贝氏隐孢子虫鸡胚气管组织培养模型建立与初步应用
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摘要
贝氏隐孢子虫(Crptosporidium baileyi,C. baileyi)是世界各地禽类感染的优势虫种,可感染30多种禽类,导致雏鸡较高发病率和一定死亡率,研究者通过扫描电镜观察发现在感染雏鸡气管上皮细胞上粘附有大量各发育阶段虫体,可引起禽类严重呼吸道感染。更重要的是C. baileyi可污染环境并通过水、食物、空气传播。有研究表明在中国上海生活废水中已发现C. baileyi卵囊。C. baileyi卵囊对环境抵抗力很强,保存在4℃2.5%重铬酸钾溶液中18个月的卵囊仍可引起雏鸡明显感染。目前,对于隐孢子虫病尚无有效防控药物,研究表明抗球虫药物单独或联合使用均不能减轻或治疗由该病原引起的呼吸道症状。此外,通过免疫印迹和免疫荧光检测,C. baileyi和C. parvum在抗原上的相似性高于C. muris。因此,研究C. baileyi具有重要的医学和兽医学价值,并且对C. baileyi生物学等特性研究可作为其他隐孢虫研究模型。雏鸡感染C. baileyi后繁殖大量卵囊用于生物学试验研究,有助于阐明C. parvum的生物学特性。
自1983年开始,人们开始尝试体外培养隐孢子虫,但至目前为止,关于禽类隐孢子虫体外细胞培养研究较少。有研究证明:完整的C. baileyi体外发育只在鸡、火鸡、鸭、鹧鸪、鹌鹑胚内完成。由于在实验感染雏鸡呼吸道内通过电镜观察可看到C. baileyi有性和无性内生发育阶段,并且C. baileyi在一些禽类和哺乳动物的原代细胞和一些哺乳动物传代细胞系内不发育,而在鸡胚内发育但不利于实验观察,所以本研究尝试用鸡胚气管组织培养模型培养C. baileyi。本试验将C. baileyi卵囊和子孢子混合物或子孢子接种鸡胚气管环,通过观察气管环接种虫体后纤毛摆动强弱、虫体内生发育过程,了解C. baileyi在鸡胚气管环培养模型中虫体的发育状况及繁殖数量,并建立了鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法。此外,应用建立的气管环培养体系试验性比较了硝唑尼特(Nitazoxanide, NTZ)和巴龙霉素(Paromomycin ,PRM)对气管环和鸡胚培养模型中C. baileyi的作用,为今后研究C. baileyi毒力、感染性、代谢途径、内源和外源基因表达、治疗药物筛选等奠定了基础。
一、C. baileyi卵囊纯化、脱囊技术研究
将自河南林州自然感染隐孢子虫病鸡分离的虫体基于18S rRNA基因巢式PCR-RFLP鉴定后卵囊经雏鸡传代扩增。将鸡粪收集混匀,采用饱和蔗糖溶液漂浮法收集卵囊,用1:1.2、1:1.5、1:2三个梯度不连续蔗糖溶液密度梯度离心法(饱和蔗糖溶液和蒸馏水的体积比)进行纯化。纯化后卵囊用0.5%商用次氯酸钠(PBS工作液10倍稀释)4℃处理10 min后用灭菌并含200 IU/mL双抗的PBS工作液无菌清洗4次除去次氯酸钠,加入含200 IU/mL双抗的DMEM培养基计数后用于气管环培养。同时,本试验比较了由不同浓度胰酶和胆酸盐组成的5种脱囊液分别在38～40℃水浴中脱囊不同时间的脱囊率。子孢子用孔径为5μm双层聚碳酸酯过滤纯化,血细胞计数板计数后使用。结果显示:用1:1.2、1:1.5、1:2(2次)蔗糖溶液纯化的贝氏隐孢子虫卵囊在不含胰酶和胆酸盐的DMEM培养液中40℃水浴脱囊率最高,达77%。
二、C. baileyi鸡胚气管环体外培养模型建立
将C. baileyi卵囊和子孢子感染鸡胚气管环,气管环置于24孔培养板内,每孔1个环,每孔加1mL含2.5%热灭活血清DMEM培养液,于40℃5% CO2培养,每组10个气管环。第Ⅰ组接种约5×104个C. baileyi卵囊40℃水浴脱囊后混合物,第Ⅱ、Ⅲ、Ⅳ组分别接种5×105、1×106、2×106个纯化子孢子,第Ⅴ组不接种虫体作为空白对照。经过特定时间培养后结果显示:(1)鸡胚气管环感染鸡源贝氏隐孢子虫后6~9 d,各感染组(第Ⅰ、Ⅱ、Ⅲ、Ⅳ组)气管环纤毛脱落明显比空白对照组多,纤毛摆动活性减弱。统计结果显示:各感染组气管环纤毛上皮细胞纤毛摆动相对活力与空白对照组比较有统计学意义(P<0.05)。(2)接种虫体后第4 d,在第Ⅰ、Ⅱ、Ⅲ、Ⅳ各感染组培养液内发现平均大小为6.5μm×0.9μm的子孢子或裂殖子样左右摆动虫体。接种虫体后第6 d,可观察到平均大小约为3.5μm×3.5μm的裂殖体样虫体。在接种子孢子的第Ⅱ、Ⅲ、Ⅳ各感染组培养液内没有发现新生卵囊(第Ⅰ组因接种卵囊脱囊后混合物未进行观察)。(3)通过电镜和间接免疫荧光检查发现:虫体寄生在气管环纤毛上皮细胞边缘,虫体寄生部位纤毛倒伏,甚至脱落,通过PCR-RFLP分析寄生虫体为C. baileyi。
