Monitoring of animal species and source tracking of fecal bacteria in surface water by detection of e(nvironmental)dna

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Gervais King
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Monitoring of animal species and source tracking of fecal bacteria in surface water by detection of e(nvironmental)dna Bart Wullings, Leo Heijnen

water Pathogens of fecal origin Opportunistic pathogens which can grow in drinking water Classic methods Molecular methods Analysis of unique

1 Monitoring of animal species and source tracking of fecal bacteria in surface water by detection of e(nvironmental)dna Bart Wullings, Leo Heijnen en Edwin Kardinaal Kennisnetwerk Milieu, 27 September 2013, Wageningen Microbiological methods to analyse the microbiological quality of drinking water Pathogens of fecal origin Opportunistic pathogens which can grow in drinking water Classic methods Molecular methods Analysis of unique DNA/RNA Selective en specific Time-consuming: days! Results within a few hours Not all organisms (culturable) 2 Applicable for all organisms kwr

Available methods for water research after 10 years of development and implementation: Routine quality control Research: risk analysis Also for organisms

2 Available methods for water research after 10 years of development and implementation: Routine quality control Research: risk analysis Also for organisms which are not detectable with classic methods Optimisation of water treatment processes 3 European guidelines for microbial quality of bathing water 4 kwr

organisms which are not detectable with classic methods Optimisation of water

3 Monitoring microbial quality of bathing water Regular monitoring of fecal contamination Standardised ISO culture methods Sampling, anaysis and report takes 2/3 days In case of positive detection, resampling, again 2/3 days Quicker results are required! Monitoring Cyanobacteria Fluoroproob and microscopic counting Analysis are less relaible, sensitive for temperature change (fluoroproob), subjective and expensive (microscopic counts) Foto Waterproef qpcr Quick(<24 hours), reliable, objective, reproducible and cheap(er) 5 qpcr methods developed for most common potential toxic cyanobacteria Microcystis Aphanizomenon Planktothrix Anabaena 6 kwr

Monitoring Cyanobacteria Fluoroproob and microscopic counting Analysis are less relaible, sensitive for temperature change (fluoroproob), subjective and expensive

4 Development of specific primers and probes Research started in 2009 Target gen fycocyanine operon (cpcba) Unique for cyanobacteria and large collection of reference sequences available (total >1000 cpcba sequences) Microcystis and Planktothrix genus specific primers and probes developed Genus of Anabaena and Aphanizomenon are not mono phylogenetic but intermixed Discrepancies between morphologic and phylogenetic classification Correction factor to convert qpcr results to celequivilants 7 Field study locations Each location 7 samplings Total 232 samples 8 kwr

developed Genus of Anabaena and Aphanizomenon are not mono phylogenetic but intermixed Discrepancies between morphologic and phylogenetic

5 cellen / DNA kopie aantallen per ml cellen / DNA kopie aantallen per ml 9/30/2013 Results field study qpcr (corrected) en cell count Camping De Watermolen Anabaena C Aphanizomenon D Microcystis Planktotrix Anabaena sp. Aphanizomenon Microcystis sp. Planktothrix sp kwr 2013 Lecture DAN in the Dutch Delta Weeknummers - edan 9 Results field study qpcr (corrected) en cell count Bovenwater strand zeilplas Anabaena C Aphanizomenon D Microcystis Planktotrix Anabaena sp. Aphanizomenon Microcystis sp. Planktothrix sp Weeknummer 10 kwr

1000000 100000 10000 1000 100 10 1 25 27 29 31 33 35 37 39 kwr 2013 Lecture DAN in the Dutch Delta Weeknummers - edan 9 Results field study qpcr (corrected) en cell

6 Detection of Cyanobacteria using qpcr qpcr methods available for quick screening of potential toxic Cyanobacteria; More reliable and quicker evaluation of bathing water for the possible risk of Cyanobacteria with qpcr; Detection of 4 genera separately; Higher sensitivity and therefore possible early detection of a proliferation; Prize per sample can be lower in comparison to microscopic counting Methods ready for routine analysis Implementation of methods in routine laboratoria (this year); Approach extended to other genera: Woronichinia / Phormidium/ Trichobilharzia 11 Environmental DNA Monitoring of animal species in water by detection traces of DNA Convential monitoring methods: Labor intensive and time consuming Can disturb animal habitats Not effective for animals with a hidden lifestyle Traces of DNA from skin cells, mucous and feces Indirect detection with qpcr Density is indicative and requires additional research 12 kwr

counting Methods ready for routine analysis Implementation of methods in routine laboratoria (this year); Approach extended to other genera: Woronichinia / Phormidium/ Trichobilharzia 11

7 Target species 2013 Together with Bureau Waardenburg Weatherfish (Misgurnus fossilis) Great crested newt (Triturus cristatus) 7 freshwater crayfish (Astacidae and Cambaridae) 13 Primer and probe design 14 kwr

(Triturus cristatus) 7 freshwater crayfish (Astacidae

8 Collection of target DNA species 15 Sensitivity analysis 16 kwr

9 Conclusions and futher research edna detection methods are successfully developed for M. fossilis and T. cristatus species: In silico specific In vitro specific In general, field study showed amplification of target species DNA for locations where species were observed Sensitivity analysis Early spring and late spring sample collections Research will be continued in 2014 together with Naturalis 17 Search for the source of the fecal contamination in bathing water location Fecal contamination: Detection of E. coli and enterococces Question: What are the sources of the fecal contamination? Method: Detection of animal specific gut bacteria (like Bacteroides) or mitochondrial DNA 18 kwr

Sensitivity analysis Early spring and late spring sample collections Research will be continued in 2014 together with Naturalis 17 Search for the source of the fecal

10 Selectivity of the methods qpcr s availible for detection of DNA from feces of different animal species: General fecal contamination/positive control Human Ruminants Cows Pigs Birds Dogs Methods showed to be selective Sporadic cross contaminations, concentrations are very low kwr Lecture DAN in the Dutch Delta - edan 19 Location: Het Zeetje (Zwolle) E. coli Humaan Vogels Enterococcen All Bact 10 1 kwr Lecture DAN in the Dutch Delta - edan 20 kwr

contaminations, concentrations are very low kwr 2013 2012 Lecture DAN in the Dutch Delta - edan 19 Location: Het Zeetje

11 Conclusions Application of detection of (e)dna has many advantages: Cyanobacteria: quantitative, quick and cheap Trichobilharzia: sensitive monitoring in bathing water in stead of snail analysis edna of animal species: larger detection change, no habitat disturb ion and Source tracking: only possible with molecular method Future: Cyanobacteria: routine analysis 2013 Trichobilharzia: research needed (partners) edna of animal species: research continued Source tracking: research continued and applied 21 kwr

disturb ion and Source tracking: only possible with molecular method Future: Cyanobacteria: routine analysis 2013

How To Monitor Cyanohab

How To Monitor Cyanohab Application of molecular tools for routine water quality monitoring MWQI Technical Meeting May 27, 2015 Tim Otten, PhD, MPH Bend Genetics, LLC T: 541 600 4363 ottentim@bendgenetics.com www.bendgenetics.com