David M. Rollins

Campylobacter jejuni in broiler chickens: colonization and humoral immunity following oral vaccination and experimental infection

A formalin inactivated, Campylobacter jejuni whole cell vaccine, either with or without Escherichia coli heat labile toxin (LT) as a mucosal adjuvant, was administered orally to broiler chickens. Three vaccine trials were performed, differing in the number of vaccinations, and time of administration, as well as the inclusion and dose of LT. The overall reductions of C. jejuni colonization in the vaccinated chickens ranged from 16 to 93% compared with non-vaccinated controls. Enhanced levels of anti-C. jejuni secretory IgA antibodies were demonstrated in vaccinated chickens. Vaccination also appeared to induce an anamnestic response to C. jejuni antigens in the 14-33 kDa range, as demonstrated by Western immunoblots. Interestingly, the inclusion of LT in the vaccine regimen did not appear to boost the immunogenicity of the vaccine. These results are encouraging and suggest that future development of successful oral vaccines for the control of enteropathogenic Campylobacter in poultry is feasible.

Safety and immunogenicity of a prototype oral whole-cell killed Campylobacter vaccine administered with a mucosal adjuvant in non-human primates

The safety and immunogenicity of two prototype oral Campylobacter killed whole-cell (CWC) vaccines were tested in rhesus monkeys. Animals were immunized with a primary two-dose series (days 0 and 14) of vaccine consisting of CWC (10(10) particles/dose) given alone or in combination with 0.5-1000 micrograms of the heat-labile enterotoxin of Escherichia coli as an oral adjuvant (OA). A booster vaccination, 4 weeks after primary immunization, was given to animals receiving CWC alone or supplemented with 0.5, 5 or 50 micrograms of OA. Both CWC and CWC-OA were well tolerated, with no adverse side-effects noted. Campylobacter-specific as well as adjuvant-specific antibody-secreting cells (ASCs) were determined in peripheral blood collected 7 days after each vaccine dose. Campylobacter-specific IgA ASC responses were enhanced by OA in a dose-dependent manner (p = 0.025), while IgG ASC responses were not. Seroconversions (both IgA and IgG) to Campylobacter antigens were also enhanced in monkeys receiving adjuvanted vaccine. No significant booster vaccination effect was observed in circulating ASCs in any of the immunization groups. In vitro T-cell proliferative responses to Campylobacter jejuni antigens were somewhat enhanced in both the CWC and CWC-OA immunization groups. These results demonstrate that CWC-OA is safe and superior to CWC alone in its ability to stimulate both local and systemic Campylobacter-specific IgA and IgG responses in primates and they support its further evaluation in human clinical studies.

Killed Campylobacter elicits immune response and protection when administered with an oral adjuvant

The heat-labile toxin (HLT) of enterotoxigenic Escherichia coli (ETEC) is a potent oral adjuvant. We determined whether the ETEC HLT could be mixed with killed campylobacter to induce an immune response protective upon subsequent challenge with live pathogens. Mice were immunized orally three times with 10(9) sonicated campylobacter with or without 25 micrograms of ETEC HLT, and humoral immune responses in intestinal lavage fluids measured by ELISA. Whereas 10(9) live bacteria induced strong intestinal IgA responses, killed bacteria did not unless ETEC HLT was also added. The magnitude of the antibody response was dependent on the amount of antigen given. The ETEC HLT given with bacteria also induced a potent cross-reaction with cholera toxin. The latter had an adjuvant effect in mice similar to that of ETEC HLT. Protection against colonization was studied in mice and rabbits. In contrast to non-immune animals, those given live organisms or sonicated cells mixed with ETEC HLT quickly cleared homologous, but not heterologous, Lior serotypes of Campylobacter upon challenge. These data show for the first time that ETEC HLT can potentiate an immune response to killed campylobacter that promotes a rapid clearance of live pathogens from the intestine.

Assessment of the Duration of Protection in Campylobacter jejuni Experimental Infection in Humans

ABSTRACT A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 105, 107, or 109 CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 109-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 109-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.

Phylogenetic diversity and position of the genus Campylobacter.

RNA sequence analysis has been used to examine the phylogenetic position and structure of the genus Campylobacter. A complete 5S rRNA sequence was determined for two strains of Campylobacter jejuni and extensive partial sequences of the 16S rRNA were obtained for several strains of C. jejuni and Wolinella succinogenes. In addition limited partial sequence data were obtained from the 16S rRNAs of isolates of C. coli, C. laridis, C. fetus, C. fecalis, and C. pyloridis. It was found that W. succinogenes is specifically related to, but not included, in the genus Campylobacter as presently constituted. Within the genus significant diversity was noted. C. jejuni, C. coli and C. laridis are very closely related but the other species are distinctly different from one another. C. pyloridis is without question the most divergent of the Campylobacter isolates examined here and is sufficiently distinct to warrant inclusion in a separate genus. In terms of overall position in bacterial phylogeny, the Campylobacter/Wolinella cluster represents a deep branching most probably located within an expanded version of the Division containing the purple photosynthetic bacteria and their relatives. The Campylobacter/Wolinella cluster is not specifically includable in either the alpha, beta or gamma subdivisions of the purple bacteria.

