火鸡组织滴虫体外分离培养体系和动物感染模型的建立及中药体外抑制研究

发布时间：2019-05-27 11:37

【摘要】：禽组织滴虫病(Histomoniasis)又称为传染性盲肠肝炎或“黑头病”,是由火鸡组织滴虫(Histomonas meleagridis)引起的鸡形目禽类的一种原生寄生虫病,以肝脏坏死、盲肠肿大和排硫磺样粪便为主要特征,在火鸡中尤为严重,可引起严重的经济损失。目前,由于能够有效治疗和预防该病的化学药物具有潜在的致癌性而大多被禁止使用,导致该病近年发生大规模传播和流行,给养禽业造成了严重的危害。近年来,随着国内规模化生态圈养和放养等健康养殖技术的示范和推广,组织滴虫病在我国的流行和发生也越来越严重,而国内对相关研究并不多且不系统,因此,开展对火鸡组织滴虫及组织滴虫病的研究,研究该病的发病机制、药物防治及疫苗对于养禽业的健康发展具有重要意义。本研究首先对江苏地区的火鸡组织滴虫进行了分离,成功建立了火鸡组织滴虫体外分离培养的体系,并对该体系进行了优化；其次对分离的火鸡组织滴虫虫体进行单克隆培养并对纯化的虫株进行了冻存和复苏；用单克隆培养的虫体成功建立了动物感染模型；在体外研究了中药产品对火鸡组织滴虫的抑制作用。1.火鸡组织滴虫体外培养方法的建立为了研究火鸡组织滴虫的生物学特性,建立一种简单有效的火鸡组织滴虫体外培养方法非常重要。火鸡组织滴虫感染的主要器官是盲肠和肝脏,是一种非常脆弱的微生物,在脏器中和环境温度中裸露的虫体一旦随着粪便排出体外,只能够存活很短的时间,所以火鸡组织滴虫的体外分离非常困难。本研究采用M199培养基可作为火鸡组织滴虫体外培养的营养培养基,再加入胎牛血清或者灭活马血清以及米粉等组成基础培养基,分别从盲肠和肝脏中分离虫体。将临床上剪碎的肝脏组织或盲肠内容物接种培养48 h后,首先对培养产物进行形态学判定,再经PCR鉴定。研究结果表明,用盲肠内容物分离培养时得到3种盲肠寄生原虫：火鸡组织滴虫,芽囊原虫和鸡四毛滴虫,三者混合感染,难以获得单一种类虫体；从肝脏中分离时能获得单一种类的火鸡组织滴虫虫体,火鸡组织滴虫的体外分离培养宜从带有病变的新鲜肝脏组织中进行。2.火鸡组织滴虫体外培养体系的优化火鸡组织滴虫培养基包含M199培养基、米粉、灭活马血清及盲肠细菌,为建立最优的培养体系,本研究对培养条件及培养液的组成进行了系列优化。在40℃恒温培养箱中厌氧培养2-3天后火鸡组织滴虫的群体数量将会达到5×105个/ml,随后便会开始快速减少。实验过程中改变每9 ml基础培养基中米粉的量0 mg、10 mg、20 mgN、40 mg、60 mg、80 mg 100 mg;血清的种类：胎牛血清与灭活马血清；血清比例0、5%、10%、15%、20%、30%、40%；以及有氧与无氧培养条件的比较。再利用新鲜的病变肝脏组织进行火鸡组织滴虫分离培养,比较有盲肠细菌和无盲肠细菌的差别,最终获得了火鸡组织滴虫的最佳体外培养体系。火鸡组织滴虫在9 ml含90%M199培养基,10%灭活马血清,10 mg米粉及含盲肠细菌的基础培养基中生长良好,可培养获得稳定和高产量的火鸡组织滴虫虫体。3.火鸡组织滴虫的体外单克隆及保存复苏采用体外分离培养技术对江苏地区采集的两例典型病变的新鲜肝脏组织中火鸡组织滴虫进行了体外分离培养,获得了两个不同地区火鸡组织滴虫的虫体。进一步参照球虫单卵囊分离技术中的悬液法对火鸡组织滴虫进行了单克隆纯化,获得两株火鸡组织滴虫,分别命名为JSYZ-A和JSYZ-B。然后将扩大培养的单克隆虫株放入液氮中长期冻存,并且对冻存的虫株成功进行了复苏。研究结果表明,建立的火鸡组织滴虫体外单克隆培养、冻存及复苏的方法切实可行,为开展虫体的病原学、致病机理以及燕窝和疫苗的研制奠定了很好的基础。4.火鸡组织滴虫体外动物感染模型的建立采用单克隆培养的火鸡组织滴虫JSYZ-A感染了SPF黄羽鸡。研究了不同感染剂量、不同感染途径对火鸡组织滴虫体外感染动物模型的影响。实验中用JSYZ-A虫株以104、105和106个虫体经口感染3组15日龄的黄羽鸡；再经泄殖腔以104、105和106个虫体感染3组15日龄的黄羽鸡；并设对照组,每组10只鸡。感染15天后实验结束,对鸡的死亡率、平均增重、盲肠和肝脏的损伤等进行统计分析,评价人工感染的效果。研究结果表明,黄羽鸡经泄殖腔感染105个虫体时就可以成功建立火鸡组织滴虫的体外感染动物模型。5.不同中草药对火鸡组织滴虫体外抑制效果的研究出于食品安全方面的考虑,能够有效预防和治疗禽组织滴虫病的化学药物都被禁止使用,这使得禽组织滴虫病在禽养殖业中大量爆发,带来了重大经济损失,迫切需要寻找一种能够替代化学药物且能有效控制禽组织滴虫病的药物。本实验比较了白头翁、金莲花、鸡冠花、荆芥、常山、香薷、花椒、骆驼蓬、苦参、千里光、大蒜素、青蒿素、蛇床子素13种中草药及提取物在体外对火鸡组织滴虫的抑制作用。将药物加入到预准备M199基础培养基中后,然后再接种火鸡组织滴虫进行培养,在不同培养时间观察计数虫体的生长繁殖状况,得到药物对体外培养的火鸡组织滴虫的抑制效果,所选白头翁、金莲花、鸡冠花等10种中草药在体外对火鸡组织滴虫有较好的抑制作用,其中以金莲花、常山和骆驼蓬的抑制作用最好；大蒜素、青蒿素等3种中草药提取物在体外对火鸡组织滴虫的抑制作用优于中草药,以青蒿素的作用效果最为明显。
[Abstract]:The poultry tissue trichomoniasis, also known as infectious cecum hepatitis or "black head disease", is a primary parasitic disease of the chicken-shaped poultry caused by the tissue of the turkeys, which is characterized by liver necrosis, cecum enlargement and sulfur-like feces, especially in the turkeys, And can cause serious economic loss. At present, because of the potential carcinogenicity of the chemical drugs which can effectively treat and prevent the disease, most of them are prohibited from being used, leading to the large-scale transmission and epidemic of the disease in recent years, causing serious harm to the poultry industry. In recent years, with the demonstration and promotion of the domestic large-scale ecological breeding and breeding technology, the prevalence and the occurrence of the trichomoniasis in China are becoming more and more serious, and the domestic study of the related research is not much and the system is not systematic, so, It is of great significance to study the mechanism of the disease and the prevention