Molecular Detection and Subtyping of Blastocystis in Korean Pigs.

Blastocystis is one of the most commonly detected genera of protozoan parasites in the human intestines as well as the intestines of many other species such as pigs in several geographical regions worldwide. However, no studies have examined Blastocystis in pigs in Korea. In this study, PCR and nucleotide sequencing were performed to evaluate the genetic diversity and zoonotic potential of Blastocystis using pig fecal samples. We obtained 646 stool samples from groups of piglets, weaners, growers, finishers, and sows in Korea. A total of 390 Blastocystis-positive samples were identified, and the infection rate was 60.4%. The infection rates were significantly related to age and region. The 4 subtypes (STs) of Blastocystis confirmed by phylogenetic analysis were ST1, ST2, ST3, and ST5, indicating the high genetic diversity of Blastocystis in Korean pigs. ST5 was highly distributed in Korean pigs among detected STs in this study. Some sequences were closely related to those of Blastocystis isolated from humans. This is the first study of Blastocystis in pigs in Korea. Based on the results, Blastocystis is prevalent in Korean pigs. Although a small number of samples were obtained in some areas, the clinical development of Blastocystis infection in pigs and potential for human transmission should be further examined.

Prevalence of Campylobacter species in wild birds of South Korea.

Campylobacter species cause human gastrointestinal infections worldwide. They commonly inhabit intestines of avian species including wild birds. They might play a role in the spread of infections to humans and other bird species. The prevalence of Campylobacter species in 2164 faecal samples of wild birds (representing 71 species and 28 families) captured across the Korean peninsula was evaluated in this study. The overall prevalence was 15.3% (332/2164). Bird species belonging to the family Charadriidae had the highest isolation rate (30.0%), followed by those belonging to the families Ardeidae (26.4%), Turdidae (21.9%), and Anatidae (15.3%). The prevalence of Campylobacter spp. differed significantly according to migratory habit. Stopover birds were the most commonly infected (19.0%), followed by winter migratory (16.7%) and summer migratory birds (12.3%). However, indigenous birds showed very low prevalence (2.7%). Antimicrobial susceptibility tests were performed for 213 isolates. Results showed that Campylobacter jejuni isolates (n = 169) exhibited resistance to nalidixic acid (5.3%), ciprofloxacin (3.0%), and tetracycline (1.8%), while Campylobacter lari (n = 1) displayed resistance to nalidixic acid and ciprofloxacin. However, all Campylobacter coli isolates (n = 20) were susceptible to all antimicrobials tested. This is the first report on the prevalence of Campylobacter species in wild birds that seasonally or indigenously inhabit the Korean peninsula. Our results indicate that the overall prevalence of Campylobacter in wild birds is moderate. Therefore, birds might serve as significant reservoirs for Campylobacter pathogens.

Seroprevalence and risk factors of Besnoitia besnoiti infection in Korean cattle - short communication.

Besnoitia besnoiti is an obligate intracellular parasite that is transmitted by direct contact or via mechanical transmission by flies as vectors. Besnoitiosis causes economic losses in the cattle industry and is regarded as a re-emerging disease in Europe. This study evaluated the seroprevalence of B. besnoiti in Korean cattle using a commercial ELISA kit. Among 558 serum samples, 19 (3.4%) tested seropositive for B. besnoiti. The statistically significant risk factors included age (≥ 2 years), sex (castrated males), and region (lower latitudes) (P < 0.05). The overall seroprevalence suggested a wide distribution of B. besnoiti infection in cattle reared in Korea. Thus, the practice of intensive cattle husbandry and the regionally different seroprevalence of B. besnoiti infection in cattle in Korea warrant routine monitoring and vector control to reduce economical losses due to bovine besnoitiosis in the country.

