Document Type

Thesis - Open Access

Award Date

2024

Degree Name

Master of Science (MS)

Department / School

Natural Resource Management

First Advisor

Jonathan Jenks

Abstract

Respiratory disease has historically been recognized as a limiting factor to wild bighorn sheep (Ovis canadensis) herds throughout North America with epizootics resulting in large-scale die offs and reductions in recruitment. The primary etiologic agent associated with pneumonia outbreaks in bighorn sheep is Mycoplasma ovipneumoniae. This bacterial pathogen is frequently carried asymptomatically by domestic sheep (Ovis aries) and goats (Capra hircus); however, when these domestic hosts come in contact with and spread this pathogen to bighorns, the disease can cause epizootics and large-scale mortality. Domestic sheep strains are more commonly detected in bighorn sheep than domestic goat strains, and previous experimental exposure of bighorn sheep to goat strains has been observed to cause less severe, often nonfatal pneumonia. The first part of this study aimed to better understand the potential for exposing bighorn sheep to domestic goat strains of M. ovipneumoniae to clear chronic infections of domestic sheep strains. We hypothesized that exposing bighorn sheep, chronically infected with domestic sheep strains, to a strain from domestic goats would induce nonfatal disease and boost strain specific immune response, the goat strain would replace the existing persistently carried sheep strain, or the goat strain would not persist in bighorn sheep and chronic carriers would clear M. ovipneumoniae. We found the goat strain of M. ovipneumoniae persisted following inoculation (~2 to 4 months), but ultimately the strain from the sheep clade resumed dominance. We also observed pneumonia-related deaths with the detected strain being of the domestic goat clade, which all suggests that this method may not be a suitable tool to clear infection in bighorn sheep herds. Our study then considered the possibility of immunizing lambs with a M. ovipneumoniae-specific vaccine as a disease management tool and hypothesized that it would result in an immune response that would protect against pneumonia. We found that this prophylactic measure did not result in a protective immune response and that implementation of vaccination in a field setting may not be practical due to the variability in strains, the host’s apparent lack of cross-strain immunity, and logistic feasibility. While pneumonia-related fatalities in bighorn sheep are often linked to M. ovipneumoniae, our investigations also describe and discuss a commingling study of bighorn and domestic sheep in captivity in which M. ovipneumoniae was deliberately absent from all experimental animals with the objective being to test whether or not respiratory disease could still occur in bighorn sheep. We found that respiratory disease and pneumonia-related deaths in bighorn sheep did occur when housed in captivity with domestic sheep in absence of M. ovipneumoniae. We observed a respiratory disease process different from that typically seen with Mycoplasma pneumonia and concluded that the close contact with domestic sheep can still be hazardous to bighorn sheep health despite absence of M. ovipneumoniae, the usual predisposing bacterial pathogen. The final aspect of our overall investigation considered the use and sensitivity and specificity of a mobile qPCR machine, the Biomeme FranklinÔ Real-Time PCR Thermocycler that could potentially be used to rapidly identify M. ovipneumoniae-positive sheep in the field. We used known-positive bighorn and domestic sheep nasal swab samples to assess the diagnostic characteristics of the Biomeme, and found sensitivity to be low and specificity to be high. We then tested the impact that PCR inhibitors could be having on sensitivity by assessing the use of three inhibition-reducing agents (dilution with PCR-grade water, BSA, and Biomeme’s Inhibition Enhancer Mix) relative to the standard protocol. Our analysis revealed that sensitivity was still low (baseline: 57%, 95% CI [50%, 64%]) and not improved with the inhibition-reducing agents. We then conducted further experiments with this device and known-positive domestic sheep nasal swab samples by assessing the effect of adding a homogenization and filtration step to the extraction process (updated extraction protocol) and assessing the impact that using expired M1 Sample Prep Cartridges had on DNA extraction and M. ovipneumoniae detection; due to the COVID-19 pandemic and impacts to the supply chain, supplies for this device became limited, resulting in the need to use materials past their expiration date in the initial part of experimentation. For this second phase of the experiment, to assess this impact and compare results from bighorn and domestic sheep, we extracted nasal swab samples from domestic sheep via four treatments consisting of combinations of extraction protocol used and expiration status of extraction supplies; additionally, we gathered and analyzed DNA quantification data from our extractions via Qubit fluorometry. We found that nasal swab samples from domestic sheep likely contained higher levels of DNA from all sources than swabs from bighorn sheep, that using expired M1 Sample Prep Cartridges is not advisable and contributes to greater variability in qPCR results, that the Biomeme Thermocycler is better able to detect Movi from domestic sheep samples regardless of expiration status and protocol used, and the addition of sample homogenization and filtration seems to improve the purity of the extraction, which could aid in higher quality extraction in the field.

Publisher

South Dakota State University

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Rights Statement

In Copyright