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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.
OBJECTIVE: To isolate and speciate Cryptosporidium DNA from fecal samples obtained from dairy cattle in New York State and identify factors associated with whether cattle were shedding Cryptosporidium parvum versus Cryptosporidium bovis. DESIGN: Cross-sectional study. SAMPLE POPULATION: 115 fecal samples positive for DNA coding for the Cryptosporidium 18S rRNA gene from dairy cattle in New York State. PROCEDURES: A PCR assay was used to amplify DNA from fecal samples; amplification products were submitted for bidirectional DNA sequencing. Logistic regression was used to test for associations between various host factors and Cryptosporidium spp. RESULTS: 70 of the 115 (61%) fecal samples were found to have C parvum DNA, 42 (37%) were determined to have C bovis DNA, and 3 (3%) were found to have C parvum deer-type DNA. The presence of diarrhea at the time of fecal sample collection, oocyst count, and breed were associated with whether cattle were infected with C parvum or C bovis, with animals more likely to be infected with C parvum if they had diarrhea, had a high oocyst count, or were Holsteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that C parvum and C bovis can be isolated from dairy cattle in New York State and that various factors affect whether cattle infected with Cryptosporidium spp are infected with C parvum or C bovis. Findings also lend credence to the theory that C bovis may be more host adapted and thus less pathogenic to dairy cattle than C parvum.  相似文献   

3.
A preliminary molecular epidemiological study was carried out to investigate the utility of the Cryptosporidium oocyst wall protein (COWP) gene in the detection of Cryptosporidium oocysts in fecal samples. A nested polymerase chain reaction (PCR) approach using COWP gene primers was adopted for this purpose. Fecal samples were spiked with each of 1, 10, and 100 oocysts of C. parvum, four samples for each number, and the DNA was extracted from each sample using a glassbead method. The presence of oocysts was determined using the nested PCR with COWP gene primers, and the limit of detection of oocysts by the PCR was determined. The limit of detection was 100 oocysts spiked in 1 ml of fecal material (50% sold material) (four positives/four samples tested). Seventy-five percent of DNA extracted samples spiked with 1 and 10 oocysts was positive by the PCR (three positives/four samples tested). Based on this, small sample size using the COWP gene primers with a nested PCR analysis could reliably identify infected animals rather conveniently and accurately.  相似文献   

4.
OBJECTIVE: To determine the risk posed by Cryptosporidium parvum and Cryptosporidium hominis from dairy cattle in the New York City watershed (NYCW). SAMPLE POPULATION: Samples from cattle at risk for shedding Cryptosporidium organisms on randomly selected dairy farms in the NYCW. PROCEDURE: Feces were collected for 4 years from calves at risk for infection on 37 dairies. Oocysts were detected by use of centrifugation concentration-flotation microscopy. The DNA was directly isolated from fecal samples and used to amplify fragments of the small subunit ribosomal RNA and thrombospondin-related adhesion protein C-2 genes by use of nested polymerase chain reaction assays. Small subunit ribosomal RNA fragments were restriction digested by the enzyme Vspl and thrombospondin-related adhesion protein C-2 fragments were digested by Eco91l to distinguish between C hominis (formerly known as genotype 1) and C parvum (formerly known as genotype 2). RESULTS: Of 437 fecal samples examined, 214 contained oocysts. Amplicons were generated for 200 samples. We can be certain, with 95% confidence, that cattle in the NYCW did not harbor C hominis. CONCLUSIONS AND CLINICAL RELEVANCE: Cryptosporidium infections in cattle are under examination because of the potential contamination of public waters by manure. Although cattle may be the source of zoonotic infection via C parvum, they pose little risk for C hominis (the strain commonly isolated from humans in waterborne outbreaks of disease). Other sources of oocysts should be considered when investigating outbreaks attributable to contaminated urban drinking water because cattle pose only a small risk via shedding of C hominis.  相似文献   

