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Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.

Salmonella is a foodborne pathogen worldwide. Outbreaks of Salmonella are commonly associated with consumption of contaminated foods such as poultry products. Therefore, the objective of this study was to determine the occurrence, biofilm formation, antibiotic resistance, and sanitizer resistance of Salmonella enterica isolated from chicken carcasses. A total of 318 samples were collected from 15 chicken slaughterhouses in 8 provinces of Korea. They were then examined for Salmonella contamination. S. enterica isolates were tested for their susceptibilities to 15 antimicrobials by broth microdilution method. Their biofilm formation ability and resistance to sanitizers were also evaluated. Eighty-two isolates of S. enterica were obtained from the 318 samples. There were 14 serotypes and 2 untypable isolates. Fifty-seven (69.5%) isolates were resistant to at least one antibiotic while 30 (36.6%) isolates were resistant to 5 or more antibiotics. Two S. Senftenberg and 3 S. Montevideo isolates exhibited considerable biofilm formation ability (A600 >0.2) following incubation in Luria-Bertani (LB) broth for 48 h. Biofilm cell survival and recovery growth assay after sanitization showed that most isolates were highly susceptible to 2.5% lactic acid and 0.1% cetylpyridinium chloride. Therefore, lactic acid and cetylpyridinium chloride might be alternatively or additionally used in addition to chlorine-based sanitizers that are frequently used to reduce Salmonella contamination of chicken carcasses. Our results provide basic information on the distribution of Salmonella serotypes in chicken slaughterhouses. This study also highlights the necessity to improve farming practices and use antimicrobial agents cautiously. This study also suggests that sanitization during the slaughtering process might be necessary to reduce Salmonella contamination of chicken carcasses.

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne`s disease (JD) in ruminants. This is the first large scale report to estimate the herd-level prevalence of antibodies against MAP by using an ELISA to detect antibodies in bulk tank milk (BTM) samples. The samples were collected from January 2011 to November 2011, from 636 herds of the dairy farms in the Gyeonggi and Chungbuk areas of Korea. The overall apparent prevalence of MAP antibody-positive herds was 8.5%, and regional prevalence were 32/440 (7.3%) and 22/196 (11.2%) of dairy farms in the Gyeonggi and Chungbuk areas, respectively. The results did not differ significantly by region. While we have determined the prevalence rate of MAP in the Gyenoggi and Chungbuk areas in this study, there is a continuing need for well-designed studies to calculate the prevalence of MAP in dairy herds based on culture and molecular findings.

Bovine tuberculosis (bTB) is a common zoonosis prevalent in many countries with grave economic consequences. Most developed and developing countries have implemented the test-and-slaughter policy to protect public health and reduce economic losses in the cattle industry. The official diagnosis of bTB is based on assays dependent on cell-mediated immunity (CMI). CMI-based diagnosis demonstrates diagnostic incapability at late stages of infection, which could be overcome by diagnosis based on humoral immunity (HI). Therefore, there is an urgent need to identify and define the B cell antigenome of Mycobacterium bovis. In this study, the B cell antigenome of culture filtrate proteins (CFP) was defined by mass spectrometry-based proteomics technology. Four spots were detected on 2-dimensional gel electrophoresis (2-DE) against M. bovis-positive serum in an immunoblotting experiment. Twenty-one proteins were identified in four spots by proteomic tools, such as Mb2900, Mb2898, Mb0448, Mb3834c, Mb1918c, Mb0134c, Mb0358 and Mb1868c, which are known B cell antigens, including 13 new proteins, i.e. Mb3751, Mb2006c, Mb3276c, Mb2244, Mb1164c, Mb2553c, Mb2946c, Mb1849c, Mb1511c, Mb1034c, Mb2616c, Mb0854c and Mb2267. These new proteins identified by 2-DE and immunoblotting were the B cell antigens used in developing serological diagnostic methods based on HI to bTB.

Salmonella is a major food-borne pathogen in humans and a cause of local or systemic disease in animals. Therefore, rapid and reliable methods to detect these poultry-associated Salmonella serotypes are necessary for efficient control of Salmonella in poultry. The present study aimed to develop a real-time multiplex PCR (MqPCR) method to simultaneously detect and/or differentiate Salmonella sp. and poultry-associated serotypes, including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Pullorum, and Salmonella Gallinarum. A MqPCR method was designed using four specific primer pairs and probes for the detection of Salmonella sp., including S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. Additionally, a novel TaqMan-based MqPCR method combined with propidium monoazide (PMA) treatment was developed for the simultaneous quantification of viable cells of Salmonella sp. and these four Salmonella serotypes in rinse water of chicken carcasses. The MqPCR assay specifically detected Salmonella sp., S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum, showing 100% sensitivity and 100% specificity. This optimized PMA-MqPCR assay could detect live Salmonella (100–106 CFU/reaction) without enrichment in live/dead cell mixtures from spiked rinse water of chicken carcasses. The procedure for detecting live Salmonella required less than 2 h to complete. This PMA TaqMan-based MqPCR technique facilitates accurate and rapid monitoring of contamination with viable Salmonella. Also, the assay enables simultaneous identification of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum in rinse water of chicken carcasses. The assay developed in this study will be useful in diagnostic laboratories for improving Salmonella control in poultry and poultry products.

