Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats

Authors: NOLL, LANCE - Kansas State University; HIGHLAND, MARAGARET - Kansas State University; HAMILL, VAUGHN - Kansas State University; TSUI, WAI NING - Kansas State University; PORTER, ELIZABETH - Kansas State University; LU, NANYAN - Kansas State University; SEBHATU, TESFAALEM - Kansas State University; BROWN, SUSAN - Kansas State University; Herndon, David; GROSSMAN, PAIGE - Washington State University; BAI, JIANFA - Kansas State University

Submitted to: Transboundary and Emerging Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2022
Publication Date: 2/15/2022
Citation: Noll, L.W., Highland, M.A., Hamill, V.A., Tsui, W., Porter, E.P., Lu, N., Sebhatu, T., Brown, S., Herndon, D.R., Grossman, P.C., Bai, J. 2022. Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14477.
DOI: https://doi.org/10.1111/tbed.14477

Interpretive Summary: Mycoplasma ovipneumoniae is a globally recognized pathogen of sheep and goats and has been detected in a variety of non-domestic animals including bighorn sheep, Dall sheep, mountain goats, moose, Beira antelope, caribou, mule deer, white-tailed deer, and muskoxen. A novel respiratory-associated Mycoplasma species (M. sp. nov.)was recently identified that can cause false positive results with published PCR methods when screening for M. ovipneumonaie. We developed a real-time qPCR assay capable of detecting and differentiating both M. ovipneumoniae and the novel Mycoplasma, and tested the assay against samples previously determined to be positive for either bacterium. Inter-assay agreement was high for the detection of both species, 88% for M. ovipneumoniae and 89.9% for M. sp. nov. The assay was able to detect infection with both species in 4.7% of positive samples. The use of this new assay is expected to reduce the rate of false positive test results compared to other published assays.

Technical Abstract: Mycoplasma ovipneumoniae is a respiratory pathogen in small ruminants. Infection can result in subclinical to severe disease. A novel respiratory-associated Mycoplasma species (M. sp. nov.), of unknown clinical significance, was recently identified that can cause false positive results with published PCR methods for detection of M. ovipneumonaie. Our objective was to develop a real-time PCR (qPCR) assay for detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, with improved sensitivity and specificity compared to a conventional PCR assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA sequences in the GenBank database, and partial 16S rRNA sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided nucleic acid from DS (n=153) and DG (n=194) nasal swabs previously tested by partial 16S rRNA conventional PCR (cPCR) followed by sequencing, and described as positive for either M. ovipneumoniae (n=117) or M. sp. nov. (n=138), or negative for both targets (n=92); no co-positives were reported. A host 18S rRNA gene was included in this new assay to serve as an internal control for nucleic acid extraction efficiencies and possible PCR inhibition. Samples with discrepant results were analyzed by sequenced (Figure 2). For samples positive by cPCR, qPCR was in agreement for 88.0% (103/117; ' = 0.81) and 89.9% (124/138; '=0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12/255 (4.7%) cPCR positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and Mycoplasma sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. All samples that were successfully sequenced had identity matches that confirmed the qPCR result. The increased target specificity is predicted to reduce the rate of false positive test results compared to other published assays.