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1.
为快速检测并准确鉴别奶牛隐孢子虫种,以隐孢子虫18S rRNA基因的特殊区域为基础,设计内、外引物,并根据软件分析确定相应的内切酶EcoT141,采用Nested PCR-RFLP方法进行虫种种型鉴别分析。在Nested PCR两次PCR反应中,以微小隐孢子虫（Cryptos poridium parvum,C．p）和安氏隐孢子虫（Cryptosporidium andersoni,C．an）卵囊提取的DNA为模板,均能扩增出长约800bp和500bp的明亮条带,且特异性强,其他虫种不能扩增出条带,该方法最低可检测到5个卵囊／g粪便;对于由内引物扩增出的500bp的条带,C.an的PCR产物能被内切酶EcoT141酶切,酶切后的片段分别为416bp和92bp,C.p的PCR产物不能被此酶酶切。用所建立的Nested PCR-RFLP法对上海奶牛389头和进口奶牛200头的共计589份粪样进行检测,Nested PCR的结果表明上海奶牛和进口奶牛的隐孢子虫阳性率分别为19．02％和3．5％,RFLP的结果表明上海奶牛感染的主要是Cp和C．an,进口奶牛感染的主要是C．p。研究结果表明,本研究建立的检测奶牛粪便中的隐孢子虫的NestedPCR-RFLP法,可用于奶牛隐孢子虫流行病学调查并有效鉴别奶牛隐孢子虫种。 相似文献
2.
采进口奶牛粪样200份,收集每份样品中的卵囊液,采用巢式PCR检测隐孢子虫(Cryptosporidium)18S rRNA基因,并用RFLP进行虫种鉴定,将目的片段克隆到pEGM-T easy vector,测序并进行同源性分析以进一步佐证虫种鉴定结果。结果表明,进口奶牛隐孢子虫阳性率为3.5%(7/200),514bp的目的片段不能被EcoT14 Ⅰ酶切。测序分析表明,获得的18S rRNA基因序列与NCBI上公布的微小隐孢子虫(C.parvum)相应序列同源性高达99.4%~100%,与安氏隐孢子虫(C.andersoni)同源性仅为91.1%。说明进口牛粪样中检测出的隐孢子虫为微小隐孢子虫。 相似文献
3.
为阐明河南区域隐孢子虫分子流行病学特点,用PCR技术扩增分离虫株的18S rRNA基因全序列和HSP70基因序列,并对扩增片段进行测序。用PAUP 4.0和TREEPUZZLE 4.1构建进化树,试图从分子水平证明河南省不同地区不同宿主来源隐孢子虫的遗传特征,以阐明隐孢子虫病的分子流行病学特点。通过18S rRNA基因全序列和HSP70基因序列分析,其结果：河南人源隐孢子虫分离株为Cryptosporidium parvum鼠基因型;河南鹿源隐孢子虫分离株为C. parvum鹿基因型;河南猪源隐孢子虫的2个分离株均为C. parvum猪基因I型,即C. suis;河南鹌鹑源的隐孢子虫2个分离株分别为C. baileyi和C. meleagridis;河南乌鸡源隐孢子虫和鸵鸟源隐孢子虫分离株均为C. baileyi;河南牛源隐孢子虫分离株为C.andersoni。 相似文献
4.
应用巢式聚合酶链反应(Nested PCR)建立了一种检测隐孢子虫(Cryptosporidium)的方法。试验中隐孢子虫卵囊纯化采用庶糖密度梯度离心法,以液氮-热水浴反复冻融及酚-氯仿抽提冷乙醇沉淀法制备模板DNA,根据隐孢子虫18S rRNA序列高度保守区设计2对引物,建立Nested PCR诊断方法。该方法特异性强,可检出牛源微小隐孢子虫(C.parvum)、羊源微小隐孢子虫(C.parvum)、牛源安氏隐孢子虫(C.andersoni)、鸡源贝氏隐孢子虫(C.baileyi)及猪源隐孢子虫(C.suis);敏感性高,该方法最低核酸DNA检测量达到10fg。初步应用结果表明,所建立的Nested PCR方法适合于隐孢子虫病的诊断和分子流行病学调查。 相似文献
5.
