郑州地区观赏鸟类隐孢子虫病流行病学调查及分子特征研究
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摘要
隐孢子虫病是家养、笼养和野生禽类最常见的寄生虫病之一,已报道隐孢子虫至少可感染30种禽类,主要引起鸟类消化道和呼吸道症状。家养和宠物禽类可传播和流行人兽共患的隐孢子虫虫种,野生禽类和水禽可污染饮用水源和娱乐用水导致人畜感染隐孢子虫,具有重要的公共卫生意义。2008年3月至2009年5月采用饱和蔗糖溶液漂浮法和改良抗酸染色法对郑州市宠物市场宠物鸟类进行隐孢子虫病流行病学调查,采用PCR方法对阳性隐孢子虫分离株基于18Sr RNA基因位点扩增,序列比对结果经系统进化发育分析,鉴定出5种隐孢子虫虫种和基因型。
对31种鸟类共计493份粪便样本进行检查,发现13种鸟类40份为阳性粪便样本,感染率为8.1%,其中7-12月龄鸟类感染率最高,为9.6%,0-6月龄和1岁以上鸟类感染率分别为8.3%和4.7%,1岁以上鸟类仅高冠鹦鹉发现隐孢子虫感染;冬季鸟类隐孢子虫感染率最高,为12.2%;夏季感染率最低,为4.3%,春季和秋季感染率分别为6.5%和11.8%。
对40个隐孢子虫阳性样品基于18S rRNA基因位点进行巢式PCR扩增,对扩增出的35个隐孢子虫样品进行测序,在NCBI核酸序列数据库搜索同源序列,利用Clustal X1.81、Phylip3.67和DNAstar5.0等生物学软件对序列进行比对、同源性分析及构建分子进化树,发现5个隐孢子虫种类/基因型,35个分离株中包括18个C. baileyi、3个C. meleagridis、5个C. galli、3个avian genotype III和6个avian genotype V。
本研究对河南省郑州地区观赏鸟隐孢子虫流行状况进行了调查,并对其分子遗传特征进行研究,鉴定出5种隐孢子虫虫种/基因型,国内首次发现C. galli、avian genotype III和avian genotype V,并发现12种鸟类为隐孢子虫新宿主,掌握了观赏鸟隐孢子虫的分子遗传特征,为今后鸟类隐孢子虫的研究奠定了基础。
Cryptosporidiosis is one of the most common parasitosis in domestic, caged and wild birds. Cryptosporidium has been reported in more than 30 avian species worldwide and the clinical signs of digestive tract and respiratory tract are commonly seen. The domestic and pet avians can transmit zoonotic Cryptosporidium spp., and the wild birds and waterfowl can contaminate the drinking and amusement water, therefore, the public health significance is obvious. Between March 2008 and May 2009, a survey was conducted in pet birds in Zhengzhou pet shops by Shether’s sugar flotation and modified acid-fast stain methods.
A total of 493 fecal samples from 31 species of pet birds were examined, 40 Cryptosporidum-positive samples from 13 birds species were found, with the overall infection rate was 8.1%. The infection rate of Cryptosporidium spp. in < 6 months, 7-12 months, and more than 12 months were 8.3%, 9.6%, and 4.7%. Among the birds aged more than 12 months, only the Cockatiel (Nymphicus hollandicus) was found Cryptosporidium infection. In season, winter had the highest infection (12.2%), the lowest prevalence (4.3%) was observed in summer, whereas the infection rates in spring and autumn were 6.5% and 11.8%, respectively.
A nested-PCR method based on 18S rRNA gene was used to amplify the 40 Cryptosporidium-positive sample, 35 samples were successfully amplified and sequenced. The sequences obtained were conducted alignment analysis, similarity analysis, and phylogenetic relationship analysis by ClustalX 1.81, DNAstar 5.0, and Phylip 3.67 softwares, respectively. Five Cryptosporidium species or genotypes were identified, including 18 C. baileyi, three C. meleagridis, five C. galli, three avian genotype III, and six avian genotype V.
