Pathogens and Indicators

1. Pathogens and Indicators

2. Why are we interested in pathogens associated with AFOs? AFOs house a large number of animals in a small area => large amount of fecal waste • Swine may produce 5 to 10 times the amount of fecal waste as an equal number of humans Fecal waste from AFOs can contain high numbers of microbial pathogens • These can be zoonotic (capable of infecting both humans and animals) • Ex. E. coli 0157, Salmonella, Campylobacter, Hepatitis E (?), Cryptosporidium AFOs are largely unregulated for pathogens • New CAFO regulations > no provisions for regulation of pathogens

3. Other Considerations for Environmental Monitoring Programs for AFO Pathogens: Type and efficiency of AFO waste treatment system • Conventional system • anaerobic lagoon systems followed by land application (swine – cattle) • land application of solid waste/litter (turkey – poultry) • Alternative systems (other treatment technologies) • “Smithfield Foods/PSF/NC Attorney General” Agreement Microbial Methods: • Initial sampling, concentration, or recovery methods • Pathogen detection and isolation methods • Pathogen confirmation and further characterization • Where did the fecal waste come from?? (source tracking)

4. Considerations for Pathogen Detection Associated with AFOs: Salmonella Hepatitis E Virus Cryptosporidium • Use of indicator organisms? • Pathogens • Class of pathogen • Environmental Media • Water and wastewater • Biosolids and litter • Soils and vegetation • Air • Vectors

5. Pathogen Analysis, Monitoring, and Surveillance • Pathogen detection from environmental samples is technically demanding, often tedious, slow to produce results, sometimes unreliable, and expensive • Done routinely in the health care field (clinical diagnostic microbiology): • often essential to patient treatment and care • provides national surveillance of infectious disease epidemiology • Regularly for some pathogens in some foods (meat & poultry) • Sometimes for human (municipal) biosolids • Rarely for monitoring or managing environmental waters • pathogen occurrence surveys: • ICR (Information Collection Rule): survey (18 months) for Giardia, Cryptosporidium and enteric viruses in larger drinking water supplies using surface water sources • GWDR (Ground Water Disinfection Rule): enteric virus survey in ground water sources of drinking water

6. Microbial Indicators of Fecal Contamination • Traditional approach to assess the "sanitary" quality of water with respect to fecal contamination. • Quantify bacteria commonly present in intestines of warm blooded animals: • high numbers • easy to measure • surrogates for pathogens, especially bacterial pathogens. • May not be reliable indicators of • viruses and parasites

7. Bacterial Indicators of Fecal Contamination in Water Total coliforms: standards for drinking waters; not feces‑specific (environmental sources). Fecal ("thermotolerant") coliforms: standards for wastewater effluents and biosolids, ambient surface waters and shellfish harvest waters; not feces-specific. E. coli: the "fecal" coliform; may occur naturally in tropics. Enterococci:Streptococcus faecalis and S. faecium; a sub‑set of the fecal streptococci considered more feces‑specific; EPA standards for bathing water quality. Clostridium perfringens: anaerobe; feces‑specific?; very (too?) resistant spores; candidate indicator for protozoan cysts.

8. E. coli Famp F+ F+ Coliphage Viral Indicators of Fecal Contamination in Water • Coliphages: viruses (bacteriophages) infecting E. coli or other coliforms; attach directly to cell wall (somatic) • heterogeneous group • may not be feces-specific • host-dependent detection • Male-specific (F+) coliphages: coliphages infecting "male" strains of E. coli (posses pili) • may be feces-specific • may distinguish human from animal fecal contamination by group classification • II & III human; I & IV animal • PROBLEM: swine may harbor groups II & III also E. coli C Somatic Coliphage F+ Coliphage Somatic Coliphage

9. Concentration of EnvironmentalPathogens from Water Few initial concentration methods can efficiently concentrate all classes of pathogens Most methods directed at one class of pathogen • USEPA 1MDS Filter method for viruses • USEPA Method 1623 for Cryptosporidium and Giardia

