Salmonella carriage and change in serovar distribution in broiler giblets at slaughterhouse level in Turkiye: first report using ISO 6579-1:2017 and ISO 6579-3:2014.

This study aimed to determine the prevalence and serovar distribution of salmonellae in liver, heart, and spleen (LHS) and gizzard (G) of slaughtered broilers. For this, a total of 60 sample units, comprised of 30 LHS and 30 G collected from 3 slaughterhouses, were analysed by reference methods for detection and serotyping as revised ISO 6579-1:2017 and ISO 6579-3:2014, respectively. Also, Salmonella-specific real-time PCR (Salm-PCR) was used for species confirmation, while Salmonella Enteritidis (S. Enteritidis) and Salmonella Typhimurium (S. Typhimurium) specific real-time PCR (SE/ST-PCR) was evaluated to determine its efficiency for rapid detection of the serovars mandated in current legal regulations compared to standard serotyping. All LHS (100%-30/30) and 90% (27/30) of G samples harbored Salmonella with an overall prevalence of 95% (57/60) in samples examined, where all isolates were confirmed as Salmonella by Salm-PCR. The most prevalent serovar in broiler giblets was S. Virchow (80.70%-46/57) followed by S. Enteritidis (19.30%-11/57). SE/ST-PCR (%17.54-10/57) could not detect one G isolate, which was serotyped as S. Enteritidis by standard serotyping. High relative accuracy (98.25%), sensitivity (100%) and specificity (100%), and agreement between methods (κ: 0.94) verified SE/ST-PCR's potential to be used as an alternative in rapid detection of S. Enteritidis and S. Typhimurium. Data on high Salmonella prevalence in broiler giblets of slaughterhouse origin, and detection of the pathogen by the implementation of all requirements indicated in the revised ISO 6579-1:2017 standard method, enabling the determination of actual prevalence in the samples with high sensitivity and specificity is of significance for public health. Additionally, identification of S. Virchow as the dominant serovar followed by S. Enteritidis with a relatively lower prevalence, and absence of S. Typhimurium in broiler giblets are important findings for Turkiye. This up to date data, obtained by strict application of ISO 6579-3:2014 procedures, indicated a shift in circulating serovars in the broiler industry. The objective findings in this study would bring awareness to national/international literature, and may be of use in future improvements in legal regulations.

High Salmonella load with serovar virchow dominance pose major public safety risk in postchill broiler carcasses.

The objective of this study was to determine Salmonella contamination levels, presence and serovar distribution in broiler carcasses before and after chilling, as well as to evaluate the effectiveness of chilling process. A total of 96 pooled neck skin samples (PNSS) of 48 prechill (PreC) and 48 postchill (PosC) carcasses, representing 480 broilers collected in 6 mo' period were analyzed using ISO 6579-2:2012 Miniaturized Most Probable Number (ISO-mMPN) technique. Species confirmation and serovar identification was performed by Salmonella-specific real-time PCR (Salm-PCR) and conventional serotyping, respectively. Mean Salmonella count was 1.84 log10 MPN/g in PreC, and 1.48 log10 MPN/g in PosC samples, indicating a statistically significant reduction of 0.36 log10 MPN/g (p < 0.05) in the counts by plant's air chill system. Salmonella positivity reduced from 97.9% (47/48) in PreC to 85.42% (41/48) in PosC samples, confirmed by Salm-PCR with identified serovars as S. Virchow (89.77 %) followed by S. Schwarzengrund (9.09%) and S. Bredeney (1.14%). Persistence of high load and prevalence of Salmonella with serovar Virchow dominance (other than the ones mandated in current guidelines) in the final product contributes significant and up to date data to relevant literature, and provides unbiased epidemiological reference to legal authorities for future relevant revisions.

Salmonella detection in poultry meat and meat products by the Vitek immunodiagnostic assay system easy Salmonella method, a LightCycler polymerase chain reaction system, and the International Organization for Standardization method 6579.

