Veterinary Science - Theses
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ItemMolecular epidemiology of avian pathogenic Escherichia coli( 2013)Avian pathogenic Escherichia coli (APEC) are responsible for respiratory tract disease and colisepticaemia in poultry, resulting in large economic losses. A range of genes, in different combinations, is involved in the virulence of APEC, and a unique combination of virulence factors or a specific genotype to define APEC have not been defined. The pathogenicity of the Australian APEC strain E3, together with the contribution of the conserved portion of plasmid pVM01 to virulence of strain E3, have been established previously. The study described in Chapter 3 compared E. coli isolated from diseased birds with that isolated from healthy birds and diseased and healthy mammalian species on the basis of the presence of nine APEC plasmid-associated virulence genes, sitA, iroB, iroN, iucA, iutA, iss, tsh, hlyF and ompT, and the replicon RepFIB, using digoxigenin (DIG) labelled DNA probes for colony blot hybridisation. Most of the virulence genes and the plasmid replicon were found significantly more often in E. coli isolated from diseased birds than in those isolated from healthy birds and diseased mammalian species. The APEC pathotype was defined as one that contains either the combination of iroB or iroN, iucA or iutA, and hlyF or ompT. A combination of four genes, sitA, iroB, iroN and iss, was found to be significantly associated with the E. coli isolated from the urogenital tract of diseased mammals. Being a respiratory pathogen, the main route of entry of APEC into its hosts is inhalation of contaminated dust from the poultry shed environment. In the study described in Chapter 4, air sampling was established as a novel method for rapidly determining the concentration of airborne E. coli, as well as E. coli carrying APEC virulence genes, in the broiler shed environment. Air sampling was conducted using an air monitoring system and a selective medium allowing recovery of Gram-negative bacteria, including E. coli. Colony blot hybridisation using three DIG-labelled DNA probes for iroB, iucA and hlyF was used for identification of APEC and differentiation of APEC from other bacterial species, as well as from commensal E. coli. In Chapter 5, Air sampling at different time points during a commercial broiler production cycle showed that the mean concentrations of all airborne E. coli and of E. coli carrying either the iroB, iucA or hlyF gene, or all three genes increased over the first 2 weeks of life of the birds and then subsequently decreased. The proportions of probe positive E. coli were highest at the initial time point and consecutively decreased with the age of the birds. The peak mortality was observed a week after the peak concentrations of airborne E. coli carrying APEC virulence genes, suggesting a time lag between colonisation and disease induction in birds. Pathotypic characterisation using colony blot hybridisation and genotypic characterisation using Southern blot hybridisation to EcoRV and BglI digests of genomic DNA from isolates recovered from the air, diseased tissues and the cloacae revealed greater diversity in E. coli isolated from the air and cloacae, and lower diversity in E. coli isolated from lesions, suggesting infected birds may transmit E. coli to neighbouring birds. The presence of a single E. coli genotype in a low proportion of E. coli isolated from the air, in lesions and in the intestinal tract suggested that inhalation of faecally contaminated aerosol may also cause the disease in birds. Further investigation on another 4 individual broiler rearing facilities, as described in Chapter 6, established the association between the concentrations of all airborne E. coli and the stages of broiler production, with the concentration increasing significantly between day 2 or 3 to day 14 or 15 and then decreasing significantly by day 26 or 27. However, a definitive association was not found between the concentrations and proportions of airborne E. coli carrying APEC virulence genes and the age of the broiler birds. The daily mortality, temperature and humidity in the broiler sheds were not correlated with the concentrations of airborne E. coli carrying APEC virulence genes over the rearing cycle. Southern blot hybridisation to EcoRV and BglI digests of genomic DNA from a selection of E. coli isolates recovered from the air, and from the internal organs of dead birds further identified genetic diversity in the airborne population and demonstrated the genetic relatedness of lesion isolates, suggesting that the air was not the only source of pathogenic E. coli and that the respiratory tract of infected birds may transmit pathogenic E. coli to neighbouring birds through exhaled air or coughed up droplets. In an outbreak of colibacillosis, as described in Chapter 6, the mean concentrations of all airborne E. coli and of E. coli positive to either the iroB, iucA or hlyF probe, or all three probes, were up to 5 times higher compared to those found in a broiler shed not suffering an outbreak. The proportions of the airborne E. coli positive to any individual gene, except for iucA, or all three genes, were also higher in the broiler flock suffering the outbreak of colibacillosis than in the unaffected flock. Genotypic characterisation using Southern blot hybridisation to EcoRV and BglI digests of genomic DNA from isolates recovered from the air and lesions typical of colibacillosis identified a unique genotype in most of the E. coli recovered from the lesions in dead birds, suggesting contagious transmission of pathogenic E. coli. Future investigations on the possibility of horizontal transmission of APEC and disease potential of APEC genotypes frequently isolated from the lesions will broaden our knowledge and will assist in the control and prevention of colibacillosis in poultry.