Jin-San Moon

Validation of PCR and ELISA Test Kits for Identification of Domestic Animal Species in Raw Meat and Meat Products in Korea

In this study, two commercial PCR and ELISA test kits were examined for identification of eight animal species (beef, pork, chicken, duck, turkey, goat, lamb, and horse) from raw meat and meat products in Korea. The detection limit in RAW meat ELISA kit ® on three types of meat samples blended with beef, pork and chicken, demonstrated that all meat species were differentiable down to 0.2%. RAW meat ELISA kit ® on animal species resulted in differentiation rate of 94.5% for beef, 93.3% for pork, 90% for lamb, and 100% for chicken, duck, turkey, goat, and horse. In contrast, Powercheck Animal Species ID PCR kit TM resulted in 100% specificity at 0.05% limit of detection for all meat species. The detection limit of Cooked Meat ELISA kit ® on mixed meat samples heat-treated with different temperatures and times, resulted in 0.1% for all heat-treated mixed meat except for chicken at 1.0%. Additionally, ELISA kit on sixty meat products resulted in specificity of 31.8% for ham, 13.6% for sausages, and 12.5% for ground processed products, and relatively low rate for more than 2 types of mixed meats. On the contrary, meat species differentiation using PCR kit showed higher percentage than that using ELISA kit ® : 50.0% for ham, 41.7% for sausages, and 28.6% for ground processed meat. Futhermore, PCR kit on 54 dried beef meats detected pork genes in 13 products whereas ELISA kit showed negative results for all products. Hence, the possibility of cross-con- tamination during manufacturing process was investigated, and it was found that identical tumblers, straining trays, cutters and dryers were used in both beef and pork jerky production line, suggesting the inclusion of pork genes in beef products due to cross-contamination. In this study, PCR and ELISA test kits were found to be excellent methods for meat species differentiation in raw meat and heat-processed mixed meat. However, lower differentiation rate demon- strated in case of meat processed products raised the possibility of inclusion of other species due to cross-contamina- tion during manufacturing process.

Traceback Investigation for Salmonella Contamination at Egg Processing Plants in South Korea: Prevalence, Antibiotic Resistance, and Epidemiological Tracing by Rep‐PCR Fingerprinting

We conducted a survey of Salmonella from 8 egg-breaking plants and a farm to determine the prevalence and the source of the bacteria. The contents of 2400 shell eggs (20 eggs per pool), 75 pasteurized liquid egg products, and 120 unpasteurized liquid egg products from 8 egg-breaking plants in South Korea were examined. In liquid egg samples, 4 Salmonella-positive samples from 120 unpasteurized ones (3.3%) and 5 positive samples from 75 pasteurized ones (6.7%) were identified; no eggs were positive for Salmonella among shell egg samples. To trace the source of Salmonella, we revisited the 2 Salmonella-positive plants (plants A and C). We investigated the equipment and environments of the plants and a henhouse (farm A) that supplied shell eggs to plant A, and collected additional liquid eggs and shell eggs from plants A and C. All Salmonella isolates from plant A and the associated farm A, except for a single Typhimurium strain from farm A, were serotyped as Bareilly. Three serovars, including one Bareilly, four Tennessee, and one Richmond, were isolated from plant C. Most Salmonella isolates were susceptible to tested antibiotics. To identify differences between isolates, molecular subtyping by using the automated rep-PCR system was conducted. All Salmonella Bareilly (S. Bareilly) strains from plant A exhibited high similarity, indicating possible contamination by Salmonella strains from the henhouse A. Meanwhile, 2 S. Bareilly strains from plant C, one from liquid egg at the 1st visit and the other from container at the 2nd visit, exhibited identical antibiotic resistance and similar subtyping pattern, but clearly discriminated from the ones of plant A.

Prevalence Analysis and Molecular Characterization of Salmonella at Different Processing Steps in Broiler Slaughter Plants in South Korea

In this study, changes in the prevalence of Salmonella during the processing of broiler chicken carcasses were investigated. A total of 1040 fecal swabs and chicken carcasses samples were collected from 2 processing plants at the 4 stages of broiler processing, which included live birds in slaughter line, postevisceration/prewashing, postwashing/prechilling, and postchilling, respectively. The intraspecific biodiversity of the Salmonella isolates was determined using a DiversiLab automated repetitive sequence-based PCR (rep-PCR) system. In both plants, the prevalence of Salmonella increased considerably after evisceration (from 4.6% to 30.8%, P < 0.05) and decreased after washing (from 30.8% to 25.4%, P < 0.05). However, the chilling step had little effect on Salmonella prevalence (from 25.4% to 22.7%, P > 0.05). The most frequent Salmonella serovar in plant A was Infantis (35.8%), followed by Enteritidis (26.2%) and Montevideo (15.0%), while Montevideo (43.6%) and Enteritidis (35.9%) were most prevalent in plant B. A difference in the rep-PCR banding pattern was found to be related to the processing plant origin and serovar rather than sampling point or sampling day, although there were some exceptional strains.

