Detection of Mycoplasma ovipneumoniae and M. arginini in Bighorn Sheep Using Enrichment Culture Coupled with Genus- and Species-Specific Polymerase Chain Reaction

Abstract

ABSTRACT:Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO 2 -enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and speciesspecific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul TM tubes, cryoprotectant in liquid N 2 and Fisher Transport System) gave similar results under our study conditions.

PCR ASSAY DETECTSMANNHEIMIA HAEMOLYTICAIN CULTURE-NEGATIVE PNEUMONIC LUNG TISSUES OF BIGHORN SHEEP (OVIS CANADENSIS) FROM OUTBREAKS IN THE WESTERN USA, 2009–2010

“…data). Furthermore, Weiser et al (2012) found 50% of healthy bighorns tested were positive for M. ovipneumoniae in the nasopharynx. Their finding is supported by the detection of the presence and shedding of M. ovipneumoniae in healthy bighorns in a Colorado herd (L. Wolfe, Colorado Division of Wildlife, pers.…”

Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey

“…Mycoplasmas are identified by biochemical and serological testing (Erdağ and Türkaslan 1989; Stalheim 1985; Giangaspero et al 2012). Even though cultures are used for diagnosis in most cases, they are cumbersome and need a long time (Lin et al 2008; Weiser et al 2012). The most recent and rapid molecular methods to identify mycoplasmas are 16S rDNA PCR and denaturing gradient gel electrophoresis (McAuliffe et al 2003b).…”

Genetic diversity of Mycoplasma arginini isolates based on multilocus sequence typing

“…While no defined pathology has been associated with infection with M. arginini, reports from some experimental infections and diagnostic investigations have suggested that M. arginini may play a role in atypical pneumonia of ruminants (Foggie and Angus, 1972;Goltz et al, 1986;Nicholas et al, 2008;Weiser et al, 2012). However, the inability to establish its pathogenic role and its ubiquitous presence has led to the general assumption that M. arginini is a commensal of small ruminants.…”

“…In order to confirm the species identity of the isolates, a PCR targeting the 16S rRNA of Mycoplasma species was performed on all samples using the primers GPO3 (5' -GGG AGC AAA CAG GAT TAG ATA CCC T -3') and MGSO (5'-TGC ACC ATC TGT CAC TCT GTT AAC CTC -3') (van Kuppeveld et al, 1992). This was followed by a M. arginini-specific PCR using the primers MAGF (5'-GCA TGG AAT CGC ATG ATT CCT-3') and GP4R (5'-GGT GTT CTT CCT TAT ATC TAC GC-3') as described previously (Weiser et al, 2012). DNA sequencing of the 16S rRNA PCR product was performed on a selected set of isolates to confirm the specificity of the PCR assay for species identification.…”

“…As a consequence it is often considered to be a commensal of warm-blooded animals (Nicholas et al, 2008). Although several reports have suggested that some strains may influence the severity of respiratory disease in small ruminants, experimental and epidemiological studies in small ruminants are yet to establish their pathogenicity (Goltz et al, 1986;Stipkovits et al, 2013;Weiser et al, 2012). Molecular epidemiological typing methods for M. arginini would facilitate strain differentiation and thus help in resolving some of the uncertainty surrounding the clinical significance of this Mycoplasma species.…”