# Presentation: 877397

## State of the Science – Analytical Techniques forAnimal Feeding Operation (AFO)Pathogens and Indicators 

**Otto (Chip) D. Simmons, III**
**School of Public Health **
**Department of Environmental Sciences and Engineering**
**The University of North Carolina at Chapel Hill**
**CB# 7431**
**Chapel Hill, NC 27599-7431**
**osimmons@email.unc.edu**

## 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**

## 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)**

## Considerations for Pathogen Detection Associated with AFOs:

**Use of indicator organisms?**
**Pathogens**
**Class of pathogen**
**Environmental Media**
**Water and wastewater**
**Biosolids and litter**
**Soils and vegetation**
**Air**
**Vectors**

***Salmonella***

***Cryptosporidium***

***Hepatitis E Virus***

## Pathogen Analysis, Monitoring, and Surveillance

**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**

## Microbial Indicators of Fecal Contamination

**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**

## Bacterial Indicators of Fecal Contamination in Water

**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.**

## Viral Indicators of Fecal Contamination in Water

**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**

**Somatic**
**Coliphage**

***E. coli *****C**

**Somatic**
**Coliphage**

**F+ Coliphage**

## Concentration of EnvironmentalPathogens 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***

## One-step Concentration - Ultrafiltration Method for All Classes of Pathogens from Water

## 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)**
**Helminth***** *****ova (< 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 (26****o****C)**
**Microscopic evaluation for viability**

## 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. **

## Air Samplers - Andersen samplerand 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**

## 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.**

## 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**

## Detection of Total and Fecal Coliform Bacteria and E. coli

**Membrane Filter**
**Nutrient agar + MUG **
**EC medium + MUG**
**Multiple Fermentation Tube**
**EC broth + MUG**
**Biochemical Assay**
**defined substrate**
**Detect product (colored)**
**Colilert****TM**** or Colisure****TM**

**MI Agar under ambient and longwave UV light**

## 		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

**	**** ****1. Inoculate with *****E. coli***
**	****	****    ****and nutrients**
**	**
**	**
**	****2. Spot 10 μl to *****E. coli*****              ****	****    lawn in agar medium**
**	**
**    ****3. Look for lysis zones**

**Coliphage Detection Method 1601: Two-step Enrichment Method **

**37oC **
**overnight**

**37oC 6 to**
**12 hours       **

**Notes:**
Because this method involves tow incubation procedures, it is also call 2 step enrichment P/A method.

##   100 mL of water  	
+ 100 ml of 2X 
   trypticase soy
   agar
+ E. coli 
+ MgCl2 (optional)		

Pour the mixture on
        multiple plates

Look for plaques

**+ 100 ml of 2X **
**   ****trypticase soy**
**   ****agar**
**+ *****E. coli***** **
**+ MgCl****2**** (optional)****	****	**
**	****	**
**Pour the mixture on**
**        ****multiple plates**
**	**
**Look for plaques**

**Coliphage Detection Method 1602: Single****Agar Layer (SAL) **

**37oC**
**overnight**

## Pathogen Detection - Microscopy

**Light Microscopy**
**	****Fluorescent Microscopy**
**Electron Microscopy**

## Pathogen Detection - Hemagglutination Assay for Viruses

**Dilution**

**2**

**4**

**8**

**16**

**32**

**64**

**128**

**256**

**512**

**1024**

**No**
**virus**

**Notes:**
advantages
sensitive and specific
simple to do, relatively fast compared to virus isolation
quantitative
disadvantages
requires laboratory facilities

## Pathogen Detection - Enzyme linked Immunobsorbant Assay (ELISA)

**Sample to be tested**

**virus**

**Capturing antibody**

**Detecting**
**antibody**

**Enzyme -> color**

**Notes:**
advantages
fast
can be done in clinic or stall-side
disadvantages
not very sensitive

## Pathogen Detection - Cytopathic Effects of Viruses in Infected Cell Cultures

**Pathogen Detection - Cytopathic Effects of Viruses in Infected Cell Cultures**

** ****uninfected**

**Late cytophathic effects:**
**Enlarged cells**
**Nuclear inclusions**

**Cell degeneration**

**Plaque formation**

## Pathogen Detection - Polymerase Chain Reaction (PCR)

**Viral DNA**

**primers**

**First cycle**

**Second cycle**

**20-40 cycles**

**many copies**
**of amplicon **_**(DNA)**_

**gel**

**Notes:**
advantages:
very fast - results can be delivered same day
very sensitive and if done properly - very specific
can be adapted to distinguish between variants of same virus (different serotypes)
disadvantages:
requires laboratory facilities
proper control needed, there can be false positives if 

## 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)**

## Summary and Conclusions

**Many methods to collect, concentrate, recover, detect, and further characterize microbial pathogens in environmental samples**
**Methods chosen should take into account:**
**Effective use of indicator organisms**
**Pathogen class**
**Environmental media or matrix**
**Method Efficacy**
**Sample size, sampling frequency, and location**
**Have specific objectives when designing an environmental monitoring plan, choose the methods accordingly, and use the information collected to achieve project objectives**
**If you look for and find microbes in samples what does it mean and how will you use the results for a management decision?**

## Questions and Comments

**Questions and Comments**