保存在自来水中的微小隐孢子虫卵囊活性及感染性研究

微小隐孢子虫卵囊(CPO)保存在4℃自来水中1~30个月,通过体外脱囊技术检测CPO的脱囊率评价其活性,通过检测CPO感染免疫抑制BALB/c小鼠的潜伏期、排卵囊数量和末端回肠绒毛中的隐孢子虫数量来评价其感染性。结果表明,保存在自来水中1~13个月的CPO出现脱囊;小鼠在感染保存1~13个月的CPO后3~8 d开始排出大量的CPO,在末端回肠绒毛中寄生有大量的隐孢子虫;CPO的保存时间与潜伏期之间存在强烈的相关性(r2=0.98)。因此,CPO在自来水中能保持活性和感染性至少13个月,水是保存CPO的良好介质,水中活性CPO的长期存在对饮用水工业是一个严重的挑战。     

用核酸染色和小鼠感染力方法评估脱囊和热处理后的隐孢子虫卵囊的活力

温度是影响隐孢子虫活力的重要环境因素之一.对于疾病暴发的风险评估需要有效的方法来准确分析卵囊的活力.本试验应用核酸染色和小鼠感染力2种方法研究了脱囊和热处理后完整卵囊和不完整卵囊的活力,并与新鲜卵囊进行了对比.结果表明,脱囊和中性温度热处理后的完整卵囊保持活力.然而,高温处理后的完整卵囊以及脱囊和热处理后的不完整卵囊完全丧失活力.对于新鲜卵囊,灭活卵囊,以及在脱囊后或在40℃和70℃处理后的完整和非完整卵囊,核酸染色法和小鼠感染力法的结果相对应;但是对于50℃和60℃处理后的完整卵囊这2种方法不对应.     

Viability and infectivity of two Cryptosporidium parvum bovine isolates from different geographical location

The viability of two Cryptosporidium parvum bovine isolates from Spain and Colombia was evaluated by in vitro excystation, inclusion/exclusion of two fluorogenic vital dyes (DAPI and PI) and infectivity assay in a suckling murine model. Excystation percentages were similar for both Spain and Colombia isolates (83% and 87%, respectively). The total viability of the Spain isolate, measured by inclusion/exclusion of two fluorogenic vital dyes, was 71% in comparison with that detected for oocysts of the Colombia isolate, 32.3%. The bovine C. parvum oocysts of both isolates were viable and infectious for suckling Swiss CD-1 mice. However, infectivity percentage and the mean intensity of infection were consistently higher in the Spain isolate than those from Colombia isolate. It was not possible to obtain a good correlation between in vitro excystation, inclusion/exclusion of vital dyes and in vivo infectivity for the Colombia isolate, while data obtained with the Spain isolate indicated that there was an apparent strong correlation between excystation efficiency, total viability and the infectivity. Although a comparative analysis of genetic variation among these isolates from different geographical location is necessary, variations observed between the both isolates seemed to be a result of parasite adaptation to environmental stresses such as temperature which appears to have a direct effect on the permeability of the oocysts.     

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido,Japan

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.     

Viability of Sarcocystis neurona sporocysts after long-term storage

The effect of long-term storage on the viability and infectivity of Sarcocystis neurona sporocysts was investigated. S. neurona sporocysts were harvested from the small intestine of Virginia opossums from 1996 to 2002 and stored at 4 degrees C. Viability of sporocysts was assessed by propidium iodide (PI) exclusion assay, in vitro excystation and development in tissue cultures, and bioassay in gamma-interferon gene knockout (gamma-IFN-KO) mice. The rate of excystation was apparently unaffected by long-term storage; sporocysts retained their ability to excyst after 7 years of storage at 4 degrees C. However, the ability of sporocysts to exclude PI stain, to invade and proliferate in cells in vitro, and to cause disease and lesions in gamma-IFN-KO mice appeared to decline as sporocysts age. The results demonstrated that sporocysts of S. neurona were able to survive and maintain moderate to high viability for up to 7 years when stored in phosphate buffered saline and Hank's balanced salt solution containing antibiotic-antimycotic mixture at 4 degrees C.     

