Salmonella detection in poultry samples. Comparison of two commercial real-time PCR systems with culture methods for the detection of Salmonella spp. in environmental and fecal samples of poultry.

STUDY: The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 - Annex D) for the detection of Salmonella spp. in poultry samples. MATERIAL AND METHODS: Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. RESULTS: Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100cfu/25g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14cfu/25g. Both real- time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. CLINICAL RELEVANCE: In general the advantage of PCR analyses over the culture method is the reduction of working time from 4-5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO6579:2002 - Annex D.

Rapid monitoring of Campylobacter in high-shedding flocks for targeted disease control.

Broilers excreting Campylobacter spp. at high levels (>7 log CFU/g of feces) were described in the Dutch Campylobacter Risk Management and Assessment project as an important source of carcass contamination. The researchers concluded that the risk of infection to humans could be economically and efficiently minimized by eliminating these flocks from fresh poultry meat chains. In the present study, we evaluated a simple and rapid gold-labeled immunosorbent assay (GLISA) for the identification of Campylobacter spp. in flocks shedding high levels of the pathogen. Results were obtained within 2 h. Pooled samples from 102 of the 114 Campylobacter-positive flocks produced positive results, resulting in a test sensitivity of 89.5% (95% confidence interval, 82.6 to 94.2%) and a test specificity of 94.5% (86.7 to 98.2%). Given a GLISA detection limit of 7.3 log CFU/g of feces, nearly all Campylobacter-positive flocks were identified as "high shedders." Therefore, reduction of the incidence of Campylobacter infections by elimination of high-shedding flocks from fresh meat production is an unrealistic approach. Under the constraints given, a reduction in the incidence of Campylobacter spp. in Austria will require either improved hygiene or an intensive carcass decontamination strategy in fresh meat production facilities.

Easy-to-use rapid test for direct detection of Campylobacter spp. in chicken feces.

Human campylobacteriosis is the leading cause of acute bacterial gastroenteritis in developed countries. One important source of infection is poultry. Results from the Dutch Campylobacter Risk Management and Assessment project indicate that meat from broiler flocks shedding >or=7 log CFU Campylobacter per g of feces poses the greatest risk of transmitting campylobacteriosis. The objective of this study was to develop a simple and rapid test that would identify chicken flocks shedding high numbers of Campylobacter. We used lateral flow technology as the alternative test method, and selected the culture method according to International Organization for Standardization guidelines. To evaluate the test under field conditions, we sampled either chicken droppings at farms or cecal contents at the slaughterhouse. PCR was used to confirm presumptive Campylobacter spp. colonies. Under laboratory conditions, chicken feces containing >or=6.7 log CFU/g Campylobacter jejuni or >or=7.1 log CFU/g Campylobacter coli were identified by the lateral flow test. Overall, 3 (33%) of 10, and 29 (85%) of 34 C. jejuni- or C. coli-positive chicken flocks were identified at farms and slaughterhouses, respectively, by using the lateral flow test. Fecal samples containing >or=7.3 log of C. jejuni or C. coli CFU/g as determined by plating were always positive when using the lateral flow test. A single person testing seven flocks at a time could obtain test results within 2 h of sampling. This simple and rapid lateral flow test may contribute significantly to the identification of chicken flocks shedding high numbers of Campylobacter.

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.