奶牛隐孢子虫病流行病学调查及初生犊牛感染试验 总被引：5，自引：0，他引：5
应用饱和糖溶液漂浮法和改良抗酸染色法调查郑州、商丘和洛阳3个地区的5个奶牛场、2个专业村的582份粪样，查出阳性样品64份，隐孢子虫总阳性率11％（64／582），发现两种不同形态的卵囊，根据其形态结构等特点鉴定为小球隐孢子虫（C．parvum）和安氏隐孢子虫（C．andersoni）。其中2个场奶牛感染安氏隐孢子虫，有1个场的奶牛感染小球隐孢子虫，且感染强度较小。犊牛感染率较育成牛、成年牛高。并进行小球隐孢子虫分离株对初生犊牛的致病性试验，其结果为潜隐期7天，排卵囊高峰出现在感染后第16天，高峰期5天。剖检后消化道黏膜经抗酸染色鉴定，仅在回肠中段发现卵囊。 相似文献
6.
安氏隐孢子虫PCR检测方法的建立 总被引：1，自引：1，他引：1
经BLAST检索,以HSP70基因设计一对引物(5'-CAATCGAATTGGATTCTTTGTC-3'和5'-CACCTTCAAAT-ACTTGAATAAGT-3')对奶牛安氏隐孢子虫进行了PCR试验.结果显示所建立的PCR检测方法只能特异扩增隐孢子虫GD株DNA,而对照样本如微小隐孢子虫、弓形虫、圆孢子虫、纤毛虫、肝片吸虫、血矛线虫、莫尼茨绦虫、牛粪便以及大肠杆菌均为阴性;通过对6个浓度梯度的虫体DNA进行PCR反应,结果表明当样本中含有445个隐孢子虫卵囊的DNA时,即可扩增产生清晰可辩的条带.测得该序列长度为494bp,序列分析为牛型C.andersoni.表明该引物能特异扩增C.andersoni,敏感性较高,适合于奶牛安氏隐孢子虫的检测. 相似文献
7.
隐孢子虫病免疫学研究进展 总被引：2，自引：0，他引：2
隐孢子虫病（Cryptosporidiosis）是一种全球性的人畜共患病。其病原体为一种寄生性原虫，即隐孢子虫。隐孢子虫属于真球虫目、隐孢科、隐孢属，目前认为其有效种有6个，即感染鱼类的鼻隐抱子虫（C．nasorum）、感染爬行类的响尾蛇隐池子虫（C．crotati）、感染鸟类的火鸡隐抱子虫（C．meleagridis）和贝氏隐抱子虫（C．baileyi）、感染哺乳动物的鼠隐抱子虫（C．muris）和小隐子虫（C．parvum）。现已发现隐抱子虫可寄生于多种脊椎动物，下面就其引起的局部病理变化、免疫器官的反应、免疫反应和免疫诊断等方面述之。1寄生部位与所引起… 相似文献
8.
利用18S rRNA巢式聚合酶链反应(Nested PCR)-限制片段长度多态性(Restriction fragment length polymorphism,RFLP)鉴定吉林、大庆地区牛源隐孢子虫分离株.采取吉林、大庆地区断奶前犊牛粪便,提取DNA后经18S rRNA基因巢式PCR扩增,扩增产物测序后用Blast和MEGA4.0软件进行同源性和系统发育树分析.同时扩增产物分别用Ssp Ⅰ、Vsp Ⅰ和Mbo Ⅱ酶切后进行RFLP分析.通过18S rRNA基因PCR-RFLP分析和测序比对分析表明,吉林分离株包括2种隐孢子虫,分别为C.bovis和C.ryanae,大庆分离株包括3种,分别为C.boris、C.ryanae和C.andersoni. 相似文献
9.
PCR-RFLP鉴定隐孢子虫种类研究 总被引：15，自引：2，他引：15
为快速、准确鉴别人畜隐孢子虫种类，建立了巢式PCR扩增隐孢子虫18S rRNA基因的特殊区域，扩增片段测序结果表明：安徽牛源分离株(Cryptosporidium muris)781bp；北京鸡源分离株(C.baileyi)776bp；北京牛源分离株(C.muris)781bp；河南牛源分离株(C.muris)725bp；长春牛源分离株(C.muris)776bp；宁夏鸡源分离株(C.baileyi)725bp.该片段位于18SrRNA全序列271～1103bp之间。使用Ssp I限制性内切酶消化发现C.muris产生418～420bp和305～363bp两个片段，C.baileyi产生544～545bp和185～231bp两个片段。所检测的6个分离株可以显著区分为C.muris和C.baileyi两个种，所建立的PCR-RFLP可以有效鉴别隐孢子虫种类。 相似文献
10.