5.
以隐孢子虫(Cryptosporidium spp.)18S rRNA为靶基因,通过巢氏PCR检测发现在采集的101份新鲜粪便样品中18份样本为阳性。不同地区羊场的隐孢子虫感染率分别为：李集镇36%(18/50),新安镇0%(0/30)、堆沟港镇0%(0/21)未检出隐孢子虫。但通过对4羊场的分析发现,有2个羊场(50%)为隐孢子虫感染阳性,且不同的羊场感染率差异显著,因此单纯的以地区来评价隐孢子虫的感染率,是值得商榷的。山羊隐孢子虫的感染率为33.3%,湖羊隐孢子虫的感染率为2%。2～6月龄的育肥羊隐孢子虫的感染率为36%,6～10月龄的育成羊(0%)。对检测为阳性的样品进行了隐孢子虫18S rRNA基因片段序列分析,发现18个样品全部为肖氏隐孢子虫(Cryptosporidium xiaoi),不存在泛在隐孢子虫(Cryptosporidium ubiquitum)。在检测隐孢子虫感染阳性的1个山羊场和1个羊湖羊场均存在肖氏隐孢子虫感染,不存在泛在隐孢子虫,更未发现肖氏隐孢子虫和泛在隐孢子虫的混合感染。目前的数据提示肖氏隐孢子虫对2～6月龄山羊(33.3%)和湖羊(2%)具有更高的...  相似文献   

6.
A nested multiplex polymerase chain reaction (PCR) approach was adopted for the simultaneous detection of 4 human infective genotypes of the protozoan parasite Cryptosporidium. Specific PCR primers were designed for the heat shock protein 70 gene of 2 genotypes of Cryptosporidium parvum (human and bovine types), Cryptosporidium canis, and Cryptosporidium felis. These 4 genotypes have all been found in human fecal samples. The primers amplified DNA fragments of specific sizes, each representing a unique genotype. The limit of detection of the method was found to vary between 10 and 100 oocysts per 1 ml fecal material. There appeared to be no cross-reactivity with other organisms commonly present in feces and soil, and the approach has a high specificity. The rapid identification of various human infective Cryptosporidium isolates is a part of the authors' long-term aim of determining the routes of infection with oocysts and thereby increase their epidemiological understanding of Cryptosporidium infection in humans and animals.  相似文献   

7.
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   

8.
OBJECTIVE: To evaluate 2 rapid, patient-side assays for detection of Cryptosporidium parvum in feces from neonatal calves with diarrhea. DESIGN: Diagnostic test evaluation Sample Population-Fecal samples from 96 neonatal (1 to 30 days old) calves with diarrhea. PROCEDURE: Results of the rapid assays were compared with results of microscopic examination of fecal smears that had been stained with diamant fuchsin stain. RESULTS: One of the rapid assays correctly identified 56 of 62 (90%) fecal samples positive for C. parvum oocysts and 33 of 34 (97%) fecal samples negative for oocysts. The other assay correctly identified 53 of 62 (85%) fecal samples positive for oocysts and 33 of 34 (97%) fecal samples negative for oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that these 2 rapid assays are accurate when used to detect C. parvum in fecal samples from neonatal calves with diarrhea.  相似文献   

9.
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   

10.
Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.  相似文献   

11.
Lin Z  Zhang Y  Zhang H  Zhou Y  Cao J  Zhou J 《Veterinary parasitology》2012,185(2-4):296-300
Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.  相似文献   

12.
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13.
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   

14.
套式PCR检测奶牛粪便中隐孢子虫   总被引：8，自引：1，他引：7  
为了检测样品中的微量隐孢子虫，从含有不同数量隐孢子虫卵囊的奶牛粪便中，直接提取DNA用作初始PCR（Initial-PCR）模板，以稀释的初始PCR的产物为模板进行套式PCR（Nested-PCR），用两对人工合成寡核苷酸分别作为两PCR的引物，扩增大小分别为540pb、258pb的特异片段。PCR产物经电泳鉴定，可从阳性粪便标本DNA抽提物中扩增出目的片段，而阴性对照不能扩增目的片段；初始PCR、套式PCR的敏感小生最低可分别检测到含卵囊100、5个／g粪便。初步应用结果表明某奶牛场的奶牛自然感染率为16.4％。试验表明套式PCR的敏感性比普通PCR约高100倍，能用于奶牛隐孢子虫感染情况的调查。  相似文献   