This study was conducted to evaluate the capability of the Vitek immunodiagnostic assay system easy Salmonella (VIDAS ESLM) method and a specific real-time PCR system (LightCycler, LCPCR) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella from a total of 105 naturally contaminated samples comprised of poultry meat and poultry meat products. The detection limit of ISO and LCPCR was 9 cfu/mL for both poultry meat and poultry meat products, whereas that of VIDAS ESLM with both sample types was determined to be 90 cfu/mL. Twelve (33.33%), 11 (30.55%), and 18 (50.00%) out of 36 poultry meat samples were positive for Salmonella by ISO, VIDAS ESLM, and LCPCR, respectively. Salmonella detection rates from poultry meat products were 5.80% for ISO and 8.69% for LCPCR, whereas none of these products tested positive by VIDAS ESLM. In poultry meat samples, VIDAS ESLM and LCPCR detection results were in substantial agreement with ISO, with the relative accuracy, sensitivity, and specificity rates of 97.2, 91.7, and 100%, respectively, for VIDAS ESLM and 83.3, 100, and 75%, respectively, for LCPCR. This is the first report on the evaluation of both VIDAS ESLM and LCPCR to complement ISO for the rapid detection of Salmonella in poultry meat and meat products. We determined that both VIDAS ESLM and LCPCR have the potential to complement the ISO standard culture method in the rapid screening of Salmonella from naturally contaminated poultry meats. For the poultry meat products, VIDAS ESLM and LCPCR can be used for rapid primary screening, and they should be complemented absolutely by ISO. Although LCPCR can preferentially be used for initial screening poultry meat products, the results should definitely be confirmed by ISO. Also, the VIDAS ESLM did not seem to be a suitable method for detecting Salmonella in poultry meat products.

Incidence of Salmonella Enteritidis in chicken layer flocks in Turkey: results by real-time polymerase chain reaction and International Organization for Standardization culture methods.

This study presents Salmonella Enteritidis incidence in chicken layer flocks in Turkey determined by real-time PCR (rPCR) and by International Organization for Standardization (ISO) method 6579:2002/Amd 1:2007. A total of 259 samples, composed of 1,036 individual samples each pooled into 4, including 175 cloacal swab, 14 intestine, 35 gizzard swab, and 35 cecal swab samples, belonging to 6 major companies, were collected from 50 layer flocks and tested by rPCR and ISO culture methods. Overall incidence of Salmonella in layer flocks by rPCR and culture was 61.0 and 55.6%, respectively, where 70.1% of these Salmonella isolates were determined as Salmonella Enteritidis. Incidences of Salmonella Enteritidis in culture-positive samples were 65.3% in cloacal swabs, 50.0% in intestines, 73.9% in gizzard swabs, and 87.5% in cecal swabs. The rPCR results were in 100% agreement (100% sensitivity and specificity) with culture results when cecal swabs were selected as the sample type. The relative accuracy of rPCR was 92.4, 91.4, and 84% for intestine, gizzard, and cloacal swab samples, respectively. As a result, by using rPCR and ISO culture, we determined that the Salmonella Enteritidis incidence in layer flocks in Turkey was high and that the use of cecal swab and intestine samples in Salmonella detection would yield reliable results. To reduce this high Salmonella Enteritidis incidence in layer flocks, Salmonella Enteritidis-specific vaccination should be implemented properly in conjunction with a well-designed biosecurity plan, including verifiable corrective actions.

A capillary polymerase chain reaction for Salmonella detection from poultry meat.

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.

Implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry.

AIMS: The present study describes the implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. METHODS AND RESULTS: Real-time PCR with Salmonella invA-specific primers and a standard bacteriological method was applied to detect Salmonella in tetrathionate enrichment cultures of 492 intestinal homogenates and 27 drag swabs from 47 poultry flocks. The number of positive individual samples by real-time PCR and culture method was 65 (12.5%) and 35 (6.8%), respectively. The number of Salmonella-positive flocks was 13 (27.7%) by both methods. PCR detection required 25 min for up to 32 samples. Melting curve analysis revealed the Tm for Salmonella-specific PCR product as 87 +/- 1 degrees C. CONCLUSIONS: Implementation of real-time PCR to tetrathionate broth enrichment step reduces the Salmonella detection time to 18 h and 25 min. Isolation of Salmonella should be carried out with PCR to determine the serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a powerful tool in rapid and accurate Salmonella monitoring in poultry companies, together with standard bacteriology.