Characterization of Salmonella enterica Serovar 4,[5],12:i:- Isolates from Korean Food Animals.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as one of the most common serotypes related to human salmonellosis. In this study, the 22 isolates of S. 4,[5],12:i:- from food animals were identified by a specific multiplex polymerase chain reaction between 2009 and 2012. The isolation rate of S. 4,[5],12:i:- accounted for 1.7% (22/1271) of Salmonella spp. isolates from food animal origins: more specifically, 7.6% (18/235) from pigs and 0.6% (4/686) from chickens. The predominant S. 4,[5],12:i:- isolates in Korea belonged to phage type DT193 (12/22) with ampicillin-streptomycin-sulfonamide-tetracycline (ASSuT) resistance pattern (9/22). The XbaI-pulsed-field gel electrophoresis (PFGE) analysis revealed 11 different pulsotypes, and the major X-1 pattern was shared by 8 isolates. The isolates belonging to pattern X-1 were further subdivided into three BlnI-PFGE patterns and four variable-number tandem-repeat analysis (MLVA) allele combinations. The combining of MLVA and PFGE data could be valuable in characterizing highly clonal strains and discriminating their epidemiological relationship.

Serology and clinical relevance of Corynebacterium pseudotuberculosis in native Korean goats (Capra hircus coreanae).

This study was conducted to assess the seroprevalence and clinical relevance of Corynebacterium pseudotuberculosis, which is the causative agent of caseous lymphadenitis (CLA), in native Korean goats (Capra hircus coreanae). A total of 466 native Korean goats from 40 herds (11 to 12 samples per herd) were randomly selected throughout the nation and evaluated by direct palpation, bacterial isolation, ELISA, and PCR. In serological examinations, 267 (57.3 %) of the goats tested were positive against C. pseudotuberculosis. When seroprevalence was analyzed according to age, region, and season, statistically significant differences were observed in relation to all three parameters (P < 0.05). For clinical examination, the superficial lymph nodes of all goats were palpated to diagnose CLA. Pus samples taken from superficial abscesses were used for bacterial isolation. Among the 466 goats tested, 34 (7.3 %) were presumptively diagnosed with CLA, and C. pseudotuberculosis was isolated from 24 goats (70.6 % of goats with CLA lesions) whose infections were confirmed by PCR. Considering the high seroprevalence and bacterial isolation rate from most of the superficial CLA lesions, it is suspected that many internal CLA lesions exist in this goat population. These results suggest that C. pseudotuberculosis infection is widespread in native Korean goats, and appropriate control programs need to be established.

First isolation of Clostridium perfringens type E from a goat with diarrhea.

A 2-day-old goat died suddenly after the onset of severe diarrhea. No specific gross lesions were observed except for a remarkably thin intestinal wall and watery intestinal contents. Histopathological analysis revealed large numbers of Gram-positive bacilli layered upon the intestinal epithelia of the small intestine. Heavy growth of only Clostridium perfringens type E, and no detection of the other enteric pathogens in the small intestine, suggests that C. perfringens type E contributed to the death of this kid. To our knowledge, this is the first isolation of C. perfringens type E from a goat with diarrhea.

Plasmid typing and resistance profiling of Escherichia fergusonii and other Enterobacteriaceae isolates from South Korean farm animals.

In this study, we focused on determining the distribution and prevalence of major plasmid replicons in ß-lactam-resistant Escherichia fergusonii and Enterobacteriaceae of animal and human origin. A high degree of plasmid variability and multiple plasmid replicons were observed among the isolates. The IncF and IncI1 replicons were the most prevalent in E. fergusonii and Salmonella enterica serovar Indiana isolated from swine and poultry in South Korea, respectively. The presence of broad-host-range plasmid replicons such as IncN, IncA/C, IncHI1, and IncHI2 that are associated with important virulence genes and toxins as well as antimicrobial resistance determinants indicates that E. fergusonii has the potential to become an important pig pathogen and possible emerging opportunistic zoonotic pathogen.

Antibiotic resistance patterns and detection of blaDHA-1 in Salmonella species isolates from chicken farms in South Korea.

