The aim of this study was to investigate the prevalence of antimicrobial resistance in Escherichia coli isolated from pigs and their farmhouse environment in Korea. A total of 585 E. coli were isolated in this study during 2006 2007: 426 isolates from 492 pigs and 159 isolates from 312 farmhouse environment samples from 16 different pig farms. The most frequently observed resistance in pig fecal samples was antimicrobials such as tetracycline, streptomycin, and ampicillin. However, resistance to cephalosproins, β-lactam / β-lactam inhibitor combination, and colistin was low. We found an inverse relationship between prevalence of resistant E. coli and animal age. Resistance rate and multi-drug resistance (MDR) was greater in young pigs (piglet and nursery) than for those from adult pigs (grower-finish and sow). Resistance of E. coli from farm environment such as floor, Iron partition, and ventilation was similar with those from pig fecal samples. Farm environment contaminated with MDR bacteria might be a possible source of infections to pigs. Thus, to control and reduce the antimicrobial resistance in pigs, we must also pay attention to the environment.

Virus infections of the honeybee(Apis mellifera) have been increasingly investigated during the last decade. In general, honeybee viruses are widespread and most of them persist as inapparent infections. We screened honeybee colonies for the presence of several bee viruses, including deformed wing virus(DWV), black queen virus(BQCV), Kashmir bee virus(KBV), Israeli acute paralysis virus (IAPV), sacbrood virus(SBV), acute bee paralysis virus(ABPV), using uniplex RT-PCR. Frequently simultaneous infections with different viruses are diagnosed in seemingly healthy bee colonies. Therefore we developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses.

Sacbrood virus (SBV) is one of the most destructive honey bee virus. The virus causes failure to pupate and kills honey bee larvae. The infacted larvae`s color is change to brown. At the end, honey bee colony is destructed. Recently Korean Scabrood virus(KSBV) caused a great loss of Korean honey bee(Apis cerena) colonies for short period. Therefore, We need a highly rapid diagnosis method for rapid detection of KSBV. In this study, We need amicro-scale chip-based real-time PCR system (GeneChecker®). This system was developed for rapid, specific PCR based diagnosis. This system has uncommonly fast heating and cooling system. So We was able to detecting of KSBV in Apis cerena in short time. This system needs small reaction volume(total 10ul). This volume include SsoFast™ Evagreen Supermix and serially diluted cDNA templates showed a high sensitivity of 101copies.That machine can setting each PCR stage time. A specific detection primer set (KSBV-123-F/R) was used to amplify a unique 123bp DNA fragment. This PCR assays using serially diluted cDNA templates showed a high sensitivity of 101 copies. When applied to KSBV-positve samples, the result showed high specifity. The minimum diagnosis time was 9m 47s (30cycle). The amplied positive samples appear red fluorescent color. This novel detection method could be used a PCR-based diagnositic tool (GeneChecker®). The results showed high sensitivity and specifity in short time. And this diagnosis method is expected to be applied to rapidly detect various pathogens.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, Korean Sacbrood virus (KSBV) caused a great loss in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a novel micro PCR-based detection method, termed ultra-rapid real-time PCR (URRT-PCR) were applied for rapid detection for korean sacbrood virus (KSBV) from honeybees (Apis cerana) infected with SBV in Korea. The LAMP could be detect the virus in RT-LAMP reactions containing 102copies of pBX-KSBV within 30 min, which was 10 times more sensitive than a RT-PCR assay. The URRT-PCR showed high sensitivities which were able to detect 10 copies in the standard assays. In the application of URRT-PCR detection to an KSBV-infected honeybee, the shortest detection time was 10 min 12 sec, including reverse transcription. In addition, these methods could be distinguished between KSBV and other closely-related SBV strains, These rapid methods were rapid molecular-based diagnostic tools and useful tool for the rapid and sensitive diagnosis of KSBV infection of honeybees.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of KSBV in cell cultures. The growth of KSBV was demonstrated by RT-PCR, quantitative real-time PCR, TEM and nucleotide sequence analysis. The results demonstrated that SBVshowed the replication signals in mammalian cell lines, including Vero cells without any signs of cytopathic effect (CPE). The results of RT-PCR, quantitative real-time PCR and in vivo infection with KSBV were also indicated the replication. Phylogenetic tree analysis shows our sequence included in distinct group with other SBV strains from China and Korea. It clearly showed the differenciation between field strain and attenuated strain through cell culture. The results of present study demonstrated for the first time that SBV like other animal viruses could be adapted and attenuated in cells through the sequential passages. The sequential adaptation through cell culture could result in discrepancy of pathogenicity of virus and morphological characterization. For this reason, the present results indicated that the cell adapted SBV could be a valuable tool to study the general properties of this emerging virus, including pathogenicity in the future.

