• 한국동물위생학회지
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• 제45권2호
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• pp.87-99
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• 2022

A novel porcine circovirus 4 (PCV4) was recently emerged in Chinese and Korean pig herds, which provided epidemiological situation where three pathogenic PCVs, PCV2, PCV3, and newly emerged PCV4, could co-infect pig herds in these countries. In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) method was developed for the rapid and differential detection of these viruses. The assay specifically amplified each viral capsid gene, whereas no other porcine pathogenic genes were detected. The detection limit of the assay was below 10 copies/µL and the assay showed high repeatability and reproducibility. In the clinical evaluation using 1476 clinical samples from 198 Korean pig farms, the detection rates of PCV2, PCV3 and PCV4 by the tqPCR assay were 13.8%, 25.4%, and 3.8%, respectively, which were 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4, with a κ value (95% CI) of 1 (1.00~1.00). The prevalence of PCV2, PCV3, and PCV4 at the farm levels were 46.5%, 63.6%, and 19.7%, respectively. The co-infection analysis for tested pig farms showed that single infection rates for PCV2, PCV3, and PCV4 were 28.8%, 44.4%, and 9.6%, respectively, the dual infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 12.6%, 3.5%, and 5.1%, respectively, and the triple infection rate for PCV2, PCV3, and PCV4 was 1.5%. These results demonstrate that three pathogenic PCVs are widely spread, and their co-infections are common in Korean pig herds, and the newly developed tqPCR assay will be useful for etiological and epidemiological studies of these pathogenic PCVs.

• 한국동물위생학회지
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• 제45권4호
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• pp.305-316
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• 2022

Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

• 한국동물위생학회지
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• 제46권1호
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• pp.15-27
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• 2023

Bordetella (B.) bronchiseptica, Mycoplasma (M.) cynos, and M. canis are the major bacterial pathogens that cause canine infectious respiratory disease complex (CIRDC). In this study, we developed a triplex real-time polymerase chain reaction (tqPCR) assay for the differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra- and inter-assay variations of less than 1%. The diagnostic results of the assay using 94 clinical samples from household dogs with CIRDC clinical signs, the prevalence of B. bronchiseptica, M. cynos, and M. canis was 22.3%, 18.1%, and 20.2%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported qPCR assays. The dual infection rate of B. bronchiseptica and M. cynos, B. bronchiseptica and M. canis, and M. cynos and M. canis was 5.3%, 7.4%, and 3.1%, respectively. Moreover, the triple infection rate of B. bronchiseptica, M. cynos, and M. canis was 2.1%. These results indicate that coinfections with B. bronchiseptica, M. cynos, and M. canis have frequently occurred in the Korean dog population. The newly developed tqPCR assay in the present study will be a useful tool for etiological and epidemiological studies on these three CIRDC-associated bacterial pathogens. The prevalence and coinfection data revealed through this study will contribute to expanding knowledge on the epidemiology of CIRDC in the recent Korean dog population.

• 한국동물위생학회지
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• 제46권4호
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• pp.269-281
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• 2023

In this study, a new triplex real-time quantitative reverse transcription polymerase chain reaction (tqRT-PCR) assay was developed for the rapid and differential detection of three feline viral pathogens including feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and influenza A virus (IAV) in a single reaction. The assay specifically amplified three targeted viral genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 1%. Based on the diagnostic results of the assay using 120 clinical samples obtained from cats with feline respiratory disease complex (FRDC)-suspected signs, the prevalence of FCV, FHV-1, or IAV was 43.3%, 22.5%, or 0%, respectively, indicating that the diagnostic sensitivity was comparable or superior to those of previously reported monoplex qRT-PCR/qPCR assays. The dual infection rate for FCV and FHV-1 was 8.3%. These results indicate that FCV and FHV-1 are widespread and that co-infection with FCV and FHV-1 frequently occur in the Korean cat population. The developed tqRT-PCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens, and the prevalence data for three feline viruses obtained in this study will contribute to expanding knowledge about the epidemiology of FRDC in the current Korean cat population.

• 한국초지조사료학회지
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• 제42권3호
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• pp.201-207
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• 2022

Fescues, which are widely cultivated as grasses and forages around the world, are often naturally infected with the endophyte, Epichloë. This fungus, transmitted through seeds, imparts resistance to drying and herbivorous insects in its host without causing any external damage, thereby contributing to the adaptation of the host to the environment and maintaining a symbiosis. However, some endophytes, such as E. coenophialum synthesize ergovaline or lolitrem B, which accumulate in the plant and impart anti-mammalian properties. For example, when livestock consume excessive amounts of grass containing toxic endophytes, problems associated with neuromuscular abnormalities, such as convulsions, paralysis, high fever, decreased milk production, reproductive disorders, and even death, can occur. Therefore, pre-inoculation with non-toxic endogenous fungi or management with endophyte-free grass is important in preventing damage to livestock and producing high-quality forage. To date, the diagnosis of endophytes has been mainly performed by observation under a microscope following staining, or by performing an immune blot assay using a monoclonal antibody. Recently, the polymerase chain reaction (PCR)-based molecular diagnostic method is gaining importance in the fields of agriculture, livestock, and healthcare given the method's advantages. These include faster results, with greater accuracy and sensitivity than those obtained using conventional diagnostic methods. For the diagnosis of endophytes, the nested PCR method is the only available option developed; however, it is limited by the fact that the level of toxic alkaloid synthesis cannot be estimated. Therefore, in this study, we aimed to develop a triplex real-time PCR diagnostic method that can determine the presence or absence of endophyte infection using DNA extracted from seeds within 1 h, while simultaneously detecting easD and LtmC genes, which are related to toxic alkaloid synthesis. This new method was then also applied to real field samples.