Carcass Swabs Research Articles

Occurrence and characteristics of fastidious Campylobacteraceae species in porcine samples

This study investigated the prevalence and characteristics of Campylobacteraceae including a range of fastidious species in porcine samples. Over a thirteen month period caecal contents (n=402) and pork carcass swabs (n=401) were collected from three pork abattoirs and pork products (n=399) were purchased at point of sale in the Republic of Ireland. Campylobacteraceae isolates were recovered by enrichment, membrane filtration and incubation in antibiotic free media under a modified atmosphere (3% O2, 5% H2, 10% CO2 and 82% N2). Campylobacteraceae isolates were identified as either genus Campylobacter or Arcobacter and then selected species were identified by Polymerase Chain Reaction (PCR). Campylobacteraceae were isolated from 103 (26%) caecal samples, 42 (10%) carcass swabs, and 59 (15%) pork products. Campylobacter coli was the most commonly isolated species found in (37%) all sample types but many fastidious species were also isolated including Campylobacter concisus (10%), Arcobacter butzleri (8%), Campylobacter helveticus (8%), Campylobacter mucosalis (6%), Arcobacter cryaerophilus (3%), Campylobacter fetus subsp. fetus (1%), Campylobacter jejuni subsp. jejuni (1%), Campylobacter lari (0.5%), Campylobacter curvus (0.5%) and Arcobacter skirrowii (0.5%). Among all isolates, 83% contained cadF and 98% flaA. In this study 35% of porcine C. coli were resistant to ciprofloxacin but none of the fastidious species demonstrated any resistance to this drug. The level of resistance to erythromycin was very high (up to 100%) in C. concisus and C. helveticus and this is a real concern as this is the current empiric drug of choice for treatment of severe gastroenteritic Campylobacter infections. The study shows that there is a much wider range of fastidious Campylobacteraceae present in porcine samples than previously assumed with C. concisus the second most common species isolated. The majority of fastidious Campylobacteraceae isolates obtained contained virulence genes and antibiotic resistance indicating potential public health significance.

Disinfectant and Antibiotic Susceptibility Profiles of Escherichia coli O157:H7 Strains from Cattle Carcasses, Feces, and Hides and Ground Beef from the United States

The disinfectant and antibiotic susceptibility profiles of 344 Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States were determined. A low prevalence of antibiotic resistance was observed (14%). The highest prevalences of resistance were to sulfisoxazole (10.5%), tetracycline (9.9%), streptomycin (7%), and chloramphenicol (4.9%). Four strains were resistant to eight antibiotics (two strains from ground beef and one strain each from hide and preevisceration carcass swabs of cull cattle at harvest). Pulsed-field gel electrophoresis analysis of the E. coli O157:H7 strains revealed two major groups (designated 1 and 2) composed of 17 and 20 clusters, respectively. Clusters 1A, 1B, 1C, and 1G.1 were associated with multidrug-resistant strains. There was no observed correlation between disinfectant resistance and antibiotic resistance. Sixty-nine (20%) of the 344 strains were resistant to chlorhexidine or benzalkonium chloride or the MICs of benzyldimethyldodecylammonium chloride were elevated. Inducible resistance was observed at elevated concentrations of antibiotics (1.4%) and disinfectants (6.1%). The highest rate of disinfectant inducible resistance was to OdoBan, quaternary ammonium chlorides, and the surface disinfectants F25, FS512, and MG, which are used in dairies, restaurants, and food processing plants. High MICs (1,024 to 4,096 m g/ml) of acetic, lactic, and citric acids were found. The decreasing order of acid potency based on molar MICs (MICs(molar)) was acetic, citric, and lactic acid. The correlation of the concentration of dissociated organic acids and MICs(molar) strongly suggests that the observed inhibition of E. coli O157:H7 was primarily due to dissociated forms of the acids.