During January-November 2012, a total of 2,041 quarter milk samples were collected from dairy cattle of 82 dairy farms nationwide. About 42% (870/2,041) of the samples that had somatic cell counts (SCC) of ≥ 200,000 cells/ml were subjected to microbiological examination. No bacteria was isolated from 95 of 870 (10.9%) samples. Among 1,237 bacteria isolated from the rest 775 samples, 1,085 were identified with VITEK: more than half (52.1%, 645/1,237) of the isolates were gram negative bacillus. Gram positive cocci including Staphylococcus accounted for 35% of the isolates and almost none of gram positive bacilli isolated. Excluding Coagulase-negative Staphylococcus (CNS), the most frequently isolated bacterial species was Escherichia coli (11.2%, 138/1, 237), followed by Klebsiella pneumoniae (8.1%,100/1,237), Staphylococcus aureus (7.1%, 88/1, 237), Enterobacter cloacae (6.0%, 74/1, 237), and Serratia marcescens (3.5%, 43/1, 237). The most common resistance of S. aureus was to penicillin (77.4%) and ampicillin (73.0%), while no resistance was observed against gentamicin and cephalothin. Although CNS presented resistance to all antimicrobials tested but the most prevalent resistance was to penicillin (35.6%) and ampicillin (37.0%). The pattern of antimicrobial resistance observed in CNS was similar to that of S. aureus, but the rates were much lower than those of S. aureus. E. coli also showed resistance to all the antimicrobials tested, although the rates were not very high. The highest resistance of E. coli was to cephalothin (39.4%) and ampicillin (36.2%), while most of the strains (98.0%) showed sensitivity to amikacin. The results of this study provide information on current situation of bovine mastitis in Korea.

The current standard solutions for somatic cells used for calibration of electronic somatic cell counts as reference material in raw milk are preserved with bronopol, boric acid, sodium azide, or potassium dichromate, and have a shelf-life of only up to 6 days at 4 ± 2℃. In the present study, a set of somatic cell standard solutions (SCSS) with a stability of 5 months for calibration of electronic instruments was developed. Somatic cells collected from cow’s milk and stored in a bulk tank at a dairy plant were treated with 10% formaldehyde in order to improve stability, and then separated by centrifugation. The resulting somatic cell suspension was preserved with glycerin, thimerosal, and dimethyl sulfoxide, and diluted in 3% processed skim milk solution ranging from 200,000～250,000 (low level), 350,000～ 450,000 (medium level), and 550,000～650,000 (high level) cells/㎖. Each SCSS was verified by direct microscope somatic cell counting (DMSCC), C-reader, and commercial standard samples. The average somatic cell count determined by DMSCC was 248, 214, 226 × 103 cells/㎖, 436, 382, 420 × 103 cells/㎖, and 612, 595, 609 × 103 cells/㎖. The coefficient of variation representing the repeatability of DMSCC decreased as the number of cells increased, and was <10.0% in almost all SCSS samples (range 4.6～7.1%). No statistically significant difference in somatic cell concentration was observed after storage at refrigeration temperature (2～6℃) over a period of 22 weeks (5 months). The stabilized SCSS may be useful as a reference material for determination of somatic cell count and quality control in testing of bovine raw milk.

Bovine brucellosis causes abortion and infertility. The authors conducted this study in order to determine pathological lesions of Korean native cows and fetuses who received experimental vaccination with Brucella abortus RB51 and were challenged with Brucella abortus 2308. Gross and histopathological lesions in endometrium and placenta were observed in cows of the vaccinated group. Twenty-five percent of pregnant cattle in the vaccinated group showed endometritis and placentitis, which was three times lower, compared with the non-vaccinated group. The pathological lesions in the uterus and placenta in both groups were consistent with previous reports. Therefore, vaccination in heifers using Brucella abortus RB51 may not provide adequate protection against infection with Brucella abortus virulent strain.

