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.     

Comparison of viability and infectivity of Cryptosporidium parvum oocysts stored in potassium dichromate solution and chlorinated tap water

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.     

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.     

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.     

Evaluation of fenbendazole for treatment of Giardia infection in cats concurrently infected with Cryptosporidium parvum

OBJECTIVE: To determine whether fenbendazole effectively eliminates Giardia organisms from chronically infected cats that have a concurrent Cryptosporidium parvum infection. ANIMALS: 16 clinically normal cats. PROCEDURE: Eight cats with chronic concurrent Giardia and C parvum infections received fenbendazole (50 mg/kg, PO, q 24 h) for 5 days (treatment-group cats). Feces from each cat were collected and processed 3 days weekly for 23 days after treatment. By use of an immunofluorescent assay for detection of Giardia lamblia cysts and C parvum oocysts, organism numbers were counted and scored. Fecal results from treatment-group cats were compared with those of 8 untreated cats with Giardia infection but no C parvum infection (control-group cats). RESULTS: Four of 8 treatment-group cats had consistently negative results for Giardia infection after treatment. These 4 cats had consistently positive results for C parvum oocysts prior to treatment and consistently negative results after treatment. One treatment-group cat had positive results for cysts on all fecal samples, and 3 treatment-group cats had 1 to 3 negative results and then resumed shedding large numbers of cysts; each of these cats had consistently positive results for C parvum oocysts. When compared with control-group cats, treatment-group cats shed less Giardia cysts during week 1 after treatment but not during week 2. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of fenbendazole decreases Giardia cyst shedding to less than detectable numbers in some cats. In our study, persistent C parvum infection may have been associated with failure of fenbendazole to eliminate Giardia infection.     

Evaluation of two rapid assays for detecting Cryptosporidium parvum in calf feces

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.     

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.     

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.     

Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods

Cryptosporidium spp. are monoxenous protozoan parasites that cause gastrointestinal diseases in humans and animals. Shellfish harvesting areas can become contaminated by the infectious stage of the parasite and humans are therefore at risk of infection either by consumption of shellfish, or by taking part in recreational activities in these areas. In the present study we determined the levels of detection, by IFA and PCR techniques, of Cryptosporidium oocysts in mussels experimentally contaminated with a theoretical number of oocysts. There was a significant correlation between the results obtained by both techniques (P<0.05). IFA and PCR were also applied to a total of 222 samples of mussels (Mytilus galloprovincialis) destined for human consumption. In the naturally contaminated samples, we detected a 31.1% of contamination and only Cryptosporidium parvum (previously denominated C. parvum genotype II) was identified.     

Peri-parturient rise of Cryptosporidium oocysts in cows: New insights provided by duplex quantitative real-time PCR

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.     

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。     

A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age

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.     

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil

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.     

Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea

OBJECTIVE: To determine duration of infection and association of infection with diarrhea for dairy calves with naturally acquired cryptosporidiosis and giardiosis. DESIGN: Cohort study. ANIMALS: 20 Holstein calves on a single dairy farm. PROCEDURE: Fecal samples were collected 3 times/wk for the first 45 days after birth, then weekly until calves were 120 days old and examined for Giardia duodenalis cysts and Cryptosporidium parvum oocysts. Calves were monitored for diarrhea during the first 45 days after birth; during each episode of diarrhea, fecal samples were examined for parasitic, bacterial, and viral pathogens. RESULTS: All 20 calves shed Giardia cysts and Cryptosporidium oocysts at some time during the study. Mean ages at which Giardia cysts and Cryptosporidium oocysts were first detected were 31.5 and 16.3 days, respectively. Mean number of Giardia cysts in feces remained high throughout the study, whereas Cryptosporidium occysts decreased to low or undetectable numbers 2 weeks after infection. Eighteen calves had a total of 38 episodes of diarrhea during the first 45 days after birth. Giardia duodenalis was the only pathogen identified during 6 (16%) episodes, C parvum was the only pathogen identified during 9 (24%) episodes, and G duodenalis and C parvum were identified together during 10 (26%) episodes. CONCLUSIONS: Prevalences of giardiosis and cryptosporidiosis were high in these calves, and both parasites were associated with development of diarrhea. Cryptosporidium parvum was an important pathogen when calves were < 1 month old, but G duodenalis was more important when calves were older. Calves cleared C parvum infections within 2 weeks; however, G duodenalis infections became chronic in these calves.     

Activity of decoquinate against Cryptosporidium parvum in cell cultures and neonatal mice

Cryptosporidium parvum is an apicomplexan parasite that is an important cause of diarrhea in neonatal calves and humans. No treatment is currently available for neonatal calves. We have recently learned from colleagues in the pharmaceutical industry that dairy practitioners are sometimes using decoquinate for the treatment of neonatal bovine cryptosporidiosis. Therefore, the present study was undertaken to determine whether the clinical observations in calves can be substantiated by laboratory investigation. Oocysts of the KSU-1 isolate of C. parvum were used to infect human ileocecal epithelial cells in vitro to measure the efficacy of treatment using an ELISA based assay. No activity was observed at 10 or 50microM decoquinate, but at 100microM an 8% inhibition of development was seen. Oocysts of the AUCp-1 isolate of C. parvum were then used to infect suckling mice. The numbers of oocysts observed in suckling mice treated with 2.5 or 5.0mg/kg decoquinate were not significantly different from untreated control suckling mice (p0.05). The results of our study suggest that decoquinate should have little efficacy for treatment of neonatal bovine cryptosporidiosis if administered once per day and that any clinical improvement observed in treated calves may be due to factors unrelated to decoquinate's effect on C. parvum.     

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.     

Recombinant bovine interleukin-12 stimulates a gut immune response but does not provide resistance to Cryptosporidium parvum infection in neonatal calves

This study was undertaken to determine if administration of recombinant bovine interleukin-12 (rBoIL-12) could stimulate a cellular immune response that protected calves from an oral challenge inoculation with Cryptosporidium parvum oocysts. In a first experiment, rBoIL-12 intraperitoneally administered as a single dose 1 day before challenge inoculation, did not alter the course of infection. The percentage of immune competent cells and levels of cytokine gene expression in the ileo-cecal mucosa and in the draining lymph nodes of treated calves were similar to those of untreated control calves. However, when rBoIL-12 was subcutaneously administered daily from 2 days before infection to 2 days after infection, a consistent increase of T lymphocytes and an higher expression of interferon-gamma (IFN-gamma) was detected. Again, treatment did not alter the course of infection. Similar results were obtained when rBoIL-12 was administered daily for 4 days beginning 2 days after oral inoculation. These data indicate that although rBoIL-12 stimulated a strong immune response in the gut of neonatal calves, the response was not able to provide protection from challenge inoculation with C. parvum oocysts.     

Cryptosporidium parvum in diarrheic calves detected by microscopy and identified by immunochromatographic and molecular methods

Cryptosporidium is an important protozoan parasite that causes diarrhea in neonates and young bovines. The objective of the present study was to determine the frequency of Cryptosporidium infection in animals of dairy farms of the Metropolitan Region (Santiago), Chile. Fecal samples of 205 newborn calves with diarrhea were studied and used for comparing the efficiency of two microscopic staining methods for diagnosis of the parasite, the auramine (AU) and a modified Ziehl-Neelsen (ZN) procedure. Out of the 205 fecal samples, we detected oocysts in 115 (56.1%) with AU and 102 (49.8%) with ZN. Comparison of results obtained with the two microscopic techniques showed significant difference (p<0.05), AU being more sensitive. On the other hand, concordance between the two methods was almost perfect (kappa value of 0.83). The results with these two operator dependent methods were confirmed using an operator independent immunochromatographic (IC) method. The IC method also enabled us to determine the identity of the parasite species as that of Cryptosporidium parvum. Identification of the parasite species was further corroborated by performing a Cryptosporidium species-specific polymerase chain reaction (PCR) test on few samples taken at random. Overall, the results showed a high number of infected animals suggesting the parasite C. parvum as a major parasitic disease agent of neonatal calves with diarrhea in dairy farms of the Metropolitan Region (Santiago) of Chile.     

Genotypic characterization of cryptosporidium oocysts isolated from healthy people in three different counties of Korea

To investigate Cryptosporidium infection among healthy people, we collected stool samples from 150 healthy individuals in Gokseong, Muan, and Imshil Counties, southwest Korea, where neighbors on both an animal farm and a river respectively. In 12 of 150 samples, Cryptosporidium oocysts were detected by means of modified acid-fast staining. The bovine genotype, Cryptosporidium parvum, was identified by PCR/RFLP and 18S rRNA sequencing. C. parvum existed endemically in these areas, and the residents showed a relatively higher infection rate for C. parvum than that for C. hominis. Our results indicate that countermeasures against Cryptosporidium infection must be taken in these areas to ensure human health.     

Comparison of staining and concentration techniques for detection of Cryptosporidium oocysts in cat faecal specimens.

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.