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1.
In November 1997, Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998. C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted C. andersoni, while 25 (16.7%) were positive for C. parvum. Oocysts of the Danish C. andersoni isolate were ovoid, 7.3(6.5-8.0) x 5.7(5.0-7.0) microm(2) (n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14-1.40, n=25). The identification was verified by sequencing of a 246bp fragment of the rDNA, which was identical to Cryptosporidium muris, the calf genotype (AF093496). The Danish C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of C. andersoni in Scandinavia.  相似文献   

2.
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   

3.
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   

4.
应用巢式聚合酶链反应（Nested PCR）-限制片段长度多态性 （restriction fragment length polymorphism, RFLP）方法对微小隐孢子虫（C．parvum）、安氏隐孢子虫（C．andersoni）和火鸡隐孢子虫（C．meleagrides）的鉴别进行了研究。结果显示C．parvum BOCC2、C．andesoni BOCC2和C．meleagrides CHCC1扩增产物片段大小分别为830bp、828bp和828bp，扩增产物分别经VspI酶切后形成3种不同的RFLP图谱，根据RFLP图谱可鉴别C．parvum、C．andersoni和C．meleagrides。本研究为我国隐孢子虫的分类和隐孢子虫病的分子流行病学研究打下了良好基础。  相似文献   

5.
The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.  相似文献   

6.
Faecal specimens from 305 horses and mules used as packstock at one of 17 commercial or governmental (National Park Service, US Forest Service) operations were examined for Giardia duodenalis and Cryptosporidium parvum using immunofluorescent microscopy. Fourteen packstock (4.6%) were shedding G. duodenalis cysts, with herd-level prevalences ranging 0-22%. Number of packstock in the corral, size of corral and density of packstock in the corral were associated with the odds of shedding G. duodenalis cysts. None of the horses had detectable C. parvum oocysts. Assuming a sensitivity of at least 43% and a specificity of 100% for our assay, the estimated maximum true prevalence of shedding of C. parvum for packstock would be < or = 2.3% of the population. These data suggest that faecal dispersal of C. parvum on back country watersheds is unlikely with packstock.  相似文献   

7.
Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll(?) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. Results: (a) Diagnostic methods: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(?) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.  相似文献   

8.
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   

9.
安氏隐孢子虫PCR诊断试剂盒的初步应用   总被引：2，自引：0，他引：2  
运用首次研制的安氏隐孢子虫PCR诊断试剂盒对广东省4个奶牛场和河南省1个奶牛场共234份样品,进行了安氏隐孢子虫感染的实际检测,并与常规检测方法饱和蔗糖漂浮法、改良抗酸染色法进行了比较。本试剂盒的检出率比常规检测方法提高了2%～13%,显示该试剂盒具有特异、敏感等优点,对开展隐孢子虫病的鉴别诊断和分子流行病学调查具有重要的应用价值。  相似文献   

10.
以筛选安氏隐孢子虫T7噬菌体展示文库获得的肌动蛋白（CA42）部分编码基因的序列设计特异性引物,扩增目的基因CA42,构建重组表达载体pET-28a-CA42,通过大肠杆菌BL21的诱导表达,产物经SDS-PAGE和Western-blotting鉴定。然后纯化重组蛋白,免疫BALB／c进行体液和细胞免疫水平的检测,并观察重组蛋白对微小隐孢子虫的交叉保护性效果。结果显示,成功构建重组原核表达质粒pET-28a-CA42,Western-blotting显示重组蛋白约为19ku,能被安氏隐孢子虫免疫小鼠的多克隆抗体识别。重组蛋白免疫BALB／c小鼠的抗体水平差异显著（P〈0．05）,CD4^＋T淋巴细胞数差异均显著,CD4^4／CD8^＋T淋巴细胞数的值与佐剂对照组和空白对照组相比差异均显著（P〈0．05）。各组小鼠经接种微小隐孢子虫卵囊后,试验组与各对照组相比卵囊减少率为32．2％,差异均显著（P〈0．05）。结果表明,成功表达了重组安氏隐孢子虫肌动蛋白,纯化的重组蛋白具有一定的免疫原性和交叉保护性。  相似文献   

11.
A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium andersoni (C. muris) [corrected] in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.  相似文献   

12.
To study the diversity of Cryptosporidium spp. in various hosts, we used the variability of the small-subunit rRNA gene and the Cryptosporidium oocyst wall protein genes. Oocysts from humans, cattle, horses, dogs, field mice, chickens, reptiles, deer, goat, cat, antelope and from a sample of water reservoir were assayed. The zoonotic C. parvum bovine genotype sequence was found to be present in the most of isolates. This study shows a complex epidemiology pattern for C. parvum bovine genotype infections. The identification of cattle, horse, and deer isolates emphasizes a transmission route for C. parvum via these hosts, and identifies a potential source for human infection in the Czech Republic. Furthermore, C. andersoni from a cow, C. baileyi from a chicken, C. felis from a cat, C. meleagridis from a dog, and C. saurophilum and C. serpentis from reptiles were also identified in the isolates from the Czech Republic.  相似文献   

