Cryptosporidiosis is an important zoonosis caused by the Cryptosporidium species. To develop a PCR diagnostic kit for molecular detection and differential diagnosis of Cryptosporidium spp., a portion of ITS-1 sequence...Cryptosporidiosis is an important zoonosis caused by the Cryptosporidium species. To develop a PCR diagnostic kit for molecular detection and differential diagnosis of Cryptosporidium spp., a portion of ITS-1 sequence of Cryptosporidium. andersoni was chosen as the target DNA for designing the species-specific primers （ZRQF/ZR）. The kit components were determined after the PCR amplification conditions were serially optimized. A series of tests were conducted in the specificity, sensitivity, stability, reproducibility, and stored period of the kit, respectively. The results showed that only C. andersoni were amplified specific band of about 500 bp, while Cryptosporidium. parvum, Cryptosporidium. baileyi, Eimeria sp of dairy cow, Toxoplasma gondii, Eimeria sp of pig, Ascaris suum, Cyclospora sp, and E. coli could not be amplified. 254 oocysts of C. andersoni was the lowest number that could be detected using the kit. The kit worked well after being stored at room temperature, 4 and -20℃ for nine months. Fecal specimens, which were collected from a total of 243 calves on four different dairy farms in Guangdong Province, China, and one dairy farm in Henan Province, China, were examined using the kit; the positive rate of the kit was 2-13% higher than that of the routine methods. The results indicated that the kit can detect fecal samples faster, more sensitively, and conveniently, and can provide a useful tool for the identification and differentiation of C. andersoni from the other Cryptosporidium species; it also has implications for further studies on molecular epidemiology and differential diagnostics of cryptosporidiosis in animals.展开更多
Objective:To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between counlries depending on the terrain surrounding livestock farms and investigati...Objective:To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between counlries depending on the terrain surrounding livestock farms and investigations in Indonesia have never been performed.Methods:Fecal samples from cattle at 35 farms in 7 districts in West Java,Indonesia,has been examined using the floatation or sedimentation methods,and a immunofluorescence assay and experimentally inoculation to mice for Cryptosporidium or Giardia spp.Results:153 of 394 examined cattle(38.8%)were infected with gastrointestinal parasites.The prevalence of Eimeria spp.,Nematoda spp.(including Oesophagustomum and Bunostomum-like),Fasciola gigantica and Paramphistomum spp.was 22.4%,11.2%,12.5%and 3.8%,respectively.Cryptosporidium andersoni(C.andersoni)was also found in two samples.One isolate of this parasite was confirmed to be transmitted to mice,in contrast to the isolates from other countries.Conclusions:although this survey is preliminary,the results shows that the infection of gastrointestinal parasites in Indonesia was not high,but these infected cattle could be as a potential source leading to economic losses in livestock production.展开更多
根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便...根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便进行了检测。结果表明,此次建立的real timePCR对微小隐孢子虫和安氏隐孢子虫均能扩增出曲线,且其他寄生虫(鸡贝氏隐孢子虫、刚地弓形虫、犬新孢子虫)和大肠杆菌均未检测到;标准基因组DNA的检测阈值达到5个拷贝,牛粪中卵囊的最低检测量为每克粪便5个卵囊,乳牛粪便阳性率为15%(6/40)。表明,建立的荧光定量PCR快速、特异、敏感,可用于乳牛隐孢子虫病的流行病学调查。展开更多
文摘Cryptosporidiosis is an important zoonosis caused by the Cryptosporidium species. To develop a PCR diagnostic kit for molecular detection and differential diagnosis of Cryptosporidium spp., a portion of ITS-1 sequence of Cryptosporidium. andersoni was chosen as the target DNA for designing the species-specific primers （ZRQF/ZR）. The kit components were determined after the PCR amplification conditions were serially optimized. A series of tests were conducted in the specificity, sensitivity, stability, reproducibility, and stored period of the kit, respectively. The results showed that only C. andersoni were amplified specific band of about 500 bp, while Cryptosporidium. parvum, Cryptosporidium. baileyi, Eimeria sp of dairy cow, Toxoplasma gondii, Eimeria sp of pig, Ascaris suum, Cyclospora sp, and E. coli could not be amplified. 254 oocysts of C. andersoni was the lowest number that could be detected using the kit. The kit worked well after being stored at room temperature, 4 and -20℃ for nine months. Fecal specimens, which were collected from a total of 243 calves on four different dairy farms in Guangdong Province, China, and one dairy farm in Henan Province, China, were examined using the kit; the positive rate of the kit was 2-13% higher than that of the routine methods. The results indicated that the kit can detect fecal samples faster, more sensitively, and conveniently, and can provide a useful tool for the identification and differentiation of C. andersoni from the other Cryptosporidium species; it also has implications for further studies on molecular epidemiology and differential diagnostics of cryptosporidiosis in animals.
基金partly supported by the JICA Project of Capacity Development of Animal Health Laboratory and Grants-in-Aid for Scientifie Research from the Ministry of Education.Culture.Sports.Science and Technology.Japan(No.22700773 to M.M.)
文摘Objective:To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between counlries depending on the terrain surrounding livestock farms and investigations in Indonesia have never been performed.Methods:Fecal samples from cattle at 35 farms in 7 districts in West Java,Indonesia,has been examined using the floatation or sedimentation methods,and a immunofluorescence assay and experimentally inoculation to mice for Cryptosporidium or Giardia spp.Results:153 of 394 examined cattle(38.8%)were infected with gastrointestinal parasites.The prevalence of Eimeria spp.,Nematoda spp.(including Oesophagustomum and Bunostomum-like),Fasciola gigantica and Paramphistomum spp.was 22.4%,11.2%,12.5%and 3.8%,respectively.Cryptosporidium andersoni(C.andersoni)was also found in two samples.One isolate of this parasite was confirmed to be transmitted to mice,in contrast to the isolates from other countries.Conclusions:although this survey is preliminary,the results shows that the infection of gastrointestinal parasites in Indonesia was not high,but these infected cattle could be as a potential source leading to economic losses in livestock production.
文摘根据GenBank中登录的隐孢子虫小亚基rRNA基因序列设计1对引物,并以标准基因组DNA为模板,初步建立了检测乳牛微小隐孢子虫和安氏隐孢子虫的SYBR Green real time PCR方法,并对乳牛微小隐孢子虫和安氏隐孢子虫阳性样品和上海40份乳牛粪便进行了检测。结果表明,此次建立的real timePCR对微小隐孢子虫和安氏隐孢子虫均能扩增出曲线,且其他寄生虫(鸡贝氏隐孢子虫、刚地弓形虫、犬新孢子虫)和大肠杆菌均未检测到;标准基因组DNA的检测阈值达到5个拷贝,牛粪中卵囊的最低检测量为每克粪便5个卵囊,乳牛粪便阳性率为15%(6/40)。表明,建立的荧光定量PCR快速、特异、敏感,可用于乳牛隐孢子虫病的流行病学调查。
