United States
Environmental Protection
     Detecting and Mitigating the
     Environmental Impact of
     Fecal Pathogens
     Originating from Confined
     Animal Feeding Operations:

                                          September 2005
Detecting and Mitigating the Environmental
Impact of Fecal Pathogens Originating from
   Confined Animal Feeding Operations:
                   Dr. Shane Rogers
                   Dr. John Haines
       Land Remediation and Pollution Control Division
       National Risk Management Research Laboratory
                 Cincinnati, OH 45268
        National Risk Management Research Laboratory
            Office of Research and Development
        United States Environmental Protection Agency
                 Cincinnati, OH 45268


The U.S. Environmental Protection Agency through its Office of Research and Development
funded and managed the research described here. It has been subjected to the Agency's review
and has been approved for publication as an EPA document.


The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
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This publication has been produced as part of the Laboratory's strategic long-term research plan.
It is published and made available by EPA's Office of Research and Development to assist the
user community and to link researchers with their clients.
Sally Gutierrez, Director
National Risk Management Research Laboratory


                               Table of Contents
Table of Contents	v
Table of Figures	vii
Table of Tables	viii
1. Introduction and Overview	1
2. Pathogens	4
3. Antimicrobial Resistance	11
  3.1 Mechanisms of bacterial resistance	11
  3.2 Antimicrobial resistance in livestock animals	16
  3.3 Risk to public health	17
4. Survival of Pathogens in the Environment	18
  4.1 Manure and manure slurries	18
  4.2 Natural waters	21
  4.3 Manure-amended soil	23
  4.4 Discussion	26
5. Pathogen Movement — An Ecological Perspective	27
  5.1 CAFOs and Abattoirs	27
  5.2 Food	32
  5.3 Air	34
  5.4 Recreational and drinking water	35
  5.5 Hydrologic events	40
6. Public Health Outcomes	42
  6.1 Waterborne and foodborne outbreaks	42
  6.2 Specific cases	45
  6.3 Antimicrobial resistance	48
  6.4 Hydrologic events	49
  6.5 Economic considerations	50
  6.6 Discussion	52
7. Emerging Technologies: Monitoring Pathogens in the Environment	53
  7.1 Sample processing	54
  7.2 Conventional cultivation and nucleic acids approaches	55
  7.3 Pathogen viability	60
  7.4 Emerging surveillance technologies	61

  7.5 Discussion	62
8. Microbial Source Tracking.	64
  8.1 Antibiotic Resistance Analysis (ARA)	65
  8.2 Ribotyping	67
  8.3 Amplified Fragment Length Polymorphisms (AFLP)	68
  8.4 Host-specific molecular biomarkers	70
  8.5 Discussion	71
9. Treatment Technologies and Management Practices	 73
  9.1 Manure management: active and passive systems	74
  9.2Disussion	80
10. Ongoing research at the EPA and Other Federal Agencies	82
11. Summary and Outstanding Issues	90
  11.1 General recommendations	91
  11.2 Recommendations for future research	92
12. References	99

                                List of Figures
Figure 1.   Confined swine, poultry, dairy cattle, and feed cattle per county in 1997  	   2
Figure 2.   Movement of pathogens - an ecological perspective  	  28
Figure 3.   The impact of confined animal feeding operations on agricultural watershed ....  36
Figure 4   Distribution of livestock animals in regions impacted by Hurrican Katrina,
           August, 2005	  51

                                 List of Tables
Table 1.     Selected zoonotic pathogens zoonoses that may be of concern for water
            quality near CAFOs	     6
Table 2.     Selected Antimicrobial Agents Approved for Use in Animal Agriculture	   12
Table 3.     Estimates of the use of antimicrobial agents in livestock animal production.    14
Table 4.     Survival of pathogenic zoonoses in livestock manures and manure slurries...   19
Table 5.     Survival of pathogenic zoonoses in soils, contaminated water-irrigated soils,
            and manure-amended soils	   22
Table 6.     Survival of pathogenic zoonoses in drinking water, livestock rinse waters,
            surface fresh waters, surface salt waters, surface water sediments, soils
            irrigated with livestock rinse waters, and ground waters	   24
Table 7.     Water and foodborne outbreaks in the U.S. reported by the CDC (1991-
            1997)	   44
Table 8.     Estimated number of total cases, hospitalizations, and fatalities that may
            occur annually in the U.S. by selected etiological agent as reported by Mead
            et a/., (1999)	   45
Table 9.     Sample times and detection limits of several nucleic acids-based techniques
            for detecting pathogens in different matrices without enrichment	   56
Table 10.   Sample times and detection limits of several nucleic acids-based techniques
            for detecting pathogens in different matrices following enrichment	   58
Table 11.   Sample times and detection limits of several nucleic acids-based techniques
            for detecting pathogens in different matrices following enrichment	   76
Table 12.   Bacterial decimation times in aerated and non-aerated manure slurries in
            weeks	   77
Table 13    Microorganism inactivation by different management techniques	   78
Table 14.   Bacterial decimation times in anaerobic digesters	   79
Table 15    Studies carried out or in progress in the United States Geological Survey....   83
Table 16.   Studies carried out or in progress by the United States Department of
            Agriculture, National Program 206	   86
Table 17.   Studies carried out or in progress by USDA or cooperating Universities
            listed in the CRIS database	   87

1. Introduction and Overview
The trend in animal production has shown a dramatic shift in the last 50-60 years from small
family farms and grazing operations towards large commercial confinement operations.  Since
1982, animal production at these facilities has nearly doubled while at the same time they have
become more spatially concentrated (U.S. Department of Agriculture, National Resources
Conservation Services (USDA-NRCS), 2000). Recently, the U.S. Department of Agriculture
(USDA) reported that more than 80% of all livestock revenues are generated in confinement
facilities that account for a scant 18% of all livestock operations (USDA-NRCS, 2002).  In fact,
more than 43% of all beef cattle, dairy cattle, swine,  and poultry are raised in the largest two
percent of operations (Goellehon etal., 2001).  The concentration of animals into confinement
facilities poses many environmental challenges, among which pathogenic microorganisms of
fecal origin are of concern.

The U.S. Environmental Protection Agency (USEPA) defines a concentrated animal feeding
operation (CAFO) as an animal feeding facility that houses more than 1,000 animal units (AU),
has 300 to 1000 AU but meets certain conditions, or  is designated a CAFO by the state (USEPA,
2001). The number of animal units are based on an equivalent number of beef cattle.  Therefore,
1,000 AU equals 1,000 beef cattle, 700 mature dairy  cattle, 2,500 swine, 5,000 ducks, 10,000
sheep, 55,000 turkeys, or between 30,000 and 100,000 laying hens or broilers depending on the
animal waste management system employed.  According to National Resources Conservation
Service (NRCS) estimates, 11,398 CAFOs (>1000 AU) were in operation in the U.S. in  1997,
and comprised five percent of all livestock facilities (USDA-NRCS, 2002). These CAFOs were
largely commercial operations (94%) with large revenues.  Total agricultural sales for 97.9% of
CAFO owners exceeded $500,000 per year. In comparison, non-commercial livestock facilities
(intermediate or rural-residence farms) earned 22.3% of all livestock revenue, generating on
average $18,500 per farm. Figure 1 shows the distribution of confined poultry, swine, dairy
cattle, and feed cattle operations in the U.S. in 1997.

Animal agriculture results in the production of copious amounts of manure, much of which is
ultimately used as fertilizer for crops or spread onto land. On a per weight basis, livestock
animals produce between 13 and 25 times more manure than humans. Comparing the most
recent U.S.  census data and USDA livestock reports, it can be estimated that animals produce
somewhere between 3 and 20 times more manure than people in the U.S. each year, as much as
1.2 - 1.37 billion tons (wet weight) (American Society for Microbiology (ASM), 1998; USEPA,
2003; USEPA, 2004). This is enough to cover a land mass the size of Rhode Island with more
than twelve inches of manure. Even moderate livestock operations can produce as much manure
as a small sized city. For example, a 2,500-head dairy cattle operation can produce a waste load
similar to a city of 61,000 people.  Two important differences are that livestock CAFO animal
wastes can be as much as 100 times more concentrated than human wastes, and the treatment of
human wastes is required by law prior to  discharge into the environment (USEPA, 2001).

Animal wastes contain zoonotic pathogens, which are viruses, bacteria, and parasites of animal
origin that cause disease in humans. Diseases that can be caused by zoonotic pathogens include
Salmonellosis, Tuberculosis, Leptospirosis, infantile  diarrheal disease, Q-Fever, Trichinosis,
Cryptosporidiosis, and Giardiasis to name a few.  These diseases typically present  as mild

              Confined Poultry, 1997
                                                      Confined Swine, 1997
               Confined Milk Cows, 1997
                                                      Confined Fattened Cattle, 1997
Figure 1.
Confined swine, poultry, dairy cattle, and feed cattle per county in 1997 (adapted from USDA-NRCS, 2002).

diarrhea, fever, headaches, vomiting, and muscle cramps.  In more severe cases, however, these
diseases may cause meningitis, hepatitis, reactive arthritis, mental retardation, miscarriages, and
even death, particularly in the immunocompromised.  The dosing of livestock animals with
copious amounts of antimicrobial agents for growth promotion and prophylaxis may promote
antimicrobial resistance in pathogens, increasing the severity of disease and limiting treatment
options for sickened individuals (Lee et al., 1994; Marano et al., 2000).

Zoonotic diseases from livestock animals, transmitted through air, water, and food, cause
significant human suffering and economic losses in the U.S. every year (Schlech et al., 1983;
Bessere^a/., 1993; MacKenzie etal., 1994; Solo-Gabriele and Neumeister, 1996; Hoxie etal.,
1997; Mead et al., 1999; Valcour et al., 2002; Clark et al., 2003). Increasing the concentration
of animals in confinement facilities amplifies the potential for localized runoff and
contamination, increasing the probability for accidental exposure of susceptible individuals.  In
fact, living near CAFO operations has been associated with significant deterioration in human
health including increased gastrointestinal illness, headaches, sore throats, sinusitis, and
childhood asthma (Wing and Wolf, 2000; Merchant et al., 2005). There is increasing evidence
that impoverished and nonwhite communities may be burdened with a disproportionate share of
not only these negative health outcomes, but also pollution and offensive odors emanating from
CAFO facilities (Wing et al., 2000; Wilson et al., 2002; Wing et al., 2002). Based on studies in
North Carolina, operations run by cooperate investors may be more likely to be concentrated in
poor and nonwhite areas than operations run by independent growers (Wing et al, 2000).

The USEPA recognizes the need to improve manure management practices at confined animal
feeding operations (USEPA, 2003). Several other U.S. governmental entities, including the U.S.
Department of Agriculture (USDA), U.S. Geological Survey (USGS), and Centers for Disease
Control and Prevention (CDC), have also recognized the need for control  of pathogens at CAFOs
and have robust research and surveillance activities to improve the outcomes for public health
and welfare in the U.S. Several recent advances in the fields of medicine, molecular
microbiology, engineering, agronomy, and epidemiology are addressing issues pertinent to the
control of pathogens from CAFOs at a rapid pace. However, reported literature and research
activities can in some cases be divergent between some disciplines and repetitive between others.
There is a lack of integration of both knowledge and skills necessary to drive the research in an
appropriate direction.  As stated by Landry  and Wolfe (1999):

       The range of disciplines conducting fecal bacteria research and the diverse nature of the
       literature are obstacles  to application and synthesis of existing knowledge by animal
       waste managers and scientists.

In this report, we synthesize the current state of knowledge regarding pathogen research as it
relates to livestock CAFOs, including a summary of research ongoing at USDA and other federal
agencies.  Pathways for the release of zoonotic agents and antimicrobial-resistant bacteria
endemic in animals raised in confinement and their potential to persist in different milieus are
reviewed. We discuss the impact to the environment and public health and welfare posed by the
release of these agents from CAFOs, as well as manure management practices that are employed
to mitigate their release into the environment.  The objectives of this review are to summarize
pathogen issues with regard to livestock CAFOs and identify and discuss gaps in the research
that need to be addressed to improve public health.

 2. Pathogens
 Livestock animals can harbor and shed viruses, bacteria, protozoan parasites, and helminthes that
 are pathogenic for humans, other domestic animals, and wildlife.  Pathogens present in animal
 carcasses or shed in animal wastes may include rotaviruses, hepatitis E virus, Salmonella spp., E.
 coli O157:H7, Yersinia enterocolitica, Campylobacter spp., Cryptosporidiumparvum, and
 Giardia lamblia to name a few (Sobsey et al., 2002).  These zoonotic pathogens can exceed
 millions to billions per gram of feces, and may infect humans through various routes such as
 contaminated air, contact with livestock animals or their waste products, swimming in water
 impacted by animal feces,  exposure to potential vectors (such as flies, mosquitoes, water fowl,
 and rodents), or consumption of food or water contaminated by animal wastes (Schlech et al.,
 1983; Bezanson et al, 1983; Hawker et al, 1998; Valcour et al., 2002; Armand-LeFevre et al.,
 2005).  The consequences  of infection by pathogens originating from animal wastes can range
 from temporary morbidity to mortality, especially in high-risk individuals. Antimicrobial use in
 animal agriculture may exacerbate the problem by increasing the resistance of these pathogens to
 therapeutic drugs used to treat human disease.

 It has been estimated that 61% of all human pathogens and 75% of emerging human pathogens
 are zoonotic (Mahy and Murphy, 1998; Murphy, 1998;  Taylor et al., 2001; Woolhouse et al.,
 2002).  The overwhelming majority of these pathogenic zoonoses that commonly infect humans
 are related to animal husbandry practices.  Table 1 lists some of the zoonotic pathogens that may
 be of concern in animal  agriculture.  Many of these pathogens are endemic in livestock and
 difficult to eradicate from the animals or their production facilities (Sobsey et  al., 2002).  For
 instance, a study of healthy swine on eight farms in Iowa and North Carolina revealed greater
 than 90% incidence of Campylobacter coli in all three growth stages (nursery, grower, and
 finisher) (Wesley et al.,  1998). Similarly, the prevalence of Salmonella spp. and Campylobacter
jejuni have been reported to be as high as  100% in poultry operations, Yersinia enterocolitica as
 high as 18% in swine operations, and Giardia lamblia and Cryptosporidium spp. as high as
 100% in cattle operations (Olson, 2003).  The primary reservoir for E. coli O157:H7 was
 determined to be healthy cattle in one study in Canada,  although this bacterium is also endemic
 to swine and sheep (Jackson  et al., 1998).  In the U.S., E. coli O157:H7 infection was widely
 distributed across all 13  states at an average rate of 1.61% of all cattle when tested in  1994
 (Dargatz, 1996). At slaughter, the prevalence of E. coli  O157:H7 in Scottish cattle may be
 greater than 13% (Low et al., 2005). Fratamico et al., (2004) tested 687 swine fecal samples
 from swine operations in 13  of the top 17 swine-producing states and determined that 70% of the
 samples were positive for shiga-toxin (stx 1 and stx 2) genes.  Due to the endemic nature of
 zoonotic pathogens in livestock, there is a clear need for appropriate management practices at
 livestock facilities that are protective of human health and the environment and firmly grounded
 in risk analyses.

 The risk of contracting disease following exposure to livestock wastes is dependent on the
 properties of the infectious agent, the exposed individual, the route of exposure, and the dose.
 There are a wide range of infective doses for different pathogens as shown (Table  1).  For
 instance, severe gastrointestinal illness may require the  ingestion of millions of Yersinia
 enterocolitica bacteria, or as little as 5 to 10 E. coli O157:H7 cells (PHAC, 2005).  The
 infectious doses listed in Table 1  were established based on infectivity studies in healthy

individuals, and therefore, may not be particularly useful for establishing safe exposure limits for
human health. Particularly susceptible individuals such as children, the elderly, or the
immunocompromised, which represent nearly 25% of the U.S. population may succumb to
infection at much lower doses than the general population (Naumova et a/., 2003). For instance,
approximately 70% of the diarrhea-associated deaths in the U.S. each year occur among
individuals 55 or older.