DEVELOPMENT AND EVALUATION OF A 24 HOUR METHOD FOR THE DETECTION AND QUANTIFICATION OF LISTERIA MONOCYTOGENES IN MEAT PRODUCTS

A 24-h filter monitor-based test, Listeria-SELeCT, has been developed to quantify Listeria monocytogenes organisms in meat samples with a sensitivity of < or = 1.0 CFU/g. The technique comprises a filter monitor-based system and a colony lift immunoassay to identify and enumerate the target organism. Meat homogenates were centrifuged and the eluate was filtered to trap and immobilize the microorganisms on the filter. Fraser broth was then added to the filter apparatus to allow the organisms to become established overnight and to inhibit contaminants, after which the filters were transferred onto Modified Oxford medium agar, a selective medium for L. monocytogenes. After 10 to 12 h, a colony lift immunoassay was used to confirm and enumerate suspect colonies on the filter. A correlation study between the Listeria-SELeCT method and the most probable number technique showed the Listeria-SELeCT to be considerably more accurate than the most probable number for quantitatively determining the number of viable organisms in meat samples. Because of ease and speed of testing, the Listeria-SELeCT system also provided major advantages over the most probable number technology.

Campylobacter: the new leader in food-borne disease aetiology

In the genus Campylobacter there are 15 species, three of which are significant human pathogens. The organism is a uniquely small, Gram-negative rod found primarily in zoonotic reservoirs from which humans can be infected. Different clinical features ranging from watery diarrhoea to dysentery and subsequent possible sequelae result from infection. Several immunodominant antigens are now recognized and these have been exploited for use in epidemiological typing methods and are gradually being analyzed to determine their role in pathogenesis. Although the nature of Campylobacter pathogenesis remains elusive, some information is now available on adherence, invasion and toxicity. Fortunately, clinical laboratory diagnosis is now routine and numerous molecular characterization procedures are potentially available. Vaccine development is underway, but thus far, significant protection has not been demonstrated. Fluoroquinolones remain the drugs of choice for severe gastroenteritis, although resistance to these antibiotics and to the macrolides, is beginning to emerge.

Development of a Quantitative Methodology to Estimate the Number of Enteropathogenic Campylobacter on Fresh Poultry Products

While there are numerous infectious agents associated with food-borne infection and intoxication, the major pathogens of concern to consumer safety, i.e., cause the greatest threat from disease for humans, are Campylobacter jejuni/coli, Salmonella spp., Escherichia coli 0157:H7, and Listeria monocytogenes 3–6,10,12–14. The currently accepted methodologies for qualitative (and, where available, quantitative) analyses are at best: slow, cumbersome, costly, and manpower intensive. For example, the currently accepted “gold standard” method for quantitation of Salmonella spp. is the most probable number test (MPN), an analytical method with which additional drawbacks are recognized13–15. At best, the MPN provides an estimate of a range of statistically probable numbers, not an exact count, of specific viable target microorganisms contained in a given unit sample. Also, the MPN is only as reliable as the ability of the selective broth medium to support detectable growth of as few as one cell. To obtain this MPN estimate, the sample has to be serially diluted in such a manner that a more dilute sample will result in fewer positive tubes as indicated by growth and specific isolation and confirmation tests for the target organism. These requirements are not readily achievable for the enteropathogenic campylobacters, given their fastidious nature1. Direct spread plating and filtration techniques frequently have been applied to the task of isolating and semi-quantifying campylobacters from environmental sources1,2,7–9,16, but applications for rapid detection and enumeration from food sources have not been developed. Our laboratories’ objective was to develop an alternative approach(es) to the existing methodologies, e.g., MPN, in an effort to quantify the four target pathogens, Campylobacter jejuni/coli, Salmonella spp., Escherichia coli 0157:H7, and Listeria monocytogenes in raw meat and poultry samples.

Evaluation of Colony Lift Immunoblot Methodologies for Specific Enumeration of Enteropathogenic Campylobacter

Continued advances and refinements in isolation techniques combined with increased surveillance and epidemiologic studies, have clearly demonstrated that Campylobacter is one of the leading causes of human diarrhoeal disease3, 13. The vast majority of human cases of campylobacteriosis are a result of sporadic infection3, 10, 12, 14. Case control studies in the United States have estimated that 48 to 70% of these sporadic infections are a direct result of the handling or consumption of raw or undercooked poultry4, 7. Thus, growing public and governmental concern for the safety of retail meat and poultry products has spurred the need for rapid, quantitative methods for detecting human pathogens. However, methodologies that allow for the enumeration of the fastidious Campylobacter spp. from food samples have not been adequately explored. We have developed a colony lift immunoassay (CLI) for the identification and enumeration of Campylobacter jejuni/coli/lari grown on the surface of solid agar medium or filter membranes. With the impending U.S. government implementation of the science-based method of safe food production known as Hazard Analysis and Critical Control Points (HACCP), the CLI utilized in combination with improved retail meat and poultry bacterial sampling techniques (Rollins et al., 1–29), would be an efficient means of monitoring the bioload of C. jejuni/coli/lari in large scale commercial operations.