Recovery, Bioaccumulation, and Inactivation of Human Waterborne Pathogens by the Chesapeake Bay Nonnative Oyster, Crassostrea ariakensis

The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 × 10⁵ transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters.     

Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Effectiveness of Standard UV Depuration at Inactivating Cryptosporidium parvum Recovered from Spiked Pacific Oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 × 10⁶ oocysts liter ⁻¹) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Development and validation of a predictive model for the growth of Vibrio vulnificus in postharvest shellstock oysters

Postharvest growth of Vibrio vulnificus in oysters can increase risk of human infection. Unfortunately, limited information is available regarding V. vulnificus growth and survival patterns over a wide range of storage temperatures in oysters harvested from different estuaries and in different oyster species. In this study, we developed a predictive model for V. vulnificus growth in Eastern oysters (Crassostrea virginica) harvested from Chesapeake Bay, MD, over a temperature range of 5 to 30°C and then validated the model against V. vulnificus growth rates (GRs) in Eastern and Asian oysters (Crassostrea ariakensis) harvested from Mobile Bay, AL, and Chesapeake Bay, VA, respectively. In the model development studies, V. vulnificus was slowly inactivated at 5 and 10°C with average GRs of -0.0045 and -0.0043 log most probable number (MPN)/h, respectively. Estimated average growth rates at 15, 20, 25, and 30°C were 0.022, 0.042, 0.087, and 0.093 log MPN/h, respectively. With respect to Eastern oysters, bias (B(f)) and accuracy (A(f)) factors for model-dependent and -independent data were 1.02 and 1.25 and 1.67 and 1.98, respectively. For Asian oysters, B(f) and A(f) were 0.29 and 3.40. Residual variations in growth rate about the fitted model were not explained by season, region, water temperature, or salinity at harvest. Growth rate estimates for Chesapeake Bay and Mobile Bay oysters stored at 25 and 30°C showed relatively high variability and were lower than Food and Agricultural Organization (FAO)/WHO V. vulnificus quantitative risk assessment model predictions. The model provides an improved tool for designing and implementing food safety plans that minimize the risk associated with V. vulnificus in oysters.     

Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.     

Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.     

Survival of Infectious Cryptosporidium parvum Oocysts in Seawater and Eastern Oysters (Crassostrea virginica) in the Chesapeake Bay

Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.     

Discriminatory predation by three invertebrates on eastern oysters (Crassostrea virginica) compared with non-native Suminoe oysters (C. ariakensis)

Abstract. Diminished populations of eastern oysters Crassostrea virginica in Chesapeake Bay have stimulated proposals to introduce Crassostrea ariakensis from Asia to restore oyster stocks. As part of a program evaluating possible ramifications of such an introduction, we studied how invertebrate predators responded to this non-native oyster. We compared predation activity under laboratory conditions by oyster drills ( Urosalpinx cinerea; Eupleura caudata ) that bore through an oyster's shell and by the seastar Asterias forbesi that pulls shell valves apart. These three predators preyed significantly (p<0.05) more on the familiar C. virginica than on the novel C. ariakensis . We previously reported that five crab species preyed significantly more on C. ariakensis than on C. virginica , with predation by polyclad flatworms similar between oyster species. Thus, the drills and the seastar differed from the crabs and the flatworms in their response to novel prey. When Urosalpinx cinerea was placed in a Y-maze after being held for 40 d with oysters of one species or the other, the drills moved toward C. virginica effluent more than toward C. ariakensis effluent. This response did not depend on the species of oyster the drills had been held with, suggesting that the drills were responding to more familiar infochemicals from eastern oysters than from the non-native oysters.     

Quantitative Evaluation of the Impact of Bather Density on Levels of Human-Virulent Microsporidian Spores in Recreational Water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.     

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.     

Co‐localized Crassostrea virginica and Crassostrea ariakensis Oysters differ in bioaccumulation,retention and depuration of microbial indicators and human enteropathogens

Aims: To evaluate the bioaccumulation, retention and depuration rates of nine pathogens and surrogates when two oyster species were co‐localized in tanks of seawater. Methods and Results: Crassostrea ariakensis (n = 52) and Crassostrea virginica (n = 52) were exposed to five virus types, two protozoan and two microsporidian species for 24 h. Oysters were then placed in depuration tanks, and subsets were removed and analysed for micro‐organisms at weekly intervals. The odds of C. ariakensis oysters harbouring mouse norovirus‐1 (MNV‐1), human norovirus (NoV) or haepatitis A virus (HAV) were significantly greater than the odds of C. virginica oysters harbouring the same viruses (MNV‐1 OR = 5·05, P = 0·03; NoV OR = 6·97, P = 0·01; HAV OR = 7·40, P < 0·001). Additionally, compared to C. virginica, C. ariakensis retained significantly higher numbers of transmissive stages of all protozoan and microsporidian species (P < 0·01). Crassostrea ariakensis oysters are also capable of retaining multiple human pathogens for at least 1 month. Conclusions: Crassostrea ariakensis oysters were statistically more likely to harbour enteropathogens and microbial indicators, compared to C. virginica. Individual C. ariakensis were also statistically more likely to retain multiple viruses, protozoa and microsporidia than C. virginica, highlighting the role the species may play in the transmission of multiple diseases. Significance and Impact of the Study: Nonnative Crassostrea ariakensis oysters are under review for their introduction into the Chesapeake Bay. The results of this study suggest that nonnative C. ariakensis oysters may present a serious public health threat to people consuming the oysters raw from contaminated sites.     

Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine.

Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants. Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water. In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants. Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h. The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms. A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h. Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h. The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts.     

Concentration of Cryptosporidium, microsporidia and other water-borne pathogens by continuous separation channel centrifugation

AIMS: The aim of this study was to determine the effectiveness of continuous separation channel centrifugation for concentrating water-borne pathogens of various taxa and sizes. METHODS AND RESULTS: Cryptosporidium parvum oocysts, Giardia lamblia cysts, Encephalitozoon intestinalis spores and Escherichia coli were seeded into different water matrices at densities ranging from 5 to 10 000 organisms l(-1) and recovered using continuous separation channel centrifugation. All pathogens were enumerated on membrane filters using microscopy. Recovery efficiencies were usually > 90%. Oocyst recovery did not vary with source water turbidity or with centrifuge flow rate up to 250 ml min(-1). Based on excystation, this concentration method did not alter oocyst viability. CONCLUSIONS: Continuous separation channel centrifugation is an effective means of concentrating water-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods are needed for detecting pathogens in drinking water to ensure public health. The first step for any pathogen detection procedure is concentration. However, this step has been problematic because recovery efficiencies of conventional methods, like filtration, are often low and variable, which may lead to false negatives. Continuous separation channel centrifugation can simultaneously concentrate multiple pathogens as small as 1 microm with high and reproducible efficiency in a variety of water matrices.     

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water

AIM: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. METHODS AND RESULTS: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. CONCLUSIONS: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring of waterborne pathogens in surface waters in amsterdam, the Netherlands, and the potential health risk associated with exposure to cryptosporidium and giardia in these waters

The water in the canals and some recreational lakes in Amsterdam is microbiologically contaminated through the discharge of raw sewage from houseboats, sewage effluent, and dog and bird feces. Exposure to these waters may have negative health effects. During two successive 1-year study periods, the water quality in two canals (2003 to 2004) and five recreational lakes (2004 to 2005) in Amsterdam was tested with regard to the presence of fecal indicators and waterborne pathogens. According to Bathing Water Directive 2006/7/EC, based on Escherichia coli and intestinal enterococcus counts, water quality in the canals was poor but was classified as excellent in the recreational lakes. Campylobacter, Salmonella, Cryptosporidium, and Giardia were detected in the canals, as was rotavirus, norovirus, and enterovirus RNA. Low numbers of Cryptosporidium oocysts and Giardia cysts were detected in the recreational lakes, despite compliance with European bathing water legislation. The estimated risk of infection with Cryptosporidium and Giardia per exposure event ranged from 0.0002 to 0.007% and 0.04 to 0.2%, respectively, for occupational divers professionally exposed to canal water. The estimated risk of infection at exposure to incidental peak concentrations of Cryptosporidium and Giardia may be up to 0.01% and 1%, respectively, for people who accidentally swallow larger volumes of the canal water than the divers. Low levels of viable waterborne pathogens, such as Cryptosporidium and Giardia, pose a possible health risk from occupational, accidental, and recreational exposure to surface waters in Amsterdam.     

Temporal and Spatial Variability in the Distribution of Vibrio vulnificus in the Chesapeake Bay: A Hindcast Study

Vibrio vulnificus, an estuarine bacterium, is the causative agent of seafood-related gastroenteritis, primary septicemia, and wound infections worldwide. It occurs as part of the normal microflora of coastal marine environments and can be isolated from water, sediment, and oysters. Hindcast prediction was undertaken to determine spatial and temporal variability in the likelihood of occurrence of V. vulnificus in surface waters of the Chesapeake Bay. Hindcast predictions were achieved by forcing a multivariate habitat suitability model with simulated sea surface temperature and salinity in the Bay for the period between 1991 and 2005 and the potential hotspots of occurrence of V. vulnificus in the Chesapeake Bay were identified. The likelihood of occurrence of V. vulnificus during high and low rainfall years was analyzed. From results of the study, it is concluded that hindcast prediction yields an improved understanding of environmental conditions associated with occurrence of V. vulnificus in the Chesapeake Bay.     

Molecular markers and sentinel organisms for environmental monitoring

Molecular methods are useful for both to monitor anthropogenic viral, bacterial, and protozoan enteropathogens, and to track pathogen specific markers in a complex environment in order to reveal sources of these pathogens. Molecular genetic markers for fecal viruses, bacteria, and protozoans hold promise for monitoring environmental pollution and water quality. The demand for microbiologically safe waters grows exponentially due to the global demographic rise of the human population. Economically important shellfish, such as oysters, which are harvested commercially and preferentially consumed raw can be of public health importance if contaminated with human waterborne pathogens. However, feral molluscan shellfish which do not have an apparent economic value serve as indicators in monitoring aquatic environments for pollution with human waterborne pathogens and for sanitary assessment of water quality. Current technology allows for multiplexed species-specific identification, genotyping, enumeration, viability assessment, and source-tracking of human enteropathogens which considerably enhances the pathogen source-tracking efforts.     

Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number-cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21 degrees C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg. min/liter were needed to inactivate approximately 0.5 log(10) and 2.0 log(10) units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg. min/liter were required to achieve approximately 2.0 log(10) units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)