Regulatory limits on the concentrations of pathogens in the environment protective of human
health have not been established. As such, pathogenic organisms are rarely monitored in waste
streams from animal feeding operations.  Difficulty in quantifying pathogens at relevant
concentrations in environmental matrices, the large number of analytical tests that would be
required to measure all of the zoonotic pathogens shed in livestock feces, and a lack of
epidemiological data to establish appropriate and safe levels of pathogens in the environment
have all led to this deficiency. Due to the difficulties in quantifying pathogens, indicators of
fecal pollution, including  coliform bacteria, fecal coliforms, E. coli, and/or Enterococci have
been monitored in lieu of overt pathogens for more than 100 years (Smith, 1893; Allen etal.,
1952; Kirschner etal., 2004; Byamukama etal., 2005). Epidemiological evidence supports the
relationship between the fecal indicator bacteria E. coli and enterococci and incidence of
gastrointestinal illness following recreational water exposure, and provides the basis for local,
state, and federal water quality regulations (USEPA,  1986). However, the works of several
researchers has shown that these indicators are not reliable surrogates for many pathogens,
including bacteria and most viruses and parasites (Seligmann and Reitler,  1965; Boring et al.,
1971; Wetzlere^a/., 1979; Carter ef al., 1987; Geldreich, 1996; Ashboltetal., 2001; Grabow,
2001; Leclerc etal., 2001; Tillett etal., 2001; Herman etal., 2004; Harwood etal., 2005).  New
approaches for detecting pathogens are needed to improve monitoring systems.  There also
remains a need for epidemiological data to enable the identification of appropriate and safe limits
of pathogens in the air, drinking water, recreational water,  and in food. Based on  surveillance of
water and foodborne outbreaks in the U.S., priority for standard methods and recreational and
drinking water guidelines should be given to Salmonella spp., Campylobacterjejuni, E. coli
O157:H7, Cryptosporidium, Giardia, and selected viral agents indicative of viral contamination.
Priority should be established on the incidence of a particular illness due to a pathogen or the
severity of the illness or possibly both.

Table 1.    Selected zoonotic pathogens zoonoses that may be of concern for water quality near CAFOs1
Infectious Agent
Bacillus anthracis
Brucella spp.
Campy lobacterjejun i
Clostridium tetani
Coxiella burnetii


(by inhalation)
(by ingestion)
Toxin is
(by inhalation)

Incubation Disease
Period Symptoms
2-5 days Anthrax, Wool sorter's disease
Cutaneous - skin lesions, death (5-20%)
Inhalation - respiratory distress, fever, shock,
Intestinal - abdominal distress, fever,
septicemia, death (rare)
Highly Variable Brucellosis, Undulant Fever, Bang's Disease,
5-60 days Malta Fever, Mediterranean Fever
Intermittent fever, headache, weakness, profuse
sweating, chills, arthralgia
1-10 days Campylobacter enteritis, Vibrionic enteritis,
Traveler's Diarrhea
Diarrhea, abdominal pain, malaise, fever,
nausea, vomiting, septicemia, meningitis,
Guillain-Barre syndrome, death (rare)
3-21 days Lockjaw, Tetanus
Painful muscular contractions, abdominal
rigidity, spasm, death (30-90%)
2-3 weeks Q fever, Query Fever, Rickettsia
Acute febrile disease - chills, headache,
weakness, malaise, severe sweats, pneumonitis,
pericarditis, hepatitis
generalized infections - endocarditis
Host Range
Humans, cattle,
swine, goats,
sheep, horses
Humans, cattle,
swine, goats,
sheep, deer,
caribou, elk,
dogs, coyotes
Humans, cattle,
swine, goats,
sheep, poultry,
rodents, birds,
household pets,
Humans, animals
Humans, cattle,
sheep, goats

Spores remain
viable in soil
contaminated by
animal wastes for
Cattle most
Cattle, swine,
sheep, poultry
household pets,
rodents, birds
Intestine of
animals and
humans, soil
contaminated with
animal feces
Sheep, cattle,
goats, especially at

       Hazen and Toranaos, 1990; WHO, 1993; DuPont et al., 1995; Morris and Levin, 1995; Geldrich, 1996; ASM, 1998; Haines et al, 2004; PHAC, 2005

Table 1.   Selected pathogenic zoonoses that may be of concern for water quality near CAFOs (Continued)
Infectious Agent
Bacterial (Cont.)
Escherichia coli
(E. coli O157:H7 and
Escherichia coli
Leptospira spp.
Listeria monocytogenes
Mycobacterium bovis
M. tuberculosis
Salmonella spp.
(non-typhi mparatyphi)
Yersinia enterocolitica
Infectious Incubation
Dose Period
5-10 2-8 days
108-1010 in 0.5-3 days
Unknown in
Unknown, but 4-19 days
may be as low
as 3
Unknown, but 3-70 days
likely less than (mean = 21)
10 4-12 weeks
(by inhalation)
100-1000 0.25-3 days
(by ingestion)
106 3-7 days
EHEC, Verotoxin-produding E. coli, VTEC,
Shiga toxin-producing E. coli, STEC
Hemorrhagic colitis, abdominal pain, bloody
diarrhea, fever, hemolytic uremic syndrome,
thrombocytopenic purpura, death (in children)
Attaching and effacing E. coli, enteroadherant
E. coli, infantile diarrheal disease
Watery diarrhea, fever, cramps, vomiting, bloody
stool in some cases, serious disease in infants
Leptospirosis, Weil's Disease, Canicola fever,
Hemorrhagic jaundice, Mud fever,
Swineherd's disease
Fever, headache, chills, muscle aches, vomiting,
meningitis, rash, jaundice death (rare)
Listeriosis, Listerella
Fever, muscle aches, nausea, diarrhea,
headache, stiff neck, confusion, loss of balance,
convulsions miscarriage or stillbirth, premature
delivery, death in about 20% of all cases
Tuberculosis, TB
Fatigue, fever, cough, chest pain, hemoptysis
fibrosis, irreversible damage to lungs
Salmonellosis, Acute Gatroenteritis
Abdominal pain, diarrhea, nausea, vomiting,
dehydration, septicemia, reactive arthritis
Yersiniosis, enterocolitis, pseudotuberculosis
Diarrhea, acute mesenteric lymphadenitis
mimicking appendicitis, fever, headache,
anorexia, vomiting, pharyngitis, reactive arthritis
Host Range
Humans, cattle,
swine, goats,
sheep, poultry
Humans (esp.
infants), cattle,
swine, goats,
sheep, poultry
Humans, cattle,
swine, horses,
dogs, rats, wild
Mammals, birds,
fish, crustaceans,
and insects
Humans, cattle,
swine, other
Humans, cattle,
swine, poultry,
horses, rodents,
household pets
Humans, swine,
household pets
Humans and
livestock animals
Humans and
livestock animals
Farm and pet
animals, rats and
rodents (urine and
abortion products)
Domestic and wild
mammals, fowl,
and humans
(aborted fetuses of
livestock animals)
Humans, diseased
cattle, swine, and
other mammals
Humans, cattle,
swine, poultry,
horses, rodents,
domestic pets
Primarily swine

         Table 1.    Selected pathogenic zoonoses that may be of concern for water quality near CAFOs (Continued)
Infectious Agent
Balantidium coli

Cryptosporidium parvum

Giardia lamblia

Toxoplasma gondii

Infectious Incubation
dose Period

Unknown, 4-5
may be as low
as 10-100

132 1-12

1-10 3-25
(by ingestion)

Unknown 10-23
(by ingestion)


Balantidiasis, Balantidiosis, Balantidial
Diarrhea, dysentery, abdominal colic, tenesmus,
nausea, vomiting, bloody and mucoid stools
Diarrhea, cramping, abdominal pain, weight
loss, nausea, vomiting, fever
Prolonged symptoms and in some instances
death in immunocompromised host
Giardiasis, Lambliasis, "Beaver Fever"
Diarrhea, abdominal cramps, bloating, fatigue,
weight loss, severe hypothyroidism, lactose
intolerance, chronic joint pain
Mild cases - diarrhea, localized
lymphadenopathy, fever, sore throat, and rash
Severe cases - stillbirths, abortion, newborn
syndrome, hearing and visual loss, mental
retardation, dementia and/or seizures
Host Range

Humans, swine

Humans, small
and large
poultry, fish,
. "I
Humans, wild
and domestic
household pets
Humans, felines,
most warm
blooded animals
and birds


Primarily swine,
also rodents

Humans, cattle,
and other domestic

Humans, wild and
domestic animals

Cats, cattle, swine,
chicken, sheep,
goats, rodents, and

Table 1.   Selected pathogenic zoonoses that may be of concern for water quality near CAFOs (Continued)
Infectious Agent
Schistosoma spp.

Trichinella spiralis

Infectious Incubation
dose Period

Unknown 14-42

Unknown 1-2 days for
2-4 weeks for

Schistosomiasis, Bilharziasis, Snail Fever,
Swimmer's Itch
S. mansoni and S. japonicum -diarrhea,
abdominal pain, and hepatosplenomegaly
S. haematobium - urinary manifestation
including dysuria and hematuria
Chronic infections may lead to liver fibrosis,
portal hypertension, or colorectal malignancy
Trichinelosis, Trichinosis, Trichiniasis
Malaise, nausea, diarrhea, abdominal cramping,
muscular soreness, edema of upper eyelids,
eosinophila, ocular pain, photophobi,
pneumonitis, remittent fever, cardiac and
neurologic complications or death
Host Range

Humans, cattle,
swine, water
buffalo, horses,
rodents, and
household pets

Humans, swine,
household pets,
rodents, wild
mammals, and
marine mammals


Humans, cattle,
swine, water
buffalo, horses,
rodents, household

Swine, household
pets, rodents, wild

Table 1.
Selected pathogenic zoonoses that may be of concern for water quality near CAFOs (Continued)
Infectious Agent
Hepatitis E Virus

Influenza A virus

choriomeningitis virus

SARS Coronavirus

West Nile Virus

Infectious Incubation Disease
dose Period Symptoms

Unknown 14-63 HEV
Jaundice, anorexia, hepatomegaly, abdominal
pain, nausea, vomiting, fever, Liver Failure;
most severe hepatitis during pregnancy of all
hepatitis viruses
2-790 1-4 Flu
Acute fever, chills, headache, myalgia, weakness,
runny nose, sore throat, cough

Unknown 8-21 LCM, Lymphocytic meningitis
Mild influenza-like illness or maningeal or
meningoencephalomyelitic symptoms, Guillain-
Barre type syndrome, orchitis or parotitis.
In more severe cases, temporary or permanent
neurological damage, abortion, congenital
hydrocephalus, and mental retardation
Unknown 6.4 (mean) SARS
High fever, dry cough, dyspnoea, myalgia,
diarrhea, vomiting, death (13.2% for infected
individuals under 60, 43.3% for those over 60)
Unknown 3-14 West Nile Encephalitis, Viral Encephalitis
Sudden onset of flu-like illness, malaise,
anorexia, nausea, vomiting, rash, and
More severe infections can result in aseptic
meningitis or encephalitis, mental status
changes, seizures, coma, severe neurologic
disease, and death (4-11%)
Host Range

Humans, swine,
rodents, chicken

Humans, swine,
horses, domestic
and wild avian

Humans, swine,
household pets,

Humans, swine
chickens, ferrets,
cats, macaques

reptilian, and
avian hosts.
considered dead-
j -i .
end hosts


Unknown -
possibly in swine

Humans, animal
(particularly swine)
are suspected as
sources of new
human subtypes
Rodents, swine,
household pets

Unknown - but
animal reservoir is

Birds are the
amplifying host

3. Antimicrobial Resistance
Antimicrobial agents include all types of natural or synthetic substances capable of killing or
inhibiting the growth of microorganisms. Antimicrobials include antibiotics, antivirals,
antifungals, probiotics, disinfectants, sanitizers, food preservatives, antimicrobial
pesticides/biocides, and wood preservatives among others (Health Canada, 2002). The proper
use of antimicrobial agents is an integral component of good animal agriculture practices.
However, their use may be exacerbated in large confinement facilities where animals are raised
in close quarters and infection in one animal can rapidly spread through hundreds or even
thousands of animals. Many times, infection of one animal leads to the treatment of many
animals within the facility prophytactically (Shea, 2004). Additionally, antimicrobial agents
have long been administered in sub-therapeutic (non-lethal) doses to livestock animals in their
feed, with the ultimate goal of increased animal growth rates. Table 2 lists the antimicrobials
used therapeutically and non-therapeutically (prophylaxis and growth promotion) in livestock
animals in the United States.

The use of antimicrobial compounds in animal feed has increased more than 10-fold since the
1950s, as total U.S. production of antimicrobials increased from approximately 1 million pounds
in 1950 to as much as 44 million pounds in  1986 (Levy, 1992; U.S. Congress, OTA, 1995;
McEwen and Fedorka-Cray, 2002). The rise in agricultural use of antimicrobial agents is
certainly related to changes in their production and availability, improvements in animal health
practices, increasing need for therapeutic use as animals are confined into smaller and more
densely packed housing units, a perceived need for prophylactic use due to close confinement
and increased risk of the spread of disease, and realization of the financial benefits of shortening
the time to reach market weight. According to Dewet et a/., (1997) farmers with large operations
are much more likely than those with small farms to use antibiotics in feed supplements for
growth promotion and prophylaxis. Of the large confinement operations, those working with
veterinary consultants were twice as likely to use such feed additives. In a recent survey of
antimicrobial treatment practices, approximately 83% of feedlots administered at least one
antimicrobial to cattle in feed or water for prophylaxis or growth promotion (Animal and Plant
Health Inspection Service,  1999). Precise figures on the use of antibiotics in animal agriculture
are not available, but Table 3 shows some recent estimates by various sources.  Although
estimates shown in Table 3 vary, three facts remain: the use of antimicrobials in animal
agriculture has increased substantially since the 1950s, copious amounts of antibiotics are used
every year in livestock animals, and most of the antimicrobials used are for growth promotion
and prophylaxis, not for the treatment of sickened animals.
3.1 Mechanisms of bacterial resistance
Each class of antimicrobial compound operates at a specific site within the bacterial cell.
Bacitracin, cephalosporins, penicillins, ionophores, and polymyxins attack cell walls and
membranes.  Aminoglycosides, chloramphenicols, and tetracyclines act on cellular components
responsible for protein synthesis.  Rifamycins, nalidixic acid, and quinolones act upon nucleic
acids, and methotrezate and sulfonamides interrupt important biochemical pathways within the
cell (Khachatourians, 1998).  To combat the action of antimicrobial compounds, bacterial cells
have adapted three primary mechanisms including reducing the accumulation of antimicrobial

Table 2.
              Selected Antimicrobial Agents Approved for Use in Animal Agriculture*
Class and Drug
                                                 Use in Animal Agriculture
                           Animal Species
                                                                     Therapeutic   Non-therapeuticf
                             Analogs Used for
                             Human Therapy ^

Cattle (Beef and Dairy), Horses, Swine, Poultry5
Cattle (Beef and Dairy), Sheep, Swine, Poultry
Beef Cattle, Swine, Poultry
Cattle (Beef and Dairy), Swine, Poultry

Cattle (Beef and Dairy), Horses, Swine
Cattle (Beef and Dairy), Swine




Amikacin, Gentamicin,
Neomycin, Streptomycin

Amoxicillin, Ampicillin,
Amoxicillin-clavulanic acid,
 Cephalosporins (3rd generation)
                          Cattle (Beef and Dairy), Horses,  Swine,
                          Poultry, Sheep
                          Beef Cattle, Poultry
 Lincomycin hydrochloride

                          Swine, Poultry
                          Cattle (Beef and Dairy), Swine, Poultry, Layers
                          Cattle (Beef and Dairy), Swine, Poultry
                          Cattle (Beef and Dairy), Sheep, Swine

Ceftriaxone, Cefixime,
Cefotaxime, Ceftazidime,

Ciprofloxacin, Difloxacin,
Gatifloxacin, Levofloxacin,
Moxifloxacin, Norfloxacin,
Ofloxacin, Trovafloacin-
Nalidixic acid

Clindamycin, Lincomycin

Erythromycin, Azithromycin
       US Congress OTA, 1995; Khachatourians, 1998; US GAO, 1999; NRC, 1999; Mellon et ai, 2001; Shea, 2003; Sayan et al., 2005; USFDA, 2005
       Non-therapeutic uses include prophylaxis and/or growth promotion.
       Antimicrobials used in human medicine that are similar to or the same as antimicrobials used in animal agriculture.
       Poultry = Broilers and/or turkeys; Fowl = Quail, pheasant, duck, and/or geese; Sheep = sheep and/or goats.

