Transmission Dynamics of African Swine Fever Virus, South Korea, 2019.

African swine fever (ASF) is a substantial concern for global food production and security. However, lack of epidemiologic data in affected areas has limited the knowledge of the main drivers of ASF virus (ASFV) transmission. To assess the role of vehicle movements and wild boar populations in spreading ASFV to pig farms in South Korea, we combined data generated by ASF surveillance on pig farms and of wild boars with nationwide global positioning system-based tracking data for vehicles involved in farming activities. Vehicle movements from infected premises were associated with a higher probability of ASFV incursion into a farm than was geographic proximity to ASFV-infected wild boar populations. Although ASFV can spill over from infected wild boars into domestic pigs, vehicles played a substantial role in spreading infection between farms, despite rapid on-farm detection and culling. This finding highlights the need for interventions targeting farm-to-farm and wildlife-to-farm interfaces.

Arthropods as potential vectors of African swine fever virus outbreaks in pig farms in the Republic of Korea.

The seasonality of African swine fever (ASF) in the summers and outbreaks in farms with high biosecurity levels suggest that the ASF virus (ASFV) may be transmitted by arthropod vectors. Arthropods were collected in this study from 14 pig farms with ASF outbreaks in Korea in 2019 to explore the role of arthropods as potential ASFV vectors. A total of 28,729 arthropods, including 28,508 (99.2%) Diptera, were collected using blacklight traps, insect nets and yellow sticky strips. All arthropod samples were negative for ASFV genomic DNA according to laboratory tests using real-time polymerase chain reaction. This result may reflect the effects of immediate control measures following the detection of farms with ASF outbreaks in the early phase of infection in Korea.

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis.

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

Epidemiological Characteristics of Foot-and-Mouth Disease in the Republic of Korea, 2014-2019.

This study describes the epidemiological characteristics of six epidemics of foot-and-mouth disease (FMD) in the Republic of Korea between 2014 and 2019. A total of 223 outbreaks had been confirmed in 40 municipalities across nine provinces. Most farms with FMD (194, 87%) were located in three densely populated livestock areas (Chungcheongnam-do, Gyeonggi-do, and Chungcheongbuk-do). More cases of FMD were found in farms with more than 1,000 pigs or 50 cattle (risk ratios = 1.27 for pigs; 9.46 for Korean native cattle) and fattening pigs. In farms affected by FMD, the proportion of animals with vaccine antibodies was low (5%-50% for Korean native beef cattle farms with FMD in 2017 vs. 97.5% in the surveillance in 2016). Effective control of FMD can be achieved through strict biosecurity measures, proper vaccination, regionalized management, and instilling awareness of FMD prevention in farmers.

Probe-based real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay for rapid and specific detection of foot-and-mouth disease virus.

Rapid and specific detection of foot-and-mouth disease virus (FMDV) is a key factor for promoting prompt control of FMD outbreaks. In this study, a real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay with high sensitivity, rapidity and reliability was developed using a targeted gene-specific assimilating probe for real-time detection of seven FMDV serotypes. Positive assay signals were generated within 15 min for the lowest concentration of a standard RNA sample at 62°C; this was substantially faster than that achieved by the OIE (World Organisation for Animal Health)-recommended real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The new assay specifically amplified the 3D gene of all seven FMDV serotypes and did not amplify other viral nucleic acids. The detection limit of the assay was 102  copies/µl which is comparable to that achieved by qRT-PCR. Furthermore, using clinical samples, the results of the RRT-LAMP assay were largely in agreement with those from the qRT-PCR assay with a kappa value (95% confidence interval [CI]) of 0.94 (0.86-1.02). The established RRT-LAMP assay that features assimilating probes is an advanced molecular diagnostic tool that is easily applicable to a wide range of circumstances and has high potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of FMDVs in clinical samples.

Prevalence and Antimicrobial Characteristics of Shiga Toxin-Producing Escherichia coli Isolates from Pork in Korea.

Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens that can be transmitted through the consumption of food products derived from pigs. Moreover, antimicrobial resistance in STEC has been a matter of increasing concern. The aim of this study was to investigate the prevalence and antimicrobial characteristics of STEC isolates from pork in Korea. We isolated 131 isolates of E. coli from 334 pork samples collected from slaughterhouses and retail markets from 2008 to 2009. Among the 131 isolates, 6 (4.58%) were confirmed to belong to 6 different serotypes of STEC. All six STEC isolates contained stx1 and eaeA virulence genes, and four of them additionally carried the hly gene. The minimum inhibitory concentration (MIC) of 15 antibiotics (amoxicillin/clavulanic acid, ampicillin, cephalothin, cefoxitin, ceftiofur, gentamicin, neomycin, streptomycin, nalidixic acid, ciprofloxacin, colistin, chloramphenicol, florfenicol, tetracycline and sulfamethoxazole/trimethoprim) toward the STEC isolates was determined. As a result, three strains were associated with high MICs for florfenicol and chloramphenicol (64 µg/mL). Furthermore, all three strains were found to contain the florfenicol-resistant gene (floR) but not the chloramphenicol-resistant gene (cat). Sequence alignment and BLAST analysis of the polymerase chain reaction products of the floR gene indicated that they contained sequences with homology to the floR gene of E. coli or Salmonella enterica serovar, Heidelberg. This is the first report on the detection of floR in STEC isolated from pork obtained from retail markets in Korea.

Clinical symptoms of African swine fever in domestic pig farms in the Republic of Korea, 2019.

This study describes the clinical characteristics of the African swine fever (ASF) outbreaks in 14 domestic pig farms in the Republic of Korea. ASF outbreak was identified by farmers' notifications in 11 farms and by active surveillance in the remaining three. At the time of notification, farmers reported sudden death, abortion and anorexia in sows. Death was the primary symptom identified by farmers in fattener pigs. The number of animals exhibiting clinical symptoms did not exceed four heads at notification, and the number of asymptomatic virus positives was four heads per farm on average. As ASF virus was detected only in the same pig house (in a pen for fattener pigs) in each of 14 ASF outbreak farms, there has been no evidence of house-to-house viral spread within any of the ASF outbreak farms. This in turn supports our hypothesis that infection was successfully detected during its initial phase.

Development of Monoclonal Antibody Specific to Foot-and-Mouth Disease Virus Type A for Serodiagnosis.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. FMD virus (FMDV) type A is one of the most common causes of FMD outbreaks among the seven FMDV serotypes, and its serological diagnosis is therefore important to confirm FMDV type A infection and to determine FMD vaccine efficacy. Here, we generated monoclonal antibodies (mAbs) specific to FMDV type A via hybridoma systems using an inactivated FMDV type A (A22/Iraq/1964) and found 4 monoclones (#29, #106, #108, and #109) with high binding reactivity to FMDV type A among 594 primary clones. In particular, the #106 mAb had a higher binding reactivity to the inactivated FMDV type A than the other mAbs and a commercial mAb. Moreover, the #106 mAb showed no cross-reactivity to inactivated FMDV type South African territories 1, 2, and 3, and low reactivity to inactivated FMDV type O (O1 Manisa). Importantly, the solid-phase competitive ELISA (SPCE) using horseradish peroxidase (HRP)-conjugated #106 mAb detected FMDV type A-specific Abs in sera from FMD type A-vaccinated cattle more effectively than a commercial SPCE. These results suggest that the newly developed FMDV type A-specific mAb might be useful for diagnostic approaches for detecting Abs against FMDV type A.

Complete Genome Sequence of O/VN1/2014, a Foot-and-Mouth Disease Virus of Serotype O Isolated in Vietnam in 2014.

In this article, we report the complete genome sequence of foot-and-mouth disease virus (FMDV) strain O/VN1/2014 isolated in Vietnam (Lao Cai) in 2014. The virus belongs to serotype O, topotype South East Asia (SEA), and genotype Mya-98 (O/SEA/Mya-98). It is the latest complete genome information for the genotype O/SEA/Mya-98 in Vietnam since 2009.

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

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 (B f , 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.

An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of serotype O foot-and-mouth disease virus.

A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 102 TCID50/mL or 103 RNA copies/µL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.