Fifteen nonrepetitive ampicillin-resistant Salmonella spp. were identified among 91 Salmonella sp. isolates during nationwide surveillance of Salmonella in waste from 131 chicken farms during 2006 and 2007. Additional phenotyping and genetic characterization of these 15 isolates by using indicator cephalosporins demonstrated that resistance to ampicillin and reduced susceptibility to cefoxitin in three isolates was caused by TEM-1 and DHA-1 beta-lactamases. Plasmid profiling and Southern blot analysis of these three DHA-1-positive Salmonella serovar Indiana isolates and previously reported unrelated clinical isolates of DHA-1-positive Salmonella serovar Montevideo, Klebsiella pneumoniae, and Escherichia coli from humans and swine indicated the involvement of the large-size plasmid. Restriction enzyme digestion of the plasmids from the transconjugants showed variable restriction patterns except for the two Salmonella serovar Indiana isolates identified in this study. To the best of our knowledge, this is the first report of the presence of the DHA-1 gene among Salmonella spp. of animal origin.

Seroprevalence of Leptospira spp. in clinically healthy racing horses in Korea.

Leptospirosis is a zoonotic disease of global importance, and has a worldwide distribution. The present study aimed to determine leptospiral seroprevalence in clinically healthy racing horses from all three racecourses in Korea. Serum samples from 1,226 racing horses were examined using a microscopic agglutination test to detect the presence of antibodies against 18 Leptospira serovars. Of the tested samples, 307 (25.0%) were found to be positive. The distribution of seroprevalence differed significantly by racecourse (P=0.004); the Jeju course had the highest incidence (31.1%), followed by the Seoul (25.2%) and Busan (19.5%) racecourses. Seasonal variation in seropositivity was also apparent (P=0.000), being lower in spring (13.0%) and winter (12.5%), and higher in summer (36.7%) and autumn (34.7%). No significant age- or gender-related difference in seroprevalence was noted in this study (P>0.05). Seroprevalence was higher (P=0.006) among ponies than among thoroughbreds. Sejroe was the most frequently detected serovar (n=236), followed by Bratislava (n=35), Ballum (n=16), Autumnalis (n=10), and Canicola (n=10). The majority of serum titers were relatively low; most values ranged from 1:100 (n=217) to 1:200 (n=69). These results suggest that the Sejroe serovar may be maintained in the racing horse population in Korea.

Emergence of mcr-3 carrying Escherichia coli in Diseased Pigs in South Korea.

We examined the prevalence and molecular characteristics of mcr-3 carrying colistin-resistant Escherichia coli among cattle, pig, and chicken isolates in South Korea. Among a total of 185 colistin-resistant E. coli isolates determined in this study (47 from cattle, 90 from pigs, and 48 from chicken), PCR amplification detected mcr-3 genes in 17 isolates predominantly from diseased pigs. The mcr-3 genes were characterized as mcr-3.1 in 15 isolates and mcr-3.5 in 2 isolates. The mcr-3 gene was transferred to the E. coli J53 recipient strain from more than 50% of the mcr-3-carrying isolates. The mcr-3.1 and mcr-3.5 genes were identified predominantly in IncHI2 and IncP plasmids, respectively. Multi-locus sequence typing analysis revealed eight previously reported sequence types (ST), including ST1, ST10, and ST42. We identified isolates with similar pulsed-field gel electrophoresis patterns from diseased pigs in three farms. Besides, the isolates carried various virulence factors and demonstrated resistance to multiple antimicrobials, including ß-lactams and quinolones. Further, the mcr-3.5 encodes three amino acid substitutions compared with mcr-3.1. To the best of our knowledge, this is the first report of pathogenic E. coli carrying mcr-3.5 in South Korea, which implies that mcr-3 variants may have already been widely spread in the pig industry.

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats.

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 +/- 0.09 and 0.65 +/- 0.07 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.

Evaluation of the broad-spectrum lytic capability of bacteriophage cocktails against various Salmonella serovars and their effects on weaned pigs infected with Salmonella Typhimurium.

The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥109 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.

Seroprevalence of leptospirosis in Korean municipal zoo animals.

