Browsing by Author "Knight, Cameron G."

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    Investigation into Equine Papillomavirus Type 2 Prevalence and Diagnosis
    (2018-09-28) Wachoski-Dark, Garrett Louis; Knight, Cameron G.; Klein, C.; Czub, Markus; Munday, John S.
    Equine papillomavirus type 2 (EcPV-2) has been consistently associated with most equine genital cancers and a subset of cancers at other locations. The prevalence of EcPV-2 in healthy horses has been estimated to be <20% for DNA presence, and 10-36% for anti-EcPV-2-L1 seroprevalence. However, no study has thus far examined the prevalence of EcPV-2 at the herd-level or over multiple timepoints. This thesis examined EcPV-2 prevalence in a predominantly closed herd with a known EcPV-2 infected horse over three timepoints. We found that DNA prevalence (0-4%) was equivalent to the general horse population, but that seroprevalence (51.5%) was higher. Additionally, no study has examined the distribution of EcPV-2 infection in situ, nor compared such a technique to PCR. We found that RNA ISH concorded with PCR in 80% of cases. We also recapitulated previous results concerning EcPV-2 distribution in situ as well as found EcPV-2 in numerous novel locations.
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    Porcine Circovirus Cap-Induced Apoptosis of Non-Infected PK15 Cells
    (2019-07-16) Rowell, Jared S.; Czub, Markus; Zheng, Xilong; Jirik, Frank Robert; Knight, Cameron G.; Corcoran, Jennifer A.
    Porcine Circovirus 2 (PCV2) is a pathogen of major importance for swine production around the world. Despite development of several vaccines and robust vaccination programs, PCV2 continues to persistently transmit within and between swine herds causing infections marked by immunosuppression via lymphocyte depletion. This study aims to improve understanding of porcine circovirus diseases (PCVD) pathogenesis caused by PCV capsid cytotoxicity which activates apoptosis in non-infected bystander cells. Putatively non-pathogenic Porcine Circovirus 1 (PCV1) was also included to help understand why there is a difference in pathogenicity between PCV1 and PCV2. Porcine Circovirus 3 (PCV3) has recently emerged worldwide as a possible etiological agent of clinical syndromes observed in swine that are similar to PCVD caused by PCV2. The capsid protein of PCV3 (PCV 3 Cap) was also included in this study to provide initial information regarding the cytotoxicity of this protein. Results were obtained through flow cytometric analysis of apoptosis using Annexin V-FITC and 7-AAD to measure apoptosis markers and cell death respectively. Flow cytometry results were reinforced by also utilizing a TUNEL assay in conjunction with confocal imaging to detect cells undergoing DNA fragmentation leading to apoptosis. Results show that monomeric PCV2 Cap, 1% formaldehyde inactivated and dialyzed PCV2, PCV1 virus-like particles (VLPs), PCV2 VLPs, and PCV3 VLPs are all able to activate apoptosis in porcine kidney (PK15) cells at a rate of ~%30 after a 24-hour exposure; this apoptotic similar to apoptosis caused by 50 µM etoposide, which is a strong chemical inducer of apoptosis. These results coupled with preliminary data obtained by pre-treating PK15 cells with caspase-8 or caspase-9 inhibitors suggest the involvement of caspase-8 and caspase-9 in PCV2 Cap induced apoptosis and lend weight to the idea of PCV2 Cap as a primary virulence factor during infections. In contrast, PCV1 Cap induced apoptosis does not appear to involve caspase-8 or caspase-9 indicating an alternate pathway to PK15 cell apoptosis, while PCV3 Cap appears to induce PK15 cell apoptosis with some involvement of caspase-9.