犬隐孢子虫的鉴定及隐孢子虫感染对小鼠免疫指标的影响
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摘要
隐孢子虫病是由隐孢子虫(Cryptosporidium)引起的一种以腹泻为主要临床表现的呈世界性分布的人畜共患寄生虫病,该病严重危害人类健康和畜牧业生产,也是人类艾滋病患者的主要致死因素之一。美国疾病预防控制中心将其列为B类病原。2003年该病被列为我国重点防范的两种重要寄生虫病之一。近年来,隐孢子虫病研究在病原生物学、流行病学、分子生物学、诊断及综合防治技术等方面取得许多重要成果,但是由于隐孢子虫虫种间形态差异不明显,加之近年来分子生物学技术的应用,隐孢子虫新基因型的报道不断出现,造成了隐孢子虫明确分类一直不很清楚,这给预防和控制该病带来了极大困难。目前隐孢子虫的致病机理还不十分清楚,隐孢子虫病的治疗仍是目前世界范围尚未攻克的难题。本课题旨在通过对合肥地区犬粪样中隐孢子虫的检测,获取隐孢子虫感染强阳性犬,从强阳性犬粪样中分离隐孢子虫卵囊,采用形态学和分子生物学方法对所获卵囊进行鉴定;然后用犬源隐孢子虫构建动物(小鼠)模型,并对小鼠感染隐孢子虫后免疫指标的变化进行研究,以期为阐明隐孢子虫致病机理和研究隐孢子虫病的防控等提供理论依据。
首先,采用饱和白糖溶液漂浮法对合肥地区232份犬粪样进行检测,根据所获阳性粪样中隐孢子虫卵囊的形态、结构、大小、卵形指数等特征,初步鉴定合肥地区犬隐孢子虫为微小隐孢子虫(Cryptosporidium parvum)。对不同年龄段犬隐孢子虫感染情况进行分析后发现:各个年龄段犬均可感染隐孢子虫,但不同年龄段犬隐孢子虫感染情况不同,幼犬感染率显著高于其他年龄阶段的犬。同时发现各年龄段犬隐孢子虫感染强度高低不等。
其次,采用蔗糖密度梯度离心法对分离的隐孢子虫卵囊进行纯化。取纯化的卵囊,先用漩涡振荡器震荡15min,加裂解液(2%DTT,1×TE),再将卵囊放入-70℃冰箱反复冻融三次,90℃水浴20min,最后提取模板DNA。根据隐孢子虫18SrRNA序列,设计并合成隐孢子虫特异性引物,扩增出540bp的片段。根据隐孢子虫HSP70基因序列,设计合成隐孢子虫特异性引物,扩增出448bp的片段。使用PCR产物回收试剂盒对扩增产物进行回收,纯化后送上海生物工程技术有限公司测序。然后根据GenBank相关序列,以DNAStar软件进行序列分析,构建种系进化树,从分子水平确定合肥地区犬源隐孢子虫为微小隐孢子虫(C.parvum)。
最后,构建动物模型,将15只BALB/C小鼠随机分成A、B、C、D和E5个组,分笼饲养,每组分别为3只。A组接种卵囊,用移液器吸取60μL卵囊液经口灌入每只小鼠体内,并以饮水的方式用地塞米松1.5mg/100ml,每天换水,自由饮用直至本次试验结束;B组接种卵囊,免疫抑制药物、方法、人工感染方法同于A组,添加免疫抑制药物剂量为1.0mg/100ml;C组为非免疫抑制组,同A、B组方法接种卵囊,饮水中不添加免疫抑制剂;D组为免疫抑制对照组,饮水中添加免疫抑制药物剂量为1.0mg/100ml;E组为非免疫抑制对照组。对小鼠粪便中的卵囊进行计数,并记录排出卵囊规律,排出卵囊强度等情况。对死亡的小鼠进行剖检,观察体内免疫器官变化情况。在饲养实验小鼠期间,每隔10天进行眼眶采血,分离血清,并测定血清总蛋白、白蛋白和球蛋白的含量,了解感染动物的机体免疫指标的变化。
综上所述,本课题得出的犬隐孢子虫鉴定结果为今后合肥地区犬隐孢子虫病的防治提供了理论依据;对小鼠感染隐孢子虫后机体免疫指标变化的研究,为今后隐孢子虫致病机理和治疗药物的筛选等研究提供了一些参考资料。
Cryptosporidium was one of the important prozotoan parasite,which infected people and animals together. It was found in experimental mice gland in 1907. Cryptosporidisis is a worldwild distribution anthropozoonosis with the main clinical signs of diarrhea.The disease has severely harm to human health and animal production and been recognized as one of the cause of mortality in association with the acquired immunodeficiency syndrome(AIDS).The control and prevention centre of USA has classified the disease as B etiology. This disease was also classified as one of the two kinds of important parasitic disease that our country took precautions against especially in 2003. In recent years,cryptosporidium was studied and made a lot of important achievements in many respects such as aetiology,morphology,epidemiology, pathogenic molecular biology,diagnosis and integrated control technology ;But as the morphologic features among differential genus often do not markedly,And in present lack of consistency in the clasiification of protozoan parasite in general in domestic and abroad.With the development of the technology of molecular biology,these reports constantly rush about the new genetic of Cryptosporidium,Which make the precise number of Cryptosporidium unknown.We confronted great difficult in preventive and control this disease.the host immune function appears to be less present report, therefore characteristics of the host immunity has been our concern.
