| Cryptosporidium baileyi is one of three avian Cryptosporidium spp., and probably the most common avian species, it has been reported in a wide range of avian hosts. Sexual stages of C. baileyi were seen attached to the tracheal epithelium and free in the tracheal lumen by Scanning electron microscopy, which can cause severe respiratory infections. and result in high morbidity and mortality of birds, especially broiler chickens. More importantly , C. baileyi is an ubiquitous contaminant of water and food which serves as an excellent vehicle for transmission and C. baileyi oocysts have been identified in domestic wastewater in Shanghai, China. C. baileyi oocysts appear to be environmentally robust as studies have shown that chickens inoculated with C. baileyi oocysts that had been stored in 2.5% potassium dichromate at 4℃for 1 to 18 months developed patent infections. At present, no effective chemotherapy is available for the treatment of avian cryptosporidiosis. Experimental studies have shown that most commonly used anticoccidials when used alone or in combination do not prevent or reduce respiratory disease in chickens inoculated with C. baileyi oocysts . Moreover, C. baileyi and C. parvum were more closely related antigenically to one another than to C. muris examined by western blotting and immunofluorescence, and is of medical and veterinary importance. To study characterization of C. baileyi can serve as a model for other species. Furthermore, chicken infected with C. baileyi can produce prodigious numbers of oocysts, which can be used for experimental study and developed in C. parvum or other species.Since 1983, attempts have been made to cultivate Cryptosporidium species in vitro, yet, Little research has been conducted on the cell culture of avian Cryptosporidium species. Complete development of C. baileyi has only been described from the chorioallantoic membrane (CAM) of chicken, turkey, duck, partridge and quail embryos. Considering sexual development and other stages of C. baileyi were found in the respiratory tract of experimentally infected broiler chickens on an ultrastructural level , and C. baileyi had failed in development in primary cell from either avian or mammalian hosts, or in mammalian cell lines and the inconvenience observation of embryos cultivation model ,the cultivation in vitro with tracheal organ culture (TOC) for C. baileyi should be carried out.Here we report on a new method for cultivation of C.baileyi in vitro by inoculation infective oocysts and, or sporozoites to chick embryo tracheal rings, and a real-time qPCR-based with SYBR GreenⅠmethod has been introduced for assessment of C. baileyi cultured in chick embryo tracheal rings by detecting the level of parasite 18S rRNA gene. Comparative effecte of nitazoxanide (NTZ) and paromomycin(PRM) against C.baileyi cultured in chick embryo tracheal rings and cultured in chick embryo were carried out, respectively. The primary aim of this study was to produce in vitro culture models for C. baileyi in chick embryo tracheal organ, and also sought to determine precisely a variety of biological characteristics of this intracellular parasite. These findings will contribute to investigators for further study on its antigens, virulence, infectivity, metabolize, endogenous or exogenous gene expression, and assessing potential drug therapies.1. C. baileyi oocyst purification and excystationC.baileyi oocysts originally derived from naturally infected chicken, from Linzhou city, Henan province, China, genotyped according to the method described by Xiao et al based on nested polymerase chain reaction(PCR)analysis and sequencing of the 18S rRNA and subsequently been passaged through chicken. Chicken feces were homogenized and floated with Sheather’s solution and then applied to classical discontinuous sucrose gradient centrifugation to purify. Sheather’s solution was diluted with distilled water to yield 1:1.2, 1:1.5, 1:2 (Sheather’s solution : distilled water by volume =1:1.2, 1:1.5, 1:2respectively) solutions for use in density centrifugation with the modified method as previously described. The purified oocysts were“sanitized”with 0.5% sodium hypochlorite for 10min at 4°C and then washed 4 times (3000 r/min, 10 min) with PBS containing penicillin (200 IU/ml) and streptomycin (200 μg/ml). For chick embryo tracheal organ culture studies, five excystation protocols have been developed with different concentration trypsin and sodium taurocholate solution for the