From 2002 to 2005, we collected 118 serum samples from 34 species belonging to 13 families of zoo animals in Korea and determined the prevalence of antibodies for 18 serovars of Leptospira spp. using the microscopic agglutination test (MAT). Twenty-nine (25%) of the serum samples tested were positive for one or more of the serovars. There were no significant differences in relation to genders: 23% and 26% of positives occurring in male and female animals, respectively (P>0.05). However, the seroprevalence for the Leptospira spp. was significantly higher (P<0.05) in herbivores (45%) than in either carnivores (17%) or omnivores (17%). Among the 5 serovars detected in this study, the most common was sejroe (n=27; 87% of all positive reactions). All positive reactions showed low titers (< or = 1:200) and the positives were most frequently detected in 1:25 (58%) and 1:50 (23%) serum dilutions. The highest antibody titer (1:200) was observed for the serovars sejroe (n=1) and bratislava (n=1). We conclude that the exposure of zoo animals to Leptospira spp. is relatively common in Korea and produces low MAT titers, with sejroe being the most commonly encountered serovar.

Capsular serotypes, virulence-associated genes and antimicrobial susceptibility of Streptococcus suis isolates from pigs in Korea.

Streptococcus suis is an important pig pathogen with potential for human transmission. The serotype distributions and phenotypic characteristics vary over time and among regions; however, little is known about the characteristics of S. suis isolates in Korea. In this study, 240 S. suis isolates collected from pigs in Korea in 2009-2010 were serotyped by coagglutination tests, subsequently screened for three virulence-associated genes (mrp, epf and sly) and tested for antimicrobial susceptibility. As for 80 isolates, the serotypes of which were relevant to human infections, clonal complexes (CCs) were further identified by PCR. Serotype 3 was the most prevalent (15.8%), followed by serotype 2 (15.0%), with geographical variation for each serotype. Overall, 55.4% of the isolates carried mrp, whereas only 3.8% carried epf. CC25 was the most prevalent (41.3%) and was related to serotypes 2 and 9. The isolates showed higher susceptibility to ampicillin (93.4%) and ceftiofur (90.8%) than to the other antimicrobial agents tested. The highest resistance rate was observed to tetracycline (98.0%), followed by erythromycin (88.8%). In addition, the resistance to certain antimicrobials was significantly associated, in part, with virulence-associated genes or serotypes. Therefore, continuous characterization of S. suis is essential for the benefit of veterinary and human medicine.

The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells.

An investigation was undertaken to assess whether polyclonal convalescent and hyperimmune sera obtained from pigs inhibit Mycoplasma hyopneumoniae induced increases in intracellular calcium [Ca2+](i) in ciliated porcine tracheal cells. Basal [Ca2+](i) in the tracheal cells was 97+/-13 nM (n=22 cells in four experiments) and after exposure to M. hyopneumoniae (300 micro g/mL or 10(11) CCU/mL), [Ca2+](i) increased by 246+/-56 nM within 100 s. After pre-treatment with hyperimmune or convalescent serum, M. hyopneumoniae increased [Ca2+](i) by 196+/-43 and 223+/-65 nM, respectively. It was found that neither hyperimmune nor convalescent serum significantly prevented the increase in [Ca2+](i) compared with M. hyopneumoniae alone. It was concluded that polyclonal antibodies produced by mycoplasma vaccination or exposure to the pathogen do not prevent M. hyopneumoniae-induced increase in [Ca2+](i).

Seroprevalence of Mycobacterium avium subspecies paratuberculosis in Korean black goats (Capra hircus aegagrus).

In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (chi=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.

Development of a rapid immunochromatographic strip for detection of Escherichia coli O157.

We developed an immunochromatographic (IC) strip for the rapid detection of Escherichia coli O157 in enriched samples. Murine monoclonal antibody to E. coli O157:H7 lipopolysaccharide was conjugated with 40 nm of colloidal gold particles by the citrate method. The specificity of the IC strip was determined using 48 pure-cultured bacteria, including 32 E. coli strains and 16 non-E. coli strains. Regardless of H serotype, E. coli O157 strains produced a positive signal, whereas the others, representing 29 E. coli serotypes, did not. Among 16 non-E. coli strains, only Citrobacter amalonaticus yielded a positive signal. The sensitivity of the IC strip was determined using 10-fold diluted E. coli O157:H7, with a range of 1.8 X 10(7) to 1.8 CFU/ml in enriched raw beef. E. coli O157 could be detected at a minimum of 1.8 x 10(5) CFU/ml without enrichment and 1.8 CFU/ml after enrichment. Various samples were enriched to detect E. coli O157 using the IC strip and to isolate E. coli O157:H7 using traditional culture procedures. The IC strip test results exhibited 100% agreement with traditional methods after selective enrichment, since E. coli O157:H7 was also isolated from all the samples with positive strip test results. However, the specificity of the strip was somewhat higher with pork (98.8%) than with bovine feces (87.9%) and swine feces (93.4%). These results indicated that the IC strip exhibits high specificity and sensitivity in the detection of E. coli O157, and this assay is rapid, economical, and simple, without requirement of complicated equipment.