Table 2 (cont.) Selected Antimicrobial Agents Approved for Animal Agriculture*
 Class and Drug
                      Use in Animal Agriculture
                           Animal Species
                                           Therapeutic  Non-therapeutic^
                              Used for Human
                              Therapy *
 Cloxacillin sodium
 Penicillin G procaine

 Penicillin G benzathine


Dairy Cattle
Cattle (Beef and Dairy), Horses, Sheep, Swine,
Poultry, Fowl
Beef Cattle, Horses
Cattle (Beef and Dairy), Sheep, Swine, Poultry,

Cattle (Beef and Dairy), Horse, Poultry, Fowl,

Ampicillin sublactam,
Cloxacillin sodium, Penicillin G
benzathine, Penicillin G
potassium, Piperacillin


Tetracycline hydrochloride

Cattle (Beef and Dairy), Swine, Poultry
Cattle (Beef and Dairy), Poultry

Beef Cattle, Swine, Poultry

Cattle (Beef and Dairy), Sheep, Swine, Poultry
Cattle (Beef and Dairy), Sheep, Swine, Poultry,
Fish, Honey bees
Cattle (Beef and Dairy), Horses, Sheep, Swine,





X Quinipristin, Dalfopristin

X Tetracycline hydrochloride,
X Doxycycline


       US Congress OTA, 1995; Khachatourians, 1998; US GAO, 1999; NRC, 1999; Mellon et al, 2001; Shea, 2003; Sayah et al, 2005; USFDA, 2005
       Non-therapeutic uses include prophylaxis and/or growth promotion.
       Antimicrobials used in human medicine that are similar to or the same as antimicrobials used in animal agriculture.
       Poultry = Broilers and/or turkeys; Fowl = Quail, pheasant, duck, and/or geese; Sheep = sheep and/or goats.

Table 3.       Estimates of the use of antimicrobial agents in livestock animal production
 Total Mass Used
Specific Use
 20 million pounds used annually
 18 million pounds used in 1985
 17.8 million pounds used in 1998
 29.5 million pounds used annually
 14.4 million pounds used in 1997
20% for treating disease
80% for growth promotion and
12.2% for treating disease
63.2% for prophylaxis
24.6% for growth promotion
83% for prophylaxis and treating
17% for growth promotion
7% for treating disease
93% for growth promotion and
Not Reported
Swartz, 1989
U.S. Congress, OTA, 1995
Animal Health Institute, 2000
Mellon et al, 2001
Silbergeld, 2004
agents within the cell, attacking and inactivating the antimicrobial compounds enzymatically, or
altering, protecting, or replacing target cellular structures.  Bacteria may gain these resistance
mechanisms in three ways: (1) acquire resistance genes from the DNA of antibiotic producers
and modify them such that they are optimized for resistance to the antimicrobial agent (2) mutate
genes whose products play a role in physiological cell metabolism such that they attack or
inactivate the antimicrobial agent, and/or (3) mutate genes whose products are the target
structures of the antimicrobial compounds such that the target structures become resistant to the
inhibitory effects of the respective antimicrobials (Schwartz and Chaslus-Dancla, 2001).

The initial development of antimicrobial resistance may be relatively slow as single point
mutations that give rise to resistance genes are rare events (10"9 to 10"8 per cell per generation)
(Kelly et al, 1986; Freifelder, 1987; Smith et al, 1999). Once acquired, antimicrobial resistance
traits can be rapidly transferred vertically through division of the host cell, and/or horizontally
between different bacteria (both commensal and pathogenic) via transduction (a bacteriophage-
mediated process), conjugation/mobilization (requiring contact between donor and recipient
cell), or transformation (transfer of free DNA into competent recipient cells). In the mixed
bacterial populations of animal and human skin and mucosa, conjugation and mobilization are
considered to be of primary importance for the spread of resistance genes (Schwartz and
Chaslus-Dancla, 2001) and may occur on the  order of 10"5 to 10"4 per cell per generation
(Summers, 2002). Transduction only  occurs between bacteria  of very similar species and genera
as it is limited by host-specificity of bacteriophages, and therefore plays a lesser role in the
spread of resistance traits in these milieus. Spread of resistance traits via transformation is
considered to be very limited (Bennett, 1995).

The primary genetic elements involved in horizontal gene transfer include plasmids, transposons,
and integrons/gene cassettes. Aside from the antimicrobial-resistance traits,  plasmids and
transposons may also carry genes (such as the tra gene complex) which allow them to move
from one bacterial cell to another via conjugation or mobilization. Plasmids  may serve as
vectors for transposons and integrons/gene cassettes facilitating their horizontal transfer to
competent cells.  Transposons and integrons/gene cassettes can be transferred via transduction

when resistance genes are co-located with prophage genes that are not excised precisely from
chromosomal DNA prior to packing into phage heads.  Small plasmids may also be transferred
via transduction if they are packed into bacteriophage heads instead of phage DNA during phage
assembly (pseudophages), however this process is limited compared to conjugation and
mobilization (Schwartz and Chaslus-Dancla, 2001).  Once established, resistance genes may
persist in commensal bacteria serving as a reservoir for rapid acquisition of antimicrobial
resistance for any new pathogen that may inhabit the intestinal tract (Barza, 2002).  Of particular
interest are enterococci and E. coli that can play a major role in the transmission of mobile
resistance genes (Salyers, 1995).

Antimicrobial-resistance in bacteria  may be conferred by tandem arrays of genetically linked
resistance genes borne by integrons or other transposons that can reside in the chromosome and
on conjugative or mobilizable plasmids (O'Brien et al,  1985; Zhao et a/., 2001; Roe et a/., 2003).
Adaptation of a bacterial cell to any  given antimicrobial via gene transfer can thus result in
selection for resistance to not only that specific agent, but also, by genetic linkage of resistance
genes, to other antimicrobials (Summers, 2002).  Antimicrobial resistance determinants are also
often co-located with virulence determinants on mobile genetic elements. Treatment with
antimicrobials for which resistance is conferred may result in the enrichment of more virulent
bacterial strains in the selective environment. Epidemiological evidence from reported
Salmonella and Campylobacter infections suggest that resistant strains are somewhat  more
virulent than susceptible strains, exhibiting prolonged or more severe illness (Travers and Barza,
2002). In a study of 67 individuals not treated with antimicrobials, diarrhea lasted longer when
the isolates were ciprofloxacin-resistant (12 days) than when they were ciprofloxacin  susceptible
(6 days) (P=0.02) (Marano et al., 2000). The likelihood of hospitalization and average length of
hospital stay are significantly higher in those infected with antimicrobial-resistant organisms
than those with susceptible stains (Lee et al., 1994).

Resistance to one antimicrobial compound may also confer resistance to other antimicrobial
compounds through similarity of the antimicrobial agents (Khachatourians, 1998).  Cases of
multi-drug resistance in bacterial zoonoses caused by structural similarity of human-use
antimicrobials to those used in animal agriculture have been documented. Virginiamycin-
resistant bacterial isolates from turkeys were found to be resistant to the structurally similar and
clinically important human-use drugs quinipristin and dalfopristin (Feinman, 1998; Chadwick
and Goode, 1997). Tylosin-resistant streptococci and staphylococci-resistant animal isolates
were determined to be resistant to the structurally similar and clinically important human-use
drug erythromycin, and were found not only in the livestock animals, but in their caretakers as
well (Feinman, 1998; Chadwick and Goode, 1997). Virginiamycin and Tylosin are both used
prophytactically and/or for growth promotion in beef and dairy cattle, swine, broilers, and
turkeys.  Table 2 lists human-use drugs that are structurally similar to several antimicrobial
compounds used in animal agriculture.

3.2 Antimicrobial resistance in livestock animals
The occurrence of antimicrobial-resistant bacteria tends to be rapid following introduction of
antimicrobial agents into clinical or agricultural use. For instance, occurrence of tetracycline
resistant bacteria was reported in 1956, four years following its introduction to clinical use and
only eight years following its initial discovery.  The time lag between introduction to clinical use
and occurrence of antimicrobial resistant bacteria was  15 years for vancomycin, 4 years for
nalidixic acid, 3 years for gentamicin, 3 years for fluoroquinolones, one year for erythromycin,
and less than one year for streptomycin (Schwartz and Chaslus-Dancla, 2001). Although the
latent period between the introduction of an antimicrobial and the emergence  of resistance may
vary, once the prevalence of resistance in a population reaches a certain level, reversal of the
problem may be extremely difficult (Swartz, 2002). For example, fluoroquinolone-resistant
Campylobacter were detected in 43-96% of market chickens from two producers more than one
year after fluoroquinolones were no longer used in their poultry production (Price et a/., 2005).

Repeated exposure of bacteria to antimicrobial agents and access of bacteria to increasingly large
pools of antimicrobial resistance genes in mixed bacterial populations are the  primary driving
forces for emerging antimicrobial resistance (Schwartz and Chaslus-Dancla, 2001).  Resistance
of both commensal and pathogenic bacteria in livestock animals to antimicrobials  of clinical
importance is now commonplace and is related to their increased use for growth promotion and
prophylaxis over the last 50 years (Shere et al, 1998; Maynard et al., 2003). Hayes et al (2004)
surveyed 541 Enterococcus faecium isolates from 82 farms within a poultry production region in
the eastern United States. Sixty-three percent were resistant to quinipristin-dalfopristin and
52.7% were resistant to four or more antimicrobials. In a study of several swine farms in the
United States, Jackson et al., (2004) determined that Tylosin use for growth promotion resulted
in erythromycin-resistance in  59% of enterococci isolates, compared to 28%  at a farm where
Tylosin was used  for treatment of disease only,  and 2% at a farm that did not use Tylosin.
National surveillance of Salmonella in swine in the U.S.  has revealed resistance to several
important antimicrobials including tetracycline (50%), ampicillin (12%), sulfamethoxazole
(23%), and streptomycin (23%) (NARMS,  1998). Hoyle etal., (2004) studied ampicillin-
resistant E. coli in calves in the United Kingdom and determined that ampicillin resistance
peaked over 80%  within 4 months, steadily declining to less than 10% as the calves aged to 8
months.  Schroeder et al., (2002) tested 752 E. coli isolates from humans and  animals for
resistance to several antimicrobials of clinical importance.  Approximately half of the isolates
displayed resistance to one or more antimicrobials including penicillins, sulfonamides,
cephalosporins, tetracyclines, and Aminoglycosides, with the highest frequencies of
antimicrobial resistance in humans and turkeys and the lowest in non-food animals.  Sayah et al.,
(2005) studied antimicrobial resistance patterns in livestock, companion animals, human septage,
wildlife, farm environments (manure storage facilities, lagoons, and livestock holding areas) and
surface water in the Red Cedar Watershed in Michigan.  E. coli isolates from livestock showed
resistance to the largest number of antimicrobials and multidrug resistance was most common in
swine fecal samples.  Resistance was demonstrated most frequently to tetracycline, cephalothin,
sulfisoxazole, and streptomycin. Similarities in patterns  of resistance in E. coli were observed in
livestock animals  and  environmental samples taken from their respective farms. These authors
suggest that farm  environment samples may best describe potential contamination of nearby
waters with antimicrobial-resistant bacteria.

3.3 Risk to public health
Much concern surrounds the elevated use of antimicrobial agents in confinement facilities and, in
particular, the use of antimicrobial agents at non-therapeutic doses in animal agriculture
(American Academy of Microbiology, 1999, Mellon et al., 2001). The use of antimicrobial
agents inevitably selects for resistance of both commensal and pathogenic microorganisms
exposed to the agents (Linton et al, 1975; Dawson et al., 1984; Levy et al, 1976; Dunlop et al,
1998;Endtzera/., 1991; Jacob-Rietsmae^a/., 1994; Eager et al., 1997'; Low et al., 1997; Tauxe,
1997; Gynn et al., 1998; McEwen and Fadorka-Cray, 2002; Vasil' et al., 2002). The conditions
of widespread, prolonged exposure to antimicrobial compounds at sublethal doses with little
dose control in CAFOs may exacerbate their development. Once established, the movement of
antimicrobial-resistant microorganisms from animal to animal or animal to animal care worker
may be facilitated by the crowding of animals into confinements, often with suboptimal hygiene.
The co-colonization of animal gastrointestinal tracts by antimicrobial-resistant commensal
bacteria and bacterial pathogens may lead to further development of antimicrobial-resistant
bacterial zoonoses (Kruse et al., 1999).  As much as 75-80% of an antibiotic may pass
undigested through an animal, thus its waste may not only harbor high concentrations of
antimicrobial-resistant bacteria, but also their resistance genes and raw (undigested)
antimicrobial compounds (Campagnolo and Rubin,  1998). This waste is often stored in open air
lagoons and/or spread on fields where these compounds, resistant organisms, and antimicrobial-
resistance gene reservoirs may move into the environment via aerosolization, infiltration into the
groundwater, or runoff into surface water resources.

Antimicrobial  resistance in zoonotic pathogens is a  serious threat to human health (Ghidan et al.,
2000; Cheng et al, 2002; Travers and Barza, 2002). Many of the drugs are used in animal
agriculture and human medicine are the same or very similar including, but not limited to, beta-
lactams (penicillin, ampicillin, cloxacillin), tetracyclines, sulfonamides and potentiated
sulfonamides,  cephalosporins, and fluoroquinolones (McEwen and Fadorka-Cray, 2002).
Exposure to zoonotic pathogens harboring resistance to antimicrobials of clinical importance
may lead to diseases with few or no treatment options in humans. In cases where pathogens are
resistant to administered antimicrobial compounds,  vulnerability to infection can increase up to
three-fold, primarily resulting from a transient decrease in an individual's  resistance to
colonization by the pathogen  (Barza and Travers, 2002).  Antimicrobial-resistant pathogens tend
to be more virulent than their susceptible counterparts, causing more prolonged or severe
illnesses (Marano et al, 2000; Travers and Barza, 2002; Swartz et al, 2002).  There is
circumstantial  evidence that increased prevalence of antimicrobial resistance in human isolates
may be linked  to the use of antimicrobial agents in animal agriculture (Levy et al, 1976; Jensen
etal, 1998; Swartz etal, 2002; Silbergeld, 2004).  Many cases  of severe  human disease caused
by acquisition  of antimicrobial-resistant zoonotic pathogens from animal agriculture have been
documented (Levy etal, 1978; Schlech etal, 1983; Holmberg etal, 1984; Morgan etal,  1988;
Bessere^a/., 1993; Cieslak et al, 1993; Isaacson etal, 1993; LeeetaL, 1994; MacKenzie etal.,
1994; Millard et al, 1994; Tschape et al., 1995; Centers for Disease Control and Prevention,
1998; Jackson  etal., 1998; Crampin etal., 1999; Huovinen, 1999; Wegner etal., 1999; Franklin,
1999; Kruse, 1999; Health Canada, 2000; Licensee^/., 2001; Clark et al, 2003).

4. Survival of Pathogens in the Environment
Pathogens at concentrated animal feeding operations may be present in animal wastes, water
used for maintenance of livestock and animal housing units, soils where animal manures and
wastewaters are spread, on crops grown in soils where manures were applied or where
contaminated irrigation waters are used, and groundwater and surface waters contaminated by
manure runoff.  The survival of pathogenic organisms in the environment varies widely
depending on the pathogen, environmental conditions, and the chemical, physical, and biological
composition of milieu of interest. Enteric bacterial, viral, and protozoan pathogen inactivation in
soil, water, crops, or manure may be affected by predation, competition, water stress/osmotic
potential, temperature, UV radiation, pH, inorganic ammonia, and organic nutrients (Geldreich et
al., 1968; Davenport et al., 1976; Crane and Moore, 1986; Hurst et al., 1989; Davies andEvison,
1991; Olson et al., 1999; Sattar et al., 1999; Burkhardt et al., 2000; Davies-Colley et al., 2000;
Wait and Sobsey, 2001; Jamieson etal., 2002; Ferguson et al., 2003). The importance of each
factor is strongly related to the milieu of interest. In general, the survival of pathogens is
inversely related to predation, competition, temperature, UV radiation, water stress, and
inorganic ammonia, except for Cryptosporidium oocysts and Giardia cysts, which have low
survival at sub-zero (<-20°C) temperatures (Van Donsel et al., 1967; Zibilske and Weaver, 1978;
Reddy et al., 1981; Jamieson et al., 2002; Ferguson et al., 2003). The relationship of pathogen
survival to pH and organic nutrients may be more complex. Under the right conditions,
pathogens are capable of surviving in the environment for days to more than a year.
4.1 Manure and manure slurries
Table 4 summarizes the survival of bacterial and parasitic pathogens noted in literature in
manures and manure slurries. These nutrient rich environments may offer protection from
environmental insults such as solar UV radiation, desiccation, and temperature fluctuations,
promoting survival or even regrowth of pathogenic zoonoses. For instance, Muirhead et al.,
(2005) determined that within cowpats, E. coll grew for 6 to 14 days instead of following a
traditional logarithmic die off curve and Olson (2003) noted that the eggs of Ascaris suum, a
common parasite in swine, are highly resistant to inactivation in feces, potentially remaining
infectious for years. However, these environments may also me hostile, as they may harbor
predators and competitors, or produce toxic components that may reduce pathogen viability.  For
instance, inorganic ammonia, naturally produced by hydrolysis of urea and in decomposing
manure, can be biocidal at high  concentrations, and has been exhibited to be directly proportional
to Cryptosporidium oocyst inactivation (Jenkins et al., 1998; Jenkins et al, 1999). As seen in
Table 4, animal manures and manure  slurries may remain significant reservoirs for
environmental contamination by zoonotic pathogens for many months.