In conclusion, this study investigated the prevalence status of Cryptosporidium spp. in pet birds in Zhengzhou area. And molecular analysis was conducted and identified five Cryptosporidium species/genotypes. This is the first findings of C. galli, avian genotype III, and avian genotype V in China. Moreover, the 12 species of birds represented the new hosts for Cryptosporidium infection. The molecular characterization of pet birds in this study lay a solid foundation for further studing the Cryptosporidium spp. in birds.
对31种鸟类共计493份粪便样本进行检查,发现13种鸟类40份为阳性粪便样本,感染率为8.1%,其中7-12月龄鸟类感染率最高,为9.6%,0-6月龄和1岁以上鸟类感染率分别为8.3%和4.7%,1岁以上鸟类仅高冠鹦鹉发现隐孢子虫感染;冬季鸟类隐孢子虫感染率最高,为12.2%;夏季感染率最低,为4.3%,春季和秋季感染率分别为6.5%和11.8%。
对40个隐孢子虫阳性样品基于18S rRNA基因位点进行巢式PCR扩增,对扩增出的35个隐孢子虫样品进行测序,在NCBI核酸序列数据库搜索同源序列,利用Clustal X1.81、Phylip3.67和DNAstar5.0等生物学软件对序列进行比对、同源性分析及构建分子进化树,发现5个隐孢子虫种类/基因型,35个分离株中包括18个C. baileyi、3个C. meleagridis、5个C. galli、3个avian genotype III和6个avian genotype V。
本研究对河南省郑州地区观赏鸟隐孢子虫流行状况进行了调查,并对其分子遗传特征进行研究,鉴定出5种隐孢子虫虫种/基因型,国内首次发现C. galli、avian genotype III和avian genotype V,并发现12种鸟类为隐孢子虫新宿主,掌握了观赏鸟隐孢子虫的分子遗传特征,为今后鸟类隐孢子虫的研究奠定了基础。
Cryptosporidiosis is one of the most common parasitosis in domestic, caged and wild birds. Cryptosporidium has been reported in more than 30 avian species worldwide and the clinical signs of digestive tract and respiratory tract are commonly seen. The domestic and pet avians can transmit zoonotic Cryptosporidium spp., and the wild birds and waterfowl can contaminate the drinking and amusement water, therefore, the public health significance is obvious. Between March 2008 and May 2009, a survey was conducted in pet birds in Zhengzhou pet shops by Shether’s sugar flotation and modified acid-fast stain methods.
A total of 493 fecal samples from 31 species of pet birds were examined, 40 Cryptosporidum-positive samples from 13 birds species were found, with the overall infection rate was 8.1%. The infection rate of Cryptosporidium spp. in < 6 months, 7-12 months, and more than 12 months were 8.3%, 9.6%, and 4.7%. Among the birds aged more than 12 months, only the Cockatiel (Nymphicus hollandicus) was found Cryptosporidium infection. In season, winter had the highest infection (12.2%), the lowest prevalence (4.3%) was observed in summer, whereas the infection rates in spring and autumn were 6.5% and 11.8%, respectively.
A nested-PCR method based on 18S rRNA gene was used to amplify the 40 Cryptosporidium-positive sample, 35 samples were successfully amplified and sequenced. The sequences obtained were conducted alignment analysis, similarity analysis, and phylogenetic relationship analysis by ClustalX 1.81, DNAstar 5.0, and Phylip 3.67 softwares, respectively. Five Cryptosporidium species or genotypes were identified, including 18 C. baileyi, three C. meleagridis, five C. galli, three avian genotype III, and six avian genotype V.
In conclusion, this study investigated the prevalence status of Cryptosporidium spp. in pet birds in Zhengzhou area. And molecular analysis was conducted and identified five Cryptosporidium species/genotypes. This is the first findings of C. galli, avian genotype III, and avian genotype V in China. Moreover, the 12 species of birds represented the new hosts for Cryptosporidium infection. The molecular characterization of pet birds in this study lay a solid foundation for further studing the Cryptosporidium spp. in birds.
引文
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[3] Sréter T, Varga I. Cryptosporidiosis in birds-a review[J]. Vet Parasitol, 2000, 87(4): 261-279.
