【作者】 陈甫；

【导师】 黄克和；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2008， 博士

【摘要】 隐孢子虫病(cryptosporidiosis)是由隐孢子虫(Cryptosporidium)寄生于人或动物胃肠道和呼吸道上皮细胞内引起的一种全球性的人兽共患原虫病。当前,隐孢子虫病已被认为是世界最常见的6种腹泻病之一,世界卫生组织(WHO)更将其列为艾滋病(AIDS)的怀疑指标。迄今为止,隐孢子虫的致病机理仍不清楚,也没有有效的治疗药物。而且目前尚未见到对华东地区动物隐孢子虫基因分型和隐孢子虫病流行病学进行系统的研究。因此,本文对华东地区动物隐孢子虫进行了分子流行病学调查和基因型鉴定,并从病理学和凋亡基因表达的角度研究了隐孢子虫的致病机理,探索了黄芪多糖、苦参碱与硝唑尼特对隐孢子虫感染的抑制作用。1、微小隐孢子虫卵囊分离纯化方法的建立与形态学观察为探索一种实用、纯度和回收率高且卵囊活力强的分离纯化微小隐孢子虫(C. parvum)卵囊的方法,本文分别采用不连续蔗糖密度梯度离心法(DSGC)、改良饱和盐水飘浮法(INaCl)、氯化铯密度梯度离心法(CsCl)和Percoll密度梯度离心法(Percoll)分离纯化C57BL/6小鼠粪中隐孢子虫卵囊,并将4种方法分离纯化获得的卵囊各105个体外感染牛输卵管上皮细胞(BFTE),48 h和72 h后油镜下观察各分离卵囊的发育情况,以比较卵囊的纯度和活性。结果表明,DSGC分离获得的卵囊数与INaCl无差异(P>0.05),但高于Percoll(P<0.01)和CsCl(P<0.05)。4种方法分离纯化的卵囊体外感染BFTE48 h和72 h后隐孢子虫数无差异(P>0.05)。但CsCl纯化的隐孢子虫卵囊纯度明显高于DSGC,且纯化的卵囊不作任何处理不会使BFTE发生污染。INaCl较DSGC操作简单、快速,纯化的卵囊活性无差异；CsCl能够获得纯度极高的隐孢子虫卵囊。2、微小隐孢子虫感染小鼠模型的建立探索适合隐孢子虫传代和扩增模型的最佳隐孢子虫种类、小鼠的品系、性别、日龄、免疫抑制时间和免疫抑制剂地塞米松磷酸钠(DEXp)的最佳剂量。结果表明,C. andersoni卵囊不能感染BALB/c小鼠；与Kunming鼠比,BALB/c小鼠感染C. parvum后卵囊增殖较多；雌性小鼠排卵囊强度均较同日龄雄性小鼠大,而不同日龄小鼠中又以50日龄小鼠排卵囊量最多；各剂量组小鼠中,15 mg·L-1组排卵囊强度较其他各组大；免疫抑制时间组中,免疫抑制3d感染组排卵囊强度最大。因此,适合于C. parvum扩增和传代的最佳鼠模型是50日龄雌性BALB/c小鼠：DEXp的最佳剂量是15 mg·L-1；最佳免疫抑制时间是感染前3 d。3、微小隐孢子虫体外感染MDCK模型的建立及生长发育过程的观察本文利用MDCK细胞为隐孢子虫感染对象,优化了隐孢子虫感染细胞的培养条件,探索了隐孢子虫在细胞中的生长发育过程。结果表明,在含有5%胎牛血清DMEM培养基的6孔培养板中,用1×105个隐孢子虫卵囊感染培养12h的2.0×105个细胞为最佳培养条件；在感染细胞的72 h中,隐孢子虫出现了连续的发育阶段：脱囊、子孢子、裂殖子、裂殖体、滋养体、配子体和卵囊,并在60～72 h之间观察到卵囊的生成。上清液接种小鼠结果表明,在感染48 h时排出有感染性的隐孢子虫。这为进一步研究隐孢子虫奠定了坚实的基础。4、保存在水和重铬酸钾中的微小隐孢子虫卵囊活性及感染性比较应用体外脱囊技术评价保存在水中和重铬酸钾(K2Cr207)中1、4、7、10、13、16、20、25和30个月的C. parvum卵囊的活性差异,通过检测隐孢子虫感染BALB/c小鼠的潜伏期、排卵囊数量和末端回肠绒毛中隐孢子虫数量来评价保存在水中和K2Cr207中卵囊的感染性差异。结果表明,保存在水中1～13个月和K2Cr207中1-16个月的卵囊能出现脱囊和感染小鼠。两种方法保存的卵囊体外脱囊率之间和体内感染小鼠的潜伏期之间均差异不显著(P>0.05),但是两种方法保存时间与潜伏期之间存在强烈的相关性(K2Cr207：R2=0.92；自来水：R2=0.98)；两种方法保存的卵囊感染小鼠的排卵囊强度之间和末端回肠的隐孢子虫数量之间差异不显著(P>0.05)。因此,在普通实验室中可以水代替K2Cr207保存C. parvum卵囊；水中活性卵囊的长期存在是饮用水处理工业的一个严重的威胁。5、检测隐孢子虫卵囊MAFS法的改进及与IFA-McAb和PCR法的比较通过检测小鼠和奶牛粪便中的C. parvum卵囊改进改良抗酸染色技术(MAFS),并与IFA-McAb和PCR法进行比较。结果表明,粪便直接涂片法和加血清法的检测效率优于粪便纯化后的涂片法。MAFS、IFA-McAb和PCR法的检测效率分别为每10 g粪便中有2.0×104、2.0×104和2.O×102个卵囊,操作一次分别会花费5 min、60 min和3 h,检测鼠粪样品的阳性率分别为65.0%、70.0%和85.0%,检测牛粪样品的阳性率分别为21.21%、18.18%和27.27%,三种方法之间差异不显著(P>0.05)。总之,粪便直接涂片法和加血清法两者互相结合的MAFS简便、快速、敏感性强,条件要求不高,适合大量样品的检测；IFA-McAb和PCR更适合于隐孢子虫虫种或基因型的鉴定。6、实时荧光PCR定量检测微小隐孢子虫方法的建立与初步应用根据GenBank公布的C. parvum18s rRNA基因序列设计一对引物,通过质粒DNA和卵囊DNA标准曲线的绘制和实时荧光定量PCR (Real-time PCR)法检测C. parvum特异性和重复性的研究,建立了检测C. parvum的Real-time PCR方法,并对奶牛粪便进行了检测。结果表明,设计的引物对检测C. parvum具有很高的特异性,质粒DNA和卵囊的检测阈值分别达到1个拷贝和10个卵囊,奶牛粪便阳性率为25.93%(21/81)。建立的Real-time PCR方法简便、快速,特异性强,敏感度高,可用于C. parvum的快速定量检测。7、华东地区动物隐孢子虫的流行病学调查应用MAFS技术对华东地区动物隐孢子虫感染情况进行调查。结果表明：奶牛、猪、兔、犬、水牛、鸡和鹅样品的阳性率分别为19.09%、11.96%、14.10%、21.05%、19.79%、16.67%和7.74%。奶牛感染率显著高于猪、兔、鹅(P<0.05),鹅隐孢子虫感染率与蛋鸡之间差异显著(χ2=5.12,P<0.05)。不同日龄奶牛之间的感染率在16.93～23.74%之间,不同日龄猪之间的感染率在10.34～13.31%之间；感染强度为“+”和“++”的猪占了总感染率的84.75%,奶牛占了总感染率的88.45%；腹泻牛(33.76%)与猪(23.61%)阳性率显著高于非腹泻奶牛(17.10%)(χ2=41.48,P<0.01)和猪(10.81%)(χ2=14.97,P<0.01),；产仔母畜与仔畜的感染率之间差异不显著；月份之间差异不显著(χ2=6.68,P>0.05)。说明华东地区动物隐孢子虫感染率普遍较高,大多数动物是低强度感染；隐孢子虫是动物发生腹泻的重要病原之一；母畜是仔畜感染隐孢子虫的重要传播源。8、华东地区隐孢子虫卵囊的分离与分子鉴定对华东地区猪与奶牛隐孢子虫卵囊的形态学、密度和感染性进行研究,并应用RT-PCR扩增部分18s rRNA序列进行RFLP分析和系统发育进化分析。结果表明：安徽、上海和江苏地区隐孢子虫卵囊的形态有3种,卵囊长、短径和卵囊指数之问差异显著(P<0.01),卵囊密度之间差异不显著；RFLP与序列同源性和系统发育进化分析结果表明,上海、安徽地区奶牛感染的是C. andersoni和C.parvum,江苏地区猪、奶牛隐孢子虫为C. parvum;其中,C. parvum与"mouse"基因型有99.8～100%的同源性,且能感染小鼠；而C. andersoni与已知C. andersoni同源性达99～100%,不能感染小鼠。