The subject has taking an epidemic investigate of cryptosporidium to in part farm of hefei in Anhui province , survey the postive dogs of infected cryptosporidium , observing the morphologic oocyst of Cryptosporidium. Morphology and Technology of molecular biology were applied to identify the havested oocysts: Attempt to determine genotypes of all Cryptosporidium in dogs to expound mechanism of Cryptosporidium molecule epidemiology; Take the oocyst to construct an animal model in order to offer the theoretical base for elucidating the pathogenic mechanism and researching the prevention and regulation.
Firstly,the 232 fecal specimen in dogs was detected by satturated white sugar, we preliminaryly classified the Cryptosporidium harvested in hefei as Cryptosporidium parvum by the oocyst characteristics such as shape constitution、 size and the index number of size. The infection rates of the other three dairy farms is greatly different. Counted and analyied infection of dog in different age,each age dog could infect cryptosporidium, but different age dog with different situation. Puppies's infection rate was higher than the other age of dogs notably.Meanwhile, different age dogs are different in infected intensity.
sencondly,we applied the discontinous sucrose gradients to purified oocysts, which was shaked by shaker for 15 minutes, added plitting liquid(2%DTT,1×TE),freezed and melted for three times in -70℃, water-bath for 20 minutes in 90℃, extracted DNA from oocysts finally. We designed and synthetized the specific primer of cryptosporidium according to the crytposporidium 18SrRNA then amplifing the specific 540bp fragment. In the same time, we designed and synthetized the specific primer of cryptosporidium according to the crytposporidium HSP70 then amplifing the specific 448bp fragment The reagent box of PCR was used to purified PCR products and the products was sequenced by shanghai sangon company. According to the homology sequence in NCBI, we take the biological sofe such as DNAStar analyze the sequence and construct phylogentic trees, confiring that the Cryptosporidium is Cryptosporidium parvum in hefei for the molecular level.
Lastly ,we constructed animal models, 15 BALB / C mice were randomly divided into five groups , A, B, C, D and E meanwhile every group was feed in different cages. The number of mice in every groups is 3. The belly of mice in group A were inoculated 60μL Cryptosporidium with syringe by the channel of mouth ,those mice drinked water with dexamethasone and the does is 1.5mg/100ml , renoveatured water every day until the end of this study . The group B are same as A,excepting to add the drug of immunosuppression and the does is suppression, 1.0mg/100ml; The group C as an non-immunosuppressive group, the way of inoculation Cryptosporidium as A and B,meawile ,the way of drinking without the drug of immunosuppression. The group D as an immunosuppressive group add the drug of immunosuppression, and the does is 1.0mg/100ml; The group E is an non-immunosuppressive group.