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[5] Graczyk TK, Majewska AC, Schwab KJ.The role of birds in dissemination of human waterborne enteropathogens[J]. Trends Parasitol, 2008, 24(2):55-59.
[6] Ng J, Pavlásek I, Ryan U. Identification of novel Cryptosporidium genotypes from avian hosts[J]. Appl Environ Microbiol, 2006, 72(12):7548-7553.
[7] Lee HY, Stephen A, Sushela D,etal.Detection of protozoan and bacterial pathogens of public health importance in faeces of Corvus spp. (large-billed crow) [J]. Trop Biomed. 2008 ,25(2):134- 139.
[8] Xiao L, Fayer R, Ryan U, Upton SJ. et al.Cryptosporidium taxonomy : recent advance and implications for public health[J]. Clin Microbiol Rev, 2004, 17 (1):72-97.
[9] Plutzer J, Karanis P. Genetic polymorphism in Cryptosporidium species: An update[J]. Vet Parasitol,2009, 165(3-4):187-199.
[10] Lv C, Zhang L, Wang R, etal. Cryptosporidium spp. in Wild, Laboratory, and Pet Rodents in China:Prevalence and Molecular Characterization[J].Appl Environ Microbiol,2009,75(24): 7692 -7699.
[11] Slavin D. Cryptosporidium meleagridis (sp. nov.)[J]. J Comp Pathol, 1955, 65(3):262-266.
[12] Current WL, Upton S, Haynes TB .The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens[J]. J Protozool, 1986, 33(2):289-296.
[13] Pavlásek I. Cryptosporidia: biology, diagnosis, host spectrum specificity and the environment [J]. Remedia-Klin Mikrobiol,1999,3:290-301.
[14] Ryan U.Cryptosporidium in birds, fish and amphibians[J].Exp Parasitol,2010,124(1):113- 120.
[15] Meireles MV, Soares RM, dos Santos MM, etal. Biological studies and molecular characterization of a Cryptosporidium isolate from ostriches (Struthio camelus)[J]. J Parasitol,2006,92(3): 623-626.
[16] Jellison KL, Distel DL, Hemond HF, et al. Phylogenetic analysis of the hypervariable region of t he 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes[J]. Appl Environ Microbiol, 2004, 70(1):452-458.
[17] Morgan UM, Monis PT, Xiao L,et al. Molecular and phylogenetic characterisation of Cryptosporidium from birds[J].Int J Parasitol, 2001, 31(3):289-296.
[18] Ryan U, Xiao L, Read C, et al. Identification of novel Cryptosporidium genotypes from the Czech Republic[J]. Appl Environ Microbiol,2003,69(7):4302-4307.
[19] Zhou L, Kassa H, Tischler ML, et al. Host-adapted Cryptosporidium spp. in Canada geese (Branta canadensis)[J]. Appl Environ Microbiol,2004, 70(7): 4211-4215.
[20] Xiao L, Fayer R, Ryan U, et al. Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health[J]. Clin Microbiol Rev, 2004, 17(1):72-97.
[21]张龙现,宁长申,蒋金书.鸭源隐孢子虫种类鉴定及对鸡鸭致病性研究[J].中国农业大学学报,2002,7(3):107-112.
[22] Cheadle MA, Toivio-Kinnucan M, Blagburn BL. The ultrastructure of gametogenesis of Cryptosporidium baileyi (Eimeriorina; Cryptosporidiidae) in the respiratory tract of broiler chickens (Gallus domesticus) [J]. J Parasitol, 1999,85(4):609-615.
[23]高庚渠,齐萌,王岩,等.不同禽源贝氏隐孢子虫分离株对鹌鹑致病性比较研究[J].畜牧兽医学报,2010,41(2):200-207.
[24] Lindsay DS, Blagburn BL. Cryptosporidiosis in birds[M]. Boca Raton: CRC Press, 1990, 133-148.
[25] Chvala S, Fragner K, Hackl R, et al. Cryptosporidium infection in domestic geese (Anser anser f. domestica) detected by in situ hybridization[J]. J Comp Pathol, 2006,134(2-3): 211-218.
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