Prevalent toxin types of Clostridium botulinum in South Korean cattle farms.

Clostridium botulinum produces neurotoxic substrates that can cause fatal flaccid paralysis called botulism. These neurotoxins are classified into types A-G. Several botulism cases were recorded in 2012-2013 in the Gyeonggi province, South Korea. We assessed the distribution of C. botulinum types B, C, and D in several South Korean farms. A total of 184 samples collected in 2012-2013, including feces (n = 72), hay and silage (n = 50), soil (n = 26), water trough (n = 21), and stomach contents (n = 15), were subjected to multiplex polymerase chain reaction (PCR) to screen for types B, C, and D. Twenty-four samples tested PCR-positive as follows: type B (n = 11), type C/D (n = 4), and type D (n = 18). Eight of the 11 type B samples were detected in hay and silage. Sixteen of the 18 type D samples were detected in fecal and stomach content samples. PCR-positivity was observed in fecal (n = 9, 12.5%), hay and silage (n = 10, 20.0%), water trough (n = 2, 9.5%), and stomach content (n = 12, 80.0%) samples. Fourteen (42.4%) C. botulinum-positive samples were isolated from the PCR-positive samples (type B [n = 8], type C/D [n = 1], and type D [n = 5]). Our findings demonstrate that C. botulinum types B, C/D, and D were prevalent in South Korean cattle farms between 2012 and 2013.

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.

Isolation and characterization of Avibacterium paragallinarum with different nicotinamide adenine dinucleotide requirements.

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.

Erratum to: Draft Genome Sequences of a Unique t324-ST541-V Methicillin-Resistant Staphylococcus aureus Strain from a Pig.

This erratum is being published to correct the errors of the words in the section of introduction and title. The words of 't324'(left column, line 20, 21; right column, line 4, 14) in page 799 and title should be corrected as 't034'.

Detection of novel oxazolidinone and phenicol resistance gene optrA in enterococcal isolates from food animals and animal carcasses.

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.

Antimicrobial susceptibility and characterization of extended-spectrum ß-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015.

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum ß-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.

Comparison of the Antimicrobial and Sanitizer Resistance of Salmonella Isolates from Chicken Slaughter Processes in Korea.

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.

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

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.

Experimental infection of mandarin duck with highly pathogenic avian influenza A (H5N8 and H5N1) viruses.

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.

Macrolide resistance mechanisms and virulence factors in erythromycin-resistant Campylobacter species isolated from chicken and swine feces and carcasses.

Resistance to antimicrobials was measured in 73 isolates of Campylobacter jejuni (C. jejuni) and 121 isolates of Campylobacter coli (C. coli) from chicken and swine feces and carcasses in Korea. Both bacterial species showed the highest resistance to (fluoro) quinolones (ciprofloxacin and nalidixic acid) out of the nine antimicrobials tested. Erythromycin resistance was much higher in C. coli (19.0%, 23/121) than in C. jejuni (6.8%, 5/73). The mutation in the 23S rRNA gene was primarily responsible for macrolide resistance in Campylobacter isolates. Several amino acid substitutions in the L4 and L22 ribosomal proteins may play a role in the mechanism of resistance, but the role requires further evaluation. A total of eight virulence genes were detected in 28 erythromycin-resistant Campylobacter isolates. All C. jejuni isolates carried more than four such genes, while C. coli isolates carried fewer than three such genes. The high rate of resistance highlights the need to employ more prudent use of critically important antimicrobials, such as fluoroquinolones and macrolides, in swine and poultry production, and to more carefully monitor antimicrobial resistance in Campylobacter isolates in food animals.

Epidemiological relationships of Campylobacter jejuni strains isolated from humans and chickens in South Korea.

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.

Ostrich ( Struthio camelus ) Infected with H5N8 Highly Pathogenic Avian Influenza Virus in South Korea in 2014.

Highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype was isolated from a young ostrich in South Korea in March 2014. Clinical signs characterized by anorexia, depression, and signs of nervousness were observed. The isolated A/ostrich/Korea/H829/2014 (H5N8) virus had a cleavage site motif containing multiple basic amino acids, typical of HPAI virus. The phylogenetic tree of the hemagglutinin gene of the H5 HPAI virus showed that this ostrich H5N8 virus belongs to clade 2.3.4.4 viruses together with H5N8 strains isolated from ducks and wild birds in South Korea in 2014. Pathologically, redness of pancreas, enlargement and hemorrhage of spleen, friability of brain, and hydropericardium were prominently found. Histologic legions were observed in pancreas, spleen, liver, lung, heart, and brain, and influenza A nucleoproteins were detected in the same organs by immunohistochemistry. Other ostriches farmed together in open camps were not infected with HPAI virus based on the serologic and virologic tests. The findings indicate that ostriches are susceptible to H5N8 HPAI virus, but this virus does not spread efficiently among ratites.

Experimental infection of SPF and Korean native chickens with highly pathogenic avian influenza virus (H5N8).

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.

Genome Sequence of a Unique t2247-ST692-III Livestock-Associated Methicillin-Resistant Staphylococcus aureus Strain from Chicken Carcass.

We report the draft genome sequence of a novel livestock-associated t2247-ST692-III methicillin-resistant Staphylococcus aureus strain designated K12S0375, which was isolated from a chicken carcass in South Korea. The K12S0375 strain contains uncommon genes, including antimicrobial resistance genes (tetL and tetS) and leukotoxin (lukED), and the genomic distance indicates a single lineage in a genome-based phylogenetic tree compared with 459 S. aureus genome sequences. This genome sequence will contribute to understanding epidemiological and genomic features of the ST692 lineage, including antimicrobial resistance and virulence genes.

Mycobacterium bovis infection in a wild sow (Sus scrofa): 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.

Genetic characterization and evolutionary analysis of Newcastle disease virus isolated from domestic duck in South Korea.

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.

First Isolation of Streptococcus halichoeri and Streptococcus phocae from a Steller Sea Lion (Eumetopias jubatus) in South Korea.

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.

Supplemented vaccination with tandem repeat M2e virus-like particles enhances protection against homologous and heterologous HPAI H5 viruses in chickens.

Highly pathogenic avian influenza (HPAI) H5 viruses derived from A/Goose/Guangdong/1/96 have been continuously circulating globally, severely affecting the public health and poultry industries. The matrix 2 protein ectodomain (M2e) is considered a promising candidate for a universal cross-protective influenza vaccine that provides more effective control over HPAI H5 viruses harboring variant hemagglutinin (HA)-antigens. Here, we evaluated the protective efficacy of a tandem repeat construct of heterologous M2e presented on virus-like particles (M2e5x VLPs) either alone or as a supplement against HPAI H5 viruses in a chicken model. Chickens immunized with M2e5x VLPs alone induced M2e-specific antibodies but were not protected against HPAI H5. The homo- and cross-protective efficacy of M2e5x VLP-supplemented vaccination of chickens was also examined. Importantly, supplementation with M2e5x VLPs induced significantly higher levels of antibodies specific for M2e and different viruses as well as provided improved protection against homologous and heterologous HPAI H5 viruses. Considering the limited efficacy of inactivated vaccines, supplement vaccination with M2e5x VLPs may be an effective measure for preventing outbreaks of HPAI viruses that have the ability to constantly change their antigenic properties in poultry.

Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms.

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.