• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.86-93
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• 2000

Paratuberculosis (Johne's disease), a chronic enteritis produced by Mycobacterium paratuberculosis, affects a large proportion of ruminants in all continents and causes important economic losses. The identification of well-characterized and species-specific components of M paratuberculosis would provide the means to improve the specificity and sensitivity of immunodiagnostic assays for Johne's disease. The aims of this study were to express the recombinant C-terminal of 34kDa protein (rC34P) of M paratuberculosis in E coli and to investigate the effectiveness of this protein in detecting antibodies to the native protein in sera from paratuberculosis infected cattle. The C-terminal of the gene encoding the 34kDa protein was amplified by polymerase chain reaction from the chromosomal DNA of M paratuberculosis (ATCC 19698) and cloned into vector pGEX-4T-2. Then, cloned plasmid was transformed into E coli DH5${\alpha}$ and the rC34P was overexpressed. The rC34P was purified by affinity chromatography and gel filtration. The rC34P was examined antigenicity by Western blot. The rC34P was reactive with culture positive bovine serum and hyperimmune rabbit anti-M paratuberculosis serum but was not reactive with culture negative bovine serum and tuberculin positive bovine serum in Western blot. In conclusion, the rC34P produced in this study is expected as a useful candidate for antigen in serological diagnosis of Johne's disease.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.81-88
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• 2002

The purpose of this study was to conduct diagnosis of bovine paratuberculosis in Kangwon area. Blood samples were collected from 2,261 dairy cows of 162 herds, and the ELISA and immunoblotting using recombinant 34KDa protein of M. paratuberculosis were conducted. The feces collected from dairy cows were cultured on HEY medium with mycobactin-J and PCR was conducted with washing solution of medium 4 weeks after culture. The ELISA had sensitivity of 83.3% and specificity of 96.7%. And the immunoblotting had sensitivity of 83.3% and specificity of 100%. Of the 2,261 dairy cows, 371 cows(16.4%) were positive in ELISA and 75 cows(3.3%) were positive in immunoblotting. And of the 162 herds, 109 herds(67.3%) had an apparent paratuberculosis prevalence by ELISA and 40 herds(24.7%) by immunoblotting. The geographic distribution of herds with paratuberculosis was not uniform. Of the 241 feces samples including 110 feces from ELISA positive cow, 9 feces were positive in culture and PCR. PCR was able to detect the growth of M. paratuberculosis as early as 4 weeks of culture.

• Korean Journal of Veterinary Research
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• v.46 no.3
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• pp.271-274
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• 2006

A 2-years-old female domesticated mouflon with a clinical history of chronic diarrhea and emaciation was submitted to NVRQS. Grossly, there were severe thickening of small intestine wall and enlargement of mesenteric lymph nodes. Microscopically, severe granulomatous inflammation was found in small and large intestine, mesenteric lymph nodes, spleen and liver. By Ziehl-Neelsen stain, innumerable acid-fast rod bacteria were found in the cytoplasm of epitheloid and Langhans type giant cells present in these organs. By PCR assay with primer pair specific for Mycobacterium avium subspecies paratuberculosis(IS 900) with small intestine sample, strong positive reaction was detected, although the organism was not isolated from this organ. Based on the results of histopathology and PCR, we concluded that the case was a typical paratuberculosis in mouflon. As far as we know, this is the first case report of paratuberculosis in mouflon Korea.

• Korean Journal of Veterinary Service
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• v.36 no.2
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• pp.133-137
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• 2013

The purpose of this study was to investigate the seroprevalence of paratuberculosis in Korean cattle in western Jeonbuk area. From February 2012 to January 2013, blood samples were collected from 2,606 Korean cattle of 263 farms. As a result, 60 (2.3%) heads of 46 (17.5%) farms were positive on the ELISA test for paratuberculosis. Based on regional analysis, 18 (19.6%) out of 92 farms and 24 (2.5%) out of 941 heads in Iksan area, 28 (22.0%) out of 127 farms and 36 (2.8%) out of 1,291 heads in Kimje area were positive but samples from Gunsan area were all negative. According to scale breeding, small scale (below 50 heads) breeding showed the most high prevalence rate compared to middle (50 to 99 heads) or large (over 100 heads) scale breeding. To clarify the relation between number of heads and paratuberculosis prevalence, some additional analysis would be required in further, though.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.349-358
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• 1997

Mycobacterium paratuberculosis is the etiologic agent of Johne's disease, a chronic inflammatory bowel syndrome in ruminants. The attempts to control or eradicate the disease were severely hampered by the inadequacies of present diagnostic methods. The first purpose of this study was to detect Johne's disease out of 577 cows in the province of Kyunggi, Chungchong, Gangweon and the second purpose was to compare the results of non-absorbed ELISA, absorbed ELISA, PCR, and conventional culture methods. The third purpose was to increase diagnostic specificity, accuracy and rapidity. When non-absorbed ELISA test was conducted with Mycobacterium paratuberculosis antigen, the prevalence of positive was 10.9%. To increase diagnostic specificity, absorbed ELISA test with Mycobacterium phlei was used. In this test, the positive prevalence was 1.7%. For the specific detection of Mycobacterium paratuberculosis, PCR was applied to bacterial culture obtained from fecal samples of cattle. The DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of Mycobacterium paratuberculosis by PCR. PCR for M paratuberculosis isolated from fecal cultures amplified specific target DNA. PCR was much more rapid than that obtained by conventional culture technique in diagnosis of Johne's disease.

