Detection and quantitative assessment of viral and bacterial pathogens in bovine respiratory diseases by real-time-QPCR

Bovine respiratory diseases are widespread in all countries with intensive animal husbandry and cause significant economic damage. They are the result of a synergistic interaction of several viruses and bacteria, predominantly of the Pasteurellaceae family. Clinical signs and pathological changes in internal organs depend on the presence or absence of a particular pathogen. Mass outbreaks occur when animals from different sources are mixed. The etiological structure of such outbreaks has been sufficiently studied, however, there is insufficient data on the distribution of bacteria and viruses in the respiratory tract and their quantitative determination. The article presents the results of studying the etiological structure of the outbreak of respiratory diseases in the big dairy farm after the import of cattle, during which more than 400 animals of different age and sex groups died. Samples of internal organs of 58 dead animals of different ages were examined. When studying the etiological structure of the outbreak, standard bacteriological methods were used, viral agents were identified by PCR by gel electrophoresis, and real-time PCR was used to quantify all detected infectious agents. In total, 9 viruses and bacteria were identified, of which the respiratory syncytial virus of cattle (BRSV, Bovine Respiratory Syncytial Virus, genus Pneumovirus , family Paramyxoviridae ) and bacteria of the Pasteurellaceae family played a leading etiological role. Using quantitative PCR, the concentrations of the virus and bacteria Pasteurella multocida and Mannheimia haemolytica were determined in the respiratory tract organs of 13 calves of different ages with similar clinical signs, pathological changes and the presence of three pathogens in the respiratory tract organs. The concentration of agents ranged from 0.1±0.03 to 4.8±0.47 log10 genomic equivalents (GE)/ml for BRSV, from 1.3±0.60 to 4.1±0.30 log10 GE/ml for P. multocida , and from 1.9±0.03 to 4.9±0.67 log10 GE/ml for M. haemolytica . The concentration and distribution of pathogens in the organs of calves of different ages differed. BRSV was detected in a wider range of respiratory organs, both free from bacteria and colonized by them. In the lungs, the concentration of the virus was higher than in the tracheal and bronchial exudate. P. multocida was present only in the upper and middle lobes of the lungs of 2.5-4-month-old calves at approximately equal concentrations in acute bronchopneumonia. The degree of colonization of the lungs by this bacterium increased with age and in calves at the age of 6 months its number reached maximum values in the upper and middle lobes of the lungs, pulmonary lymph nodes and washes from the mucous membranes in chronic bronchopneumonia. M. haemolytica was detected in acute bronchopneumonia in calves at the age of 2.5 months in a minimum amount in the middle lobes of the lungs, in a maximum amount in tracheal and bronchial exudates. The results showed that the virus and bacteria multiply in different parts of the lungs without suppressing each other, which confirms the effect of their synergistic interaction and leads to an increase in the severity of the course of pneumonia. Quantification of viruses and bacteria by real-time PCR can be a useful tool for studying the pathogenesis of mixed viral-bacterial infections in vivo. The results obtained underline the role of the BRSV in the development of pulmonary pasteurellosis.