Bacterial  pathogens may persist for long periods  in animal manures under typical environmental
conditions. This may be exacerbated  when the temperatures are low, moisture remains optimal,
and aeration is not used. For instance, Salmonella and E. coll O157:H7 have been noted to
survive for 4-6 months in animal manures and manure slurries kept at  1-9°C, up to 49 times
longer than at 40-60°C.  Nicholson et al, (2002)  studied the survival ofE. coli O157:H7,

Table 4.
Survival of pathogenic zoonoses in livestock manures and manure slurries

Survival1 (days)
(°C) Bacterial Pathogens *

Broiler Litter
Cattle, beef or dairy


Manure slurries
Cattle, beef or dairy


-20 to -4

-20 to -4
On farm (5-20)
Salmonella sp.






Campylobacter sp.







Yersinia enterocolitica E. co//O157:H7 Listeria sp.

4 8
>365 >100
100 130*
30 49
8 4
32 4

93 185

Paras ites§
Giardia Cryptosporidium

<1 >365
7 56

7 28
7 28

        Longest survival time reported
        Bolton et al., (1999); Kudva et al, (1998); Wang et al., (1996); Himathongkham et al., (1999); Mitscherlich and Marth (1984); Guan and Holley (2003);
        Olson (2003); Tauxe (1997); Plym-Forshell (1993); Nicholson et al., (2002)
        Cole et al., (1999); Robertson et al., (1992); Payer et al., (1998); Olson (2003); Olson et al., (1999)
        Calculated as 7 times the reported decimal reduction time (time required for 1-log reduction in pathogen concentration) assuming logarithmic die-off
        and based on a reported initial inocula of 106-108 organisms per gram manure or milliliter slurry.

Salmonella, Listeria, and Campylobacter in dairy cattle, swine, and poultry manures stored at
40-60°C and determined that aeration of the solid manures decreased survival times for E. coli
O157:H7 and Salmonella by as much as 88%.  These researchers noted a decrease in the survival
of E. coli O157:H7 and Salmonella sp. when a higher dry matter content was maintained in the
slurry. Kudva et al, (1998) noted similar changes in E. coli O157:H7 in sheep manure, which
survived for 630 days at temperatures below 23°C when not aerated versus 120 days when
aerated, the difference likely due to drying of the aerated manure.

Parasitic protozoan survival in animal manures may also be related to temperature, but the trends
are not as strong as those reported for bacterial pathogens. This is likely due to their ability to
form cysts and oocysts for protection from environmental pressures under the range of
temperatures reported in Table 4.  These parasites have been shown to be susceptible to
temperature extremes, with reported survival of Cryptosporidium oocysts ranging from 1 hour at
-70°C, 1 day at -20°C, one  or more years at 4°C, 3-4 months at 25°C, 1-2 weeks at 35°C, and just
minutes at 64°C (Payer and Nerad, 1996; Finstein, 2004). Cryptosporidium oocysts in manures
may also be susceptible to  desiccation and bacterial degradation whereby warmer temperatures
may accelerate the degradation process. A similar pattern exists for Giardia cysts, but they are
inactivated more rapidly than Cryptosporidium oocysts and are less resistant to temperature

Information regarding the survival of zoonotic viruses in animal wastes is sparse.  Although not
shown in Table 4, zoonotic viruses in animal manures and manure slurries may exhibit long
inactivation times that extend for weeks to months. Karetnyi et al, (1999) determined that swine
hepatitis E was detectable in positive stool samples for more than 2 weeks, regardless of whether
the samples were maintained at -85°C, 4°C, or room temperature. Pesaro et al (1995) studied the
survival of several viruses  including picnoraviruses, rotaviruses, parvoviruses, adenoviruses, and
herpes viruses as well as the coliphage f2 in nonaerated liquid and semisolid animal wastes.
Ninety percent reduction in virus titer ranged from  less than 1 week for herpes virus to more than
6 months for rotavirus, suggesting that a 4-logio reduction in viruses may require storage for as
much as two years for some pathogens. Although little information exists regarding the survival
of viral pathogens in fecal  environments, these studies show that under non-aerated conditions
viruses may exhibit prolonged persistence in manure and manure slurries, suggesting a strong
potential for viral pathogen contamination when manure is spread on land.

In general, pathogen survival in animal manures is  dictated by the effects of aeration and
temperature, whereby increased aeration and higher temperatures lead to more rapid die-off.  Of
the pathogens listed in Table 4, E.  coli O157:H7, Listeria sp., and Salmonella sp. were the most
persistent in manure and manure slurries regardless of the temperature. However, considering
the work of Pesaro et al., (1995), viral pathogens may persist much longer than the bacterial
pathogens,  and should be given more consideration in future studies.

Much of the work to date has concentrated on the survival of pathogens  in cattle manures and
manure slurries.  However, based on the summary presented in Table 4,  there seems to be
dissimilarities in the survival of pathogens in different animal feces.  This may be due to
differences in the physical, chemical, or biological  properties of the various animal manures, but
could also be a result of the low numbers of studies on swine and poultry manures versus those

of cattle. The lack of studies on pathogen survival in swine and poultry manures impedes the
development of safe management practices.

The survival of pathogens in animal manures and manure slurries is typically studied under
controlled laboratory conditions. Kudva et a/., (1998) noted that survival of pathogens in
laboratory studies were generally lower than those observed in field studies. For instance, these
researchers determined thatE1. coli O157:H7 survived in sheep manure for lOOd under a
controlled (4-10°C) laboratory setting versus 630 days when exposed to environmental (ambient)
conditions (<23°C). Based on their observations, laboratory experiments may not provide a
reasonable estimate of pathogen survival in on-farm conditions.  Future efforts should
concentrate on measuring the survival of pathogens in-situ.
4.2 Natural Waters
Manure runoff and wastewaters from concentrated animal feeding operations may contain
pathogenic zoonoses and antimicrobial-resistant bacteria that can survive and proliferate in
nearby natural waters. Runoff and wastewater discharges may also contribute both organic and
inorganic nutrients that may encourage the growth and proliferation of indigenous or introduced
pathogens (Grimes etal., 1986).  Table 5 summarizes the survival of bacterial and parasitic
pathogens in dirty waters from livestock operations, natural waters, and drinking water as
reported in literature. In these milieus, UV radiation, disinfectants, temperature, predators, and
toxin producers generally challenge the survival of pathogenic zoonoses and antimicrobial-
resistant bacteria (Chao etal., 1988;  Johnson et a/., 1997).

The survival of bacteria in natural waters may be longer than exhibited in manures or manure
slurries.  Yersinia enterocolitica exhibited the greatest survival among all bacterial pathogens
considered in Table 5 whereas Campylobacter was the least. However, in a viable but not
cultivable state, Campylobacter may survive for as much as 120 days at 4°C.  E. coli O157:H7
has also been noted to enter a viable  but not cultivable state in water increasing the survival time
over that reported in Table 5 (Wang  and Doyle, 1998).  Pathogens may also settle into streambed
sediments, decreasing exposure to UV radiation and predators and increasing survival times over
those reported in Table 5.  For instance, Anderson et a/., (2005) determined that a 90% reduction
in fecal coliforms in fresh waters required 4.2 days, whereas 50 days was required to achieve the
same reduction in the underlying sediments. Although differences in the survival of
Enterococcus spp. was observed, the trend was the same (1.4 days in water and 4.5 days in the
underlying sediments), and held true for salt water environments.

Cryptosporidium oocysts may also be especially resistant in environmental waters,  surviving for
more than a year in optimal (low temperature)  conditions.  For instance, Robertson et a/., (1992)
studied oocyst infectivity during incubation in  cold river water and reported up to 66% viability
at 33 days and 11% viability at 176 days.  In another study, Medema et a/., (1997) determined
that the time required for one-log reduction in Cryptosporidium oocyst infectivity in river water
at 15°C was 40-160d, whereas at 5°C it was  lOOd.  Even more extreme are viruses, which can
persist for several years in the subsurface. Azadpour-Keeley et a/., (2003) reviewed the
movement and longevity of viruses in the subsurface and suggested that soil environments may
actually enhance viral survival.  They report a wide variation in inactivation rates in different

          Table 5.
Survival of pathogenic zoonoses in soils, contaminated water-irrigated soils, and manure-amended soils
Survival1 (days)

(°C) Bacterial Pathogens *


-20 to -4
Salmonella sp.

Campylobacter sp.

Yersinia enterocolitica E. co//O157:H7

>365 >300
>365 100
10 >56
Listeria sp.

Paras ites§
Giardia Cryptosporidium

<1 >365
49 56
14 28
Dirty Water-Irrigated Soils

Farm-yard manure-amended soil
Poultry litter
Broilers & layers






Manure slurry-amended soil






                  Longest survival time reported
                  Mubiru et al., (2000); Mitscherlich and Marth (1984); Zibilske and Weaver (1978); Quo et al., (2002); Chao et al., (1988); Guan and Holley (2003);
                  Olson (2003); Ciesak et al., (1993); Nicholson et al., (2002); Hutchinson et al., (2004); Hutchinson et al., (2005)
                  Cole etal, (1999); Robertson et al., (1992); Payer et al., (1998); Olson (2003); Olson et al., (1999)
                  Dirty water from livestock operations.

soils at near-neutral pH suggesting it may take as little as 0.8 days to as many as 11 years to
achieve 99.99% (4-logio) die off of some viruses in aquifers. Keswick et al., (1982) report that
survival of enteric bacteria and viruses were longer in groundwater than surface water,
presumably due to lower temperatures and protection from sunlight and microbial antagonism.

Maintenance of antimicrobial-resistance in natural waters has not been studied extensively.  In
untreated seawater suspensions, Guardabassi and Dalsgaard (2002) noted that multiple antibiotic
resistant E. coll and Citrobacter freundii  survived and maintained their multiple resistance
properties for more than 30 days, whereas a multi-antibiotic resistant Acinetobacter johnsonii
survived and maintained its multiple resistance properties for 14 days. In untreated pond water
suspensions, these authors noted survival times of 21 days (E. coli and A. johnsonii) and 28 days
(C. freundii), while maintaining multiple resistance properties.  This suggests that antimicrobial
resistant microorganisms may survive for long periods upon discharge to aquatic environments
and that stress and nutrient depletion may not affect the stability of their resistance  phenotypes.
The effect of low concentrations of antimicrobial compounds discharging to surface or ground
waters via manure runoff, lagoon leakage, or wastewater discharge on the maintenance of
antimicrobial-resistant phenotypes or genotypes has not been studied.
4.3 Manure-amended soil
Table 6 summarizes the survival of bacterial and protozoan pathogens in soils. The survival
times reported in Table 6 are more similar to those of manures and manure slurries and less than
those exhibited in water. In general, it has been reported that survival of pathogens in soil
increases when manures are incorporated into soils rather than unincorporated. For instance,
Hutchison et al (2004) studied the die off of Salmonella, Listeria, Campylobacter, and E. coli
O157 following application of manure to soil and incorporation of the manure upon application,
one week following application, or no incorporation. The authors noted that die-off was similar
in summer and winter months, but more rapid when  the manure was not incorporated into the
soil. The increased survival of pathogens incorporated into soils may be related to decreased
exposure to UV radiation, temperature extremes, and desiccation and increased availability of
nutrients.  However, soils may harbor competitor organisms and predators that can reduce
pathogen survival. Survival of pathogenic bacteria in soils may also be limited by low soil pH
(Jamieson et al., 2002)  or freeze-thaw cycling. Jenkins et al., (1999) determined that
Cryptosporidium oocyst infectivity decreased from greater than 50% to less than 1% when
exposed to freeze-thaw cycles in a soil environment. Walker et al (2001) noted that inactivation
of Cryptosporidium oocysts during freeze-thaw cycling or heating was enhanced by increased
osmotic stress (decreased water potential).

The most important factor affecting the survival of enteric pathogens in  soils systems may be the
moisture status, which is influenced not only by precipitation, but also by moisture retaining
properties such as particle size distribution and organic matter content (Gerba et al., 1975; Tate
etal, 1978;  Kibbey etal, 1978; Chandler and Craven, 1980; Crane etal, 1981;  Reddy etal,
1981; Faust, 1982; Mubiru etal, 2000, Entry etal,  2000b; Jamieson etal, 2002). For instance,
Nicholson et al, (2002) studied the survival of bacterial pathogens following land spreading and
determined that there are some indications that pathogen survival is longer in clay loam
grassland soil than in sandy arable soil. Burton et al (1983) determined that Salmonella newport

          Table 6.       Survival of pathogenic zoonoses in drinking water, livestock rinse waters, surface fresh waters, surface salt waters, surface water
                         sediments, soils irrigated with livestock rinse waters, and ground waters
           Environment     Temperature                                            Survival (days)
                                                     Bacterial Pathogens:


Ground or Spring


Dirty water tt

-20 to -4
-20 to -4
Salmonella sp.





Campylobacter sp.




Yersinia enterocolitica E. coli O157:H7 Listeriasp.

90 90


>365 >300
>365 >300

10 84
16 93
Giardia Cryptosporidium


<7 >365
77 >365

14 70

Longest survival time reported
Wang and Doyle (1998); Bolton et al, (1999); Santo Domingo et al, (2000); Mitscherlich and Marth (1984); Karapinar and Gonul (1991); Chao et al,
(1988); Buswell et al, (1998); Rollins and Colwell (1986); Blaser et al, (1980); Guan and Holley (2003); Olson (2003); Payer et al, (1998); Kenneth et
al, (1998); Ford, 1999; Nicholson et al, (2002)
Cole et al, (1999); Olson (2003); Robertson et al, (1992); Payer et al, (1998); Olson et al, (1999)
In the presence of a biofilm, survival was as much as 29 days at 4°C and 11 days at 30°C
Survival may be more than 120 days in a viable but not cultivable (VBNC) state
Dirty water from livestock operations

survived longer in soils with higher clay content, potentially owing to a higher concentration of
organic matter and nutrients. Mubiro etal., (2000) suggested that survival of E. coli O157:H7
may also be enhanced in soils of higher matric potential not only due to enhanced water holding
capabilities, but also because these soils better retained nutrients.  The addition of manure to the
soils may enhance survival of pathogens such as Campylobacter spp. or E. coli O157:H7,
possibly due to increased organic and inorganic nutrient availability (Gagliardi and Karns, 2000).

The effects of water/osmotic potential on microbial stress in soil environments may be
exacerbated by specific properties of the pathogen of interest.  Bacteria and viruses with a
hydrophobic envelope tend to accumulate at the air water interface leading to increased
inactivation (Johnson and Gregory,  1993; Thompson etal., 1998; Thompson and Yates, 1999).
The lack of a hydrophobic envelope may reduce attraction to the air-water interface, and thus
may afford some protection from viral inactivation due to osmotic stress (Ferguson et a/.,  2003).
In soils, osmotic stress typically increases near the soil surface, and may lead to reduced
pathogen survival (Gerba, 1999).

Even when not incorporated into soils, the survival of pathogens following application of
manures to land may be lengthy.  Hutchinson et al., (2005) determined decimal  reduction times
(the time required for 1-logio reduction) for E. coli O157:H7, Listeria monocytogenes,
Salmonella spp., and C.jejuni of 1.31 - 3.20 days (mean) and  Cryptosporidiumparvum oocysts
of 8-31  days following the application of livestock waste onto fescue plots (no incorporation).
Most zoonotic agents declined below detectible levels by 64 days, except for L.  monocytogenes,
which persisted for up to 128 days in some plots. Potential mechanisms for pathogen reduction
may have included, among others, desiccation, UV radiation, and runoff from the grasslands to
nearby receiving waters.