We count the number of oocyste get from the mice feacal and record the law of discharge oocysts and others like the intensity of oocysts. When the mice died,we dissect dead body to observe the changes in endosomatic immune organ. During of feeding ,we took a little blood from eyes, separating serum form blood and knowing the total amount protein, albumin and globulin in serum, knowing changes of immunosuppressive agents in infected animals immune function.
Berifly ,the result we get froming the study provided with some significant reference for prevention and cure Cryptosporidium in hefei; and monitored Cryptosporidiosis. And provied some improtant reference for studying the changes in endosomatic immune organ when was infected and studying the pathogenic mechanism of Cryptosporidium and choosing drug for curing the disease.
首先,采用饱和白糖溶液漂浮法对合肥地区232份犬粪样进行检测,根据所获阳性粪样中隐孢子虫卵囊的形态、结构、大小、卵形指数等特征,初步鉴定合肥地区犬隐孢子虫为微小隐孢子虫(Cryptosporidium parvum)。对不同年龄段犬隐孢子虫感染情况进行分析后发现:各个年龄段犬均可感染隐孢子虫,但不同年龄段犬隐孢子虫感染情况不同,幼犬感染率显著高于其他年龄阶段的犬。同时发现各年龄段犬隐孢子虫感染强度高低不等。
其次,采用蔗糖密度梯度离心法对分离的隐孢子虫卵囊进行纯化。取纯化的卵囊,先用漩涡振荡器震荡15min,加裂解液(2%DTT,1×TE),再将卵囊放入-70℃冰箱反复冻融三次,90℃水浴20min,最后提取模板DNA。根据隐孢子虫18SrRNA序列,设计并合成隐孢子虫特异性引物,扩增出540bp的片段。根据隐孢子虫HSP70基因序列,设计合成隐孢子虫特异性引物,扩增出448bp的片段。使用PCR产物回收试剂盒对扩增产物进行回收,纯化后送上海生物工程技术有限公司测序。然后根据GenBank相关序列,以DNAStar软件进行序列分析,构建种系进化树,从分子水平确定合肥地区犬源隐孢子虫为微小隐孢子虫(C.parvum)。
最后,构建动物模型,将15只BALB/C小鼠随机分成A、B、C、D和E5个组,分笼饲养,每组分别为3只。A组接种卵囊,用移液器吸取60μL卵囊液经口灌入每只小鼠体内,并以饮水的方式用地塞米松1.5mg/100ml,每天换水,自由饮用直至本次试验结束;B组接种卵囊,免疫抑制药物、方法、人工感染方法同于A组,添加免疫抑制药物剂量为1.0mg/100ml;C组为非免疫抑制组,同A、B组方法接种卵囊,饮水中不添加免疫抑制剂;D组为免疫抑制对照组,饮水中添加免疫抑制药物剂量为1.0mg/100ml;E组为非免疫抑制对照组。对小鼠粪便中的卵囊进行计数,并记录排出卵囊规律,排出卵囊强度等情况。对死亡的小鼠进行剖检,观察体内免疫器官变化情况。在饲养实验小鼠期间,每隔10天进行眼眶采血,分离血清,并测定血清总蛋白、白蛋白和球蛋白的含量,了解感染动物的机体免疫指标的变化。
综上所述,本课题得出的犬隐孢子虫鉴定结果为今后合肥地区犬隐孢子虫病的防治提供了理论依据;对小鼠感染隐孢子虫后机体免疫指标变化的研究,为今后隐孢子虫致病机理和治疗药物的筛选等研究提供了一些参考资料。
Cryptosporidium was one of the important prozotoan parasite,which infected people and animals together. It was found in experimental mice gland in 1907. Cryptosporidisis is a worldwild distribution anthropozoonosis with the main clinical signs of diarrhea.The disease has severely harm to human health and animal production and been recognized as one of the cause of mortality in association with the acquired immunodeficiency syndrome(AIDS).The control and prevention centre of USA has classified the disease as B etiology. This disease was also classified as one of the two kinds of important parasitic disease that our country took precautions against especially in 2003. In recent years,cryptosporidium was studied and made a lot of important achievements in many respects such as aetiology,morphology,epidemiology, pathogenic molecular biology,diagnosis and integrated control technology ;But as the morphologic features among differential genus often do not markedly,And in present lack of consistency in the clasiification of protozoan parasite in general in domestic and abroad.With the development of the technology of molecular biology,these reports constantly rush about the new genetic of Cryptosporidium,Which make the precise number of Cryptosporidium unknown.We confronted great difficult in preventive and control this disease.the host immune function appears to be less present report, therefore characteristics of the host immunity has been our concern.
