Gil-Jae Cho

PARENTAGE TESTING OF THOROUGHBRED HORSE IN KOREA USING MICROSATELLITE DNA TYPING

The present study was to construct a parentage testing system for Thoroughbred (TB) horse. A total number of 1,285 TB horse samples including 962 foals for parentage testing, 9 sires and 314 dams for individual identification were genotyped. Genomic DNA was extracted from 5 hair roots and genotyped by using 14 microsatellite markers (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS3, HMS6, HMS7, HTG4, HTG10, LEX3 and VHL20). This method consisted of multiplexing PCR procedure and showed reasonable amplification of all PCR products. Genotypes were determined by genetic analyzer. The number of alleles per locus varied from 3 to 9 with a mean value of 6.36 in TB horse. The expected heterozygosity was ranged from 0.548 to 0.831 (mean 0.699), and the total exclusion probability of 14 microstellite loci was 0.9998. Of the 14 markers, ASB2, ASB17, ASB23, HMS7 and HTG10 loci have relatively high PIC value (> 0.7). Of the 962 foals, 960 foals were qualified by compatibility according to the Mendelism. These results suggest that the DNA typing method has high potential for parentage verification and individual identification of TB horses.

Seroprevalence of equine piroplasms in the Republic of Korea

Equine piroplasms include two tick-borne protozoan parasites, Babesia caballi and Theileria equi. Although no clinical equine piroplasmosis has been reported in the Republic of Korea, the possible existence of the disease has been proposed due to a nationwide distribution of the vector ticks. To determine if the antibodies against B. caballi and T. equi were present, 184 sera of horses (Equus caballus) raised in the Republic of Korea from 2007 to 2010 were assessed using cELISA kits. Two (1.1%) out of 184 sera were positive for T. equi, but none were seropositive for B. caballi. Both samples tested positive came from one region (Gyeonggi province). The accuracy of the cELISA was confirmed by PCR using primers specific to the 18S rRNA of T. equi. This study presents for the first time horses infected by T. equi in the Republic of Korea. Since the infection of T. equi occurred in horses raised in the Republic of Korea, further studies with continuous monitoring of the vector ticks for equine piroplasms and appropriate control programs need to be established.

Detection and Genotyping of Coxiella burnetii and Coxiella-Like Bacteria in Horses in South Korea

Coxiella burnetii and Coxiella-like bacteria (CLB) are genetically and ecologically distinct despite some genetic similarities. Furthermore, CLB are exceptionally diverse and widespread in ticks, but rarely detected in domestic animals. Since Coxiella bacteria can be transmitted from infected horses by inhalation or by coming in contact with ticks during activities such as horseback riding, it is necessary to study their prevalence. To the best of our knowledge, this is the first large-scale nationwide investigation of the prevalence of C. burnetii and CLB among horses reared in South Korea. Of 816 blood samples collected between 2007 and 2013, 11 (1.3%) were identified as C. burnetii by ELISA, and six (0.7%) as CLB by 16S rRNA sequencing. While a sequence from Jeju Island was similar (97.9–100%) to those within clade B, five sequences obtained from the northern region were categorized into a new clade, indicating the sequence diversity of the genus Coxiella. Studies until date had detected CLB only in ticks; here, we describe their detection in mammals. Given their zoonotic potential, strategic monitoring and appropriate control programs for Coxiella species need to be established.

Evidence of Toxoplasma gondii exposure among horses in Korea.

The present study investigated the seroprevalence of Toxoplasma gondii (T. gondii) antibodies by ELISA in horses reared in Korea. Serum samples were collected from 2009 through 2013 from 816 horses reared in Korea. Analysis was performed using a commercial toxoplasmosis ELISA kit to detect anti-T. gondii antibodies. Overall, 24 out of 816 horses (2.9%) were seropositive for T. gondii. The result was analyzed by age, gender, breed and region. Significant differences were observed according to breed and region (P<0.05). This is the first nationwide serological investigation of T. gondii in horses reared in Korea. The study results reveal that T. gondii occurs nationwide in Korean horses.

Molecular and phylogenetic analysis of equine piroplasms in the Republic of Korea.

This study was conducted to screen out horses infected with piroplasms using PCR and to assess the phylogenetic variations of the piroplasm isolates. From 2007 to 2010, a total of 224 blood samples of horses were collected from three provinces of Korea and analyzed by PCR using primers specific to the 18S rRNA of piroplasms. Out of 224 samples analyzed, only two (0.9%) horses were found positive for Theileria equi. Sequencing of the complete 18S rRNA of T. equi from the two horses (GG-7 and GG-14) whose information was submitted to the GenBank (accession nos. HM229407 and HM229408, respectively) showed 100% identity. Alignment of the complete sequences of T. equi 18S rRNA with the GenBank databases of T. equi showed a high degree of homology (98.6-99.8%). The phylogenetic analysis showed T. equi GG-7 and GG-14 clustered together with T. equi isolates from Spain, Sudan, Jordan and South Africa, indicating the possibility of a close epidemiological link among these isolates.