说明江苏地区猪、奶牛源隐孢子虫为C.parvum "mouse"型,上海、安徽奶牛源隐孢子虫为C. andersoni和C. parvum "mouse"型,提示猪和奶牛与鼠之间存在交叉传播的可能。9、微小隐孢子虫感染对细胞损伤及肠黏膜屏障的影响应用C. parvum感染BALB/c小鼠与MDCK细胞模型,研究隐孢子虫对细胞损伤及对小鼠肠黏膜屏障的影响。结果发现,第3 d,感染小鼠的小肠对葡萄糖、谷氨酸的吸收显著低于对照组(P<0.05),第6～15d,感染组均显著高于对照组(P<0.05)；隐孢子虫感染MDCK细胞12 h,总氨基酸、葡萄糖的吸收显著低于对照组(P<0.05)。40%的感染组小鼠血液检测到大肠杆菌,极显著高于对照组(χ2=9.32,P<0.01)；感染组血清D-乳酸含量显著高于对照组(P<0.05),感染组较对照组能显著提高血清和细胞上清中的NO含量与血清中iNOS活性(P<0.05),感染组上清中D-乳酸含量在感染后12～36 h明显高于对照组(P<0.05)。隐孢子虫感染细胞12h明显升高了细胞中iNOS活性(P<0.05)；iNOS活性在感染后24～36 h显著低于对照组(P<0.05)。感染组小鼠胃壁皱褶及肠道绒毛明显较对照组排列疏松。因此,在感染初期, C. parvum侵入细胞,使细胞发生损伤,完整性下降；在感染后期,破坏了肠黏膜屏障,使肠道的通透性增加。10、微小隐孢子虫感染对MDBK细胞凋亡相关基因mRNA表达的影响以(3-actin为内参,建立并应用Real-time PCR定量检测MDBK细胞不同感染时期凋亡相关基因Fas、FasL、TNF-a、Bcl-2和Caspase-3 mRNA的表达情况。结果表明,在感染的12-36 h,与对照组相比C. parvum能引起MDBK细胞诱导凋亡基因Fas、FasL与Caspase-3 mRNA的明显表达(P<0.05或P<0.01)；在感染的36～60 h,感染组TNF-a基因mRNA的表达量极显著高于对照组(P<0.01)；而在感染60 h,感染组Fas、FasL、Caspase-3、TNF-a与Bcl-2 mRNA的表达与对照组之间差异显著(P<0.05)。说明C. parvum感染MDBK细胞引起了凋亡基因Fas、FasL、Caspase、TNF-a和Bcl-2 mRNA的表达异常。11、APS、MT与NTZ对C. parvum活性及感染性的抑制影响应用MDBK细胞体外感染模型和BALB/c小鼠体内模型,研究了黄芪多糖(APS)、苦参碱(MT)与硝唑尼特(NTZ)对C. parvum感染的抑制作用。结果表明,APS、MT与NTZ各浓度组的隐孢子虫感染数量均显著或极显著低于感染组(P<0.05或P<0.01)；NTZ治疗组的抑虫效率均极显著高于NTZ感染前处理组(P<0.05),APS感染前处理组的抑虫效率均极显著高于自身感染后处理组(P<0.05),MT感染前处理组体内抑虫效率均显著低于MT感染后处理组(P<0.05),体外抑虫效率之间差异不显著。体外试验表明,各药物组和感染组iNOS活性与NO水平均显著高于对照组(P<0.05)；体内试验表明,各药物组血清D-乳酸水平与血液细菌的检出率均显著低于感染组(P<0.05),NTZ感染后处理组、MT组和APS感染前处理组血清D-乳酸水平和血液细菌的检出率较感染组显著降低(P<0.05)。APS、MT与NTZ均能不同程度的抑制隐孢子虫对MDBK细胞和BALB/c小鼠的感染性；APS、MT与NTZ通过降低隐孢子虫对细胞的损伤和肠黏膜屏障的破坏而抑制了隐孢子虫对MDBK细胞和BALB/c小鼠的感染活性。更多还原

【Abstract】 Cryptosporidiosis is caused by the obligate intracellar protozoan parasites, Cryptosporidium, which infects the epithelium of gastrointestinal and respiratory tract of animals and humans, has recently emerged as a global public health problem. Nowadays, cryptosporidiosis was considered as one of the suspicious index of acquired immunodeficiency syndrome (AIDS) and one of the six diseases leading to diarrhea worldly. However, up to the present, the pathogenesis of cryptosporidiosis is not distinctly understood. Furthermore, there is no effective treatment available for cryptosporidiosis in human and animals, although large numbers of antiparasitic, antifungal, antibiotic, or antiviral agents were investigated. Furthermore, the epidemiology and genotypes of Cryptosporidium parasites were not investigated systematically in animals in eastern China. Consequently, the present study was undertaken to investigate the molecular epidemiology, the pathogenesis in the respect of pathogenesis and the expression level of apoptosis gene mRNA, and screening of anti-infection drugs against cryptosporidiosis on the basis of establishing the in vivo and in vitro infected models.1. Morphology and methods for isolation and purification of C. parvum oocysts.The study was designed to explore an applicable method for isolation and purification of C. parvum oocysts with high purity, recovery and better living of oocysts from mice feces, and investigate the morphology of oocysts. Improved saturated salt water flotation (INaCl), percoll gradient centrifugation (Percoll) and CsCl gradient centrifugation (CsCl) after INaCl were compared with the classical discontinuous sucrose gradient centrifugation (DSGC). The results