新发现家禽病原体Chlamydia gallinacea传播方式的研究

发布时间：2019-06-01 16:41

【摘要】：衣原体(Chlamydia spp.)是一种严格胞内寄生的原核细胞微生物,革兰氏染色阴性,镜下外形呈现球状;其能够在鸡胚和易感的细胞系中良好地生长、繁殖,生活史分为原体和始体两个时期。衣原体不仅在自然界广泛存在,在美洲、欧洲、亚洲、非洲和大洋洲均有感染的报道。它的宿主也十分多样,可感染多种禽类和哺乳动物。衣原体门包括1个衣原体纲(Chlamydiia),1个衣原体目(Chlamydiales),衣原体目下设8个科,其中衣原体科(Chlamydiaceae)是与引起人和动物患病的相关衣原体种,目前总共有12个衣原体种:沙眼衣原体(C.trachomatis)、肺炎衣原体(C.pneumonia)、流产衣原体(C.abortus)、鹦鹉热衣原体(C.psittaci)、猪衣原体(C.suis)、家畜衣原体(C.pecorum).猫衣原体(C.felis)、鼠衣原体(C.muridarum)、豚鼠衣原体(C.caviae)、朱鹭衣原体(C.ibidis)、鸟衣原体(C.avium)和家禽衣原体(C.gallinacea)。鹦鹉热衣原体一直被认为是引起禽衣原体病的唯一病原,直到近几年C.galliacea和鸟衣原体在欧洲、中国等地区被发现。根据当前的调查发现,鸟衣原体在鸽子中检出较多,而C.gallinacea在鸡、火鸡中广泛流行。目前C.gallinace 在家禽中的流行率甚至超过了鹦鹉热衣原体,关于C.galliacea的报道主要是不同地区的流行状况、宿主专嗜性和基因组学研究,但其传播方式仍然未知。为了对C.gallinace 在鸡群中的水平传播方式进行探究,本研究利用禽用隔离器建立了水平传播实验模型,使用通气管连接排气口和进气口,让SPF鸡通过粪口途径和空气吸入途径分别接触经阳性鸡群排泄物污染的食物、饮水和空气。Ⅰ号隔离器中的SPF鸡可以直接接触人工接种的带毒鸡群及其粪便,Ⅱ号隔离器为对照组,Ⅲ号隔离器中SPF鸡吸入的气体全部来自于人工接种的带毒鸡群所在的隔离器。实验结果表明,第15天时粪口途径感染组中所有的SPF鸡都感染了 C.gallinacea,而空气传播组在第5、10、15、20、25天所采集的喉拭子与泄殖腔拭子样品检测C.galliacea均为阴性,对照组中两种拭子样品检测也均为阴性,因此证明C.galliacea能够通过粪口途径传播,但不能够通过空气传播。随后,分别从人工感染组、直接接触组和空气传播组中随机选取5只鸡检测脏器中C.gallinacea分布。结果显示空气传播组中所有脏器均为阴性,而人工感染组和直接接触组中脏器C.gallinacea阳性个数如下:心脏(2/10)、肺脏(2/10)、脾脏(3/10)、肾脏(1/10)、腺胃(3/10)、十二指肠(7/10)、直肠(10/10)、泄殖腔(10/10)、卵巢(1/10),其中十二指肠、直肠、泄殖腔C.gallinacea的阳性率和平均基因拷贝数均高于其他脏器。为了对C.gallinacea在鸡群中的垂直传播方式进行研究,本研究采集了江苏省邵伯镇某种鸡场100只种母鸡泄殖腔拭子样品、28只种公鸡泄殖腔拭子及精液样品;采集其所产种蛋的蛋壳拭子样品294份,其中128枚种蛋(18枚为孵化9日后经照蛋发现未受精蛋)收集蛋清、蛋黄样品;55枚种蛋经孵化得到19日龄的鸡胚采集心、肝、脾、肺、肾、肠等脏器样品,应用基于衣原体23S rRNA基因的FRET-qPCR方法进行C.gallinacea的检测。研究表明,在该种鸡场种母鸡泄殖腔拭子样品的阳性率为75%(75/100),而公鸡精液中样品的阳性率为0%(0/28),公鸡泄殖腔拭子样品阳性率为0%(0/28)。294枚种蛋样品中,蛋壳拭子的阳性率为97.62%(287/294),蛋清阳性率为7%(9/128),蛋黄阳性率为5.4%(7/128),并且所有蛋黄样品检测为阳性的种蛋,其蛋清也是阳性。所检鸡胚脏器中,心脏的阳性率为9%(5/55),肝脏为5.5%(3/55),脾脏为12.7%(7/55),肺脏为11%(6/55),肾脏为 14.5%(8/55),肠为 7.3%(4/55)。本研究是首次对C.gallinacea传播方式进行的探索,通过建立水平传播实验模型,验证了粪口途径为Cgalinacea在鸡群中水平传播的主要方式。同时,通过检测种鸡场中种鸡喉拭子、泄殖腔拭子、种蛋、鸡胚脏器等样品,提示C.gallinacea可能通过蛋壳渗透等方式在亲代和子代之间发生垂直传播。
[Abstract]:Chlamydia spp. ) It is a strictly intracellularly parasitic prokaryotic cell microorganism which is negative in gram staining and has a spherical shape under the microscopic shape, and can be well grown and propagated in the chicken embryo and the easy-to-sense cell line, and the life history is divided into a plasma and a starting body for two periods. Chlamydia is not only widely present in nature, but also in the Americas, Europe, Asia, Africa and Oceania. Its host is also very diverse and can infect a variety of birds and mammals. Chlamydia trachomatis (Chlamydia trachomatis),1 Chlamydia,1 Chlamydia, and 8 families in the order of chlamydia, of which the family of Chlamydia is the type of chlamydia associated with the cause of the disease of humans and animals, and there are currently 12 Chlamydia species: Chlamydia trachomatis, and C. pneumonia. Chlamydia trachomatis (C. abnorus), Chlamydia psittaci, C. suis, and C. pecium. Chlamydia trachomatis (C. felis), C. muridum, C. caviae, C. bialis, C. avium and C. golinacea. Chlamydia psittaci has been thought to be the only pathogen that causes the disease of the birds, and it has been found in the regions of Europe, China, and the like in the last few years. According to the current survey, the number of chlamydia is detected in pigeons, and C. golinacea is widely used in chickens and turkeys. At present, the prevalence of C. gallinace in poultry is even higher than that of Chlamydia psittaci, and the report on C. galactia is mainly the prevailing situation in different regions, the specificity of the host and the study of genomics, but the mode of transmission is still unknown. In order to explore the way of the horizontal propagation of C. gallace in the chicken group, the present study uses the isolator to establish the horizontal propagation