Study on Carcass Contaminating Escherichia coli in Apparently Healthy Slaughtered Cattle in Haramaya University Slaughter House with Special Emphasis on Escherichia coli O157:H7, Ethiopia

A cross sectional study was conducted from October 2010 to March 2011, on apparently healthy slaughtered cattle in Haramaya University Slaughter House to determine prevalence of Escherichia coli, with special emphasis on Escherichia coli O157:H7, and its antimicrobial susceptibility pattern. Carcass swab samples were collected and pre-enriched in buffered peptone water and plated on MacConkey agar plate. Presumptive colonies were confirmed by biochemical tests. Further identification of Escherichia coli O157:H7 was done by plating the isolatedm bacteria on Sorbitol MacConkey agar; and then colorless colonies were subjected to Escherichia coli O157:H7 Latex agglutination test. From a total of 113 samples collected, Escherichia coli was isolated from 35 (30.97%) and out of these, 3 (2.65%) were found to be Escherichia coli O157:H7. The difference in prevalence was not statistically significant (P>0.05) between local and cross breeds (χ2=0.11, df =4), among age group of animals (χ2=2.56, df=1) and origin of animals (χ2=2.56, df=2). The isolated bacteria were subjected to antimicrobial susceptibility testing and the majority were found to be susceptible to Chloramphenicol (30 μg), Kanamycin (30 μg), Spectinomycin (SH, 100 μg). The presence of E. coli O157:H7 in raw meats reaching to consumers indicated possible risks of infection to people through the consumption of raw (undercooked) meat and cross contamination of other food products. Therefore, control measures at all stages of food chain was recommended.

Transfer of verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 from fleece to carcass during sheep slaughter in an Irish export abattoir

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from fleece to dressed carcasses of 500 sheep, and to establish the virulence potential of recovered VTEC. Individual sheep were tracked and sampled (10 g fleece, full carcass swab) through the slaughter process. Samples were examined for the presence of verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay and positive samples were further screened for the presence of the above five serogroups by real-time PCR. VTEC cells were recovered from PCR positive samples by serogroup specific immunomagnetic separation and confirmed by serogroup specific latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR and isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). VTEC O26 was recovered from 5/500 (1.0%) fleece and 2/500 (0.4%) carcass samples. VTEC O157 was isolated from 4/500 (0.8%) fleece samples and 3/500 (0.6%) carcass samples. E. coli O103 was recovered from 84/500 (16.8%) fleece and 68/500 (13.6%) carcasses, but only one E. coli O103 isolate (0.2%) carried vt genes. E. coli O145 was recovered from one fleece sample, but did not carry vt genes. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from fleece to carcass was not observed with PFGE showing that VTEC O26 isolates from a matched fleece/carcass “pair” were not identical. This study shows that while VTEC O157 are being carried by sheep presented for slaughter in Ireland, other potentially clinically significant verotoxin producing strains (particularly VTEC O26) are emerging.

Detecção de fatores de virulência em estirpes de Campylobacter spp. isoladas de carcaças de suínos abatidos em frigoríficos

Isolaram-se estirpes de Campylobacter spp. em amostras de carcaças (n=65), fezes (n=65) e linfonodos mesentéricos (n=65) de suínos abatidos em frigoríficos do estado de São Paulo e detectaram, pela técnica da Multiplex-PCR, a presença do complexo de genes cdt, responsáveis pela expressão do fator de virulência da toxina CDT. Do total de 195 amostras de origem suína, Campylobacter spp. foi isolado de 31 (15,9%), sendo 29 (93,6%) de amostras de suabe retal, 1/65 (3,2%) de suabe de carcaça e um (3,2%) de linfonodo. Vinte e oito estirpes de C. coli foram positivas para a detecção dos genes cdt, e três estirpes de C. jejuni foram negativas para a detecção desses genes. Foi detectada, pela primeira vez no estado de São Paulo, a presença dos genes cdt em 100% das estirpes de Campylobacter coli provenientes de suínos abatidos em frigoríficos.

