1. Environmental Transmission of Pathogenic Microbes – why they are where they are when they are Dr. Julie Ann Kase Public Health Scientist – Bioterrorism and Emerging Pathogens Unit NC State Laboratory of Public Health Julie.Kase@ncmail.net

2. Why There is Job Security in Infectious Disease Work

3. 1976 Legionnaires' disease – Outbreak of pneumonia occurred among persons attending a convention of the American Legion in Philadelphia –~ 8,000 to 18,000 cases in the US each year; 5% to 30% die 1981 AIDS –report in MMWR of 5 CA men with severe immunodeficency (1983 HIV recovered from lymph node) 1993 Cryptosporidiosis –400,000 people sick and killed more than 100 – worst waterborne disease outbreak in the United States 1995 Ebola Hemorrhagic Fever –Ebola Virus initial recognition in 1976 –Occurred in Kikwit and surrounding area –315 cases 81% death of cases 2002 (November) Severe acute respiratory syndrome (SARS) –Viral respiratory illness that was recognized as a global threat in March 2003 –first appearing in Southern China in November –November 2002 - July 2003, a total of 8,098 people worldwide became sick; 774 died –By late July 2003, no new cases, and WHO declared the global outbreak to be over Late 2003 and Early 2004 Avian influenza –Outbreaks of influenza H5N1 occurred among poultry in eight countries in Asia (Cambodia, China, Indonesia, Japan, Laos, South Korea, Thailand, and Vietnam) –By March 2004, the outbreak was reported to be under control until late June 2004 –Human infections (H5N1) have been reported in Thailand, Vietnam and Cambodia

4. Seek and Ye Shall Find Environmental Transmission –Presence Cover our planet –Several 100m deep in glacial ice sheets –Deep-ocean thermal vents –Survival and transport Media –Water –Soil –Air –Vectors/Animals Natural fate + influence of man’s actions

5. Microbial Survival in the Environment Pathogen survival Differs widely among microbes: –Bacteria: Spores survive better than vegetative cell –Viruses: non-enveloped viruses survive better than enveloped viruses under most environmental conditions –Envelopes are relatively fragile compared to outer capsids (protein coats) –Parasites: protozoan (oo)cysts and spores and helminth ova survive better than active life stages of these parasites or than those with no resting or special environmental forms –Fungi: spores survive better than other forms

6. Environmental Factors Influencing Survival or Proliferation of Infectious Agents Temperature –Greater inactivation/death rates at higher temps –Environmental temperatures influence pathogen spread by insect vectors pH –Extreme pH inactivates microbes –Important exceptions: enteric pathogens survive pH 3.0 Moisture content –Drying or low moisture inactivates /kills some microbes –Ex. Preserving food by desiccation Sunlight (UV) –Nucleic acids absorb the UV energy and is damaged

7. Environmental Factors Influencing Survival or Proliferation of Infectious Agents Pressure (atmospheric & hydrostatic) –Typical atmospheric pressure –Use of high hydrostatic pressure in shellfish Weather –Microbes levels may increase or decrease –Warmer weather increases some microbes (ex. Vibrio bacteria in NC coastal waters during warmer months) and vectors, like mosquitoes –Wet weather carries microbes also resuspension in water resources Chemicals & nutrients –Levels influence microbe survival –Ex. Lack of nutrients (e.g. carbon, nitrogen) will limit proliferation Biological factors –Predation, vectors, reservoirs (animals), microbe species/type

8. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Microbes can be found anywhere in which water is maintained in a liquid state Most human illness associated with water – infectious, usually acute in nature but some have chronic sequelae (e.g. stomach cancer (Helicobacter pylori), myocarditis & diabetes (coxsackieviruses )) Highly effective means of introducing an infectious microbe to a large population –Person-to-person vs. fecal-oral

9. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Reservoirs for pathogenic microbes found in water: humans, animals, environment –Source tracking –Human sources – important contributor of pathogens in water Recreational activities (e.g. water theme parks) Domestic wastewater –Discharge of wastewater into surface waters –Aquifer contamination

10. Infectious Disease Water Transmission Model

11. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Microbial agents associated with waterborne diseases –Enteric, non-enteric,and aquatic bacteria –Enteric viruses –Enteric protozoa

12. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Enteric bacteria –Shigellosis: Shigella spp. (classical bacillary dysentery) occur around the world, with most victims being children in developing countries 18,000 cases of shigellosis are reported in the United States Shigella dysenteriae type 1 – deadly epidemics in dev. world Spread can also be via vector, food and water, and fomites –Cholera: Vibrio cholerae serogroup O1 or O139 (1992) that produces toxin Endemic poverty, summer rains, poor health & living conditions 56 countries, 101,383 cases (2345 deaths) in 2004 Africa - 94% cases in 2004 Easily treatable –Infectious dose variable: usually high - 10 2 to 10 8 orgs

13. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Aeromonas hydrophila Commonly found in water but recently implicated as a cause of GI illness, also wound infection Potential to grow in water distribution systems, especially in biofilms, where it may be resistant to chlorination EPA list (candidate contaminant list) for drinking water Legionella pneumophila Ubiquitous in water, aerosol transmission Proliferate at high temperatures (hot water systems, cooling towers and evaporative condensers) Etiologic agent of Legionnaires’disease, Pontiac fever Leptospira spp. Species not enteric or aquatic - Urine from dom. & wild animals Outbreak during Eco-Challenge - grueling 2-week contest in late Aug and early Sept [2000]

14. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Enteric viruses –Generally more persistent than enteric bacteria but can not multiply in environment –Survival influenced by physical, chemical, and microbial factors as well as virus type Non-enveloped viruses most persistent – protein coat offers stability and resistant barrier to stressors Majority of enteric viruses are non-enveloped –Require more sophisticated detection methods Tissue cultivation, electron microscopy, molecular techniques (e.g. PCR, nucleic acid hybridization) Limit microbe ID from clinical & environmental samples Distinguish between infectious/non-infectious - MAYBE

15. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air –Implicated in majority of AGI outbreaks in US based upon clinical & epidemiological evidence Rotavirus - the virus is stable in the environment, transmission can occur through ingestion of contaminated water, global problem Norovirus - Among the 232 outbreaks of illness reported to CDC from July 1997 to June 2000, 3% were waterborne; in 23% of outbreaks, the cause of transmission was not determined –Infectious dose: typically low, maybe 1 to 10 infectious units –Developing countries: Rotaviruses, Hepatitis A virus, Hepatitis E virus

16. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Proposed EPA drinking water contaminant list The Safe Drinking Water Act directs that periodically publish a CCL 2003 - regulatory determinations for nine contaminants from the first CCL (60 contaminants in March 1998) Summer of 2005 - second cycle of preliminary regulatory determinations - continue research on the list of contaminants on the first CCL Make final regulatory determinations in August of 2006 –Adenoviruses –Noroviruses –Coxsackieviruses –Echoviruses

17. “Payment Intervention Studies”: References Payment P., et al, (1991). A Randomized Trial to Evaluate the Risk of Gastrointestinal Disease due to Consumption of Drinking Water Meeting Current Microbiological Standards. American Journal of Public Health 81 (6) 703-708. Payment P., et al (1997). A prospective epidemiological study of gastrointestinal health effects due to the consumption of drinking water. International Journal of Environmental Health Research. 7(1). 1997. 5-31

18. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Payment et al. intervention studies Wanted to know risk of GI illness associated w/consumption of municipal drinking H 2 O –Municipal H 2 O source = river polluted with human sewage –1 st study: 606 households; ~half had RO filters, others no intervention; 15mo 35% higher rate of GI symptoms households w/o intervention; symptoms & serologic evidence pointed to enteric viruses –2 nd study: 1400 households; 3 groups w/tap H 2 O, 1 group purified bottle H 2 O; 16 mo ~20% more GI illness in tap H 2 O consumers

19. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Enteric protozoa –Recently recognized as water borne pathogens –Thick-walled protozoan cysts and oocysts are environmentally resistant –Recovery and detection technically challenging Filtering liters of water Immunofluorescence microscopy May not distinguish between nonviable/viable orgs –Infectious dose is low C. parvum ~ 100 oocysts

20. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Enteric protozoa (cont.) –Ex. Cryptosporidium, Giardia Size: Cryptosporidium oocysts (4-6 µm) Giardia cysts (7-14 µm) Animal reservoirs Size limits movement thru soil, thus surface waters contamination mostly (esp. unfiltered) Greater than half of 300 surface water supplies in US contained Cryptosporidium oocysts –Ex. Toxoplasma gondii Matures only inside cats and spreads through their feces Lethal parasite that has infected or killed hundreds of California sea otters Parasite can concentrate inside clams and other bivalves, favorite of otters Flushable cat litter, affecting the ecosystem, including the clams that otters eat

21. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Other aquatic microbes Cyanobacteria – algal blooms, dermatitis Naegleria fowleri – meningoencephalitis, usually fatal –2003 case in North Carolina Acanthamoeba species – subcutaneous abscesses, conjunctivitis –Contact lens wearers, EPA published warning: Acanthamoeba Guidance Document EPA-822-B-04-001 –EPA determined not to regulate because “regulation would not present a meaningful opportunity for health risk reduction” Schistosoma species – dermatitis, swimmer’s itch –Feces and urine of infected animals and birds

22. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Categories of water-related infectious hazards –Waterborne –Water contact –Water related –Water washed

23. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Waterborne –Physical contact w/microbially contaminated H 2 O –Bathing, recreational activities, ingesting water (also ice) –Example: cholera

24. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Water Contact –Infections caused by aquatic organisms Pathogenic microorganisms or macroorganisms (worms) Naturally spend part of life cycle in aquatic envr or within a host that resides in water –Ingestion or immersion in water –Example: Schistosomiasis

25. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Water Related –Disease as a consequence of being bitten by an invertebrate vector whose life cycle requires access to H 2 O –Influences of season, rain events (monsoon, flooding) –Example: Malaria (parasite) and yellow fever, west nile infection (viruses)

26. Environmental Transmission of Pathogenic Microbes : Water, Soil, Air Water Washed –Infections associated with inadequate hygiene –Eliminate hazard by using microbe-free water for cleaning of eating and cooking utensils, plates, self, etc. –Example: Shigellosis

27. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Improve microbiological water quality, reduce diarrhea morbidity and mortality associated with waterborne disease Standards for water quality –EPA –World Health Organization (WHO) Total and fecal coliforms

28. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Methods to measure water quality –Accurate and reproducible –Rapid –Relatively simple techniques, applicable to most laboratories –Common reagents, inexpensive

29. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Detection of infectious agents in water –Many are difficult to detect/quantify May require expensive equipment Complex techniques – specialized reference laboratories –Newly recognized agents, methods are still being developed –Pathogen occurrence surveys and special studies

30. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Detection of infectious agents in water –Laboratory investigations of disease outbreaks often never detect the etiologic agent Contamination was temporary (e.g. problem was discovered and fixed before investigation) Microbe died off or flushed away Sensitivity/specificity of methods Size of event

31. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Detection of infectious agents in water –Microbial Indicator Organism Traditional approach to protect/assess the "sanitary" quality of water (food) with respect to fecal contamination Quantify bacteria commonly present in intestines of warm blooded animals; surrogates for pathogens, especially bacterial May not be reliable indicators of viruses and parasites

32. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Criteria for an Ideal Indicator of Fecal Contamination Applicable to all types of water (and other relevant samples) Present in feces, sewage and fecally contaminated samples when pathogens are present; numbers correlate with amount of fecal contamination; outnumber pathogens No "after growth" or "regrowth" in the environment Survive/persist > than or = to pathogens Easily detected/quantified by simple lab tests in a short time Constant characteristics Harmless to humans and other animals Numbers in water are associated with risks of enteric illness in consumers (dose-response relationship)

33. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air The Quest for The Ideal Indicator(s) No single indicator (so far discovered) meets all of the criteria of an ideal indicator It is unlikely that a single organism or (taxonomic) group will meet all of the indicator criteria when applied to viruses, bacteria and parasites Current microbial indicator criteria do not address those pathogens that are not associated with fecal contamination

34. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Current and Candidate Bacterial Indicators of Fecal Contamination Total coliforms : standards for drinking, bathing and shellfish harvesting waters; not feces ‑ specific (environmental sources) Fecal ("thermotolerant") coliforms : ditto for total coliforms E. coli : the "fecal" coliform; may occur naturally in tropics Fecal streptococci : another group of enteric, fecally excreted bacteria; not feces ‑ specific (environmental sources) Enterococci : Streptococcus faecalis and S. faecium ; a sub ‑ set of the fecal streptococci considered more feces ‑ specific; EPA guide-line for bathing water quality used as standards in some states Clostridium perfringens : anaerobe; ?feces ‑ specific?; very (too?) resistant spores; candidate indicator for protozoan cysts

35. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air CANDIDATE VIRAL INDICATORS OF FECAL CONTAMINATION OF WATER Coliphages: viruses (bacteriophages) infecting E. coli and perhaps 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); but, pigs may harbor groups II & III, too. Bacteroides fragilis phages: may be human feces specific; USA studies do not show human-specificity; concentrations too low. Salmonella phages: in human and animal feces; may indicate presence of Salmonella bacteria; concentrations too low.

36. Male-specific and Somatic Coliphage Hosts and Phages Male-specific host Somatic host E. coli Famp F+ E. coli C Somatic Coliphage F+ Coliphage Somatic Coliphage F+ Coliphage

37. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Microbial Indicators of Fecal Contamination and Treatment Efficacy for Enteric Protozoan Pathogens Clostridium perfringens spores may be useful indicators of enteric protozoan parasites –Plentiful in feces, raw sewage, treated effluents and receiving waters and soils –Spores of aerobic bacilli (Bacillus spp.) may be useful indicators of water treatment efficacy –Plentiful in water and other environmental media –But, not feces-specific –Spores are reduced less than are conventional vegetative indicator bacteria by water and sewage treatment processes –Spores of C. perfringens and Bacillus spp. superficially resemble enteric protozoan parasite cysts and oocysts

38. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Sources of drinking water Surface water –Widely used as a supply for municipal DW –Need to protect from sources of contamination (land run-off, wildlife, recreational activities) Ground water –Comprises 95% of water serving rural populations in US (>100 million people) –Assumed that soil will filter out most microbes –Rarely are private wells treated, also public H 2 O supply

39. Environmental Monitoring of Pathogenic Microbes : Water, Soil, Air Traditionally, more than half waterborne disease outbreaks – groundwater responsible –From 1997-98 15/17 (88%) reported outbreaks - groundwater responsible –Most common pathogens identified = Shigella spp. and Hepatitis A virus –2/3 of the time no etiologic agent identified