Investigating molecular approaches for detection of pathogens in horses
Grout, E. K. (2018). Investigating molecular approaches for detection of pathogens in horses (Thesis, Master of Science (Research) (MSc(Research))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12094
Permanent Research Commons link: https://hdl.handle.net/10289/12094
Infectious disease of equines has significant impacts on the New Zealand equine industry, causing avoidable morbidity and mortality. The correct identification of the aetiological agents involved in such infections allows for prompt and accurate administration of an appropriate therapeutic strategy, reducing the economic impact associated with equine wastage. Traditional culturing methods are widely used for the diagnosis of infections in horses, however, conventional PCR offers a novel and rapid approach, through the targeting of unique genes for diagnosis of pathogens. PCR is an attractive alternative to microbial culturing, which can take anywhere between 24 hours to 1 week to determine the causative infectious agent, and in a number of cases the results are not as specific or as accurate as they need to be. It is the lack of sensitivity of the traditional culturing techniques that continues to contribute to the economic loss and welfare issues associated with equine infections. Initially, it was found that an SDS lysis extraction method worked best for extraction of DNA which was used to extract microbial DNA from various types of clinical samples. These were subsequently used as DNA templates within PCR alongside primers associated with up to 28 genes associated with common equine infections. From a total 146 clinical samples received for testing 66.4% returned a PCR positive for one or more microbial genera, whereas traditional bacteriological culturing produced an identification rate of 46%. In addition, PCR was significantly faster than culturing methods (6 hours vs 48 hours) and allowed more specific identification of the microbes present, which provides a significant advantage for infection management and increasing animal welfare. The results found from these experiments show rapid PCR identification holds promise as a diagnostic technique in the future, however, further development is required before this can be used as a primary diagnostic technique. Recommendations include further research into trends of infection in NZ equines and respectively increasing the diversity of PCR primers to represent the larger scope of microbial species. PCR diagnosis would also benefit from the standardisation of the DNA extraction methods used to allow consistency and increase throughput of various samples. Lastly, the simplification of the individual PCRs for each infectious agent could be developed into multiplex reactions to decrease time processing time and reduce human pipetting error.
The University of Waikato
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- Masters Degree Theses