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.

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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.

The pathogenesis of <i>C. jejuni</i> in broiler chickens is still poorly understood despite the importance of poultry meat as a source of infection in humans. The overall objective of this project was to understand the role of flagella and Campylobacter invasion antigens in mucosal and systemic colonization, and to evaluate the vaccine potential of <i>C. jejuni</i> paralyzed flagella mutants. As a first step to track <i>C. jejuni in vivo</i>, a Green Fluorescent Protein (GFP) reporter system that is constitutively expressed was constructed. The system was transformed into different <i>C. jejuni</i> strains and isolates, and their mucosal and systemic spreading was studied over the period of 7 days. <i>C. jejuni</i> NCTC11168V1 and V26 share the same background but differ in their ability to colonize chickens. <i>C. jejuni</i> 81-176 and K2-55 share the same genetic background but K2-55 has an insertion mutation in <i>pflA</i> gene that produced paralyzed flagella. Although the K2-55 flagella remained intact structurally, it did not secret <i>Campylobacter</i> invasion antigens (Cia). The reporter system was stable in all of these strains both <i>in vitro</i> and <i>in vivo</i>. Fluorescent bacteria were visualized successfully using fluorescent and confocal microscopes. C. jejuni NCTC11168V1 and 81-176 were detected in the intestinal tract and in the liver and spleen of more than 30% of the challenged birds, while V26 and K2-55 were only detected in the intestinal tract. <i>C. jejuni</i> 81-176 and K2-55 did not spread systemically to the spleen and liver of BALB/c mice challenged using the same approach, although they colonized the ceca.<p> A live attenuated vaccine based on <i>C. jejuni</i> K2-55 protected broiler chickens from <i>C. jejuni</i> 81-176 challenge in chickens following streptomycin treatment of drinking water. The same vaccine had no significant protection against a heterolgous <i>C. jejuni</i> NCTC11168V1 strain challenge. The vaccine was a poor stimulator of secretory IgA.<p> Macrophage-like HD11 cells inflammatory response to the presence of <i>C. jejuni</i> K2-55 was not significantly different from their response to wild-type 81-176 when measured by qRT-PCR. The lack of Cia secretion and motility had no effect on expression of IL-1â, IL-2, IL-6, IL-8, IL, IL-10, IL-12â, or TLR5. A <i>flgK</i> mutant expressing the flagella up to the hook had a significantly lower expression of these genes.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection.

The pathogenesis of <i>C. jejuni</i> in broiler chickens is still poorly understood despite the importance of poultry meat as a source of infection in humans. The overall objective of this project was to understand the role of flagella and Campylobacter invasion antigens in mucosal and systemic colonization, and to evaluate the vaccine potential of <i>C. jejuni</i> paralyzed flagella mutants. As a first step to track <i>C. jejuni in vivo</i>, a Green Fluorescent Protein (GFP) reporter system that is constitutively expressed was constructed. The system was transformed into different <i>C. jejuni</i> strains and isolates, and their mucosal and systemic spreading was studied over the period of 7 days. <i>C. jejuni</i> NCTC11168V1 and V26 share the same background but differ in their ability to colonize chickens. <i>C. jejuni</i> 81-176 and K2-55 share the same genetic background but K2-55 has an insertion mutation in <i>pflA</i> gene that produced paralyzed flagella. Although the K2-55 flagella remained intact structurally, it did not secret <i>Campylobacter</i> invasion antigens (Cia). The reporter system was stable in all of these strains both <i>in vitro</i> and <i>in vivo</i>. Fluorescent bacteria were visualized successfully using fluorescent and confocal microscopes. C. jejuni NCTC11168V1 and 81-176 were detected in the intestinal tract and in the liver and spleen of more than 30% of the challenged birds, while V26 and K2-55 were only detected in the intestinal tract. <i>C. jejuni</i> 81-176 and K2-55 did not spread systemically to the spleen and liver of BALB/c mice challenged using the same approach, although they colonized the ceca.<p> A live attenuated vaccine based on <i>C. jejuni</i> K2-55 protected broiler chickens from <i>C. jejuni</i> 81-176 challenge in chickens following streptomycin treatment of drinking water. The same vaccine had no significant protection against a heterolgous <i>C. jejuni</i> NCTC11168V1 strain challenge. The vaccine was a poor stimulator of secretory IgA.<p> Macrophage-like HD11 cells inflammatory response to the presence of <i>C. jejuni</i> K2-55 was not significantly different from their response to wild-type 81-176 when measured by qRT-PCR. The lack of Cia secretion and motility had no effect on expression of IL-1â, IL-2, IL-6, IL-8, IL, IL-10, IL-12â, or TLR5. A <i>flgK</i> mutant expressing the flagella up to the hook had a significantly lower expression of these genes.

Las bacterias del género Campylobacter han cobrado importancia como patógenas para el hombre, debido principalmente a su asociación con procesos gastroentéricos, a través del contacto con animales infectados y/o alimentos contaminados como fuente principal de contaminación para el hombre que provoca cuadros gastrointestinales ocasionados por este tipo de bacterias, las personas mas susceptibles son los niños menores de cinco años, las personas de edad avanzada y los inmunocomprometidos. Los objetivos del presente trabajo fueron determinar la presencia e incidencia de Campylobacter (Campylobacter jejuni, C. coli, C. lari) como agente etiológico de diarrea aguda, en niños menores de cinco años atendidos en hospitales de Lima (Perú). / The bacteria of the Campylobacter genus have become important as pathogens for humans mainly because of their association to gastroenteric processes. These are caused through contact with infected animals and/or contaminated food as the main source of contamination for humans, which cause gastrointestinal manifestations resulting from this type of bacteria. The most sensitive are children under five, the elderly and the immune deficient people. The objectives of the present work were to determine the presence and incidence of Campylobacter (Campylobacter jejuni, C. coli, C. lari) as etiological agent of acute diarrhea in children under five treated in hospitals in Lima (Perú).

Infection with Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis, causing an estimated 2.1 million cases annually. Although infections with C. jejuni resolve naturally, over 13,000 hospitalizations and 100 deaths are attributed to this organism. Despite these alarming numbers, relatively little is known about C. jejuni pathogenesis, when compared to other enteric pathogens. This dissertation outlines the identification and characterization of a novel virulence factor in C. jejuni, the protein expressed by the Cj1534c gene. Using microarray and RT real time PCR, Cj1534c was found to be greater than 10 fold over expressed in both swine and poultry. Employing immunoelectron microscopy, we determined that at least a subset of the protein is surface localized. Based on the surface localization and up regulation in poultry, colonization studies were performed. Results demonstrate a significant reduction in colonization by a Cj1534c deficient mutant as compared to wild type. In vitro binding assays using both biotic and abiotic surfaces indicate this protein is involved with attachment to surfaces, as well as the invasion of cultured epithelial cells. In vitro findings were confirmed in vivo using swine, with the Cj1534c mutant being highly attenuated as compared to wild type strains. Additionally, the Cj1534c protein was tested for its potential as a vaccine in poultry. Studies demonstrated that Cj1534c recombinantly expressed in a Salmonella expression vector partially protected chickens, reducing cecal colonization three logs as compared to wild type. Taken together, this data demonstrates a major role of Cj1534c in both chicken cecal colonization and infection of swine.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection.