Mycoplasma ovipneumoniae in Wildlife Species beyond Subfamily Caprinae

Elucidating the emergence of Mycoplasma ovipneumoniae–associated respiratory disease in ruminants requires identification of the pathogen host range. This bacterium was thought to be host restricted to subfamily Caprinae, but we describe its identification in healthy moose, caribou, and mule deer and diseased mule and white-tailed deer, all species in subfamily Capreolinae.


Figure.
Direct examination with dark-field microscopy of specimens from a patient with agammaglobulinemia who had Spiroplasma apis infection, France. A) Helical and motile bacteria in blood culture. B) Elongated and coccoid bacteria in joint fluid. C) Helical and motile bacteria in culture from joint fluid in modified SP4 broth medium. Scale bar indicates 10 µm.

RESEARCH LETTERS
Elucidating the emergence of Mycoplasma ovipneumoniae-associated respiratory disease in ruminants requires identification of the pathogen host range. This bacterium was thought to be host restricted to subfamily Caprinae, but we describe its identification in healthy moose, caribou, and mule deer and diseased mule and white-tailed deer, all species in subfamily Capreolinae.

M ycoplasma ovipneumoniae was identified in
Queensland, Australia, in 1972 as an infectious agent associated with pneumonia in domestic sheep (Ovis aries) (1). Since then, it has most frequently been identified in healthy and diseased domestic sheep, domestic goats (Capra aegagrus hircus), and bighorn sheep (Ovis canadensis). Although M. ovipneumoniae was identified in respiratory disease outbreaks in bighorn sheep as early as 1980 (2), the past decade has brought it under scrutiny because of evidence supporting its association with bighorn sheep pneumonia in western North America (3). Because most reports have described this bacterium in sheep and goats, and fewer in muskoxen (Ovibos moschatus) (4), some have concluded that M. ovipneumoniae is specific to the subfamily Caprinae (5) or has a host range limited to Caprinae (6), despite publications describing M. ovipneumoniae in non-Caprinae species, including Beira antelope (Dorcatragus megalotis) with respiratory disease in Qatar (7) and in 9 cattle (Bos taurus) in Colorado, USA (8). Unfortunately, description of the method(s) used to identify M. ovipneumoniae in those reports was limited to stating the use of PCR with no supporting sequence data.
In general, definitive claims of host range restrictions are absent from mycoplasma literature, because "assumptions about restricted host range of mycoplasmas, based on the host from which they were first or frequently isolated, are usually made in the context of nearly complete absence of representative sampling of the vast majority of potential vertebrate hosts" (9). In addition to insufficient sampling of potential hosts, the fastidious and variably culturable nature of M. ovipneumoniae often requires molecular techniques for identification. We used molecular techniques to analyze multiple species from the subfamily Capreolinae for the presence of M. ovipneumoniae.
During July 2017-January 2018, the US Department of Agriculture Agricultural Research Service in Pullman, WA, USA, received nasal swab samples from 230 moose (Alces alces) and 243 caribou (Rangifer tarandus) from Alaska and 5 mule deer (Odocoileus hemionus) from Arizona (online Technical Appendix, https://wwwnc.cdc.gov/ EID/article/24/12/18-0632-Techapp1.pdf). Also received in February 2018 was an isolate of M. ovipneumoniae that had been cultured by Newport Laboratories (Worthington, MN, USA) from lung tissue from a white-tailed deer (Odocoileus virginianus) that died during a pneumonia outbreak at a captive facility in the upper Midwest region of the United States in 2016. We extracted DNA from swab samples and from the white-tailed deer isolate, performed PCR using a modified published PCR method (10) to amplify part of the 16S rRNA gene, and sequenced amplicons of the correct size (online Technical Appendix). Forward and reverse sequences were merged, manually inspected for errors, and trimmed to 290 bp using Sequencher 5.2.2 (Gene Codes, Ann Arbor, MI, USA) corresponding to a 103-392-bp region of the 16S rRNA gene of type strain Y98 obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Sequences were blastn queried (https://www. ncbi.nlm.nih.gov/BLAST/) and identified as M. ovipneumoniae at 100% coverage and ≥97% identity to Y98 (Gen-Bank accession no. NR_025989.1). The analyzed region represents the most divergent region of the 16S rRNA gene among strains of M. ovipneumoniae and between Mycoplasma spp. of the highest percentage identity to M. ovipneumoniae (online Technical Appendix).
We detected M. ovipneumoniae in 6 moose (2.6%), including 3 from 2 captive facilities and 3 free-ranging; 5 free-ranging caribou (2.1%); and 2 of 5 mule deer, 1 of which was coughing and had nasal discharge at the time of sample collection. The identity of the lung isolate, cultured from the white-tailed deer that had died from pneumonia, was confirmed as M. ovipneumoniae. For sequence comparison, we generated a percent identity matrix with the M. ovipneumoniae sequences from the Capreolinae species, nasal swabs collected from 2 healthy M. ovipneumoniaepositive wild sheep (online Technical Appendix), Y98, and bacteria of the closest identity to Y98 and sequences generated in this study based on blastn queries (M. dispar, M. hyopneumoniae, and M. flocculare) (online Technical Appendix). The percent identity matrix revealed 2 groupings of M. ovipneumoniae and illustrates the divergence from the other Mycoplasma spp. of closest identity to M. ovipneumoniae. Sample sequences have been submitted to GenBank (online Technical Appendix).
This report describes M. ovipneumoniae carriage in multiple members of the subfamily Capreolinae (moose, caribou, and mule deer), and emergence of M. ovipneumoniae-associated respiratory disease in deer. These findings are of importance to epidemiologic investigations because current dogma regarding host specificity may dissuade laboratories from pursuing identification of this fastidious bacterium in hosts beyond the subfamily Caprinae. Improved diagnostic methods to increase detection sensitivity are warranted based on information provided in this report (online Technical Appendix). Full-length genome sequencing and phylogenetic analysis of M. ovipneumoniae isolates are necessary next steps in inferring evolutionary relationships and origin of this bacterium in identified host species.