Characterization and detection of antibodies to candidate Rhodococcus equi vaccine antigens
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The pathogenic actinomycete Rhodococcus equi is the causative agent of a severe contagious purulent bronchopneumonia in foals. R. equi is endemic in many stud farms worldwide and causes major economic losses to the equine industry. No R. equi vaccine is available and the prophylaxis -and treatment- is based on prolonged antibiotic therapy. The problem is aggravated by the lack of suitable diagnostic methods for the early detection of infected foals. The work described in this MSc thesis was developed as part of the efforts of the host group in identifying suitable vaccine and serodiagnostic targets to combat rhodococcal foal pneumonia. Through their work with the pathogen’s genome, the group had previously identified the cytoadhesive R. equi pili (Rpl) as a key surface-exposed antigen with an important role in pathogenesis. The first aim of my research involved the development of serological tests to monitor the immune response of horses to the major pilin subunit, RplB, a novel R. equi candidate vaccine antigen. Specifically, I set up and standardized indirect ELISA and western-blot assays for the detection of anti-RplB IgG and IgG(T) antibodies in equine serum. For the ELISA test, I coated 96-well plates with RplB synthetic peptide, and incubated them with different dilutions of control and test horse sera, including convalescent foal sera. As positive control, I used sera from convalescent horses, and as negative controls, sera from Icelandic horses (known to be R. equi infection-free) or pre-suckling newborn foals. The RplB western blot was developed using RplB-GST fused protein and also, bacterial cell extracts from wild-type R. equi and an rplB deletion mutant, which I constructed by introducing an in-frame deletion in the target gene, to aid in the identification of the RplB-specific band. In the second aim of my MSc, I worked on the characterization of the role of another Rpl pilus component, the putative minor pilin RplC, in pilus formation and as a potential additional component of a pilus-based vaccine. I constructed an in-frame deletion mutant in rplC and tested its phenotype and adhesion characteristics alongside those of the rplB mutant using different methods, including cell-based in vitro assays. This work was undertaken as part of a broader research programme aiming at the identification of novel surface-associated R. equi antigens which could be included in the formulation of a vaccine interfering with the early stages of rhodococcal infection in the lung. To this aim, I also contributed to the standardization of fractionation procedures that will be used later for immunoproteomic studies to identify novel R. equi antigens.