Multiplex allele specific PCR 방법을 이용한 한우고기와 젖소고기의 신속한 판별

고바라다,Koh, Ba-Ra-Da 대한수의학회 2005 大韓獸醫學會誌 Vol.45 No.3

Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.

바바리양에서 발생한 Streptococcus equi subsp. zooepidemicus 감염증

고바라다,박성도,김재익,박종태,Koh, Ba-Ra-Da,Park, Seong-Do,Kim, Jae-Ik,Park, Jong-Tae 대한수의학회 2007 大韓獸醫學會誌 Vol.47 No.4

An eight years old female barbary sheep (Ammotragus lervia), which bred at the Gwangju Uchi Park Zoo had shown anorexia, depression, respiratory problem for several weeks after parturition. In necropsy, extensive necrotizing pneumonia was found with severe immunocytes infiltration in the alveolar spaces and bronchioles. Pulmonary pleura were thickened with fibrin and inflammatory cells. Bacteria were isolated from lung and identified as Streptococcus equi subsp. zooepidemicus (SEZ) by biochemical tests and PCR on sodA and gusA genes, though seel gene was not detected. Isolation of zoonotic SEZ in public place such as a zoo should be emphasized for the public health mangagement.

오리에서 발생한 바이러스성 간염과 살모넬라균증의 혼합감염

고바라다,김용환,김계엽 한국수의병리학회 2001 한국수의병리학회지 Vol.5 No.2

Ducklings collected from three farms, having history of rapid onset and spread of nerve sings including kick spasmodically with legs and opisthotonos, were pathologically, bacteriologically, virologically examined. Grossly, multiple petechial to ecchymotic hemorrhages were detected in the swollen liver. Histopathologically, diffuse coagulative necrosis of hepatocytes was characteristic in acute cases. Chronic cases revealed marked bile duct hyperplasia rather than hepatocyte necrosis. Some of these cases exhibited multiple granulomas consisting of macrophages, heterophil, fibrin and necrotic cell debri. Filtered homogenate of livers sampled from ducklings caused embryo death with marked hemorrhage and swollen of liver after inoculation into chorioallantoic membrane. Three strains of Salmonella spp., S montevideo, S hadar, and S give, which were biochemically and serologically identified, were isolated from ducklings of three farms, respectively. From these results, these ducklings were concurrently infected with duck hepatitis virus and Salmonella spp.

소, 돼지, 가금육류의 신속한 동정을 위한 TaqMan^ⓡ probe를 이용한 real-time PCR 개발

고바라다,김지연,나호명,박성도,김용환,Koh, Ba-Ra-Da,Kim, Ji-Yeon,Na, Ho-Myung,Park, Seong-Do,Kim, Yong-Hwan 한국동물위생학회 2012 韓國家畜衛生學會誌 Vol.35 No.3

Species-specific $TaqMan^{(R)}$ probe-based real-time PCR assays were developed for detection of beef, pork, chicken, duck, goose and turkey. The primer and probe sets used in this study were designed to be complementary to fibroblast growth factor (FGF) for cattle and pig, mitochondrial NADH dehydrogenase (ND) subunit 3 and ND2 for chicken and duck, 12S rRNA for goose and turkey, respectively. As internal positive control we used conserved region in the ribosomal 18S RNA gene to ensure the accuracy of the detection of target DNA by real-time PCR. We confirmed that real-time PCR assays with the primer and probe sets were positive for cattle, pig and chicken intended target animal species with no cross-reactivity with other non-target animal species. Only >50 ng DNA of beef show cross-reactivity in the determination of duck. Using species-specific primer and probe sets, it was possible to detect amounts of 0.1 ng DNA of cattle and pig, 1.0 pg DNA of chicken, duck and turkey, and 0.1 pg DNA of goose for raw samples, respectively. The detection limits were 0.1 ng DNA of cattle, 1.0 ng DNA of pig and 1.0 pg DNA of chicken for DNA mixtures (beef, pork and chicken) extracted from heat-treated ($121^{\circ}C$/5 min) meat samples. In conclusion, it can be suggested that the $TaqMan^{(R)}$ probe-based assay developed in this study might be a rapid and specific method for the identification of meat species in raw or cooked meat products.