Caracterização molecular e fenotípica de estirpes de Escherichia coli produtoras de shiga-toxina (STEC) não-O157 de fezes e carcaças bovinas

Foram coletados 100 suabes retais e 100 suabes de carcaças bovinas em matadouros do estado de São Paulo, e um total de 326 estirpes de E. coli foram identificadas, sendo 163 de amostras retais e 163 de amostras de carcaça. Todos os isolados submetidos à PCR para detecção dos genes das toxinas Stx1 e Stx2 foram identificados como não-O157 e fenotipados pelo teste da citotoxicidade em células Vero. Das 26 estirpes que apresentaram apenas o gene stx1, das 56 que apresentaram apenas o gene stx2 e das 30 estirpes que apresentaram ambos os genes, 17 (65,4%), 42 (75%) e 22 (73,3%), respectivamente, foram positivas ao teste de citotoxicidade. Não houve diferença estatística entre os três perfis genéticos e na positividade ao teste de citotoxicidade. Os resultados mostram a alta frequência de expressão dos fatores de virulência das STEC de bovinos.

Quantification of Campylobacter in Swine before, during, and after the Slaughter Process

Campylobacter has been implicated as a major cause of foodborne illness worldwide. Pigs can be subclinically infected, and fecal contamination of meat during slaughter is a food safety risk. The objective of this study was to determine the association between the concentration of Campylobacter pre- and periharvest with postharvest contamination in swine. Samples were collected from 100 individually identified swine during the pre-, peri-, and postharvest periods. For each animal, the following phases were sampled: on farm (fecal sample), in lairage (hide swab), post-stunning and exsanguination (rectal contents), prechilling (carcass swab), and final product (rib meat) sample. The proportions of samples that were Campylobacter positive were 90, 95, 76, 100, and 49% for fecal, rectal content, hide, carcass, and rib meat samples, respectively. The mean Campylobacter concentrations for each sample were fecal sample, 1.7 × 10(6) CFU/g; rectal content, 1.2 × 10(7) CFU/g; hide swab, 1.4 CFU/cm(2); carcass swab, 1.7 × 10(3) CFU per half carcass; and rib meat, 18 CFU/g. There was a positive correlation between Campylobacter concentrations in fecal samples (R = 0.20, P = 0.065) and concentration of Campylobacter on rib meat, and between rectal content sample concentration (R = 0.20, P = 0.068) and the concentration on rib meat. There was no association between the isolation of Campylobacter on rib meat and the isolation of Campylobacter at any pre- or periharvest stage. This could indicate that the risk of a meat product being contaminated is associated with pigs that shed higher concentrations of Campylobacter before slaughter.

Detection of Salmonella in Meat: Comparative and Interlaboratory Validation of a Noncomplex and Cost-Effective Pre-PCR Protocol

Cost-effective and rapid monitoring of Salmonella in the meat production chain can contribute to food safety. The objective of this study was to validate an easy-to-use pre-PCR sample preparation method based on a simple boiling protocol for screening of Salmonella in meat and carcass swab samples using a real-time PCR method. The protocol included incubation in buffered peptone water, centrifugation of an aliquot, and a boiling procedure. The validation study included comparative and interlaboratory trials recommended by the Nordic Organization for Validation of Alternative Microbiological Methods (NordVal). The comparative trial was performed against a reference method (NMKL 187, 2007) and a PCR method previously approved by NordVal with a semiautomated magnetic bead-based DNA extraction step using 122 artificially contaminated samples. The LOD was found to be 3.0, 3.2, and 3.4 CFU/sample for the boiling, magnetic bead-based, and NMKL 187 methods, respectively. When comparing the boiling method with the magnetic beads, the relative accuracy (AC), relative sensitivity (SE), and relative specificity (SP) were 98, 102, and 98%, respectively (Cohen's kappa index 0.95). When comparing results obtained by the boiling to the culture-based method, the AC, SE, and SP were found to be 98, 102, and 98%, respectively (kappa index 0.93). In the interlaboratory trial including valid results from 11 laboratories, apart from two false-positive samples by the boiling method combined with PCR, no deviating results were obtained (SP, SE, and AC were 100, 95, and 97%, respectively). This test is under implementation by the Danish meat industry, and can be useful for screening of large number of samples in the meat production, especially for fast release of minced meat with a short shelf life.

