Investigation of bovine tuberculosis outbreaks by using a trace-back system and molecular typing in Korean Hanwoo beef cattle

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.

Mycobacterium bovis infection in a wild sow (Sus scrofa): the first case in Korea

Mycobacterium (M.) bovis causes tuberculosis and has a broad host range, including humans, livestock, and wild animals. M. bovis infection of wild boar has been reported in several European countries. We report here the first case of M. bovis infection in a domesticated wild sow in Korea. Granulomatous and necrotizing lesions with small numbers of acid-fast bacilli were observed in nodules of the lung of wild sow. Furthermore, the M. bovis isolate from the wild sow had spoligotype SB0140 and a novel MIRU-VNTR allelic profile, which is not found in cattle and deer in Korea.

Isolation and characterization of Brucella abortus isolates from wildlife species in South Korea

A total of 782 blood and 465 tissue samples from 1,039 wild animals and 127 dairy goats were collected from January 2011 to December 2013 in 10 provinces of South Korea and tested for the presence of brucellosis. The Rose Bengal test revealed that 8.0% (52/650) of the serum samples were seropositive, while 4.2% (33/782) of the serum samples were positive for Brucella antibodies by competitive enzyme-linked immunosorbent assay. Of the 650 sera examined, only 16 (2.5%) were positive by both serological tests. Direct polymerase chain reaction (PCR) assay using B4/B5 primers for Brucella abortus (BCSP31) revealed the prevalence of Brucella to be 26.5% (129/487) in blood samples and 21% (98/465) in tissue samples while, 16S rRNA PCR detected Brucella DNA in 6.8% (33/487) and 2.6% (12/465) in blood and tissue samples, respectively. Of PCR-positive samples, only 6.2% (30/487) of blood samples and 2.4% (11/465) of tissue samples were found to be positive by both BCSP31 and 16S rRNA PCRs. However, Brucella strains were isolated by blood culture from only two out of 487 blood samples (0.4%). This characterization and identification of pathogenic Brucella isolates is the first to clearly indicate that the organisms were Brucella abortus biovar 1.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Identification of Brucella melitensis isolates originating from Mongolia and diagnostic real-time PCR evaluation using a specific SNP / 대한수의학회지

A real-time PCR assay using hybridization probe (HybProbe) has been developed to detect Brucella (B.) melitensis strains. The primer and HybProbe sets were designed based on the gap gene of chromosome I with a specific single nucleotide polymorphism of B. melitensis. Specificity of the assay was confirmed by comparison to reference Brucella species and other related strains. In the melting curve analysis, B. melitensis generated a peak at 67degrees C unlike those for other Brucella species observed at 61degrees C. Sensitivity of the assay for B. melitensis ranged from 20 ng to 200 fg of genomic DNA. The ability to identify 94 Mongolian B. melitensis isolates using the real-time PCR assay was identical to that of classical biotyping methods and differential multiplex PCR. These data showed that this new molecular technique is a simple and quick method for detecting B. melitensis, which will be important for the control and prevention of brucellosis.

Trends in antimicrobial sales for livestock and fisheries in Korea during 2003-2012 / 대한수의학회지

In this study, trends in the sales of antimicrobials for use in livestock facilities and fisheries from 2003 to 2012 were investigated with regard to antimicrobial group, antimicrobial usage, and animal species. The overall amount of antimicrobials sold each year from 2003 to 2007 was 1,500 tons, after which they decreased, with the lowest sales being 936 tons in 2012. The total volume of antimicrobials used for feed additives decreased markedly by 94% from 2003 to 2012, which was mainly attributed to banning of feed additives. However, antimicrobial consumption through self prescription by farmers for disease prevention and treatment increased by 25% from 2003 to 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 penicillins and 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 antimicrobial resistance. Thus, early establishment of veterinary prescription guidelines for prudent use of antimicrobials is urgently needed in Korea.

