Download Introduction to Waterborne Pathogens

Introduction to
Waterborne Pathogens
Marylynn V. Yates
Department of Environmental Sciences
University of California, Riverside
Waterborne Disease Associated with
Drinking Water in the U.S., 1920-2004
800
600
400
Outbreaks
Cases (1000s)
Deaths
Year
01-
91-00
81-90
71-80
61-70
51-60
41-50
31-40
0
20-30
200
Number of Waterborne Disease Outbreaks
Associated with Drinking Water, 1971-2004
CDC, 2006
Waterborne Disease Outbreaks Associated
with Drinking Water, 1986-2002
14
12
10
8
6
4
2
0
AGI
Giardia
Cryptosporidium
Shigella
Norwalk virus
Salmonella
E. coli
Naegleria
Campylobacter
1986
1988
1990
1992
1994
1996
1998
2000
2002
Enteric Pathogens
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Exposure is via ingestion
primary site of infection is
gastrointestinal tract
gastroenteritis symptoms
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nausea
vomiting
diarrhea
fever
may spread to other sites (blood,
liver, nervous system)
shed in fecal material
“fecal-oral” route of transmission
Types of Waterborne Pathogens
Viruses
Bacteria
Parasites
Salmonella
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Arrows indicate Salmonella cells
invading pig epithelium
Causes diarrhea, fever, cramps
12-72 hours after infection
illness lasts 4-7 days
can also cause typhoid fever
40,000 cases reported annually;
1000 deaths annually
0.1% population excretes
Salmonella at a given time
most common bacterial pathogen
in wastewater
primarily foodborne (beef, poultry,
milk, eggs), but also transmitted
by water
Shigella
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Macrophage infected with
Shigella
Causes diarrhea (often bloody),
fever, cramps 24-48 hours after
infection
illness lasts 5 -7 days
infect only humans
18,000 cases reported annually
primarily transmitted by direct
contact with infected individual
also transmitted by contaminated
food, water, recreation
low infectious dose (~10
organisms)
Vibrio cholerae
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Releases endotoxin that
causes mild to profuse
diarrhea --> loss of fluids->death if untreated
infects only humans
several pandemics have
occurred
Latin America: 1 million cases;
10,000 deaths (1991-1994)
primarily transmitted by water
and food; rarely by direct
contact
Pathogenic E. coli
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Enterohemorrhagic: E. coli
O157:H7 - bloody diarrhea, may
cause acute kidney failure, death
Enterotoxigenic: minor to severe
diarrhea; contaminated food and
water (poor sanitation)
Enteroinvasive: dysentery
Enteropathogenic: traveler’s
diarrhea - watery diarrhea
Enteroaggregative: persistent,
non-bloody diarrhea
Health effects (food and water)
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~ 73,000 E. coli cases each year
~ 61 deaths
Giardia
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Causes diarrhea, abdominal
cramps, nausea for 4-6 weeks
1-2 week incubation period
transmitted by contaminated
food/water
can be transmitted from
animals to humans
antibiotics are available
Cryptosporidium
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Causes diarrhea, abdominal
cramps, slight fever for 1 week
2-10 day incubation period
transmitted by contaminated
food/water, person-person
can be transmitted from animals to
humans
no antibiotics are available
can cause very severe illness in
individuals with weakened
immune systems
Rotavirus
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Most common cause of severe
diarrhea worldwide
in developing countries, 1 million
deaths/yr
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20-25% diarrhea deaths
6% deaths in kids <5 yrs.
Waterborne outbreaks
documented
very high numbers in feces
(1010/gram)
very low infectious dose (~1)
Rotavirus
CDC, 2006
Norovirus
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Causes diarrhea, vomiting (1-4
days)
1-2 day incubation period
transmitted by contaminated
food/water
may cause up to 50% food-related
gastroenteritis outbreaks
may cause 25% waterborne
outbreaks
Health effects (food and water):
23 million cases annually
Recreational Water-Associated
Outbreaks, 1978-2004
CDC, 2006
Pseudomonas
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Associated with hot tubs,
pools
¾ Typically causes dermatitis
¾ preventable by maintaining
adequate residual
disinfectant levels
Legionella
PONTIAC FEVER:
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Fever, muscle aches for 2-5
days
incubation period:hours-2
days
8,000 - 18,000 cases/year
grow in warm (90 -105 F),
stagnant water
spread through aerosols
(cooling towers, whirlpool
spas, showers)
elderly, cigarette smokers,
persons with chronic lung or
immunocompromising
disease, and persons
receiving
immunosuppressive drugs at
increased risk
Naegleria
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Found in soil and warm,
stagnant bodies of fresh
water, unchlorinated
swimming pools, and in
warm wastewater pools from
power plants
¾ Entry through nose, infection
of brain and spinal cord
¾ Headache, fever, nausea
and vomiting, stiff neck,
confusion, loss of balance
and bodily control, seizures,
and hallucinations. Infection
usually results in death
within 7-10 days.
Concentrations of Pathogens in Stools
of Infected Individuals
Organism
Giardia
Cryptosporidium
Poliovirus
Hepatitis A
Rotavirus
Concentration (per g)
5 x 106
6
7
10 - 10
3
6.5
10 - 10
8
10
8
10
10 - 10
from Gerba, 1995
Pathogen Removal During Sewage
Treatment
Viruses
Salmonella
Giardia
Cryptosporidium
Concentration
in raw sewage
(no./liter)
100,000 – 1
million
5,000 – 80,000
9,000 –
200,000
1 – 3,960
Concentration
after primary
treatment
1,700 –
500,000
160 – 3,360
72,000 –
146,000
0.7
Concentration
after secondary
treatment
80 – 470,000
3 – 1,075
6,480 –
109,500
Concentration
after advanced
secondary
treatment
0.007 – 170
0.000004 – 7
0.099 – 2951
Maier et al., 2000
Minimal Infective Doses for Some
Pathogens
Organism
Salmonella spp.
