Occurrence of waterborne pathogens and Escherichia coli at offshore drinking water intakes in lake Ontario.

The occurrence of waterborne pathogens was investigated at three drinking water intakes located about 2 km offshore in Lake Ontario. Water sampling was conducted over 3 years for Campylobacter spp., Cryptosporidium spp., Giardia spp., cultivable enteric viruses, and water quality parameters. All pathogens were detected in the offshore source water for each water treatment plant (WTP1 to WTP3), although at relatively low frequencies and concentrations. Giardia was the most common pathogen, occurring in 36% of water samples from the influent of WTP1 (n = 46), and with a maximum concentration of 0.70 cysts/liter in this influent. Cryptosporidium occurred as frequently as 15% in the WTP2 influent (n = 35), with a maximum concentration of 0.40 oocysts/liter in the WTP1 influent. The human Bacteroidales HF183 DNA marker was most common in the WTP1 influent (19%), and this was the only WTP where the Cryptosporidium hominis genotype was detected. No water quality parameter was predictive of pathogen occurrence across all three WTP influents. Escherichia coli was often below detection when pathogens were detected, and spikes in E. coli concentrations often did not coincide with pathogen occurrence. After summer rain events, river plumes had E. coli concentrations as high as 222 CFU/100 ml in surface waters 2 km offshore, without impacting drinking water intakes below the thermocline on the lake bottom. At times, prechlorination to control mussels at offshore intake cribs compromised the use of E. coli for "raw" water quality assessment, particularly for chlorine-resistant Cryptosporidium. E. coli measured by standard methods did not reliably predict pathogen occurrence at drinking water intakes in offshore ecosystems.

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Antibody response to rubella virus proteins in different physical forms.

The immunogenicity of different antigens, containing rubella virus hemagglutinating (HA) membrane protein, was studied using live virus, beta-propiolactone-inactivated virus, detergent and lipid-free octamers and virosomes. Whole virus particles, live or inactivated, induced hemagglutination inhibition (HAI) antibodies in rabbits after one subcutaneous injection of 0.16 micrograms of HA protein. Hemagglutinin rosettes or virosomes failed to induce antibodies even at a dose of 120 micrograms. Apparently, the extraction of viral membrane hemagglutinin, for the preparation of a rubella subunit vaccine, led to destruction of the antigenicity responsible for the induction of hemagglutination inhibiting antibodies. These results are discussed in the light of earlier studies on the preparation of a rubella subunit vaccine.

Rubella virosomes: preparation and ultrastructure.

Rubella virosomes were prepared from performed liposomes and detergent solubilized viral hemagglutinin. The liposomes were made from lecithin/dicetyl phosphate (3.5 : 1) films resuspended in NTE buffer and sonicated. Viral hemagglutinin was prepared from purified virus after solubilization with Triton X-100 and centrifugation through a sucrose gradient containing beta-D-octylglucoside. Electron microscopy of the rate zonal purified virosomes, showed virus-like structures of 40 - 80 nm. The virosomes retained the biological activity of the hemagglutinin and had a buoyant density of 1.2 g/cm3.

Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration.

Human respiratory syncytial virus was concentrated by polyethylene glycol or ammonium sulfate precipitation as well as by hollow ultrafiltration. Recoveries obtained were respectively 49.4%, 47.7%, and 75.2%; however, further analysis of these results by resuspension experiments showed that all the infectivity could be recovered from the different concentrates. The protein content of polyethylene glycol concentrates was much lower than those of ammonium sulfate or hollow fiber ultrafiltration. Electron microscopy revealed that the morphological integrity of virus particles was unaffected by the concentration methods used. Purified virus was obtained when polyethylene glycol concentrates were centrifuged through two successive density gradients.

Purification of infectious rubella virus by gel filtration on sepharose 2B compared to gradient centrifugation in sucrose, sodium metrizoate and metrizamide.

Rubella virus was purified by chromatography on Sepharose 2B after concentration by ultrafiltration on hollow fibers and hydroextraction with PEG 20,000. Yields of 40% infectivity and 70% hemagglutinating activity were routinely obtained. Chromatographic purification was compared to ultracentrifugation in sucrose, metrizamide and sodium metrizoate. Yields were lower in sucrose and metrizamide, while sodium metrizoate reduced the infectivity of the virus below detectable levels. These results demonstrate the advantage of Sepharose 2B for the purification of infectious rubella virus.

Comparison of hemagglutination inhibition, serum neutralization and ELISA-PVM tests for the detection of antibodies to pneumonia virus of mice in rat sera.

Recently, the seroneutralization, hemagglutination inhibition, and the ELISA procedures were used to determine the presence of serum antibody to the pneumonia virus of mice in rat sera. This study was initiated to evaluate the reliability of these procedures to detect specific antibodies in our laboratory animal species. The results obtained indicate that: 1) the seroneutralization test is not reliable since numerous false-positive sera were detected; 2) the ELISA is more sensitive than both the seroneutralization and hemagglutination inhibition procedures, and 3) the presence of low hemagglutination inhibition titers should not be considered significant.

