首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 62 毫秒
1.
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   

2.
鼠隐孢子虫卵囊及子孢子的纯化   总被引：4，自引：0，他引：4  
应用不连续蔗糖密度梯度离心法，对犊牛粪便中的鼠隐孢子虫卵囊进行了提取，经过两次漂浮和一次不连续蔗糖密度梯度离心，得到纯净的卵囊，其中含有极少量的杂质，不含其他微生物。卵囊的回收率为３７％。纯化的卵囊脱囊后，经过一次不连续蔗糖密度梯度离心，得到很纯的子孢子。子孢子的回收率为４７．６％。纯化的卵囊及于孢子可用于隐孢子虫的核酸分析、生物化学、免疫学及细胞培养等方面的研究。该法具有简便、快速、大量分离卵囊的优点。  相似文献   

3.
An epidemiological study was carried out in natural water supplies of Hokkaido, one of the largest dairy prefectures in Japan. To investigate the prevalence of Cryptosporidium parvum (C. parvum) oocysts water samples were collected from three rivers in the eastern area of Hokkaido from August 1999 to October 2001, and C. parvum oocysts were collected and purified by the ferric sulfate flocculation method. The oocysts were detected using the immunofluorescent assay test (IFAT) and 4', 6-diamidino-2-phenylindole (DAPI) staining. The seasonal change in the number of oocysts detected was observed. Oocysts increased in numbers from the late summer to the early autumn (from August to November), thereafter, they exhibited a trend to decrease until December, when no oocysts could be detected. The maximum number of oocysts detected in the three rivers was 3.50, 5.00 and 3.33 oocysts/l, respectively. The oocyst density in river water changed in relation to the season in 1999, 2000 and 2001. This report first cleared up the seasonal changes in C. parvum oocysts number in river water.  相似文献   

4.
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   

5.
Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1-8 weeks of age) (45.8%) than in post-weaned calves (3-12 months of age) (18.5%) and heifers (12-24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in pre-weaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1.  相似文献   

6.
A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive.  相似文献   

7.
The removal effects of the faucet mounted type water purifier for home use were examined against Cryptosporidium parvum oocysts. The water purifier is composed of a layer of granular activated carbon and the hollow fiber membrane filter. The cartridges were unused, 25%, 50% and 75% flow down by Arizona-dust of U. S. A. Two respective cartridges were used of the examination. The faucet and the water purifier were connected by anti-pressure tube, and 3.0 x 10(7) oocysts of Cryptosporidium parvum were injected into anti-pressure tube while water was running. Twenty liter of collected purified water was examined under the fluorescent microscope. Any oocysts in the purified water collected from all cartridges were not found. Therefore, we considered this purifier as an effective one in removing Cryptosporidium oocysts from drinking water.  相似文献   

8.
Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).  相似文献   

9.
During calving time on an experimental farm, 32 newborn calves were selected at random and monitored for infection with Cryptosporidium parvum for the first 30 days of their lives. The animals were fed pooled colostrum for 2-3 days after birth and housed in individual pens, which were washed daily using a pressure hose. Fecal smears were examined by microscopy after staining with carbol fuschin for visualization of oocysts. Oocyst shedding was scored semiquantitatively according to the average number of oocysts in 20 randomly selected fields at 1000x magnification. All the animals acquired the infection before 18 days of age. The period of maximum risk was between 9 and 12 days; 50% of the animals were infected by 9.4 days of age. It was found that the earlier the animals acquired the infection, the longer the patent period. Oocyst shedding, which did not always begin with the onset of diarrhea, lasted between 8 and 23 days (mean 12.4+/-3.3 days). Furthermore, fecal samples from 32 periparturient cows (within +/-7 days of giving birth) were filtered, concentrated and examined for oocysts using a fluorescent monoclonal antibody test, which revealed that six of the cows, although asymptomatic, were excreting C. parvum oocysts.  相似文献   

