New approaches to tracing the sources of faecal contamination in the environment

1. New approaches to tracing the sources of faecal contamination in the environment Craig Baker-Austin CEFAS Weymouth, Dorset, UK

2. Shellfisheries in the UK Shellfish make up almost 1/3rd of the total commercial fisheries catch in England and Wales. The total Shellfish industry in the UK is worth approximately £200M/year. There are legislative constraints on shellfisheries in the UK (i.e. the Shellfish Waters Directive (79/923/EEC)/ Water Framework Directive), stipulating strict microbiological controls on faecal bacteria inputs in shellfish harvesting areas. The periodic occurrence of shellfish-transmitted illness outbreaks associated with faecal contamination have contributed to a public confidence problem over shellfish safety and resulted in important economic losses by the seafood industry (Morse et al. 1986)

3. Shellfish pathogens associated with faecal contamination Filter feeding bivalves can concentrate a range of viral and bacterial pathogens associated him faecal material. Most of the more harmful bacterial and viral pathogens tend to be found in human faecal material, highlighting the need to identify sources accurately in shellfish harvesting areas.

4. Class Criteria Requirements A <230 E. coli per 100g Can be collected for direct human consumption B 90% compliance with <4600 E. coli per 100g Must be treated in a purification centre or relayed to meet class A; may also be heat-treated by an approved method. C <46000 E.coli per 100g Must be relayed for at least 2 months (whether or not combined with purification) to meet class A or B; may also be heat treated by an approved method P Greater than the above or public health risk Prohibited. Not suitable for human consumption E.coli – Classification • To ascertain the sanitary conditions of shellfish harvesting areas, classifications are based on the levels of E. coli/100g flesh, as shown in the table below.

5. Microbial source tracking (MST) Refers to techniques used to apportion faecal contamination among sources Library-dependent methods: Multiple Antibiotic Resistance testing (MAR) Carbon utilization profiles Immunological methods PFGE Ribotyping RAPD Library independent: Faecal bacteria ratios F+ coliphage serotyping Molecular indicators (i.e. 16S) Bacteriophage indicators Human Viruses Mitochondrial DNA It is generally established that no individual MST tool is accurate, sensitive and temporally and spatially reliable enough to be used in all ‘real-world’ situations. There are also concerns regarding the clinical/public health relevance of most MST approaches.

6. Why are many MST approaches problematic? • No single target has been identified that possesses similar survival characteristics to faecal pathogens, is present in greater numbers than faecal pathogens, is specific to a particular faecal source, correlates with health risk and is readily detectable. • Most MST studies tend to concentrate on a single target, limiting the usefulness of the approach. • Few studies undertake rigorous validation work prior to field applications. • Applying MST approach to shellfish matrices poses technical issues.

7. Storm sewage scheme completed (March 2003). Screening after 10 March 2003 demonstrates effectiveness of remedial works 1/9/02 6/12/99 19/4/01 14/1/04 28/5/05 22/2/08 10/10/96 Defining faecal sources is critical in shellfish water Data courtesy of Environment Agency

8. Defining faecal sources is critical in shellfish water Data courtesy of Environment Agency

9. Current source tracking capabilities at Cefas • Cefas has recently developed a range of source tracking approaches to detect, monitor and quantify a range of faecal sources into surface waters and shellfish harvesting areas. • The initial development of these molecular tools has focused on the use of mitochondrial DNA (mtDNA). We have developed pig, sheep, cow and human probes. • We have also assessed several bacterial targets to identify and confirm faecal contamination. • Several key questions need to be resolved prior to using these approaches routinely – temporal and spatial applicability of targets, robustness, sensitivity, linearity, matrices (water & shellfish) etc.

10. Some characteristics of eukaryote mtDNA • Mitochondria are organelles found in nearly all eukaryotes – essential for energy production and regulation of cellular respiration. • mtDNA is highly conserved (is species-specific). Can identify faecal source directly. • Found in high concentrations in faecal material. • Many copies per cell, and amenable using appropriate molecular methods. • We get carryover of food items identified from our molecular analysis – for example, eating a hamburger will provide a human and cow signal!