돼지고기 제품 내 닭고기 검출을 위한 TaqMan^® real-time PCR의 적용

고바라다,김지연,나호명,박성도,김용환,Koh, Ba-Ra-Da,Kim, Ji-Yeon,Na, Ho-Myung,Park, Seong-Do,Kim, Yong-Hwan 한국동물위생학회 2013 韓國家畜衛生學會誌 Vol.36 No.3

Many consumers are increasingly concerned about the meat they eat, and accurate labelling is important due to public health, economic and legal concerns. Meat species adulteration is a common problem in the retail markets. In this study, a TaqMan$^{(R)}$ quantitative real-time polymerase chain reaction (PCR) assay was applied for its ability to quantify chicken meat, which was not indicated on the label, in 79 commercial pork products (ham, sausages, bacon and ground meat) producted by 10 different manufacturers. The amplification efficiency was 82.05% and the square regression coefficient ($R^2$) was 0.995. PCR results showed that 38.6% of ham samples, 50.0% of sausages samples, and 50.0% of ground meat samples were contaminated with chicken residuals, while the bacon samples were not contaminated with chicken residuals. Only twelve pork products of one of the manufacturers were in accordance with indicated in their labels. The PCR assay reported in this work could be particularly useful in inspection programs to verify the food labelling of commercial processed meats and to gain consumers' trust.

학술발표(學術發表) 연제(演題) 및 초록(抄錄) 제이부(第二部): 미생물(微生物) 분야(分野) : 13. Listeria monocytogenes에 의한 젖소의 유방염

고바라다,김용환,배성열,김철희 한국가축위생학회 2000 학술대회논문집 Vol.2000 No.-

소, 돼지, 가금육류의 신속한 동정을 위한 TaqManⓇprobe를 이용한 real-time PCR 개발

고바라다,김지연,나호명,박성도,김용환 한국동물위생학회 2012 韓國家畜衛生學會誌 Vol.35 No.3

Species-specific TaqManⓇ probe-based real-time PCR assays were developed for detection of beef, pork, chicken, duck, goose and turkey. The primer and probe sets used in this study were designed to be complementary to fibroblast growth factor (FGF) for cattle and pig, mitochondrial NADH dehydrogenase (ND) subunit 3 and ND2 for chicken and duck, 12S rRNA for goose and turkey, respectively. As internal positive control we used conserved region in the ribosomal 18S RNA gene to ensure the accuracy of the detection of target DNA by real-time PCR. We confirmed that real-time PCR assays with the primer and probe sets were positive for cattle, pig and chicken intended target animal species with no cross-reactivity with other non-target animal species. Only >50 ng DNA of beef show cross-reactivity in the determination of duck. Using species-specific primer and probe sets, it was possible to detect amounts of 0.1 ng DNA of cattle and pig, 1.0 pg DNA of chicken, duck and turkey, and 0.1 pg DNA of goose for raw samples, respectively. The detection limits were 0.1 ng DNA of cattle, 1.0 ng DNA of pig and 1.0 pg DNA of chicken for DNA mixtures (beef, pork and chicken) extracted from heat-treated (121oC/5 min) meat samples. In conclusion, it can be suggested that the TaqManⓇ probe-based assay developed in this study might be a rapid and specific method for the identification of meat species in raw or cooked meat products.