Development and Validation of Predictive Model for Salmonella Growth in Unpasteurized Liquid Eggs

Abstract Liquid egg products can be contaminated with Salmonella spp. during processing. A predictive model for the growth of Salmonella spp. in unpasteurized liquid eggs was developed and validated. Liquid whole egg, liquid yolk, and liquid egg white samples were prepared and inoculated with Salmonella mixture (approximately 3 Log CFU/mL) containing five serovars (S. Bareilly, S. Richmond, S. Typhimurium monophasic, S. Enteritidis, and S. Gallinarum). Salmonella growth data at isothermal temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C) was collected by 960 h. The population of Salmonella in liquid whole egg and egg yolk increased at above 10°C, while Salmonella in egg white did not proliferate at all temperature. These results demonstrate that there is a difference in the growth of Salmonella depending on the types of liquid eggs (egg yolk, egg white, liquid whole egg) and storage temperature. To fit the growth data of Salmonella in liquid whole egg and egg yolk, Baranyi model was used as the primary model and the maximum growth rate and lag phase duration for each temperature were determined. A secondary model was developed with maximum growth rate as a function of temperature. The model performance measures, bias factor (Bf, 0.96-0.99) and r2 (0.96-0.99) indicated good fit for both primary and secondary models. In conclusion, it is thought that the growth model can be used usefully to predict Salmonella spp. growth in various types of unpasteurized liquid eggs when those are exposed to various temperature and time conditions during the processing.

Prevalence and toxin type of Clostridium perfringens in beef from four different types of meat markets in Seoul, Korea

Beef is the primary source of foodborne poisoning caused by Clostridium perfringens. We investigated the prevalence of C. perfringens in retail beef from four different types of meat markets in Seoul using a standard culture method and real-time PCR assay. From June to September 2015, 82 beef samples were collected from 6 department stores (n=12), 14 butcher shops (n=28), 16 traditional markets (n=32), and 5 supermarkets (n=10). The culture method and real-time PCR assay revealed that 4 (4.88%) and 10 (12.20%) samples were positive for C. perfringens, respectively. The beef purchased from the department store showed the highest prevalence (16.67%), followed by the traditional market (3.12%), butcher shop (3.57%), and supermarket (0%) (p>0.05). All isolates were type A and negative for the enterotoxin gene. In conclusion, the real-time PCR assay used in this study could be useful for rapid detection and screening of C. perfringens in beef.

Genotypic characterization of ESBL-producing E. coli from imported meat in South Korea

Twenty extended-spectrum β-lactamase (ESBL)-producing E. coli strains were isolated from imported meat in South Korea. ESBL strains of E. coli were detected in chicken (14/20) more often than in pork (6/20) and beef (0/20); the highest number (12/20) was detected in Brazilian meats. The blaCTX-M genes were predominant in meats from many countries. E. coli from pork imported from France produced the blaCTX-M-58 enzyme, which has never been documented previously in ESBL-producing bacteria from clinical or environmental sources. Additionally, the coexistence of the blaCTX-M-2 and blaOXA-1 enzymes in EC12-5 isolate was found for the first time in an ESBL E. coli isolate. A rare blaCTX-M type, blaCTX-M-25, was found in 40% of ESBL E. coli isolates. Phenotypic susceptibility testing showed that E. coli isolates were resistant to up to eleven antibiotics, including ciprofloxacin. For the first time, a new combination in an integron gene cassette, aacA4-cmlA6-qacEΔ1, was found in an E. coli isolate from poultry imported from Brazil. Three E. coli ST117 isolates, from an avian pathogenic lineage producing CTX-M-94, harbored fimH, fyuA, iutA, papC, rfc, and traT virulence genes and were not susceptible to quinolones. For the first time, rfc and papG virulence factors were detected in ESBL E. coli strains isolated from meat products. Even though E. coli CC21 and CC22 were obtained from meats from the USA and Brazil, respectively, they had a similarity coefficient higher than 99% in rep-PCR and the same MLST type (ST117), phenotypic antibiotic resistance pattern, integron gene (qacEΔ1), and plasmid DNA profile. This study indicates that imported meat products may be a source of ESBL-producing E. coli strains in South Korea.

Comparison of the Microsatellite and Single Nucleotide Polymorphism Methods for Discriminating among Hanwoo (Korean Native Cattle), Imported, and Crossbred Beef in Korea.

The identity of 45 Hanwo and 47 imported beef (non-Hanwoo) samples from USA and Australia were verified using the microsatellite (MS) marker and single nucleotide polymorphism (SNP) methods. Samples were collected from 19 supermarkets located in the city of Seoul and Gyeonggi province, South Korea, from 2009 to 2011. As a result, we obtained a 100% concordance rate between the MS and SNP methods for identifying Hanwoo and non-Hanwoo beef. The MS method presented a 95% higher individual discriminating value for Hanwoo (97.8%) than for non-Hanwoo (61.7%) beef. For further comparison of the MS and SNP methods, blood samples were collected and tested from 54 Hanwoo × Holstein crossbred cattle (first, second, and third generations). By using the SNP and MS methods, we correctly identified all of the first-generation crossbred cattle as non-Hanwoo; in addition, among the second and third generation crossbreds, the ratio identified as Hanwoo was 20% and 10%, respectively. The MS method used in our study provides more information, but requires sophisticated techniques during each experimental process. By contrast, the SNP method is simple and has a lower error rate. Our results suggest that the MS and SNP methods are useful for discriminating Hanwoo from non-Hanwoo breeds.