Evaluation of two commercial disinfectants on the viability and infectivity of Cryptosporidium parvum oocysts

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.     

The effect of heating against Cryptosporidium oocysts

The effect of heat treatment was examined against oocysts of Cryptosporidium parvum, Cryptosporidium muris and chicken Cryptosporidium sp. isolated in Japan. The oocysts of these species were exposed at 50, 55, 60 and 70 degrees C for 5, 15, 30 and 60 sec in water bath, respectively. To determine the infectivity of heated oocysts, the nice and chickens were inoculated with the treated oocysts and the oocyst output in the feces after inoculation was examined. In C. parvum and chicken Cryptosporidium sp., the oocysts were not detected from mice or chickens which were received oocysts heated at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. In C. muris, the oocysts were not detected from mice which were received oocysts heated at 55 degrees C for 15 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. Consequently, it was clarified that the infectivity of Cryptosporidium oocysts to mice and chickens was lost by heating at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec.     

Effects of low and high temperatures on infectivity of Cryptosporidium muris oocysts suspended in water

Cryptosporidium muris oocysts suspended in 200 microl of water were pipetted into plastic microcentrifuge tubes which were stored at 4 degrees C or frozen at -5 degrees C for 1, 3, 5, 7, and 10 days and at -20 degrees C for 1, 3, 5, and 8h, respectively. Other samples of C. muris oocysts suspended in water were heated in the metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 70 degrees C. At each high temperature setting microcentrifuge tubes containing C. muris oocysts were exposed for 1 min. Both, frozen and heated oocyst suspensions as well as untreated control oocyst suspensions were then inoculated into each of four ICR mice by gastric intubation. Untreated, freeze-thawed or heated oocysts were considered infectious when oocysts of C. muris were found microscopically in the faeces of mice after inoculation. All inoculated mice that received oocysts frozen at -5 degrees C for 3, 5, 7, and 10 days and -20 degrees C for 1, 3, 5, and 8h had no oocysts in faeces. In contrast, C. muris oocysts frozen at -5 degrees C for 1 day remained infective for inoculated mice. Our results also indicated that when water containing C. muris oocysts was exposed at a temperature of 55 degrees C or higher for 1 min, the infectivity of oocysts was lost.     

Comparison of viability assays for Cryptosporidium parvum oocysts after disinfection

In order to test various viability assays for Cryptosporidium parvum oocysts were used to infect HCT-8 cells in vitro or baby mice. Infected cells were either stained with fluorescent anti-Cryptosporidium-antibody or lysed and subjected to C. parvum-specific PCR after 48 h. Titrations with infective oocysts were performed and compared to oocysts disinfected with Neopredisan for 2 h at varying concentrations. Caecal smears and histological sections from infected animals were examined in parallel. The number of foci of parasite development in vitro after immunofluorescent staining correlated well with the infection dose. PCR was less quantifiable and the results were not always reproducible, especially when low infection doses were used. Disinfection resulted in a dose-dependent reduction of oocyst infectivity when compared to the controls in all three assays. The infection of cells cultured in vitro with oocysts of C. parvum provides a suitable tool for the estimation of viability after treatment with chemical disinfectants. Immunofluorescence is easy to perform and gives quantitative results, while PCR-based detection of parasite DNA, although possible, requires the use of more sophisticated tools for quantification.     

In vitro and in vivo efficacy of alpha-cyclodextrin for treatment of experimental cryptosporidiosis

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.     

Efficacy of amine-based disinfectant KENO™COX on the infectivity of Cryptosporidium parvum oocysts

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.     