showed that, in the aspect of oocyst numbers, DSGC was significantly higher than Pecoll and CsCl, but not significantly differenf from INaCl. Primary BFTE cell cultures were prepared from bovine fallopian tubes and infected with oocysts obtained by the purification methods, and no significant differences were found in the aspect of oocyst numbers at 48 and 72 hours postinoculation. In the aspect of oocyst purifty, CsCl was better than others. After infected with oocysts purified using CsCl and without treatment, the BFTE cell cultures were not polluted. In conclusion, comparing to the DSGC, the INaCl is operated more simply and quickly, and of no difference from others in the oocyst liveness. The purity of oocysts obtained from CsCl was higher than others.2. Establishment of mice model infected with C. parvum oocysts.The present study was designed to explore the optimal species of Cryptosporidium, the optimal gender and age of mice, the optimal dose of the dexamethasone phosphate (DEXp) and the optimal time of immunosuppression in the mice model applied for C. parvum infection and reproduction. The results showed that C. andersoni oocysts could not be reproduced in the BALB/c mice, and BALB/c mice could reproduce more oocysts than others. In the aspect of oocyst intensity discharged, femal mice were more than the male, the 50 day-old mice were more than the 25 and 75 day-old,15 mg·L-1 group were more than other dosage groups, and mice infected after immunosuppressed for 3 days were more than others. In conclusion, the optimal gender and age of BALB/c mice as model of reproducing C. parvum oocysts are female and 50 day-old. The optimal dosage of DEXp is 15 mg·L-1, the optimal time of inoculation is on the third day after immunosuppression.3. Cultivation and development of C. parvum in MDCK cells.It is great important to develop culture system in vitro in which C. parvum can complete its entire life cycle. An in vitro culture system using Madin-Darby bovine and canine kidney (MDCK) cells as the host cell was developed, and the growth and development of C. parvum in cells were investigated. The results showed that 2.0×105 cells cultured for 12 h and infected by 1×105·C. parvum oocysts in the Dulbecco’s Modified Eagle Medium with 5 percent of heat-inactivated fetal bovine serum in 6 well tissue culture plates was the optimal condition for culturing C. parvum in cells. Sequential development of desquamate, sporozoite, trophozoites, meronts, microgametocytes, macrogametocytes, zygote, thin-wall oocyst, and thick-wall oocyst were observed over a 72-h period of culture. Between 60 and 72 h in MDCK, the formation of oocyst walls was observed. C. parvum with infectivity were firstly extracted when MDCK cells were infected for 48 h. This culture system provides access to C. parvum investigation.4. Comparison of viability and infectivity of C. parvum oocysts stored in potassium dichromate solution and chlorinated tap waterThe present study was undertaken to compare the viability and infectivity of C. parvum oocysts that had been stored for 1.4,7,10,13,16,20,25 and 30 months at 4℃in 2.5% potassium dichromate (Cr) and chlorinated tap water, respectively. An excystation protocol was performed in vitro to evaluate the viability of oocysts. BALB/c mice were used to evaluate the infectivity of oocysts