Serological monitoring on brucellosis in livestock of Korea / 대한수의학회지

In Korea, brucellosis has been reported periodically in cattle and rarely in dogs; however, it has not previously been screened in domestic animals such as elk, pigs and goats. To investigate the serological prevalence, serum samples were 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 three elk that were shown to be positive serologically in 2008. There was no evidence of brucellosis in pigs. Based on serological 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 livestock exists; therefore, extensive and continuous serological monitoring is required to maintain their brucellosis-free status.

Comparisons of CTX-M-Producing Escherichia coli Isolates from Humans and Animals in South Korea

To investigate the possibility of transmission of CTX-M-producing Escherichia coli isolates among humans and animals, we compared CTX-M-producing E. coli isolates showing the same genotype from humans and dogs in Korea. Sixteen CTX-M-producing E. coli isolates from animals were selected and their genotypes were identified using MLST. Among clinical CTX-M-producing E. coli isolates from humans, which have been identified in previous studies, 12 isolates showing the same STs with those of E. coli isolates from animals were selected. For these 28 CTX-M-producing E. coli isolates, identification of bla CTX-M genes and their genetic environments, antimicrobial susceptibility testing, extended MLST, and PFGE were performed. Some CTX-M-producing E. coli isolates from humans showed the same genotypes, such as ST10, ST38, ST58, and ST95, but different CTX-M enzymes and PFGE patterns. Thus, it can be concluded that dissemination of ESBL-producing E. coli isolates between humans and animals is rare so far.

Epidemiology of brucellosis among cattle in Korea from 2001 to 2011

In the present study, the outbreak patterns of bovine brucellosis in Korea from 2000 to 2011 were analyzed to understand the epidemiological evolution of this disease in the country. A total of 85,521 brucella reactor animals were identified during 14,215 outbreaks over the 12-year study period. The number of bovine brucellosis cases increased after 2003 and peaked in 2006 before decreasing thereafter. The majority of the bovine brucellosis cases were Korean native cattle, Han Woo. The numbers of human brucellosis cases and cattle outbreaks increased and decreased in the same pattern. The correlation coefficient for human and bovine cases per year was 0.96 (95% confidence interval = 0.86~0.99; p < 10(-3)). The epidemiological characteristics of bovine brucellosis appeared to be affected by the intensity of eradication programs that mainly involved a test-and-slaughter policy. Findings from the present study were based on freely available statistics from web pages maintained by government agencies. This unlimited access to information demonstrates the usefulness of government statistics for continually monitoring the health of animal populations.

Seroprevalence of Q-fever in Korean native cattle / 대한수의학회지

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 = 6.2%). Interestingly, Jeju province had a seropositivity rate six times greater than that of other provinces (18.9% vs. 3.2%). High seroprevalence might be caused by wide distribution of ticks in Jeju province compared to other regions. Based on these data, extensive monitoring of C. burnetii infection in cattle, tick distribution, and climate changes is required.

Seroprevalence of Q-fever in Korean native cattle / 대한수의학회지

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 = 6.2%). Interestingly, Jeju province had a seropositivity rate six times greater than that of other provinces (18.9% vs. 3.2%). High seroprevalence might be caused by wide distribution of ticks in Jeju province compared to other regions. Based on these data, extensive monitoring of C. burnetii infection in cattle, tick distribution, and climate changes is required.

Antimicrobial resistance of Escherichia coli isolated from healthy animals during 2010-2012 / 대한수의학회지

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 > or = 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.

Changes of characterization of Salmonella Typhimurium isolate following sequential exposures to porcine neutrophil / 대한수의학회지

To develop a live vaccine candidate using an attenuated strain of Salmonella Typhimurium (ST), biochemical properties, plasmid profile, PFGE patterns and pathogenic analysis of the ST isolate were carried out after sequential passage of the ST isolate in porcine neutrophils. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the ability of the strain to ferment trehalose was delayed after 2 or more days of the culture. Also, changes including deletion of the gene fragments were observed in PFGE analysis of the neutrophil-adapted isolates. Two plasmids, 105kb and 50kb, were cured in the strain passaged over 15 times in porcine neutrophils. The 50% of lethal dose (LD50) of the parent strain was changed from 1 x 10(5) LD50 to 6 x 10(6) LD50 by the passage in intraperitoneal injection of the strains into mice. These results suggested that bacterial genotypic and phenotypic responses might be globally altered depending on the inside environment of neutrophils.

