本文選題：弓形蟲 + 微孢子蟲　； 參考：《吉林農(nóng)業(yè)大學》2016年碩士論文

【摘要】：弓形蟲(Toxoplasma gondii)是一種重要的專性細胞內(nèi)原蟲,可以感染包括狐貍在內(nèi)的所有恒溫動物。據(jù)估計全世界有三分之一的人呈慢性感染弓形蟲,其中部分人表現(xiàn)出了癥狀。人類通過食用未煮熟的含有卵囊的組織肉類和飲用被終末宿主污染的含有卵囊的水而感染。弓形蟲感染能導致先天性感染弓形蟲病的胎兒嚴重的疾病,并且對免疫缺陷的病人有致命的傷害,例如艾滋病病人和器官移植癌癥患者。隱孢子蟲(Cryptosporidium)作為一種重要的人與動物的腸道寄生蟲,在世界范圍內(nèi)呈多樣性分布,截至目前至少發(fā)現(xiàn)有26種蟲種以及73種有宿主特異性的未確定基因型。犬隱孢子蟲是寄生于犬體內(nèi)的一種常見寄生蟲,并且已經(jīng)被證實發(fā)現(xiàn)于狐貍體內(nèi)。從全世界各國感染的病人的癥狀來看,犬隱孢子蟲已經(jīng)確定為一種潛在的人獸共患的隱孢子蟲蟲種。微孢子蟲病是一種在動物和人群中常見的疾病。微孢子蟲(Enterocytozoon bieneusi)是人群中常見的孢子蟲屬的蟲種,許多動物物種都可以成為微孢子蟲感染的潛在感染對象。然而,關(guān)于養(yǎng)殖狐貍的微孢子蟲感染率和基因型信息幾乎還沒有報道。因此,本研究使用基因檢測方法對我國東部地區(qū)養(yǎng)殖狐貍的微孢子蟲陽性率,風險因素以及基因型進行了調(diào)查。本研究第一部分對我國東部地區(qū)的養(yǎng)殖狐貍進行了弓形蟲血清學調(diào)查,使用改良凝集試驗法對1346份養(yǎng)殖狐貍樣品進行了檢測,其中滴度在1:25判定為陽性,陽性樣品數(shù)為113個(8.39%),其中滴度在1:25的為66個,1:50的為26個,1:100的為9個,1:200的為7個,1:400的有3個,1:800的有2個。雄狐的弓形蟲陽性率為8.68%,高于母狐的7.95%,在白狐中的陽性率為7.07%,低于藍狐的感染率(9.75%)。在黑龍江,吉林,遼寧,山東的弓形蟲感染率分別為:85%,9.21%,7.37%和8.68%。經(jīng)過二元邏輯回歸分析后,亞種、性別、地區(qū)因素與血清陽性率之間均沒有顯著的差異性。這項調(diào)查結(jié)果表明,養(yǎng)殖狐貍的弓形蟲感染情況是比較嚴重的,這是我國關(guān)于北極狐的弓形蟲血清學研究方面的首篇報道。本研究第二部分檢測了北極狐的微孢子蟲的感染率、風險因素以及基因型。在302份養(yǎng)殖狐貍糞便樣品中,經(jīng)過巢式PCR的檢測之后37份為微孢子蟲陽性(12.25%,95%CI 8.55-15.95),這個結(jié)果只與北極狐的養(yǎng)殖模式有顯著相關(guān)性:處于戶外養(yǎng)殖模式的北極狐微孢子蟲陽性率(26.03%positive,95%CI=18.91-33.15)明顯高于室內(nèi)的(5.77%,95%CI=2.11-9.43),共鑒定出11種ITS基因型,其中4種為常見的基因型:Peru8,Types IV,CHN-DC1 and D;7種新的基因型:NCF1-NCF7。NCF2基因型是陽性個數(shù)中占比最多的,存在于三個省份的五個養(yǎng)殖場中。所有的基因型屬于系統(tǒng)發(fā)育第一種群。本研究第三部分采用巢式PCR方法檢測北極狐的隱孢子蟲陽性率為15.9%(48/302),其中雄性感染率為12.9%(18/139),雌性感染率為18.4(30/163),來自于各個不同養(yǎng)殖場的北極狐隱孢子蟲感染率為0到31.4%不等。不同的年齡段的北極狐隱孢子蟲的感染率也在14.1%到19.0%變化。此外,河北養(yǎng)殖場的北極狐隱孢子蟲感染率(7.8%,11/141)明顯的低于黑龍江省(22.9%,16/70)的和吉林省(23.1%,21/91)(P=0.0015)的感染率。SSU r RNA序列分析結(jié)果表明所有的分離株均為犬隱孢子蟲。本研究還首次對我國養(yǎng)殖狐貍的弓形蟲基因型進行了鑒定。264腦組織樣品分別采集于吉林、黑龍江以及山東。樣品經(jīng)過組織DNA提取后采用半巢式PCR方法檢測弓形蟲B1基因,然后再以11個遺傳標記為基礎(chǔ)(SAG1,alternative SAG2,5’-and3’-SAG2,SAG3,L358,BTUB,c22-8,GRA6,c29-2,PK1 and Apico),采用PCR-RFLP方法對陽性樣品做進一步的基因分型,264份組織樣品中有21份組織DNA的弓形蟲B1基因檢測呈陽性(7.96%)。一共鑒定兩種基因型(Toxo DB#9和Toxo DB#10)。本研究使用MAT方法檢測了我國東部地區(qū)北極狐弓形蟲感染的血清陽性率。使用巢式PCR方法對微孢子蟲和隱孢子蟲進行了流行病學調(diào)查和基因型研究。本研究使用PCR-RFLP從基因?qū)用鎸蜗x分離株進行了基因型鑒定,發(fā)現(xiàn)了一種主要流行弓形蟲基因型Toxo DB#9型和另一種基因型Toxo DB#10型。這些研究結(jié)果為我國控制和預(yù)防北極狐的弓形蟲病、微孢子蟲病和隱孢子蟲病提供了基本數(shù)據(jù),填補了相關(guān)學科空白。
[Abstract]:Toxoplasma gondii is an important specific intracellular protozoa that can infect all the thermostat animals, including foxes. It is estimated that 1/3 of the world are infected with chronic infection of Toxoplasma gondii, some of which show symptoms. Human beings eat uncooked tissue meat containing oocysts and drink the end host. Infected with the water of the oocyst. Toxoplasma infection can cause serious diseases of the fetus with congenital infection of toxoplasmosis, and can be fatal to immunodeficient patients, such as AIDS patients and organ transplant cancer patients. Cryptosporidium is an important intestinal parasite of human and animal, in the world. At least 26 species and 73 host specific unidentified genotypes have been found at least. Cryptosporidium canis is a common parasite parasitic in dogs, and has been found in foxes. From the symptoms of infected patients all over the world, Cryptosporidium canis has been confirmed. A latent zoonozoic species of Cryptosporidium. Microsporia is a common disease in animals and populations. Microspore (Enterocytozoon bieneusi) is a common species of Sporozoa in the population. Many animal species can become a potential target for infection of microspore. However, foxes are raised. The infection rate and genotype information of microspore have hardly been reported. Therefore, this study used gene detection methods to investigate the positive rate, risk factors and genotypes of Microspore in cultured foxes in eastern China. The first part