11.
Gottlieb Y Markovics A Klement E Naor S Samish M Aroch I Lavy E 《Veterinary parasitology》2011,177(3-4):378-382
A total of 750 faecal samples of dairy calves at up to 2 months of age kept in various housing systems were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method. DNA was extracted from Cryptosporidium positive samples and from 150 randomly selected microscopically negative samples. Nested PCR was performed to amplify the partial SSU rRNA gene of Cryptosporidium that was subsequently digested by SspI, VspI and MboII restriction enzymes to determine the present Cryptosporidium species and genotype. In addition, the samples characterized as Cryptosporidium parvum were subsequently analyzed at the GP60 gene to determine the distribution of zoonotic subtypes. Sequence analyses and RFLP identified C. parvum in 137, Cryptosporidium andersoni in 21 and Cryptosporidium bovis in 3 samples. Neither mixed infections nor Cryptosporidium ryanae was detected. Sequencing of the GP60 gene from C. parvum-positive samples revealed all five subtypes of family IIa (A15G2R1, A16G1R1, A22G1R1, A18G1R1, and A15G1R1). The obvious management-associated distribution of Cryptosporidium spp. was demonstrated. Direct contact with adult animals was found to be a risky factor for C. andersoni and C. bovis infection. IIaA15G2R1 and IIaA16G1R1 were detected as major subtypes, whereas only the IIaA16G1R1 subtype was found in animals kept in boxes. Three of the five detected subtypes were previously associated with human cryptosporidiosis, and moreover, the IIaA15G1R1 subtype, previously reported in humans only, was detected in calves for the first time. 相似文献
12.
Dung samples were collected from dairy calves of south Indian states viz., Andhra Pradesh, Karnataka, Kerala, Tamil Nadu and union territory, Puducherry and are subjected to nested polymerase chain reaction (PCR) targeting 18S rRNA gene for detection of Cryptosporidium infection. Of the 459 dung samples screened 182 were found positive with a prevalence of 39.65%. Highest prevalence of Cryptosporidium was observed in Puducherry (86.67%) and lowest in Kerala (17.65%). Genotyping by PCR-restriction fragment length polymorphism (RFLP) and sequence analysis revealed the presence of all the four major Cryptosporidium species of cattle viz., Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium parvum and Cryptosporidium bovis. C. andersoni was widely distributed in calves of Tamil Nadu, Karnataka and Puducherry whereas in Andhra Pradesh C. ryanae was the major species. Of the 64 samples subjected to PCR-RFLP, 39 (60.94%) could be classified as C. andersoni, 18 (28.13%) as C. ryanae, 4 (6.25%) as C. parvum and 3 (4.69%) were confirmed as C. bovis. The results were also confirmed by sequencing of 19 Cryptosporidium DNA samples. 相似文献
13.
Robinson G Thomas AL Daniel RG Hadfield SJ Elwin K Chalmers RM 《Veterinary parasitology》2006,142(1-2):163-167
Bovine cryptosporidiosis is usually an acute diarrhoeal disease of young calves caused by Cryptosporidium parvum. However, chronic infection with Cryptosporidium andersoni has been associated with gastritis, reduced milk yield and poor weight gain in adult cattle. Here we describe the first genetic confirmation and characterisation of C. andersoni from cattle in the United Kingdom and its sample prevalence within a dairy herd. Oocysts measured 7.5+/-0.4 microm x 5.5+/-0.4 microm (7.0-8.5 microm x 4.5-6.5 microm) with a length-to-width ratio of 1.37 (1.08-1.60). The within-herd sample prevalence was 16% (95% confidence intervals=10.4-21.6%). Nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequence analysis of the small subunit rDNA was used to confirm the species and characterise the isolates. Due to the lack of overt, acute, clinical symptoms, the incidence, prevalence and importance of this parasite is probably currently underestimated in cattle in the UK. The potential for zoonotic transmission is unknown. 相似文献
14.
