Inge Van Damme

Yersinia enterocolitica in slaughter pig tonsils: Enumeration and detection by enrichment versus direct plating culture

Tonsil samples from 139 slaughter pigs were examined for the presence of pathogenic Yersinia enterocolitica by enrichment procedures based on the standard method ISO 10273:2003. In addition, samples were tested by direct plating method to evaluate its efficiency compared to the enrichment culture methods and to quantify the level of contamination in porcine tonsils. In total, 52 samples (37.4%) were positive for pathogenic Y. enterocolitica, all belonging to bioserotype 4/O:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating. Enumeration showed an average concentration of 4.5 log(10) CFU g(-1) and 4.4 log(10) CFU g(-1) tonsil on Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) and cefsulodin-irgasan-novobiocin (CIN) agar plates, respectively. The enrichment procedures recommended by the ISO 10273:2003 method were not optimal for the isolation of pathogenic Y. enterocolitica from pig tonsils: two days enrichment in irgasan-ticarcillin-potassium chlorate (ITC) broth resulted in an isolation rate of 84.6%, while 5 days enrichment in peptone-sorbitol-bile (PSB) broth recovered only 59.6% of positive samples. Reducing the enrichment time in PSB from 5 to 2 days resulted in a significantly higher recovery rate (94.2%) and might serve as an appropriate enrichment protocol for the isolation of pathogenic Y. enterocolitica from pig tonsils. Compared to enrichment culture methods, results based on direct plating can be obtained in a shorter time course and provide quantitative data that might be needed for further risk assessment studies.

Campylobacter carcass contamination throughout the slaughter process of Campylobacter-positive broiler batches.

Campylobacter contamination on broiler carcasses of Campylobacter colonized flocks was quantified at seven sampling sites throughout the slaughter process. For this purpose, in four slaughterhouses samples were collected from twelve Campylobacter positive batches. Broilers from all visits carried high numbers of campylobacters in their caeca (≥7.9log10cfu/g). Campylobacter counts on feathers (up to 6.8log10cfu/g), positively associated with the breast skin contamination of incoming birds and carcasses after plucking, were identified as an additional source of carcass contamination. A high variability in Campylobacter carcass contamination on breast skin samples within batches and between batches in the same slaughterhouse and between slaughterhouses was observed. In slaughterhouses A, B, C and D Campylobacter counts exceeded a limit of 1000cfu/g on 50%, 56%, 78% and 11% of carcasses after chilling, respectively. This finding indicates that certain slaughterhouses are able to better control Campylobacter contamination than others. Overall, the present study focuses on the descriptive analysis of Campylobacter counts in different slaughterhouses, different batches within a slaughterhouse and within a batch at several sampling locations.

Evaluation of the ISO 10273:2003 method for the isolation of human pathogenic Yersinia enterocolitica from pig carcasses and minced meat.

Pig carcass swabs (n = 254) and minced meat samples (n = 82) were examined for pathogenic Yersinia enterocolitica using different routinely used enrichment protocols. All samples were obtained in the context of the official Yersinia monitoring program in Belgium. In total, 28 carcasses (11.0%) were contaminated with Y. enterocolitica bioserotype 4/O:3 and one (0.4%) with bioserotype 2/O:9. Four minced meat samples (4.9%) tested positive for Y. enterocolitica bioserotype 4/O:3. Using the ISO 10273:2003 method, eight out of the 29 Yersinia-positive carcasses (27.6%) and none of the contaminated minced meat samples (0.0%) were detected. Reducing the enrichment time in PSB from 5 to 2 days increased the number of positive samples. Overall, enrichment in PSB at 25 °C recovered more positive carcasses and minced meat samples than selective enrichment and cold enrichment. As the exclusive use of the ISO 10273:2003 method results in a strong underestimation of Y. enterocolitica positive carcasses and minced meats, efforts are needed to optimize the current version of the ISO method. In addition, isolation of pathogenic Y. enterocolitica requires experience and the use of a stereomicroscope to avoid false negative results.