by detecting the prepatent period of C. parvum infection, the quantity of oocysts excreted, and the number of parasites that colonized the villi of the ileum. The results showed that C. parvum oocysts preserved in Cr for 1-16 months and in water for 1-13 months were capable of excystation in vitro and infection of mice in vivo. The excystation rates of oocysts and the prepatent periods in mice infected by oocysts stored in Cr and water were not significantly different (P>0.05), and there was a strong correlation between prepatent period and duration of oocyst storage (Cr:R2=0.92; water:R2=0.98). There were no significant differences in oocyst shedding from feces or parasitism of the terminal ilea of mice by Cryptosporidia between the two storage media (P>0.05). In conclusion, C. parvum oocysts may be stored at 4℃in water instead of Cr for the purposes of laboratory research. However, the presence of viable C. parvum oocysts in water is a severe challenge to the drinking water treatment industry.5. Modification of MAFS and comparative study with IFA-McAb and PCR methods for detection of C. parvum oocystsThe modified acid-fast staining technique (MAFS) was modified to detect Cryptosporidium oocysts, and compared in the respect of the detecting effect of C. parvum oocysts in mice and cow feces with immunofluorescence using monoclonal antibody (IFA-McAb) immunolabeling by FITC against C. parvum and polymerase chain reaction (PCR). The results showed that in the respect of the detecting effect, the direct smear of feces was superior to the methods of smearing after feces purification, in which, smear with serum was better than that without serum. The least dosages of oocysts detected using the forenamed 3 methods in ten gram feces were 2.0×104,2.0×104 and 2.0×102, respectively. The least time spend on the three methods for detection was 5 min,60 min and 3 hours, respectively. Positive ratios in mice feces detected were 65.0%,70.0%,85.0%, and in cow feces were 21.21%,18.18%, 27.27%, respectively. No difference was found among the three methods.In conclusion, MAFS is operated simply and quickly, suited for detection of a great number of samples and spends less with lower conditions. IFA-McAb and PCR are more suited for identification of the species or genotype.6. Establishment and application of Real-time PCR assay for detection of C. parvumIt is important to develop a real-time fluorescent quantitative PCR assay (real-time PCR) for detection of Cryptosporidium. A pair of primers was designed acording to the small subunit ribosomal RNA of C. parvum in GenBank, and real-time PCR assay was established for detection of C. parvum as to detect 81 feces of cows. The results showed that the primers were highly differential to C. parvum, the least dosages detected by real-time PCR were one copies of plasmid DNA and ten oocysts, and a total of 21 (25.53%) positive cows were detected. In conclusion, real-time PCR assay based on SYBR Green, which was established for detection of C. parvum, can be used as a rapid, simple and convenient tool for the quantitative detection of C. parvum with great sensitivity and differentia.7. Epidemiology of Cryptosporidium parasites in animals in eastern China.The present study was designed to investigate the epidemiology of cryptosporidiosis by the MAFS technique in