Shigella spp.
E. coli
E. coli O157:H7
Vibrio cholerae
Campylobacter jejuni
Giardia lamblia
Cryptosporidium
Hepatitis A virus
Minimal Infective Dose
10,000 – 10 million
10 - 100
1 million – 100 million
<100
1000
~500
10 -100 cysts
10 oocysts
1 -10 pfu
from Bitton, 2006
Sampling for Viruses
Collection Apparatus
Collection on Filters
Positively-charged filters
COO- +
+
+
NH2
+
+
Elution from Filters
Positively-charged filters
COO- +
+
+
NH2
+
+
COONH2
+ Beef extract,
pH 9.5
-
Sample Concentration
1-liter sample concentrate:
¾ Add acid to decrease pH to 3.5
¾ Organic material (with viruses attached)
precipitates
¾ Centrifuge
¾ Viruses pellet out
¾ Resuspend pellet in buffer
Sample Processing
Filter sample
1000 liters
Elute from filter
1 liter
Concentrate sample
30 ml
Analyze sample:
cell culture
PCR
up to 30 ml
10 - 100 ul
Physiological Methods
Virus
Host Cells
Infection
Replication
Release
Cell Culture
Confluent Monolayer
Cytopathic Effects
Plaque assay
Advantages of Cell Culture
¾ Detects only infective particles
¾ Ability to quantify viruses
¾ High sensitivity
¾ Entire sample concentrate can be
analyzed (1000 L equivalent volume)
Limitations of Cell Culture
¾ Detects only culturable viruses
¾ Relatively non-specific
¾ Up to 2-4 weeks for analysis
¾ Cost of analysis
Biochemical Methods
+
Nucleic
Acid
Gene Probes
PCR
RT-PCR
Oligos
Ab
Coat
Y
+
RIA
ELISA
Polymerase Chain Reaction
(PCR)
Nucleic Acids
3’
5’
ACTGGTCAAGT
TGACCAGTTCA
3’
5’
Double-stranded DNA
5’
3’
ACUGGUCAAGU
mRNA
PCR: Cycles 1-4
Gel Electrophoresis
lar
u
c
le
Mo r k e r
ma
311 bp
197 bp
92 bp
tive
i
s
Po trol
con
ple
m
Sa
#1
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mp
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#2
tive
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Advantages of PCR
¾ Relatively rapid results
¾ Highly sensitive
¾ Highly specific
¾ Ability to detect non-culturable viruses
¾ Ability to tailor primers for desired
application
Limitations of PCR
¾ High potential for contamination
¾ Potential for amplification of non-target
sequences
¾ Small equivalent sample volume analyzed
(0.3 - 3 L)
¾ Inability to distinguish between infective
and non-infective particles
Virus Detection Methods
Infectivity
test?
Detection
limit
Time
no
105 – 106
< 24 h
viral antigens
no
105
<2h
antiviral antibodies
yes
105
<2h
Real-time (RT) PCR
no
100 - 101
<8h
Plaque assay
yes
100 - 101
< 21 d
Method
Electron microscopy
ELISA
Microorganisms in Untreated
Wastewater
Organism
Total coliform bacteria
Fecal coliform bacteria
Salmonella
Shigella
Enteroviruses
Rotaviruses
Giardia
Cryptosporidium
Ascaris
No. per liter
100 million – 1 billion
10 million – 100 million
1000 – 100,000
10 – 10,000
10,000 – 100,000
100 – 100,000
100 – 100,000
100 – 10,000
10 – 10,000
Characteristics of an “ideal”
indicator organism
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Present in waters contaminated by pathogens and
absent from these same waters when contamination
is not present
Exist in concentrations that outnumber pathogenic
organisms
Non-pathogenic to humans
Equal or greater resistance to treatment protocols
and environmental factors than pathogens
Unable to reproduce in the environment
Detectable by simple, rapid, and economical methods
Examples of indicator organisms
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Total coliform bacteria (in use for
approximately 75 years)
Fecal coliform bacteria (thermotolerant
coliforms)
Fecal streptococci (Enterococci)
coliphages
Detection of Indicator Organisms
Total Coliform Bacteria
Fecal Coliform Bacteria
E. coli
mTEC agar
modified mTEC agar
EPA, 2000
Total Coliform Bacteria
E. coli
Enterococci (Fecal Streptococci)
mEI agar, courtesy EPA
Somatic Phages (phiX174)
Somatic phage
Potential Applications of an
Indicator
Being an indicator of:
¾ fecal contamination
¾ the presence of domestic sewage
¾ the presence of pathogens
¾ the efficiency of a particular water or waste
treatment process
¾ the environmental fate of a pathogen of interest
¾ the movement of particles suspended in water
during subsurface transport
Relative Sizes of
Microorganisms
Bacteria: 0.1 – 10 µm
Viruses: 0.01 – 0.1 µm
Coliphage and Human Enteric
Virus Similarity
Gravel
Pore Diameter > 400 um
Sand
Pore Diameter 12-400 um
Clay
Pore Diameter <10 um
Concluding Thoughts
When designing a monitoring program:
¾ Do your homework
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Indicator vs. pathogen?
All indicators are not created equal
One size does not fit all