Maikai BV Umoh JU Kwaga JK Lawal IA Maikai VA Cama V Xiao L 《Veterinary parasitology》2011,178(3-4):241-245
Despite numerous molecular epidemiologic studies of cryptosporidiosis in dairy cattle in industrialized countries, there are very few studies on the diversity and public health significance of Cryptosporidium species in native cattle in developing countries. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium spp. in 194 fecal specimens from 2 to 365 days old calves in 20 White Fulani and Sokoto Gudali herds in Nigeria. Thirty one (16.0%) of the specimens were positive for Cryptosporidium. Restriction digestion of the PCR products showed the presence of Cryptosporidium bovis (7.2%), Cryptosporidium ryanae (4.1%), Cryptosporidium andersoni (2.5%), and concurrent occurrence of C. bovis and C. ryanae (1.5%), and C. bovis and C. andersoni (0.5%). There were no significant differences (p>0.05) in Cryptosporidium infection rates by sex, herd location, management system, breed of calves, or fecal consistency. However, calves 180 days or younger had a higher infection rate of Cryptosporidium than older calves (p=0.034). Likewise, younger calves also had higher occurrence of C. bovis and C. ryanae (p=0.022). The absence of zoonotic Cryptosporidium parvum in the calves studied suggests that native breeds of cattle may not be important in the transmission of human cryptosporidiosis in Kaduna State, Nigeria. 相似文献
15.
Zheng J Ren W Pan Q Wang Q Elhag IA Li J Li M Gong P Liu Y Zhang X 《Veterinary parasitology》2011,179(1-3):7-13
Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development. 相似文献
16.
Age-related and housing-dependence of Cryptosporidium infection of calves from dairy and beef herds in South Bohemia, Czech Republic 总被引：1，自引：0，他引：1
Data of the prevalence, age-related and housing-dependence of naturally acquired cryptosporidiosis on 11 dairy and 11 beef farms in South Bohemia (Czech Republic) were collected. The farms were visited over four consecutive years (from 2002 to 2005). The prevalence of Cryptosporidium in pre-weaned (animals until second month of age) and post-weaned (animals from the third month of age) calves was determined. A total of 7001 faecal samples were collected, concentrated by Sheather's floatation method and stained by aniline-carbol-methyl violet. All samples were examined by light microscopy. Cryptosporidium parvum and C. andersoni oocysts were differentiated on morphological criteria. Of the 7021 specimens, 1814 (25.8%) were positive for Cryptosporidium oocysts; 561 samples (8%) for C. parvum and 1253 (17.8%) for C. andersoni. Pre-weaned dairy calves had higher infection levels of C. parvum than pre-weaned beef calves. The prevalence of C. parvum ranged from 1.4 to 56.5% on dairy farms. Only three cases of C. parvum oocysts shedding in pre-weaned calves on beef farms were found. Only one case of C. andersoni infection in pre-weaned calves was detected and no infections of C. parvum in post-weaned calves were found. The prevalence of C. andersoni reached 35.5% on dairy farms and 61.7% on beef farms. Calves that were on pasture all year long, had a lower probability of C. andersoni infection than those calves kept in a cowshed during the winter season. 相似文献
17.
Recent studies have identified the novel, host adapted Cryptosporidium bovis and the deer-like genotype in dairy cattle from farms in the United States, China, India and Europe. This novel species and genotype appear to be more prevalent in older, post-weaned dairy cattle than previously thought. However, little information is available on their prevalence in beef cow-calf operations. In the present study, we determined the prevalence of Cryptosporidium species in 98 calves (6-8 months old) and 114 cows (>2 years old) in seven beef cow-calf herds in western North Dakota. DNA was extracted from fecal samples and Cryptosporidium spp. were identified by amplification of the 18S rRNA gene followed by sequencing or RFLP analysis. All seven herds tested positive for Cryptosporidium. Overall, 43/212 (20.3%) animals were positive. Only five of these positives were from cows. C. bovis, the deer-like genotype and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. C. parvum was not identified in any of the positive samples. C. bovis, the deer-like genotype and C. andersoni were detected in 6/7, 5/7 and 2/7 herds, respectively. C. bovis and the deer-like genotype were primarily detected in calves, while C. andersoni was only detected in cows. Six isolates could not be typed. These results show a relatively high prevalence of C. bovis and the deer-like genotype in 6-8-month-old beef calves compared to cows older than 2 years in the seven herds studied. 相似文献