火雞組織滴蟲體外分離培養(yǎng)體系和動物感染模型的建立及中藥體外抑制研究
發(fā)布時間：2019-05-27 11:37
【摘要】：禽組織滴蟲病(Histomoniasis)又稱為傳染性盲腸肝炎或“黑頭病”,是由火雞組織滴蟲(Histomonas meleagridis)引起的雞形目禽類的一種原生寄生蟲病,以肝臟壞死、盲腸腫大和排硫磺樣糞便為主要特征,在火雞中尤為嚴(yán)重,可引起嚴(yán)重的經(jīng)濟(jì)損失。目前,由于能夠有效治療和預(yù)防該病的化學(xué)藥物具有潛在的致癌性而大多被禁止使用,導(dǎo)致該病近年發(fā)生大規(guī)模傳播和流行,給養(yǎng)禽業(yè)造成了嚴(yán)重的危害。近年來,隨著國內(nèi)規(guī)�；鷳B(tài)圈養(yǎng)和放養(yǎng)等健康養(yǎng)殖技術(shù)的示范和推廣,組織滴蟲病在我國的流行和發(fā)生也越來越嚴(yán)重,而國內(nèi)對相關(guān)研究并不多且不系統(tǒng),因此,開展對火雞組織滴蟲及組織滴蟲病的研究,研究該病的發(fā)病機(jī)制、藥物防治及疫苗對于養(yǎng)禽業(yè)的健康發(fā)展具有重要意義。本研究首先對江蘇地區(qū)的火雞組織滴蟲進(jìn)行了分離,成功建立了火雞組織滴蟲體外分離培養(yǎng)的體系,并對該體系進(jìn)行了優(yōu)化；其次對分離的火雞組織滴蟲蟲體進(jìn)行單克隆培養(yǎng)并對純化的蟲株進(jìn)行了凍存和復(fù)蘇；用單克隆培養(yǎng)的蟲體成功建立了動物感染模型；在體外研究了中藥產(chǎn)品對火雞組織滴蟲的抑制作用。1.火雞組織滴蟲體外培養(yǎng)方法的建立為了研究火雞組織滴蟲的生物學(xué)特性,建立一種簡單有效的火雞組織滴蟲體外培養(yǎng)方法非常重要。火雞組織滴蟲感染的主要器官是盲腸和肝臟,是一種非常脆弱的微生物,在臟器中和環(huán)境溫度中裸露的蟲體一旦隨著糞便排出體外,只能夠存活很短的時間,所以火雞組織滴蟲的體外分離非常困難。本研究采用M199培養(yǎng)基可作為火雞組織滴蟲體外培養(yǎng)的營養(yǎng)培養(yǎng)基,再加入胎牛血清或者滅活馬血清以及米粉等組成基礎(chǔ)培養(yǎng)基,分別從盲腸和肝臟中分離蟲體。將臨床上剪碎的肝臟組織或盲腸內(nèi)容物接種培養(yǎng)48 h后,首先對培養(yǎng)產(chǎn)物進(jìn)行形態(tài)學(xué)判定,再經(jīng)PCR鑒定。研究結(jié)果表明,用盲腸內(nèi)容物分離培養(yǎng)時得到3種盲腸寄生原蟲：火雞組織滴蟲,芽囊原蟲和雞四毛滴蟲,三者混合感染,難以獲得單一種類蟲體；從肝臟中分離時能獲得單一種類的火雞組織滴蟲蟲體,火雞組織滴蟲的體外分離培養(yǎng)宜從帶有病變的新鮮肝臟組織中進(jìn)行。2.火雞組織滴蟲體外培養(yǎng)體系的優(yōu)化火雞組織滴蟲培養(yǎng)基包含M199培養(yǎng)基、米粉、滅活馬血清及盲腸細(xì)菌,為建立最優(yōu)的培養(yǎng)體系,本研究對培養(yǎng)條件及培養(yǎng)液的組成進(jìn)行了系列優(yōu)化。在40℃恒溫培養(yǎng)箱中厭氧培養(yǎng)2-3天后火雞組織滴蟲的群體數(shù)量將會達(dá)到5×105個/ml,隨后便會開始快速減少。實驗過程中改變每9 ml基礎(chǔ)培養(yǎng)基中米粉的量0 mg、10 mg、20 mgN、40 mg、60 mg、80 mg 100 mg;血清的種類：胎牛血清與滅活馬血清；血清比例0、5%、10%、15%、20%、30%、40%；以及有氧與無氧培養(yǎng)條件的比較。