Molecular Genetic Diversity of the Gyeongju Donggyeong Dog in Korea

ABSTRACT The present study was conducted to analyze the genetic characteristics of the Donggyeong dog and establish parentage conservation systems for it by using 10 microsatellite markers recommended by the International Society for Animal Genetics (ISAG). A total of 369 dogs from 12 dog breeds including the Donggyeong dog were genotyped using 10 microsatellite loci. The number of alleles per locus varied from 5 to 10 with a mean value of 7.6 in the Donggyeong dog. The observed heterozygosity and expected heterozygosity ranged from 0.4706 to 0.9020 (mean 0.7657) and from 0.4303 to 0.8394 (mean 0.7266), respectively. The total exclusion probability of 10 microsatellite loci was 0.99955. Of the 10 microsatellite markers, the AHT121, AHTh260 and CXX279 markers had relatively high PIC values (≥0.7). This study found that there were specific alleles, 116 allele at AHT121 in the Donggyeong dog when compared with other dog breeds. Also, the results showed two (Korean native dogs and the foreign dog breeds) distinct clusters. The closest distance (0.1184) was observed between the Donggyeong dog and Jindo dog, and the longest distance (0.3435) was observed between the Donggyeong dog and Bulgae. The Korean native dog breeds have comparatively near genetic distances between each other.

Targeted next-generation sequencing for identifying genes related to horse temperament

It is a fundamental challenge to discover the association of genetic traits with phenotypic traits. In this study, we aimed to identify possible genetic traits related to horse temperament. Based on previous findings, we selected 71 candidate genes related to temperamental trait and examined them in the human and horse reference genomes (hg38 and equCab2, respectively). We found 16 orthologous genes and, by comparing with the human reference genome, 17 homologous genes in the horse reference genome. We designed probes specific for the 33 horse genes. Using the probes, we built sequencing libraries of the genomic DNA samples from eight aggressive and eight docile horses, and sequenced the constructed libraries using the Illumina Hiseq2500 platform. Through the analysis of the targeted exome sequences, we identified single nucleotide polymorphisms (SNPs) in the genes. SNPs could be served as genetic markers to evaluate aggressive or docile levels of horses. To examine whether any genetic variants are associated with horse temperament, we performed genome-wide association study (GWAS) using the SNP data. GWAS analysis identified ten variants (p-value <0.05) which could be related to horse temperament. We validated the variants using Sanger sequencing. The most significant variants were found in MAOA (c.1164+41T>C) and AR (c.1047+27G>T) genes with 8.09 × 10−4p-value. We suggest that the variants might be used to assess horse temperament and to determine superior horses for riding or racing.

Serological Detection of Borrelia burgdorferi among Horses in Korea.

Lyme disease is a tick-borne zoonotic infectious disease caused by Borrelia burgdorferi. The present study assessed the infection status of B. burgdorferi among horses reared in Korea using ELISA and PCR. Between 2009 and 2013, blood samples were collected from 727 horses throughout Korea. Data for each animal including age, gender, breed, and region of sample collection were used for epidemiological analysis. Overall, 38 (5.2%; true prevalence: 5.5%) of 727 horses were seropositive by ELISA. There were statistically significant differences according to breed and region (P<0.001) whose differences might be attributed to the ecology of vector ticks and climate conditions. Using 2 nested PCR, none of the samples tested positive for B. burgdorferi. Thus, a positive ELISA result can indicate only that the tested horse was previously exposed to B. burgdorferi, with no certainty over the time of exposure. Since global warming is likely to increase the abundance of ticks in Korea, continuous monitoring of tick-borne diseases in Korean horses is needed.

RAPD Analysis and Antimicrobial Susceptibility of Streptococcus equi subsp. zooepidemicus Isolated from Thoroughbred Horses

A total of 68 samples were collected including vaginal mucosa (n=66) from Jangsu stud farm, an equine aborted fetus (n=1), and uterine contents (n=1) from Jeju island. Seventeen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) strains isolated from horses in Korea were identified as S. zooepidemicus by biochemical tests and sodA?seeI specific multiplex PCR. All isolated strains were divided into 4 clusters: group 1 (No. 2, 3, 5, 6, 7, 11, 12, 13, 14, 15), group 2 (No. 4, 9), group 3 (No. 10, 16, 17), and group 4 (No. 1, 8) by RAPD typing. In group 3, No. 10 isolate that was isolated from vaginal mucosa was indistinguishable from No. 16 and 17 isolates, which were isolated from the equine uterine contents and the equine aborted fetus, respectively. The results of this study suggest that a limited epidemiological relationship exists between the strains from Jangsu (No. 10) and Jeju (No. 16 and No. 17). All isolates showed a high susceptibility to ampicillin, cefoxitin, ceftiofur, cephalothin, florofenicol, gentamicin, nalidixic acid, oxacillin, penicillin, tiamulin, tylosin and vancomycin in antimicrobial susceptibility tests. These results may provide the basic information needed to establish strategies for the treatment and prevention of reproductive diseases in mares in Korea.

Characteristics and Antimicrobial Susceptibility Patterns of Pasteurella multocida Isolated from Pneumonic Lung Lesions of Swine

The present study was conducted to investigate the species-specific gene detection and antimicrobial susceptibility of Pasteurella (P.) multocida isolated from pneumonic lung lesions of Youngnam swine herds during the period from July 2006 to September 2007. A total of 91 (36.3%) strains of P. multocida were isolated from 251 pneumonic lung lesions. The species-specific P. multocida gene was detected at 460 bp amplicons by PCR. The P. multocida tested was susceptible to florofenicol (93.4％), amikacin (91.2％), cephalothin (87.9％), cefoxitin (84.6％), ofloxacin (80.2％) and norfloxacin (65.9％) in 27 antimicrobial susceptibility tests. Most of strains were resistant to more than 5 drugs.