Methicillin-resistant Staphylococcus aureus (MRSA), the major causative agent of nosocomial infection, has also been reported from non-human sources. A sequence type (ST) 541 MRSA isolate designated K12PJN53 was isolated from a healthy pig in 2012. The genome of K12PJN53 consists of 44 contiguous sequences (contigs), totalling 2,880,108 bases with 32.88% GC content. Among the annotated contigs, 14, 17, and 18 contained genes related to antimicrobial resistance, adherence, and toxin genes, respectively. The genomic distance of strain K12PJN53 was close to the ST398 strains. This is the first report of the draft genome sequence of a novel livestock-associated MRSA ST541 strain.

Domestic ducks are considered a potential reservoir of Newcastle disease virus. In the study, a Newcastle disease virus (NDV) isolated from a domestic duck during surveillance in South Korea was characterized. The complete genome of the NDV isolate was sequenced, and the phylogenetic relationship to reference strains was studied. Phylogenetic analysis revealed that the strain clustered in genotype I of Class II ND viruses, has highly phylogenetic similarity to NDV strains isolated from waterfowl in China, but was distant from the viruses isolated in chickens and vaccine strains used in South Korea. Pathogenicity experiment in chickens revealed it to be a lentogenic virus. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the avirulent motif (112)GKQGRL(117) at the cleavage site and caused no apparent disease in chickens and ducks. With phylogeographic analysis based on fusion gene, we estimate the origin of an ancestral virus of the isolate and its sister strain located in China around 1998. It highlights the need of continuous surveillance to enhance current understanding of the molecular epidemiology and evolution of the pathogenic strains.

To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.

(Received: July 23, 2014; Revised: September 16, 2014; Accepted: October 13, 2014)Abstract : In Korea, brucellosis has been reported periodically in cattle and rarely in dogs; however, it has not previouslybeen screened in domestic animals such as elk, pigs and goats. To investigate the serological prevalence, serum sampleswere taken from the aforementioned animals annually during 2007-2013 and screened by the rose-bengal test (RBT)or modified RBT, after which positive sera were evaluated by the standard tube agglutination test (STAT). Finally,RBT and STAT-positive sera were confirmed by competitive-ELISA. Brucella abortus biovar 1 was isolated from threeelk that were shown to be positive serologically in 2008. There was no evidence of brucellosis in pigs. Based onserological monitoring and investigation of etiological agents, there is no evidence of outbreak of brucellosis in elk,pigs or goats of Korea since 2008. However, the possibility for brucellosis from cattle to affect these other livestockexists; therefore, extensive and continuous serological monitoring is required to maintain their brucellosis-free status.Keywords : brucellosis, competitive-ELISA, rose-Bengal test, serological test, standard tube agglutination test

The plasmid-mediated colistin resistance gene mcr-1 was first reported in China in 2013, and has since been identified in both humans and livestock from many other countries (1-3,5).….

Streptococcus species are emerging potential pathogens in marine mammals. We report the isolation and identification of Streptococcus halichoeri and Streptococcus phocae in a Steller sea lion (Eumetopias jubatus) in South Korea.

Twenty field isolates of Avibacterium paragallinarum were obtained from chickens in South Korea during 2011-2015. The isolates were identified by a HPG-2 PCR assay specific for A. paragallinarum and by biochemical tests. Growth requirements, Page serovars, carbohydrate fermentation patterns, and antimicrobial susceptibility were also examined. Most isolates (16/20) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. One isolate needed increased levels of NAD and serum for good growth. Three isolates showed NAD-independent growth on blood agar under aerobic conditions. In terms of carbohydrate fermentation patterns, three biochemical biovars were recognized; these varied with respect to acid production from maltose and D-xylose. The 16 typical NAD-dependent isolates were serovar A while the variants, both NAD-independent isolates and the isolate with increased NAD dependency were non-typeable. All isolates were sensitive to amoxicillin-clavulanic acid, ceftiofur, gentamicin, and spectinomycin. High rates of resistance, including intermediate resistance, to lincomycin (100%), cloxacillin (75%), and erythromycin (70%) were observed. The four variant strains (the three NAD-independent isolates and the isolate showing unusual growth requirements) were more resistant to antibiotics than the typical NAD-dependent strains. The finding of NAD-independent forms of A. paragallinarum extends the known distribution of this form, previously only reported in South Africa, Mexico and Peru. There is clearly a need for increased caution in the diagnosis and, possibly, the control of infectious coryza.