三、鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法建立
为了测定鸡胚气管环体外培养模型中C. baileyi发育动态,并评价硝唑尼特(NTZ)和巴龙霉素(PRM)对该培养体系中C. baileyi的作用,本试验建立了鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法。该方法同时测定虫体和鸡胚气管环组织细胞18S rRNA基因,采用相对定量法计算组织细胞中虫体相对量。具体为将约5×104 C. baileyi个卵囊40℃水浴脱囊后混合物接种于培养在24孔培养板内鸡胚气管环,每孔1个环,加1 mL含2.5%热灭活血清DMEM培养液于40℃5% CO2培养。分别提取接种后3 h、6 h、12 h、24 h、48 h、72 h、96 h、120 h、144 h、168 h、192h、240 h以及NTZ和PRM作用后72 h、96 h、120 h、168 h气管环细胞基因组DNA,应用实时定量PCR检测方法测定虫体繁殖水平。结果显示:C. baileyi感染鸡胚气管环后72～96 h虫体量最高,96 h以后虫体量开始明显下降。NTZ和PRM对鸡胚气管环培养模型中C. baileyi具有一定抑制作用,且这2种药物的抑制作用强度差异具有统计学意义(P<0.05)。
四、鸡胚培养模型中NTZ和PRM抗C. baileyi活性研究
为了探讨NTZ和PRM对鸡胚培养模型中C. baileyi的作用,将10日龄SPF鸡胚每枚接种3×105个C. baileyi卵囊,每组10枚鸡胚,40℃培养7 d,每天记录鸡胚和虫体的发育及虫体数量来评价NTZ和PRM在鸡胚培养模型中抗隐孢子活性。结果表明:每枚鸡胚分别加入125μg NTZ和10 mg PRM,感染168 h后尿囊液中平均卵囊数分别为对照组的0.33% (0.04/12.11),2.48% (0.30/12.11),NTZ和PRM对鸡胚培养模型中C. baileyi抑制作用效果不同于气管环培养模型中效果,且药效差异比较具有统计学意义(P<0.01)。
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 excystation
C.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 Organ
The 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 cultures
A 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 Chicken
To 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).
自1983年开始,人们开始尝试体外培养隐孢子虫,但至目前为止,关于禽类隐孢子虫体外细胞培养研究较少。有研究证明:完整的C. baileyi体外发育只在鸡、火鸡、鸭、鹧鸪、鹌鹑胚内完成。由于在实验感染雏鸡呼吸道内通过电镜观察可看到C. baileyi有性和无性内生发育阶段,并且C. baileyi在一些禽类和哺乳动物的原代细胞和一些哺乳动物传代细胞系内不发育,而在鸡胚内发育但不利于实验观察,所以本研究尝试用鸡胚气管组织培养模型培养C. baileyi。本试验将C. baileyi卵囊和子孢子混合物或子孢子接种鸡胚气管环,通过观察气管环接种虫体后纤毛摆动强弱、虫体内生发育过程,了解C. baileyi在鸡胚气管环培养模型中虫体的发育状况及繁殖数量,并建立了鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法。此外,应用建立的气管环培养体系试验性比较了硝唑尼特(Nitazoxanide, NTZ)和巴龙霉素(Paromomycin ,PRM)对气管环和鸡胚培养模型中C. baileyi的作用,为今后研究C. baileyi毒力、感染性、代谢途径、内源和外源基因表达、治疗药物筛选等奠定了基础。
一、C. baileyi卵囊纯化、脱囊技术研究
将自河南林州自然感染隐孢子虫病鸡分离的虫体基于18S rRNA基因巢式PCR-RFLP鉴定后卵囊经雏鸡传代扩增。将鸡粪收集混匀,采用饱和蔗糖溶液漂浮法收集卵囊,用1:1.2、1:1.5、1:2三个梯度不连续蔗糖溶液密度梯度离心法(饱和蔗糖溶液和蒸馏水的体积比)进行纯化。纯化后卵囊用0.5%商用次氯酸钠(PBS工作液10倍稀释)4℃处理10 min后用灭菌并含200 IU/mL双抗的PBS工作液无菌清洗4次除去次氯酸钠,加入含200 IU/mL双抗的DMEM培养基计数后用于气管环培养。同时,本试验比较了由不同浓度胰酶和胆酸盐组成的5种脱囊液分别在38～40℃水浴中脱囊不同时间的脱囊率。子孢子用孔径为5μm双层聚碳酸酯过滤纯化,血细胞计数板计数后使用。结果显示:用1:1.2、1:1.5、1:2(2次)蔗糖溶液纯化的贝氏隐孢子虫卵囊在不含胰酶和胆酸盐的DMEM培养液中40℃水浴脱囊率最高,达77%。