• Korean Journal of Veterinary Service
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• v.32 no.2
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• pp.171-176
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• 2009

Johne's disease is a chronic inflammatory bowel disease of cattle, sheep, goats and other ruminants, and Mycobacterium paratuberculosis is the etiologic agent of this disease. Many studies have been carried out on paratuberculosis from daily cattle and Korean native cattle in multiple areas around nation, but there is no report in Eastern-Gyeongbuk area. The purpose of this study is to investigate the seroprevalence of bovine paratuberculosis in Eastern-Gyeongbuk area. From July to December in 2007, blood samples were collected from 363 dairy cattle of 27 farms and 281 Korean cattle of 114 farms and the ELISA was conducted. 25 (6.9%) dairy cattle of 6 (22%) farms and 19 (6.8%) Korean cattle of 8 (7.0%) farms were positive in ELISA. In regional analysis, 25 (8.3%) out of 300 dairy cattle in Gyeungju were positive and Pohang were negative in this research. 12 (16.4%) out of 73 Korean cattle in Gyeungju and 7 (9.6%) out of 73 Korean cattle in Uljin were positive. Pohang and Youngdeok of Korean cattle were negative in this research. According to raising scale of dairy cattle, 4 (66.7%) farms out of 6 farms were raising 30 below and 2 (33.3%) farms out of were raising 30$\sim$70. And there were negative raising scale more than 70. In Korean cattle, 6 (75%) farms out of 8 were raising below 10 and 2 (25%) farms were raising 10$\sim$30. And there were negative raising scale more than 30. The rate of seropositive of paratuberculosis dairy cattle and Korean cattle were similar and the positive rate of Eastern-Gyeongbuk area is reported lower than that of any other region.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.23-28
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• 2010

Mycobacterium avium ssp paratuberculosis, intracellular bacteria that can cause chronic granulomatous enteritis in cattle, continues to pose significant economic losses and health problem with high prevalence. The purpose of this study is the polymerase chain reaction (PCR)-base strategy for early detection of M. avium ssp paratuberculosis in whole blood. Blood samples were collected from korean cattles in Jeonbuk, Korea. The 16 out of 88 serum samples were detected M. partuberculosis by ELISA. Then samples of infected 8 Korean cattles were amplified by PCR. The PCR amplified targets are 16s rDNA and heat shock protein 65kDa (hsp 65). The 16s rDNA provided a highly sensitive and specific tool for the direct detection of mycobacteria. In addition M. avium was confirmed characteristically by the hsp65. Finally there were sure to M. avium ssp paratuberculosis by IS900 PCR. The restriction fragment length polymorphism was identified by PCR amplifications and subsequence restriction enzyme digestions with Pst I of a hsp65. These results indicate that confirm M. avium with 16s rDNA, hsp65 and a restriction fragment length polymorphism in the hsp65 gene can be seem the other pattern. Therefore, these results can be used for clinical direct detections of M. avium ssp paratuberculosis in whole blood of Korean cattle and also to be used epidemiological researches.

• Journal of Veterinary Clinics
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• v.32 no.3
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• pp.219-223
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• 2015

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

• Korean Journal of Veterinary Research
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• v.24 no.1
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• pp.58-63
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• 1984

Fecal material from cattle, which was confirmed to be infected with Johne's disease by clinical and pathological symptoms, was decontaminated with 4% NaOH and inoculated into the $L{\ddot{o}}wenstein$-Jensen media supplemented with 1% of heat-killed Mycobacterium bovis. After 2-4 week-incubation at $37^{\circ}C$, typical acid-fast mycobacteria was isolated. With the results of staining properties, morphological characteristics, the requirement of mycobactin for growth and the other biochemical properties, isolated mycobacteria was identified as Mycobacterium paratuberculosis. Female guinea pigs were sensitized with the isolates, and skin test was done with purified protein derivatives (PPDs) of M. avium, M. bovis and M. paratuberculosis 4 weeks after sensitization. Animals showed the largest reaction to the PPDs of M. avium and M. paratuverculosis.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.93-97
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• 1994

A immunological survey of paratuberculosis in dairy and Korean native cattles was conducted by enzyme linked immunosorbent assay(ELISA), complement fixation test(CFT), agar gel immunodiffusion test (AGID) and intradermal skin test(ID). Over all prevalence of pararuberculosis in cattles was 6.7%(109/1633) by ID, 7.5(205/2719) by AGID, 9.3% (245/2641) by CFT and 13.4%(363/2719) by ELISA. Prevalence in dairy cattle was higher than that of Korean native cattle. Of 70 ELISA-positive cattle, 23(28.6%) and 48(68.6%) cattles were classified as positive in the AGID and positive or suspect in CFT, respectively. Of 92 ELISA-suspect cattle, 32(34.9%) and 48(52.2%) cattles were classified as AGID-positive and CFT-positive or suspect, respectively. It was concluded that paratuberculosis is widespread in cattle of Korea.