experimental model, and uses the vent pipe to connect the exhaust port and the air inlet. And the SPF chickens respectively contact the food, the drinking water and the air which are polluted by the excrement of the positive chicken group through the way of the manure mouth and the air suction way. The SPF chickens in the No.1 isolator can be directly contacted with the artificially-inoculated chicken flocks and their feces, and the No. II isolator is the control group, and the gas sucked from the SPF chickens in the III-type isolator is all from the isolator with the artificial inoculation of the infected chicken group. The results of the experiment show that all SPF chickens in the faecal route infection group were infected with C. golinacea in the 15th day, while the throat swabs collected in the 5th, 10th, 15th, 20th and 25th days of the air-spreading group were negative, and both of the two swabs in the control group were negative. It has thus been shown that C. galliacea can be spread through the faecal pathway, but can not be propagated through air. Then,5 chickens were randomly selected from the artificial infection group, the direct contact group and the air propagation group to detect the distribution of C. golinacea in the organs. The results showed that all the organs in the air communication group were negative, and the number of the C. golinacea positive number in the artificial infection group and the direct contact group were as follows: heart (2/10), lung (2/10), spleen (3/10), kidney (1/10), glandular stomach (3/10), duodenum (7/10) and rectum (10/10). The positive rate and the average gene copy number of the cloaca (10/10) and the ovary (1/10) were higher than those of other organs. In order to study the vertical propagation of C. gallinacea in the chicken group, a sample of 100 hens from a certain chicken farm in the Shab town of Jiangsu province was collected,28 species of cock cloaca and semen were collected, and 294 samples of the egg shell swab samples from which the eggs were produced were collected. The method comprises the following steps: (1) collecting the egg white and the egg yolk sample by 128 seed eggs (18 of which are after hatching for 9 days), collecting the egg white and the egg yolk sample, and hatching to obtain the 19-day-old chicken embryo collecting heart, the liver, the spleen, the lung, the kidney, the intestine and the like, The FRET-qPCR method based on the 23S rRNA gene of Chlamydia trachomatis was used for the detection of C. golinacea. The positive rate of the sample was 75% (75/100), the positive rate of the sample in the rooster was 0% (0/28), and the positive rate of the sample in the cock was 0% (0/28). The positive rate of the egg shell swabs was 97.62% (287/294), the positive rate of egg white was 7% (9/128), and the positive rate of egg yolk was 5.4% (7/128). And all the egg yolk samples are detected as positive seed eggs, and the egg white is also positive. The positive rate of the heart was 9% (5/55), the liver was 5.5% (3/55), the spleen was 12.7% (7/55), the lung was 11% (6/55), the kidney was 14.5% (8/55) and the intestine was 7.3% (4/55). This study was the first to explore the mode of C. golinacea, and through the establishment of the experimental model of horizontal propagation, the main way of the level of Cgallinea in the chicken population was verified. At the same time, by detecting the samples of the chicken's throat swab, the cloaca swab, the seed egg, the chicken embryo and the like in the chicken farm, it is suggested that C. gallinacea can spread vertically between the parent and the offspring through the way of egg shell penetration.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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