animals in eastern China. The results showed that, a total of 11.96%,19.09%,14.10%,21.05%,19.79%,16.67%, and 7.74% pigs, cows, rabbits, dogs, buffaloes, geese and laying hens were detected positive. The prevalence of cows was significantly different from those of pigs, rabbits and laying hens. And no significant difference was found between the prevalence of Cryptosporidium infection in the geese and laying hens (P>0.05). No significant difference was found between the prevalence of the female and the young (P>0.05), the prevalence did not vary significantly by age and among the twelve months (P>0.05). The prevalence of the diarrhea was significantly higher than that of the undiarrhea (P<0.05). It is concluded that the prevalence of animal in eastern China was high, and lower intensity was found in the most animals. Cryptosporidiosis might be one of the pathogens causing aniaml diarrhea. The female might act as reservoirs for C. parvum infection in the younger animals.8. Isolation and molecular identification of Cryptosporidium parasites in animals in eastern China.The present study was designed to analyze the morphology, density, and infection of Cryptosporidium oocysts, and identify the genotypes of Cryptosporidium in pigs and cattle in Jiangsu, Shanghai and Anhui by RT-PCR-RFLP technique and analysis of the homologous rate and phylogenetic tree. The results showed that there were three kinds of oocysts with different morphology, and only two kind oocysts could infect mice. RFLP analysis, phylogenetic analysis and a distances matrix generated from these sequences showed that C. andersoni and C. parvum were identified in cattle in Anhui province and Shanghai city, and C. parvum was detected in the pigs and dairy cows in Jiangsu. Thereinto, the isolates belonged to the species C. parvum were most similar to the C. parvum "mouse" genotype, which has not been reported in cattle and pigs previously. It is concluded that there are two species of Cryptosporidium in eastern China, the isolates of C. parvum is C. parvum "mouse" genotype. There might be a cross-transmission between pigs, or cattles and mice.9. Effect of C. parvum infection on the cell trauma and the intestinal mucosa barrierThe MDBK cells and BA LB/c mice model were used to investigate the effect of C. parvum infection on the cell trauma and the intestinal mucosa barrier (IMB). The results showed that in vivo, in the respect of the absorbability of glucose and glutamic acid, the infected mice was significantly lower than the control on the third day (P<0.05), and higher than the control on the sixth to fifteenth day (P<0.05). In vitro, the absorb of the total content of amino acid and glucose was lower than that of control at the 12 hour after infection (P<0.05), and no significant difference was found between the control and infection at the 24-60 hour after infection. In vivo, E. coli in the blood was detected in forty percent of the infected mice, which was significantly higher than that of the control (x2=9.32,P<0.01). D-lactic aicid, nitrous oxide (NO) and nitrous oxide synthase (iNOS) in serum of the infected mice were significantly higher than those of the control (P<0.05). In vitro, D-lactic aicid of the control was significantly lower than that of the infection at 12-36 hours after infection (P<0.05). Compared with the