乳牛結核病田間調查及親緣性分析

Abstract

台灣實施牛隻皮內結核菌素檢驗 (ITT) 至今已超過五十年，但每年仍有零星的結核菌素檢驗陽性病例，且仍被世界動物衛生組織 (OIE) 認定為牛結核病疫區。本研究收集台灣地區12個牧場共137頭ITT陽性撲殺乳牛組織樣本，進行分枝桿菌分離、病理學檢查、分子生物學診斷及菌株基因型別鑑定。結果顯示，共有40頭 (40/137; 29.2%) 培養出分枝桿菌，其中有29株為Mycobacterium bovis (29/137; 21.17%)，另外11株則為M. bovis以外的Mycobacterium spp. (11/137; 8.03%)。有89頭 (89/137; 64.96%) 在肉眼或/及顯微檢查下有結核樣病灶，其中最常出現病變的位置為縱膈淋巴結，其次依序為咽背淋巴結、肺門淋巴結、腸繫膜淋巴結及肺臟。透過細菌分離及聚合酶鏈鎖反應 (PCR) 檢測，確認是因M.bovis感染引起ITT陽性反應的有69頭 (69/137; 50.37%)，其中出現結核樣病變的有59頭 (59/137; 43.07%)。Variable number tandem repeat (VNTR) 的分析，從本研究的34個M.bovis陽性樣本中，鑑別出5種不同的基因型別，其中，I型和IV有親緣關係， III型和V型亦有親緣關係。本研究揭示了台灣地區因ITT陽性而撲殺之乳牛中，結核病的確切發病情形。台灣仍有牛結核病的疫情，且仍為乳牛ITT陽性的重要致敏原。然而，亦有其他非M. bovis及M. tuberculosis的分枝桿菌感染，也可引起ITT陽性反應。此外，結果亦顯示牛隻的M. bovis有超過一個以上的VNTR菌株存在，且同一親緣性的菌株有可能感染不同縣市牧場的乳牛。VNTR基因分型技術能分辨出不同的M. bovis菌株及其間的親緣關係，展現其在台灣牛結核病疫情之流行病學調查上的應用潛力。

The tuberculin test-and-culling program for eradication of bovine tuberculosis has been carried out in Taiwan for more than 50 years; however, sporadic tuberculin test reactors still occur, and Taiwan is still defined as an epidemic country of bovine tuberculosis by OIE. In this study, a total of 137 dairy cattle with skin test-positive reaction, originated from 12 dairy farms, were euthanatized and sampled for mycobacterial isolation, pathological examination, molecular diagnosis, and bacterial strain genotyping. Mycobacterium spp. could be isolated in 40 cattle (40/137; 29.2%), 29 of them (29/137; 21.17%) were identified as Mycobacterium bovis, another 11 cattle (11/137; 8.03%) were other Mycobacterium spp. other than M. bovis. Tuberculous lesions were identified in 89 cattle (89/137; 64.96%) following gross and/or microscopic examination. The most frequently affected site was mediastinal lymph node, followed by retropharyngeal lymph node, hilar lymph node, mesenteric lymph node, and lung. Sixty nine cattle (69/137; 50.37%) were identified as M. bovis infection through mycobacterium isolation and PCR assay, 59 of them (59/137; 43.07%) developed tuberculous lesion. Five genotypes of M. bovis were found among 34 skin test-positive cattle from four different farms by a molecular genotyping method based on variable number tandem repeat (VNTR) analysis, and the phylogenetic relationships between genotype I, IV and genotype III, V were also noted. The findings uncovered the actual incident status of bovine tuberculosis in the cattle under routine tuberculin test survey, indicating that M. bovis still is the major cause of bovine tuberculosis and is also an important allergen for the positive reaction of tuberculin test in Taiwan. However, there are Mycobacterium spp. other than M. bovis and M. tuberculosis that may trigger the tuberculin test reaction of dairy cattle in Taiwan. Based on the data obtained from the 12 dairy farms, there are more than one VNTR strains of M. bovis present in Taiwan, and the same VNTR strain may infect cattle from different counties. The present study further supports that VNTR genotyping can be used to differentiate different strains of M. bovis and can be a powerful tool for epidemiological investigation of bovine tuberculosis in Taiwan.