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    Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) producing Escherichia coli in dairy calves from the Canterbury region : a dissertation presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Veterinary Public Health at Massey University, Manawatū Campus Palmerston North, New Zealand
    (Massey University, 2020) Mwenifumbo, Merning
    Antimicrobial resistance (AMR) has become a global public health concern threatening current effective prevention and treatment options. Infections caused by antimicrobial resistance strains are costly, often harder to treat and sometimes fatal. One group of bacteria that contributes to AMR are the extended beta lactamase (ESBL) and AmpC beta lactamase (AmpC) producing E. coli. These are on the critical list of important antibiotic-resistant bacteria of human importance compiled by the World Health Organisation and are a public health concern due to their resistance to an extended range of beta lactams. The main driver for the spread of AMR is the use of antimicrobials in both human and animals. One potential spread is by the feeding of waste milk to calves. Waste milk is the milk that contains antibiotics or other drugs. Waste milk is not recommended as feed for calves due to its association with the development of antibiotic resistance bacteria. Using the culture depended methods and whole genome sequencing, this study aimed to determine the prevalence of ESBL and AmpC producing E. coli isolated from recto-anal mucosal swabs (RAMS) from waste milk fed dairy calves, and to phenotypically and genotypically characterise ESBL and AmpC producing isolates. Recto-anal mucosal swabs samples (n = 40) from waste milk fed dairy calves collected from Canterbury region, New Zealand were screened for antimicrobial resistant E. coli. Fifty-eight percent (23/40) of the calves harboured antibiotic resistant E. coli. 25% (10/40) calves were positive for AmpC producing E. coli and none of the calves were positive for ESBL producing E. coli. The highest prevalence of resistance was observed for tetracycline. PCR and Sanger sequencing revealed that all the AmpC positive E. coli were chromosomal mediated with four mutations in the promoter region of the ampC gene. Whole genome sequencing of eight isolates resistant to both tetracycline and streptomycin revealed additional resistance genes that were not tested phenotypically. Using the Clermont phylogrouping method of E. coli, the AmpC positive, the tetracycline and streptomycin resistant E. coli isolates were distributed among phylogroups B1, C and D. In conclusion, this study revealed the presence of AmpC producing E. coli and other resistance genes in E. coli isolated from waste milk fed calves. Further epidemiological studies are required to determine whether these antibiotic resistant E. coli are associated with waste milk.
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    Persistent contamination of Salmonella, Campylobacter, Escherichia coli and Staphylococcus aureus at a broiler farm in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Albany, New Zealand
    (Massey University, 2017) Castaneda, Kristine Mejia
    The public demand for poultry products has increased over the years due to their health benefits and relatively low cost. Intensive production of poultry in broiler farms gives an opportunity for contamination of the birds, thus creating potential foodborne hazards to consumers. Foodborne cases are therefore extensively monitored to implement mitigating strategies to control the outbreaks. Therefore, the main aim of this project was to determine the prevalence and microbial loads of contaminating Campylobacter spp., Salmonella spp., S. aureus and E. coli, in different locations of four broiler sheds at a selected poultry farm in Auckland New Zealand. Standard microbiological methods and multiplex quantitative polymerase chain reaction (qPCR) were used in the analyses. Swab samples were collected in three cycles from March 2016 to June 2016. During each cycle of the cleaning and disinfection regime, 248 swab samples were collected from feeders, feed loaders, drinkers, fans, vents, annex floor, and wall crevices to determine the extent of contamination before cleaning and after disinfection. The collected samples (n = 744) were analysed for the presence of Salmonella spp. and Campylobacter spp. using standard microbiological methods. Suspected isolates of Salmonella spp. were confirmed by latex agglutination test, whilst Campylobacter spp. was confirmed by both latex agglutination and oxidase tests. The swab samples were also analysed for viable S. aureus and E. coli cell counts using Petrifilm™ plates. Multiplex qPCR was developed and validated to enumerate Salmonella spp. and Campylobacter spp. positive samples. Results of this study showed that all collected samples were contaminated with Salmonella spp., Campylobacter spp., S. aureus and E. coli before performing cleaning. After disinfection, different areas of the shed were still contaminated, posing real danger for infection of the new flock. Crevices and drinkers were the most contaminated