10. USEPA 503 Regulations/Methods for Biosolids • Fecal Coliform (FC) bacteria (< 1,000 / g) and Salmonella (< 3 / 4g) • FC: Multiple fermentation tube method (MPN) • Salmonella: broth enrich, isolate colony, biochemical tests • Total Culturable Viruses (< 1 / 4g) • Elute biosolids with beef extract • Acid precipitation > raise pH to neutral • Cell culture plaque assay method on “Buffalo Green monkey Kidney cells” (BGMK) • Helminthova (< 1 / 4g) • elute with buffered water (+ surfactant) • zinc sulfate flotation method • acid-alcohol/solvent extraction • embryonate with (0.1 N) sulfuric acid or formaldehyde water at RT (26oC) • Microscopic evaluation for viability

11. Air Samplers: Membrane Filters and Slit Impaction Samplers Membrane filter: Air sampled through a porous filter that retains microbes • Organisms may lose viability due to desiccation • Collection efficiency varies with particle size, shape, density, pore size, and flow rate. Slit samplers: Sample of air is directed through a slit against a rotating collection surface. • Rotation is intermittent so that each impaction area represents a specific volume of sampled air and a time series of samples can be collected.

12. Air Samplers - Andersen samplerand Liquid Impinger Andersen Sampler: Microbes are separated by size and impact onto a a series of solid or semi-solid collection surfaces (agar medium plates) Liquid Impinger: Air sampled through a limiting orifice into an impinger filled with a liquid, typically a dilute buffer solution

13. High Volume Air Samplers – Liquid Cyclone Scrubbers and Electrostatic Precipitators Liquid Cyclone Scrubbers: Particles in air travel at high speeds through a progressively smaller, helical passageway impinge against the container walls and are collected into a re-circulating collection fluid Electrostatic Precipitation: Air is drawn over electrically charged collection plates so that charged particles are attracted to and collected on either a positively or negatively charged, wetted surface. • Collected particles are washed off into the circulating collection fluid on the charged plate surface.

14. Vector (Houseflies) Collection and Sample Processing Methods Sampling methods: both methods use a pheromone based attractant • Alive (Fly Terminator) • Dead (QuikStrike) • Houseflies are separated, counted, and weighed • Appropriate numbers of flies are blended with a beef extract eluting solution • Liquid eluting solution is used for subsequent assay methods

15. Detection of Total and Fecal Coliform Bacteria and E. coli MI Agar under ambient and longwave UV light • Membrane Filter • Nutrient agar + MUG • EC medium + MUG • Multiple Fermentation Tube • EC broth + MUG • Biochemical Assay • defined substrate • Detect product (colored) • Colilert™ or Colisure™

16. Coliphage Detection Method 1601: Two-step Enrichment Method 37ºC overnight 37ºC 6 to 12 hours 100-1000 mL of water 1. Inoculate with E. coli and nutrients 2. Spot 10 µl to E. coli lawn in agar medium 3. Look for lysis zones

17. Coliphage Detection Method 1602: SingleAgar Layer (SAL) 37ºC overnight 100 mL of water + 100 ml of 2X trypticase soy agar +E. coli + MgCl2 (optional) Pour the mixture on multiple plates Look for plaques

18. Pathogen Detection - Microscopy Light Microscopy Fluorescent Microscopy Electron Microscopy

19. Pathogen Detection - Hemagglutination Assay for Viruses Dilution No virus 2 4 8 16 32 64 128 512 1024 256

20. Pathogen Detection - Enzyme linked Immunobsorbant Assay (ELISA) Enzyme -> color Sample to be tested virus Detecting antibody Capturing antibody

21. Pathogen Detection - Cytopathic Effects of Viruses in Infected Cell Cultures uninfected Late cytophathic effects: Enlarged cells Nuclear inclusions Cell degeneration Plaque formation

22. Pathogen Detection - Polymerase Chain Reaction (PCR) primers Viral DNA First cycle Second cycle 20-40 cycles many copies of amplicon (DNA) gel

23. Further Characterization Methods Bacterial confirmation: • Biochemical test kits Antimicrobial resistance patterning: • Micro-dilution method • Disk diffusion method Microbial Source Tracking: • Male-specific (F+) coliphage grouping • Molecular methods for bacteria • Ribotyping; PFGE • Rep-PCR or Multi-locus PCR • Bacterial toxin genes (Betty Olson – UC Irvine)