Where food crops are grown in manured soils or using contaminated irrigation waters, pathogens
can contaminate produce surfaces.  The level  and persistence of contamination may be related to
the irrigation method (spray irrigation or surface irrigation) and time of contact of produce with
contaminated soils. For instance, Ingham etal., (2004) identified E. coli contamination on
carrots, lettuce, and radishes up to 120 days following application of non-composted bovine
manure as a fertilizer in fields in Wisconsin. Following growth in E. coli O157:H7 contaminated
manure-fertilized soil; Johannessen et al., (2005) detected E. coli O157:H7 on the stems, but not
on the edible parts of lettuce. Solomon et a/., (2002) noted that spray irrigation  following a
single exposure to E. coli O157:H7  resulted in 90% of the lettuce being contaminated with E.
coli O157:H7 and the contamination persisted for more than 20 days in 82% of the plants.
Where surface irrigation was used under the same circumstances, only 19% of the lettuce was
contaminated. Immersion of harvested lettuce heads in 200ppm chlorine solution for 1 minute
did not eliminate all E. coli O157:H7 cells from infected lettuce, regardless of irrigation method.
Guo etal., (2002) investigated water and soil  as reservoirs of Salmonella for contaminating
mature green tomatoes, and determined that the population of Salmonella on tomatoes in contact
with contaminated soil increased over 4 days by 2.5 logio  CFU per tomato during storage at
20°C, and remained constant for an  additional 10 days. In contrast, where tomatoes were not in
contact with soil, but Salmonella were inoculated onto the fruit surface, the number of cells
declined over 14 days by 4 logio CFU per tomato when held at 20°C. At day one, Salmonella
was associated with the skin surface. As time of storage increased, more Salmonella cells were

associated with less accessible stem scar and subsurface areas of the tomatoes, which may render
the fruits more resistant to disinfection with sanitizing agents.
4.4 Discussion
Relatively few studies are available describing the survival of pathogenic zoonoses in
environmental milieu, especially considering the broad range of properties of soils, manures, and
waters that may potentially be contaminated. Much of the emphasis has been placed on cattle
manures, manure-amended soils, and surface waters, with less emphasis on ground waters and
manures from other livestock animals such as swine and poultry.  In general, pathogenic
zoonoses tend to survive longer cooler rather than warmer temperatures and in water rather than
in manures or soils. This may be problematic as manures and soils are stationary whereas water
is a significant transport medium for pathogens.  Further, very few studies have been reported on
the survival of viruses which is troubling because the relatively few studies that are available
suggest that viruses are more persistent than bacteria and parasitic protozoa and can travel vast
distances in both surface and ground waters. A significant limitation is the lack of information
regarding the survival of antimicrobial-resistant bacteria in various milieus including the
persistence of phenotypic and genotypic antimicrobial-resistance traits. Most studies reported in
Tables 4-6 were carried out in the laboratory instead ofin-situ, and only a few examined more
than one environmental stressor simultaneously. The combined effects of multiple stressors in
the natural environment or presence of additional growth and maintenance factors may limit or
enhance pathogen survival in reference to lab-scale studies of single stressors (Crane and Moore,
1986; Robertson et al, 1992; Kudva etal., 1998; Jenkins etal, 1999; Friere-Santos etal, 2000;
Walker etal, 2001).

Current methods for detecting pathogens in environmental systems may limit the ability to
determine accurate survival times in difficult milieu.  Specific soil or manure properties or
survival strategies of the various pathogens may limit their detection with cultivation techniques.
For instance, upon being stressed, bacteria may die or adapt using a number of mechanisms
including formation of spores, formation of ultramicrobacteria, or entering viable but not
cultivable (VBNC) states. Many of the bacterial pathogens can survive for much longer periods
of time than indicated in Tables 4-6 in VBNC states (Wang and Doyle, 1998; Santo Domingo et
a/., 2000; Rollins and Colwell, 1986).  Better and more sensitive methods for pathogen detection
in different media need to be developed to determine more accurately pathogen survival.
Accurate information regarding the survival of pathogenic zoonoses and antimicrobial resistant
bacteria is necessary  for modeling their fate and transport from confined animal feeding
operations.  Based on available information, ensuring the safety of food crops and water
resources may require management practices that eliminate pathogens in manures and other
CAFO wastes prior to land application or discharge to natural waters.

5. Pathogen  Movement - An Ecological Perspective
Figure 2 provides a partial picture of the potential routes of transmission of zoonotic pathogens
from confined livestock animals to humans and the environment. The movement of pathogens
onto, within, and off farms is a complex ecological issue owing to the continuous exchange of
microbes between human and animal hosts and environmental reservoirs (Sobsey et al., 2002;
Summers, 2002). For instance, Herriott et al., (1996) tested twelve herds and their feeds and
water troughs as well as co-located (non-bovine) livestock, companion animals, wild birds,
rodents and flies at dairies and feedlots in Idaho,  Oregon, and Washington for the presence of E.
coli O157:H7.  E. coll O157:H7-positive cattle were identified in all 12 herds with a prevalence
of 1.1-4.4% in dairies and 1.5-6.1% in feedlots. It was also detected in 1.3% of trough water
samples, 2.0% of trough water biofilm samples, in a nearby horse, two dogs, pooled bird
droppings, and composite fly samples.  Considering antimicrobial resistance, the issue becomes
more complicated as mobile genetic elements conferring resistance provide a distinct selective
advantage in stressed environments such as the colonic tract of humans and animals being treated
with antimicrobials. In these environments, proliferation of resistance traits among bacteria can
be rapid and have lasting effects (O'Brien, 2002; Summers, 2002).  Addressing the movement  of
pathogens between intensive livestock operations and the environment will require
understanding of the ecological principle that everything is connected to everything else. The
following is a discussion of some of the potential pathways for movement of zoonotic pathogens
from livestock animals raised in confinement to humans and the environment.
5.1 CAFOs and Abattoirs
The presence of zoonotic pathogens in CAFO environments may begin with the stocking of
infected animals or with the use of selected feed products on the farm.  Animal feeds and
drinking water containing antimicrobial compounds may lead to the development and persistence
of resistant bacterial zoonoses in livestock animals which may proliferate through the farm
environment. Animal feeds can also be a direct source of zoonotic pathogens and antimicrobial-
resistant bacteria for livestock animals (Curtain, 1984; Durand et a/., 1990; Izat and Waldroup,
1990; Gabis, 1991; Veldman etal,  1995; Davies and Wray, 1997; Primm, 1998; Shirota etal,
2001a,b).  For instance, often feed ingredient piles at 12 commodity dairy feeding farms, Kidd et
al., (1999) identified two feeds contaminated with Salmonella enteritidis.  Sixty two percent of
Enterobactedaceae isolates from the ten piles were ampicillin-resistant and 10% were
tetracycline-resistant.  Although feed can be contaminated on-farm, it may also arrive
contaminated, as shown in a recent  survey of 629 feed samples from 3 feed mills where 8.8% of
feed mash samples and 4.2% of pelleted feed samples were positive for Salmonella (Jones and
Richardson, 2004). Antimicrobial-resistant bacteria and other pathogens can also be present in
trough waters (Marshall et al., 1990; Herri ott et al, 2002; Kemp et al, 2005) potentially
resulting from either stocking troughs with contaminated water or through deposition of
contaminated material into the water from an animal harboring the disease (via the saliva,
mucosa, or feces). Antimicrobial compounds in the water and the presence of biofilms in which
bacteria are in close contact may lead to proliferation of antimicrobial resistance within these
microbial communities. The confinement of animals into dense units where trough waters are
shared and where animals have increased contact with each other and their fecal matter may
exacerbate the spread of pathogens from animal to animal.

                                                                                                                       Migratory Birds
                                                                                                    Land application   .•
                                                                                                      afmanure  Exposure to
                                                                                                                animal manure
                                 Water Treatment Plant
                          Treatment Plant
,         Recreational
 '•„        Water           "Contaminated
 Drinking       \         /Produce  r-
                                                                                                                        -1    gm*	      t
                                                                                                                  *w-O      •-$
                Airborne pathogens


                                                       Exposure to
                                                       animal manure
                                                                                            Wild Animals
                                                                         1  4e^\
 Contaminated   **..          house air and
. groundwater  Exposure to    airborne pathogens
 (well water)    animal manure  from other farm
 (trough water)         *•„     operations
                              Waste water
                                                                                                            Insect, bird, and
                                                                                                            rodent vectors
                                                                                                                           Animal Feed
*Sickened  ^ '\
 Individuals *   *.
                                           T!^  ct.
                                              Predatory and
                                              animals  T--.
                                                                                               Slaughterers, tnei
                                                                                               families, and pets
                                                                                     * Livestock
                                                                          .**      Cv Animals
                                                                         «         V1
                                                                                                                      Direct contact with live and/or
                                                                                                                      dead livestock animals
                                                                                                                                        Farm workers, theicf*
                                                                                                                                        families, and pets
            Figure 2.        Movement of pathogens - an ecological perspective

Standing trough waters and animal feeds laden with antimicrobial compounds and pathogenic
zoonoses as well as unsanitary conditions and poor manure management practices pose other
problems for controlling the spread of disease. Animal and insect vectors may be attracted to
feed piles, trough waters, fecal matter, manure treatment lagoons, treatment wetlands, or the
dense animal populations present in CAFOs resulting in movement of pathogens on and between
farms as well as off of farms and into human populations.  Several studies have supported the
movement of pathogens and antimicrobial resistant bacteria through animal vectoring. In a
longitudinal study on Wisconsin farms,  Shere et al., (1998) reported that the use of
antimicrobials subtherapeutically in animal feeds or trough waters and therapeutically for
treatment of diarrhea correlated  well to the emergence of antimicrobial-resistant E.  coli
O157:H7, which may have been transmitted through birds eating the animal feed and drinking
contaminated trough waters. Nielsen et al (2004) screened 446 fecal samples at eight Danish
cattle and swine farms and detected stxl and stx2 genes in production animals, wild birds, and
rodents suggesting transmission between the livestock animals and vectors.  Halos et al., (2004)
detected the Bartonella citrate synthase  gene in Hippoboscidae flies on wild roe deer, cattle,
horses, and sheep in France suggesting that these flies may act as a vector for transmission of
Bartonella between wild and domestic ruminants. Waldenstrom et  al (2005) observed
antimicrobial resistant Campylobacter jejuni in wild thrushes, shorebirds, and raptors in Sweden
suggesting the spread of antimicrobial-resistant pathogens to wild birds. Raptors had the highest
prevalence of antimicrobial-resistant strains, potentially from predation on infected animal
vectors. On 12 diary and beef feedlots in Idaho, Oregon, and Washington, Herriott etal, (2002)
identified E. coli O157:H7 in composite fly samples and pooled bird droppings. Marshall et al.,
(1990) inoculated pigs with an antimicrobial-resistant strain of E. coli and within a four month
period was able to isolate the same strain from trough water,  bedding materials, mice, flies, and a
human caretaker.

Other studies may point to broader ecological implications of animal vectoring in the
environment. Cole et al (2005)  compared free-living Canadian geese in Craven county, Georgia
that were using swine waste lagoons and surface waters adjacent to farm fields to Canadian
geese in Griffin, Georgia, where there were crop fields, but no nearby animal production
facilities.  The proportion of E. coli isolates resistant to antimicrobial agents was significantly
greater (p=0.0004) among Craven county geese (72%), where interaction with swine waste
lagoons was observed, than in Griffin geese (19%).  These researchers proposed that Canadian
Geese may be acting as vectors for antimicrobial-resistance and resistance genes in agricultural
animal-production  environments. Their findings suggest the spread of pathogens and
antimicrobial resistant bacteria from livestock operations may be vast considering potential
migration of Canadian geese over hundreds of kilometers.

Other factors unique to concentrated animal feeding operations may encourage the  spread of
disease  on farms.  Several researchers have detected high levels of airborne bacteria (2xl03 -
8xl05 CFU/m3) in confinement house air including antimicrobial-resistant bacteria and other
zoonotic pathogens such as Enterococcus, Staphylococcus, Pseudomonas, Bacillus, Listeria,
Salmonella, Campylobacter, andE. coli (Cormier et al., 1990; Cazwala etal, 1990; Crooked
al., 1991; Heederick et al., 1991; Predicala et al., 2002). In houses of experimentally infected
broiler chickens, Gast et al (2004) were  able to detect Salmonella spp. in the air for four weeks
post-infection, even when the litter was  cleaned from the floors weekly. In a study of three

mechanically ventilated swine CAFOs, Zahn et al., (2001) detected tylosin resistance in 80% of
cultivable airborne bacteria.  Chapin et al., (2005) isolated 137 Enterococcus and staphylococci
from the air within a concentrated swine feeding operation and screened them for resistance to
erythromycin, clindamycin, virginiamycin, tetracycline, and vancomycin. 88% of the isolates
expressed high-level resistance to at least two antibiotics and 84% to at least three antibiotics
commonly used in swine production, but none were resistant to vancomycin,  an antibiotic that
has never been approved for use in livestock in the United States. Thirty seven percent of the
isolates were resistant to virginiamycin, an analog to quinipristin-dalfopristin which is a drug of
last resort for multidrug-resistant gram-positive infections characterized by glycopeptide-
resistant E. facium and coagulase-negative staphylococci.

These findings have significant implications for the health of livestock animals, animal care
workers, their families, and casual farm visitors, and to a lesser extent to nearby communities
that may be susceptible to secondary infections via exposure to sickened animal care workers
and their families.  Chapin et al., (2005) proposed a scenario by which airborne pathogenic
zoonoses resistant to clinically important antimicrobials may spread from confined livestock
animals to the public through exposure to  sickened animal care workers and their families.

        "Bacteria resistant to virginiamycin are often cross-resistant to quinipristin-dalfopristin,
       and a previous study has shown that transfer of streptogramin-resistant Enterococcus
       can occur between animals and humans in the livestock environment (Jensen et al,
       1998)... Inhalation of air contaminated with multidrug resistant Enterococcus or
       streptococci could lead to colonization of both the nasal passages (Aubry-Damon, 2004)
       and the lungs of swine CAFO workers, potentially making the workers themselves
       reservoirs of antibiotic-resistant organisms. Co-exposures to other aerosols and gases in
       the swine environment such as organic dusts, molds, and ammonia have been shown to
       induce symptoms associated with chronic bronchitis, including a persistent cough
       characterized by expectoration (Mackiewicz, 1998). The presence of this type of cough
       can increase the potential for secondary spread of antibiotic-resistant organisms into the
       community, where additional individuals could serve as reservoirs of multidrug-resistant
       bacteria... Thus, the inhalation of virginiamycin-resistantgram-positive bacteria in the
       swine environment could contribute to the appearance of quinipristin-dalfopristin-
       resistant gram-positive infections in humans, leaving few or no treatment options for the
       affected individual(s) " - Chapin et al., (2005)

The more recent work of Armand-LeFevre et al., (2005), who determined that a number of
Staphylococcus aureus strains that caused infections in swine populations (including four
methicillin-resistant strains) were also present in healthy swine farmer nasal cavities, but not in
the nasal cavities of healthy non-farmer controls, further supports their hypothesis.

