Infection by Campylobacter is the most notified gastrointestinal disease in New Zealand. Reliable recovery and identification of campylobacters is challenging. Improved and validated methods are needed to increase the power of subtyping and epidemiological studies to trace the sources and transmission routes of Campylobacter. An enrichment-PCR method for the isolation and detection of C. jejuni and C. coli was developed and sensitivity levels determined in 13 environmental matrices, including animal faeces, food and water. Less than ten cells per sample of either C. jejuni or C. coli could be detected, except for rabbit faeces where the minimum number of cells detected per sample was greater than ten cells for C. coli (range 3-32 cells). The sensitivity of the method was comparable to that determined for the conventional methods in the same matrices. Application of the method to retail chicken carcasses (n =204) determined a prevalence of 27.5% C. jejuni and 1% C. coli. River water assays (n = 293) found 55.3% of samples to contain C. jejuni and 4.1% C. coli. Furthermore, the enrichment-PCR assay was shown to identify up to three subtypes in individual water samples. It was proposed that the identification of non-dominant subtypes carried by a chicken carcass may aid the identification of subtypes implicated in human cases of campylobacteriosis. An average of twenty-three C. jejuni isolates from each of ten retail chicken carcass were subtyped by PFGE using the two restriction enzymes SmaI and KpnI. Fifteen subtypes, in total, were identified from the ten carcasses. One subtype was identified on three carcasses. Five carcasses carried a single subtype, three carcasses carried two subtypes each and two carcasses carried three subtypes each. Some of the subtypes carried by an individual carcass were shown to be clonally related raising the question of in vivo recombination events during host passage. Comparison of C. jejuni subtypes from chickens with those isolated from human clinical cases revealed three of the fifteen subtypes correlated with those from human cases. None of the minority subtypes were identified in human case isolate data, suggesting that the lack of identification of non-dominant subtypes from chicken carcasses may not hinder the investigation of campylobacteriosis outbreaks.

No description available.

In a previous study, twenty-five flaA types were detected among 200 Campylobacter jejuni isolates obtained from clinical and poultry meat sources. The most common flaA type detected among poultry isolates was flaA-3 at a frequency of 23%. In contrast, flaA-3 constituted 5% of the clinical isolates. FlaA-15 was detected most frequently among clinical isolates (31%) but rarely among poultry isolates (5%). Purchasers of poultry meat were therefore commonly exposed to flaA-3 yet most of the human infections were due to flaA type 15. The prevalence of different flaA types in poultry and humans might have been due to: FlaA-15 was more virulent for humans than flaA-3 (infection more likely to result). There were more C. jejuni flaA-15 cells on poultry meat (dose effect). Better survival of flaA-15 cells when freeze/thawed or when stored at +4�C (survival in kitchen). Ecological performance of flaA-3 strains in chicken gut better than that of flaA-15 (more flaA -3 cells in gut therefore greater chance of carcass contamination)? Eleven strains representing flaA types 3, 13, and 15 were tested for their ability to invade cultured human epithelial cells (HEp-2). Invasiveness was considered to reflect virulence. FlaA-15 isolates were more invasive in comparison to flaA-3 and flaA-13 isolates (p<0.0001). Washings from chicken portions were cultured to enumerate Campylobacter cells present on the meat. C. jejuni isolates were flaA typed and the numbers were related to FlaA type. A correlation was not detected. The eleven representative strains were used to inoculate 1 cm� sections of chicken skin which were stored at -20�C or +4�C over a five day period. The samples stored at -20�C were thawed and held either overnight at 25�C, overnight at +4�C or for thirty minutes at 25�C. The numbers of viable Campylobacter cells on the sections were determined. Survival ability differed from strain to strain but was not associated with flaA type. The most invasive C. jejuni strain (T1016; flaA-15) and the least invasive strain (Pstau; flaA-3) were assessed for their ability to colonise the intestinal tract of one-day-old chicks. The dynamics of colonisation, after inoculation of the birds with pure cultures or with mixtures, was monitored by real-time quantitative PCR. Strain-specific primers based on the variable region of the nucleotide base sequence of flaA genes were derived for this work. This enabled the individual strains to be enumerated in gut contents from colonized chickens. Both strains could colonise the chick intestinal tract but C. jejuni strain T1016 (flaA-15) could competitively exclude PStau (flaA-3). It was concluded that the higher prevalence of flaA-15 strains among the clinical isolates was due to its higher virulence for humans. In other words, despite a low prevalence of flaA-15 on poultry meat, infection was more likely to result when C. jejuni flaA-15 cells were consumed.