再利用新鮮的病變肝臟組織進(jìn)行火雞組織滴蟲分離培養(yǎng),比較有盲腸細(xì)菌和無盲腸細(xì)菌的差別,最終獲得了火雞組織滴蟲的最佳體外培養(yǎng)體系�；痣u組織滴蟲在9 ml含90%M199培養(yǎng)基,10%滅活馬血清,10 mg米粉及含盲腸細(xì)菌的基礎(chǔ)培養(yǎng)基中生長良好,可培養(yǎng)獲得穩(wěn)定和高產(chǎn)量的火雞組織滴蟲蟲體。3.火雞組織滴蟲的體外單克隆及保存復(fù)蘇采用體外分離培養(yǎng)技術(shù)對江蘇地區(qū)采集的兩例典型病變的新鮮肝臟組織中火雞組織滴蟲進(jìn)行了體外分離培養(yǎng),獲得了兩個不同地區(qū)火雞組織滴蟲的蟲體。進(jìn)一步參照球蟲單卵囊分離技術(shù)中的懸液法對火雞組織滴蟲進(jìn)行了單克隆純化,獲得兩株火雞組織滴蟲,分別命名為JSYZ-A和JSYZ-B。然后將擴(kuò)大培養(yǎng)的單克隆蟲株放入液氮中長期凍存,并且對凍存的蟲株成功進(jìn)行了復(fù)蘇。研究結(jié)果表明,建立的火雞組織滴蟲體外單克隆培養(yǎng)、凍存及復(fù)蘇的方法切實可行,為開展蟲體的病原學(xué)、致病機(jī)理以及燕窩和疫苗的研制奠定了很好的基礎(chǔ)。4.火雞組織滴蟲體外動物感染模型的建立采用單克隆培養(yǎng)的火雞組織滴蟲JSYZ-A感染了SPF黃羽雞。研究了不同感染劑量、不同感染途徑對火雞組織滴蟲體外感染動物模型的影響。實驗中用JSYZ-A蟲株以104、105和106個蟲體經(jīng)口感染3組15日齡的黃羽雞；再經(jīng)泄殖腔以104、105和106個蟲體感染3組15日齡的黃羽雞；并設(shè)對照組,每組10只雞。感染15天后實驗結(jié)束,對雞的死亡率、平均增重、盲腸和肝臟的損傷等進(jìn)行統(tǒng)計分析,評價人工感染的效果。研究結(jié)果表明,黃羽雞經(jīng)泄殖腔感染105個蟲體時就可以成功建立火雞組織滴蟲的體外感染動物模型。5.不同中草藥對火雞組織滴蟲體外抑制效果的研究出于食品安全方面的考慮,能夠有效預(yù)防和治療禽組織滴蟲病的化學(xué)藥物都被禁止使用,這使得禽組織滴蟲病在禽養(yǎng)殖業(yè)中大量爆發(fā),帶來了重大經(jīng)濟(jì)損失,迫切需要尋找一種能夠替代化學(xué)藥物且能有效控制禽組織滴蟲病的藥物。本實驗比較了白頭翁、金蓮花、雞冠花、荊芥、常山、香薷、花椒、駱駝蓬、苦參、千里光、大蒜素、青蒿素、蛇床子素13種中草藥及提取物在體外對火雞組織滴蟲的抑制作用。將藥物加入到預(yù)準(zhǔn)備M199基礎(chǔ)培養(yǎng)基中后,然后再接種火雞組織滴蟲進(jìn)行培養(yǎng),在不同培養(yǎng)時間觀察計數(shù)蟲體的生長繁殖狀況,得到藥物對體外培養(yǎng)的火雞組織滴蟲的抑制效果,所選白頭翁、金蓮花、雞冠花等10種中草藥在體外對火雞組織滴蟲有較好的抑制作用,其中以金蓮花、常山和駱駝蓬的抑制作用最好；大蒜素、青蒿素等3種中草藥提取物在體外對火雞組織滴蟲的抑制作用優(yōu)于中草藥,以青蒿素的作用效果最為明顯。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.9
本文編號：2486115
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.9
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