Studies of the pathogenesis of canine pyometra

Abstract

© 2011 Dr. Natali KrekelerPyometra, a prevalent uterine infection that most commonly affects intact-middle aged to aged bitches, is typically associated with the bacterial pathogen Escherichia coli (E. coli) bacteria. Dogs are particularly susceptible to infection during dioestrus, when uterine tissues are under the influence of progesterone. The pathogenesis of pyometra is not thoroughly understood. Many host factors, such as age of the bitch, ovarian hormones, breed and parity seem to play a role, as well as bacterial factors. Pathogenic E. coli strains carry different uropathogenic virulence factors (UVF), which facilitate infection in the urogenital tract. Adhesive proteins, at the tip of bacterial pili (fimbriae), bind to receptors on epithelial cells of the urogenital tract. Three different types of adhesins (FimH, PapGIII and Sfa) have been identified in E. coli strains isolated from bitches with pyometra. The objectives of the studies in this thesis were to investigate the role of bacterial adhesins in the disease process and to investigate differences in host immune defences during different stages of the oestrous cycle. The primary objective of the first study was to investigate whether the adhesin FimH, which can be found in all E. coli pyometra isolates, facilitates the binding of E. coli to the canine endometrial epithelium. An E. coli strain (strain P4), isolated from the uterus of a bitch with pyometra, that only carried the adhesin gene fimH was used. In order to study the effect of the adhesin FimH, a mutant knock-out strain of P4 (P4-∆fimH::kan) was generated from the P4-wildtype strain (P4-wt) by insertionally inactivating fimH. An in vitro assay that provided quantitative and qualitative assessment of bacterial binding was developed and used to assess the binding properties of the mutant and wildtype strains. The second objective was to examine the role of different stages of the canine oestrous cycle on bacterial binding. The stages of the canine oestrous cycle were simulated in ovariectomised greyhound bitches using a previously established protocol and uterine biopsies from these bitches were used to examine the effect of the stage of the oestrous cycle on bacterial binding. The amount of bacterial binding did not vary significantly with the stage of simulated oestrous cycle. The objective of the second study, described in Chapter 4, was to investigate the role of each adhesin gene product, acting alone or expressed in combination, in the bacterial binding of a strain (P3) carrying all three known adhesin genes to the canine endometrium. Knockout (KO) mutants of this wildtype (P3-wt) strain were generated using insertional inactivation. Single (P3-∆fimH::Kan; P3-∆pap::Cm; P3-∆sfa::Kan), double (P3-∆fimH::Kan-∆pap::Cm; P3-∆fimH::Kan-∆sfa::Kan; P3-∆pap::Cm-∆sfa::Kan), and triple (P3-∆fimH::Kan-∆pap::Cm-∆sfa::Kan) mutants were produced. Adhesion assay were conducted on anoestrous uteri of three post-pubertal bitches. Overall, the number of bacteria adhering to canine endometrial biopsies was comparable and no significant difference in the number of bound bacteria was found between the P3-wt strain and the single or double KO-strains. However, the triple knockout strain (P3-∆fimH::Kan-∆pap::Cm-∆sfa::Kan) displayed less binding to the canine endometrium compared with the P3-wt strain. This study showed that a pathogenic E. coli strain (P3) isolated from the uterus of a bitch with pyometra was able to fully compensate for the loss of two of its three known adhesin genes. It was necessary to inactivate all three known adhesin genes in order to see a significant decrease in binding to canine endometrium. However, the triple knockout mutant still retained 42% of its binding capacity compared with the P3-wt strain. This retained binding contrasted with the analyses of strain P4, in which functional loss of a single adhesin gene (fimH) eliminated >99% of bacterial binding. These combined studies suggest that pathogenicity varies between E. coli strains and that there may be additional adhesions in some E. coli strains that play a role in the pathogenesis of pyometra. The objective of the final study was to investigate if differences in innate immunity account for the differential susceptibility to pyometra during different stages of the oestrous cycle. The expression of the antimicrobial peptide β-defensin 1 was assessed during different stages of the simulated oestrous cycle and in response to pathogenic E. coli (P4 strain). A 100-fold increase in canine β-defensin 1 mRNA expression was seen during dioestrus when compared to anoestrus or oestrus. Canine β-defensin 1 mRNA expression remained unchanged during incubation with or without bacteria over 3 or 8 hours, suggesting constitutive expression. In summary, the studies presented in this thesis illustrate the interaction of bacterial virulence and host immunity. The novel in vitro model developed will allow future research in this area in order to further elucidate the pathogenesis of canine pyometra