Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India

Abstract

The present study was planned to detect the genetic elements of Mannheimia haemolytica and Pasteurella multocida inpneumonic sheep lungs. Pneumonia was diagnosed on the basis of gross pathological lesions. Lung tissues were collected at necropsyof sheep (n = 96) and subjected to isolation of total DNA. The M. haemolytica-specific PHSSA and Rpt2 genes, and the P. multocidaspecific KMT1 and the Omp87 genes were amplified using polymerase chain reaction (PCR). A housekeeping gene targeting the sheepcellular mitochondrial 12S ribosomal DNA was used as an internal control. PCR reactions were optimized using the positive andnegative controls. Gene-specific PCR products were subjected to nucleotide sequencing for confirmation. The pneumonic lungs showedcongestion and hemorrhagic changes with consolidation, which was most evident in the whole of the apical lobes and parts of thediaphragmatic lobes. PCR amplification showed detection of PHSSA (327 bp) and Rpt2 (~1022 bp) genes specific to M. haemolytica in52 (54.1%), and the KMT1 (457 bp) and Omp87 (2627 bp) genes specific to P. multocida in 16 (16.6%) lung samples. Sequence analysisconfirmed the PCR products for specific genes. This study highlighted the culture-independent, rapid, and confirmatory diagnosis ofovine pneumonic pasteurellosis caused by M. haemolytica and/or P. multocida.