Detection and genotyping Pasteurella multocida of five capsular groups in real time polymerase chain reaction

Respiratory diseases in calves cause significant economic losses in livestock. Bacterium Pasteurella multocida plays important role in the etiology of these diseases. It is known that five identified P. multocida capsular groups (A, B, D, E and F) differently affect animal epizooty. Identification of bacteria based on the cultural, morphological, biochemical properties is very labor-intensive and time-consuming. Molecular biology techniques, in particular, the polymerase chain reaction (PCR), quickly detect and identify microorganisms directly in samples of biological material, mixed or pure cultures. In this regard, the purpose of our research was to develop multiplex real-time PCR for the detection, genotyping and discrimination of five P. multocida capsular groups (A, B, D, E and F) in cattle. The target primers and probes to the highly conserved gene kmt1 and the genes in the loci of capsule synthesis ( hyaD, fcbD, dcbF, bcbD and ecbJ ) specific to the capsular groups have been designed. The sensitivity of DNA detection for different bacterial groups ranged from 1.6·10 to 5.9·102 genomic equivalents per reaction, non-specific reactions were not observed. The diagnostic sensitivity of the test was 103 CFU/ml for pure cultures and 105 CFU/g for biological material. The developed PCR protocol allowed us to type 11 bacterial cultures which were previously characterized serologically and bacteriologically and related to capsular groups A, B, and D. The kmt1 gene sequencing confirmed the results of PCR analysis. PCR analysis of 260 samples from died calves detected P. multocida in lung (63.3 %), the lymph nodes (42.6 %), and spleen (8.8 %). We did not revealed the circulation of P. multocida В and E capsular groups among the tested livestock, the majority of the samples contained P. multocida group A, in some cases, there was group D, and, in one case, group F.