Detection of Enteric Viruses in Groundwater in Alberta

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1 Detection of Enteric Viruses in Groundwater in Alberta Xiaoli Pang 1,2, Yuanyuan Qiu 2, Niamh Caffrey 3, Tiejun Gao 2, Bonita Lee 4, Norman Neumann 5, Jessica Popadynetz 6, Sylvia Checkley 1, 3 1 Provincial laboratory for Public Health, Edmonton, AB, Canada 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB Canada, 3 Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary 4 Department of Pediatrics, University of Alberta, Edmonton, AB, Canada 5 School of Public Health, University of Alberta, Edmonton, AB, Canada 6 Healthy Physical Environments, Alberta Health Services, Edmonton, AB, Canada Environ Tech, Calgary, April 3-5, 2018

2 Background Enteric viruses have increasing public health impact. The US Environmental Protection Agency s (EPA) recently updated the list of enteric viruses that are candidates for regulatory consideration in drinking water safety. Are groundwater pristine free of viruses? There was few studies on the occurrence of enteric viruses in groundwater sources Conflicting findings on enteric viruses in groundwater were reported (Bradbury et al., 2013)

3 Background There is no water treatment system in rural area. Groundwater contamination with enteric viruses may expose a high risk to population relying on well water for drinking (Borchardt et al. reported the presence of enteric viruses in almost 25% of the over 1,200 tap water samples analyzed from 14 communities, relying on untreated groundwater) Septic system on-site of premise with well and condensed livestock operations might increase a chance for viruses contamination of groundwater Weather conditions (high precipitation or snowmelt) may increase virus migration via groundwater recharge event (Corsi et al., 2014)

4 Purposes To assess if extent well water is contaminated by enteric viruses To study whether the presence of viruses correlates well with routine bacteriological indicators of water quality (i.e., Escherichia coli) To provide the first evidence of viruses contaminated in Alberta ground water

5 Project Overview Whole project includes two major components: A. Questionnaire survey on demographics, water well characteristics, the septic system and other possible contaminants, livestock production, risk management, etc. B. Laboratory testing for enteric viruses and bacteriological indicators in well water Focused area: FoodNet Sentinel Site of Albert Duration: 3 years (2015 to 2018) Study recruitment: Part A. 104 premises (wells) and Part B. 51 of 104 premises Team: University of Calgary, University of Alberta and Alberta Provincial Laboratory for Public Health

Sentinel Site of Alberta * (Calgary and central Zones of AHS) The site comprises part of the City of Calgary and an area predominantly north and east of the city.

6 Sentinel Site of Alberta * (Calgary and central Zones of AHS) The site comprises part of the City of Calgary and an area predominantly north and east of the city. FoodNet Canada s criteria for sentinel sites: a population of 500,000 to 1,000,000; an urban/rural mix representative of major geographic areas; private and public health laboratory capacity; innovation in local public health and water services; and willingness to participate. *

7 Site map of well water samples

Testing Approach Groundwater samples from the premise The filter

Presence and levels of human enteric viruses (rotavirus, reovirus,

8 Testing Approach Groundwater samples from the premise The filter samples (>500 liters) from well discharge Duplicated samples (250ml each) from kitchen tap Dr. Pang s Res Lab in the ProvLab, Edmonton Provlab Water-lab in Calgary Presence and levels of human enteric viruses (rotavirus, reovirus, adenovirus, norovirus, enterovirus, sapovirus, astrovirus and JC virus) Presence and levels of Escherichia coli and faecal Coliforms

9 Methods On-site training and well flow calibration Sample collection (Monthly x 12 per site): 500 liters of groundwater for enteric viruses 250 ml x 2 for bacterial indicators The filter sample (500 L) was used for elution of viruses, concentration, and analysis using established method (Pang et al, 2012) 250 ml samples were tested for coliform (TC) and E. coli (EC) using Colilert (IDEXX Laboratories, Inc.) Data acquisition and analysis

10 Enteric viruses Human Viruses Targets Human caliciviruses (Norovirus & Sapovirus): +ssrna viruses, non-cultivable Human rotavirus: dsrna virus, cultivable but grows extremely slowly Reovirus: dsrna virus, grown in BGM and MA104 cell lines Astrovirus: +ssrna virus, need special cell lines Human adenoviruses: dsdna virus, grown well in BGM and MA104 cell lines Enteroviruses Human coxsackievirus: +ssrna viruses, grown in the BGM cell line Human echoviruses: +ssrna viruses, grown in the BGM cell line Polyomavirus JC virus and BK virus: double-stranded circular DNA viruses. JC virus is a human virus causing latent and chronic infections. Recently JC virus was found in 98% to 100% of sewage samples and was proposed to be an indicator of human fecal contamination (Bofill-Mas et all 2006)

11 Water Sampling Device (WSD) WSD was designed and assembled for collecting a large quantity of water sample for viruses concentration and testing. WSD can be used to collect water sample from river, lake and well

Personal Collecting Tool (PCT) for well water

Water is pre-running through for 2 minutes.