광주지역 한우 분변 내 설사병 병원체 조사

고바라다,김효중,오아름,정보람,박재성,이재기,나호명,김용환 한국동물위생학회 2019 韓國家畜衛生學會誌 Vol.42 No.2

Calf diarrhea is a common disease in young claves and is still a major cause of productivity and eco-nomic loss in livestock farms. Fecal samples from Korean native cattle (n=100) with diarrhea from 64 farms in Gwangju area, Korea from september 2017 to December 2018 were examined for shedding of important protozoan parasitic, viral and bacterial pathogens using culture, rapid test kit and PCR methods. Of 57 (89.1%) of the 64 Korean native cattle farms examined had samples infected with at least one of the investigated pathogens. Among 100 fecal samples, 88 samples were positive for at least one the twelve pathogens and 51 samples were simultaneously positive for two or more pathogens by culture and PCR assay. Bovine group A rotavirus (BRV) was the most common pathogen, found in 43/100 (43.0%) samples on 32/64 (50.0%) farms. Subsequently, kobuvirus (30.0%), pathogenic E. coli (29.0%), bovine parvovirus (17.0%), Giardia spp. (13.0%), Eimeria spp. (10.0%), Clostridium per-fringens type A (8.0%), bovine torovirus (8.0%), bovine viral diarrhea virus (6.0%), bovine coronavirus (5.0%), bovine norovirus (2.0%) and Cryptosporidium spp. (2.0%) were detected. Nebovirus, kırklareli virus, bovine adenovirus, Salmonella spp. and intestinal parasites were not detected. Of the 72 calves sampled in this age group, 64 (88.9%) samples were positive for at least one enteropathogen. BRV was identified in 34/72 (47.2%) samples from 27/48 (56.3%) farms. Subsequently, pathogenic E. coli (30.6%), kobuvirus (29.2%), BPaV (22.2%), Giardia spp. (15.3%), Eimeria spp. (9.7%), BVDV (6.9%), Cl. per-fringens type A (6.9%), BCoV (4.6%) and Cryptosporidium spp. (2.8%) were detected in fecal samples. A total of ninety-six strains of E. coli were isolated from one hundred fecal samples collected from Korean native cattle with diarrhea. The presence of stx1, stx2, eaeA, LT, STa, STb, ehxA, saa, F4, F5(K99), F6, F17, F18 and F41 genes in the isolates was investigated by PCR. Out of ninety-six E. coli isolates screened for specific genes, 30 strains E. coli were identified to harbor shiga toxin-produc-ing E. coli (STEC) 7 (7.3%), enterohemorrhagic E. coli (EHEC) 8 (8.3%), enteropathogenic E. coli (EPEC) 6 (6.3%), enterotoxigenic E. coli (ETEC) 2 (2.1%) and STEC/ETEC hybrid 7 (7.3%). This study pro-vides epidemiological estimates of the prevalence of Korean native cattle’s enteropathogens in Gwangju area, Korea, which would be used for cattle farmers and veterinarians to select appropriate therapeutic method.

광주광역시동물보호소 입양 대상 유기견의 호흡기 질병 실태 조사

고바라다,김한나,김효중,오아름,정보람,박재성,이재기,나호명,김용환 한국동물위생학회 2020 韓國家畜衛生學會誌 Vol.43 No.2

Canine infectious respiratory disease (CIRD), also known as infectious tracheobronchitis or kennel cough occurs in a multiple-dog environment such as a shelter. In this study, we were collected 300 of nasal swab samples from dogs and 145 of environmental samples from a shelter to investigate respira-tory pathogens of dogs in the Gwangju metropolitan city animal shelter from February to October, 2019. Bacteria cultures for isolation of Bordetella (B.) bronchiseptica and polymerase chain reaction (PCR) tests were performed for detection of eleven canine respiratory pathogens, namely Mycoplasma (M.) cynos, canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine respiratory coronavirus (CRCoV), alpha-coronavirus (CCoV), canine pneumovirus (CnPnV), ca-nine hepacivirus (CHeV), canine adenovirus type 2 (CAdV-2), canine herpesvirus-1 (CHV-1) and canine bocavirus (CBoV). Among 300 nasal swab samples, 148 samples (49.3%) were positive for at least one pathogens. CHV-1 was the most common pathogen, found in 95/300 (31.7%) samples. Subsequently, M. cynos (22.0%), B. bronchiseptica (2.3%), CPIV (2.0%), CBoV (1.7%), CCoV (0.7%) were detected. The detection rates of M. cynos and CHV-1 according to the duration of stay in the shelter were statisti-cally significant. Among environmental samples, M. cynos, CCoV, CBoV and CHV-1 were detected in 45/145 (31.0%). These results indicated the need for disease control and prevention systems in the shelter.

광주지역 동물보호소내 유기견의 개심장사상충과 개 브루셀라병 감염 실태조사

고바라다 ( Ba Ra Da Koh ),나호명 ( Ho Myung Na ),장미선 ( Mi Sun Jang ),김지연 ( Ji Yeon Kim ),박성도 ( Seong Do Park ) 한국가축위생학회 2007 韓國家畜衛生學會誌 Vol.30 No.1

This study was conducted to investigate the prevalence of canine heartworm infections, canine brucellosis and hematologic values from 153 free roaming dogs in the area of Gwangju city from March to November 2006. Nineteen (12.4%) of 153 samples tested with modified Knott`s technique showed positive reaction for microfilariae. Polymerase chain reaction using specific primers for D immitis amplified the expected product from all samples of 19 microfilaremic canine blood samples as determined by the modified Knott`s test for microfilariae. The seasonal infection rates of microfilariae were higher in the spring season (10/19, 52.6%) than in the other seasons. The major hematological findings in microfilaremic dogs were mild leukocytosis and mild monocytosis. A total of 100 dogs randomly selected from 153 free roaming dogs were negative for canine brucellosis by serological test using immunochromatographic antibody test kit.