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 in pneumonic sheep lungs. Pneumonia was diagnosed on the basis of gross pathological lesions. Lung tissues were collected at necropsy of 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 sheep cellular mitochondrial 12S ribosomal DNA was used as an internal control. PCR reactions were optimized using the positive and negative controls. Gene-specific PCR products were subjected to nucleotide sequencing for confirmation. The pneumonic lungs showed congestion and hemorrhagic changes with consolidation, which was most evident in the whole of the apical lobes and parts of the diaphragmatic lobes. PCR amplification showed detection of PHSSA (327 bp) and Rpt2 (~ 1022 bp) genes specific to M. haemolytica in 52 (54.1%), and the KMT1 (457 bp) and Omp87 (2627 bp) genes specific to P. multocida in 16 (16.6%) lung samples. Sequence analysis confirmed the PCR products for specific genes. This study highlighted the culture-independent, rapid, and confirmatory diagnosis of ovine pneumonic pasteurellosis caused by M. haemolytica and/or P. multocida.