【摘要】：寄生蟲在野生鳥類中感染相當普遍,在寄生蟲的影響下鳥類可能發(fā)生疾病,造成生長發(fā)育停滯,甚至發(fā)生致死性的嚴重疾病等,因此成為保護生物學研究的熱點問題之一。白頭鶴(Grus monacha)是一種脆弱的大型遷徙水鳥,種群保護的形勢十分嚴峻,考慮其瀕危性在進行寄生蟲感染狀況調(diào)查時大多采取最簡單傷害最小的非損傷性取樣法取其糞便樣品。據(jù)以往研究表明,在白頭鶴糞便中能檢出大量的腸道寄生蟲,主要包括球蟲,線蟲和吸蟲等。但是由于傳統(tǒng)的形態(tài)學鑒別方法的局限性,寄生蟲的種類鑒別研究并不透徹,建立白頭鶴腸道寄生蟲的分子檢測方法對寄生蟲進行快速、準確的鑒別尤為重要。本研究主要探討野生白頭鶴腸道寄生線蟲感染的分子檢測方法。于2013年11月至2015年2月期間,收集了在長江中下游地區(qū)鄱陽湖、菜子湖和升金湖越冬的白頭鶴糞便樣本共908份。采用飽和鹽水漂浮法進行白頭鶴腸道寄生蟲形態(tài)學鑒定,白頭鶴糞便樣品中共檢測出5類寄生線蟲,即毛細線蟲(Capillaria spp.)、類圓線蟲(Strongyloides spp.)、禽蛔蟲(Ascaridia spp.)、毛圓線蟲(Trichostrongylus spp.)、鉤蟲(Ancylostomatidae spp.)等。線蟲感染率最高的種類為禽蛔蟲(6.9%),其次為毛細線蟲(4.6%)、類圓線蟲(4.1%)。在有限稀釋法對越冬白頭鶴糞便樣品中寄生蟲卵分離的基礎(chǔ)上,進行了白頭鶴種群腸道寄生蟲分子檢測方法的探索。通過對毛細線蟲單蟲卵分別進行液氮/85℃水浴、超聲破碎、裂解液消化等三種前處理后進行熒光PCR檢測,結(jié)果表明單蟲卵經(jīng)裂解液消化后用于熒光PCR檢測的效果明顯而穩(wěn)定。所建立的寄生蟲卵熒光PCR檢測方法敏感性強,可以滿足微量模板高檢出率的要求。糞便經(jīng)漂浮和短暫的離心沉淀后即可進行熒光PCR檢測。檢測過程中鏡檢步驟的省略使得非專業(yè)人員即可操作,減少了工作量,并能夠滿足快速、批量的寄生蟲檢測需要。本研究建立的方法中,寄生蟲蟲卵經(jīng)一步預處理即可,RT-PCR能夠快速準確地檢測出陽性樣品,顯著提高檢測效率,且經(jīng)實驗驗證檢測效率高于鏡檢處理。本研究借助熒光PCR檢測的高度特異性,設(shè)計毛細線蟲ITS區(qū)引物,經(jīng)特異性實驗驗證表明與鉤蟲、毛圓線蟲等鶴類常見寄生蟲無交叉反應。此方法的探索可為今后毛細線蟲的分子檢測提供可靠的方法,并為糞便中其他寄生蟲的檢測提供了一條有效途徑。
[Abstract]:Parasites infection is very common in wild birds. Under the influence of parasites, birds may suffer from diseases, resulting in stagnation of growth and development, and even fatal serious diseases, so it has become one of the hot issues in the research of conservation biology. (Grus monacha) is a kind of fragile large migratory waterfowl, and the situation of population protection is very serious. When investigating the parasite infection status, the most simple non-invasive sampling method is used to collect the fecal samples. According to previous studies, a large number of intestinal parasites, including coccidia, nematode and trematode, can be detected in the droppings of white-headed cranes. However, due to the limitations of traditional morphological identification methods, the study of parasite species identification is not thorough. It is very important to establish a molecular detection method to identify parasites quickly and accurately. In this study, molecular detection methods of parasitic nematode infection in the intestinal tract of wild cranes were studied. From November 2013 to February 2015, 908 samples of white-headed cranes were collected from Poyang Lake, Caizi Lake and Shengjin Lake in the middle and lower reaches of the Yangtze River. The morphology of intestinal parasites was identified by saturated saline floating method. Five parasitic nematode species, (Capillaria spp.), nematode, (Strongyloides spp.), roundworm, Ascaris lumbricoides (Ascaridia spp.), were detected in fecal samples of Crane Whitehead Crane. (Trichostrongylus spp.), hookworm (Ancylostomatidae spp.) Etc. The highest infection rate of nematodes was avian roundworm (6.9%), followed by nematode capillaris (4.6%) and nematoid nematode (4.1%). Based on the separation of parasite eggs from the feces of overwintering white-headed cranes by finite dilution method, the molecular detection method of intestinal parasites of white-headed cranes was explored. Three kinds of pre-treatment, such as liquid nitrogen / 85 鈩，

本文編號：2378902