A tailored reverse transcription loop-mediated isothermal amplification for sensitive and specific detection of serotype A foot-and-mouth disease virus circulating in pool 1 region countries.

Rapid and accurate diagnosis of foot-and-mouth disease viruses (FMDV) is essential for the prompt control of FMD outbreaks. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) are used for routine FMDV diagnosis as World Organisation for Animal Health-recommended diagnostic assays. However, these PCR-based assays require sophisticated equipment, specialized labour, and complicated procedures for the detection of amplified products, making them unsuitable for under-equipped laboratories in developing countries. In this study, to overcome these shortcomings, a simple, rapid, and cost-effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of serotype A FMDV circulating in the pool 1 region. The amplification could be completed in 40 min at 62°C, and the results could be visually detected by the naked eye without any additional detection systems. The assay specifically amplified the VP1 gene of the Sea-97 genotype of serotype A FMDV, but it did not amplify other viral nucleic acids. The limit of detection of the assay was 102 TCID50 /ml, which is 10 times more sensitive than RT-PCR and is comparable to the sensitivity of qRT-PCR. Evaluation of the assay using different FMDV strain serotypes showed 100% agreement with the results of RT-PCR. Surprisingly, the previously reported RT-LAMP assay did not detect all eight tested strains of serotype A FMDVs, due to multiple mismatches between primer and template sequences, demonstrating that it is not suitable for detecting serotype A FMDVs circulating in pool 1-region countries. Conversely, the newly developed RT-LAMP assay using improved primers can rapidly and accurately diagnose the genotype of Sea-97 strains of serotype A FMDVs from the pool 1 region. The established RT-LAMP assay in this study is a simple, rapid, specific, sensitive, and cost-effective tool for the detection of serotype A FMDV in the resource-limited pool 1-region countries.

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

Twenty extended-spectrum ß-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.

Complete Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype O, Isolated from Gochang, Republic of Korea, in 2016.

The complete genome sequence of a foot-and-mouth disease (FMD) serotype O virus isolated from Gochang, Republic of Korea, is reported here.

Complete Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype O Isolated from Gimje, Republic of Korea, in 2016.

The complete genome sequence of a foot-and-mouth disease (FMD) serotype O virus isolated from Gimje, Republic of Korea, is reported here.

Distribution and Molecular Characterization of Campylobacter Species at Different Processing Stages in Two Poultry Processing Plants.

The present study analyzed the prevalence and molecular characterization of Campylobacter at different processing steps in poultry slaughterhouses to determine where contamination mainly occurs. A total of 1,040 samples were collected at four different stages (preprocessing cloacal swabs, postevisceration, postwashing, and postchilling) in two processing plants. Campylobacter was detected in 5.8% (15 of 260) of the cloacal swabs and in 13.3% (104 of 780) of the processing samples. In both plants, the sampling points with the greatest contamination rates were after evisceration (20.5% and 15.4% for plants A and B, respectively) and significantly decreased after chilling (p < 0.05, from 20.5% to 10.9%) in plant A and after washing (from 15.4% to 2.9%) in plants B. In the result, however, the reduction in Campylobacter contamination was achieved through the sequential processing procedures in both plants. Campylobacter loads (>103 colony-forming units [CFUs]/mL) also decreased from 41.7% at evisceration to 20.0% in final carcasses. The genetic relationships of isolates were analyzed by the automated repetitive sequence-based polymerase chain reaction (rep-PCR) system, and the rep-PCR banding pattern was found to be unrelated to the processing plants, species, sampling point, or sampling day. As the gap in the intervention efficacy remains between plant A and B despite several consistencies, a national program for monitoring critical processing stages in poultry processing plants is recommended for the successful exportation of Korean-processed white mini broiler meat.

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.

Characterization of Escherichia coli-Producing Extended-Spectrum ß-Lactamase (ESBL) Isolated from Chicken Slaughterhouses in South Korea.

