Biomedical subjects

M J Arrowood

Publications and source records attributed to M J Arrowood.

At least 19 recordsLinked to original sources

PubMed2002

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

Animals↗

PubMed2001

Cryptosporidium parvum-specific antibody responses among children residing in Milwaukee during the 1993 waterborne outbreak.

A major gastroenteritis outbreak among >400,000 residents of Milwaukee, Wisconsin, in April 1993 was attributed to Cryptosporidium parvum oocysts in drinking water. Plasma specimens obtained from children (6 months to 12 years old) for routine blood lead level surveillance March-May 1993 were assayed by ELISA for levels of IgG antibody against the immunodominant Triton-17 and 27-kDa C. parvum antigens. Over a 5-week period, the seroprevalence for antibodies to the 2 antigens increased from 15% to 82% and from 17% to 87%, respectively, in samples from children living in southern ZIP code areas (n=218), whereas smaller increases (20% to 43% and 22% to 46%, respectively) were noted among samples from children living in northern ZIP code areas (n=335; P<.0001). The results demonstrate that C. parvum infection was much more widespread than previously appreciated and confirm that infection was associated with residence in the area served by the southern water treatment plant.

Animals↗

PubMed2001

Outbreak of cyclosporiasis associated with basil in Missouri in 1999.

During the summer of 1999, an outbreak of cyclosporiasis occurred among attendees of 2 events held on 24 July in different counties in Missouri. We conducted retrospective cohort studies of the 2 clusters of cases, which comprised 62 case patients. The chicken pasta salad served at one event (relative risk [RR], 4.25; 95% confidence interval [CI], 1.80-10.01) and the tomato basil salad served at the other event (RR, 2.95; 95% CI, 1.72-5.07) were most strongly associated with illness. The most likely vehicle of infection was fresh basil, which was included in both salads and could have been grown either in Mexico or the United States. Leftover chicken pasta salad was found to be positive for Cyclospora DNA by means of polymerase chain reaction analysis, and 1 sporulated Cyclospora oocyst was found by use of microscopy. This is the second documented outbreak of cyclosporiasis in the United States linked to fresh basil and the first US outbreak for which Cyclospora has been detected in an epidemiologically implicated food item.

Adolescent↗

PubMed2001

The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored.

Cryptosporidium parvum is a protozoan parasite of the intestinal epithelium that has caused numerous outbreaks of diarrheal illness in humans. During our studies of the host immune response to C. parvum infection, we noted that two of the immunodominant surface antigens of the sporozoite stage of the parasite readily extract into Triton X-114. We recently cloned the immunodominant 17-kDa surface antigen and suggested that the carboxy-terminal peptide sequence may satisfy the requirements for GPI anchor addition. In the work presented here, we were able to show that the 17-kDa antigen could be metabolically labeled in vitro with tritiated ethanolamine and that the antigen contained myo-inositol. The antigen was cleaved by GPI-PLD but not by PI-PLC and it could be converted to a water soluble form by chemical deglycosylation. We suggest that the 17-kDa antigen is indeed GPI anchored and that the anchor contains an acylated inositol and either a lyso-acyl- or a diacyl-glycerol. We are currently working to determine what role the anchor may play in the human immune response to this antigen.

Animals↗

PubMed2001

Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts.

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 purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) 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.

Animals↗

PubMed2001

Enzyme immunoassay detection of antigen-specific immunoglobulin g antibodies in longitudinal serum samples from patients with cryptosporidiosis.

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.

Adult↗

PubMed2001

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry.

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

Animals↗

PubMed2001

Development and application of a quantitative, specific assay for Cryptosporidium parvum oocyst detection in high-turbidity environmental water samples.

Chlorine-resistant Cryptosporidium parvum oocysts in drinking water play an important role in the epidemiology of cryptosporidiosis. Current methods of detecting these organisms in water are insensitive, labor-intensive, highly subjective, and severely limited by sample turbidity. We describe here an alternative technique utilizing electrochemiluminescence (ECL) technology for detecting C. parvum oocysts in environmental water samples. This method is quantitative, reproducible, and requires only minimal sample processing. Currently, the ECL assay can detect as few as one oocyst in one milliliter of concentrated test sample with sample turbidity of up to 10,000 nephelometric turbidity units. Water and sewer samples collected during a cryptosporidiosis outbreak were tested by ECL assay. Cryptosporidium parvum oocysts were found in the source water at the time of outbreak, and a sharply decreasing level of oocysts in sewer samples was observed over a three-month period following the outbreak.

Animals↗

PubMed2000

Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum.

Infection with Cryptosporidium parvum causes a self-limiting diarrheal illness in immunocompetent humans and is associated with the development of a serum IgG antibody response dominated by the 27-kDa and 17-kDa parasite surface antigens. Antibodies against the 27-kDa and 17-kDa antigens may serve as useful markers for past infection in population-based studies of the risk factors associated with Cryptosporidium infection. A recombinant form of the 17-kDa antigen would be useful both in epidemiologic studies and in studies of the role of the humoral response in immunity. We have partially purified and sequenced the immunodominant 17-kDa surface antigen from sporozoites, and we have cloned a 975 bp open reading frame from C. parvum that includes all of the 17-kDa antigen peptide sequences. We show immunologic identity between a recombinant form of the protein and the native 17-kDa antigen. We conclude that the carboxy-terminal fragment of the cloned protein is the authentic 17-kDa antigen.

Amino Acid Sequence↗

PubMed2000

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.

Adolescent↗

PubMed2000

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Animals↗

PubMed2000

Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

Animals↗

PubMed1999

Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens.

Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.

Animals↗

PubMed1999

Biallelic polymorphism in the intron region of beta-tubulin gene of Cryptosporidium parasites.

Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.

Alleles↗

PubMed1998

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.

Animals↗

PubMed1998

In-vitro activities of paromomycin and lasalocid evaluated in combination against Cryptosporidium parvum.

Using a chemiluminescence immunoassay, paromomycin and lasalocid were shown to inhibit Cryptosporidium parvum growth in Madin-Darby canine kidney cells in a concentration-dependent manner. The median effective concentrations (EC50s) for paromomycin and lasalocid were 1184 mg/L and 0.4 mg/L, respectively. Neither drug was cytotoxic to host cells at concentrations up to five times their EC50s. Drug combination studies were conducted and the resulting data were analysed by the median-effect principle and combination index method. Statistically significant synergy was observed when combinations of paromomycin and lasalocid were used at ratios of 5000:1 and 2500:1. A possible mechanism for synergy is discussed.

Animals↗

PubMed1998

Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.

Symptoms consistent with an outbreak of cryptosporidiosis (diarrhea, vomiting, nausea, and abdominal cramps) occurred on a U.S. Coast Guard cutter within 0-18 days after the cutter filled its tanks with Milwaukee, Wisconsin city water in March 1993. At three-weeks postdocking (PD), the suspected water was removed, and serum samples and stool specimens were collected from 47 of the 58 crew members, as well as questionnaire data on their water consumption and symptoms aboard the cutter. At 10-weeks PD and/or at 28-weeks PD, additional serum specimens were collected. Intensitometric data from enzyme-linked immunoelectrotransfer blot (EITB) were obtained on IgA responses to a 17-kD antigen group, IgM responses to a 27-kD antigen group, and IgG responses to 27-, 17-, and 15-kD antigen groups extracted from oocysts. In addition, IgG responses to crude oocyst antigens were obtained by ELISA. Based on reported symptoms, EITB results, and stool examination, the crew members were classified as confirmed (10), probable (10), suspected (22), and noncases (16). Of the 10 confirmed cases (all symptomatic) and the 10 probable cases (eight symptomatic) whose stools were positive and negative, respectively, for Cryptosporidium oocysts by microscopy, all showed changes in EITB intensities to the antigen groups and were considered EITB positive. The remaining 38 crew members, 22 suspected cases (all symptomatic), and 16 noncases (all asymptomatic), if tested, had negative stool examinations and were considered EITB negative. Of the 10 confirmed cases, only four showed a significant change in IgG responses (P < 0.05) between three-weeks PD and follow-up serum specimens by ELISA. Crew members considered confirmed cases consumed significantly more water (P < or = 0.005) aboard the cutter than noncases. Crew members considered EITB positive consumed more water (P < or = 0.04) than crew members considered EITB negative while there was no significant difference in water consumption (P > or = 0.19) between crew members considered ELISA positive and ELISA negative. Using the EITB, the observation of changes in intensity of IgA responses to the 17-kD antigen group, IgM responses to the 27-kD antigen group, and IgG responses to the 27- 17-, and 15-kD antigen groups from C. parvum oocysts between acute and convalescent serum specimens appears useful for immunodiagnosis of Cryptosporidium infection and for prospective epidemiologic studies designed to monitor infection risk.

Animals↗

PubMed1998

An outbreak of cyclosporiasis in Florida in 1995: a harbinger of multistate outbreaks in 1996 and 1997.

Before 1995, only one outbreak of cyclosporiasis had been reported in the United States. To identify risk factors for Cyclospora infection acquired in Florida in 1995, we conducted a matched case-control study (24 sporadic cases and 69 controls) and retrospective cohort studies of clusters of cases associated with two May social events (attack rates = 15.4% [8 of 52] and 54.5% [6 of 11]). In univariate analysis of data from the case-control study, consumption of fresh raspberries (odds ratio [OR] = 6.0, 95% confidence interval [CI] = 1.1-31.7) and bare-handed contact with soil (OR = 5.4, 95% CI = 1.4-20.7) were associated with infection; soil contact was also implicated in multivariate analysis. For the events, mixed-fruit items that had only fresh raspberries and strawberries in common had elevated relative risks (3.7 and 4.2), but the confidence intervals overlapped 1.0. The raspberries eaten at the events and by sporadic case-patients were imported. Given the cumulative evidence of the three studies and the occurrence in 1996 and 1997 of outbreaks in North America associated with consumption of Guatemalan raspberries, food-borne transmission of Cyclospora was likely in 1995 in Florida as well.

Adolescent↗