Prevalence of Salmonella serovars in chickens in Turkey.

In this study, 151 (18.6%) of 814 ceca obtained during in-line processing of 28 broiler (Hybro G, Avian, Arbor acres, and Cobb breeds) and 5 layer (Ross, Tetra SL, Isa Brown, and Brown Nick breeds) flocks in Turkey were found to be contaminated with four different Salmonella serovars. Only Salmonella enterica subsp. enterica Serovar Enteritidis (Salmonella Enteritidis) was recovered from layer birds, whereas Salmonella Enteritidis (81.5%). Salmonella Agona (7.6%), Salmonella Thompson (10.1%), and Salmonella Sarajane (0.8%) were isolated from broiler birds. Isolations of Salmonella Agona and Salmonella Thompson from poultry are reported for the first time in Turkey. The isolation of Salmonella Sarajane from chickens is the first report in the world. The standard method of National Poultry Improvement Plan, U.S. Department of Agriculture, was used to detect Salmonella from chicken cecal samples. Primary and delayed secondary enrichments (PE and DSE) were done in tetrathionate-Hajna broth (TTHB). Two different agar media, xylose lysine tergitol 4 (XLT4) and brilliant green with novobiocin (BGN) were used to observe, and compared for their isolation and selective differentiation of, Salmonella-suspected colonies. Isolated salmonellae were then biotyped and serotyped. Ninety-one and 151 salmonellae were isolated with XLT4 agar after PE and DSE, respectively. From the same samples, BGN agar was able to detect only 50 and 131 Salmonella after PE and DSE, respectively. The isolation rate with XLT4 was 11.2% (P < 0.01) with PE, and this rate increased to 18.6% after DSE. Also, the PE isolation rate (11.2%) with XLT4 agar was significantly higher (P < 0.01) than PE with BGN agar (6.1%). Salmonella was isolated from 39.3% (11 of 28) of the broiler flocks and from 60.0% (3 of 5) of the layers. The detection sensitivity of the isolation method was determined as 1 CFU g(-1) experimentally. These data demonstrate the presence of Salmonella Enteritidis, Salmonella Thompson, Salmonella Agona, and Salmonella Sarajane in chicken flocks in Turkey.

Detection of salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis.

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml(-1), respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml(-1), respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.

Cytolethal distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates: detection and analysis by PCR.

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of Campylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni and Campylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between the cdt genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate C. jejuni and C. coli isolates on the basis of restriction patterns within their cdt genes.

Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses.

This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses.

Motile aeromonads in the feces and carcasses of broiler chickens in turkey.

Motile Aeromonas spp. were surveyed in chicken feces, carcasses, scalding water, and chilling water during slaughtering in a commercial processing plant in Turkey. Fecal and carcass samples of 351 chickens from 15 different flocks were examined by selective isolation techniques. All of the 15 flocks were positive for motile aeromonads. Motile aeromonads were detected in 14.8%, 90.5%, and 100% of fecal, carcass, and chilling water samples, respectively, while scalding water samples were negative for these organisms. Aeromonas hydrophila was the predominant species in both feces (51.9%) and carcass (66.9%) samples, followed by A. caviae and A. sobria. These results suggested that during the slaughtering process the spread of motile aeromonads from the intestinal contents to carcasses via processing water caused a heavy contamination of chicken carcasses.

Sulphate-reducing bacteria in bovine faeces.

Sulphate-reducing bacteria (SRB) were found in all of 200 bovine faeces examined. The number of SRB in bovine faeces ranged from 5 x 10(2) to 6 x 10(8) bacteria g-1. Of 50 isolates identified, all were assigned to the genus Desulfovibrio.