Tracking verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 in Irish cattle

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge.

Prevalence ofClostridium difficileToxin Genes in the Feces of Veal Calves and Incidence of Ground Veal Contamination

A study was conducted in two parts to determine the prevalence of toxigenic Clostridium difficile in veal calves and retail meat. The first part of the study focused on the veal production continuum (farm to abattoir). Fifty calves from 4 veal herds (n=200) were followed for 18-22 weeks from the time of arrival on the veal farm to the time of slaughter. Fecal samples were collected from calves every 4-6 weeks. Half of the calves included in the study (n=100) were followed to the abattoir where carcass swabs were collected post slaughter. Fecal samples and carcass swabs were screened for genes encoding C. difficile toxins TcdA, TcdB, and CDT by using real-time polymerase chain reaction (PCR). Carcass swabs were also screened for toxigenic C. difficile by using traditional culture methods. In the second part of the study, ground veal products (n=50 samples) purchased from local grocery stores were examined for toxigenic C. difficile by using real-time PCR and traditional culture methods. Fecal samples from 56 of 200 (28%) calves tested positive for C. difficile toxin genes at least once over the course of the study. Calf age (p=0.011) influenced prevalence of C. difficile toxin genes in calf feces. Toxin genes of C. difficile were detected in one carcass swab by multiplex real-time PCR only. Toxigenic C. difficile was detected by PCR and culture in four (8%) and three (6%) ground veal samples, respectively. The findings of the study reveal that toxigenic C. difficile was most prevalent in veal calves (12%) just before slaughter, although viable toxigenic C. difficile was not recovered from veal carcasses. On the contrary, viable toxigenic C. difficle was recovered from 6% retail meat, thus suggesting that contamination occurs either during or after veal fabrication.

Prevalence and antimicrobial resistance profiles of Salmonella enterica serovars isolated from slaughtered cattle in Bahir Dar, Ethiopia

A study was undertaken from October 2006 to March 2007 to determine the prevalence and antimicrobial resistance patterns of Salmonella serovars. Liver, mesenteric lymph nodes, intestinal content, and carcass swab samples (each n = 186) were collected from 186 apparently healthy slaughtered cattle at Bahir Dar abattoir. Bacteriological analysis was done according to the International Organization for Standardization (ISO 6579 2002). Isolates were serotyped at Agence Française de Securite Sanitaire des Aliments, Cedex, France. Twenty-eight isolates consisting of Salmonella Typhimurium, Salmonella Newport, Salmonella Haifa, Salmonella Heidelberg, Salmonella Infantis, and Salmonella Mishmarhaemek were identified. Salmonella Typhimurium and Salmonella Newport were most frequently isolated while Salmonella Heidelberg and Salmonella Mishmarhaemek were isolated least. Eleven of the 28 (39.3%) were resistant to one or more of the antimicrobials tested. Resistance was shown to ampicillin, chloramphenicol, gentamycin, norfloxacin, polymyxin-B, streptomycin, tetracycline, and trimethoprim. Four of 11 (36.4%) were multiple antimicrobial resistant. All the isolates tested were susceptible to the antimicrobial effects of gentamycin, norfloxacin, and trimethoprim. Eleven, four, and two isolates of the 28 were resistant to streptomycin, tetracycline, and ampicillin, respectively. All isolates of Salmonella Infantis, Salmonella Typhimurium (except one), and Salmonella Mishmarhaemek were susceptible to the tested antimicrobials. One Typhimurium isolate was resistant to chloramphenicol, streptomycin, and tetracycline. Salmonella Haifa was multiply antimicrobial resistant to ampicillin, tetracycline, and streptomycin. All isolates of Salmonella Heidelberg were resistant to streptomycin. Results of this study indicated high level of carcass contamination with antimicrobial-resistant Salmonella serovars which could pose public health risk; suggests need for hygienic slaughtering operations and proper cooking of meat before consumption. Further detailed studies involving different abattoirs, animal products, food items, and animals on different settings were recommended in the study area.

