Krista Allred

Microbial Diversity In Bighorn Sheep Revealed By Culture-Independent Methods

Bighorn sheep have been dying in North America due to pneumonia caused by
the Pasteurellaceae-Proteobacterias Mannheimia Haemolytica and Pasteurella
Trehalosi. These bacterias are transferred from domestic sheep and goats to the
Bighorn sheep. The bacteria is not always harmful because some sheep with the
bacteria are healthy. The purpose of the study was to determine the effectiveness of
culture-independent molecular methods to discover host-associated microbial diversity
in bighorn sheep and to see if the methods can overcome the limitations of traditional
approaches. The result of the study can enhance the understanding of microbial
communities associated with bighorn sheep respiratory diseases.
In 2004, 42 nasal and oropharyngeal swab samples were collected from bighorn
sheep in Colorado, California, and Oregon to determine their microbial diversity. The
samples were sent to the Washington Animal Disease and Diagnostic Laboratory and
the San Diego State University. The samples collected were cultured and determined to
be M. haemolytica and P. trehalosi. The DNA was extracted to ascern what primer
combinations the samples had. The samples were purified and cloned and sequenced
in one direction using the 8F primer by the San Diego State University Microchemical
Core Facility. The researchers then found all known bacterial rDNA sequences and
grouped the 99% similar sequences together. They then did phylogenetic and
population genetic analyses to determine the composition of any two microbial

communities, such as the nasal and oropharyngeal communities within one sheep to
see if they contained bacteria from different phylogenetic groups.
All of the methods the researchers did were successful. The DNA extractions,
cloning, and sequencing were effective in understanding more about microbial diversity
in bighorn sheep. The pasteurellaceae species associated with the disease, such as
Pasteurella trehalosi and Mannheimia haemolytica were found in healthy sheep and
Pseudomonas, Moraxella, Streptococcus, and Neisseria were found in healthy and
dead sheep. The phylogenetic and population genetic analyses uncovered a diverse
array of bacterial species and found that there is no difference in phylogenetic diversity
of bacterial communities in the nasal and oropharyngeal samples.
The results demonstrate that host-associated microbial diversity in the respiratory
tracts of bighorn sheep can be determined by molecular methods. They also found that
molecular techniques can detect toxin genes in a disease. In this study some of the
samples were mishandled and stored wrong causing them to be inviable. Cost restraints
and a small variety of samples caused limitations in the study. They conclude that many
more culture-independent studies need to be done to understand the microbial diversity
of bighorn sheep.