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KMcD Dissertation Final 122116.pdf (1.93 MB)

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Mycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation

McDonald, Kristina Marie
http://rave.ohiolink.edu/etdc/view?acc_num=osu1482317197896844

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Comparative and Veterinary Medicine.
Mycoplasma bovigenitalium (Mbvg) is a common inhabitant of the reproductive tract of diseased and apparently normal cattle. The bovine mycoplasma has been associated with multiple reproductive tract disorders in both male and female cattle. Additionally, Mbvg can colonize the prepuce and distal urethra of bulls, often without producing disease, resulting in Mbvg contaminated semen. The use of Mbvg-contaminated semen for AI has been reported as a potential source of infection for the bovine female genital tract. Although the pathogenic nature of Mbvg has been reproduced experimentally, it is not evident what triggers certain animals to exhibit disease while others remain asymptomatic carriers. Therefore, it is critical to increase the sensitivity of Mbvg detection as well as improve our knowledge regarding the pathogenicity and epidemiology of Mbvg. The first objective of this study was to develop and validate a TaqMan qPCR for the rapid detection of Mbvg in bovine semen. The assay exclusively amplified 81 field and laboratory strains of Mbvg when tested against a panel 15 bovine mycoplasma species. Fresh bovine semen samples (n=77) were assessed for Mbvg by qPCR and culture. M. bovigenitalium was recovered in 57 (74%) semen cultures and detected by qPCR in 62 (80.5%) samples. The rapid nature of the assay (results obtained in 5 h) may provide an alternative diagnostic and research tool for the detection and quantification of Mbvg in bovine semen samples as opposed to traditional culture, which requires up to 10 days. The second objective of this study was to develop a Multilocus Sequence Typing (MLST) assay for Mbvg strain differentiation. The sequences of three targeted loci (ppa, tpiA, rpoB) were analyzed from a collection of 86 Mbvg isolates from around the world. A high degree of nucleotide variability was observed and the isolates were organized into 61 strain types (ST). A phylogenetic tree constructed from the concatenated sequences displayed coherent relationships between branching and the geographic and species origin of the isolates. The third objective of the study was to assess Mbvg in bovine semen using MLST to identify potentially pathogenic strains. Semen samples from 41 bulls were cultured for mycoplasma. A total of 33 Mbvg isolates were recovered (80.5%) and subjected to MLST. Despite isolates originating from the same location, a high degree of nucleotide variability was present resulting in 24 STs. Phylogenetic analysis of the concatenated sequences revealed four distinct clusters (strain family A-D). Fertility potential (SCR) and six months (± 3 months from collection date) semen quantity/quality data for each bull were compared to strain family. A statistically significant association was observed between strain family and SCR (P = 0.002). Further analysis revealed that bulls carrying strain family C had significantly lower SCR than bulls negative for Mbvg (P = 0.006). These findings may allow the development and refinement of additional hypotheses for future research studies to identify additional carriers of Mbvg strain family C, assess the impact of Mbvg on bull fertility, and intervention strategies to control and prevent its propagation in cattle.
Thomas Wittum (Advisor)
Gustavo Schuenemann (Advisor)
Gregory Habing (Committee Member)
Christopher Premanandan (Committee Member)
James M. DeJarnette (Committee Member)
166 p.
Animal Sciences; Epidemiology; Microbiology; Molecular Biology
Mycoplasma bovigenitalium; qPCR; MLST; bovine semen; genotyping; fertility

Recommended Citations

Citations

  • McDonald, K. M. (2017). Mycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1482317197896844

    APA Style (7th edition)

  • McDonald, Kristina. Mycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1482317197896844.

    MLA Style (8th edition)

  • McDonald, Kristina. "Mycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1482317197896844

    Chicago Manual of Style (17th edition)

osu1482317197896844
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This open access ETD is published by The Ohio State University and OhioLINK.