Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2017, V. 52, № 2, pp. 401-408
(how to cite this article)

doi: 10.15389/agrobiology.2017.2.401eng

UDC 619:579.843.95:616-078

Acknowledgements:
Supported in part by Russian Science Foundation (project № 14-16-00114)

 

DETECTION AND GENOTYPING Pasteurella multocida OF
FIVE CAPSULAR GROUPS IN REAL TIME POLYMERASE
CHAIN REACTION

A.V. Nefedchenko1, A.N. Shikov 2, A.G. Glotov1, T.I. Glotova1,
V.А. Ternovoy 2, R.A. Maksyutov1, 2, A.P. Agafonov 2,
A.N. Sergeev 2

1Siberian Federal Scientific Centre of Agro-BioTechnologies RAS, Federal Agency of Scientific Organizations, p/b 463, r.p. Krasnoobsk, Novosibirsk Region, Novosibirsk Province, 630501 Russia, e-mail nav-vet@mail.ru, glotov_vet@mail.ru, t-glotova@mail.ru (corresponding author);
2State Research Center of Virology and Biotechnology Vector, Koltsovo, Novosibirsk Region, Novosibirsk Province, 630559 Russia, e-mail shikov_nsk@mail.ru, tern@vector.nsc.ru, agafonov@vector.nsc.ru, sergeev@vector.nsc.ru, maksyutov_ra@vector.nsc.ru

The authors declare no conflict of interests

ORCID:

Nefedchenko A.V. orcid.org/0000-0002-4181-4268

Ternovoy V.А. orcid.org/0000-0003-1275-171X

Shikov A.N. orcid.org/0000-0002-7051-6091

Maksyutov R.A. orcid.org/0000-0003-1314-281X

Glotov A.G. orcid.org/0000-0002-2006-0196

Agafonov A.P. orcid.org/0000-0003-2577-0434

Glotova T.I. orcid.org/0000-0003-3538-8749

Sergeev A.N. orcid.org/0000-0001-7883-6017

Received May 30, 2016

 

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 1.6x10 to 5.9x102 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.

Keywords: bacteria, Pasteurella multocida, real-time PCR, genes, capsular groups.

 

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