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¹ Biology Department, San Diego State University, San Diego, California 92182, USA
² Caine Veterinary Teaching Center, University of Idaho, Caldwell, Idaho 83607, USA
³ Idaho Department of Fish and Game, Lewiston, Idaho 83501, USA
⁴ Denver Museum of Nature and Science, Denver, Colorado 80205, USA
⁵ Corresponding author (email: skelley{at}sciences.sdsu.edu)

ABSTRACT:   We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.
  Key words:  16S rDNA, bighorn sheep, leukotoxin, Mannheimia, Pasteurella, polymerase chain reaction.

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