and control of the disease and the healthy development of the poultry industry. In this study, the turkeys of the turkeys in Jiangsu area were separated, and the system of the in vitro separation and culture of the tissue of the turkeys was successfully established and the system was optimized. Secondly, the isolated turkey tissue trichomonas is subjected to the monoclonal culture and the purified worm strain is frozen and recovered; the animal infection model is successfully established by using the monoclonal-cultured insect body; and the inhibition effect of the traditional Chinese medicine product on the trichomonas hominis is studied in vitro. In order to study the biological characteristics of the trichomonas hominis, a simple and effective method for the in vitro culture of the tissue of the turkeys is very important. The main organ of the turkey tissue trichomonas infection is the cecum and the liver, is a very fragile microorganism, and once the exposed insect body in the internal organs and the environment temperature is discharged in the body with the excrement, it can only survive for a short time, so the in vitro separation of the turkey tissue trichomonas is very difficult. In this study, M199 medium can be used as a nutrient medium for the in-vitro culture of the trichomonas hominis, and then the basal medium such as fetal bovine serum or inactivated horse serum and rice flour is added, and the insect body is separated from the cecum and the liver, respectively. After the liver tissue or cecum contents were inoculated and cultured for 48 h, the culture product was first determined by morphology and then identified by PCR. The results showed that the cecum contents were isolated and cultured to obtain three cecum parasitic protozoa: the turkeys, the trichomonas, the trichomonas and the trichomonas, which were mixed and infected, and it was difficult to obtain a single type of worm. The in vitro separation culture of the turkey tissue trichomonas is suitable to be carried out from the fresh liver tissue with the lesion. In order to establish the optimal culture system, the culture conditions and the composition of the culture medium were optimized. After 2-3 days of anaerobic culture in a 40 & deg; C constant-temperature incubator, the number of groups of turkeys in the turkeys will reach 5 to 105/ ml after 2-3 days of anaerobic culture, and then it will begin to decrease rapidly. In the course of the experiment, the amount of rice flour in every 9 ml basal medium was changed to 0 mg,10 mg,20 mg N,40 mg,60 mg,80 mg 100 mg; the type of serum: fetal bovine serum and inactivated horse serum; the serum ratio of 0,5%,10%,15%,20%,30%,40%; and the comparison of the aerobic and anaerobic culture conditions. Using fresh diseased liver tissue to separate and culture the turkeys, the difference of the cecum bacteria and the non-cecum bacteria was compared, and the optimal in vitro culture system of the turkey tissue trichomonas was finally obtained. A total of 9 ml of 90% M199 medium,10% inactivated horse serum,10 mg of rice flour and caecum-containing bacteria were grown in 9 ml. In vitro, the method of in-vitro isolation and culture was used to separate and culture the trichomonas hominis in the fresh liver tissues of two typical lesions, which were