Many other pathways  for infection of animal care workers with pathogenic zoonoses and
antimicrobial-resistant bacteria exist including, but not limited to direct contact with infected
animals, increased exposure to insect and wild animal vectors on the farm, exposure to animal
excreta, handling animal carcasses, exposure to contaminated air from manure spreading, and
drinking water from fecally-contaminated wells (Skilbeck and Miller, 1986; Everard et al.,  1989;
Seuri and Granfors, 1992;Thomas etal, 1994; Hogue etal, 1997; Cole etal, 2000; Barkocy-

Gallagher et al, 2001; Chomel,
2004). These exposures have
manifested in increased illness
in animal care workers, their
families and pets, and casual
farm visitors (Levy et al., 1976;
CDC, 2000), and have
potentially spread into nearby
communities based on
empirical evidence reported in
literature.  For instance,
McDonald etal, (1997)
genotyped vancomycin-
resistant fecal bacterial isolates
from swine,  poultry, farm
workers and their pets in
Denmark and concluded that
transmission had occurred
between livestock animals,
humans, and household pets.
Hummel etal, (1986) detected
nourseothricin-resistance traits
in 33% of fecal isolates of
swine exhibiting diarrhea, 18%
of fecal isolates from swine
farmers and  their families, and
16% fecal isolates from
outpatients exhibiting diarrhea
                                The presence of antimicrobial-resistant bacteria may occur
                                rapidly following the introduction of antimicrobials as
                                growth promoters in feed animals. Levy et al (1976)
                                determined that tetracycline-resistance in fecal isolates from
                                chickens increased rapidly from 10% of animals excreting
                                less than 0.1% of organisms resistant to tetracycline
                                (baseline) to 90% of animals excreting 100% of organisms
                                resistant to tetracycline within 2 weeks of introducing
                                tetracycline to chicken feed, whereas no increase was
                                observed in the control group. Further, multidrug resistance
                                developed, even though only tetracycline was being
                                supplemented in the feed.  By 12 weeks, more than 60% of
                                the animals from the experimental group excreted bacteria
                                resistant to tetracycline plus one or more other antimicrobial
                                compounds. More then 25% were resistant to 4 or more
                                antimicrobials.  After 4 months, antimicrobial resistance had
                                spread from the experimental group to the control group,
                                where a third of the chickens excreted bacteria of which more
                                than 50% of isolates were tetracycline resistant. Within 6
                                months, antimicrobial resistance had also spread to the farm
                                workers and their immediate families. More than 30% of
                                fecal samples form farm workers and their families contained
                                more than 80% tetracycline-resistant organisms, versus 6.8%
                                from their neighbors.  A 4-drug resistance pattern similar to
                                that observed in the experimental chickens was observed in
                                the farm workers and their families. Stopping the feed
                                additives eventually reduced the incidence of tetracycline-
                                resistant bacteria in the farm dwellers.
in communities adjacent to the swine farms. Nourseothricin was not used for treatment of
human disease in the region, but was used for two years for promoting growth of swine on the

Livestock animals can also be a source of antimicrobial-resistant bacteria and pathogenic
zoonoses such as E. coli O157:H7, Salmonella sp., and Staphylococcus aureus in abattoirs,
which may slowly die off or in some instances regrow in the waste products (Hepburn et al.,
2002).  When improperly handled, these wastes may potentially contaminate adjacent land and
nearby watercourses or infect slaughters, and through secondary infections, their families and
pets (Crawford et al., 1969; Nesbakken, 1988; Molin et al, 1989; Reboli and Farrar, 1989;
Merilahti et al., 1991;  Seuri and Granfors, 1992; Huys et al., 2005). In fact, as with animal care
workers, epidemiological evidence supports transmission of these pathogens from livestock
animals to humans in the abattoir environment, but suggests the infection rate is lower than that
observed in farmers and their families. For instance, van den Bogaard et al., (1997) phenotyped
fecal Enterococcus spp. isolates from turkeys, turkey farmers, turkey slaughterers, and nearby
residents of the turkey farms in Europe. Vancomycin-resistant Enterococcus (VRE) was
detected in half of turkey samples, 39% of turkey farmers, 20% of turkey slaughterers, and 14%

 In a recent study, van den Bogaard etal., (2001) surveyed
 three poultry operations (broilers, turkeys, and laying hens)
 and five human populations (turkey farmers, broiler
 farmers, laying-hen farmers, broilers slaughterers, and
 turkey slaughterers) in the Netherlands for antimicrobial-
 resistant fecal E. coli. These researchers determined that
 35% of isolates from laying hens were antimicrobial-
 resistant, as compared to 84% of the isolates from turkeys
 and 80% of the isolates from broilers. Similarly, 66% of E.
 coli isolates from turkey farmers, 60% from broiler
 farmers, 67% from turkey slaughterers, and 59% from
 poultry slaughterers were antimicrobial-resistant whereas
 only 45% of isolates from laying hen farmers were
 antimicrobial-resistant. Antimicrobial resistance patterns
 of the isolates were similar between turkeys, turkey
 farmers and turkey slaughterers, and in broilers, broiler
 farmers, and broiler slaughterers. Pulsed-field gel
 electrophoresis (PFGE) "fingerprinting" patterns of anE.
 coli isolate from a turkey was identical to one from a
 turkey farmer.  Similarly, one isolate from a broiler
 chicken was identical to an isolate found in a broiler
 chicken farmer.  Their results strongly indicate
 transmission of antimicrobial resistant bacteria between
 humans and poultry commonly occurs.
of area residents. VRE is one of
the leading causes of nosocomial
infections in the hospital
environment. Nij sten et al.,
(1994) determined that the
resistance of fecal isolates to
 antimicrobial compounds was
 more prevalent in swine farmers
 than slaughterhouse workers and
 suburban residents in the same
 geographic region.  In Japan,
 antimicrobial resistance of fecal
 microbes was also noted to be
 highest in swine farmers and
 elevated in slaughterhouse
 workers when compared to urban
 control cohorts (Saida et al.,
 1981). Others have realized
 similar trends supporting the
 movement of antimicrobial-
 resistant bacteria from farm
 animal to farmer or slaughterer
 (Ozanneetal.,  1987; Levy, 1978;
 Marshall etal, 1990).
 5.2 Food
 It is well established that pathogenic zoonoses can cause human disease via consumption of
 contaminated meat products (Corpet, 1993; U.S. Congress, Office of Technology and
 Assessment, 1995; Milleman et al., 2000).  The amplified use of antimicrobial compounds in
 confinement animals for growth promotion and prophylaxis may exacerbate disease by reducing
 treatment options and potentially increasing the virulence of bacterial pathogens in meats. For
 instance, in 1995 fluoroquinolone antibiotics were approved for use in poultry for growth
 promotion and prophylaxis. In 1997, Smith et al., (1999) screened chicken obtained from
 Minnesota shopping markets that originated from 15 abattoirs in nine states for Campylobacter
jejuni and resistance to ciprofloxacin, an important human-use fluoroquinolone antibiotic of
 choice for presumptively treating severe bacterial food poisoning. Fourteen percent of the
 samples were contaminated with ciprofloxacin-resistant C. jejuni. During a similar period,
 statewide-surveillance indicated that fluoroquinolone-resistance increased from 1.3% of all
 human C. jejuni infections in 1992 to 10.2% in 1998 (Smith et al, 1999).  In a more recent study,
 Wallinga et al., (2002)  surveyed 200 fresh whole market chickens and 200 packages of ground
 turkey from stores in Iowa and Minnesota and determined that 95% of whole chickens were
 contaminated with Campylobacter and 18% with Salmonella. Two percent of ground turkey
 samples and were contaminated with Campylobacter and 45% with Salmonella. Six percent of
 the Salmonella isolates were resistant to 4 or more antimicrobials, while 62%  of the
 Campylobacter isolates were resistant to 1 or more antimicrobial compound including an 8%

prevalence of resistance to ciprofloxacin.  Greater than ninety percent of enterococci isolated
from the chicken or turkey were resistant to quinipristin-dalfopristin, an important antibiotic for
the control of VRE infections in hospitals.  In a similar study, Hayes etal., (2003) screened 981
samples of raw retail meats including chicken, turkey, pork, and beef from 263 grocery stores in
Iowa and found high levels of resistance to several antimicrobials in Enterococcus isolates.
Their results indicate that antimicrobial-resistant Enterococcus spp. commonly contaminate retail
meat products and that the antimicrobial resistance pattern of isolates from each meat product
(poultry, pork, and beef) reflected well the use of approved agents in each food animal
production class (broilers, swine, and beef cattle).

Dairy products may also be contaminated with pathogenic zoonoses and antimicrobial-resistant
bacteria following direct contact of dairy cattle to contaminated sources in the farm environment
and subsequent excretion from the udders of infected animals (Oliver et a/., 2005). For instance,
Van Kessel et al, (2004) surveyed 861 bulk tank milks on farms  in 21  states and detected
Listeria monocytogenes (6.5%) and several Salmonella serotypes (2.6%) including Montevideo,
Newport, Muentster, Meleagris, Cerro, Dublin, and Anatum. Kim etal, (2005) tested 316 bulk
milk tank samples across the U.S. between January 2001 and December 2003 for Coxiella
burnetii, the causative agent for Q-fever. These researchers detected C. burnetii in greater than
94% of bulk tank  milk. Jayarao and Henning (2001) surveyed bulk tank milks from 131 dairy
herds in South Dakota and Minnesota and detected Campylobacter jejuni (9.2%), shiga-toxin
producing Escherichia coll (3.8%), Listeria monocytogenes (4.6%), Salmonella spp. (6.1%), and
Yersinia enterocolitica (6.1%), with one or more species of pathogenic bacteria in 26.7% of the
samples. Although pasteurization may reduce the incidence of disease in humans attributable to
contaminated milk, Oliver etal, (2005) argue that outbreaks of disease have been traced back to
both unpasteurized and pasteurized milk, and that unpasteurized milk is often consumed directly
by dairy producers, farm employees, and their families, as well as by their neighbors and raw
milk advocates. In their bulk tank-milk study, Jayarao and Henning (2001) observed that 60% of
the dairy producers drank unpasteurized milk, 27% of which contained one or more types of
pathogenic bacteria. According to the model presented in Figure  1, disease contracted via this
route may be spread to nearby communities via contact with infected individuals. It has also
been noted that an even larger segment of the population may be  directly exposed to
contaminated dairy products via consumption of cheeses made from unpasteurized milk (Oliver
etal., 2005).

CAFOs produce massive quantities of manure, much of which is  spread onto agricultural fields
as fertilizer. Fecally-contaminted water, potentially resulting from runoff from manure-treated
fields or discharge of wastes from agricultural operations, may be used to irrigate crops in  arid
regions of the United States.  Direct contact with soils on which manure was applied and/or
irrigation with fecally-contaminated water may result in contamination of produce such as
lettuce, radishes, apples, and sprouts with pathogenic zoonoses including antimicrobial-resistant
bacteria, especially where the edible parts are exposed to the soil  or water (Besser et al, 1993;
Tschape etal., 1995; Nelson,  1997; Taormina etal., 1999). In a recent study, Ingham et al,
(2004) identified E. coli contamination on carrots, lettuce, and radishes up to  120 days following
application of non-composted bovine manure as a fertilizer in fields in Wisconsin. In contrast,
Johannessen etal., (2005) did not detect E. coli O157:H7 on the edible parts of lettuce after
growth in E. coli O157:H7-contaminated manure fertilized soil.  Solomon et al., (2002) noted

that spray irrigation following a single exposure to E. coll O157:H7 resulted in 90% of the
lettuce being contaminated, persisting for more than 20 days in 82% of the plants. Where
surface irrigation was used under the same circumstances, only 19% contamination was observed
on the lettuce.  Immersion of harvested lettuce heads in 200ppm chlorine solution for 1 minute
did not eliminate all E. coli O157:H7 cells from infected lettuce, regardless of irrigation method.
It has been suggested that some produce may absorb pathogens into their internal tissues through
the root system, protecting them from cleaning procedures such as washing or irradiation. In a
survey of fresh domestic produce conducted in the spring of 2000, the US Food and Drug
Administration detected Salmonella on 2.6% of cantaloupe, 1.6% of cilantro, and 1.8% of lettuce
originating from U.S. farms (US FDA, 2001).
5.3 Air
Vast quantities of manure produced at CAFOs containing high levels of pathogenic
microorganisms and antimicrobial-resistant bacteria are applied to agricultural lands each year.
Viable bacteria and viruses become airborne from agricultural sprayers, pasturelands, and farm
fields treated with manure, ultimately decreasing the quality of air near CAFOs. The upward
flux of viable bacteria may be strongly related to plant cover and soil moisture condition. For
instance, upward flux of viable bacteria from bare soil and various crops has been reported to
increase an order of magnitude in dry soil over young corn in wet soil, another order of
magnitude in a closed wheat canopy over dry soil, and four orders of magnitude between bare
soil and an alfalfa field (Lindemann et a/., 1982).  Although plants have been found to be a
stronger source of bacteria than soil (Lindemann and Upper, 1985), specific agricultural practices
that increase particle  emissions may  significantly impact bacterial loading to an airshed.  Strong
vertical temperature gradients, low relative humidity and low soil moisture may lead to increased
emission of PM10 from agricultural fields during tilling (Holmen etal., 2000; Clausnitzer and
Singer, 2000). As dust may harbor viable bacteria, these factors may increase pathogen loading
to an airshed. If pathogens survive in soils until harvest, it is possible that significant airborne
spread may occur.  It has been estimated  that during harvest, up to 42% of bacterial loading in an
airshed can be attributed to harvesting activities (Lighthart, 1984; long and Lighthart,  2000).

Upward flux of viable bacteria may also be related to temperature, exposure to solar radiation,
protection from associated soil particles,  and wind speed. Lindeman and Upper (1985) report
that upward flux of bacteria over bean plants in Wisconsin occurred during the warmest parts of
the day with a maximum around noon,  especially on windy days, and was observed to  cease
when wind speeds were less than Im/s. Tong and Lighthart (1999) suggest that peak
concentrations of viable bacteria over agricultural lands in Oregon may occur in late afternoon,
presumably due to less exposure to solar  radiation or association with larger particles protective
from the effects of the sun. Upward flux of viable bacteria over a high desert chaparral have
been observed to peak late in the evening, with minimum viable bacterial concentrations at 13:20
hours and a maximum at 22:00 hours, presumably due to the strong effects of solar radiation
(Lighthart and Schaffer, 1994). The effects of temperature on upward flux of bacteria  may
overcome the effects  of solar radiation. Summer months have been associated with higher
incidence of airborne viable bacteria, even though increased solar radiation may negatively
influence bacterial viability due to UV  damage or desiccation of bacterial cells (Tong and
Lighthart,  1997; Tong and Lighthart, 2000).

Several studies have documented increased airborne pathogens directly attributable to the spread
of human or animal manure on agricultural lands through spray irrigation with contaminated
waters or deposition of animal placental and fecal wastes and subsequent distribution to
downwind animal or human receptors (Boutin et al., 1988; Hughes, 2003; Donnison et aL, 2005;
Brooks et a/., 2005). Boutin et a/., (1988) identified bacterial counts as high as 2000 viable
particles per cubic meter at the edge of applied areas following land spreading of cattle and pig
slurry. Donnison et al., (2005) studied the survival of Bacillus subtilis and Serratia entomophila
in irrigation aerosols in spring and summer in New Zealand. Viable B. subtilis and S.
entomophila corresponding to the respirable fraction of inhaled air were recovered at 100 m from
a low pressure sprayer and 200 m from a high pressure sprayer. Brooks et al (2005) studied the
aerosolization of E. coli and coliphage MS-2 from liquid biosolids applied from a spray tanker
under hot (22-37.5C) and arid (5-15% relative humidity) conditions. At wind speeds between
0.7-6 m/s, these researchers could not detect aerosolized E.  coli at distances as low as 2 m, but
detected coliphage MS-2 at distances as far as 60 m. Paez-Rubio et al (2005) identified
aerosolization as a potential mechanism for the dissemination of wastewater bacteria and other
microorganisms at flood irrigation wastewater reuse sites. These researchers identified more
than 1 billion enteric bacteria per cubic meter in downwind air samples.  Airborne  Coxiella
burnetii associated with sheep operations and Picnoravirus from swine operations have been
estimated to travel several kilometers in the air in concentrations sufficient to cause infectious
disease in humans and animals (Henderson, 1969; Hugh-Jones and Wright, 1970; Smith et al..,
1993; Hawker etal., 1998; Lyytikainen etal, 1998).
5.4 Recreational and Drinking Water
The USEPA's 1998 National Water Quality Inventory indicates that agricultural operations,
including animal feeding operations, are the most common polluters of rivers and streams,
contributing to the impairment of 59% of those surveyed.  Agricultural operations also have
significant impacts on lakes, ponds, reservoirs, and estuaries, contributing to the impairment of
more than 3,590,000 acres of these valuable water resources (USEPA-NACAC, 2005). Figure 3
illustrates the relationship between confined livestock animals in the U.S. in 1997 and
impairment of surface waters indicated in the  1998 National Water Quality Inventory. In 1998,
pathogens (microbial indicators, not overt pathogens) were the most common water pollutant
contributing to 7,742 impairments (14.24% of surveyed waters).  Sources of these
microorganisms may have included wastewater and storm water outflows, the spreading of
biosolids and animal manures on agricultural lands, and wild animals. However, the sheer
quantities of animal wastes generated and spread onto land compared to those of other sources
suggests animal agriculture may be the dominant contributor. Pathogenic microorganisms
continue to pose a major challenge to the quality of U.S. waters, contributing to a total of 7,894
impairments (13.16% of surveyed waters) on the USEPA's 2002 National Water Quality

A survey of literature regarding overt pathogens in agricultural waters and drinking water
sources suggest that the National Section 303(d) listings may underscore the actual extent of
microbial contamination in agricultural watersheds resulting from livestock activities. For
instance, two surveys of source waters for surface water treatment plants in 29 states resulted in
detection of  Cryptosporidium oocysts in 55%  and 87% of the waters tested. Similarly, Giardia

      Percent of surface waters listed as impaired, 1998
       Confined animal units per acre for operations
             with confined livestock, 1997
Percent of Farms Operated by Corporation,
Figure 3.      The impact of confined animal feeding operations on agricultural watersheds (adapted
              from USDA-NRCS, 2002; USEPA, 1998)

cysts were detected in 16% and 81% of the waters tested (LeChevallier et a/., 1991; Rose et
a/.,1991). Mycobacterium avium, potentially from cattle, swine, and broiler operations, have
been detected in several marine waters, rivers, lakes, streams, ponds, and spring waters
(AWWARF, 1997; Ichiyama etal., 1988; Falkinham etal., 2001; LeChevallier, 1999).  A high
prevalence of Campylobacter spp. in environmental water samples in a dairy farming area in the
United Kingdom including 56.7 % of running waters (streams and ditches) and 45.9% of
standing waters (ponds) was recently noted (Kemp et a/., 2005). Swine farming activities have
been implicated in the contamination of at least one major Canadian river by enteroviruses
(Payment, 1989), and have also been correlated to the presence of Cryptosporidium parvum
oocysts, Yersinia enterocolitica, and Salmonella spp. in nearby drainage canals, groundwater
wells, and surface waters where lagoon and spray systems are used (CDC, 1998).  Groundwater
surveys in Ontario Canada indicated that wells located near manure application areas were at
higher risk for fecal bacterial contamination, and the level of contamination was inversely
correlated to the distance the wells were from animal feedlots or exercise yards (Conboy and
Goss, 2002).