The purpose of present study was to investigate the antimicrobial resistance in Escherichia coli isolated from healthy animals in all provinces of the Republic of Korea. A total of 2,085 E. coli strains isolated from 11,336 fecal samples of healthy animals during 2010-2012 were examined for antimicrobial resistance. Comparison of average resistance rate through the years revealed that tetracycline (47.0% and 76.1%) and streptomycin resistance (42.6% and 64.6%) was most frequently observed in cattle and pigs, respectively. Whereas, in chicken isolates, resistance against nalidixic acid (90.9%) was highest among the antimicrobials tested. Percentage of E. coli that showed multidrug resistance (resistance against ≥ three subclasses of antimicrobial agents) was 17.6% (151/860) in cattle, 69.4% (506/ 729) in pigs, and 86.1% (427/496) in chickens. Overall, the rates of resistance are apparently different between animal species and, in particular, resistance was less prevalent in cattle than in pigs and chickens. In conclusion, this study showed higher prevalence of resistance in commensal E. coli strains to antimicrobial agents in Korean livestock and highlighted the urgent need for measures to regulate the abuse of antimicrobial agents.

Q-fever is a vector-borne (Coxiella [C.] burnetii) zoonotic disease that is an increasing public health concern. To date, some research about Q-fever prevalence in dairy herds and human patients has been reported in Korea, but information about Korean native cattle is scarce. To measure the prevalence rates of C. burnetii in Korean native cattle, a total of 1,095 bovine serum samples collected during 2010~2013 were analyzed with an enzyme-linked immunosorbent assay. Sixty-eight heads of cattle were diagnosed as positive and while 19 heads were suspected (positive rate

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.

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis . The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10 3 colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis . Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis , and the new assay will provide a valuable tool for the diagnosis of B. canis infection.

This study compared the antimicrobial susceptibility and prevalence of virulence genes in Salmonella enterica Typhimurium isolated from healthy and diseased pigs in Korea. A total of 456 Salmonella Typhimurium isolated from healthy (n = 238) and diseased (n = 218) pigs between 1998 and 2011 were investigated. In total, 93.4% of the Salmonella Typhimurium isolates were resistant to at least one antimicrobial agent tested. The isolates were most often resistant to tetracycline (85.7%), followed by streptomycin (83.6%), nalidixic acid (67.3%), ampicillin (49.3%), chloramphenicol (42.8%), and gentamicin (37.1%). Moreover, multidrug resistance phenotype and resistance to ampicillin, florfenicol, gentamicin, nalidixic acid, neomycin, streptomycin, and tetracycline were significantly higher (P < 0.01) among Salmonella Typhimurium isolates from the diseased pigs compared with those from the healthy pigs. The most common resistance pattern observed in both groups of isolates was streptomycin-tetracycline. Overall, more than 96% of the isolates tested possessed invA, spiA, msgA, sipB, prgH, spaN, tolC, lpfC, sifA, sitC, and sopB virulence genes. The prevalence of orgA, pagC, and iroN were 50.2, 74.1, and 91.0%, respectively, whereas isolates carrying cdtB (1.5%), pefA (7.0%), and spvB (14.9%) were identified much less frequently. Furthermore, the prevalence of invA, lpfC, orgA, pagC, and iroN was significantly higher (P < 0.01) among the isolates from the diseased pigs than in isolates from the healthy pigs. Our results demonstrated that, among diseased pigs, there was significantly higher resistance to some antimicrobials and greater prevalence of some virulence genes than in healthy pigs, indicating the role these factors play in pathogenesis. Multidrug-resistant Salmonella isolates that carry virulence-associated genes are potentially more dangerous and constitute a public health concern. Thus, continuous surveillance of antimicrobial resistance and virulence characteristics in Salmonella is essential.

