Brucella spp., the etiologic agents of brucellosis, are Gram-negative, non-motile, facultative, intracellular coccobacilli that can infect a wide range of mammalian species, including humans and some amphibians. It remains one of the major neglected diseases in this world. Since its discovery in 1887 by Sir David Bruce, many diagnostics have been developed to make inroads into the control or eradication of this disease. The current gold-standard diagnostic is bacterial culture. However, only 30–50% of sero-positive animals will be culture positive. A novel diagnostic assay (polymerase chain reaction; PCR) is needed to identify infected individuals within a population. Our objectives were: 1) the identification of optimal techniques for the extraction of relatively pure and high yield DNA is needed for amplification; 2) identification of novel targets for primer/probe for exploitation in a PCR assay; 3) characterizing the performance of the novel assay against serology and the gold-standard of culture; and 4) conducting a genome wide association study to identify single nucleotide polymorphism that differentiate biovars.
For objective 1, we compared the performance of various DNA extraction kits for their ability to recover Brucella abortus Strain 19 inoculated into Brucella-free bovine tissues. Tissues were homogenized and extracted in triplicate using one of five kits (Qiagen DNeasy, GE Illustra™, Omega Bio-tek E.Z.N.A.®, Quanta Extracta™, and IBI Science Mini Genomic DNA Tissue Kit). Whole blood was also taken from animals, aliquoted, and then fractioned into buffy coat, red blood cells, and plasma. Whole blood, buffy coat, and plasma were run with four kits (Qiagen DNeasy, Omega Bio-tek E.Z.N.A.®, IBI Science Mini Genomic DNA Blood Kit, and 5PRIME PerfectPure). A fifth kit, Quanta Extracta™, was run on buffy coat and plasma only. Previously published primers targeting the erythritol catabolism (eryC) gene in Strain 19 were used to amplify extracted DNA and identify the optimal extraction kit. Real-time PCR was performed using a SYBR® Green platform and kits were compared for statistical differences using quantification cycles as an outcome measure. Omega Bio-tek E.Z.N.A.® was statistically superior in lower quantification cycle values (P ≤ 0.0068) across all kits. Blood and its fractions (plasma, buffy coat, and red blood cells) samples were also pooled, the IBI Science Mini Genomic DNA Blood Kit was statistically superior to Qiagen DNeasy, 5PRIME PerfectPure, and Quanta Extracta™ (P < 0.0001; P = 0.0004; P = 0.0013) but was not different than Omega Bio-tek E.Z.N.A.® (P = 1.0). Based on these results, the optimal extraction kit for B. abortus Strain 19 for tissue is Omega Bio-tek E.Z.N.A.® and the optimal kit for blood and its fractions is the IBI Science Mini Genomic DNA Kit. Eluted DNA was also concentrated using the Zymo Research DNA Clean & Concentrator™–25 kit. The concentration of the eluted DNA with target was superior to the not concentrated elution of DNA (P ≤ 0.0001).
For objective 2, an in-silico analysis utilized 103 isolates (88 field and 16 reference isolates) obtained from the USDA-National Veterinary Services Laboratory (USDA-NVSL), and the NCBI. Single nucleotide polymorphisms (SNPs) were identified and used to design primers and/or probe for use in a novel real-time PCR assay. There was no single SNP locus that would differentiate species of Brucella and the current vaccine strains. Therefore, three preferred primer sets were chosen based on their PCR efficiency, limit of detection, and limit of quantification. These primer sets were termed pan_82 (pan B. abortus), RB51-_151 (does not amplify RB51), and S19-_116 (does not amplify S19). Using 42 serologic and culture-positive animals, and 127 serologic and culture-negative animals, preferred primer sets were tested against tissues to determine assay statistics. Sensitivity and specificity point estimates for all the preferred primer sets were 100%. Additionally, the preferred primer sets identified animals that were serologically-positive and culture-negative, as PCR-positive. This indicates that culture may underestimate the true prevalence of brucellosis within populations. Additionally, DNA concentration and bacterial localization within tissues was found to be important factors that impacted the sensitivity of this novel assay.
For objective 3, the novel real-time polymerase chain reaction assay was utilized to determine apparent disease prevalence, demographic risk factors for infection, and to reevaluate serologic cutoff values in wild bison. Yellowstone National Park bison (n = 159) were sampled in the winter of 2017 from slaughterhouses in Montana. For evaluating cutoff values, Yellowstone National Park bison (n = 63) from the 2016 cull were included in the analysis. Fluorescence polarization assay was used to determine the apparent seroprevalence of 39.6% in the animals sampled. Culture prevalence was 20.1% (n = 32). Males were more likely to be infected as defined by culture. (Abstract shortened by ProQuest.)
|Advisor:||Schumaker, Brant A.|
|Commitee:||Andrews, Gerard P., Gigley, Jason P., Laegreid, William W., Miller, Myrna M.|
|School:||University of Wyoming|
|School Location:||United States -- Wyoming|
|Source:||DAI-B 79/09(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Epidemiology, Veterinary services|
|Keywords:||Brucellosis, Infectious disease epidemiology, Molecular diagnostics, PCR, Yellowstone|
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