of this study conducted a serological survey on the cultured foxes in eastern China. The modified agglutination test was used to detect 1346 cultured foxes, of which the titer was judged positive at 1:25, and the number of positive samples was 113 (8.39%), of which the titer was 66 at 1:25, 26 for 1:100, 7 for 1:200, 3 for 1:400 and 2 for 1:800. The positive rate of Toxoplasma gondii in male fox was 8.68%, higher than 7.95% of the female fox. The positive rate in white fox was 7.07%, lower than the infection rate of blue fox (9.75%). The infection rates of Toxoplasma gondii in Heilongjiang, Jilin, Liaoning and Shandong were respectively 85%, 9.21%, 7.37% and 8.68%. after two yuan logical regression analysis. There were no significant differences between subspecies, sex, regional factors and serum positive rates. The infection of Toxoplasma gondii is very serious in fox. This is the first report on the serological study of the Toxoplasma gondii in the Arctic fox. The second part of this study examined the infection rate, risk factors and genotypes of the microspore worm in the Arctic fox. In 302 foxes feces samples, 37 were detected by nested PCR. Sporozoite positive (12.25%, 95%CI 8.55-15.95), this result is only related to the Arctic Fox culture model: the positive rate of the Arctic Fox microspore (26.03%positive, 95%CI=18.91-33.15) in the outdoor culture model is significantly higher than that in the indoor (5.77%, 95%CI= 2.11-9.43), and 11 kinds of ITS genotypes are identified, of which 4 are common genotypes. Peru8, Types IV, CHN-DC1 and D, 7 new genotypes: the NCF1-NCF7.NCF2 genotype is the most positive in the positive number, and exists in five farms in three provinces. All genotypes belong to the first phylogeny population. The third part of this study uses the nested PCR method to detect the positive rate of Cryptosporidium in arctic fox 15.9% (48/302). The male infection rate was 12.9% (18/139) and the female infection rate was 18.4 (30/163). The infection rate of Cryptosporidium from arctic fox was 0 to 31.4% from different farms. The infection rate of Cryptosporidium of arctic fox was 14.1% to 19% in different age groups. In addition, the infection rate of Cryptosporidium of arctic fox in Hebei farm was 7.8%, 11/141). Significantly lower than Heilongjiang (22.9%, 16/70) and Jilin province (23.1%, 21/91) (P=0.0015) infection rate.SSU R RNA sequence analysis results showed that all the isolates were canine Cryptosporidium. This study also identified the Toxoplasma gondii genotypes of cultured foxes in China for the first time,.264 brain tissue samples were collected in Jilin, Heilongjiang and mountains, respectively. The B1 gene of Toxoplasma gondii was detected by the semi nested PCR method after the tissue DNA extraction. Then 11 genetic markers were used as the basis (SAG1, alternative SAG2,5 '-and3' -SAG2, SAG3, L358, BTUB, c22-8). The positive samples were further genotyping, and 21 of the 264 tissue samples were used. The B1 gene of Toxoplasma gondii in DNA was positive (7.96%). A total of two genotypes (Toxo DB#9 and Toxo DB#10) were identified. The serum positive rate of Toxoplasma gondii infection in the eastern region of China was detected by MAT method. The epidemiological investigation and genotyping of microspore and cryptospora were investigated by nested PCR method. This study used PCR-RFLP to identify the genotypes of Toxoplasma gondii isolates from the gene layer, and found a major genotype Toxo DB#9 and another genotype Toxo DB#10. These results provide basic data for the control and prevention of toxoplasmosis, sporozoon and cryptosporidiosis in the Arctic fox in China. It fills the gap in the related disciplines.

【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.92

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