12 Personal Collecting Tool (PCT) for well water sampling on the sites PCT (one for each site of participants) Cartridge and filter insert WSD is connected with outlet of well on-site. Water is pre-running through for 2 minutes. The filter is inserted and timer started for sampling process.

13 Lab processing: concentration of viruses Virus Concentration hr Elution 1.0 L of 1.5% BE, ph hrs Flocculation 15 ml concentrated viral specimen

CT value Detection and quantification ABI 7500 Concentrated Specimen 45.00 40.00 35.00 30.00 25.00 Extraction of viral nucleic acid 20.

14 CT value Detection and quantification ABI 7500 Concentrated Specimen Extraction of viral nucleic acid Hours Standard Curve for coxsackievirus Amplification and detection Cycle Number

15 Integrated viral culture with PCR (ICC-PCR) To enhance the sensitivity for infectious virus To confirm the presence of specific viruses

16 Viral cell culture Concentrated Specimen Identifying of infectious viruses CPE positive - detection of infectious viruses CPE negative - no infectious viruses

17 Results - Viruses 51 wells have been sampled from May 2015 to March 2018 A total of the 535 samples were tested with reported results up to Nov samples (4%) from 17 wells were positive for enteric viruses 15 wells tested positive only once, one well tested positive twice and one well tested positive three times during the 12-month period The most commonly detected virus was reovirus (n=8) with 2 confirmed to be animal reoviruses, followed by adenovirus (n=6), rotavirus (n=5) and JC virus (n=1)

18 Results Bacterial indicators 76 samples were positive for TC (14%) from 31 wells and 16 wells showed more than once positive/well 11 samples were positive for both TC and EC (2%) from 10 wells with twice positive in 1 well only. 2 wells each had 8 and 5 samples tested positive for TC, respectively, and one well had 7 samples test positive for TC.

19 Results No seasonal distribution of detected viruses in well water, indicating viral contamination of well water might be an occasional event TC and EC positive samples were mostly seen in the months of June to Oct. None of wells showed both detected viruses and TC/EC in the samples taken at the same time, indicating that contaminations may derive from different sources or routes

20 Viral nucleic acid level /liter Results ( Stimson creek site) 12 The period switched groundwater to surface water reservoir 10 8 Virus positive/w copy numbers/l TC positve

25 20 15 10 5 Entero JCV Sapo Astro Noro AdV Rota 0 Rota AdV Noro Astro Sapo JCV

21 Entero JCV Sapo Astro Noro AdV Rota 0 Rota AdV Noro Astro Sapo JCV Entero Occurrence of enteric viruses in major rivers of Alberta (unpublished data)

22 Conclusions The study demonstrates that the groundwater in Alberta s sentinel site has low-level contamination with enteric viruses There was no association of the presence of enteric viruses in the well water samples and TC or EC positivity in tap water samples at the same premise, which was supported by previous evidencing the lack of correlation between presence of enteric viruses and bacteriological indicators (Wu et al, 2011)

23 Conclusions There was no trend of correlation between contamination of enteric viruses in groundwater and livestock operations Correlation between well characteristics, on-site septic system and viral occurrence will be analyzed when all data are available

24 Acknowledgment This study was supported by Alberta Innovates and Alberta Agriculture and Forestry Staffs in Water Lab of Provlab Calgary location Min Cao and Charlet Liu for their technical supports all the way for this project FoodNet Canada for assistance in study recruitment All individuals, businesses and institutes for participating and supporting the study

25 Thanks

Detection of Enteric Viruses in Surface (Source) Water and Groundwater in Alberta, Canada

Detection of Enteric Viruses in Surface (Source) Water and Groundwater in Alberta, Canada XL Pang 1,2, BE Lee 2, D Mooney 3, S Gabos 2, S Craik 4, R Zurawell 5, and N Neumann 1,2 1 Provincial Public Health