Assessing shared respiratory pathogens between domestic (Ovis aries) and bighorn (Ovis canadensis) sheep; methods for multiplex PCR, amplicon sequencing, and bioinformatics to characterize respiratory flora.

Respiratory disease is responsible for dramatic population declines in bighorn sheep (Ovis canadensis), and respiratory pathogen diagnostics contribute to the management of bighorn populations. To create a comprehensive and consistent approach to bighorn sheep respiratory diagnostics, we created a culture-independent assay to detect and strain type Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae. The assay also detects and characterizes the Pasteurellaceae leukotoxin A gene, and broadly assesses the bacterial composition of each sample based on 16S rRNA sequences. The assay is based on a three-step approach: 1) Multiplex PCR to amplify targets including eight loci for each bacterial species, the Pasteurellaceae lktA gene, and the 16S rRNA gene 2) Library preparation, barcoding, and short-read Illumina sequencing to determine the genetic sequences of each target, and 3) Bioinformatics in the form of automated software to analyze genetic sequences. The assay was designed to assess shared pathogens between domestic and bighorn sheep, but could be useful for many applications in bighorn sheep respiratory disease research and management.

NOVEL AVIBACTERIUM SPECIES ASSOCIATED WITH SINUSITIS AND CONJUNCTIVITIS IN A MERRIAM'S WILD TURKEY (MELEAGRIS GALLOPAVO MERRIAMI) FLOCK IN COLORADO, USA.

A Merriam's Wild Turkey (Meleagris gallopavo merriami) with periocular swelling and periocular skin crusting in Pueblo County, Colorado, USA, was diagnosed with severe catarrhal and fibrinous sinusitis and conjunctivitis. A novel clade of Avibacterium was detected in the exudate from this bird. Although eight additional turkeys culled from the affected flock did not have clinical signs or gross lesions, histologically all had mild-to-moderate chronic sinusitis, and infraorbital cultures yielded the same novel clade of Avibacterium that was found in the symptomatic turkey. The presence of this Avibacterium species in the absence of significant disease in some birds suggested that other factors might have been involved in the development of severe sinusitis and conjunctivitis in the symptomatic Wild Turkey. Negative culture results from a distant flock of Wild Turkeys, acquired with similar methods to the affected flock, suggested that this novel species of Avibacterium was not widespread throughout Wild Turkeys in Colorado.