(Received: January 6, 2014; Revised: April 2, 2014; Accepted: April 9, 2014)Abstract : In this study, trends in the sales of antimicrobials for use in livestock facilities and fisheries from 2003to 2012 were investigated with regard to antimicrobial group, antimicrobial usage, and animal species. The overallamount of antimicrobials sold each year from 2003 to 2007 was 1,500 tons, after which they decreased, with thelowest sales being 936 tons in 2012. The total volume of antimicrobials used for feed additives decreased markedlyby 94% from 2003 to 2012, which was mainly attributed to banning of feed additives. However, antimicrobialconsumption through self prescription by farmers for disease prevention and treatment increased by 25% from 2003to 2012. The largest volume of antimicrobials sold was for use in pigs (48~57%), followed by poultry (18~24%),fisheries (11~25%), and cattle (5~8%). Tetracycline was the highest selling antimicrobial, followed by penicillinsand sulfonamides, although the overall sale of all three antimicrobials gradually decreased over the study period.This study demonstrated that the total consumption of antimicrobials has gradually decreased since 2008.Nevertheless, usage by nonprofessionals increased, which can ultimately cause emergence and spread of antimicrobialresistance. Thus, early establishment of veterinary prescription guidelines for prudent use of antimicrobials is urgentlyneeded in Korea. Keywords : antimicrobials, fisheries, livestock, sales

3 Research Unit, BIONOTE, Hwaseong 445-170, Republic of Korea Abstract: The aim of the present study was to evaluate the use of an immunochromatographic test (ICT) strip using recombinant MPB70 (rMPB70) protein as a complementary tool for the diagnosis of naturally occurring tuberculosis in cattle. The study was performed on 249 cattle from populations known to be free from Mycobacterium bovis (M. bovis) and 119 cattle with M. bovis infection, confirmed postmortem. Compared to reference standards (culture isolation and/or visible lesion), the sensitivity of ICT was 94.12% (95% CI: 89.89 98.35%) while the specificity was 96.80% (95% CI; 94.62 96.82%). The findings indicate that the ICT strip is efficient for diagnosing bovine tuberculosis in cattle from Korea.

Kashmir bee virus (KBV) is one of the most common viral infections in honeybees. In this study, a phylogenetic analysis was performed using nine partial nucleotide sequences of RdRp and the structural polyprotein regions of South Korean KBV genotypes, as well as nine previously reported KBV genotypes from various countries and two closely related genotypes of Israeli acute paralysis virus (IAPV) and Acute bee paralysis virus (ABPV). The Korean KBV genotypes were highly conserved with 94-99 % shared identity, but they also shared 88-95 % identity with genotypes from various countries, and they formed a separate KBV cluster in the phylogenetic tree. The complete genome sequence of Korean KBV was also determined and aligned with previously reported complete reference genome sequences of KBV, IAPV, and ABPV to compare different genomic regions. The complete Korean KBV genome shared 93, 79, and 71 % similarity with the complete reference genomes of KBV, IAPV, and ABPV, respectively. The Korean KBV was highly conserved relative to the reference KBV genomes in the intergenic and 3' untranslated region (UTR), but it had a highly variable 5' UTR, whereas there was little divergence in the helicase and 3C-protease of the nonstructural protein, and the external domains of the structural polyprotein region. Thus, genetic recombination and geographical distance may explain the genomic variations between the Korean and reference KBV genotypes.

Deformed wing virus (DWV) is one of the most common viral infection in honeybees. Phylogenetic trees were constructed for 16 partial nucleotide sequences of the structural polyprotein region and the RNA helicase region of South Korean DWVs. The sequences were compared with 10 previously reported DWV sequences from different countries and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus-1 (VDV-1). The phylogeny based on these two regions, the Korean DWV genomes were highly conserved with 95-100% identity, while they also shared 93-97% similarity with genotypes from other countries, although they formed a separate cluster. To investigate this phenomenon in more detail, the complete DWV genome sequences of Korea-1 and Korea-2 were determined and aligned with six previously reported complete DWV genome sequences from different countries, as well as KGV and VDV-1, and a phylogenetic tree was constructed. The two Korean DWVs shared 96.4% similarity. Interestingly, the Korea-2 genome was more similar to the USA (96.5%) genome than the Korea-1. The Korean genotypes highly conserved with USA (96%) but low similarity with the United Kingdom3 (UK3) genome (89%). The end of the 5' untranslated region (UTR), the start of the open reading frame (ORF) region, and the 3' UTR were variable and contained several substitutions/transitions. This phenomenon may be explained by intramolecular recombination between the Korean and other DWV genotypes.

