以PCR-DGGE方法進行臺灣長鬃山羊(Capricornis swinhoei)圈養族群糞便細菌相變化之探討

臺灣長鬃山羊 (Formosan serow, Capricornis swinhoei）屬珍貴稀有保育動物，為了維持其族群數量，我們需握有相關訊息，達到預防疾病並減少其死亡率。臺灣長鬃山羊如同其他反芻獸，其腸胃道中的微生物對於健康及生產性能扮演著重要的角色，所有的微生物當中，細菌為最主要幫忙宿主有效吸收養分及維持正常生理功能的角色；當宿主的年齡、健康情況、飼糧配方改變、生長環境中的季節變換、棲息地變動等，皆會造成腸胃道菌相的改變，觀察腸胃道菌相的變化為監控反芻動物本身生理情況的重要環節，而瘤胃及糞樣中之微生物相為目前較廣泛作為偵測反芻動物體健康情況的一項工具。然而，瘤胃樣本收集的困難度較糞樣收集高出許多，且屬於一種侵入式之採集方法，故糞樣之收集是較為方便的方法。因此，本試驗之目的為建立自圈養之臺灣長鬃山羊糞樣萃取 DNA 之非侵入式分析方法，並建立其糞樣菌相變化圖譜。本試驗共採集 7 頭臺北市立動物園臺灣長鬃山羊糞便樣本，4頭公羊，3頭母羊，採樣時間自2012年9月至2013年4月，共計8個月，每週定期採樣一次；該園供給受試動物的日糧主要為桑葉與山麻黃葉，精料則為胡蘿蔔、甘藷及有蹄類動物低蛋白顆粒精料。所有自糞樣萃取之 DNA 利用 37.5-50% 之聚合酶鏈鎖反應-變性梯度膠體電泳(polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE) 增幅及分析16S rDNA V3 區域，以 BioNumerics 6.6 分析軟體，比較採樣期間菌相之變化並鑑定主要糞樣細菌種類。根據 PCR-DGGE分析結果， Clostridium populeti與Papillibacter cinnamivorans在臺灣長鬃山羊的糞樣中為常駐菌種；此外，糞樣中之細菌有 73.9% 屬於 Firmicutes，此為最主要之菌門 (phylum)，第二群主要菌門屬於 Bacteroidetes，佔有 16.5%，其他則分屬於 Proteobacteria、Synergistetes、Actinobacteria與Spirochaetes 等菌門，其所佔比例皆小於 5%。菌相相似度 (similarity) 分析方面，所有樣本相似度達 44.5%，且從中可以發現群集 (cluster) 的分布大致依照性別進行分群。至於單一羊隻菌相相似度樹狀圖中，群集的產生及菌相的變化與糞樣採集時間點有關，顯示羊隻的糞樣菌相隨著季節做變化，在一至三月期間，正值季節交替時，可發現圈養之臺灣長鬃山羊菌相會有之菌相的改變。NGS 結果顯示Firmicutes為最主要菌門，佔了58.2%，接著的主要菌門屬於 Bacteroidetes，含有 28.3%。針對性別對菌相的影響，資料發現公羊含有較高比例的 Prevotella spp.，母羊則含有較多的 Campylobacter spp.。另外觀察親子間菌相的相似程度，未發現有特別的關聯，因此我們認為對於糞樣菌相而言，環境因素可能比遺傳因素更具影響力。本研究初步建立臺灣長鬃山羊糞樣菌相變化圖譜，了解糞樣中之細菌與季節轉換之間的關係，將可作為飼養管理之參考資料。在季節轉換期間應特別注意圈養羊隻之生理情況，並配合觀察糞樣細菌消長情形，可作為監控動物園內臺灣長鬃山羊生理情況之指標，減少死亡率。

關鍵字

臺灣長鬃山羊；糞樣菌相； PCR-DGGE ； NGS

並列摘要

Formosan serow (Capricornis swinhoei) is a rare species which is endemic to Taiwan. In order to maintain the populations of Formosan serow, it is important to obtain the information concerning disease prevention. As other ruminants, they depend on a complex symbiotic rumen microbial ecosystem to absorb most nutrients. The enteric microbiome of ruminant plays a critical role not only in animal health and productivity but also in pathogen shedding. In addition, bacteria are the most active micros in fermentation providing nutrition for the host. There were wide variety of bacterial strains found in ruminants at different age, health conditions, geographical locations, seasons, and diets previously indicating a potential application of monitoring the physiological conditions through examining the microflora in the gut. The microbiome in the rumen and feces of ruminants have been used as a diagnostic tool for assessing animal health. However, obtaining rumen samples is much more invasive and difficult comparing with collecting fecal samples which is non-invasive, but still provides valid materials for analyzing the enteric microbiome. The objective of this study was to establish bacterial DNA preparation procedure from fecal samples and understand the fecal bacteria community in captive population of Formosan serow. The 7 captive Formosan serows, including 3 males and 4 females, in Taipei Zoo were used in this study. Formosan serows’ feces were collected once a week from September in 2012 to April in 2013. In this period, the main forages were Morus australis leaves and Trema orientalis leaves, the concentrates were carrot, sweet potato and concentrate pellets. The dominant feces bacteria in Formosan serow were investigated using PCR-DGGE approach and NGS of V1-V3 region of 16S rDNA sequence. All samples were subjected to run on a 10% acrylamide gel with a 37.5 to 50 % linear denaturing gradient. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis V3 region of 16S rDNA sequence in DGGE profiles were combined to reveal the dominant bacteria communities and compared the differences between individual and dates. DGGE profiles revealed that Clostridium populeti and Papillibacter cinnamivoranswere were the resident bacteria in captive Formosan serow. In addition, Firmicutes was the most abundant phylum, which make up 73.9% of the total phyla, suggesting that Firmicutes in particular play a critical role in the microbial ecology of the Formosan serow gut. A secondary phylum found was Bacteroidetes (16.5%). The remaining and less abundant phyla were Proteobacteria, Synergistetes, Actinobacteria, and Spirochaetes (< 5%). The overall comparison of bacterial profiles showed a similarity of 44.5% among all individuals, and the samples tended to group according to the gender. The quality of forages changed in different collection dates had made clusters. During Jan. to Mar., time of season change, the fecal bacteria community in captive population of Formosan serows would alter. The NGS results suggested sequences related to the phyla Firmicutes (58.2%) and Bacteroidetes (28.3%) predominated all samples. Gender effects were observed higher proportions of Prevotella spp. and lower proportions of Campylobacter spp. were found in male serows. Compared with the fecal bacteria community of parent and child, we thought environmental factors are more influential than genetic factors in fecal bacteria community. In conclusion, the data suggest that there are needs for attention on animal health during seasonal change. Monitoring the physiological conditions through examining the fecal bacteria community would reduce the mortality rate in captive population of Formosan serow. These findings provided a framework to understand how microbiome can impact the health of the animals and possibly contribute to the management of Formosan serows reared in captive.