Molecular tracking, through processing, of Campylobacter strains colonizing broiler flocks.

Many of the poultry flocks produced in the United Kingdom are colonized with Campylobacter, and the intensive nature of poultry processing usually results in contaminated carcasses. In this study, a previously reported molecular oligonucleotide probe method was used to track a specific flock-colonizing strain(s) on broiler carcasses during processing in two United Kingdom commercial poultry processing plants. Five Campylobacter-positive flocks were sampled at four points along the processing line, postbleed, postpluck, prechill, and postchill, and two Campylobacter-negative flocks processed immediately after positive flocks were sampled prechill. flaA was sequenced from Campylobacter strains isolated from these flocks, and strain-specific probes were synthesized. Skin and cecal samples were plated onto selective agar to give individual colonies, which were transferred onto membranes. These were then hybridized with the strain- and genus-specific probes. For all the 5 positive flocks, there was a significant reduction in campylobacters postbleed compared to postpluck but no subsequent fall on sampling pre- and postchill, and the strain(s) predominating on the carcasses throughout processing came from the flock being processed. This indicates that strains from the abattoir environment were not a significant cause of carcass contamination in flocks with well-established campylobacter colonization. However, negative flocks that were preceded by positive flocks were contaminated by strains that did not generally originate from the predominating strains recovered from the ceca of the previous positive flocks. This suggests that the abattoir environment has a significant role in the contamination of carcasses from negative but not fully colonized flocks.

Longitudinal molecular epidemiological study of thermophilic campylobacters on one conventional broiler chicken farm.

Improved understanding of the ecology and epidemiology of Campylobacter in the poultry farm environment is key to developing appropriate farm-based strategies for preventing flock colonization. The sources of Campylobacter causing broiler flock colonization were investigated on one poultry farm and its environment, from which samples were obtained on three occasions during each of 15 crop cycles. The farm was adjacent to a dairy farm, with which there was a shared concreted area and secondary entrance. There was considerable variation in the Campylobacter status of flocks at the various sampling times, at median ages of 20, 26, and 35 days, with 3 of the 15 flocks remaining negative at slaughter. Campylobacters were recoverable from various locations around the farm, even while the flock was Campylobacter negative, but the degree of environmental contamination increased substantially once the flock was positive. Molecular typing showed that strains from house surroundings and the dairy farm were similar to those subsequently detected in the flock and that several strains intermittently persisted through multiple crop cycles. The longitudinal nature of the study suggested that bovine fecal Campylobacter strains, initially recovered from the dairy yard, may subsequently colonize poultry. One such strain, despite being repeatedly recovered from the dairy areas, failed to colonize the concomitant flock during later crop cycles. The possibility of host adaptation of this strain was investigated with 16-day-old chickens experimentally exposed to this strain naturally present in, or spiked into, bovine feces. Although the birds became colonized by this infection model, the strain may preferentially infect cattle. The presence of Campylobacter genotypes in the external environment of the poultry farm, prior to their detection in broiler chickens, confirms the horizontal transmission of these bacteria into the flock and highlights the risk from multispecies farms.

Chemical treatment of animal feed and water for the control of Salmonella.

The control of Salmonella in animal feedstuffs is important, principally to protect the human food chain from contamination by Salmonella derived from infected animals. The transmission of Salmonella from animal feeds to animals, and onward to human food products, has been convincingly documented. This is especially important for chicken breeding and laying flocks and pigs, in view of the consequences of recent or imminent control legislation in the European Union. Animal feed ingredients, particularly animal and plant-derived protein meals, are frequently contaminated with Salmonella either from source or from processing plant, and recontamination in compounding mills is an additional problem. Several complementary strategies have been used to control this feed contamination, and these include a range of chemical treatments. The principal agents used are as follows: organic acids and their salts, formaldehyde, and bacterial membrane disruptors such as terpenes and essential oils. Experimental agents include chlorate compounds. Many products use blends of agents from the same or different chemical groups to achieve synergistic or combination effects. The present review draws upon published and company data to describe the various modes of action and efficacies of different chemical agents delivered in feed or in drinking water against Salmonella occurring in feed or in livestock environments. Reasons for the failure of protection are explored, along with problems in usage such as corrosion and reduced palatability. Given the wide array of products available with contrasting modes of action, the need for standardized tests of efficacy is also discussed.

Real-time PCR approach for detection of environmental sources of Campylobacter strains colonizing broiler flocks.

Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 10(2) to 3.7 x 10(2) CFU/ml in bacterial cultures and 6.6 x 10(2) CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.

Development of a strain-specific molecular method for quantitating individual campylobacter strains in mixed populations.

The identification of sites resulting in cross-contamination of poultry flocks in the abattoir and determination of the survival and persistence of campylobacters at these sites are essential for the development of intervention strategies aimed at reducing the microbial burden on poultry at retail. A novel molecule-based method, using strain- and genus-specific oligonucleotide probes, was developed to detect and enumerate specific campylobacter strains in mixed populations. Strain-specific oligonucleotide probes were designed for the short variable regions (SVR) of the flaA gene in individual Campylobacter jejuni strains. A 16S rRNA Campylobacter genus-specific probe was also used. Both types of probes were used to investigate populations of campylobacters by colony lift hybridization. The specificity and proof of principle of the method were tested using strains with closely related SVR sequences and mixtures of these strains. Colony lifts of campylobacters were hybridized sequentially with up to two labeled strain-specific probes, followed by the generic 16S rRNA probe. SVR probes were highly specific, differentiating down to 1 nucleotide in the target sequence, and were sufficiently sensitive to detect colonies of a single strain in a mixed population. The 16S rRNA probe detected all Campylobacter spp. tested but not closely related species, such as Arcobacter skirrowi and Helicobacter pullorum. Preliminary field studies demonstrated the application of this technique to target strains isolated from poultry transport crate wash tank water. This method is quantitative, sensitive, and highly specific and allows the identification and enumeration of selected strains among all of the campylobacters in environmental samples.