The delivery of zoonotic pathogens to environmental waters following manure application is
dependent on several factors including, but not limited to, the initial and persisting pathogen
load, properties of the pathogen of interest, the soil and vegetation type, travel distance/time to
the receiving water, pathogen inactivation by various environmental stressors, and potential
engineered or natural barriers to pathogen transport. When manure is spread onto land, overland
flow of pathogens to water bodies may occur via attachment to applied waste products,
attachment to soil particles, or movement in the free form (Tyrrel and Quinton, 2003, Muirhead
et al, 2005). Leachate from manure-amended fields and poorly designed manure holding
lagoons may inundate natural soil barriers resulting in contamination of underlying groundwater
(Jones, 1980; Kowel, 1982; Natsch etal, 1996; Jogbloed and Lenis,  1998). Within the soil
profile, movement of pathogenic  zoonoses may be limited by soil moisture and solid-phase
interactions (adsorption-desorption) or facilitated by the presence of macropores from burrowing
animals, fractured media, or plant roots (Ferguson et al., 2003).  Contaminated groundwater may
be captured by drainage tiles that discharge to surface waters, bypassing overland treatment in
natural or engineered vegetative or riparian buffers. Alternatively, pathogens may enter
groundwater where they may potentially  migrate towards wells or natural springs that may be
used for drinking water. The delivery of an infective dose to a susceptible individual will depend
not only on the transport properties of pathogens, but also on the time required for pathogen
inactivation due to environmental stressors or predation in surface and groundwater resources.

The concentration of pathogens reaching subsurface tile drains that discharge to nearby streams
often exceeds drinking water supply and  recreational use standards (Warnemuende and Kanwar
2000).  Tile drainage has been noted to be a significant pathway for pathogens to enter surface
waters from manure-treated fields, especially during periods of wet weather (Dean and Foran,
1992; Joy etal.,  1998; Geohring etal., 1999; Hunter et al., 2000; Monaghan and Smith, 2005).
Evans and Owens (1972) noted that approximately 0.05% if E.  coli applied with swine manure
applications to a  sandy clay loam pasture could be recovered in the tile drainage water.  In a
study of swine operations in Iowa and Missouri, Karetnyi etal., (1999) identified swine hepatitis
E in a tile outlet draining a field to which manure had been applied. Evans and Owens (1972)
determined that fecal bacteria present in swine waste slurries could be detected in the tiles
draining the pasture to which the  waste was applied within a few hours of application.

Where pathogens bypass tile drainage systems or tile drainage is non-existent, significant
contamination of groundwater resources may occur.  The transport of pathogens that have
infiltrated the soil profile depends strongly on adsorption-desorption interactions.  Key
characteristics of pathogenic zoonoses related to adsorption-desorption phenomena include  size,
surface electrostatic properties, cell wall hydrophobicity, and the presence of flagella (Gerba,
1984; Dowd et al., 1998; Heise and Gust, 1999). For viruses, attachment to soil particles is
rapid, and may be increased by low pH or high ionic strength groundwater or by high soil
organic carbon content (Gerba, 1981; Gerba etal.,  1978; Goyal and Gerba, 1979; Taylor etal.,
1980; Moore etal, 1981; Taylor  et al, 1981; Moore,  1982; Singh et al, 1986; Bales et al, 1991;
Bales etal, 1993; Sakoda etal, 1997).  At the neutral pH of most groundwater, organic carbon
content of the soil may dominate  retardation of viral particles.  Retardation of bacterial particles
in saturated porous media may  also be dominated by organic-carbon partitioning, but can also be
a product of straining or simple filtration (Heise and Gust, 1999). Straining and filtration may be

even more significant for the
larger protozoan parasites such as
Cryptosporidium oocysts and
Giardia cysts.  Once
contaminated, restoration of
water quality in contaminated
aquifers is very slow (Olson,

In packed sand columns, it has
been demonstrated that
Cryptosporidium oocysts,
although initially filtered, may
exhibit time-dependent
detachment leading to a constant
low-level elution from porous
media (Harter et al., 2000).  Free
oocysts have been observed to
move in pore water without
retardation suggesting potential
for considerable transport in
aquifers considering their long
survival times (Brush et al., 1999;
Harter etal, 2000).  Similarly,
viruses become attached to
sediments near the source of
contamination  and leach slowly
into the groundwater.  Therefore,
even single contamination events
may provide a  lingering source of
viral contamination to
groundwater (de Borde et al,
1998b). Viruses have been shown
to be able to travel considerable
distances through the subsurface
depending on their size,
In a collaborative study performed at 9 Swine CAFOs in
Iowa employing lagoon and spray systems, the CDC
tested the swine waste lagoons and several selected
points near the agricultural facilities for pathogenic
zoonoses. They identified elevated concentrations of E.
coli (<380,000 per lOOmL), Enterococcus sp.
(<1,900,000 per 100 mL), Salmonella sp. (<9,300 per
100 mL), and Cryptosporidiumparvum oocysts (<2250
per liter) in the  swine waste lagoons. C. parvum oocysts
were detected in monitoring wells nears the swine waste
lagoons of three CAFOs (9-15 oocysts per L) and in the
river adjacent one CAFO (6 oocysts per L). A single
Yersinia sp. was detected in an agricultural drainage
ditch draining the spray field at one facility.  Elevated E.
coli were detected in the agricultural drainage wells
(300-740/1 OOmL), drainage ditches (520-3,700/1 OOmL),
monitoring wells (10-390/1 OOmL), and drainage tile
inlet/outlets (10-2,900/1 OOmL).  Similarly,
Enterococcus sp. were detected in the agricultural
drainage wells (4,500/1 OOmL), drainage ditches (610-
13,000/lOOmL), monitoring wells (80-910/1 OOmL), and
drainage tile inlet/outlets (30-2,400/100mL).
Campylobacter sp. were not detected at any of the
sampling points. Of the  18 E. coli, 3 Salmonella sp.,
and 20 Enterococcus sp. isolates tested for antimicrobial
resistance, 16 E. coli, and all 3 Salmonella and 20
Enterococcus sp. were resistant to one or more
antimicrobials commonly used in swine management
practice as feed supplements and therapeutics (16 total
including fluorfenicol, tetracycline, sulfamethoxazole,
ampicillin, streptomycin, apramycin, bacitracin,
lincomycin, penicillin, synercid, kanamycin,
cephalothin, amoxicillin-clavulanic acid,  ceftiofur,
chloramphenicol,  and gentamicin). Eight E1. coli and all
3 Salmonella sp. and 20 Enterococcus  sp. were multi-
drug resistant (2-11 antimicrobials) (CDC, 1998).
adsorption characteristics, and
degree of inactivation (Keswick and Gerba, 1980; Dowd etal., 1998). For instance, enteric
viruses, some of which may remain infective for more than 9 months, have been observed to
move up to 1000-1600 m per year in channelized limestones and several hundred meters per year
in glacial silt-sand aquifers with travel times similar to bromide tracers (Skilton and Wheeler,
1988; Bales et al, 1995; Bosch, 1998; de Borde et al; 1998a; de Borde et al; 1999). Bacterial
pathogens may similarly move considerable distances as indicated in a study by Withers etal.,
(1997), who identified groundwater contamination by E. coli from an unlined cattle waste lagoon
76 m below ground surface and 80 m downstream the lagoon in the United Kingdom.

A significant limitation of the National 303(d) listings is the lack of monitoring for
antimicrobial-resistant bacteria. Antimicrobial-resistant bacteria are generally shed in animal
feces, but may also be present in the mucosa of livestock animals. The massive use of antibiotics
in animal agriculture pose a great risk as antimicrobial-resistant bacteria shed in animal wastes
and stored in lagoons or spread onto land may eventually find their way to the aquatic
environment (CDC, 1998; Levy, 1998; Chee-sanford et al, 2001).  For instance, Chee-Sanford et
al., (2001) detected all eight classes of tetracycline-resistance genes in two swine waste lagoons
and the underlying groundwater up to 250 meters down-gradient the lagoons. Tetracycline
resistant bacterial isolates from groundwater harbored a tet(M) gene identical to that detected in
the swine waste lagoons. Resistance genes from antimicrobial-resistant bacteria in contaminated
discharge waters can be transferred to otherwise susceptible bacteria living in unpolluted aquatic
habitats, encouraging the spread of antimicrobial resistance in environmental waters
(Guardabassi and Dalsgaard, 2002; Gurdin et al., 2002).  The extent of proliferation may be
limited by the distance from the discharge point.

Antimicrobials in livestock animals are primarily removed in the urine and bile, either
unchanged or in metabolite form, and therefore can directly contaminate environmental waters.
Once in the environment, antimicrobial compounds and their metabolites may degrade rapidly
(tetracyclines, penicillins, and fluoroquinolones) or persist (macrolides and sulfonamides),
resulting in long-term contamination near animal confinement operations. For instance,
Campagnolo et al., (2002) detected several antimicrobials used in animal agriculture in animal
waste lagoons (2.5-1000 |ig/L) and in monitoring wells, field drainage tiles, springs, streams, and
rivers (0.06-7.6 |ig/L) proximal to confined animal feeding operations in Iowa and Ohio. The
use of antimicrobials in  animal agriculture most certainly contributed to the frequent detection of
antimicrobials in a recent U.S.G.S. survey of rivers and streams of the United States (Kolpin et
al., 2002). Although the presence of a pharmaceutical residues and their metabolites in potable
water sources present their own ecological challenges (Goni-Urriza et al., 2000; Zuccato et al.,
2000; Hirsch et al., 1999; Hailing-Sorensen et al., 1998; Daughton et al., 1999), their typical
concentrations in  environmental waters are usually far below (approximately 1000-fold) those
that would selectively enrich for resistant bacteria. Resistant bacteria found in surface waters are
likely to have originated from wastewater or manure runoff from antimicrobial-rich settings such
as animal feeding operations or wastewater treatment plants or subsequently  contaminated
animal vectors (Levetin, 1997; Stetzenbach, 1997; Summers, 2002).

Although low environmental concentrations of antimicrobials may not be adequate to enrich for
resistant strains of bacteria, their role in the proliferation and maintenance of antimicrobial-
resistance genes in these complex  milieus is uncertain.  For example,  Gurdin et al (2002)
screened isolates of E. coli and enterococci from swine farm wastes, and environmental isolates
of E. coli, enterococci, Kleibsiella, andAeromonas in surface waters upstream and downstream
of study farms for antimicrobial resistance.  These researchers observed that the diverse
resistance patterns exhibited by rural background surface water isolates likely reflected human
and animal impacts.  In contrast, bacteria isolated downstream from swine farms exhibited
increased antimicrobial-resistance that reflected the swine waste isolates.  Sixty seven percent of
Aeromonas and 12% of enterococci isolates upstream the study farms were resistant to
erythromycin, whereas 91% of Aeromonas and 30% of enterococci isolates down-stream the
study farms were  resistant.  Antimicrobial residues were also more likely to be detected

downstream rather than upstream swine farms. However, antimicrobial resistance did not always
correlate to detection of residues. Swine farms were shown to be capable of contributing
resistant enteric bacteria that act as reservoirs for the spread of resistance traits to susceptible
bacteria, and antimicrobial residues which may encourage the maintenance and spread of the
resistance traits.  More work is needed to clearly identify threshold concentrations of
antimicrobial residues in environmental waters that encourage the spread of antimicrobial
5.5 Hydrologic events
Once in natural water bodies, viral particles, bacteria, and protozoan cysts and oocysts may
attach to larger particles such as organic matter or soils and settle into the sediments of streams
or reservoirs. Due to their size, settling of free particles may be limited. Their association with
sediments may offer some protection from environmental stressors such as solar and UV
radiation, pH extremes, desiccation, antibiotics,  and predators leading to increased survival
(Gerba and McLeod, 1976; Smith etal, 1978; Roper and Marshall, 1979; Bitton and Marshall,
1980;LaBelleandGerba, 1980; Schaiberger etal., 1982; Metcalf etal, 1984; Rao etal, 1984;
Long and Davies, 1993). .As such, the sediments of natural water bodies may act as reservoirs
for pathogenic zoonoses and antimicrobial-resistant bacteria discharged from CAFOs
(Hendricks, 1971; Grimes, 1975; Gerba etal., 1977; Davies etal., 1995). For instance, in
estuary waters, Metcalf et al., (1984) detected enteroviruses and rotaviruses in 14 and 50% of
two water samples but 72 and 78% of their respective sediments contained these viruses. In 20-
70% of surface waters, it has been observed that viruses occur as solid-associated particles, and
may be present in high concentrations in bed sediments when compared to overlying water even
at vast distances from the original source of contamination (Ferguson etal., 2003).

The movement of pathogens from CAFO operations can be exacerbated by rainfall, which may
stimulate the release of pathogens from otherwise stable manure-treated fields or fecal pats
leading to increased overland transport, discharge to surface waters by drainage tiles, or
infiltration into groundwater resources (Kress and Gifford, 1984; Mawdsley et al, 1996a,b;
Hunter etal, 2000; Ogden etal, 2001; Davies etal, 2004; Monaghan and Smith, 2005).  Often,
stream flow increases significantly during hydrologic events, stirring up bedded sediments and
further increasing pathogen concentrations, especially in shallow surface waters (Ferguson etal,
2003). For example, Ferguson (1994) determined that an increase of 1-cm in rainfall increased
Cryptosporidium oocysts in the Georges River by 24%. Atherholt et al, (1998) demonstrated a
positive correlation between parasitic protozoan concentrations in the Delaware River Watershed
and precipitation events. Kistemann etal, (2002) measuredE. coli, fecal streptococci,
Clostridium perfringens, Cryptosporidium, and Giardia in the tributaries of 3 drinking water
reservoirs during normal and wet weather events and noted a 1-2 logic increase in bacterial and
parasitic microbial concentrations during runoff compared to normal conditions.  Crowther et al,
(2002) observed highly significant positive correlations between concentrations of coliforms, E.
coli, and enterococci in two watersheds in the United Kingdom during hydrologic (high flow)
events and land use/management variables associated with intensive livestock farming.  High
flow conditions were associated with a greater than 10-fold increase in geometric mean fecal
indicator concentrations (coliforms, E.  coli, and enterococci) potentially due to storage and
resuspension of viable organisms in channel bed sediments. Kunkle (1972) noted a marked

dependence of bacterial concentrations on stream flow in the Sleepers River Basin near St.
Johnsbury, Vermont, and emphasized the importance of stream surveillance that accounts for the
hydrology involved. Joy et a/., (1998) reported bacterial contamination of surface water due to
the application of liquid manure by accepted practices over a two year period. Drainage tiles
were determined to deliver significant amounts of bacteria to surface waters, which was
exacerbated by rainfall shortly following manure application.

