摘要
寄生于禽类的隐孢子虫有效种为3个即C. meleagridis, C. baileyi, C. galli,其中C. meleagridis作为人隐孢子虫病的第三大感染种类已引起广泛的关注。基于此,作者对河南省部分地区的禽类(鸵鸟、鸭、鸡、鹌鹑)进行了隐孢子虫流行病学调查,掌握了其流行特点。并利用收集获得的鸡源C. baileyi卵囊进行了卵黄抗体的制备与鉴定研究,为进一步开展应用抗隐孢子虫特异性IgY抗体进行隐孢子虫病的治疗与诊断提供基础。
2006年6月～2007年7月,对河南地区6个鸵鸟养殖场、8个鸭场、8个规模化鸡场和37个中小规模鸡养殖户、47个鹌鹑养殖场的禽类进行了调查。粪便样品中隐孢子虫的检测用饱和蔗糖溶液漂浮法和改良抗酸染色法进行。结果显示鸵鸟隐孢子虫感染率为1.7%(14/829),鸭隐孢子虫阳性率为16.31%(92/564),蛋鸡、种鸡隐孢子虫感染率为10.57%(163/1542)、肉鸡感染率为3.17%(15/473),鹌鹑隐孢子虫感染率为13.15%(239/1818)。统计数据分析表明幼龄鸵鸟和雏鸭较易感染隐孢子虫,对于鸡,0～31、31～60d的雏鸡和121～180d初产蛋鸡感染率相对较高,育成鸡(60～120d)和产蛋鸡(180d以后)的感染率相对较低。根据形态学鉴定,寄生于鸵鸟、鸭、鸡的隐孢子虫为C. baileyi,而寄生于鹌鹑的隐孢子虫除了C. baileyi外,尚感染有C. meleagridis。
利用实验室扩增后的鸡源C. baileyi卵囊,以改进的Sheather蔗糖梯度离心法提纯卵囊,低速离心结合滤膜微滤除菌后,通过反复冻融和超声波处理的全卵囊抗原,以福氏完全和非完全佐剂制备免疫抗原,设定4种免疫程序,免疫5月龄开产健康母鸡。结果显示首免、二免剂量均为5×107个/只,免疫间隔6周,2次免疫的第一组,每次免疫剂量均为1×107个/只,免疫间隔15～20天, 4次免疫的第三组不能取得好的免疫效果,应用免疫抗原量梯度增加的免疫方法的第二组,取得了满意的抗体水平,一免、二免抗原量分别为1×107个/只,2×107个/只,三免、四免抗原量均为3×107个/只,五免、六免抗原量均为5×107个/只,每次免疫间隔15～20d,共免6次。采用间接ELISA法和免疫荧光法对获得的卵黄抗体进行初步鉴定,其具有隐孢子虫属特异性。以水稀释-盐析法纯化本次实验获得的IgY抗体,经SDS-PAGE分析后,卵黄抗体未提纯前呈现12条以上清晰蛋白条带,主要的有136KDa、74KDa、62KDa、49KDa和33KDa蛋白条带,提纯后的IgY抗体,主要有2条蛋白条带,即62KDa左右的重链,33KDa左右的轻链。对获得的IgY抗体进行了免疫荧光定位,显示抗体与卵囊顶部的荧光反应最强且与脱出囊外的子孢子的荧光反应强于卵囊壁。
Three valid Cryptosporidium species C. meleagridis, C. baileyi and C. galli are found in bird, in which, C. meleagridis is the third species infected humans and have made extensive concerntration worldwide. Based on it, we conducted an investigation on the prevalence of Cryptosporidium spp. in birds (ostrich, duck, chicken and quail) in Henan province, and the results made us understand the prevalence characterization of Cryptosporidium spp. Moreover, the product of egg yolk antibodies was also performed using C. baileyi from chicken. The results provides a basic research for diagnosing and curing cryptosporidiosis by specific IgY antibodies against Cryptosporidium.
From June 2006 to July 2007, six ostrich farms, eight duck farms, eight scale and 37 moderate chicken farms, and 47 quail farms were checked to understand the prevalence of Cryptosporidium spp. The fecal samples were examined using the Sheather’s sugar flotation technique and the modified acid-fast stain method. The results showed that the infection rate of ostrich was 1.7% (14/829), 16.31% (92/564) for the duck, the prevalence of laying hens was 10.57%(163/1542)and the broilers was 3.17%(15/473), and 13.15%(239/1818) for the quail. The statistical data showed that the young ostrich and duck are more susceptible to Cryptosporidium. To chicken, with a more higher infection rate for the 0-30d, 31-60d and 121-180d chicken, however, 60-120d and >180d chichen had a lower prevalence. According to the shape of oocysts, the parasites found in ostrich, duck and chichen was C. baileyi,. However, C. baileyi and C. meleagridis were also discovered in quail.
The oocysts of C. baileyi were purified by amelioration discontinuous sucrose gradients, then eliminated bacterium by filtration and low speed centrifugation, and after several freeze-thawed cycles and treatment by ultrasonic wave, the antigen of whole oocysts were mixed with Freund compiete and incompiete adjuvant to obtain the immune-antigen. Four immune procedure were designed to immunize the five-month-old laying hens, so to choice the best procedure. The results confirmed that the best immunity procedure was gradually augment the dosage of antigen: the dosage of antigen in the 1st time and the 2nd time are all 1×107 oocysts per hen, 3×107 oocysts per hen in the 3rd time and the 4th time, 5×107 oocysts per hen in the 5th time and the 6th time,and 15～20 days arfter the next immunity injection. The egg yolk antibody were examined with indirect immunofluorescence assay and indirect ELISA assay that has been set up, and testified it was against Cryptosporidium. The egg yolk antibodies were purified with water dilution method, and passed through SDS- PAGE electrophoresis:the result shown that egg yolk show more than twelve protein codes,there were 136KDa,74KDa,62KDa,49KDa and 33KDa,and the purified egg yolk show heavy chains were approximately 62 kDa and light chains was about 33 kDa. The reaction between IgY antibody and oocyst was located by immunofluorescence test. The results showed that the reaction intension was the highest in the top of oocysts, and the intension with sporozoite was higher than that of oocyst wall.
引文
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