11. Quantitative Real-Time PCR (TaqMan) • In real-time PCR, primers and probes are specifically designed to hybridise with specific genetic targets. • PCR exponentially replicates the sequence of interest and uses a fluorescent probe to measure the increase in copies of DNA in real time. • This gives a cycle threshold value (Ct) and is displayed • graphically. • By running a dilution series of a known amount of DNA a standard curve can be produced, allowing quantification of target DNA in a given sample.

12. Fluorescence Fluorescence R Cycle number Taqman probe Forward Primer Taq Q How does TaqMan work? Target DNA sequence A T G C

13. True positive False positive CT Negative/undetected Threshold Typical TaqMan results plot

14. aProbable human contamination. Accurate source differentiation (85%)

15. Degradation plots Pig, Sheep, Cow and Human mtDNA signal lasts up to 14 days in aquatic microcosms.The mtDNA T90 values (time taken to lose 90% of mtDNA) based on these and other experiments were: Cow (53 h), Sheep (86 h), Pig (122 h) and Human (~90 h). Corresponds well with T90 values for other faecal pathogens, such as E. coli (30 h), Norovirus (50.4 h) and poliovirus (23.3 h). Important performance characteristic – animal and human mtDNA survives for long enough to detect faecal contamination, but not too long to limit biological relevancy.But can it be used on shellfish marices?

16. Degradation plots Degradation (PCR units) Degradation (PCR units) Degradation (PCR units) Degradation (PCR units)

17. Sensitivity and limits of detection • Serial dilutions of faecal material in both seawater and freshwater suggest that with appropriate sample preparation, we can detect as little as 2 mg/l faecal material. • Pig >1 mg/l faecal material • Sheep >2 mg/l faecal material • Human >2 mg/l faecal material • Pig >6 mg/l faecal material • Have found significant inter and intra-species variation • in detection limits of mtDNA, hwoever these are among • the most sensitive MST methods available.

18. Faecal samples: Human faecal Agricultural faecal Controls Fluorescence Cycle number Field study sites Faecal amendment Faecal samples Human Pig Sheep Cow Shucking and glanding Source identification DNA extraction & RT-PCR Using mtDNA analysis on shellfish matrices

19. Cow probe Cow Cow Sheep Sheep Sheep probe Pig Pig Human Human Pig probe Cow Sheep Pig Human PCR units PCR units PCR units Sample Sample Sample Specific detection of faecal source in shellfish water and shellfish flesh

20. Cow probe Cow Sheep Pig Human PCR units Sample Specific detection of faecal source in shellfish water and shellfish flesh

21. Cow Sheep Sheep probe Pig Human PCR units Sample Specific detection of faecal source in shellfish water and shellfish flesh

22. Pig probe Cow Sheep Pig Human PCR units Sample Specific detection of faecal source in shellfish water and shellfish flesh

23. Summary of TaqMan analysis for E. coli and mtDNA targets • We have been able to successfully detect CytB from both water and shellfish matrices in faecally amended water and shellfish samples. • Using this outlined approach we were able to unambiguously detect each source (i.e. pig, sheep, cow and human) in around 85% of water samples, and 100% of shellfish samples. • Very sensitive - we can accurately detect as little as 1 mg/100ml of faecal material from spiked water samples. • Early indications are that mtDNA is degraded relatively quickly in shellfish matrices (< 2 days), but may remain longer in water samples (up to 14 days).

24. Future work • We are currently expanding the range of RT-PCR probes to cover other potential sources of faecal contamination in shellfish harvesting areas (dog, cat, wild birds). • Future work will involve using complex mixtures of faecal material to establish whether the outlined approach can accurately determine relative proportions akin to ‘real world’ scenarios. • Some technical limitations of this approach are currently being tackled, such as meal carry-over and non-specific contamination (i.e. human skin cells leading to false-positive results). • Current work involves absolute quantification of mtDNA and assessing it’s relative quantities with other markers, such as E. coli.

25. Acknowledgements • Dr Rachel Rangdale, Dr David Lees & Dr James Lowther (Cefas) • Dr Jonathan Porter (Environment Agency Starcross Laboratory) • Joseph Morris (MSc Student, Kings College, London) • Razvan Doleaand Joanna Sier (University of Surrey)

26. Questions?