste trabalho foi realizar a validação de uma reação em cadeia da polimerase em tempo real com o sistema Plexor® (qPCR) para o diagnóstico da Leucose Enzoótica Bovina (LEB), por meio da comparação com testes de diagnóstico recomendados pela Organização Mundial de Saúde Animal (OIE). A qPCR foi comparada com duas outras técnicas: a PCR nested (nPCR) e a imunodifusão em gel de ágar (IDGA). Das 82 amostras analisadas pela qPCR e nPCR, 79 apresentaram resultados concordantes, sendo a concordância, classificada pelo Índice Kappa, como alta. Entre as PCRs e a IDGA, o número de resultados concordantes foi de 71 e 69, respectivamente, para qPCR e nPCR, sendo a concordância classificada como considerável. A qPCR apresentou altos valores de sensibilidade e especificidade. Os valores preditivos da qPCR observados demonstraram a alta capacidade de classificação dos casos positivos e negativos. A qPCR não foi capaz de detectar três amostras positivas e tem custo ligeiramente superior que a nPCR. Entretanto, a qPCR é uma técnica mais rápida, menos susceptível a contaminações, tem alta sensibilidade, não utiliza e não gera resíduos carcinogênicos. Concluímos que a qPCR pode substituir a nPCR recomendada pela OIE no diagnóstico de rotina em áreas em que a LEB é endêmica, como no Brasil.
The goal of this research was to validate a Plexor® real time Polymerase Chain Reaction (qPCR) for Enzootic Bovine Leukosis (EBL) diagnosis by comparison with methods recommend by the World Animal Health Organization (OIE). The qPCR was compared with two other techniques: the nested PCR (nPCR) and to the agar gel immunodiffusion (AGID). Of 82 qPCR and nPCR analysed samples, 79 presented concordant results, being the concordance classified by Kappa Index as high. Between the PCRs and AGID, the number of concordant results was 71 and 69, out of 82, to qPCR and nPCR, respectively, being the concordance classified as considerable, in both cases. The qPCR presented high specificity and sensitivity values. The observed qPCR negative and positive predictive values show that the qPCR has a high capacity to correctly classify positive and negative results. The qPCR was not able to detect three positive animals and has a lightly higher cost than the nPCR. However, the qPCR its faster, less prone to contamination, has a high sensitivity and does not use or generate carcinogenic residues. We conclude that the qPCR may be used to replace the OIE nPCR technique in routine diagnosis in areas where EBL is endemic, as is the case of Brazil.
