Norman J. Stern

Biosecurity-Based Interventions and Strategies To Reduce Campylobacter spp. on Poultry Farms

ABSTRACT The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.

Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens.

Campylobacter spp. cause numerous foodborne diseases. Poultry is thought to be a significant source of this zoonosis. Although many interventions designed to control this agent have been researched, none have succeeded. We evaluated a bacteriocin-based treatment to reduce Campylobacter jejuni colonization in poultry. A previously described purified bacteriocin (class IIa; molecular mass, 3,864 Da), secreted by Paenibacillus polymyxa NRRL-B-30509, was microencapsulated in polyvinylpyrrolidone, and 0.25 g of the purified bacteriocin was incorporated into 1 kg of chicken feed. One-day-old chickens were orally challenged and colonized with one of four isolates of C. jejuni, then reared in isolation facilities. Birds were provided ad libitum access to standard broiler starter feed and water for 7 days until 3 days before sampling, when only the treated groups of birds were provided the bacteriocin-emended feed described. In each of the eight (four by two replicates) trials, significant reductions in colonization by C. jejuni were observed (P < or = 0.05). As an example of this highly consistent data, in the first trial, 10 untreated 10-day-old chickens were colonized at a mean log 7.2 + 0.3 CFU/g of feces, whereas none of the 10 bacteriocin-treated 10-day-old chickens were colonized with detectable numbers of C. jejuni. Bacteriocin treatment dramatically reduced both intestinal levels and frequency of chicken colonization by C. jejuni. Feeding bacteriocins before poultry slaughter appears to provide control of C. jejuni to effectively reduce human exposure. This advance is directed toward on-farm control of pathogens, as opposed to the currently used chemical disinfection of contaminated carcasses.

Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin.

An effective bacteriocin was identified and characterized. Lactic acid bacteria were screened against Campylobacter jejuni. One bacteriocin producer, Enterococcus faecium (NRRL B-30746), was studied. The isolate was grown, and the bacteriocin was purified to single-band homogeneity. Biochemical traits indicated that the peptide was a Class IIa bacteriocin, and it was named E 50-52. The bacteriocin had a molecular weight of 3339.7 and an isoelectric point of 8.0. The minimal inhibitory concentrations of E 50-52 against C. jejuni, Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. ranged from 0.025 to 32 microg/mL. In therapeutic broiler trials, oral treatment with E 50-52 reduced both C. jejuni and Salmonella enteritidis by more than 100,000-fold in the ceca, and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens provides a promising alternative to antibiotics.

Identification of a New Source of Campylobacter Contamination in Poultry: Transmission from Breeder Hens to Broiler Chickens

SUMMARY. Campylobacter jejuni, a foodborne pathogen closely associated with market poultry, is considered to be the most frequent agent of human gastroenteritis in the United States. The pathways involved in the contamination of poultry flocks, vertical transmission and/or horizontal transmission, are unclear. In this study, Campylobacter isolates from two independent commercial broiler breeder flocks, as well as from their respective progeny, were characterized and compared by PstI ribotype analysis and by DNA sequence analysis of the short variable region (SVR) of the flaA gene (flaA SVR). Campylobacter isolates originating from one set of breeder hens and the feces from their respective progeny demonstrated identical ribotype patterns as well as identical flaA SVR DNA sequences, thereby suggesting that these isolates were clonal in origin. Ribotype analysis of Campylobacter isolates from the second set of breeder hens and processed carcasses from their offspring resulted in two patterns. Sequence analysis placed these isolates into two closely related groups and one distant group, similar to the ribotype analysis. These results demonstrate that Campylobacter isolates from commercial broiler breeder flocks and from the respective broiler progeny may be of clonal origin and that breeder hens can serve as a source for Campylobacter contamination in poultry flocks.

Isolation of Bacillus circulans and Paenibacillus polymyxa Strains Inhibitory to Campylobacter jejuni and Characterization of Associated Bacteriocins

We evaluated anti-Campylobacter activity among 365 Bacillus and Paenibacillus isolates from poultry production environments. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and further studied. Cell-free ammonium sulfate precipitate (crude antimicrobial preparation) was obtained from each candidate culture. Zones of Campylobacter growth inhibition surrounding 10 microl of this crude antimicrobial preparation were quantified using a spot test. Campylobacter growth resumed when the preparation was preincubated with selected protease enzymes, demonstrating peptide characteristics consistent with a bacteriocin. These peptides were further purified using combinations of molecular mass resolution and ion exchange chromatography. Molecular masses of the peptides were estimated at approximately 3,500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing was used to determine the pI values of the peptides. Amino acid sequences of the bacteriocins and more precise molecular masses were obtained by matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) analysis. The bacteriocin from P. polymyxa NRRL B-30507 had a pI of 4.8, that from P. polymyxa NRRL B-30509 had a pI of 7.2, that from P. polymyxa NRRL B-30508 had a pI of 4.8, and that from B. circulans NRRL B-30644 had a pI of 7.8. The amino acid sequences were consistent with those of class IIa bacteriocins. These antagonists and the corresponding bacteriocins may be useful in the control of Campylobacter infection in poultry.

Lack of evidence for vertical transmission of Campylobacter spp. in chickens.