During the collection of boar semen, bacterial contamination usually occurs. The contamination has deleterious effects both on semen quality and on sow fertility. The majority of contaminants are gram-negative bacteria, especially Serratia marcescens. In this study, we developed a PCR assay for the identification of S. marcescens targeting the luxS gene (GenBank no. EF164926). S. marcescens yielded a specific 306 bp PCR product. However, no amplification was observed in the other strains tested. The detection limit of PCR was 50 pg/μl of template DNA of S. marcescens. The antimicrobial susceptibility patterns of S. marcescens isolated from boar semen were tested using the disk diffusion method. Gentamicin, ceftiofur, florfenicol, and neomycin showed high sensitivity in this test. The minimum inhibitory concentration (MIC) was also determined by the broth microdilution method. The MIC90 values of ceftiofur, enrofloxacin, gentamicin, and neomycin were 8, 8, 8, and 16 μg/ml, respectively. These results indicate that PCR amplification of the luxS gene is a reliable and effective method for the identification of S. marcescens and that ceftiofur, enrofloxacin, gentamicin, and neomycin are effective semen extenders for controlling S. marcescens.

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.

Apramycin resistance was observed in 22.8% (81 of 355) of Salmonella Typhimurium isolates collected from pigs from 1998 through 2009 in Korea. All apramycin-resistant Salmonella Typhimurium isolates also were cross-resistant to gentamicin and tobramycin. Among the seven types of aminoglycoside resistance genes tested, only four types were detected in the apramycin-resistant Salmonella Typhimurium isolates: aac (3)-IV, aac (3)-II, aac (3)-III, and ant (2'')-I. Although the aac (3)-IV gene was found in all apramycin-resistant Salmonella Typhimurium isolates, aac (3)-II, aac (3)-III, and ant (2'')-I genes were detected in five (6.2%), two (2.5%), and three (3.7%) isolates, respectively. The apramycin-resistant isolates comprised six phage types, of which PT193 (16 of 81 isolates, 19.8%) was most commonly observed. To our knowledge, this is the first report describing characteristics of apramycin-resistant Salmonella Typhimurium isolates in Korea. Further study is warranted to determine whether apramycin use in animals results in cross-resistance to gentamicin, which may affect public health when gentamicin is required for disease treatment in humans.

The aim of this study was to determine the presence and persistence of methicillin-resistant Staphylococcus aureus (MRSA) in milk, farm environment, and farmers on 22 dairy cattle farms in Korea during 2008-2009. Genetic relatedness among the MRSA isolates was also investigated. Of 1146 samples examined, 35 of 559 (6.3%) quarter milk samples from 371 cows, four of 86 (4.7%) hand and nose samples from 43 farmers, and 6 of 501 (1.2%) farm environment samples were MRSA positive. Except for three isolates, all MRSA were classified into ST72-spa t324-SCCmec IV with PVL negative, the most predominant clonal type among community-associated MRSA in South Korea. All 35 MRSA-positive milk samples from 19 cows were obtained from a single farm (Farm G) out of 22 (4.5%) farms tested. The farm G was revisited 1 year later and milk samples were collected for examination of MRSA again. Two of six previous MRSA-positive cattle that had been kept on the farm still harbored MRSA genetically identical to MRSA strains, which were isolated from the same farm a year ago. The results of this study provide the evidence of transmission of MRSA among cattle, farm environment, and farmers and also long-term persistence of MRSA in animals.

A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla CTX-M genes that belonged to the CTX-M-1 ( n = 35) or CTX-M-9 ( n = 49) family. The most predominant CTX-M type identified was CTX-M-14 ( n = 49), followed by CTX-M-32 ( n = 26). The bla CTX-M genes were identified most commonly in E. coli isolates from feces ( n = 29), teats ( n = 25), and milk ( n = 14). A bla CTX-M-14 gene was also detected in an E. coli isolate from a farmer's hand. Transfer of the bla CTX-M gene from 60 bla CTX-M -positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla CTX-M -positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla CTX-M genes possessed an insertion sequence, IS Ecp1 , upstream of the bla CTX-M gene. Only in the case of the CTX-M-14 genes was IS 903 downstream of the gene. The bla CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC , hok-sok , and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.

This study was carried out to identify the tick species that infest grazing cattle and to determine the presence of tick-borne pathogens transmitted by these ticks in Korea. A total of 903 ticks (categorized into 566 tick pools) were collected from five provinces during 2010-2011. The most prevalent tick species was Haemaphysalis longicornis, followed by three Ixodes spp. ticks. The collected ticks were infected with both rickettsial and protozoan pathogens. In all, 469 (82.9%) tick pools tested positive for the Anaplasma/Ehrlichia 16S rRNA gene, whereas 67 (11.8%) were positive for the Babesia/Theileria 18S rRNA gene. Among the rickettsial pathogens, E. canis was detected with the highest rate (22.3%), followed by A. platys (20%), E. chaffeensis (19.4%), E. ewingii (19.3%), Rickettsia sp. (12.4%), A. phagocytophilum (5.5%) and E. muris (0.5%). Among the protozoan pathogens, T. equi was detected with the highest rate (7.2%), followed by T. sergenti/T. buffeli (3.7%) and B. caballi (0.35%). Simultaneous infections with up to seven pathogens were also identified. In particular, ticks infected with rickettsial pathogens were also infected with protozoan pathogens (22 samples). All five provinces investigated infected with tick-borne pathogens.