In South Korea, few reports have indicated the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in food-producing animals, particularly in poultry slaughterhouses. In this study, we investigated the occurrence and antibiotic resistance of ESBL-producing E. coli from whole chicken carcasses (n=156) and fecal samples (n=39) of chickens obtained from 2 slaughterhouses. Each sample enriched in buffered peptone water was cultured on MacConkey agar with 2 mg/L cefotaxime and ESBL agar. ESBL production and antibiotic susceptibility were determined using the Trek Diagnostics system. The ESBL genotypes were determined by polymerase chain reaction (PCR) using the bla(SHV), bla(TEM), and bla(CTX-M) gene sequences. Subtyping using a repetitive sequence-based PCR system (DiversiLab™) and multilocus sequence typing (MLST) were used to assess the interspecific biodiversity of isolates. Sixty-two ESBL-producing E. coli isolates were obtained from 156 samples (39.7%). No bla(SHV) genes were detected in any of the isolates, whereas all contained the bla(TEM) gene. Twenty-five strains (40.3%) harbored the CTX-M group 1 gene. The most prevalent MLST sequence type (ST) was ST 93 (14.5%), followed by ST 117 (9.7%) and ST 2303 (8.1%). This study reveals a high occurrence and ß-lactams resistance rate of E. coli in fecal samples and whole chickens collected from slaughterhouses in South Korea.

Antibiotic Resistance and Virulence Potentials of Shiga Toxin-Producing Escherichia coli Isolates from Raw Meats of Slaughterhouses and Retail Markets in Korea.

In this study, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated among raw meat or meat products from slaughterhouses and retail markets in South Korea, and their potential for antibiotic resistance and virulence was further analyzed. A total of 912 raw meats, including beef, pork, and chicken, were collected from 2008 to 2009. E. coli strains were frequently isolated in chicken meats (176/233, 75.9%), beef (102/217, 42.3%), and pork (109/235, 39.2%). Putative STEC isolates were further categorized, based on the presence or absence of the Shiga toxin (stx) genes, followed by standard O-serotyping. Polymerase chain reaction assays were used to detect the previously defined virulence genes in STEC, including Shiga toxins 1 and Shiga toxin 2 (stx1 and 2), enterohemolysin (ehxA), intimin (eaeA), STEC autoagglutination adhesion (saa), and subtilase cytotoxin (subAB). All carried both stx1 and eae genes, but none of them had the stx2, saa, or subAB genes. Six (50.0%) STEC isolates possessed the ehxA gene, which is known to be encoded by the 60-megadalton virulence plasmid. Our antibiogram profiling demonstrated that some STEC strains, particularly pork and chicken isolates, displayed a multiple drug-resistance phenotype. RPLA analysis revealed that all the stx1-positive STEC isolates produced Stx1 only at the undetectable level. Altogether, these results imply that the locus of enterocyte and effacement (LEE)-positive strains STEC are predominant among raw meats or meat products from slaughterhouses or retail markets in Korea.

Molecular Characterization, Antibiotic Resistance, and Virulence Factors of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Imported and Domestic Meat in Korea.

During a nationwide surveillance in Korea, 13 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated from imported and domestic meat between 2009 and 2011. The predominant MRSA genotype was SCCmec type V, and only two agr types (I and II) were found. Unexpectedly, sequence type ST72 comprised more than 50% of the isolates; this is the first instance of type ST72 in food from Canada. Two Spanish pork isolates were ST398, which caused human disease in Europe, and they carried leukotoxin genes, lukS, lukF, and lukE-lukD. Furthermore, P71 and P6 harbored all of the known leukocidin genes, lukS-lukF-lukE-lukD-lukM. Our collected MRSA strains were multidrug resistant with various antimicrobial and heavy-metal resistance genes. Toxin genes that are commonly found in clinical MRSA also were detected in our meat strains. One MRSA strain exhibited an uncommon type of enterotoxin, sec-see-seg-sei-sel-sem-sen-seo-sep. Plasmids (1.5-15.0 kb) were found in 12 of the 13 MRSA isolates. Repetitive sequence-based polymerase chain reaction of the genomic DNA showed 3 clusters with 95% similarity. The presence of multidrug-resistant and toxigenic MRSA in meat products suggests that comprehensive surveillance should be continued for imported meats in Korea.