The role of transport, lairage and slaughter processes in the dissemination of Salmonella spp. in pigs in Ireland

Salmonella is an important foodborne pathogen worldwide and is commonly isolated from pigs and pig products in Ireland. Pigs, reared in an environment free of Salmonella spp. or with low levels of infection, may acquire infection or become contaminated during transport, lairage or post-slaughter. The main objective of this study was to determine the role of the abattoir as a potential factor that contributes to the dissemination of Salmonella spp. in slaughter pigs from herds with a low Salmonella seroprevalence (≤10%). A total of 128 pigs from eight herds were monitored from farm through the slaughter process in three separate abattoirs. The prevalence of Salmonella spp. was determined in samples collected from trucks, lairage pens and the slaughterline before pigs entered, from pigs after slaughter (caecal contents and ileocaecal lymph nodes) and carcass surfaces post-evisceration. Isolates were characterised by serotype, phage type and pulsed-field gel electrophoresis (PFGE) patterns. Of the swabs taken from the trucks, lairage and slaughterline, before the pigs entered, 4.3% (3/70), 80% (64/80) and 16.7% (4/27) were positive for Salmonella spp., respectively. The proportion of pigs showing serological evidence of infection was 3.1% (4/128). Salmonella spp. were isolated from the ileocaecal lymph nodes and caecal contents of 14.8% (19/128) and 11.7% (15/128) of pigs, respectively, and 13/128 (10.2%), 5/128 (3.9%), 2/111 (1.8%) and 8/111 (7.2%) carcass swabs pre wash, post wash, post chill and belly-strip samples, respectively, were Salmonella-positive. There was only slight agreement between serological and bacteriological data at the pig level. Salmonella isolates from 45% of all positive pig samples and 82% of positive carcass samples were indistinguishable, based on PFGE patterns, from salmonellae isolated from the lairage and slaughterline. Based on these results it is concluded that the lairage and the slaughterline provide a substantial source for Salmonella contamination of pigs and carcasses.

Salmonella Serovars and Antimicrobial Resistance Profiles in Beef Cattle, Slaughterhouse Personnel and Slaughterhouse Environment in Ethiopia

The present study was undertaken to determine the occurrence, distribution and antimicrobial resistance profiles of Salmonella serovars in slaughter beef cattle, slaughterhouse environment and personnel engaged in flaying and evisceration during slaughtering process. A total of 800 samples (each sample type, n = 100) consisting of swabs from hides, slaughterhouse personnel hands at flaying and evisceration, rumen and caecal contents, mesenteric lymph nodes, carcasses and holding pens were collected. Of the total 100 beef cattle examined, 14% were Salmonella positive in caecal content and/or mesenteric lymph nodes. Of the various samples analysed, Salmonella was detected in 31% of hides, 19% of rumen contents, 8% of mesenteric lymph nodes, 6% of caecal contents, 2% of carcass swabs, 9% of palm swabs taken from the hands of personnel in the slaughterhouse during flaying (7%) and evisceration (2%), and in 12% of holding pen swabs. The Salmonella isolates (n = 87) belonged to eight different serovars of which S. Anatum (n = 54) and S. Newport (19) were the major serovars and both serovars were detected in all sample sources except in carcass swabs. Eighteen of the 87 (20.7%) Salmonella serovars consisting of Newport (n = 14), Anatum (n = 3) and Eastbourne (n = 1) were resistant to one or more antimicrobials. Among the antimicrobial resistant Salmonella serovars, S. Newport was multidrug resistant (15.6%) and exhibited resistance to streptomycin, sulphisoxazole and tetracycline.

Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 experimentally inoculated in beef carcass swabs, beef trim, and ground beef.

Numerous foodborne outbreaks are attributed to Shiga toxin-producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 10(2), and 10(3) CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 10(2), and 10(3) CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 10(2) CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STEC in irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n=407) and pooled carcass swabs (n=407) from 5 animals belonging to the same herd or flock and minced meat (n=91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n=77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection.

Phenotypic and Genotypic Heterogeneity of Campylobacter coli Within Individual Pigs at Farm and Slaughter in the US

The aim of this study was to determine the phenotypic and genotypic diversity of multiple Campylobacter isolates (n = 3 per sample) present within individual (heterogeneity) pig faecal and carcass samples at farm and slaughter, respectively. We isolated 1459 Campylobacter coli (1110 on farm and 349 from slaughter) from 908 pigs and 757 carcasses and characterized them for their antimicrobial susceptibility profile to a panel of six antimicrobials using the agar dilution method. Overall, we detected a significantly higher Campylobacter prevalence at the farm (54.7%) than at slaughter (19%) level (P < 0.05). C. coli isolates were resistant most commonly to tetracycline (66.2%) and erythromycin (53.6%) while fluoroquinolone resistance was detected in isolates (n = 17) only from the farm level. Phenotypic diversity of C. coli isolates at the 4-fold minimum inhibitory concentration levels within the same sample was detected in 38.6% (n = 192) pigs and 40.2% (n = 58) carcass swabs with no significant difference between the two sources (P = 0.72). Phenotypic heterogeneity based on the antimicrobial resistance patterns was observed in 32.5% (n = 162) of the farm samples and in 30.5% (n = 44) carcass swabs at slaughter (P = 0.64). A subset of 40 isolates representing ten pigs and eight carcass samples (originating from separate pigs) were further genotyped by multi locus sequence typing. The observation of phenotypic diversity was replicated at the genotypic level, as it was highlighted by the 22 sequence types which represented the 40 isolates. In conclusion, we detected multiple C. coli subtypes from individual pig or carcass samples indicating unprecedented level of heterogeneity. Our study clearly signifies the importance of testing multiple colonies to make appropriate and valid conclusions in epidemiological-based studies.

Description of Extended Pre‐Harvest Pig Salmonella Surveillance‐and‐Control Programme and its Estimated Effect on Food Safety Related to Pork

Salmonella in pork can be combated during pre- or post-harvest. For large slaughterhouses, post-harvest measures like decontamination might be cost-effective while this is less likely with small-to-medium sized slaughterhouses. In this study, pre-harvest measures might be more relevant. We describe an extended surveillance-and-control programme for Salmonella in finisher pigs, which, to establish equivalence to the Swedish control programme, is intended for implementation on the Danish island, Bornholm. The effect of the programme on food safety was estimated by analysing Salmonella data from pig carcasses originating from herds that would have qualified for the programme during 2006-2008. Food safety was interpreted as prevalence of Salmonella on carcasses as well as the estimated number of human cases of salmonellosis related to pork produced within the programme. Data from the Danish Salmonella programme were obtained from Bornholm. We used a simulation model developed to estimate the number of human cases based on the prevalence of Salmonella on carcass swabs. Herds are only accepted in the programme if they have one or less seropositive sample within the previous 6 months. In this way, the Salmonella load is kept to a minimum. The programme is not yet in operation and pigs that qualify for the programme are currently mixed at slaughter with those that do not qualify. Therefore, we had to assess the impact on the carcass prevalence indirectly. The prevalence of Salmonella in carcass swabs among qualifying herds was 0.46% for the 3 years as a whole, with 2006 as the year with highest prevalence. According to the simulation the expected number of human cases relating to pork produced within the programme was below 10. When the programme is in operation, an extra effect of separating pigs within the programme from those outside is expected to lower the prevalence of Salmonella even further.

