Michael J. Arrowood

Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients.

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls.

A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium

Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.

OCCURRENCE OF CRYPTOSPORIDIUM OOCYSTS IN SEWAGE EFFLUENTS AND SELECTED SURFACE WATERS

An existing method for the detection of Cryptosporidium oocysts in water was modified to investigate oocyst prevalence in large volumes of water. Surface waters and sewage effluents were filtered, eluted from the filter, and concentrated using centrifugation. The resultant pellet was then homogenized, sonicated, and placed on a sucrose gradient to separate oocysts from the sediment. The uppermost gradient layer was then examined by immunofluorescence using a labeled monoclonal antibody. Using this technique, average numbers of oocysts detected in raw and treated sewage were 5.18 X 10(3) and 1.30 X 10(3)/L, respectively. Filtered sewage effluents had significantly lower numbers of oocysts (10.0/L). These data show that sand filtration may reduce the concentrations of this parasite in waste waters. Highly variable oocyst numbers were encountered in surface waters. Since Cryptosporidium oocysts are frequently present in environmental waters, they could be responsible for waterborne outbreaks of disease.

Improved Purification Methods for Calf-Derived Cryptosporidium parvum Oocysts Using Discontinuous Sucrose and Cesium Chloride Gradients

Cryptosporidium parvum oocysts can be generated in low to moderate numbers in rodent animal models, but large-scale production is generally accomplished in neonatal livestock, especially calves. We have introduced a number of improvements in the production and purification process for calf-derived oocysts to minimize labor, consumption of materials, and generation of hazardous waste. C. parvum oocysts (IOWA isolate) were generated in neonatal (36-72 hr old) Holstein calves following inoculation of lo8 oocysts. Stool collection began on day 4 and was continued through day 12 post infection. Calf caging utilized a customized chimpanzee cage modified by installing Plexiglas barriers around the inner walls to direct scouring fecal matter to drop into a custom stainless steel pan positioned beneath the cage floor. The original cage floor was overlaid with rubber-coated expanded metal. This material provided a compact, but open surface that supported the calfs hooves while allowing urine and fecal matter to drop through into the collection pan. Calf cage surfaces were cleaned with a detergent/bleach-based solution (F364, Rochester Midland) and the pooled feces-contaminated waste was heat inactivated (see below).

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

ABSTRACT Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purifiedC. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvuminfectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm2 (=30 J/m2), the reduction reached the cell culture assay detection limit of ∼3 log10. At UV doses of 1.2 and 3 mJ/cm2, the log10 reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.

A Waterborne Outbreak of Gastroenteritis with Multiple Etiologies among Resort Island Visitors and Residents: Ohio, 2004

BACKGROUNDnThe implementation of treated municipal water systems in the 20th century led to a dramatic decrease in waterborne disease in the United States. However, communities with deficient water systems still experience waterborne outbreaks. In August 2004, we investigated an outbreak of gastroenteritis on South Bass Island, Ohio, an island of 900 residents that is visited by >500,000 persons each year.nnnMETHODSnTo identify the source of illness, we conducted a case-control study and an environmental investigation. A case was defined as diarrhea in a person who traveled to the island during the period from May 1 through 30 September 2004 and became ill within 2 weeks after the visit. Healthy travel companions served as matched control subjects. We also performed an environmental assessment and extensive testing of island water sources.nnnRESULTSnAmong the 1450 persons reporting illness, Campylobacter jejuni, norovirus, Giardia intestinalis, and Salmonella enterica serotype Typhimurium were identified in 16, 9, 3, and 1 persons, respectively. We interviewed 100 case patients and 117 matched control subjects. Case patients were more likely to drink water on the island than control subjects (68% vs. 35%; matched odds ratio, 4.3; 95% confidence interval, 2.2-9.3). Sampling of ground water wells indicated contamination with multiple fecal microbes, including Escherichia coli, C. jejuni, Salmonella species, and Giardia species. Irregularities in sewage disposal practices that could have contaminated the underground aquifer were noted.nnnCONCLUSIONSnThe combined epidemiological and environmental investigation indicated that sewage-contaminated ground water was the likely source of this large outbreak. Long-term changes to the islands water supply and sewage management infrastructure are needed.

An Outbreak of Cryptosporidiosis Linked to a Foodhandler

In September and October 1998, a cryptosporidiosis outbreak occurred on a Washington, DC, university campus. In a case-control study of 88 case patients and 67 control subjects, eating in 1 of 2 cafeterias was associated with diarrheal illness (P<.001). Morbidity was associated with eating dinner on 22 September (odds ratio, 8.1; 95% confidence interval, 3.4-19.5); weaker associations were found for 6 other meals. Cryptosporidium parvum was detected in stool specimens of 16 (70%) of 23 ill students and 2 of 4 ill employees. One ill foodhandler with laboratory-confirmed C. parvum prepared raw produce on 20-22 September. All 25 Cryptosporidium isolates submitted for DNA analysis, including 3 from the ill foodhandler, were genotype 1. This outbreak illustrates the potential for cryptosporidiosis to cause foodborne illness. Epidemiologic and molecular evidence indicate that an ill foodhandler was the likely outbreak source.

The Antibody Response to 27-, 17-, and 15-kDa Cryptosporidium Antigens following Experimental Infection in Humans

Previous studies have suggested that persons infected with Cryptosporidium parvum develop antibody responses to 27-, 17-, and 15-kDa C. parvum antigens. Studies of volunteers infected with Cryptosporidium species provided an opportunity to evaluate the relationship between antibody reactivity to these antigens and infection outcome. As monitored by immunoblot, increases in specific antibody reactivity were more prevalent among volunteers who developed signs and symptoms of cryptosporidiosis (n = 11) than among asymptomatic infected (n = 7; P = .05) or oocyst-negative volunteers (n = 11; P = .02). Volunteers with preexisting IgG antibody to the 27-kDa antigen excreted fewer oocysts than volunteers without this antibody (P = .003). IgG reactivity to the 17-kDa antigens and IgM reactivity to the 27-kDa antigens were higher at day 0 for asymptomatic infected persons than for those who developed symptoms (P = .03 and P = .04, respectively). These results suggest that characteristic antibody responses develop following C. parvum infection and that persons with preexisting antibodies may be less likely to develop illness.

ANTIGENS OF CRYPTOSPORIDIUM SPOROZOITES RECOGNIZED BY IMMUNE SERA OF INFECTED ANIMALS AND HUMANS

The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.

Genetic diversity of Cryptosporidium spp. in captive reptiles.

ABSTRACT The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.