The purpose of this study was to determine the prevalence and characteristics of CTX-M β-lactamases in Escherichia coli among healthy swine and cattle in Korea. A total of 1212 fecal samples obtained from healthy pigs (n=558) and cattle (n=654) were screened for CTX-M-type extended spectrum β-lactamase (ESBL)-producing E. coli isolates. One hundred and twenty-one E. coli that produced ESBL were subjected to phenotypic and genotypic characterization. A high number (120/558, 21.5%) of swine fecal samples showed the presence of CTX-M β-lactamase-producing E. coli compared to cattle samples (1/654, 0.2%). The most predominant CTX-M-type identified was CTX-M-14 (n=82), followed by CTX-M-15 (n=16). Isolates producing CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 were also identified. Overall, the bla(TEM-1) gene was associated with CTX-M β-lactamase in 55 E. coli isolates. Transfer of bla(CTX-M) gene was demonstrated from 76 out of 121 bla(CTX-M)-positive E. coli isolates to the recipient E. coli J53 by conjugation. Plasmid DNA isolation from the transconjugants revealed a large (90-120 Kb) conjugative plasmid. ISEcp1 and IS903 were detected upstream and downstream of bla(CTX-M) genes in 117 and 91 E. coli isolates, respectively. Our results demonstrated that a combination of clonal expansion and horizontal transmission is spreading bla(CTX-M) genes among swine E. coli. The horizontal dissemination of bla(CTX-M) genes among E. coli was mostly mediated by IncF or IncI1-Iγ plasmids. To the best of our knowledge, this study represents the first report of CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 β-lactamases in bacterial isolates from food animals in Korea. This study revealed that the CTX-M β-lactamase-producing E. coli are widely disseminated among healthy pigs but very rare in cattle in Korea. Increasing prevalence of bla(CTX-M) genes in intestinal E. coli of food animals is a matter of concern and should be carefully monitored.

The purpose of this study was to investigate the prevalence of Brachyspira species and antimicrobial susceptibility of Brachyspira (B.) hyodysenteriae isolates in Korea. A total of fifty-five Brachyspira species were isolated; five (1.0%) beta-hemolytic Brachyspira species and 50 (10.4%) weak hemolytic Brachyspira species from 116 different diarrheic pig samples and 367 apparently normal pig samples. In farm level, beta hemolytic and weak hemolytic Brachyspira species were detected in 7.4% (5/68) and 19.1% (13/68) of tested pig farms, respectively. By phenotypic and genotypic characterization, all beta hemolytic Brachyspira isolates was classified as group I (B. hyodysenteriae), whereas weak hemolytic Brachyspira species isolates were group III (B. innocens or B. murdochii). B. hyodysenteriae isolates showed high level of minimum inhibition concentrations to macrolide antimicrobials. This study shows that the prevalence of pathogenic B. hyodysenteriae in pigs is low but antimicrobial resistance of the pathogens is high in Korea. This is the first report of the prevalence of Brachyspira group III and antimicrobial susceptibility of B. hyodysenteriae in pigs in Korea. Our results could provide basic data for the management and treatment guidelines of Brachyspira infection.

A confirmatory serological test, the standard tube agglutination test (STAT) is evaluated for the diag-nostic efficiency in brucellosis Korea. A total of 345 bovine samples were collected from regional vet-erinary branch under national brucellosis monitoring program from January 2010 to June 2012 in Korea. These samples were diagnosed as suspected serum and brucellosis positive by the Rose Bengal test (RBT) and the STAT, respectively. The STAT was compared and evaluated with three serological test such as the indirect-enzyme linked immunosorbent assay (I-ELISA), competitive-enzyme linked im-munosorbent assay (C-ELISA) and fluorescence polarisation assay (FPA) prescribed for international trade by OIE. Among the 345 bovine serum samples, 302 (87.5%) were diagnosed as positive in the STAT, while 215 (62.3%), 223 (64.6%) and 194 (56.2%) serum samples were diagnosed as positive for brucellosis in the I-ELISA, C-ELISA and FPA, respectively. The STAT showed quite high positive results as compared with three prescribed tests of OIE. FPA, I-ELISA and C-ELISA have shown 60.6%, 64.9% and 67.2% correlation, respectively as compared to the STAT. However correlations of three pre-scribed tests ranged high 84.1∼97.7%. Especially, correlation between I-ELISA and C-ELISA is quite high, 97.7%. These results suggest that the STAT has shown many false-positive reactions. Therefore, additional serological test, such as ELISAs and FPA, would be necessary to adopt as a confirmatory test in the national surveillance program of bovine brucellosis in Korea.