Rapid identification and serotyping of poliovirus isolates by an immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for the identification and typing of polioviruses is described. Polioviruses could be rapidly detected and typed from cell culture supernatants by a double antibody sandwich technique. The assay is valuable for the rapid screening of a large number of viral isolates from water samples submitted for virological analysis. Several hundred isolates per day can be typed and only the remainder have to be tested by the conventional serum neutralization test.

A simple and rapid microassay for the titration of human respiratory syncytial virus.

A microassay, using tissue culture microplates for the titration of human respiratory syncytial virus by syncytium formation, is described. Virus titers obtained agreed well with those obtained in a larger assay system; the microassay, however, is more rapid and economical. Large numbers of virus samples are easily and rapidly processed as the assay necessitates an incubation period of only three days.

Characterisation of rubella virus hemagglutinin rosettes.

Purified rubella virus treated with Triton X-100 was subjected to centrifugation in a sucrose density gradient containing nonionic detergent beta-D-octylglucoside. The result of this treatment was the formation of hemagglutinating rosettes containing viral glycoproteins VP2 (50,000 mol. wt.) and VP3 (63,000 mol. wt.). The rosettes have a 26 S sedimentation coefficient and a density of 1.2 g/cm3 in sucrose. Electron microscopy revealed 15 nm rosettes with a hollow center. The molecular weight of the rosettes was extrapolated at 850,000.

Modified immunoprecipitation procedure for the identification of human respiratory syncytial virus polypeptides.

When analysed by polyacrylamide gel electrophoresis, human respiratory syncytial virus harvested after a one step growth cycle and purified through a continuous sucrose density gradient was shown to be composed of nine structural proteins of 90, 68, 49, 42, 34, 28, 25, 19 an 13 kd. The 90, 49 and 19 kd polypeptides were identified as glycopolypeptides by glucosamine incorporation. A modified immunoprecipitation procedure confirmed the viral specificity of the 49, 42, 28, 25 and 19 kd polypeptides.

Rapid titration of bovine, caprine and human RS virus by a micro-immunoperoxidase assay using a monoclonal antibody and a permissive ovine kidney cell line.

An indirect immunoperoxidase micro-assay, using a continuous cell line derived from ovine kidney cells (OK) and a previously characterized monoclonal antibody (7C2), specific for an exposed and highly conserved epitope of the fusion protein of different strains of RS virus, was used advantageously to rapidly titrate bovine, caprine and human strains of RSV by either quantal (TCID50) or plaque forming assays. Virus titers, obtained in less than 36 h, were in agreement with those obtained by the conventional plaque assays which required an incubation period of 4 days or more. This assay is also applicable to micro-neutralization of fusion inhibition assays for testing serum or screening monoclonal antibodies.

Rapid virus subunit visualization by direct sedimentation of samples on electron microscope grids.

Airfuge direct ultracentrifugation of viral samples on electron microscope grids offers a rapid way for concentrating viral particles or subunits to facilitate their detection and study. Using the A-100 fixed angle rotor (30 degrees) with a K factor of 19 at maximum speed (95,000 rpm), samples up to 240 microliters can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10- to 40-fold increase in sensitivity depending on the size of particles. Application of this technique to rubella virus permitted better visualization of viral membrane subunits on the particles. Rubella hemagglutinin immuno-stimulating complexes preparations were also better visualized and their morphology conserved after direct ultracentrifugation on the specimen grids. Similar observations are reported for respiratory syncytial virus associated subunits.

Identification of rubella virus structural proteins by immunoprecipitation.

Immunoprecipitation of [3H]amino acid labelled virus with monoclonal or human convalescent rubella sera and subsequent analysis by electrophoresis and fluorography, revealed three structural proteins of rubella virus: VP3: 59,000; VP2: 44,800; and VP1: 33,000.

E1 glycoprotein of rubella virus carries an epitope that binds a neutralizing antibody.

We identified by immunoprecipitation and Western blot analysis, using a monoclonal antibody that neutralizes rubella virus, that E1 glycoprotein carries an epitope linked with neutralization. Glycosidase treatment of virus does not prevent blotting of this monoclonal antibody with the E1 glycoprotein, dissociating this epitope from the hemagglutination epitope which is linked with the oligosaccharide side chains. We also investigated by Western blot analysis human serum reactivity toward E1 glycoprotein and the two other structural proteins of rubella virus, E2 and C: all positive sera detected E1 and C, irrespective of their titers, indicating the importance of glycoprotein E1 in immunity. Frequent lack of reactivity against E2 might suggest that this glycoprotein is either less exposed or less immunogenic.

A fatal waterborne disease epidemic in Walkerton, Ontario: comparison with other waterborne outbreaks in the developed world.

