Comparison of three methods of enumeration for Mycoplasma ovipneumoniae

Authors: ACKERMAN, MELISSA - Washington State University; Schneider, David; BAKER, KATHERINE - Washington State University; BESSER, THOMAS - Washington State University

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2019
Publication Date: 10/1/2019
Citation: Ackerman, M., Schneider, D.A., Baker, K.N., Besser, T.E. 2019. Comparison of three methods of enumeration for Mycoplasma ovipneumoniae. Journal of Microbiological Methods. 165. https://doi.org/10.1016/j.mimet.2019.105700.
DOI: https://doi.org/10.1016/j.mimet.2019.105700

Interpretive Summary: Mycoplasma ovipneumoniae is a respiratory pathogen that affects bighorn sheep as well as domestic sheep. Recurring outbreaks of pneumonia in bighorn lambs can be particularly devastating and limit population growth. Unique characteristics of Mycoplasma ovipneumoniae make it difficult to quantify by standard methods, which impedes accurate study of its prevalence and virulence as well as the susceptibility of various host species. To improve methodological application and interpretation, this study determined the relative accuracy of three assay methods used to enumerate M. ovipneumoniae growth and provides a comparison of the advantages and limitations of each assay method.

Technical Abstract: Mycoplasma ovipneumoniae is a respiratory pathogen that contributes to pneumonia in domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis). In bighorn lambs, recurrent pneumonia epizootics caused by M. ovipneumoniae can limit population growth. Therefore, studies that explore M. ovipneumoniae virulence, infectivity potential, and prevalence are of interest and accurate quantitation is necessary. Methods relying on growth and viability like colony forming units (CFU) and color changing units (CCU) assays are commonly utilized for bacterial enumeration, but are challenging with M. ovipneumoniae due its small size and prolonged growth time. Thus, the objective of this study was to compare M. ovipneumoniae growth over time using three known methods of enumeration: quantitative PCR (qPCR), flow cytometry (FC), and color changing units 50% endpoint (CCU50) assay, to evaluate the relative accuracy, advantages, and limitations of each method. Both FC and qPCR were strongly correlated and linear during log phase growth compared with CCU50, however, FC consistently underestimated concentration. Both FC and qPCR provided rapid enumeration (<2 hours and <2 days, respectively) compared to CCU50 (<3 weeks). However, qPCR was the only method specific for M. ovipneumoniae detection, so in mixed cultures it would be the method of choice. The wide dynamic ranges of CCU50 and qPCR tests make either suitable for studies necessitating quantification of standardized inoculums or exploring minimum infective dose. However, studies necessitating information about cell viability are limited to the CCU50 method.