Detection of Mannheimia Haemolytica in culture and lung tissue of lambs by real-time polymerase chain reaction assay

Abstract

Mannheimia haemolytica is one of the bacterial species involved in cases of ovine respiratory complex, causing significant economic loss to sheep production worldwide. In the year 2016–17, a study was undertaken with the aim to evaluate a SYBR Green dye based real-time polymerase chain reaction (PCR) assay targeting O-sialo glycoprotein endopeptidase (gcp) gene for the detection of M. haemolytica in the DNA isolated from culture and pneumonic lung tissue of neonatal lambs. The test showed thousand times more sensitivity than conventional PCR and detected down to 100 fg of genomic DNA of pure M. haemolytica. The real-time PCR was found specific for gcp gene of M. haemolytica, as no cross reactivity was detected with a variety of known bacterial isolates characterized previously by species specific or 16S rRNA PCR. The real-time PCR was employed to screen M. haemolytica in 41 ovine lung tissues collected from neonatal lambs, which showed increased level of detection as compared to the conventional PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products of 227 bp size that showed 99–100% homology with the published sequences available in the NCBI database. It was concluded that the assay may be used as a valuable diagnostic tool for the rapid and specific detection of M. haemolytica in clinical samples.