achievement of high excystation rates in water bath at 38-40°C. Sporozoites purified by filtration through double layer 5μm polycarbonate track etch membranes, followed by counting with hemocytometer. The results indicate that 77% excystation rate of C. baileyi oocysts purified with 1:1.2, 1:1.5, 1:2 Sheather’s solution can be reached incubated in free trypsin and sodium taurocholate DMEM culture medium for 90 min at 40℃.2. Producing in vitro culture models for C. baileyi in chicken embryo tracheal OrganThe study describes the protocol of in vitro cultivation in chick embryo tracheal organ by oocysts or sporozoites of C. baileyi. Approximately 5×104 sporozoites and oocysts mixture for groupⅠ, 5×105, 1×106 , 2×106 purified sporozoites for groupⅡ, groupⅢand groupⅣ, were inoculated respectively into one chick embryo tracheal ring maintained in DMEM supplemented with 2.5% heat-inactivated FBS ,and cultured in one well(with 1ml medium)of the 24-well culture plate at 40℃in 5% CO2. groupⅤt hat received no parasites was included in each experiment. Ten rings were used in each group for different investigation.After the given time cultivation, the results showed that:(1) In infection group (groupⅠ, groupⅡ, groupⅢ, groupⅣ), parasite caused significant ciliostasis and loss of cilia (P < 0.05) when compared with control group 6-9 days post inoculation of tracheal rings. (2) Sporozoite or merozoite-like with an average size 6.5×0.9μm and type I or II meronts–like with 3.4×3.4μm in diameter appeared in the medium 4-6d post inoculation, no newly formed oocysts were observed in sporozoites infection group medium(3)The parasites budded on the epithelial cell membrane and loss of cilia induced by C.baileyi in infected group by electron microscopy,indirect immunofluorescence and PCR-RFLP analysis.3. Application of quantitative real-time PCR in assessing drug efficacy against the C.baileyi in chick embryo tracheal organ culturesA real time quantitative PCR (q-PCR) was developed for assessing the C.baileyi infection of in vitro cultivated chick embryo tracheal organ cultures and verifying efficacy of nitazoxanide (NTZ) and paromomycin(PRM) against C.baileyi. The q-PCR assay detects 18S rRNA gene from both parasites and chick embryo tracheal organ , and evaluates the relative expression between parasite and host rRNA gene levels to minimize experimental and operational errors. Approximately 5×104 C.baileyi oocysts were inoculated into one chick embryo tracheal ring maintained in DMEM supplemented with 2.5% heat-inactivated FBS ,and each cultured in one well of the 24-well culture plate at 40℃in 5% CO2. In order to estimate efficacy of washing steps, negative controls that received equally parasites thermally inactivated at 80℃for 10 min were included in each experiment. The genomic DNA were extracted at 3, 6, 12, 24, 48, 72, 96, 120, 144 , 168, 192, 216 and 240 h ,and at 72, 96, 120 and 168 h incubated with NTZ and PRM post inoculation (P.i)and subjected to qPCR targeting the 18SrRNA gene to quantify the development of C.baileyi. The results indicate that qPCR analysis revealed the highest amplification of parasite DNA at 72-96 h P.i , the growth of parasites apparently starts to decline after cultivation in vitro for 96 h or longer. Both compounds displayed inhibitions, and there are statistically significant differences between NTZ and PRM(P<0.05).4. Evaluation of curative anticryptosporidial activity of NTZ and PRM of Potential Drugs in Chick Embryo and ChickenTo explore the effect of oocyst multiplication and pathogenicity of Cryptosporidium baileyi from chicken in chick embryo by NTZ and PRM, ten 10-day-old specific pathogen free chick embryos of each group were inoculated with C.baileyi oocysts (3×105oocysts/embryo)and incubated at 40℃up to 7 days. The develepment of chick embryo and C. baileyi were both evaluated , and the quantity of oocyst in chick embryo were counted at 24-hour intervals . The anticryptosporidial activity of NTZ and PRM were observed in chick embryo infected artificially with C. baileyi oocyst from chicken. The results showed that the number of oocysts in chick embryo treated with NTZ or PRM(125μg NTZ or 10mg PRM per chick embry)in comparison to control is 0.33% (0.04/12.11),2.48% (0.30/12.11) at 168 h post innoculation, respectively. Drug Efficacy against the intracellular pathogen C. baileyi cultured in chick embryo is significantly different to that cultured in chick embryo tracheal rings(P<0.01). |
PDF Full Text Request