control, C. parvum made the activity of iNOS in the infected cells ascending at the 12 hours, but lesser at 24-36 hours after infection significantly (P<0.05). Characteristic histopathologic changes in the gastrointestinal tract vary from severe villous atrophy. Villi are blunt, shorter, and wide than normal. In conclusion, in the initial stages, the integrality of cells is destroyed as a result of the incursion of C. parvum. In the latter stages, the enhancive permeability of intestine as a result of the disruption of epithelial cell barrier.10. Effect of C. parvum infection on the expression of apoptosis gene mRNA of MDBK cells.The study was designed to investigate the mechanism of apoptosis induced by C. parvum infection by detecting the expression level of apoptosis gene mRNA using real-time PCR. On the basis of the infected MDBK cells, real-time PCR assay withβ-actin as the internal reference gene was established, and used to detecte the expression level of apoptosis gene mRNA:Fas、FasL、TNF-α、Bcl-2和Caspase-3 in the C. parvum infection. The results showed that in comparation with the control, C. parvum caused the expression level of Fas, FasL, and Caspase-3 gene to be significantly ascended at 12-36 hours after infection (P<0.05). At 36-60 hours after infection, C. parvum caused the expression level of TNF-a gene to be much significantly ascended (P<0.01). Whereas, no difference was found between the control and the infection in the respect of the expression level of Bcl-2 gene Mrna before 48 hours (P>0.05). At 60 hours after infection, C. parvum caused the expression level of Fas, FasL, Caspase-3, TNF-a and Bcl-2 gene mRNA to be significantly ascended. Consequently, in the stage of infection, C. parvum primarily induces abnormity of apoptosis in MDBK cells by Fas/FasL-dependent, Caspase-3-dependent, TNF-a-dependent, and Bcl-2-dependent mechanism.11. The suppressive effect of APS, MT and NTZ on C. parvum infection in vitro and in vivo.The present study was designed to investigate the suppressive effect of Astrgalus Polysaccharides (APS), Matrine (MT) and Nitazoxanide (NTZ) on C. parvum infection in vitro and in vivo. APS, MT, and NTZ were used to suppress the C. parvum infection, respectively. The results showed that the C. parvum adhesion to MDBK cells were significantly suppressed by APS, MT and NTZ groups, as compared with that of phosphate-buffered saline groups (P<0.05 or P<0.01). The treatment after infection effect of NTZ was significantly higher than the treatment before infection effect, the treatment before infection effect of APS was significantly higher than the treatment after infection effect, the treatment before infection effect of MT was significantly higher than the treatment after infection effect in vivo, but no significant difference was found between the two effect in vitro. Simultaneously, the iNOS activity and NO level of the control, APS, MT and NTZ groups in vitro, were significantly lower than the positive (P<0.05). The D-lactic aicid and the detecting rate of bacilli in APS, MT and NTZ groups in vivo, were also lower than the positive (P<0.05). In conclusion, APS, MT and NTZ could effectively inhibit C. parvum infection in vitro and in vivo. APS, MT and NTZ protect the MDBK cells and the intestinal mucosa barrier from the damnification of C. parvum.更多还原