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.
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ItemDevelopment of methods to evaluate therapy for spinal cord trauma in dogs( 2020)Spinal cord injury (SCI) is a common cause of neurological deficits in dogs and a major cause of morbidity and decreased quality of life in people. In dogs, the most common causes of SCI are intervertebral disc diseases, particularly 'type I' intervertebral disc herniation knowns as intervertebral disc extrusion (IVDE), which occurs predominantly in chondrodystrophic breeds. The IVDE commonly causes contusion and compression to the spinal cord, which in most severe cases leads to complete loss of voluntary motor functions, bladder control and pain sensation distal to the level of the lesion. For decades, researchers have used animal models in their studies of SCI. However, limited progress has been achieved in translating any beneficial findings from experimental animal models to human SCI. Moreover, experimental studies in rodent models may not closely represent clinical SCI in human and canine patients. Naturally occurring compressive and contusive lesion of SCI associated with IVDE in dogs may be more analogous to human disease than induced experimental animal models. Canine naturally occurring SCI could simulate the injury in human patients including; mechanism of damage, pathophysiological events, healing processes, functional recovery, measures of evaluating outcomes, and effectiveness of the candidate therapies. Therefore, recruiting dogs of naturally occurring IVDE associated with SCI has become a common practice in clinical research trials. To date, no therapeutic method has been shown to treat SCI successfully in dogs with severe lesions. Therefore, the long-term outcome in these cases is chronic paraplegia with loss of sensation distal to the level of the lesion. Since this is true in both human and canine patients, novel therapies must be investigated, including promising neural stem cells (NSCs) therapy. The principal objective of this thesis was to study canine SCI and develop a reliable and reproducible paradigm for evaluating the effect of candidate therapies, using human neural precursor cells (hNPCs) in dogs as an example. This was approached by; 1) evaluating different MRI measures to monitor the spinal cord lesion in putatively naturally occurring canine spinal cord trauma, pre- and post- surgical or non-surgical treatments of IVDE, 2) ex vivo examination of hNPCs’ impact on inflammatory factors in stimulated whole blood (WB) samples from healthy dogs, 3) evaluating methods to measure responses to transplant of hNPCs into the spinal cord of the paraplegic dog with grade 5 SCI, as a pilot clinical study, 4) exploring the fate of hNPCs at 18 months post-xenotransplantation into spinal cord tissue (SCT) of a dog with grade 5 SCI, and 5) investigating the possibility of isolate and culture of canine NSCs from SCT of the dog. The findings of this thesis showed that the pathological and morphometric alterations of the objective and quantifiable parameters of the MRI measures of the spinal cord and vertebral canal post-SCI and following treatments are important prognostic indicators. The interaction effect of time and surgery is an important factor in decreasing spinal cord compression and vertebral canal narrowing. The results of the ex vivo study suggested that hNPCs could modulate the inflammatory responses in stimulated WB by reducing the production of tumour necrosis factor-alpha. The in vivo clinical pilot study concluded that neither adverse effects nor immunological reactions were developed following transplantation of hNPCs into the spinal cord of the dog. Although recovery of the cutaneous trunci muscle reflex was identified, no improvements of the deep pain sensation or locomotion and urinary functions were shown at the end of the 6 months observation period post-therapy. The immunohistochemical findings revealed that the grafted hNPCs had survived and migrated, after xenotransplantation into SCT of the host dog. The gross and histopathological examination showed neither significant pathological changes nor tumour formation at 18 months following xenotransplantation in the dog with grade 5 SCI. This research work reported that it is possible to isolate, and culture canine derived NSCs from SCT. Therefore, this shows promise for future research using of dog-to-dog allotransplantation of canine derived NSCs, which may minimise the associated complications of xenotransplantation. In summary, the observations of the studies in this thesis offer insight into the methods developed to evaluate potential benefits of using hNPCs for transplantation therapy in dogs with grade 5 SCI. The current research work helps understanding of the pathological and morphometrics alterations of spinal cord lesion pre and post treatment of IVDE. The findings are of value in understanding the mechanism of potential therapeutic effects of hNPCs, including the anti-inflammatory effect and the fate of the cells following transplantation into the spinal cord of the host. Finally, this research represents an initial step towards investigating the efficacy and potential benefits of hNPCs in a preclinical study in a large number of dogs with naturally occurring SCI.