Within-batch prevalence and quantification of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis in tonsils of pigs at slaughter

Yersiniosis is a common bacterial zoonosis in Europe and healthy pigs are known to be the primary reservoir of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. However, little information is available about the prevalence of these pathogens within pig batches at time of slaughter. The tonsils of 7047 fattening pigs, belonging to 100 farms, were aseptically collected immediately after evisceration in two Belgian slaughterhouses. The batch size varied between 70 and 930 pigs. On average, 70 pigs were sampled per batch. The tonsils were examined by direct plating on cefsulodin-irgasan-novobiocin (CIN) agar plates and the number of suspect Yersinia colonies was counted. Pathogenic Y. enterocolitica serotype O:3 were found in tonsils of 2009 pigs (28.5%), originating from 85 farms. The within-batch prevalence in positive farms ranged from 5.1 to 64.4%. The number of Y. enterocolitica in positive pigs varied between 2.01 and 5.98 log10 CFU g(-1) tonsil, with an average of 4.00 log10 CFU g(-1) tonsil. Y. pseudotuberculosis was found in seven farms, for which the within-batch prevalence varied from 2 to 10%. In five of these farms, both Y. enterocolitica and Y. pseudotuberculosis were simultaneously present. Human pathogenic Yersinia spp. are widespread in slaughter pig batches in Belgium as 87% of the tested batches were infected with these pathogens at the time of slaughter. The large variation of the prevalence between batches may lead to different levels of contamination of carcasses and risks for public health.

Effect of sampling and short isolation methodologies on the recovery of human pathogenic Yersinia enterocolitica from pig tonsils.

The objective of this study was to determine the effect of sampling (swab samples compared to destructive samples) on isolation rates of human pathogenic Yersinia enterocolitica from pig tonsils. Moreover, the relative efficiency of different rapid, routinely applicable isolation methods was evaluated. Therefore, swab and destructive samples from tonsils of 120 pigs at slaughter were analyzed in parallel using direct plating and different enrichment methods. Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) agar, cefsulodin-irgasan-novobiocin (CIN) agar, and Yersinia enterocolitica chromogenic medium (YeCM) were used as selective agar media. For enrichment, irgasan-ticarcillin-potassium chlorate (ITC) broth and peptone-sorbitol-bile (PSB) broth were incubated at 25°C for 48 h. Overall, 55 tonsils (45.8%) were positive for Y. enterocolitica bioserotype 4/O:3. Recovery was significantly higher using the destructive method compared to the swabbing method. Direct plating resulted in 47 and 28 Y. enterocolitica-positive destructive and swab samples, respectively. Alkali treatment of PSB and ITC enrichment broths significantly increased recovery of pathogenic Y. enterocolitica from destructive tonsil samples. The performance of YeCM for qualitative and quantitative isolation of pathogenic Y. enterocolitica from pig tonsils was equal to SSDC and CIN. In conclusion, direct plating and ISO 10273: 2003 with minor modifications are suitable and rapid methods for isolation of pathogenic Y. enterocolitica from destructive tonsil samples.

Evaluation of detection methods for non-O157 Shiga toxin-producing Escherichia coli from food.

Shiga toxin-producing Escherichia coli (STEC) remains a major foodborne pathogen of concern across the globe. Rapid detection and isolation of this pathogen is of great importance for public health reasons. In this study the detection and isolation of four non-O157 STEC strains (O26, O103, O111, O145) from different artificially contaminated matrices, namely ground (minced) beef, cattle carcass swab, lettuce mix and sprouted soy beans, were evaluated. Low amounts of STEC were used (0.25-1.40 cfu/g) to spike the samples. All samples were enriched in parallel in Buffered Peptone Water (BPW) and Brila broth. After enrichment, detection was performed using real-time PCR (qPCR), and isolation using two chromogenic agar media, CHROMagar™ STEC and ChromID™ EHEC. Inoculation on the agar media was performed either directly after enrichment or after the use of an acid treatment procedure. Furthermore, the use of this procedure was also tested on naturally contaminated food products, using 150 stx-positive samples. Although the qPCR Cycle Threshold (Ct) values were lower after enrichment in Brila broth, no significant differences in recovery were observed between both enrichment broths. Both agar media were equally suitable for the isolation of STEC, although a significantly higher recovery was obtained when using both agar media in parallel. For samples with a Ct value above 25, an acid treatment step prior to isolation ensured a significant improvement in the recovery of STEC due to the reduction in background microbiota. This acid treatment procedure proved especially useful for the isolation of STEC from sprouted soy bean samples.

