Detection of Early Infection of Mycobacterium spp. in Cattle, Caprine, and Zoo Animals with Blood and Milk by PCR

• Main Authors: Kuo Feng-Cheng, 郭峰誠
• Other Authors: 龐飛
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/38256044163594487241

碩士 === 國立臺灣大學 === 獸醫學研究所 === 91 === Tuberculosis is a worldwide zoonotic disease and causes severe economic loss in farm animal industry. In Taiwan, tuberculosis in cattle has been controlled by the policy of test-and-slaughter but the disease still exists. Previous researches based on the rabbit model revealed that mycobacteria could multiply within inactivated macrophages recruiting from the bloodstream in the early stage of infection while the intradermal tuberculin test (ITT) was still negative. Thereafter, owing to the gradually developed immune reaction the host became ITT-positive and the lesions underwent central caseous necrosis. Our previous study showed that mycobacterial DNA could be detected in the blood of both ITT-positive and negative cattle by polymerase chain reaction (PCR) from ITT-positive farms. The positive PCR result revealed in ITT-negative animals provides a possible explanation for the unsuccess in the TB eradication program of cattle in Taiwan. The objective of the present study was to further evaluate whether mycobacteria could truly be detected in the early stage of the infection by PCR along with bacterial isolation before ITT turning to positive. Whole blood and milk samples were collected from both tuberculin test positive and negative farms and screened by the general PCR with TB1-2 primers for Mycobacterium sp. along with standard bacterial isolation. The PCR positive samples were then subjected to the subsequent multiplex-PCR to identify the possible species of M. tuberculosis, M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis. A total of 2291 blood and 485 milk samples were examined. There were a total of 52 positive samples detected by general PCR, and only one of them was ITT-positive while others were ITT-negative. In addition, 8 blood samples from ITT-positive animals showed negative results by general PCR. The positive ratios of the blood and milk samples by general PCR were 2.10% (48/2291) and 0.82% (4/485), respectively. The positive ratios for the dairy cattle and caprine were 2.35% (48/2039) and 0.43% (3/705), respectively. All blood and milk samples were negative for bacterial isolation. Subsequent multiplex-PCR followed by Southern blotting revealed M. bovis infection in 10 blood and 1 milk samples, and M. tuberculosis infection in 4 blood samples. The positive PCR result but negative bacterial isolation from ITT-negative and positive animals suggest that mycobacteria may indeed present in the blood in the early stage of infection but they may have been killed by the host immune system and the positive PCR results simply represent the presence of DNA of killed mycobacteria in these ITT-negative animals; or the number of bacteria present in the blood was too low to be successfully isolated from the infected animals. The result also suggests that tuberculin test combined with blood PCR assay may provide a more sensitive detection method for the early identification of mycobacterial infection in dairy cattle and caprines.