collected in Jiangsu area by in-vitro isolation and culture technique, and the insect bodies of the two different regions of turkeys were obtained. The invention further refers to the suspension method in the single-egg-cell separation technology of the coccidiosis to carry out the monoclonal purification on the turkey tissue trichomonas, and the two turkeys are respectively named JSYZ-A and JSYZ-B, and the two turkeys are respectively named as JSYZ-A and JSYZ-B. And then the expanded cultured monoclonal insect strain is put in liquid nitrogen for medium and long time to be frozen, and the frozen worm strain is successfully recovered. The results of the study show that the method of the establishment of the monoclonal culture, freezing and recovery of the trichomonas hominis in vitro has laid a good foundation for the research and development of the pathogen, the pathogenesis and the development of the bird's nest and vaccine. The model of animal infection in the human tissue of the turkey tissue is infected with the SPF yellow-feather chicken by using the monoclonal-cultured turkey tissue trichomonas JSYZ-A. The effect of different infection dose and different infection way on the animal model of the infection of the human tissue of the turkeys in vitro was studied. In the experiment, three groups of 15-day-old yellow-feather chickens were orally infected with JSYZ-A (104,105 and 106 insect bodies). After 15 days of infection, the mortality of the chicken, the average weight gain, the cecum and the liver injury were statistically analyzed, and the effect of the artificial infection was evaluated. The results of the study show that in the case of 105 insect bodies infected by the cloaca, an animal model of the in vitro infection of the tissue of the turkeys can be successfully established. The research of the effect of different Chinese herbal medicines on the in-vitro inhibition effect of the trichomoniasis of the turkeys is taken into account in the aspect of food safety, and the chemical drugs capable of effectively preventing and treating the chicken's trichomoniasis are prohibited from being used, There is an urgent need to find a drug that can be used in place of chemical drugs and which can effectively control the chicken's trichomoniasis. The experiment has compared the inhibitory effect of Chinese herbal medicines and extracts of Radix Pulsatilla, Flos Trollii, Flos Cartillae, Herba Schizonepetae, Herba Schizonepetae, Radix Sophorae Flavescentis, Herba Senecionis, allicin, arteannuin, and Fructus Cnidii with 13 Chinese medicinal materials and extracts in vitro. adding the medicine into the pre-prepared M199 basic culture medium, then inoculating the turkey tissue trichomonas to culture, observing the growth and propagation condition of the counting worm body at different culture time, 10 Chinese herbal medicines, such as chicken crown flowers, have a good inhibition effect on the trichomonas of the turkeys in vitro, wherein the inhibition effect of the three Chinese herbal extracts, such as allicin, artemisinin and the like, is better than that of the Chinese herbal medicines in vitro, And the effect of the arteannuin is most obvious.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.9

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