title: PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010.
abstract: Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.
authors.affiliation: 1  Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040, USA.
authors.forename: Sudarvili
authors.initials: S
authors.lastname: Shanthalingam
authors.forename: Andrea
authors.initials: A
authors.lastname: Goldy
authors.forename: Jegarubee
authors.initials: J
authors.lastname: Bavananthasivam
authors.forename: Renuka
authors.initials: R
authors.lastname: Subramaniam
authors.forename: Sai Arun
authors.initials: SA
authors.lastname: Batra
authors.forename: Abirami
authors.initials: A
authors.lastname: Kugadas
authors.forename: Bindu
authors.initials: B
authors.lastname: Raghavan
authors.forename: Rohana P
authors.initials: RP
authors.lastname: Dassanayake
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Jennings-Gaines
authors.forename: Halcyon J
authors.initials: HJ
authors.lastname: Killion
authors.forename: William H
authors.initials: WH
authors.lastname: Edwards
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Ramsey
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Anderson
authors.forename: Peregrine L
authors.initials: PL
authors.lastname: Wolff
authors.forename: Kristin
authors.initials: K
authors.lastname: Mansfield
authors.forename: Darren
authors.initials: D
authors.lastname: Bruning
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Srikumaran
mesh_terms: D000818:Animals; D004196:Disease Outbreaks / Q000662:veterinary; D016978:Mannheimia haemolytica / Q000302:isolation & purification*; D012766:Pasteurellosis, Pneumonic / Q000175:diagnosis* / Q000453:epidemiology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000453:epidemiology; D034581:Sheep, Bighorn / Q000382:microbiology*; D013045:Species Specificity; D014481:United States / Q000453:epidemiology