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Role of Staphylococcus aureus GapC and GapB in immunity and pathogenesis of bovine mastitis



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Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of S. aureus is a major economically important disease affecting the dairy industry worldwide. S. aureus is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure S. aureus intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against S. aureus mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of S. aureus to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. S. aureus also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against S. aureus mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of S. uberis and S. dysgalactiae have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from S. aureus isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of S. aureus as model antigens for mastitis and to determine the regulation of expression of gapB and gapC genes and their roles in the pathogenesis of bovine S. aureus mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of S. aureus as antigens will protect against bovine S. aureus mastitis. To test this hypothesis we took the approach of using the gapB and gapC genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-γ secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by S. aureus. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of S. aureus mastitis using the S. aureus wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in S. aureus mastitis. In aim 6 we examined the role of sar and agr loci on the expression of gapC and gapB genes by qRT- PCR using S. aureus RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both gapB and gapC expression were down regulated in the mutant strains, indicating that the expression of the gapB and gapC genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of gapB and gapC using q-RT-PCR. Our results showed that the expression of gapB and gapC genes in different strains of S. aureus was not consistent under the above-mentioned environmental conditions.



Glyceraldehyde-3-phosphate dehydrogenase, Inflammation, Bovine, Mastitis



Doctor of Philosophy (Ph.D.)


Veterinary Microbiology


Veterinary Microbiology


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