13.
A longitudinal study of 2-year duration was conducted to determine the risk, as measured by incidence rate, of Cryptosporidium parvum infection among dairy cattle in the Catskill/Delaware Watershed of New York City (NYC), and the factors that predispose animals to the likelihood of infection. A proportional sampling scheme with follow up at quarterly farm visits was employed for heifers and cows. Additionally, all calves born on the 39 study farms were sampled once during the first four weeks of life and at least once more before weaning. Samples were analyzed for the presence of C. parvum using a quantitative centrifugation concentration flotation technique and a C. parvum-specific enzyme-linked immunosorbent assay (ELISA). Of the 9914 fecal samples collected, 747 were found to contain C. parvum. The average number of oocysts detected was 1.3x10(5)/g (range: 1.0/g--8.2x10(6)/g). The average age at time of first detection of the organism was 15.0 days with a standard deviation of 6.59 days. The age range of animals infected with C. parvum in the study population was 3--60 days (inclusive). The unadjusted (crude) incidence rate of C. parvum among the entire study population was 2.05 per 1000 animal-days. The unadjusted incidence rate among pre-weaned calves was 15.55 per 1000 animal-days. After controlling for age and prior protozoal risk level, no seasonal impact on the incidence of C. parvum was detected among animals less than 61 days by negative binomial regression. A seasonal impact was identified among the oocyst counts of infected animals after controlling for age and prior protozoal risk level.  相似文献   

14.
Modified Ziehl-Neelsen (MZN), auramine-phenol (A-P) and fluorescein isothiocyanate-labelled (FITC-labelled) monoclonal antibody (MAb) techniques were compared for detection of Cryptosporidium parvum oocysts in cat faecal specimens inoculated with known numbers of C. parvum oocysts. Of the three techniques, the FITC-labelled MAb technique detected more oocysts than the MZN and A-P techniques (P < 0.05), but A-P was more efficient than MZN (P < 0.05). Comparison of sucrose flotation, zinc sulphate (ZnSO4) flotation and formol-ether (F-E) sedimentation techniques revealed that F-E was the most efficient of the three (P < 0.05) for concentration of C. parvum oocysts from cat faecal specimens. On average, the F-E technique recovered 37% of oocysts from the original sample, whereas the sucrose and ZnSO4 flotation techniques recovered 33% and 11%, respectively. The findings of this study suggest that MZN and A-P staining are both useful for screening C. parvum oocysts in cat faecal materials containing 10(6) oocysts or more, but FITC-labelled MAb should be used when the number of oocysts is low. Also, the F-E sedimentation technique is recommended for concentrating oocysts in cat faecal specimens.  相似文献   

15.
Cryptosporidium andersoni has not been previously reported in feedlot beef cattle in Western Australia. Faecal samples were collected from 10 groups of cattle ranging in age from 11 to 36 months in five different feedlots in Western Australia. The incidence of C. andersoni ranged from 0% to 26%. There were no clinical signs associated with C. andersoni infection, but there was a significant reduction in rate of gain of 0.44 kg in infected animals compared with negative pen mates. Cryptosporidium andersoni is characterised by large oocysts (7.4 x 5.5 μm) and was confirmed by 18S sequencing.  相似文献   

16.
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   

17.
The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 180 7- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.  相似文献   

18.
A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. muris was rarely found in wild mammals and C. andersoni oocysts were never detected in livestock. Faecal samples from 3721 individuals gave cumulative 6-year prevalences for C. parvum as follows: bull beef, 3.6%; dairy cows, 3.5%; ewes, 6.4%; horses, 8.9%; calves (home bred), 52%; calves (bought-in) 23.2%; lambs, 12.9%; small wild mammals (rodents) living in and around farm buildings, 32.8%; small wild mammals (mainly rodents) living in areas of pasture, 29.9%. Animal categories with the highest prevalences also shed the highest average oocyst numbers per gram of faeces (ranging from 1.4 x 10(3) for bull beef to 1.1 x 10(5) for calves). Analysis of annual and seasonal data for each animal category revealed that patterns of infection were variable and sporadic; this means that short-term sampling was never likely to provide a true or representative picture. Seasonally combined data for adult livestock, young livestock and small wild mammals showed all three categories tended to have the highest Cryptosporidium prevalences in the autumn. Calves were separated from their dams within 24h of birth and yet showed high prevalence of infection in most years despite the low prevalence for the dairy herd. It is possible the coincidence of high autumn prevalence in mice with the main period for the rearing of calves contributed to the infection of the latter. The farming estate was used to teach students of agriculture and took pride in good land management and husbandry practices that produced well fed and healthy livestock. The data from this estate may represent, therefore, the baseline, the lowest possible levels to be expected, for Cryptosporidium infection and oocyst production on a lowland farm in the United Kingdom.  相似文献   

19.
Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin-ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and beta-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.  相似文献   

20.
Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.  相似文献   

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