To determine current rate of antimicrobial resistance, a total of 236 isolates from milk samples of dairy cattle with mastitis in Korea during 2010-2011 were examined against 12 antimicrobials using disc diffusion method: 67 Staphylococcus aureus, 74 coagulase-negative Staphylococcus spp. (CNS), and 95 Escherichia coli isolates. The isolates examined in this study were submitted by Local Veterinary Service Laboratories located in 13 provinces and metropolitan cities nationwide. The highest rates of resistance among S. aureus isolates were against ampicillin (56.7%) and penicillin (56.7%), followed by kanamycin (11.9%). All S. aureus isolates were sensitive to lincomycin, amikacin, and cephalothin. Only one isolate showed resistance to tetracycline and oxacillin, respectively. Less than 10% of the S. aureus isolates presented resistance to erythromycin, neomycin, and gentamicin. Among CNS isolates, the most frequently observed resistance was to lincomycin (44.5%), followed by penicillin (28.3%), ampicillin (18.9%), tetracycline (17.5%), kanamycin (13.5%), and erythromycin (9.4%). All or most of the CNS isolates were sensitive to cephalothin, amikacin, neomycin, and gentamicin. The highest rate of resistance among E. coli isolates was against tetracycline (26.3%), followed by streptomycin (21%), neomycin (15%), kanamycin (12.6%), and gentamicin (10.5%). Amikacin was the only antimicrobial to which no E. coli isolates showed resistance. Around 10% of the S. aureus isolates and 15% of the CNS isolates showed resistance against three or more antimicrobials simultaneously, while more than 30% of the E. coli isolates did.

Salmonellosis constitutes an important public health problem in both developing and developed countries, including Korea. The aims of present study were to investigate the serovar and antimicrobial resistance of Salmonella isolated from food animals and animal products in slaughterhouses and farms. A total of 323 Salmonella were isolated from food animals (n=277) and meats (n=46) during 2010. Of the isolates, 21 different serovars were identified. The predominant serovars were S. Rissen (35%) and S. Montevideo (24.3) in healthy pigs, while S. Enteritidis (25.5%) in healthy chicken. S. Typhimurium (88.8%) was predominant in disease pigs, while S. Gallinarum (29.2%) and S. Montevideo (26.9%) were in diseased chickens. Among meat samples, S. Typhimurium (57.1%) was the most common serovar in pork but S. Enteritidis (38.7%) and S. Montevideo(32.3%) were in chikcen meats. Analysis of antimicrobial resistance patterns revealed that 20.7% of the isolates were sensitive to all the 15 drugs tested. The isolates were frequently resistant to nalidixic acid (47.7%), tetracycline (38.4%), streptomycin (33.7%), and ampicillin (32.8%). The resistance to quinolone and 3rd generation cephalosporins was higher in chicken and chicken meat isolates. Of the 323 isolates, 174 (53.9%) were resistant to one or more CLSI subclass, and 117 (36.2%) showed multiple-resistance. Our findings showed that multiple resistant Salmonella organism are widespread in animals and animal products in Korea. To prevent the transmission or exposure for consumers of antimicrobial resistant Salmonella, policies and guidelines aiming at prudential use of critical antimicrobials for humans are needed.

Campylobacterosis is the most common food borne bacterial disease in many countries. Food animals and animal products are considered to be the reservoir of the Campylobacter species. The aim of this study was to investigate the antimicrobial resistance of Campylobacter spp. from food animals and raw meats in slaughterhouses. A total of 90 Campylobacter jejuni (C. jejuni) and 127 Campylobacter coli (C. coli) were isolated, for which antimicrobial susceptibility was examined using broth dilution method. Resistance to macrolide antimicrobials was higher among C. coli isolates than among C. jejuni. Among both C. jejuni and C. coli isolates, the most frequently observed resistance was to nalidixic acid, ciprofloxacin, and tetracycline. No erythromycin resistance was observed among C. jejuni isolates from cattle, pig and beef. However, 28.3% (n=13) and 25% (n=3) of C. coli isolates from pigs and pork showed resistance to erythromycin, respectively. The predominant profile of multiple resistance among C. jejuni and C. coli isolates was ciprofloxacin/tetracycline/nalidixic acid resistance (46.7%) and ciprofloxacin/nalidixic acid resistance (31.5%), respectively. This finding has important implication for food safety and public health.