An estimated 2,300 people became seriously ill and seven died from exposure to microbially contaminated drinking water in the town of Walkerton, Ontario, Canada in May 2000. The severity of this drinking water disaster resulted in the Government of Ontario calling a public inquiry by Mr. Justice Dennis O'Connor to address the cause of the outbreak, the role (if any) of government policies in contributing to this outbreak and, ultimately, the implications of this experience on the safety of drinking water across the Province of Ontario. The circumstances surrounding the Walkerton tragedy are an important reference source for those concerned with providing safe drinking water. Although some circumstances are obviously specific to this epidemic, others are uncomfortably reminiscent of waterborne outbreaks that have occurred elsewhere. These recurring themes suggested the need for attention to broad issues of drinking water security and they present the challenge for how drinking water safety can be managed to prevent such tragedies in the future.

Comparison of methods of sampling for Toxocara species and fecal coliforms in an outdoor day care environment.

OBJECTIVE: To compare three sampling methods and to pretest methods for the determination of fecal coliform (FC) counts and Toxocara species from sand in the day care outdoor environment. DESIGN: The sand samples were obtained from the play area and the sandbox of a day care centre and examined for the presence of FC and Toxocara species, the common roundworm of dogs and cats. The sampling methods included random selection and two types of judgement methods. The latter included one method where domestic animals were judged to be likely to defecate and the other where children would be likely to be playing. In addition, to obtain a global estimate of contamination, the entire areas of both the sandbox and the play area were sampled on the last day. SETTING: Outdoor day care environment. MAIN RESULTS: The most representative levels of bacterial contamination and Toxocara species originated from the combined sample of the entire surface areas rather than from any separate random or judgement method of sampling. FCs were found in all sampled areas of the sandbox (median 910 FCs/g of sand) and of the play area (median 350 FCs/g of sand). Toxocara species were recovered from a number of areas in both the sandbox and the play area. CONCLUSIONS: Research on environmental microbial contamination of outdoor day care settings would benefit from the application of standardized and validated sampling and laboratory methods.

Risk of infectious gastroenteritis in young children living in Québec rural areas with intensive animal farming: results of a case-control study (2004-2007).

This study was designed to evaluate the epidemiology of severe gastroenteritis in children living in Québec rural areas with intensive livestock activities. From September 2005 through June 2007, 165 cases of gastroenteritis in children aged from 6 months to 5 years, hospitalized or notified to the public health department were enrolled, and 326 eligible controls participated. The parents of cases and controls were asked questions about different gastroenteritis risk factors. The quality of the drinking water used by the participants was investigated for microbial indicators as well as for four zoonotic bacterial pathogens (Campylobacter spp, Escherichia coli, Salmonella spp and Yersinia spp) and two enteric parasites (Cryptosporidium spp and Giardia spp). From 134 stool specimen analysed, viruses were detected in 82 cases (61%), while 28 (21%) were found with at least one of the bacteria investigated, and five cases were infected by parasites. Campylobacteriosis was the main bacterial infection (n = 15), followed by Salmonella sp (n = 7) and E. coli O157:H7 (n = 5) among cases with bacterial gastroenteritis. No significant difference was found between cases and controls regarding the quality of water consumed; the frequency of faecal contamination of private wells was also similar between cases and controls. Considering the total cases (including those with a virus), no link was found between severe gastroenteritis and either being in contact with animals or living in a municipality with the highest animal density (4th quartile). However, when considering only cases with a bacterial or parasite infection (n = 32), there was a weak association with pig density that was not statistically significant after adjusting for potential confounders. Contact with domestic, zoo or farm animals were the only environmental factor associated with the disease.

Association between potential zoonotic enteric infections in children and environmental risk factors in Quebec, 1999-2006.

This study was designed to evaluate the association of potential zoonotic gastroenteritis in children, and specifically giardiasis, salmonellosis and campylobacteriosis, with environmental risk factors in rural areas of Quebec. Notified cases of gastroenteritis in children of 0-4 years of age reported in the period of 1999 through 2006 from municipalities in southern Quebec with <100,000 inhabitants were investigated. Negative binomial regression models accounting for overdispersion and adjusted for clustering were used to estimate relative risks (RR) associated with livestock densities and drinking water quality. Analyses revealed that, during this period, 2500 cases of gastroenteritis were reported in children of 0-4 years, including 819 cases of giardiasis, 690 of salmonellosis and 852 of campylobacteriosis. The incidence rate associated with all potential zoonotic agents reported was 163 cases/100,000 children-years and this was statistically associated with cattle density: RR Quartile 4/Quartile 1 (Q4/Q1) = 1.92, 95% CI = 1.43-2.58. When estimated specifically for each pathogen, incidence rates of giardiasis (RR Q4/Q1 = 1.79, 95% CI = 1.11-2.87), salmonellosis (RR Q4/Q1 = 1.64, 95% CI = 1.15-2.33) and campylobacteriosis (RR Q4/Q1 = 2.43, 95% CI = 1.60-3.68) were also associated with cattle density, with a monotonic increase of RR with increasing animal density. Giardiasis incidence was also positively associated with a poor drinking water quality, although no statistically significant association was found. Our results suggest that, in rural Quebec, bacterial and parasitic enteric infections in young children may be zoonoses related to environmental risk factors and especially cattle production.