title: Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.
abstract: OBJECTIVE
  This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture.
  
  
  BACKGROUND
  M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture.
  
  
  METHODS
  A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons.
  
  
  RESULTS
  All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database.
  
  
  CONCLUSIONS
  The present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.
authors.affiliation: Division of Animal Health, Central Sheep and Wool Research Institute, Avikanagar - 304 501, Rajasthan, India.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Kumar
authors.affiliation: Division of Animal Health, Central Sheep and Wool Research Institute, Avikanagar - 304 501, Rajasthan, India.
authors.forename: Shivendra Kumar
authors.initials: SK
authors.lastname: Dixit
authors.affiliation: Animal Biotechnology Section, Central Sheep and Wool Research Institute, Avikanagar - 304 501, Rajasthan, India.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Kumar
keywords: 12S ribosomal RNA; Mannheimia haemolytica; Pasteurella haemolytica serotype-1 specific antigens; Rpt2; lung tissues; multiplex polymerase chain reaction; sheep