ABSTRACT Campylobacter jejuni is a major cause of bacterial food-borne infection in the industrial world. There is evidence that C. jejuni is present in eggs and hatchery fluff, opening the possibility for vertical transmission from hens to progeny. Poultry operations in Iceland provide an excellent opportunity to study this possibility, since breeding flocks are established solely from eggs imported from grandparent flocks in Sweden. This leaves limited opportunity for grandparents and their progeny to share isolates through horizontal transmission. While Campylobacter was not detected in all grandparent flocks, 13 of the 16 egg import lots consisted of eggs gathered from one or more Campylobacter-positive grandparent flocks. No evidence of Campylobacter was found by PCR in any of the 10 relevant quarantine hatchery fluff samples examined, and no Campylobacter was isolated from the parent birds through 8 weeks, while they were still in quarantine rearing facilities. After the birds were moved to less biosecure rearing facilities, Campylobacter was isolated, and 29 alleles were observed among the 224 isolates studied. While three alleles were found in both Sweden and Iceland, in no case was the same allele found both in a particular grandparent flock and in its progeny. We could find no evidence for vertical transmission of Campylobacter to the approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks. If vertical transmission is occurring, it is not a significant source for the contamination of chicken flocks with Campylobacter spp.

Mucosal competitive exclusion to reduce Salmonella in swine.

A mucosal competitive exclusion culture has been shown to reduce or eliminate Salmonella spp. in poultry. Using similar techniques, a mucosal competitive exclusion culture from swine (MCES) was produced from the cecum of a 6-week-old pig. Suckling pigs were inoculated with 5 ml of MCES by oral gavage within 6 h postfarrowing (PF) and again at 24 h PE All pigs were challenged with 10(3) CFU of Salmonella Choleraesuis at 48 h PF by intranasal instillation, including pigs from two sows that had not been given MCES. Clinical signs and rectal swabs were monitored daily, and pigs were allowed to suckle throughout the experiment. All pigs underwent necropsy on day 7 PF, and presence of Salmonella was determined in both qualitative (10 tissues) and quantitative (two tissues) samples. Clinical signs were inapparent in all pigs throughout the experiment. Recovery of Salmonella from rectal swabs was variable. However, 28% of the gut tissues were positive from the MCES-treated pigs versus 79% from the control pigs. A 2- to 5-log10 reduction of Salmonella in the cecal contents or ileocolic junction was observed in the MCES-treated pigs when compared with the controls. These data indicate that use of MCES may be a useful approach for control of Salmonella.

Broiler Campylobacter Contamination and Human Campylobacteriosis in Iceland

ABSTRACT To examine whether there is a relationship between the degree of Campylobacter contamination observed in product lots of retail Icelandic broiler chicken carcasses and the incidence of human disease, 1,617 isolates from 327 individual product lots were genetically matched (using the flaA short variable region [SVR[) to 289 isolates from cases of human campylobacteriosis whose onset was within approximately 2 weeks from the date of processing. When there was genetic identity between broiler isolates and human isolates within the appropriate time frame, a retail product lot was classified as implicated in human disease. According to the results of this analysis, there were multiple clusters of human disease linked to the same process lot or lots. Implicated and nonimplicated retail product lots were compared for four lot descriptors: lot size, prevalence, mean contamination, and maximum contamination (as characterized by direct rinse plating). For retail product distributed fresh, Mann-Whitney U tests showed that implicated product lots had significantly (P = 0.0055) higher mean contamination than nonimplicated lots. The corresponding median values were 3.56 log CFU/carcass for implicated lots and 2.72 log CFU/carcass for nonimplicated lots. For frozen retail product, implicated lots were significantly (P = 0.0281) larger than nonimplicated lots. When the time frame was removed, retail product lots containing Campylobacter flaA SVR genotypes also seen in human disease had significantly higher mean and maximum contamination numbers than lots containing no genotypes seen in human disease for both fresh and frozen product. Our results suggest that cases of broiler-borne campylobacteriosis may occur in clusters and that the differences in mean contamination levels may provide a basis for regulatory action that is more specific than a presence-absence standard.

Counts of Campylobacter spp. on U.S. Broiler Carcasses

Foodborne Campylobacter-associated gastroenteritis remains a public health concern, and the Centers for Disease Control and Prevention suggests that improperly handled poultry is the most important source of this human disease. In response to these concerns, 10 of the largest U.S. poultry integrators cooperatively determined the incidence and counts of Campylobacter on processed broiler carcasses. Prior to conducting the survey, laboratory personnel were trained in a direct Campy-Cefex plating procedure for enumeration of the organism. Before and after the survey enumeration, consistency in reporting was compared among the participating laboratories. Participating laboratories were able to consistently estimate inoculated concentrations of Campylobacter in carcass rinses. Within the central study, we determined the potential exposure of U.S. consumers to Campylobacter spp. associated with broiler carcasses during a 13-month period. Among each of the 13 participating poultry complexes, rinses from 25 randomly selected fully processed carcasses were sampled monthly from individual flocks. Among 4200 samples, approximately 74% of the carcasses yielded no countable Campylobacter cells. Campylobacter spp. were isolated from approximately 3.6% of all commercially processed broiler carcasses at more than 10(5) CFU per carcass. Acceptable counts of these organisms on raw poultry carcasses remain to be determined. Nevertheless, this survey indicates industry recognition of its responsibility to assess and reduce public exposure to Campylobacter through broiler chickens.

Direct Polymerase Chain Reaction Detection of Campylobacter spp. in Poultry Hatchery Samples

SUMMARY. A rapid, sensitive, and specific polymerase chain reaction (PCR) assay was developed for the direct detection of Campylobacter in environmental samples from hatcheries. PCR, with a set of primers specific for the Campylobacter flaA short variable region (SVR), detected the presence of Campylobacter in both fluff and eggshell samples; however, a determination of whether the organism was living or dead could not be made. Conventional cultural methods detected no Campylobacter from the same samples. An additional benefit of the direct PCR assay is it allows for the production of a product that can be sequenced to provide further epidemiologic information.