Type of Document Dissertation Author Duhon, Lauren E URN etd-01272010-205801 Title In Vitro and In Vivo Evaluation of a Brucella Putative Hemagglutinin Degree Doctor of Philosophy (Ph.D.) Department Pathobiological Sciences (Veterinary Medical Sciences) Advisory Committee
Advisor Name Title Elzer, Philip Committee Chair Enright, Frederick Committee Member Navarre, Christine Committee Member Thune, Ronald Committee Member Laine, Roger Dean's Representative Keywords
- Polymerase Chain Reaction
Date of Defense 2009-11-03 Availability unrestricted AbstractBrucellosis, a zoonotic disease caused by Brucella spp., presents both health and economic difficulties for livestock, wildlife, and humans. While brucellosis is nearly eradicated in the United States, the disease remains detrimental in many countries worldwide. Attempts to produce a safe and effective small ruminant vaccine have been met with limited success. The current vaccine for bovine brucellosis in the United States is B. abortus RB51. This strain transiently colonizes the host and induces a cell-mediated immune response. Levels of protection have not been demonstrated in goats and thus it is considered a relatively poor caprine vaccine that probably does not survive long enough in the tissues to produce a sufficient protective immune response. This study analyzes the possibility of using RB51 containing plasmid QAE for vaccination in goats. The plasmid QAE contains a gene region of DNA from B. melitensis 16M hypothesized to encode for a putative hemagglutinin that is believed to be a host specificity protein. The region E gene sequence is not present in any strain of B. abortus, including RB51. It is proposed that the addition of region E to RB51 will enhance survivability in the caprine host to the extent where an adequate protective immune response is elicited. The QAE plasmid was electroporated into B. abortus RB51 and screened using ampicillin resistance as a selective measure. Ten goats were conjunctivally inoculated with RB51-QAE, the modified strain, and ten goats received strain RB51 in the same manner as controls. Two goats from each group were euthanized and necropsied at weekly intervals for four weeks and again at 42 days. Tissues from these animals were taken at necropsy and used to collect data for colonization and histopathology. Serum samples were also analyzed for Brucella-specific antibodies. Both strains transiently colonized the hosts without producing any detrimental pathology. However, the RB51-QAE goats demonstrated higher levels of colonization and greater humoral immune responses for longer periods of time. These are very promising findings as the levels of colonization and humoral responses may correspond with better protection. These results warrant further testing of RB51-QAE as a potential vaccine for caprine brucellosis.The putative hemagglutinin was characterized using hemagglutination assays, absorption assays, and mass spectrometry analysis following 2D gel electrophoresis. The region E protein was found to provide RB51-QAE with increased hemagglutinating and immunogenic capabilities. Furthermore, there is evidence to suggest that the protein is a cell surface protein, not excreted from the cell.
The accepted gold standard for the detection of brucellae is bacterial culture. PCR is being evaluated as a possible alternative detection method for Brucella spp. in bodily fluids such as blood and urine. Currently, there is no accepted standard for detection of brucellae using PCR. The effects of template preparation, primer selection, and PCR optimization on the limit of detection for B. abortus 2308 and B. melitensis
16M in association with whole blood, plasma, or urine were examined. Ten-fold dilutions were made from a known number of bacterial cells in each of the fluids tested. The practices of using whole killed cells as a direct template for PCR as well as two differing DNA isolation techniques were evaluated for each fluid dilution. Our findings suggest that a more extensive template preparation technique and PCR protocol elongation can greatly improve the limit of detection capabilities. Biological fluids provided dissimilar results based on the PCR inhibition properties of the fluid and DNA isolation techniques. The results of this experiment encourage further investigation into the optimization of conventional PCR techniques as a faster and more efficient diagnostic tool for Brucella spp. in humans and animals.
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