Monitoring-Pathogen-Microorganisms

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Monitoring-Pathogen-Microorganisms Powered By Docstoc
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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
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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:
   1. Initial sampling, concentration, or recovery methods
   2. Pathogen detection and isolation methods
   3. Pathogen confirmation and further characterization
        Where did the fecal waste come from?? (source tracking)
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1.   Use of indicator organisms?

2.   Pathogens
     – Class of pathogen
                                Salmonella   Hepatitis E Virus   Cryptosporidium


3.   Environmental Media
     –   Water and wastewater
     –   Biosolids and litter
     –   Soils and vegetation
     –   Air
     –   Vectors                                                                   Si
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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            Si
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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:
   1.   high numbers
   2.   easy to measure
   3.   surrogates for pathogens, especially bacterial pathogens.



May not be reliable indicators of
viruses and parasites


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   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.

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Viral Indicators of Fecal Contamination in Water
Coliphages: viruses (bacteriophages) infecting E. coli
    or other coliforms; attach directly to cell wall
    (somatic)                                               E. coli C


     • heterogeneous group
     • may not be feces-specific                                                 Somatic
                                                                                 Coliphage
     • host-dependent detection                            F+ Coliphage


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
                                                         E. coli Famp                  Somatic
                                                                                       Coliphage
                                                                          F+
        • II & III human; I & IV animal
        • PROBLEM: swine may harbor groups
          II & III also
                                                                        F+ Coliphage


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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                 Si
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   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 (26oC)
     • Microscopic evaluation for viability
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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.                         Si
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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
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.
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Sampling methods: both methods
   use a pheromone based
   attractant
     •      Alive (Fly Terminator)
     •      Dead (QuikStrike)

1.       Houseflies are separated,
         counted, and weighed
2.       Appropriate numbers of flies
         are blended with a beef
         extract eluting solution
3.       Liquid eluting solution is
         used for subsequent assay
         methods                        Si
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1.       Membrane Filter
     •     Nutrient agar + MUG
     •     EC medium + MUG
                                        MI Agar under ambient and longwave UV light

2.       Multiple Fermentation Tube
     •     EC broth + MUG

3.       Biochemical Assay
     •     defined substrate
     •     Detect product (colored)
              Colilert™ or Colisure™
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   100-1000 mL of water

   1. Inoculate with E. coli
      and nutrients

     37ºC
overnight
  2. Spot 10 µl to E. coli
     lawn in agar medium
37ºC 6 to
12 hours
 3. Look for lysis zones
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  100 mL of water
 + 100 ml of 2X
  trypticase soy
  agar
 + E. coli
 + MgCl2 (optional)

 Pour the mixture on
     multiple plates


    37ºC
 Look for
overnight plaques
Light
  Microscopy
 Fluorescent Microscopy




Electron
 Microscopy               Si
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                      Dilution                     No
                                                   virus
2   4   8   16   32    64   128   256   512 1024




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             Sample to be tested   Enzyme -> color




virus
                                               Detecting
                                               antibody




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Capturing antibody                                          m
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Pathogen Detection - Cytopathic Effects of
Viruses in Infected Cell Cultures




uninfected   Late cytophathic effects:   Cell degeneration   Plaque formation
             Enlarged cells
             Nuclear inclusions                                                 Si
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primers


                    Viral DNA

      First cycle




     Second cycle




           20-40 cycles

             many copies
                                 gel   Si
             of amplicon (DNA)          m
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Bacterial confirmation:
    •    Biochemical test kits

Antimicrobial resistance
    patterning:
    1.   Micro-dilution method
    2.   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)
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posted:9/23/2011
language:English
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