Salmonella on pig carcasses: positive pigs and cross contamination in the slaughterhouse.

AIMS: The purpose of this study was to investigate the prevalence of Salmonella in pigs at the moment of slaughter and in the slaughterhouse environment. METHODS AND RESULTS: In total, five different commercial slaughterhouses were sampled during eight slaughterhouse visits. Carcass swabs, colon content and mesenteric lymph nodes were taken to reflect the animal status and from the slaughterhouse environmental samples were taken. Salmonella was isolated from 37% of the carcass samples as a mean value. High variations were noticed between different slaughterhouses (between 0 and 70%) and sampling days in the same abattoir (between 3 and 52%). A correlation was found between the carcass contamination and the status of the delivered animals (P=0.01675). Cross contamination was estimated to account for 29% of the positive carcasses. The slaughterhouse environment was highly contaminated; before starting the slaughtering activities 25% of the samples were positive on average. The most prevalent serotypes isolated at the slaughterhouse environment and from the colon content were S. Typhimurium, S. Livingstone and S. Derby. On carcasses S. Typhimurium was predominately isolated (71%). The biggest variability of serotypes was found in the mesenteric lymph nodes. Serologically 56.3% of the pigs were found positive for Salmonella using a cut-off level of the optical density percentage higher than 10 (O.D.% > or = 10). While on individual pig level the correlation between the bacteriological and serological data was poor, because of recent Salmonella infections, a better correlation was found at the herd level on the moment of slaughtering. CONCLUSION: A high degree of carcass contamination is noticed after slaughtering. This contamination resulted from the delivery of Salmonella-positive pigs and cross-contamination from the slaughterhouse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In pigs, Salmonella carriage is high, but it is obvious that slaughterhouse hygiene is a determinative factor for managing carcass contamination.

Prevalence of Salmonella, Campylobacter and VTEC on pig farms.

Four different pig farms were sampled for the prevalence of Salmonella, Campylobacter and verotoxigenic E. coli (VTEC). Pigs of different age groups and pigs of the same age were tested by taking rectal swabs. The farm environment was tested by examining overshoes, and the feed and drinking water in the pens. From a total of 215 rectal samples of individual pigs, 15 rectal samples taken from animals at the same farm were positive for Salmonella. The Salmonella status of the pigs at this farm differed from one age group to another. S. Typhimurium was isolated from all the positive rectal samples and S. Typhimurium and S. Schwarzengrund were isolated from the environment. On two other farms Salmonella was only present in the environment with S. London and S. Typhimurium as serotypes. The presence of Campylobacter was tested in 150 rectal swabs, 51 of these, spread over the four farms, turned out to be positive. At all four pig farms Campylobacter was isolated from the environment as well. All the strains were identified as Campylobacter coli by a species-specific PCR. To determine if pigs are a reservoir of VTEC a total of 289 samples were screened for the presence of VTEC and 54 strains were isolated that each carried one virulence gene. Thirty-one strains carried the vt2e variant of the vt2 gene wich causes the endema disease in young pigs, four strains harboured the hlyA gene and 19 the eaeA gene.

Differentiation of Brucella species by random amplified polymorphic DNA analysis.

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.

Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR.

The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used.

Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR.

Listeria monocytogenes strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing L. monocytogenes strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints, L. monocytogenes strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of L. monocytogenes was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for L. monocytogenes typing.

Confirmation of presumptive Salmonella colonies by the polymerase chain reaction.

The potential of a genus-specific polymerase chain reaction (PCR) for the confirmation of Salmonella colonies was evaluated on 209 presumptive Salmonella colonies obtained by the standard method ISO 6579. The PCR method employing primers ST11 and ST15 (S. Aabo et al., Mol. Cell. Probes 7:171-178, 1993) gave results identical (100%) to those of the biochemical and serological identification, in terms of discrimination of Salmonella from non-Salmonella strains. PCR could be used directly on the colonies from selective plating media, which allowed a reduction of the time required for confirmation to a maximum of 6 h.

Unidentified Listeria-like bacteria isolated from cheese.

Unidentified Listeria-like bacteria, which lack only one of the phenotypic characteristics used to confirm Listeria spp., were isolated from cheese during routine analysis for Listeria monocytogenes. The VIDAS Listeria assay and the Listeria specific PCR or DNA probe assays used did not identify these strains as Listeria species. This group of bacteria was studied for its homogeneity using rep-PCR and PFGE. Sequence analysis of the 16S rRNA gene showed a homology of 94% to established Listeria spp., implicating a closer relationship than that between Listeria spp. and Brochothrix spp.

Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria.

Repetitive element sequence-based PCR (rep-PCR) was used to generate DNA fingerprints for Listeria spp. Two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2) used in respectively REP- and ERIC-PCR revealed that bacteria of the genus Listeria possess short repetitive extragenic palindromic elements and enterobacterial repetitive intergenic consensus sequences. Specific band profiles obtained by ERIC-PCR enabled the identification of Listeria species. With both REP- and ERIC-PCR the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3b and 4b could be clearly distinguished from each other. Within the serotype 1/2a, REP-PCR showed a higher discriminative potential than ERIC-PCR and a comparable discriminative potential as RAPD combining 3-4 primers.

Repetitive element sequence based polymerase chain reaction for typing of Brucella strains.

A collection of 40 Brucella strains, including 19 clinical B.canis isolates, was analyzed using ERIC-PCR and REP-PCR. PCR amplification using primers REP 1R-I and REP 2-I provided distinct patterns for Brucella spp. All Brucella strains could be discriminated at least to the species level. ERIC-PCR, employing ERIC 1R and ERIC 2 as primers was less discriminative for Brucella, only allowing a discrimination to the genus level with occasional discrimination between individual strains. It can be concluded that rep PCR is a promising fingerprinting method for the evaluation of an outbreak.

Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.

Simultaneous detection of Listeria spp. and Listeria monocytogenes by reverse hybridization with 16S-23S rRNA spacer probes.

Enzymatic amplification results showed that Listeria species have at least two 16S-23S rRNA spacer regions of different lengths. These spacer regions of L. monocytogenes, L. ivanovii and L. seeligeri were cloned after enzymatic amplification. Sequence analysis of the inserts revealed two spacers of 245-246 bp and 496-498 bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. One Listeria spp.-specific probe, LIS-ICG4, was deduced from the 245-bp spacer and a L. monocytogenes-specific probe, LMO-ICG5, was inferred from the 496-bp spacer. The specificity of both probes was tested in a reverse hybridization assay (Line Probe Assay, LiPA). Both LIS-ICG4 and LMO-ICG5 proved to be highly specific when hybridized to a large collection of Listeria strains and strains from other relevant taxa. The LiPA test herein described for the simultaneous detection of Listeria spp. and L. monocytogenes can be expanded to detect other foodborne pathogens.