The subject has taking an epidemic investigate of cryptosporidium to in part farm of hefei in Anhui province , survey the postive dogs of infected cryptosporidium , observing the morphologic oocyst of Cryptosporidium. Morphology and Technology of molecular biology were applied to identify the havested oocysts: Attempt to determine genotypes of all Cryptosporidium in dogs to expound mechanism of Cryptosporidium molecule epidemiology; Take the oocyst to construct an animal model in order to offer the theoretical base for elucidating the pathogenic mechanism and researching the prevention and regulation.
Firstly,the 232 fecal specimen in dogs was detected by satturated white sugar, we preliminaryly classified the Cryptosporidium harvested in hefei as Cryptosporidium parvum by the oocyst characteristics such as shape constitution、 size and the index number of size. The infection rates of the other three dairy farms is greatly different. Counted and analyied infection of dog in different age,each age dog could infect cryptosporidium, but different age dog with different situation. Puppies's infection rate was higher than the other age of dogs notably.Meanwhile, different age dogs are different in infected intensity.
sencondly,we applied the discontinous sucrose gradients to purified oocysts, which was shaked by shaker for 15 minutes, added plitting liquid(2%DTT,1×TE),freezed and melted for three times in -70℃, water-bath for 20 minutes in 90℃, extracted DNA from oocysts finally. We designed and synthetized the specific primer of cryptosporidium according to the crytposporidium 18SrRNA then amplifing the specific 540bp fragment. In the same time, we designed and synthetized the specific primer of cryptosporidium according to the crytposporidium HSP70 then amplifing the specific 448bp fragment The reagent box of PCR was used to purified PCR products and the products was sequenced by shanghai sangon company. According to the homology sequence in NCBI, we take the biological sofe such as DNAStar analyze the sequence and construct phylogentic trees, confiring that the Cryptosporidium is Cryptosporidium parvum in hefei for the molecular level.
Lastly ,we constructed animal models, 15 BALB / C mice were randomly divided into five groups , A, B, C, D and E meanwhile every group was feed in different cages. The number of mice in every groups is 3. The belly of mice in group A were inoculated 60μL Cryptosporidium with syringe by the channel of mouth ,those mice drinked water with dexamethasone and the does is 1.5mg/100ml , renoveatured water every day until the end of this study . The group B are same as A,excepting to add the drug of immunosuppression and the does is suppression, 1.0mg/100ml; The group C as an non-immunosuppressive group, the way of inoculation Cryptosporidium as A and B,meawile ,the way of drinking without the drug of immunosuppression. The group D as an immunosuppressive group add the drug of immunosuppression, and the does is 1.0mg/100ml; The group E is an non-immunosuppressive group.
We count the number of oocyste get from the mice feacal and record the law of discharge oocysts and others like the intensity of oocysts. When the mice died,we dissect dead body to observe the changes in endosomatic immune organ. During of feeding ,we took a little blood from eyes, separating serum form blood and knowing the total amount protein, albumin and globulin in serum, knowing changes of immunosuppressive agents in infected animals immune function.
Berifly ,the result we get froming the study provided with some significant reference for prevention and cure Cryptosporidium in hefei; and monitored Cryptosporidiosis. And provied some improtant reference for studying the changes in endosomatic immune organ when was infected and studying the pathogenic mechanism of Cryptosporidium and choosing drug for curing the disease.
引文
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