二、C. baileyi鸡胚气管环体外培养模型建立
将C. baileyi卵囊和子孢子感染鸡胚气管环,气管环置于24孔培养板内,每孔1个环,每孔加1mL含2.5%热灭活血清DMEM培养液,于40℃5% CO2培养,每组10个气管环。第Ⅰ组接种约5×104个C. baileyi卵囊40℃水浴脱囊后混合物,第Ⅱ、Ⅲ、Ⅳ组分别接种5×105、1×106、2×106个纯化子孢子,第Ⅴ组不接种虫体作为空白对照。经过特定时间培养后结果显示:(1)鸡胚气管环感染鸡源贝氏隐孢子虫后6~9 d,各感染组(第Ⅰ、Ⅱ、Ⅲ、Ⅳ组)气管环纤毛脱落明显比空白对照组多,纤毛摆动活性减弱。统计结果显示:各感染组气管环纤毛上皮细胞纤毛摆动相对活力与空白对照组比较有统计学意义(P<0.05)。(2)接种虫体后第4 d,在第Ⅰ、Ⅱ、Ⅲ、Ⅳ各感染组培养液内发现平均大小为6.5μm×0.9μm的子孢子或裂殖子样左右摆动虫体。接种虫体后第6 d,可观察到平均大小约为3.5μm×3.5μm的裂殖体样虫体。在接种子孢子的第Ⅱ、Ⅲ、Ⅳ各感染组培养液内没有发现新生卵囊(第Ⅰ组因接种卵囊脱囊后混合物未进行观察)。(3)通过电镜和间接免疫荧光检查发现:虫体寄生在气管环纤毛上皮细胞边缘,虫体寄生部位纤毛倒伏,甚至脱落,通过PCR-RFLP分析寄生虫体为C. baileyi。
三、鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法建立
为了测定鸡胚气管环体外培养模型中C. baileyi发育动态,并评价硝唑尼特(NTZ)和巴龙霉素(PRM)对该培养体系中C. baileyi的作用,本试验建立了鸡胚气管环体外培养模型中C. baileyi SYBR GreenⅠ实时定量PCR检测方法。该方法同时测定虫体和鸡胚气管环组织细胞18S rRNA基因,采用相对定量法计算组织细胞中虫体相对量。具体为将约5×104 C. baileyi个卵囊40℃水浴脱囊后混合物接种于培养在24孔培养板内鸡胚气管环,每孔1个环,加1 mL含2.5%热灭活血清DMEM培养液于40℃5% CO2培养。分别提取接种后3 h、6 h、12 h、24 h、48 h、72 h、96 h、120 h、144 h、168 h、192h、240 h以及NTZ和PRM作用后72 h、96 h、120 h、168 h气管环细胞基因组DNA,应用实时定量PCR检测方法测定虫体繁殖水平。结果显示:C. baileyi感染鸡胚气管环后72～96 h虫体量最高,96 h以后虫体量开始明显下降。NTZ和PRM对鸡胚气管环培养模型中C. baileyi具有一定抑制作用,且这2种药物的抑制作用强度差异具有统计学意义(P<0.05)。
四、鸡胚培养模型中NTZ和PRM抗C. baileyi活性研究
为了探讨NTZ和PRM对鸡胚培养模型中C. baileyi的作用,将10日龄SPF鸡胚每枚接种3×105个C. baileyi卵囊,每组10枚鸡胚,40℃培养7 d,每天记录鸡胚和虫体的发育及虫体数量来评价NTZ和PRM在鸡胚培养模型中抗隐孢子活性。结果表明:每枚鸡胚分别加入125μg NTZ和10 mg PRM,感染168 h后尿囊液中平均卵囊数分别为对照组的0.33% (0.04/12.11),2.48% (0.30/12.11),NTZ和PRM对鸡胚培养模型中C. baileyi抑制作用效果不同于气管环培养模型中效果,且药效差异比较具有统计学意义(P<0.01)。
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 excystation
C.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 Organ
The 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 cultures
A 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 Chicken
To 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).
引文
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