15.
OBJECTIVES: To determine whether sampling feces off the ground replicates prevalence estimates for specific pathogens obtained from fecal samples collected per rectum of adult cows, and to determine characteristics of feces on the ground (fecal pats) that are associated with subsequent identification of Campylobacter spp, Cryptosporidium parvum, and Giardia duodenalis. ANIMALS: A random sample of adult beef cattle from 25 herds located throughout California. PROCEDURE: 1,115 rectal and ground fecal samples were obtained. Samples were submitted for culture of Campylobacter spp and examined, using a direct fluorescent antibody assay, to detect C parvum oocysts and G duodenalis cysts. Characteristics of fecal pats, such as volume and consistency, were recorded. RESULTS: Prevalence of Campylobacter spp was 5.0% (20/401) for rectal fecal samples, which was significantly greater than prevalence determined for ground fecal samples (2/402; 0.5%). Most isolates were C jejuni subsp jejuni. Prevalence of C parvum was higher in rectal fecal samples (6/557; 1.1%) than in ground fecal samples (1/558; 0.2%), but this difference was not significant. Prevalence of G duodenalis did not differ for rectal (36/557; 6.5%) versus ground (26/558; 4.7%) fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of ground fecal samples may not accurately indicate the prevalence of Campylobacter spp or C parvum in cattle but may reflect prevalence of G duodenalis. Differences in prevalence estimates between the 2 methods suggest inactivation of pathogens in feces after cattle have defecated. Prevalence estimates generated by evaluation of ground fecal samples, however, may more accurately estimate environmental pathogen burden.  相似文献   

16.
A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of S?o Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C. parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C. parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife.  相似文献   

17.
A nested polymerase chain reaction (PCR) using primers from the LipL32 sequence of Leptospira spp. was used to detect shedding of pathogenic leptospires in urine from naturally infected cattle. Amplicons (497bp) were obtained from 21 pathogenic reference serovars belonging to four species (L. interrogans, L. borgpetersenii, L. santarosai, L. kirschneri). DNA was amplified from 26/30 urine samples taken from cattle with suspected leptospirosis and from leptospires cultivated from 10 of these samples. The limit of detection of DNA in the clinical samples was 200pg and the nested PCR detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified using DNA from other common bacterial species from the bovine urogenital tract or urine, or from the non-pathogenic L. biflexa Andamana serovar. The nested PCR exhibited high specificity and sensitivity for detection of pathogenic serovars in urine from cattle.  相似文献   

18.
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   

19.
Diarrheic fecal samples from 258 pre-weaned calves (1-30 day-old) from 9 dairy farms located in Banat region, Romania, were microscopically examined for the presence of Cryptosporidium oocysts. Overall, 65 (25%) samples were found positive. A higher percent of infection was recorded in calves aged between 8 and 14 days compared with other age categories (1-7, 8-14, 15-21 and 22-30 days; p<0.05). Genetic characterization was carried out on all Cryptosporidium-positive samples. After DNA extraction, Cryptosporidium species were determined by a nested PCR of the small subunit rRNA gene (18S) followed by RFLP analysis with SspI, VspI and MboII restriction enzymes. The restriction patterns showed that animals were infected with Cryptosporidium parvum. Subsequently, subtyping of 13 C. parvum isolates, based on sequence analysis of the 60 kDa glycoprotein (GP60) gene, showed 2 subtypes (IIaA15G2R1 and IIaA16G1R1) belonging to the subtype family IIa. This is the first molecular study of bovine Cryptosporidium infection in Romania.  相似文献   

20.
Of 1346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3% were from cattle (29.8% of these were from calves <6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6% of adult cattle and 11.7% of calves were infected, compared to 28.9% of adult cattle and 36.7% of calves in Thyolo. Dependent on season, between 7.8% and 37.7% (Chikwawa) and 16.7% and 39.3% (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n=225], pigs [n=92], sheep [n=6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6% of goat samples contained oocysts in Chikwawa, compared to between 16.7% and 39.3% in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7%), whereas in Thyolo, infections occurred in all three seasons (17.9% in the rainy season, 25% in the cool season and 60% in the dry season). Of ten diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and/or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.  相似文献   

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