10.
The efficacy of alpha-cyclodextrin against infection by Cryptosporidium parvum was evaluated using in vitro and in vivo models. Cyclodextrins are water-soluble cyclic hexamers of glucose units with hydrophobic cavities capable of solubilizing lipophiles and are widely used as drug excipients in the pharmaceutical industry. The viability of purified C. parvum oocysts, exposed for 30, 60, 90, 120 min and 24h to different concentrations of alpha-cyclodextrin (2.5, 5, 7.5, 10, 12.5 and 15%), was evaluated by inclusion or exclusion of two fluorogenic vital dyes and by an excystation technique. Preventive and curative efficacies against cryptosporidial infections, at different doses (2.5 and 5%) and regimes of administration of alpha-cyclodextrin, were determined in an experimental neonatal mice model. Results of the viability assay showed a decrease in oocyst viability that was associated with an increase in exposure time, for each of the concentrations used. Moreover, a high proportion of nonviable oocysts (81%) was observed when C. parvum oocysts were exposed to alpha-cyclodextrin (2.5%) for 24h. The intensity of infection, determined 7 days post-inoculation by examination of intestinal homogenates, was significantly lower (P<0.05) than in the control litters, for all the assays carried out with alpha-cyclodextrin. Only 38.8% of the animals became infected when the alpha-cyclodextrin solution (5%) was administered 2h before inoculated oocysts, and every 24h at 1 and 2 days post-inoculation.  相似文献   

11.
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   

12.
In order to clarify if a peri-parturient rise of Cryptosporidium parvum oocysts occurs in cows, faecal samples from 42 cows on two farms were collected. These samples were taken during the pre-parturient, the peri-parturient and the post-parturient periods. Two methods were used to detect the oocysts, a nested-PCR coupled with sequencing and a duplex real-time PCR (qPCR) that quantified Cryptosporidium spp. DNA concentration. The qPCR results were adjusted using a hierarchical Bayesian model taking into account within and between run variation. Generalised Estimating Equation models (GEE) were used to determine if peri-parturient cows were at greater risk of being infected than pre- or post-parturient cows. Fourteen dairy cows exhibited a peri-parturient and post-parturient rise in the excretion of Cryptosporidium spp. oocysts, other than the zoonotic C. parvum. The cows in the suckler beef farm were the only ones infected with the zoonotic species C. parvum at calving. Due to the low concentration of oocysts excreted mainly from species other than C. parvum, it would appear unlikely that cows act as a source of infection for their calves or contribute significantly to environmental contamination.  相似文献   

13.
The present study was undertaken to compare the viability and infectivity of Cryptosporidium parvum oocysts that had been stored for 1, 4, 7, 10, 13, 16, 20, 25 and 30 months at 4 degrees C in 2.5% potassium dichromate (Cr) or chlorinated tap water, respectively. An excystation protocol was performed in vitro to evaluate viability. One hundred and eighty female BABL/c mice were used to evaluate the infectivity of oocysts by investigating the prepatent period of C. parvum infection, the quantity of oocysts excreted, and the number of parasites that colonized the villi of the ileum. The results showed that C. parvum oocysts preserved in Cr for 1-16 months or in water for 1-13 months were capable of excystation in vitro and infection of mice. The excystation rates of oocysts and the prepatent periods in mice infected by oocysts stored in Cr and water were not significantly different (p>0.05), and there was a strong correlation between prepatent period and duration of oocyst storage (Cr: R2=0.92; water: R2=0.98). There were no significant differences in oocyst shedding from feces or parasitism of the terminal ilea of mice by Cryptosporidia between the two storage media (p>0.05). In conclusion, C. parvum oocysts may be stored at 4 degrees C in water instead of Cr for the purposes of laboratory research. However, the presence of viable C. parvum oocysts in water is a severe challenge to the drinking water treatment industry.  相似文献   

14.
OBJECTIVE: To evaluate the association of herd demographics, parturition variables, stocking rate, and rotational grazing practices with the probability of fecal shedding of Cryptosporidium parvum from beef cow-calf herds in California. DESIGN: Cross-sectional study. SAMPLE POPULATION: 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Association between various demographic and management factors and the probability of shedding C parvum were statistically evaluated. RESULTS: Adjusted for age and month of collection of a fecal sample, cattle from herds with a high number of young calves (< or = 2 months old) on the day of sample collection, a high stocking rate (No. of cattle/acre/mo), or a longer calving season were more likely to shed C parvum oocysts, compared with cattle from herds with fewer young calves, a lower stocking rate, or a shorter calving season. Cattle from herds with a higher number of older calves (> 2 months old) on the day of sample collection were less likely to shed C parvum oocysts, compared with cattle from herds with fewer older calves. Using our multivariate model, rotational grazing systems or season of onset of calving were not associated with shedding status for C parvum oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: Reproductive management that would result in a shorter calving season and use of a lower stocking rate for cattle may be associated with reduced risk of C parvum shedding. Intensive rotational grazing systems and time of year for onset of calving season apparently have little effect on reducing prevalence of oocyst shedding.  相似文献   