Staphylococcus aureus is an important food-borne pathogen, which is present on the skin and mucosa of animals. Some of the S. aureus strains are causative agent of food poisoning syndrome. The aim of this study was to investigate the antimicrobial resistance of S. aureus isolates from raw meats in slaughterhouses during 2010. From 17,874 raw meat samples tested, a total of 190 S. aureus were isolated, for which antimicrobial susceptibility to 17 agents was examined using broth dilution method. Among isolates from beef, chicken and pork, 20 (51.3%), 20 (24.7%) and 9 (12.9%) were sensitive to all antimicrobials tested, respectively. Isolates from pork and chicken meats showed much higher resistance, compared to isolates from beef. Penicillin resistance was the most frequent among isolates from beef (35.3%) and pork (75.7%), while tetracycline resistance was among those from chicken meats (48.1%). A total of 3 methicillin resistant S. aureus (MRSA) were detected from beef (5.1%, 2/39) and pork (1.4%, 1/70). Although the prevalence of MRSA was low, the presence of antimicrobial resistant S. aureus such as MRSA suggests that further investigation and strict surveillance on MRSA and antimicrobial resistance are needed.

A total of 222 udder-half milk samples of lactating goats were collected from two herds in Korea during 2008 and all samples were subjected to bacteriological examination. Somatic cell counts (SCC) were also determined for all samples except for 13 (5.9%), which were collected from halves of udders with clinical mastitis. A total of 85 bacteria were isolated from 82 (36.9%) of 222 milk samples tested. Staphylococci were the predominant pathogens, accounting for almost 70% of the isolates: Coagulase negative staphylococci (CNS) and S. aureus constituted 55% (47/85) and 14.1% (12/85), respectively. Among 209 samples tested for SCC, bacteria were isolated from 36 of 115 (31.3%) samples with SCC of <1×106 cells/㎖ and 38 of 94 (40.4%) samples that had SCC of ≥1×106 cells/㎖, respectively. All S. aureus were detected from samples with SCC of ≥1×106 cells/㎖, while 25 of 47 (61.0%) CNS were isolated from milk samples with SCC of <1×106 cells/㎖. Mean SCC of milk samples that harbored S. aureus and CNS was 4,787×103 cells/㎖ and >1×106 cells/㎖, respectively. All S. aureus and CNS isolates were susceptible to all antimicrobials tested except for penicillin, to which 2 (16.6%) S. aureus and 12 (25.5%) CNS isolates showed resistance.

One hundred one enterococcal isolates from feces of livestock animals in Korea were screened for the presence of bacteriocins. Sixteen of 41 (39%) E. faecalis and 4 of 56 (7.1%) E. faecium isolates showed antimicrobial activity against at least one indicator strain. Only 4 of 20 the enterococcal isolates showing antimicrobial activity possessed at least one bacteriocin gene. While entA and entB were detected in three isolates as a pair of genotype, entQ, bac31, and AS-48 were not found in the enterococcal isolates. In almost all isolates, a correlation between genotype and phenotype of these determinants was not always observed.

For the screening of Brucella antibodies in pig, 2,140 pig serum samples were collected from six slaughter house in Korea between 2006 and 2007. The Rose Bengal test (RBT) and serum agglutination test (SAT) were used for initial screening for specific antibodies to Brucella, and competitive enzyme-linked immunosorbent assay (C-ELISA) was used for confirmation of presence of serum antibody for Brucella. Overall, 575 (26.9%) samples resulted in seropositive in RBT. In SAT, 50 (2.3%) and 10 (0.5%) samples showed suspicious positive and positive reaction, respectively, however, all sera tested in this study showed a negative reaction in C-ELISA. SAT and C-ELISA might be applicable as a tool for screening of swine brucellosis.