areas after disinfection. Organic matter that accumulates in crevices and drinkers during rearing are likely to protect pathogens against disinfectants, which may then contribute to residual contamination and biofilm formation. The ventilation system of the farm was also heavily contaminated. After disinfection, dusts were trapped between the wires of the ventilation screen, making air vents a potential source of contamination in poultry sheds. Feed loaders had higher contamination rates than feeders, even though it was elevated, away from direct contact to birds. When the ventilation system was open, contaminated dusts settle into various areas of the shed, thereby increasing contamination levels before cleaning, thus affecting the efficacy of the disinfectant used. Meanwhile, fans and the annex were less contaminated, indicating that the cleaning regime could effectively disinfect these areas. However, results showed that microbial concentration in the annex was higher after disinfection. This was probably caused by the introduction of pathogens from the outside environment, highlighting the importance of erecting hygiene barriers before entering the main shed. Multiplex qPCR is an important quantification tool due to its ability to detect, identify and quantify multiple pathogens in one assay. The standard curves generated from inoculated samples determined the detection limit to be 3.24 - 8.24 Log10 CFU/mL for Salmonella spp., and 2.97 - 7.97 Log10 CFU/mL for Campylobacter spp. respectively. The agreement of results using the standard and qPCR methods was investigated by comparing S. aureus counts obtained from100 environmental samples through Bland-Altman analysis. The two methods showed agreement, but the qPCR was limited to the detection of S. aureus from 3.5 to 6 Log10 CFU/mL. The concentration of Salmonella spp. and Campylobacter spp. enumerated by multiplex qPCR, had no significant difference between the mean counts of each location before cleaning and after disinfection. Concentration of Salmonella spp. and Campylobacter spp. in the samples subjected to analysis by qPCR post-disinfection, were below the detection limit of the method. However, the qPCR method may be suitable for analysis of samples collected before cleaning. Pre-enrichment of samples analysed post-disinfection is recommended to improve the detection and enumeration of Salmonella spp. and Campylobacter spp. by qPCR analysis.
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    Epidemiological investigations of Shiga toxin-producing Escherichia coli (STEC) O157 And STEC O26 in New Zealand slaughter cattle, and the source attribution of human illness : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Jaros, Patricia
    Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) and related non-O157 STEC strains are enteric pathogens of significant public health concern worldwide, including New Zealand, causing clinical diseases ranging from diarrhoea and bloody diarrhoea to the life-threatening haemolytic uraemic syndrome. Cattle are considered the principal hosts and have been shown to be a source of STEC infection for both foodborne and environmental outbreaks of human diarrhoeal disease overseas. A series of observational studies were conducted to gain knowledge on the epidemiology of STEC O157 and STEC O26 in New Zealand slaughter cattle and assess the relative importance of cattle as a source of domestically-acquired STEC infections in humans. A repeated cross-sectional study conducted on four selected New Zealand beef slaughter plants provided detailed data on the prevalence and concentration of faecal shedding of STEC O157 and STEC O26 in 695 very young calves (4–7 days-old) and 895 adult cattle post-slaughter, identifying calves as more prevalent carriers of STEC. Findings of a subsequent cohort study, the first of its kind, provided evidence that for the 60 calves examined, transportation and lairage was not associated with increase of faecal shedding of E. coli O157 and O26 (STEC and non-STEC) but increase of cross-contamination of hides and carcasses post-slaughter. In a national prospective case-control study, 113 STEC cases and 506 random controls were interviewed for risk factor evaluation. The study findings implicate that environmental and animal contact, but not food, as significant exposure pathways for sporadic STEC infections in humans in New Zealand, and suggest ruminants as the most important source of infection. The molecular analysis of bovine and human STEC O157 isolates provided evidence for the historical introduction of a subset of the globally-circulating STEC O157 strains into New Zealand and ongoing localised transmission of STEC between cattle and humans. These findings will contribute to the development of a risk management strategy for STEC, similar to those already implemented for Campylobacter, Salmonella, and Listeria, which pose a high risk to public health and New Zealand’s access to international markets. Furthermore, risk factors identified in the case-control study will contribute to the design of public health interventions to reduce the incidence of STEC infections in New Zealand.