Veterinary Science Collected Works - Theses
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ItemDevelopment of a live-attenuated vaccine against canine pyometra( 2022)Pyometra is a disease affecting intact bitches and is fatal if left untreated. It is caused by the bacterial invasion of the uterus under the influence of progesterone resulting in the accumulation of pus. Uropathogenic E. coli (UPEC) are one of the most predominant pathogens isolated from the uteri of affected animals. UPEC carry uropathogenic virulence factor (UVF) genes which influence their virulence in the urogenital tract. The urogenital tract is a low iron and nutrient-deficient environment making the UPEC’s iron acquisition system and the oligopeptide uptake system critical for their virulence and survival in the urogenital tract. To date, there are no medical alternatives to ovariohysterectomy for the prophylaxis of pyometra. The main objective of the studies described in this thesis was to develop a bacterial vaccine targeting the TonB-mediated iron uptake system, the Fur regulon and the oligopeptide uptake pathway. The hypothesis was that targeting the iron uptake, iron homeostasis and oligopeptide uptake systems at once would allow the vaccine to provide protection despite the genetic diversity between UPEC strains and the redundancy of many of their metabolic systems. The first step was to assemble and characterise the complete genome of canine UPEC isolates using Illumina Miseq and Oxford Nanopore MinION sequencing technologies. Whole genome comparative genomics was carried out between canine isolates and other pathogenic and non-pathogenic Escherichia coli (E. coli) strains from various sources with a special focus on human UPEC isolates. The genomes of the canine UPEC isolates P4 and YP3 contained a single circular chromosome of 5.09 mega basepairs (Mbp) and 5.08 Mbp, respectively. The genomes were annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) and 18 pathogenicity-associated islands carrying virulence-associated genes were predicted in both genomes. Both canine uterine isolates belonged to the phylogroup B2, multilocus sequence type ST12 and serotype O4. The evolutionary relatedness between the canine UPEC isolates and other E. coli based on the cgMLST scheme and core single nucleotide phylogeny (SNP) showed a close phylogenetic relationship between the canine pyometra strains and human UPEC strains UTI89 and NU14. The virulence gene profiles of P4 and YP3 were nearly identical to human cystitis causing UPEC subtypes. The canine faecal isolate YF8 was identified as Escherichia marmotae and contained a single chromosome of 4.55 Mbp and an extra-chromosomal plasmid pYF8. The assembled genomes of the canine UPEC isolates will improve the understanding of the disease processes involved in pyometra and help explain similarities to urinary tract infections. They also facilitated genetic manipulation of the isolates for developing vaccine candidates in this thesis. The strain P4 was selected as the parent strain for vaccine development described in this thesis. The strain P4 was subjected to mutagenesis. The genes tonB, fur and oppD were insertional inactivated by lambda red recombination to generate single (P4-ΔtonB; P4-ΔoppD; P4-Δfur), double (P4-ΔtonB::ΔoppD) and triple (P4-ΔtonB::Δfur::ΔoppD) knockout mutant strains. In vitro growth assays of the mutants were carried out in iron limiting (2,2’ dipyridyl) and iron excess (ferric chloride) conditions and in the presence of the iron-dependent antibiotic Streptonigrin, to confirm their phenotypes. Dipyridyl sensitivity assays (200 µM) showed that the tonB mutants (P4-ΔtonB, P4-ΔtonB::ΔoppD, P4-ΔtonB::Δfur::ΔoppD) did not grow as efficiently as the field strain (P<0.05). Even in iron excess conditions, these strains grew less when compared to the field strain P4 (P<0.05), indicating the mutants’ inability to utilise environmental iron. Streptonigrin sensitivity assays (5 µg/mL) showed that the tonB mutants were more resistant to the antibiotic compared to the parent strain (P<0.05). This further confirmed the mutants’ impaired iron uptake ability. Together, these in vitro growth studies demonstrated the central role of tonB in iron starvation. Following on from this, the expression of the iron regulated outer membrane proteins (OMPs) was analysed in the vaccine candidates in comparison to the parent strain P4 under iron limiting and iron excess conditions. The outer membrane fraction was isolated by sonication and high-speed centrifugation using N-lauroylsarcosine and separated by Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). To identify and evaluate the antigenicity of the OMPs, immunoblotting was carried out using monospecific polyclonal rabbit anti-IroN antibodies. The results of this study showed that OMPs were expressed in iron limiting conditions in all the vaccine strains and in the parent strain. However, when the Fur regulon was inactivated, OMPs were expressed even in iron excess conditions. This is suggestive of successful deregulation of the Fur and that fur mutant strains are capable of expressing the OMPs irrespective of the iron levels in the media. The results of Western blotting showed that anti-IroN sera bound to a 79 kDa protein, indicating that the surface-exposed iron-regulated OMPs are strong antigenic proteins. In the final study, the safety and efficacy of the vaccine candidates were evaluated in an in vivo study. To do this, a murine model was developed in C57BL/6 mice to evaluate the pathogenicity of the canine UPEC strain P4 in the urinary tract and the reproductive tract by transurethral and intravaginal inoculation, respectively. P4 was mildly to moderately pathogenic in the urogenital tract of mice and produced mild to moderate inflammatory changes in the urinary bladder and uterus of C57BL/6 mice. This model was therefore chosen to evaluate the safety and efficacy of the vaccine candidates P4-ΔtonB, P4-ΔtonB::ΔoppD and P4-ΔtonB::Δfur::ΔoppD in the reproductive tract of female mice. Intravaginal inoculation of the vaccine candidates was found to be safe with no adverse effects recorded in the vaccinated mice. While up to 60% of mice vaccinated with the single P4-ΔtonB or the double knockout vaccine candidate P4-ΔtonB::ΔoppD were protected from challenge with the parent strain P4, the triple knockout strain did not confer any protection. The humoral immune response was poor in the mice inoculated intravaginally with any of the vaccine candidates. The total serum IgG levels did not vary between vaccinated and unvaccinated mice. Subcutaneous inoculation of the single knockout strain P4-ΔtonB produced severe systemic illness in the vaccinated mice. The double P4-ΔtonB::ΔoppD and triple knockout P4-ΔtonB::Δfur::ΔoppD strains caused severe inflammatory reaction at the injection site resulting in the premature abortion of this part of the experiment. The initial humoral immune response to P4-ΔtonB::Δfur::ΔoppD inoculated subcutaneously was promising. In summary, the studies described in this thesis characterise the whole genome of canine UPEC isolates and assess their virulence in a murine model. The live attenuated vaccine candidates developed by inactivating TonB, Fur and OppD expressed multiple antigenic OMPs and showed reduced growth potential in vitro. The preliminary outcomes of the in vivo studies are promising but more work is necessary to further evaluate their potential as vaccines.