檢測環境分枝桿菌在台灣牧場中之分布情形及對乳牛皮內結核菌素測試之影響

Abstract

目前分布在世界上的分枝桿菌約有一百多種，為數眾多的一部分存在於環境中的水或是土壤等腐生環境當中，稱為環境分枝桿菌，當牛隻暴露在環境分枝桿菌之下，有可能會造成非特異性致敏化，進而干擾結核菌素皮內注射檢測牛隻感染牛型分枝桿菌的診斷。目前台灣在乳牛的結核病根除計畫的執行上仍以結核菌素皮內注射作為標準的檢測工具，只要檢測結果為陽性的牛隻會立即撲殺。本實驗室於2009年4月至2010年8月為止，共針對115頭結核菌素陽性撲殺牛隻進行剖檢及採樣，將咽背淋巴結送往淡水家畜衛生試驗所進行培養之後，依其培養結果將各牛隻所屬的牧場進行環境採樣並分組。 本研究中使用聚合酶鏈鎖反應，針對大部分環境分枝桿菌皆有的熱休克蛋白65 (heat shock protein 65, hsp65)基因片段檢測環境樣本所含的菌叢。共計檢測16個牧場，將其分為三個組別，第一個組別為有檢出結核菌素陽性的牛隻，且牛隻剖檢後於咽背淋巴結可以培養出牛型分枝桿菌的牧場，這類牧場中所採集的環境樣本總數為105個，PCR陽性率為50.58% (53/105)；第二個組別所對應的牧場為該場有結核菌素陽性的牛隻，但是咽背淋巴結分離牛型分枝桿菌的結果為陰性，懷疑這些牛隻有結核菌素偽陽性的情形，這類牧場共採樣194個環境樣本，PCR陽性率為78.35% (152/194)；第三個組別為結核菌素測試為陰性的牧場，總環境樣本數為88個，陽性率為60.23% (53/88)；結果顯示第二個分組的環境分枝桿菌陽性率顯著高於另外兩組 (p<0.05)，牛隻在這類牧場中有較高的機率接觸到環境分枝桿菌，造成非特異性致敏化的風險也較高。另外為了要區分在牧場的欄舍當中到底是哪一個區域有特別高的風險來接觸這些環境中的分枝桿菌，以區域劃分作為分組，可發現各牧場中的運動場區的分枝桿菌檢測的陽性率顯著高於其他畜欄。 將所有的PCR陽性所偵測到的序列定序之後繪製親緣樹狀圖，發現有些菌叢僅存在於特定的牧場分組當中，其中僅B組的牧場能偵測到的菌叢數量特別多，共有14個菌叢，且B組中的6個牧場全部都有這些菌叢的分布，這一些菌叢無法在A、C組別中檢測出來，表示B組環境確實有別於其他分組之處。有些環境樣本序列和結核菌素陽性牛隻組織、血液以及乳汁中所偵測到的序列親緣分析上屬於同一菌叢，也許此菌叢的確即為造成牛隻結核菌素檢測偽陽性的病原，此牧場需特別注意此等菌的分布情形方能進行有效的預防消毒。 結核菌素偽陽性牛隻的存在會造成政府以及酪農的經濟損失，本研究依據每個不同的牧場皆有繪製地圖，評估其高風險菌叢的位置並標示出來，期望對於這些牧場防治牛隻結核菌素的偽陽性有一定的幫助。

Among the more than 100 known species of the genus Mycobacterium, majority of them are described as “environmental mycobacteria” (EM), present as saprophytes in water or soil worldwide. Several species of EM are known as opportunistic pathogens and may lead to false positive diagnosis of bovine tuberculosis by intradermal tuberculin test (ITT). In Taiwan, ITT is a standard diagnostic tool for tuberculosis eradication program in dairy herds and cattle showing positive reaction are culled immediately. However, from April of 2009 to August of 2010, approximately 32％ (37/115) of the cattle culled did not exhibit typical tubercle lesions grossly or histopathologically, nor M. bovis or M. tuberculosis was detected by polymerase chain reaction (PCR). In the present study, we used PCR to detect the heat shock protein 65 (hsp65) gene fragments of EM present in various environmental samples to evaluate the distribution of EM flora in dairy farms displaying ITT-positive result, as well as in farms with ITT-negative result as the control group. A total of 16 dairy farms were included and divided into three groups. Group A included farms which had ITT-positive history and M. bovis could be isolated from the retropharyngeal lymph node; within the 105 environmental samples of group A, the positive rate of hsp65 PCR was 50.58%. Group B were farms which had ITT-positive history but no M. bovis could be isolated from the retropharyngeal lymph nodes, indicating that these farms might be ITT false-positive; within the 194 environmental samples of group B, the positive rate of hsp65 PCR was 78.35%. Group C were farms in which dairy cattle displayed ITT-negative history; within the 88 environmental samples of group C, the positive rate of HSP65 PCR was 60.23%. The results showed that the positive rate of hsp65 PCR from Group B was clearly higher than those of group A and C (p<0.05), the cattle in these farms might have a higher chance to contact EM resulting in non-specific sensitization. To further investigate where in the farms that the cattle had higher risk of exposing to EM, each farm was divided into several different regions and it was found that the sport fields in these farms had much higher positive rates of hsp65 PCR than in other regions. The hsp65 PCR products were sequenced and subjected to phylogenetic analysis. It was found that fourteen mycobacterial flora could only be found in all of the six farms of group B, suggesting that the EM in group B was really different from group A and group C. The results of phylogenetic analysis of the sequences of the hsp65 PCR products from environmental samples and from the milk, tissue, and blood of cattle from these farms of group B strongly supported that the EM flora should contribute to the ITT-false positivity. Such false-positive reactors have caused severe, unnecessary economic losses not only to the producers but also to the government. Therefore, knowing the EM flora present in cattle farms and whether theses EM flora may have influence on the ITT results is rather important.