No abstract

A novel respiratory-associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well-known respiratory pathogen in small ruminants. This necessitates our objective to develop a real-time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR-seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided DS (n = 153) and DG (n = 194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n = 117) or M. sp. nov. (n = 138), or negative for both targets (n = 92) by cPCR-seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR-seq, qPCR agreement was 88.0% (103/117; κ = 0.81) and 89.9% (124/138; κ = 0.84) for M. ovipneumoniae and M.sp. nov., respectively; 12 of 255 (4.7%) cPCR-seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identityThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Mycoplasma ovipneumoniae is a respiratory pathogen that can impact domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). Experimental and field data have indicated BHS are more susceptible than DS to developing polymicrobial pneumonia associated with Mycoplasma ovipneumoniae infection. We hypothesized that DS and BHS have a differential immunopathologic pulmonary response to M. ovipneumoniae infection. A retrospective study was performed using formalin-fixed, paraffin-embedded (FFPE) lung tissue from DS and BHS without and with M. ovipneumoniae detected in the lung tissue (n=8 per group). While each M. ovipneumoniae positive lung sample had microscopic changes typical of infection, including hyperplasia of intrapulmonary bronchus-associated lymphoid tissue (BALT) and respiratory epithelium, DS exhibited a more robust and well-organized BALT formation as compared to BHS. Immunohistochemistry was performed with antibodies reactive in FFPE tissues and specific for leukocyte and cytokine markers: T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. Digital analysis was used to quantitate chromogen deposition in regions of interest (ROIs), including alveolar and bronchiolar areas, and bronchiolar subregions (epithelium and BALT). Main effects and interaction of species and infection status were analyzed by beta regression and Bonferroni corrections were performed on pairwise comparisons (PBon<0.05 significance). Significant species differences were identified for bronchiolar CD3 (PBon=0.0023) and CD163 (PBon=0.0224), alveolar CD163 (PBon=0.0057), and for IL-17 in each of the ROIs (alveolar: PBon=0.0009; BALT: PBon=0.0083; epithelium: PBon=0.0007). Infected BHS had a higher abundance of bronchiolar CD3 (PBon=0.0005) and CD163 (PBon=0.0162), and alveolar CD163 (PBon=0.0073). While IL-17 significantly increased with infection in BHS BALT (PBon=0.0179) and alveolar (0.0006) ROIs, abundance in DS showed an insignificant decrease in these ROIs and a significant decrease in epithelial abundance (PBon=0.0019). These findings support the hypothesis that DS and BHS have a differential immunopathologic response to M. ovipneumoniae infection.