In a previous study, twenty-five flaA types were detected among 200 Campylobacter jejuni isolates obtained from clinical and poultry meat sources. The most common flaA type detected among poultry isolates was flaA-3 at a frequency of 23%. In contrast, flaA-3 constituted 5% of the clinical isolates. FlaA-15 was detected most frequently among clinical isolates (31%) but rarely among poultry isolates (5%). Purchasers of poultry meat were therefore commonly exposed to flaA-3 yet most of the human infections were due to flaA type 15. The prevalence of different flaA types in poultry and humans might have been due to: FlaA-15 was more virulent for humans than flaA-3 (infection more likely to result). There were more C. jejuni flaA-15 cells on poultry meat (dose effect). Better survival of flaA-15 cells when freeze/thawed or when stored at +4�C (survival in kitchen). Ecological performance of flaA-3 strains in chicken gut better than that of flaA-15 (more flaA -3 cells in gut therefore greater chance of carcass contamination)? Eleven strains representing flaA types 3, 13, and 15 were tested for their ability to invade cultured human epithelial cells (HEp-2). Invasiveness was considered to reflect virulence. FlaA-15 isolates were more invasive in comparison to flaA-3 and flaA-13 isolates (p<0.0001). Washings from chicken portions were cultured to enumerate Campylobacter cells present on the meat. C. jejuni isolates were flaA typed and the numbers were related to FlaA type. A correlation was not detected. The eleven representative strains were used to inoculate 1 cm� sections of chicken skin which were stored at -20�C or +4�C over a five day period. The samples stored at -20�C were thawed and held either overnight at 25�C, overnight at +4�C or for thirty minutes at 25�C. The numbers of viable Campylobacter cells on the sections were determined. Survival ability differed from strain to strain but was not associated with flaA type. The most invasive C. jejuni strain (T1016; flaA-15) and the least invasive strain (Pstau; flaA-3) were assessed for their ability to colonise the intestinal tract of one-day-old chicks. The dynamics of colonisation, after inoculation of the birds with pure cultures or with mixtures, was monitored by real-time quantitative PCR. Strain-specific primers based on the variable region of the nucleotide base sequence of flaA genes were derived for this work. This enabled the individual strains to be enumerated in gut contents from colonized chickens. Both strains could colonise the chick intestinal tract but C. jejuni strain T1016 (flaA-15) could competitively exclude PStau (flaA-3). It was concluded that the higher prevalence of flaA-15 strains among the clinical isolates was due to its higher virulence for humans. In other words, despite a low prevalence of flaA-15 on poultry meat, infection was more likely to result when C. jejuni flaA-15 cells were consumed.

Infection by Campylobacter is the most notified gastrointestinal disease in New Zealand. Reliable recovery and identification of campylobacters is challenging. Improved and validated methods are needed to increase the power of subtyping and epidemiological studies to trace the sources and transmission routes of Campylobacter. An enrichment-PCR method for the isolation and detection of C. jejuni and C. coli was developed and sensitivity levels determined in 13 environmental matrices, including animal faeces, food and water. Less than ten cells per sample of either C. jejuni or C. coli could be detected, except for rabbit faeces where the minimum number of cells detected per sample was greater than ten cells for C. coli (range 3-32 cells). The sensitivity of the method was comparable to that determined for the conventional methods in the same matrices. Application of the method to retail chicken carcasses (n =204) determined a prevalence of 27.5% C. jejuni and 1% C. coli. River water assays (n = 293) found 55.3% of samples to contain C. jejuni and 4.1% C. coli. Furthermore, the enrichment-PCR assay was shown to identify up to three subtypes in individual water samples. It was proposed that the identification of non-dominant subtypes carried by a chicken carcass may aid the identification of subtypes implicated in human cases of campylobacteriosis. An average of twenty-three C. jejuni isolates from each of ten retail chicken carcass were subtyped by PFGE using the two restriction enzymes SmaI and KpnI. Fifteen subtypes, in total, were identified from the ten carcasses. One subtype was identified on three carcasses. Five carcasses carried a single subtype, three carcasses carried two subtypes each and two carcasses carried three subtypes each. Some of the subtypes carried by an individual carcass were shown to be clonally related raising the question of in vivo recombination events during host passage. Comparison of C. jejuni subtypes from chickens with those isolated from human clinical cases revealed three of the fifteen subtypes correlated with those from human cases. None of the minority subtypes were identified in human case isolate data, suggesting that the lack of identification of non-dominant subtypes from chicken carcasses may not hinder the investigation of campylobacteriosis outbreaks.

Thesis (Ph. D. microbiology)--Washington State University, May 2008. / Includes bibliographical references.

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (M.S. in microbiology)--Washington State University, May 2009. / Title from PDF title page (viewed on Apr. 12, 2010). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (M.S. in microbiology)--Washington State University, May 2010. / Title from PDF title page (viewed on June 16, 2010). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (M.S. in microbiology)--Washington State University, May 2007. / Includes bibliographical references (p. 32-37).