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.

Validation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica serovars

In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR technology in tandem with an adapted two-step enrichment protocol were assessed and validated against a reference culture method, ISO 6579, 2004. The DNA and RNA real-time PCR assays amplified a 270 bp region of the hilA gene of Salmonella enterica serovars, and incorporated an internal amplification control (IAC) which was co-amplified with the hilA gene to monitor potential PCR inhibitors and ensure successful amplification. The inclusivity and exclusivity of the hilA primer set was examined for both the DNA and RNA methods and detected the 30 S. enterica serovars but not the 30 non-salmonellae strains. The inoculation of meat carcass swabs with five different S. enterica serovars at five different inocula, indicated both PCR methods were able to detect between 1 and 10 CFU per carcass swab. The real-time DNA PCR assay performed as well as the traditional cultural method in detecting Salmonella sp . in artificially contaminated salad, chocolate, fish and cheese samples. The relative accuracy, relative sensitivity and relative specificity of the DNA PCR real-time method were determined to be 98.5, 98.1 and 100%, respectively. The DNA method was further validated in a collaborative inter-laboratory trial according to ISO 16140, 2003. The validated methods provide an accurate means for the rapid detection and tracking of S. enterica serovars giving equivalent results to the standard method within three days, thus providing an alternative testing method to the reference microbiological method. The real-time PCR methodology not only offers significant time-saving advantages compared to traditional methods, it can also be applied to a wide range of samples types.

Application of PCR for rapid detection and serotyping of Salmonella spp. from porcine carcass swabs following enrichment in semi-solid agar

A new procedure for the detection and serotyping of Salmonella spp. based on PCR was developed and applied to porcine carcass swabs. Detection was based on a two-step enrichment using Buffered Peptone Water (BPW) and Modified Semi-solid Rappaport Vassiliadis (MSRV) agar followed by real time PCR targeting the genus specific hilA gene. In addition, pUC19 plasmid DNA was included as an extraction and internal amplification control (IAC). Serotyping consisted of multiplex PCR and capillary electrophoresis (CE) based on the method of Leader, Frye, Hu, Fedorka-Cray, & Boyle, 2009. To validate this method, 271 porcine carcass swabs were examined in tandem by the conventional culture method using MSRV (ISO 6579:2002; Annex D) followed by serotyping using slide agglutination and classification according to the White–Kauffmann–Le Minor scheme. Results showed 100% agreement between the conventional culture method and PCR, with 27 samples positive for Salmonella spp. by both methods.Using slide agglutination, full antigenic formulas were obtained for 25 of the 27 isolates (14 S. Typhimurium, 6 S. Infantis, 3 S. Derby, 1 S. Virchow and 1 S. Livingstone), while two isolates were not fully typeable and were designated S. Unnamed. PCR serotyping discriminated 7 different molecular code patterns, of which 2 were identical to those described by Leader et al. (2009), namely S. Typhimurium (ST) and S. Virchow (SV), found in 16 and one, respectively, of the 27 isolates. Both S. Unnamed isolates were identified as ST by the PCR method, therefore improving the identification of ST over the conventional method. The remaining 10 isolates shared four PCR patterns and although consistent codes were generally observed, discrepancies were observed compared to Leader et al. (2009). MSRV-PCR followed by serotyping using multiplex PCR and CE is rapid with final results obtained in 2days from receipt of samples compared with 6–9days using the conventional methods. The inclusion of the pUC19 plasmid DNA as an extraction and IAC demonstrated the robustness of the PCR procedure, with consistent Ct values obtained for both the hilA and pUC19 targets. This procedure could be particularly useful for surveillance and outbreak investigations by tracking sources of S. Typhimurium contamination, thus improving Salmonella control activities.