Diagnosis of canine brucellosis using recombinant ribosomal protein L7/L12 / 대한수의학회지

Brucella (B.) canis is mainly transmitted by direct or indirect contact with aborted fetuses and placenta. It's also known to be able to infect human, which likely results in providing veterinarians and companion animal owners for infectious risk. To develop diagnostic ELISA, we cloned and expressed rp1L gene of B. canis, which encodes the ribosomal protein L7/L12. Using this purified recombinant protein, indirect-ELISA (iELISA) was evaluated using 78 positive and 44 negative sera. The sensitivity and the specificity of iELISA were 94% and 89%, respectively. The results indicated that indirect-ELISA using recombinant ribosomal protein L7/L12 may be useful for diagnosis of canine brucellosis.

Prevalence and antimicrobial susceptibility of Brachyspira species in pigs in Korea / 대한수의학회지

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 Korean Nationwide Surveillance Study for Non-Typhoidal Salmonella Isolated in Humans and Food Animals from 2006 to 2008: Extended-Spectrum beta-Lactamase, Plasmid-Mediated AmpC beta-Lactamase, and Plasmid-Mediated Quinolone Resistance qnr Genes / 대한임상미생물학회지

BACKGROUND: The emergence of non-typhoidal Salmonella (NTS) with decreased susceptibilities to fluoroquinolone, ampicillin, or ceftriaxone has been reported worldwide. However, current surveillance studies of resistance among NTS in Korea are limited. Thus, the antimicrobial susceptibilities; resistance mechanisms such as extended-spectrum beta-lactamase (ESBL), plasmid-mediated AmpC beta-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and molecular epidemiologic characteristics were investigated in the present study. METHODS: National Institute of Health and National Veterinary Research and Quarantine Service collected NTS strains from 219 clinical and 293 non-clinical specimens from 2006 to 2008. The antimicrobial susceptibilities were determined using the Clinical and Laboratory Standards Institute disk diffusion test. ESBL, PABL, and qnr genotyping were performed using PCR and nucleotide sequencing. Pulsed-field gel electrophoresis was used for the molecular epidemiologic study. RESULTS: The resistance to ampicillin in clinical and non-clinical NTS was 49% and 18 to 47%, respectively. The resistance rates to trimethoprim-sulfamethoxazole in clinical and non-clinical NTS were 8% and 0 to 41%, respectively. The rates to extended-spectrum cephalosporin were 0 to 1%. One CTX-M-15-producing isolate and four CMY-2-producing isolates were detected. Notably, PFGE analysis showed four isolates carrying bla CMY-2, including one non-clinical strain had high clonality. Although the rate of ciprofloxacin resistance was very low, two qnrS1-carrying NTS strains were detected in non-clinical specimens. CONCLUSION: The resistance rates to ampicillin in both clinical and non-clinical NTS were high, while those to trimethoprim-sulfamethoxazole varied depending on the specimen. NTS strains harboring CTX-M-15-type ESBL or CMY-2-type PABL were detected even though the resistance rates to cephalosporins were very low. Four NTS strains carrying the blaCMY-2-gene implied zoonotic infection. Continuous effort to minimize transfer of resistance genes in NTS is necessary.

Brucellosis in sheep and goat of Bogra and Mymensingh districts of Bangladesh / 대한수의학회지

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.