15.
Cryptosporidium parvum is a zoonotic protozoan parasite that may cause severe neonatal diarrhoea or even mortality in newborn ruminants: its oocysts are extremely resistant to normal environmental conditions and to most common disinfectants. KENO?COX, a patent pending amine-based formula, was tested for its ability to inactivate C. parvum oocysts. The Daugschies assay (2002), a standardized assay for chemical disinfection initially described for Eimeria spp., was adapted for C. parvum oocysts. KENO?COX diluted in water at 2% and 3% concentration and incubated with oocyst suspensions for 2h, allowed a significant reduction in viability, lysing 89% and 91% of oocysts respectively. Infectivity of the remaining C. parvum oocysts was assessed by inoculation to C57 Bl/6 neonatal mice. Each mouse received 2.5 μl of a suspension initially containing 500,000 oocysts before contact with KENO?COX. Six days post inoculation, the intestinal parasite load was significantly reduced by 97.5% with KENO?COX 2% compared to that of the mice inoculated with untreated parasites. KENO?COX 3% completely eliminated infectivity of oocysts. The number of oocysts remaining infectious in the inoculum treated with KENO?COX 2% was calculated from an inoculated dose-response curve: it was estimated at about 48.6 oocysts among the 500,000 oocysts initially treated corresponding to 99.99% of inhibition. These results demonstrate the high efficacy of KENO?COX against C. parvum oocysts. Combined with an appropriate method of cleaning, the application of KENO?COX may be a useful tool to reduce cryptosporidial infectious load on farm level.  相似文献   

16.
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   

17.
Fifteen dairy farms in seven states on the east coast of the US were each visited on two consecutive years to determinate the prevalence of Cryptosporidium species in pre-weaned (5 days to 2 months) and post-weaned calves (3-11 months), respectively. After each of 971 fecal specimens collected directly from each calf was sieved and subjected to density gradient centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy, and polymerase chain reaction (PCR). For all PCR-positive specimens the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified from all farms. Types of housing appeared to have no influence with regard to prevalence of infection. Of 971 calves, 345 were infected with Cryptosporidium (35.5%), but more pre-weaned calves (253 of 503; 50.3%) than post-weaned calves (92 of 468; 19.7%) were found to be infected. A total of 278 PCR-positive specimens characterized by gene sequencing revealed Cryptosporidium parvum, Cryptosporidium andersoni, and two unnamed Cryptosporidium genotypes Bovine B (AY120911) and deer-like genotype (AY120910). The prevalence of these Cryptosporidium species and genotypes appeared to be age related between pre- and post-weaned calves. C. parvum, the only zoonotic species/genotype, constituted 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves. These findings clearly demonstrate that earlier reports on the presence and prevalence of C. parvum in post-weaned cattle that were based solely on oocyst morphology must be reassessed using molecular methods to validate species and genotype. This finding also indicates that persons handling or otherwise exposed to calves under 2 months of age are at greater risk of zoonotic infection from Cryptosporidium than the risk of infection from exposure to older calves.  相似文献   

18.
Cryptosporidiosis is mainly a problem in neonatal ruminants. Not only do Cryptosporidium spp. spread ubiquitously in our environment, but the protozoa are highly resistant to harsh environmental conditions and disinfectants, and a control measure is urgently required. This study investigated the potential biocidal activity on Cryptosporidium parvum oocysts of two commercial disinfectants developed originally to be used in farms and food-processing industries. The products, containing formaldehyde and hydrogen peroxide respectively, both had some anticryptosporidial effects. The viability and infectivity of purified C. parvum oocysts exposed to both disinfectants at different concentrations and exposure times were evaluated by inclusion or exclusion of vital dye (propidium iodide), use of an excystation technique and infection of suckling mice. Viability assays showed a decrease in oocyst viability associated with an increase in exposure time for each of the concentrations used. The intensity of infection in neonatal mice was significantly lower (P<0.05) than in the control litters.  相似文献   

19.
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   

20.
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   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号