Study of the combined influence of environmental factors on viability of cryptosporidium parvum oocysts in water evaluated by fluorogenic vital dyes and excystation techniques

Using a factorial experimental design, the combined effect of salinity, temperature and storage time on the viability of Cryptosporidium parvum oocysts in water was evaluated by fluorogenic vital dyes (4',6-diamidino-2-phenylindole and propidium iodide) and an excystation technique. Salinity, storage time and their interaction seemed to be the most influential factors, whereas temperature was not a significant factor. Under unfavourable conditions (salinity 35 per thousand, storage time 40 days), even more than 20% of oocysts remain viable, indicating a high risk of infection for immunocompromised individuals.     

Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids.

An experimental study was carried out in neonatal goat kids to examine the infectivity of Cryptosporidium oocysts, pattern of oocyst shedding and morphological changes in the intestine during the infection. Cryptosporidium oocysts isolated from adult asymptomatic goats, and identified as C. parvum by polymerase chain reaction (PCR) were used in this study. Of three 4-day-old goat kids, two were orally infected with C. parvum oocysts (10(5) oocysts in 10 ml PBS/kid). One goat kid given 10 ml PBS only by the oral route served as a control. Cryptosporidium oocysts were detected in the faeces of one infected kid on day 3 post-inoculation (pi) whereas in the other 6 days pi. The faecal oocyst counts gradually increased and the peak counts in the two kids were 2 x 10(6)g(-1) (on day 12 pi) and 3.2 x 10(6)g(-1) (on day 14 pi). The increase in faecal oocyst output coincided with diarrhoea in an infected kid from days 10-17 pi. Although the oocyst excretion declined gradually after the peak, both infected kids excreted oocysts until euthanized on days 20 and 22 pi. Light and scanning electron microscopic investigations of the ileum revealed the endogenous stages on the brush border of the enterocytes, infiltration of neutrophils and mononuclear cells into the lamina propria, atrophy, stunting and fusion of villi. For purposes of comparison, goat Cryptosporidium oocysts were inoculated orally (10(3) oocysts/mouse) to eight, 1-week-old mice. All experimental mice excreted oocysts from day 3 pi, and four infected mice continued to excrete oocysts up to day 42 pi. The experimental infection described in goat kids resembled the natural disease in terms of oocyst excretion, clinical signs and intestinal pathology. The ability of oocysts excreted by asymptomatic goats, to infect goat kids and mice is likely to have a major impact on the epidemiology of cryptosporidiosis in livestock and man.     

Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxoplasma gondii oocysts

Clinical toxoplasmosis in humans has been epidemiologically linked to the consumption of drinking water contaminated by Toxoplasma gondii oocysts. We evaluated killing of T. gondii oocysts after ultraviolet (UV) or ozone treatments by bioassay in mice and/or cell culture. A 4-log inactivation of the oocyst/sporozoite infectivity was obtained for UV fluences >20 mJ cm(-2). In contrast, oocysts were not inactivated by ozone with an exposure (Ct) up to 9.4 mg min l (-1) in water at 20 degrees C. In conclusion, UV treatment can be an effective disinfection method to inactivate T. gondii oocysts in drinking water, but ozone did not show promise in this research.     

Seasonal change in the number of Cryptosporidium parvum oocysts in water samples from the rivers in Hokkaido,Japan, detected by the ferric sulfate flocculation method

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.     

In vitro and in vivo efficacy of lasalocid for treatment of experimental cryptosporidiosis

In vitro viability of purified Cryptosporidium parvum oocysts, exposed for 30, 60, 90 and 120 min to 0.27 mg/ml lasalocid suspension was evaluated by inclusion or exclusion of two fluorogenic vital dyes and an excystation technique. Continuously, preventive and curative efficacies at different doses (9, 6.75, 5.625 and 4.5 mg/kg body weight) and regimens of lasalocid against cryptosporidial infection were evaluated on an experimental neonatal mice model. In vitro assays demonstrated a decrease in the oocyst viability related to an increase in exposure time for exposure to the lasalocid suspension. The infection was eradicated when the suspension was administered with a dose of ≥6.75 mg/kg body weight. No apparent toxic effects were observed.     

