| 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'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. |
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