Cross-sectional study of Brucella spp. using real-time PCR from bovine whole blood in Colombia.

A cross-sectional study was conducted in Colombia to recover Brucella spp. DNA from bovine whole-blood samples through probe-based real-time PCR (qPCR). By an SNP-based assay, vaccine strains were differentiated from field strains. The associated factors were evaluated using logistical regression models. A total of 656 random cows from 40 herds were selected and analyzed using serology and PCR. The qPCR assay detected 9.5% (n = 62/656; 95% CI: 7.3, 12.0) of the animals with Brucella-DNA presence, while the serological test detected a 6.6% (n = 43/656; CI: 4.8, 8.7). 62.5% (n = 25/40; 95% CI: 45.8, 77.3) of positive cases were detected at the herd-level by the qPCR, while only 27.5% (n = 11/40; 95% CI: 14.6, 43.9) were detected by the serological test. All positive samples were identified as field Brucella strains employing the SNP-based assay. In the final regression model at the animal-level, five variables were associated with Brucella-DNA presence: the use of bulls for mating recorded history of reproductive problems, pregnant cows, parlor milking, and cows belonging to farms ≤200 m from the main road. At the herd-level, two variables were associated with Brucella-DNA presence: recorded history of reproductive problems and the use of bulls for mating. Given the fluctuant brucellosis prevalence in endemic areas, updated epidemiological studies are necessary to evaluate the disease dynamic and if established prevention and control measures have been effective or need to be adjusted. The increase in the prevalence of brucellosis in animal reservoirs creates an important risk of transmission in humans.

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs / Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs' whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique's performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

RESUMO: Este estudo tem como objetivo descrever um novo método de detecção de uma reação em cadeia da polimerase quantitativa em tempo real (qPCR) visando o gene da proteína da membrana externa de 28 kDa (p28), bem como comparar este método com um PCR convencional (cPCR), que visa o mesmo gene, a fim de avaliar o desempenho da técnica desenhada neste estudo na detecção de Ehrlichia canis (E. canis). As concentrações ideais de oligonucleotídeos foram alcançadas e a sensibilidade analítica e a especificidade do qPCR foram determinadas. Um total de 218 amostras de sangue total de cães foram coletadas convencionalmente para este estudo. O DNA foi extraído de cada amostra. Posteriormente, as amostras foram testadas por um cPCR estabelecido e o novo qPCR para comparar os desempenhos entre cada técnica. A curva padrão revelou 100% de eficiência e coeficiente de determinação (R2) equivalente a 99,8%. Dentre as amostras examinadas por qPCR, 24,31% foram consideradas positivas, percentual significativamente maior do que as detectadas por cPCR (15,13%). A técnica qPCR atingiu uma sensibilidade maior do que a cPCR na detecção de E. canis. A qPCR padronizada neste estudo é um método eficiente para a confirmação do diagnóstico de erliquiose monocítica canina (EMC) e pode fornecer o monitoramento de níveis de parasitemia ao longo do tratamento da doença.