Identification of risk factors for Campylobacter contamination levels on broiler carcasses during the slaughter process

Campylobacter carcass contamination was quantified across the slaughter line during processing of Campylobacter positive batches. These quantitative data were combined together with information describing slaughterhouse and batch related characteristics in order to identify risk factors for Campylobacter contamination levels on broiler carcasses. The results revealed that Campylobacter counts are influenced by the contamination of incoming birds (both the initial external carcass contamination and the colonization level of caeca) and the duration of transport and holding time that can be linked with feed withdrawal period. In addition, technical aspects of the slaughter process such as a dump based unloading system, electrical stunning, lower scalding temperature, incorrect setting of plucking, vent cutter and evisceration machines were identified as risk factors associated with increased Campylobacter counts on processed carcasses. As such the study indicates possible improvements of the slaughter process that can result in better control of Campylobacter numbers under routine processing of Campylobacter positive batches without use of chemical or physical decontamination. Moreover, all investigated factors were existing variations of the routine processing practises and therefore proposed interventions are practically and economically achievable.

Seroprevalence of enteropathogenic Yersinia spp. in pig batches at slaughter.

Enteropathogenic Yersinia spp. are one of the main causes of foodborne bacterial infections in Europe. Slaughter pigs are the main reservoir and carcasses are contaminated during a sub-optimal hygienically slaughtering-process. Serology is potentially an easy option to test for the Yersinia-status of the pig (batches) before slaughter. A study of the variation in activity values (OD%) of Yersinia spp. in pigs and pig batches when applying a serological test were therefore conducted. In this study, pieces of the diaphragm of 7047 pigs, originating from 100 farms, were collected and meat juice was gathered, where after an enzyme-linked immunosorbent assay (ELISA) Pigtype Yopscreen (Labor Diagnostik Leipzig, Qiagen, Leipzig, Germany) was performed. The results were defined positive if the activity values exceeded the proposed cut-off value of 30 OD%. Results at pig level displayed a bimodal-shaped distribution with modes at 0-10% (n=879) and 50-60% (n=667). The average OD% was 51% and 66% of the animals tested positive. The within-batch seroprevalence ranged from 0 to 100% and also showed a bimodal distribution with modes at 0% (n=7) and 85-90% (n=16). On 7 farms, no single seropositive animal was present and in 22 farms, the mean OD% was below 30%. Based on the results obtained at slaughter, 66% of the pigs had contact with enteropathogenic Yersinia spp. at farm level. The latter occurred in at least 93% of the farms indicating that most farms are harboring enteropathogenic Yersinia spp.

Relation Between Serology of Meat Juice and Bacteriology of Tonsils and Feces for the Detection of Enteropathogenic Yersinia spp. in Pigs at Slaughter

The association between positive serology and culture detection of Yersinia spp. in individual pigs was determined. Pieces of diaphragm from 370 pig carcasses were collected for serological analysis, and tonsils and feces of the same carcass were collected for bacteriological analysis. Detection of anti-Yersinia antibodies in meat juice samples was done using an indirect enzyme-linked immunosorbent assay (ELISA) based on Yops (Yersinia outer proteins). Tonsils and feces were tested for the presence of enteropathogenic Yersinia spp. by direct plating on cefsulodin-irgasan-novobiocin agar plates. Of the 370 meat juice samples, 241 (65.1%) gave a positive serological reaction using a cutoff value of 20%. Enteropathogenic Yersinia spp. (Yersinia enterocolitica serotype O:3 and Yersinia pseudotuberculosis) were found in tonsils of 161 pigs and feces of 30 pigs. Recovery of enteropathogenic Yersinia from the tonsils was highly correlated with positive serotiters, whereas no correlation was found between serology and fecal excretion. Results demonstrated that serology has an acceptable sensitivity, but a relatively low specificity for the rapid detection of enteropathogenic Yersinia spp. in tonsils of pigs at slaughter.

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

&NA; Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP‐flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP‐flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC‐828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador.