Environmental predictors of diarrhoeal infection for rural and urban communities in south India in children and adults.

Diarrhoeal diseases are major causes of morbidity and mortality in developing countries. This longitudinal study aimed to identify controllable environmental drivers of intestinal infections amidst a highly contaminated drinking water supply in urban slums and villages of Vellore, Tamil Nadu in southern India. Three hundred households with children (<5 years) residing in two semi-urban slums and three villages were visited weekly for 12-18 months to monitor gastrointestinal morbidity. Households were surveyed at baseline to obtain information on environmental and behavioural factors relevant to diarrhoea. There were 258 diarrhoeal episodes during the follow-up period, resulting in an overall incidence rate of 0·12 episodes/person-year. Incidence and longitudinal prevalence rates of diarrhoea were twofold higher in the slums compared to rural communities (P < 0·0002). Regardless of study site, diarrhoeal incidence was highest in infants (<1 year) at 1·07 episodes/person-year, and decreased gradually with increasing age. Increasing diarrhoeal rates were associated with presence of children (<5 years), domesticated animals and low socioeconomic status. In rural communities, open-field defecation was associated with diarrhoea in young children. This study demonstrates the contribution of site-specific environmental and behavioural factors in influencing endemic rates of urban and rural diarrhoea in a region with highly contaminated drinking water.

Cryptosporidiosis: environmental, therapeutic, and preventive challenges.

Cryptosporidium spp. are responsible for endemic and epidemic disease worldwide. Clinical manifestations may include acute, persistent, or chronic diarrhea, biliary, and pulmonary disease. Disease severity ranges from asymptomatic or mild to severe, intractable diarrhea with wasting depending on immune status, nutrition, and age. Transmission is fecal-oral with both human and animal reservoirs. Disease is often self limited in healthy individuals, but therapy remains a challenge in the immune-compromised. Prevention currently depends on appropriate hygiene and proper water management and treatment.

Recent diarrhea is associated with elevated salivary IgG responses to Cryptosporidium in residents of an eastern Massachusetts community.

BACKGROUND: Serological data suggest that Cryptosporidium infections are common but underreported. The invasiveness of blood sampling limits the application of serology in epidemiological surveillance. We pilot-tested a non-invasive salivary anti-Cryptosporidium antibody assay in a community survey involving children and adults. MATERIALS AND METHODS: Families with children were recruited in a Massachusetts community in July; symptoms data were collected at 3 monthly follow-up mail surveys. One saliva sample per person (n = 349) was collected via mail, with the last survey in October. Samples were analyzed for IgG and IgA responses to a recombinant C. hominis gp15 sporozoite protein using a time-resolved fluorometric immunoassay. Log-transformed assay results were regressed on age using penalized B-splines to account for the strong age-dependence of antibody reactions. Positive responses were defined as fluorescence values above the upper 99% prediction limit. RESULTS: Forty-seven (13.5%) individuals had diarrhea without concurrent respiratory symptoms during the 3-month-long follow-up; eight of them had these symptoms during the month prior to saliva sampling. Two individuals had positive IgG responses: an adult who had diarrhea during the prior month and a child who had episodes of diarrhea during each survey month (Fisher's exact test for an association between diarrhea and IgG response: p = 0.0005 for symptoms during the prior month and p = 0.02 for symptoms during the entire follow-up period). The child also had a positive IgA response, along with two asymptomatic individuals (an association between diarrhea and IgA was not significant). CONCLUSION: These results suggest that the salivary IgG specific to Cryptosporidium antigens warrants further evaluation as a potential indicator of recent infections.

MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice.

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.

Expression of Cpgp40/15 in Toxoplasma gondii: a surrogate system for the study of Cryptosporidium glycoprotein antigens.

Cryptosporidium parvum is a waterborne enteric coccidian that causes diarrheal disease in a wide range of hosts. Development of successful therapies is hampered by the inability to culture the parasite and the lack of a transfection system for genetic manipulation. The glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in C. parvum sporozoite attachment to and invasion of host cells and, as such, may be good targets for anticryptosporidial therapies. However, the function of these antigens appears to be dependent on the presence of multiple O-linked alpha-N-acetylgalactosamine (alpha-GalNAc) determinants. A eukaryotic expression system that would produce proteins bearing glycosylation patterns similar to those found on the native C. parvum glycoproteins would greatly facilitate the molecular and functional characterization of these antigens. As a unique approach to this problem, the Cpgp40/15 gene was transiently expressed in Toxoplasma gondii, and the expressed recombinant glycoproteins were characterized. Antisera to gp40 and gp15 reacted with the surface membranes of tachyzoites expressing the Cpgp40/15 construct, and this reactivity colocalized with that of antiserum to the T. gondii surface protein SAG1. Surface membrane localization was dependent on the presence of the glycophosphatidylinositol anchor attachment site present in the gp15 coding sequence. The presence of terminal O-linked alpha-GalNAc determinants on the T. gondii recombinant gp40 was confirmed by reactivity with Helix pomatia lectin and the monoclonal antibody 4E9, which recognizes alpha-GalNAc residues, and digestion with alpha-N-acetylgalactosaminidase. In addition to appropriate localization and glycosylation, T. gondii apparently processes the gp40/15 precursor into the gp40 and gp15 component glycopolypeptides, albeit inefficiently. These results suggest that a surrogate system using T. gondii for the study of Cryptosporidium biology may be useful.

Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins.

Published genomic differences between Cryptosporidium parvum genotype 1 (human-derived) and genotype 2 (animal and human-derived) isolates suggest that these may belong to two distinct species. This is of significant interest since genotype 1 isolates are associated with sporadic cases of human cryptosporidiosis in 30-40 % of cases in contrast to 60-70 % of cases caused by genotype 2. The lower genetic sequence similarity between genotype 1 and 2 surface glycoproteins (gp40/15) suggests that antigenic differences should also occur, a feature that was investigated in this study. Using immune and convalescent serum samples from gnotobiotic piglets previously inoculated with genotype 1 and 2 isolates, we demonstrated that C. parvum gp15 was immunodominant for both genotype 1 and 2 isolates. Lower genetic sequence similarity between genotype 1 and 2 Cpgp40/15 did correspond to gp15 protein differences as detected by Western blot. Moreover, we confirmed that gp15 contains epitopes that are also immunodominant. Deglycosylation of C. parvum proteins resulted in decreased ability of gp15, gp23 and gp900 to react with homologous polyclonal antibodies, suggesting that these proteins also express carbohydrate epitopes. Taken together, our data suggest that there is a high phenotypic variability between C. parvum genotype 1 and 2 isolates at the level of gp15. We contemplate that gp15 surface glycoprotein plays an important role in the biology of C. parvum as a potent inducer of immune response and a possible virulence factor.

Occupational asthma in adults in six Canadian communities.

We examined the prevalence, population attributable risk (PAR), and clinical characteristics of occupational asthma (OA) in a randomly selected population in six communities in Canada. Our study followed the European Community Respiratory Health Survey protocol. A randomly selected population of 18,701 (87% response rate) persons from the study communities, ranging in age from 20 to 44 yr, completed an initial questionnaire, of whom 2,974 (39% response rate) attended the laboratory and completed supplementary questionnaires. Of these latter individuals, 383 had asthma. Asthma was defined as physician-diagnosed asthma, and adult-onset asthma was defined as a first attack at age 15 yr or older. We used several methods for estimating OA as follows: (1) reporting of a high-risk job (occupation and industry) for OA at the time of asthma onset (Probable OA); (2) reporting of exposure to a substance that may cause OA (Possible OA) while not in a high-risk job at the time of asthma onset; and (3) combination of the PAR for high-risk jobs and exposures. The prevalence (95% confidence interval [CI]) of Probable OA and Possible OA combined was 36.1% (31.3 to 41.0%) among subjects with adult-onset asthma. The occupations most commonly reported in association with OA were nursing in the Probable OA group and clerical and food preparation in the Possible OA group. The clinical characteristics and exposures reported by both groups were similar. The PAR for adult-onset asthma in high-risk jobs and exposures was 18.2%. The assessment of occupation and industry alone, rather than of exposures, may underestimate the contribution of occupational exposures to asthma prevalence.

Mediation of Cryptosporidium parvum infection in vitro by mucin-like glycoproteins defined by a neutralizing monoclonal antibody.

The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion- and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains alpha-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and alpha-N-acetylgalactosamine-specific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.

Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15.

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.

Attachment of Cryptosporidium parvum sporozoites to human intestinal epithelial cells.

An enzyme-linked immunosorbent assay-based attachment model using the human intestinal cell line Caco-2A was developed to study attachment of Cryptosporidium parvum sporozoites in vitro and to assess potential inhibitors of sporozoite binding. In this system, attachment was related to sporozoite dose, incubation time, and host cell differentiation status. Polyclonal antibodies to C. parvum as well as glycoprotein inhibitors of a sporozoite lectin reduced attachment. This model will be a valuable tool in elucidating specific molecules and mechanisms involved in sporozoite-host cell attachment.

An in vitro model of infection of human biliary epithelial cells by Cryptosporidium parvum.

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.

Comparison of dust related respiratory effects in Dutch and Canadian grain handling industries: a pooled analysis.

OBJECTIVES: Four previously conducted epidemiological studies in more than 1200 grain workers were used to compare exposure-response relations between exposure to grain dust and respiratory health. METHODS: The studies included Dutch workers from an animal feed mill and a transfer grain elevator and Canadian workers from a terminal grain elevator and the docks. Relations between forced expiratory volume in one second (FEV1) and exposure were analysed with multiple regression analysis corrected for smoking, age, and height. Exposure variables examined included cumulative and current dust exposure and the numbers of years a subject was employed in the industry. Sampling efficiencies of the Dutch and Canadian measurement techniques were compared in a pilot study. Results of this study were used to correct slopes of exposure-response relations for differences in dust fractions sampled by Dutch and Canadian personal dust samplers. RESULTS: Negative exposure-response relations were shown for regressions of FEV1 on cumulative and current exposure and years employed. Slopes of the exposure-response relations differed by a factor of three to five between industries, apart from results for cumulative exposure. Here the variation in slopes differed by a factor of 100, from -1 to -0.009 ml/mg.y/m3. The variation in slopes between industries reduced to between twofold to fivefold when the Dutch transfer elevator workers were not considered. There was evidence that the small exposure-response slope found for this group is caused by misclassification of exposure and a strong healthy worker effect. Alternative, but less likely explanations for the variation in slopes were differences in exposure concentrations, composition of grain dust, exposure characteristics, and measurement techniques. CONCLUSION: In conclusion, this study showed moderately similar negative exposure-response relations for four different populations from different countries, despite differences in methods of exposure assessment and exposure estimation.

Evaluation of the respiratory health of dock workers who load grain cargoes in British Columbia.

OBJECTIVES: To investigate the respiratory health of dock workers who load grain cargoes. METHODS: The respiratory health of 118 dock workers who load grain cargoes in the ports of Vancouver and Prince Rupert was compared with that of 555 grain elevator workers from the same regions. 128 civic workers were used as an unexposed control group. RESULTS: The prevalences of chronic cough and phlegm were at least as high in dock workers as those found in the elevator workers, and when adjusted for differences in duration of employment and smoking, dock workers had an eightfold higher risk of developing chronic phlegm than did civic workers. Symptoms of eye and skin irritation that were experienced at least monthly were highest for dock workers. Average percentage of the predicted FEV1 and FVC for dock workers (mean 100.6% and 105.3% respectively) were similar to the civic workers but significantly higher than those found for elevator workers. Higher subjective estimates of duration of exposure to grain dust (hours/day) were associated with lower values of FEV1. CONCLUSIONS: The more intermittent grain dust exposure patterns of dock workers may have allowed for some recovery of lung function, but chronic respiratory symptoms were less labile.

Growth inhibition of the intestinal parasite Giardia lamblia by a dietary lectin is associated with arrest of the cell cycle.

Giardia lamblia, a cause of diarrheal disease throughout the world, is a protozoan parasite that thrives in the small intestine. It is shown here that wheat germ agglutinin (WGA), a naturally occurring lectin widely consumed in normal human diets, reversibly inhibits the growth of G. lamblia trophozoites in vitro, and reduces infection by G. muris in the adult mouse model of giardiasis. The inhibitory effect was dose related, not associated with cytotoxicity and reversed by N-acetyl-D-glucosamine in accordance with the known specificity of the lectin and in agreement with the presence of GlcNAc residues on the surface membrane of G. lamblia trophozoites. Cell cycle analysis revealed that parasites grown in the presence of WGA are arrested in the G2/M phase, providing an explanation for the lectin-induced inhibition of cell proliferation. Comparison of electrophoretic profiles by lectin blot analysis revealed both glycoprotein induction and suppression in growth-arrested organisms. Our findings raise the possibility that blocking trophozoite growth with naturally occurring dietary lectins may influence the course of giardiasis. In addition, the study of cell cycle arrest by WGA may provide a model to study the regulation of cell division in lower eukaryotes.

Developmentally regulated lectins in Eimeria species and their role in avian coccidiosis.

Avian coccidiosis caused by Eimeria species is characterized by rather specific site infections of the intestine. We used hemagglutination and hemagglutination inhibition assays on various developmental stages of Eimeria tenella and on sporozoites of Eimeria acervulina and Eimeria maxima to assay for parasite lectins. Various monosaccharides, polysaccharides, and glycoproteins were used to demonstrate differences in sugar specificity of the lectins between these species. Surface lectins were found on the primary infective stage, i.e., sporozoites, but not on merozoites or unsporulated oocysts. Also, there were differences in the specificities of the various sugar lectins among the different parasite species. Furthermore, there was a dose-dependent reduction of infection of tissue culture cells by sporozoites of E. tenella that were continuously exposed to fetuin, 1 of the specific inhibitors of the lectin. The results of our study are unique in that in 3 species of avian Eimeria all have a lectin on their sporozoites, but the lectins for each species have different sugar specificities. We hypothesize that these lectins found on the surface of the sporozoites may play a role in determining the site of infection within the intestine of the host.

Giardia lamblia: absence of cyst antigens and reduced secretory vesicle formation and bile salt uptake in an encystation-deficient subline.

Encystation of Giardia lamblia entails the appearance of a number of new antigens, as well as formation of a novel class of large encystation-specific secretory vesicles (ESV) that transport stage-specific proteins to the nascent cyst wall. The monoclonal antibody GCSA-1, which was raised against purified cyst walls, recognizes protein species of approximately 26-46 kDa that are regulated by exposure to bile (plus lactic acid) and alkaline pH, the factors that induce encystation. The GCSA-1 epitope is maximally expressed after approximately 14 hr of encystation and localizes to the interior, but not the membrane of the ESV as shown by frozen section immunoelectron microscopy. To further understand the process of encystation, we compared two sublines of strain WB that differ in their ability to encyst in vitro. Water-resistant cysts were not detected in subline A6 under conditions in which subline C6 formed approximately 2 x 10(5) cysts/ml. Moreover, subline A6 did not form ESV efficiently or detectably express antigens recognized by mAb GCSA-1 or by polyclonal anti-cyst sera. Finally, uptake of the bile salt taurocholate by A6 was reduced 4- to 20-fold, compared with that of C6, although transport by both strains was sodium-dependent and regulated by bile salt starvation. The decrease in bile salt uptake by A6 may be related to its defect in encystation.

In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency.

A method for obtaining large numbers of Giardia lamblia cysts in vitro was developed based on modification of earlier methods of in vitro encystation. Maximal numbers of cysts were obtained by growing trophozoites to confluence in TYI-S-33 growth medium containing 0.5 mg/ml of bovine bile, followed by incubation in medium containing 10 mg/ml of bovine bile, at pH 7.8 for 96 h at 37 C. Up to 4 x 10(5) cysts were obtained per milliliter of encystation medium. Cysts thus obtained were similar in structure to those in vivo, were resistant to hypotonic lysis, and reacted with a cyst-specific monoclonal antibody. Further modification of this method by returning the trophozoites to growth medium after 24 hr of exposure to encystation medium resulted in production of cysts that were shown to be viable by fluorogenic dye staining and ability to excyst. This method was scaled up using roller bottles, which resulted in production of up to 1.6 x 10(8) cysts per roller bottle. In addition, of 4 strains tested, the LT strain yielded the highest number of cysts. Of 4 clones of the WB strain, clone A consistently produced the largest number of cysts.

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody.

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.