东北虎源等孢球虫在猫体内的卵囊排出规律观察

为研究猫体内的卵囊排出规律,本研究分离的东北虎源等孢球虫纯种卵囊,在猫体内多次传代后获得充足的卵囊.对猫进行人工感染30、10~3和10~4个孢子化卵囊,观察猫的卵囊排出规律,包括潜隐期、显露期、排卵囊高峰期及每克粪便中卵囊数(OPG 值).通过对比,验证了潜隐期、显露期、OPG值与感染剂量存在相关关系,即感染剂量越小,潜隐期越长,显露期越长,OPG值越小,反之亦然.     

Infectivity and oocyst excretion patterns of Cryptosporidium muris in slightly infected mice

To determine the infectivity of Cryptosporidium to hosts in slight infections, we examined the infectivity and oocyst output patterns of Cryptosporidium muris in mice inoculated with small numbers of oocysts. One of the 25 ICR mice inoculated with 2.4 x 10(1) oocysts and 19 of the 25 mice inoculated with 2.4 x 10(2) oocysts shed oocysts in the feces after inoculation. Four of the 50 mice inoculated with 2.4 x 10(1) oocysts for 10 consecutive days also shed oocysts and their OPG values were similar to that of the mice which received 2.4 x 10(2) oocysts. Consequently, it is clear that less than 10% of the mice which received 2.4 x 10(1) C. muris oocysts for 10 consecutive days.     

The prepatent and patent periods of Eimeria alabamensis and further description of the exogenous stages

Eimeria alabamensis infections were established in calves 5 to 6 weeks of age by adminestering 10 million, 80 million, or 100 million sporulated oocysts. The prepatent period was 6 to 8 days (mean 6.6). Oocyst discharges usually lasted for 2 to 3 days although a few calves passed oocysts throughout the rest of the 3-week observation period. Calves with oocyst discharge exceeding 1 million oocysts per g of feces had a moderate diarrhea at the time of peak oocyst discharge. No other clinical signs were observed in any of the infected calves. Reinoculations with 100 million sporulated oocysts given 3 weeks after the initial inoculations of 10 million or 80 million oocysts resulted in infections characterized by greatly reduced oocyst discharges. Sporulated oocysts of E. alabamensis were 16 to 24 μ by 12 to 16 μ and were usually ovoid. The oocyst wals consisted of two layers. Sporocysts were elongate-ellipsoid, had a distinct Stieda body, and were 10 to 12 μ by 4 to 6 μ. Completely sporulated oocysts were first observed after 5 days at 25 °C, and most were sporulated after 8 days. Oocysts did not sporulate at 4 °C, 33 °C, and 37 °C.     

The survival of Sarcocystis gigantea sporocysts following exposure to various chemical and physical agents.

Using in vitro excystation as a measure of viability, it was found that at 4 degrees C Sarcocystis gigantea sporocysts survived considerably better in tap water (85% excystation after 174 days) than in either 2.5% potassium dichromate (15% excystation after 174 days) or 2% sulphuric acid (0% excystation after 5 days). Although they were able to resist 48 h suspension at room temperature in most laboratory reagents and disinfectants tested, six (sulphuric acid, ammonia, methanol, ethanol, potassium hydroxide, sodium hydroxide, Medol) had substantial sporocysticidal properties. Further investigation with three of these showed that sporocyst excystation was reduced from 65% to less than 10% following contact with 2.5% sulphuric acid for 1 h or with 2% ammonia or 4% Medol for 4 h. Sporocysts were either killed or had their ability to excyst severely impaired by heating to 60 degrees C and 55 degrees C for 5 and 60 min, respectively, by exposure to ultraviolet radiation at a dose of 4000 ET, or by prolonged storage in water at 24 degrees C. Sporocysts exposed to either constant or intermittent freezing at -18 degrees C suffered a comparatively slow decline in excystation rate with time, as did those subjected to desiccation. The duration of survival of desiccated sporocysts was inversely related to relative humidity and after 245 days at 33% relative humidity and temperatures of 15 degrees C or 24 degrees C, 60% of such sporocysts excysted.