From the end of July 2012, several cases of abortion have been happened at the Korean indigenous cattle farm with 124 heads in Chungbuk province, Korea. Serological tests such as Rose-bengal test (RBT) and standard tube agglutination test (STAT) have been performed according to the standard official protocols of bovine brucellosis and 41 cattle turned out to be brucellosis-positive simultaneously. To find out the main factors of brucellosis outbreaks and spreads, additional serological, etiological and molecular investigation were applied. Totally, 11 B. abortus were isolated from 10 cattle's specimens including lymph-nodes and/or testis, and drinking water in cowhouse. In genotyping by multi-locus VNTR assay (MLVA) using 17 loci markers, the present B. abortus isolates were shown all the same pattern, D1 genotype, which has been reported in Gyeonggi and Gangwon province, Korea. These results suggest that the input of brucellosis might come from neighboring farms directly or indirectly, even if by unknown factor and expansion within farm would accelerate by materials related with aborting cows.

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum β-lactamase (ESBL)-producing isolates from bovine mastitic milk from 2012 to 2015. A total of 374 Escherichia coli isolates were analyzed (154 in 2012, 113 in 2013, 76 in 2014, and 31 in 2015). No consistent trends in antimicrobial resistance of E. coli isolates occurred during the 4-yr period. The most frequently observed resistance was tetracycline (23.3%), followed by streptomycin (17.1%), ampicillin (16.6%), neomycin (11.8%), and trimethoprim/sulfamethoxazole (11.2%). Multidrug resistance was observed in 15.5% of isolates. Among these isolates, 15 (4.0%) carried one or more blaCTX-M and AmpC ESBL genes from 11 different farms, including blaCTX-M-15 at 4 farms, blaCTX-M-3 at 2 farms, blaCTX-M-1 at 3 farms, and blaCMY-2 at 3 farms. This study is the first report of blaCTX-M-3-producing E. coli in dairy milk. Transfer of ESBL was observed in 3 blaCTX-M-3-producing isolates, 1 blaCTX-M-1-producing isolate, and all 3 blaCMY-2-producing isolates. Almost all blaCTX-M-15 and blaCTX-M-1 genes possessed an insertion sequence, ISECP1, upstream of the blaCTX-M gene. Identical pulsed-field gel electrophoresis profiles were also observed in blaCTX-M-producing E. coli from the same farm. These results suggested that ESBL might spread by both clonal and horizontal spread in dairy farms in South Korea. Although no significant changes occurred in the antimicrobial resistance of E. coli during the 4-yr study period, the resistance rates and presence of ESBL were high compared with those in other countries. Thus, these findings suggest the importance of control measures for E. coli, particularly ESBL-producing bacteria, on dairy farms to reduce treatment failure and transmission to humans.

Altogether 7720 Enterococcus faecalis and 3939 E. faecium isolated from food animals and animal carcasses during 2003-2014 in Korea were investigated to determine if linezolid-resistant (LR) enterococci (≥8μg/ml) are present. Overall, 12 E. faecalis and 27 E. faecium recovered from chickens (n=32), pigs (n=6), and cattle (n=1) were resistant to linezolid and were further characterized using molecular methods Most LR isolates were also resistant to chloramphenicol (97.44%) and florfenicol (92.31%). Molecular analysis showed no mutations in the 23S ribosomal RNA and in the ribosomal protein L3. The optrA gene was found in 89.74% of the LR enterococci, including 12 E. faecalis and 23 E. faecium isolates. Among them, 30 optrA-positive isolates co-carried phenicol exporter gene fexA. Seven LR E. faecium isolates had Asn130Lys mutations in the ribosomal protein L4, of which six also carried optrA gene. None of the isolates carried the mutliresistance gene cfr. Transfer of optrA gene was observed in 16 of the 35 optrA-positive isolates by conjugation. Pulsed-field gel electrophoresis demonstrated that the vast majority of Enterococcus strains carrying optrA gene were genetically heterogeneous. Multi-locus sequence typing revealed eight novel Sequence types among E. faecalis and E. faecium strains. To our knowledge, this is the first report of optrA gene in isolates from cattle and animal carcasses. This is also the first report of optrA gene in Korea. Active surveillance of optrA in enterococci is urgently warranted.

A highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in poultry and wild birds in South Korea in January 2014. Here, we determined the pathogenicity and transmissibility of three different clades of H5 viruses in mandarin ducks to examine the potential for wild bird infection. H5N8 (clade 2.3.4.4) replicated more efficiently in the upper and lower respiratory tract of mandarin ducks than two previously identified H5N1 virus clades (clades 2.2 and 2.3.2.1). However, none of the mandarin ducks infected with H5N8 and H5N1 viruses showed severe clinical signs or mortality, and gross lesions were only observed in a few tissues. Viral replication and shedding were greater in H5N8-infected ducks than in H5N1-infected ducks. Recovery of all viruses from control duck in contact with infected ducks indicated that the highly pathogenic H5 viruses spread horizontally through contact. Taken together, these results suggest that H5N8 viruses spread efficiently in mandarin ducks. Further studies of pathogenicity in wild birds are required to examine possible long-distance dissemination via migration routes.

Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007–2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009–2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.

A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried bla CTX-M genes. The most common CTX-M types were CTX-M-14 ( n = 5) and CTX-M-24 ( n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry bla CMY-2 genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to bla TEM-1 , two isolates carried bla DHA-1 , and one of them cocarried bla CMY-2 . Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence IS Ecp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS 903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of bla CTX-M and bla CMY-2 genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of bla CMY-2 was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of bla DHA-1 in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea.

In vitro activities of 13 antibiotics were assessed against 85 Brucella abortus isolates from naturally infected cattle in the Republic of Korea during 1998-2006, using broth microdilution test. Tetracyclines showed the most excellent activity against B. abortus, displaying MIC values of 0.5 μg/ml or below. In particular, minocycline showed the lowest MIC 50/90 values (0.125/0.125 μg/ml) in this study. Among four fluoroquinolones tested, ciprofloxacin (MIC 50/90 , 0.5/1 μg/ml) and norfloxacin (MIC 50/90 , 8/8 μg/ml) had the most and the least activities, respectively. Gentamicin (MIC 50/90 , 1/1 μg/ml) was more effective than streptomycin, erythromycin, rifampin, and chloramphenicol (MIC 50/90 , 2/2 μg/ml).

In 2014, an H5N8 outbreak of highly pathogenic avian influenza (HPAI) occurred in South Korea. The H5N8 strain produced mild to moderate clinical signs and mortality rates in commercial chicken farms, especially Korean native chicken farms. To understand the differences between their pathogenicity in SPF chicken and Korean native chicken., we evaluated the mean bird lethal doses (BLD50) of the Korean representative H5N8 virus (A/broiler duck/Korea/Buan2/2014) The BLD50values of the H5N8 virus were 10(5.3)EID50 and 10(6.7)EID50 in SPF and Korean native chickens, respectively. In addition, the mean death time was much longer, and the viral titers in tissues of H5N8-infected chickens were significantly lower, in the Korean group than in the SPF group. These features of the H5N8 virus likely account for its mild-to-moderate pathogenicity in commercial chicken farms, especially Korean native chicken flocks, despite the fact that it is a highly pathogenic virus according to the OIE criteria. To improve current understanding and management of HPAI, pathogenic characterization of novel emerging viruses should be performed by natural route in major poultry species in each country.

Brucellosis is the most important bacterial disease of livestock in Bangladesh. The present study was conducted to determine the seroprevalence of brucellosis in goat and sheep in Mymensingh and Bogra districts of Bangladesh using slow agglutination test and Rose Bengal test as screening test and indirect enzyme linked immunosorbent assay as confirmatory test. Questionnaire based data on age, gender, area, client's complaint, number of animals in herds, disease history, reproductive problems such as abnormal uterine discharge, abortion or previous abortion in sheep and goat and their reproductive diseases were recorded. A total of 200 sera samples were collected from 80 sheep and 120 goats. The prevalence of brucellosis in goat was 2.50% and 1.25% in sheep. Positive reactors were only detected in female of both goat and sheep. In this study, there existed a significant association among abortion and the prevalence of brucellosis (p < 0.01). The prevalence of brucellosis in sheep and goat in Bangladesh is not negligible, and it is therefore worth considering the adoption of preventive measures.

Forty-one Enterococcus faecalis (E. faecalis) isolates from feces of pigs and chickens in Korea were screened for the presence of virulence factors. Gelatinase activity (85.4%, 35/41) was the more commonly observed phenotype of virulence in E. faecalis, compared with hemolytic activity (12.2%, 5/41). Thirty-one of 35 (88.6%) gelatinase-positive E. faecalis isolates harbored the gelE and fsrABC genes. A gene encoding for the enterococcal surface protein (Esp) was detected in 24.4% (10/41) of the isolates. All betahemolysin- producing isolates harbored the esp gene.