Comparison of tuberculin skin test with Interferon-gamma assay for the diagnosis of bovine tuberculosis in Korean cattle / 대한수의학회지

Bovine tuberculosis (bTB), caused primarily by Mycobacterium bovis, continues to exert an economic loss, even in countries with active control measures, and is one of zoonotic diseases enable to be transmitted to human. The control and eradication of bTB are mainly based on a test and slaughter policy and/or abattoir surveillance. Various factors including limitation of diagnostic tests have been considered as major constraints to eradication. Single intradermal test (SIT) is the official diagnostic test. New diagnostic methods are needed to be developed, because of limitations of the test. In the present study SIT was compared with single intradermal comparative cervical test (SICCT) and interferon (IFN)-gamma assay. There was very low correlation between SIT and SICCT. However, high correlation was shown between SIT and IFN-gamma assay while no correlation was observed between SICCT and IFN-gamma assay. Therefore, our results suggest the possibility of replacement of SIT with IFN-gamma assay for the diagnosis of bovine tuberculosis.

Characterization of Brachyspira hyodysenteriae isolates from Korea

This study was done to characterize diversity in 10 Brachyspira hyodysenteriae isolates in Korea. The isolates were compared with 14 well-characterized non-Korean strains of various Brachyspira species. All Korean isolates showed strong beta haemolysis and had blunt cell ends with 7~14 periplasmic flagella. They produced indole, and did not ferment fructose. They were alpha-glucosidase positive and alpha-galatosidase negative using the APIZYM kit. Using polyclonal antisera raised in rabbits against recognized serotypes, all isolates showed a strong reaction to B. hyodysenteriae antisera E, A and B. Using multilocus enzyme electrophoresis (MLEE) with 15 enzymes and 5 buffer systems, the Korean and non-Korean isolates were divided into 22 electrophoretic types (ETs) and 5 divisions (A, B, C, D and E). Division A corresponded to B. hyodysenteriae, B to B. innocens, C to B. intermedia, D to B. murdochii and E to B. pilosicoli. The 10 Korean isolates of B. hyodysenteriae were relatively diverse, being divided into 9 ETs within MLEE division A. They were all distinct from the non-Korean strains.

A study on epidemiological characteristics and control methods of EHEC infection in Korea / 한국역학회지

E. coli is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless. But some strains such as Enterohaemorrhagic E. coli(EHEC), can cause severe food borne disease. It is transmitted to humans primarily through consumption of contaminated foods, such as raw or undercooked ground meat. There is no widely agreed definition of when a shiga-toxin producing E. coli is considered to be an EHEC. But in Korea, the word "EHEC", "STEC", "VTEC" are often used as same meaning, which refer to the E.coli those producing shiga-toxin. We suggest the term STEC refers to those E. coli produce one or more shiga-toxins(stx), and the term EHEC refers only to STEC that cause a clinical illness. EHEC infection were designated as the class 1 notifiable disease in Korea in 2000. Although EHEC/STEC cases were not common in Korea, the number of STEC infection cases reported has increased since 2001. From 2001 to 2004, the number of STEC infection cases in Korea were 11, 8, 52, 118 respectively. These cases included 17 due to E. coli O157, 136 due to E. coli, serogroup non-O157, and 15 due to E. coli that were not serogrouped. The most common serotype implicated is E. coli O91 without virulent factor and clinical symptoms. But those cases involve in one epidemic in primary school in 2004. STEC infections in Korea occur in all age groups, with the highest frequencies in children less than 5 years old. Healthy cattle are the main animal reservoir for STEC and they harbor the organism as part of the bowel flora. The proportion of STEC in E. coli in animal feces was examined by using stool samples from 283 Korean beef cattle on 27 farms, 169 milk cattle on 28 frams, 455 swine on 50 farms. As determined by culture and toxin assay, the proportion of STEC was 25.8%(16 STEC/62 E. coli) in milk cattle, 18.8%(19 STEC/101 E.coli) in Korean beef cattle, 14.0%(25 STEC/178 E. coli) in swine. Effective surveillance of EHEC/STEC in humans is essential in order to protect the public health. EHEC infection is notifiable in many countries including USA, Japan, and Belgium, Finland, Italy, Netherlands, and the United Kingdom(UK), have sentinel systems. England, Wales, and Scotland have comprehensive national laboratory reporting schemes for STEC. And there has been an increase in the number of reported cases and outbreaks during the past decades in many countries Prevention of STEC infection requires control measures at all stages of surveillace, investigations and special pathogen tracing such as PulseNet.