Molecular and serologic detection of Coxiella burnetii in native Korean goats (Capra hircus coreanae).

The occurrence of Q fever in native Korean goats (Capra hircus coreanae) was investigated for the first time in the country using ELISA and PCR. A total of 597 blood samples were collected from goats belonging to five different provinces of Korea. To detect Coxiella burnetii, sera were separated from the whole blood and analysed by ELISA; DNA was extracted directly from the whole blood and analysed by PCR. Overall, 114 (19.1%, 95% C.I.=16.1-22.4) and 57 goats (9.5%, 95% C.I.=7.5-12.2) tested positive for C. burnetii in the ELISA- and PCR-based screening, respectively, while 18 goats (3.0%, 95% C.I.=1.9-4.7) tested positive in both the assays. There was a significant difference between the number of ELISA- and PCR-positive goats (P<0.05). The seroprevalence of Q fever was significantly higher among the adult goats (≥1y, 22.0%) than among the young goats (<1y, 13.8%) (P<0.05). While the results of the serologic analysis showed no seasonal variation, data from the PCR-based assay indicated that there were a higher number of positive cases during the cold seasons. Because Q fever infection has high rates of prevalence in native Korean goats, further studies on humans at a high risk of contracting this disease should be conducted. The PCR-based assay used in this study is a useful method for the direct detection of C. burnetii in blood samples from small ruminants.

Detection and determination of Toxoplasma gondii seroprevalence in native Korean goats (Capra hircus coreanae).

Toxoplasma gondii is an important zoonotic protozoan pathogen that causes serious illness in immunocompromised humans and infection in animals worldwide. The current study was conducted for detection of T. gondii infection and determination of the seroprevalence of the pathogen in native Korean goats (Capra hircus coreanae). Analysis of a total of 610 sera samples collected from 60 herds between 2009 and 2011 were performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of anti-T. gondii IgG antibodies. Among the animals tested, 5.1% (31/610) showed seropositivity for anti-T. gondii antibodies, and 38.3% (23/60) of the herds were seropositive. The prevalence rates between young (<1 year) and adult (≥1 and ≤3 years) goats were 7.0% and 4.1%, respectively, without statistical significance (p>0.05). Likewise, the prevalence rates observed during cold season (October-March) and warm season (April-September) were 2.9% and 5.5%, respectively, without statistical significance. Seroprevalence rates observed in the northern, central, and southern regions were 7.9%, 3.8%, and 4.2%, respectively. In conclusion, we report for the first time on the seroprevalence of anti-T. gondii antibodies in native Korean goats (Capra hircus coreanae). The results of this study also indicate that there is a nationwide distribution of T. gondii infection among goats. Therefore, the implementation of integrated control strategies as well as measures for prevention and control of T. gondii infection within goats is recommended.

Isolation and characterization of orf viruses from Korean black goats.

Five cases of orf virus infection in Korean black goats were diagnosed in our laboratory between 2010 and 2011. One orf virus (ORF/2011) was isolated from an ovine testis cell line (OA3.Ts) for use as a vaccine candidate. Sequences of the major envelope protein and orf virus interferon resistance genes were determined and compared with published reference sequences. Phylogenetic analyses revealed that orf viruses from Korean black goats were most closely related to an isolate (ORF/09/Korea) from dairy goats in Korea. This result indicates that the orf viruses might have been introduced from dairy goats into the Korean black goat population.