Extreme precipitation may pose more significant problems for CAFO operators as lagoons and
other engineered manure management systems such as vegetative buffers, infiltration basins, and
constructed wetlands may be challenged by the level of flooding associated with these events.
Passive manure management systems are typically designed for 20-50 year flood events, and
may be overtopped during more rigorous flooding.  Flood waters may engulf vegetative buffers
allowing direct contact with animal wastes applied to fields. Flooding may also engulf animal
confinement houses drowning animals and transporting raw fecal material and animal carcasses
downstream.  Waste management systems that do not fail will  experience elevated discharge,
reducing their efficacy as a barrier to pathogens. Because of the potential liability associated
with overtopping or failing waste lagoons during flooding, many CAFO operators opt to spray
down their lagoons during heavy rainfall in lieu of violating freeboard limits (Wing etal., 2000).
Significant environmental damage associated with intentional and accidental release of manures
and other potentially infectious materials from CAFO operations during flooding events has been
documented and the danger still persists (Taylor, 1999; Mallin, 2000; Scmidt, 2000; Wing et a/.,
2002).  In  1999, Hurricane Floyd flooded several CAFOs and caused extensive environmental
damage to river and coastal waters in North Carolina. During this event, it was estimated that
dozens of animal waste lagoons were breached and more than  100,000 hogs, 2.4 million
chickens, and 500,000 turkeys drown in the flood waters. Wing et a/., (2002) estimate that
greater than 240 CAFOs still operate within the region flooded by this category 3 hurricane.

6. Public Health  Outcomes
Pathogens may enter and proliferate in a farm environment through the stocking of new animals,
exposure to airborne pathogens from an upwind source, contaminated trough water or feed,
insect or rodent vectors, human-to-animal and animal-to-animal transmission, to name a few.
Concentrating animals in confinement with suboptimal hygiene may encourage the spread of
disease within farms.  As discussed earlier (Section 4: Survival of Pathogens in the
Environment), the survival of zoonotic pathogens in animal manures and the environment can
range from days to years depending on the pathogen, the medium, and environmental conditions.
Where animal wastes are improperly managed, there exists potential for the movement of
pathogens off farms and into nearby water, land, and air. Uncontrolled releases of pathogens
may occur via runoff, aerosolization, or infiltration into soils and groundwater, especially when
manure is spread onto land. Stored animal feeds and manure can attract animal vectors that can
spread disease within a farm, to nearby farms or communities, or, in the case of migratory birds,
over large distances spanning hundreds of kilometers. Animal care workers are exposed to
elevated levels of pathogens in confinement house air and through direct contact with livestock
and animal manures, leading to an increased incidence of illness and spread of disease to their
families and communities. A similar trend is seen in abattoir workers and their families due to
the proliferation of pathogens within slaughterhouse environments. Contamination of produce or
meat products with zoonotic pathogens may further spread disease within human populations.
Even where extensive management practices are in place, exposures can and do occur. The
outcomes of these exposures are animal and human disease, sometimes with serious
6.1 Waterborne and Foodborne Outbreaks
The impacts animal feeding operations may have on public health are evident in surveillance of
waterborne and foodborne outbreaks in the U.S. reported by the Centers for Disease Control and
Prevention (CDC).  Table 7 summarizes the CDC outbreak data between 1991 and 1997.  During
this period, there were more than 3,900 reported outbreaks infecting more than 500,000
individuals. Based on reported data, foodborne outbreaks were 8.3 times more likely to be
reported than waterborne outbreaks.  However, waterborne outbreaks tend to affect larger
numbers of individuals per incident, most likely because communities share drinking water
resources and recreational waters.  Between 1991 and 1997, the number of infected individuals
per waterborne outbreak was 35 times larger than for foodborne outbreaks (2-3 times larger
discounting the Cryptosporidium outbreak in Milwaukee in  1993 that infected more than
400,000 individuals).  Of the outbreaks of
known etiology reported from 1991-1997,
slightly less than half (48%) of the
recreational water outbreaks and nearly two
thirds (66%) of the outbreaks associated
with untreated drinking water were caused
by zoonotic pathogens. During the same
period, 82% of the foodborne outbreaks of
known etiology were caused by zoonotic
pathogens.  The pathogens most often
Of the outbreaks of known etiology
reported from 1991-1997, slightly less than
half (48%) of the recreational water
outbreaks and nearly two thirds (66%) of
the outbreaks associated with untreated
drinking water were caused by zoonotic
pathogens. During the same period, 82% of
the foodborne outbreaks of known etiology
were caused by zoonotic pathogens.

associated with outbreaks include Giardia, Cryptosporidium, Campylobacter, Salmonella, and
toxigenicE. coli (including E. coli O157:H7, E. coli O126:NM, andE. coli O121:H19).  As
noted above, all of these microbial agents are endemic in cattle, swine, and poultry flocks, and all
are characterized by a low infectious dose.

Although the number of outbreaks and cases of illness reported to the CDC due to recreational
and drinking water exposure, as well as foodborne sources, are massive, they greatly underscore
the true incidence of disease caused by these sources.  A complex chain of events must occur in
order for a foodborne or waterborne disease outbreak to be reported to the CDC's foodborne and
waterborne outbreak surveillance systems. A break at any point in the chain results in an
unreported incident. Significant limitations to the reporting system begin at infection, as there is
a continuum of disease from asymptomatic infection and mild illness to death. Illness can be
sporadic  in the population following exposure, and most sickened individuals seek medical
attention only in severe cases. Outbreaks that are most likely to be brought to the attention of
public health authorities include those that are large, such as interstate or restaurant-associated
outbreaks, or those that can cause serious illness, hospitalization, or death.  The identification  of
the source of infection in many cases is difficult and may be compounded by the long incubation
periods of some agents, as noted in Table 1 (Section 2: Pathogens).  For instance, the illness
following exposure to Brucella spp. may manifest in as little as five days or as much as 60 days,
a time in which the number of potential vehicles of transmission may be massive. Even where
cases may be simple, reporting may be limited. Reporting of outbreak data is at the discretion of
the states, many of which do not have adequate monitoring and reporting systems in place,
primarily due to lack of financial resources to implement such systems.  Outbreaks reported in
the foodborne and waterborne outbreak surveillance summaries are a small and variable fraction
of all outbreaks and cases that occur in the U.S. every year.  They do not include those caused by
secondary infections, animal contact infections, airborne infections, or many of the other
pathways discussed above. As a result, the true incidence of illness that may be caused by
zoonotic  pathogens remains largely unknown. Table 8 shows the estimated total yearly
incidence of disease caused by selected pathogens in the U.S. (Mead etal, 1999).  Based on
these estimates, zoonotic pathogens may be responsible for as much as 90% of bacterial and
parasitic  infections of known etiology.

The actual incidence of waterborne and foodborne disease is certainly much higher than that
reported in annual surveillance activities.  For instance, Mead etal., (1999) estimated that
foodborne disease causes 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the
U.S. each year.  The American Society for Microbiology (1998) reported that 900,000 illnesses
and 900 deaths each year may be caused by waterborne microbial infections following
recreational water contact. Morris and Levin (1996) estimated  that disease-causing microbes in
drinking  water alone may cause 7.66 million illnesses and 1,200 deaths each year.  Based on
these estimates, acquiring infection by a foodborne organism may be 10-84 times more likely
than for waterborne infections (either through recreation or drinking contaminated water).
However, actual studies  suggest that the risks associated with drinking water that meets federal
standards are understated.  The reasons for this are unclear, but may be related to the perception
that water is "clean". There may be a tendency of individuals and medical practitioners to
identify food as a source of contamination when the vehicle of  transmission is unclear, especially
when the etiological agent is not identified.  In any case, evidence from studies of several water

Table 7. Water and foodborne outbreaks in the U.S. reported by the CDC (1991-1997).
Etiologic Agent

Bacillus spp.
Brucella spp.
Campylobacter spp.
Clostridium spp.
E. coli (toxigenic) f
Legionella spp.
Leptospira spp.
Listeria monocytogenes
Mycobacteria spp.
Plesiomonas shigelloides
Pseudomonas spp.
Salmonella spp.
Shigella spp.
Staphylococcus spp.
Streptococcus spp.
Vibrio spp.
Yersinia enterocolitica
Other bacterial
Cryptosporidia spp.
Giardia spp.
Niagleria fowleri
Schistosoma spp.
Trichinella spiralis
Adenovirus 3
Hepatitis A
AGI and Other Unknown*


5 (253)




2 (141)

6 (882)

21 (2737)
Drinking Water
Treated Unknown

1 (32) 2 (274)

5 (90) 2 (9)


2 (749) 1 (84)
4 (7 OP)

2 (114)

8 (407701) 3 (762)
16 (227S) 1 (O

4 (1804) 1 (665)
34 (11997) 6 (634)
Recreational Water

32 (476)

3 (402)


13 (1256)

6 (654)
29 (29)

11 (234)

1 (5P5)

8 (3P7)




1 (149)


63 (70PO)
5 (720)

45 (12494)


10 (26S)



9 (565)

43 (7322)
7 (229)
3 (402)

64 (1140)
26 (1981)

2 (114)

64 (421152)
36 (2555)
30 (37)


1 (5P5)
23 (3S02)
87 (16806)

22 (969)
38 (773)
107 (4991)
90 (3372)

3 (700)

560 (35861)
48 (7677)
57 (1950)
3 (22S)
6 (60P)



38 (7262)
10 (1483)
24 (2104)

23 (989)
107 (4991)
133 (4634)
7 (229)
3 (402)
3 (100)
564 (36697)
74 (3652)
3 (228)
6 (609)

43 (2634)


1 (595)
33 (5285)
25 (2174)
2548 (68537)
        Includes E. coli O157:H7, E. coli O12LH19, and£. coli O26:NM
        AGI= Acute gastrointestinal illness of unknown etiology; also includes other illnesses of unknown etiology

Table 8.   Estimated number of total cases, hospitalizations, and fatalities that may occur annually in
          the U.S. by selected etiological agent as reported by Mead et a/., (1999).
 Etiologic Agent                  Total Cases     Hospitalizations    Fatalities
Brucella spp.
Campylobacter spp.
Escherichia coli O157:H7
Enterohemorrhagic Escherichia coli
(non-O157:H7 STEC)
Listeria monocytogenes
Salmonella spp.
Yersinia enterocolitica
Protozoans and Helminthes
Cryptosporidium parvum
Giardia lamblia
Toxoplasma gondii
Trichinella spiralis










systems meeting federal drinking water standards suggests that as much as 6-40% of
gastrointestinal illness in the U.S. may be drinking water related (Payment etal., 1991; Golstein
etal, 1996; Cottle etal., 1999; Morris etal., 1996; Schwartz etal., 1997; Schwartz etal., 2000;
Levin et al, 2002). For instance, in a study conducted in Contra Costa County, California,
reverse osmosis drinking water treatment systems (half sham and half real) were installed on the
taps of more than 400 participants (50% sham and 50% real).  Participants with true systems had
20.4% less gastrointestinal illness episodes than those who used tap water meeting all federal and
state drinking water treatment standards (Colford et al, 2002). These results are similar to those
of earlier Canadian studies (Payment et al., 199la,b; Payment, 1994; Payment et al., 1994;
Payment etal., 1997), and indicate that infections acquired through contaminated drinking water
may approach those acquired through consumption of tainted food.
6.2 Specific Cases
Although the scale of infections caused by zoonotic pathogens remains unclear, the transmission
of pathogenic zoonoses from livestock animals to humans and other negative public health
outcomes resulting from living in proximity to confinement animals has been clearly
documented in reported literature. Both epidemiological studies (See sidelights.) and specific
incidences reported in the U.S. and other high income countries, such as the United Kingdom
(UK), Canada, The Netherlands, and Japan,  have implicated livestock animals and their wastes
as the source of illness and other health outcomes.  Animal manures in particular have been
implicated as the source of pathogens in several waterborne outbreaks (Jackson et al., 1998;
Crampin et al., 1999; License et al., 2001; Health Canada; 2001). When manure has been
implicated as the source of outbreak, the consequences have been severe. For instance, manure
runoff contaminating groundwater near a municipal well in Walkerton, Ontario, Canada resulted
in an outbreak of E. coli O157:H7 and Campylobacter spp. in May, 2000 that caused 2,300
illnesses and 6 deaths (Valcour etal., 2002;  Clark etal., 2003; Federal-Provincial-Territorial

                                   Merchant et al. (2005) studied the association between
                                   farm living and the prevalence of asthma outcomes.
                                   Children living on swine farms were more likely to
                                   have asthma outcomes, and the prevalence was more
                                   dramatic where antibiotics were added to feed. Nearly
                                   43% of children on farms with less than 500 pigs had
                                   asthma or asthma indicators.  This number climbed to
                                   46% on farms with more than 500 pigs.  However,
                                   55.8% of children living on hog farms where
                                   antibiotics were added to feed experienced asthma or
                                   asthma indicators . This compared to 26.2%
                                   prevalence in children on farms that did not raise
                                   hogs. The study indicated that 33.6% of children not
                                   living on a farm and not around swine had at least one
                                   indicator of asthma. Although farms that use
                                   antibiotics tended to be larger, the research team
                                   concluded that antibiotic exposure may also have
                                   played a role in the development of childhood asthma.
Committee on Drinking Water,
2005). Solo-Gabriele and
Neumeister (1996) describe a
Cryptosporidium outbreak in
Corrollton, GA in 1989 in which
manure runoff was suspected to have
been the cause of over 13,000
illnesses. Richardson et al., (1991)
and Atherton et al., (1995) describe
Cryptosporidium outbreaks in
Swindon, Oxfordshire, and Bradford
UK in 1989 and 1994, respectively,
in which storm runoff from farm
fields was suspected to have been
the cause of 641 illnesses.
MacKenzie et al., (1994) describe a
Cryptosporidium outbreak in
Milwaukee, Wisconsin in 1994 in
which 87 deaths and over 400,000
illnesses were attributed to animal manure and/or human excrement contaminating the water

Animal manure has also been implicated as the source of many foodborne outbreaks, mostly
resulting from contaminated produce (Schlech et al., 1983; Morgan et al., 1988; Besser etal.,
1993; Cieslak et al, 1993; Millard et al, 1994; Tschape etal., 1995). Manure-contaminated
produce (fruit and vegetables, including juices and salads) tends to result in more illnesses per
outbreak than those associated with contaminated meat. This is because fertilizing fields with
manure or irrigating with fecally-contaminated water results in larger numbers of potentially
infectious products that are eaten raw in most cases.  For instance, Fukushima et al., (1995)
                                                   describe an outbreak of E.  coli O157:H7
                                                   in Sakai  City, Japan in which animal
                                                   manure-contaminated alfalfa sprouts
                                                   were suspected of causing 12,680
                                                   illnesses, 425 hospitalizations, and 3
                                                   deaths. Outbreaks of E.  co//O157:H7
                                                   have also been associated with the
                                                   consumption of manure-contaminated
                                                   apple cider (Bresser et al., 1993) and
                                                   potatoes (Levy et al., 1978). In contrast,
                                                   contaminated meats, which may result
                                                   from infected animals or contamination
                                                   at the abattoir, are generally cooked,
Wing and Wolf (2000) surveyed residents of three
rural communities, one in the vicinity of a 6000-
head hog operation, one in the vicinity of two
intensive cattle operations, and a third without
livestock operations using liquid waste
management systems. Residents in the vicinity of
the hog operation were 7.6 times more likely to
report occurrences of headaches, 5.2 times more
likely to experience runny noses, 3.6  times more
likely to have sore throats, 4.7 times more likely to
excessively cough, 3.0 times more likely to have
bouts of diarrhea, and 5.6 times more likely to
have burning eyes than residents of the  community
without intensive livestock operations.  All results
were adjusted for sex, age, smoking,  and work
outside the home.
                                                   destroying pathogens and leading to
                                                   more sporadic incidence of illness per
                                                   outbreak. However, the number of
                                                   outbreaks and total number of cases

associated with contaminated meat are higher than produce. According to surveillance of
outbreaks in the U.S. between 1990 and 1998, contaminated produce accounted for about 24% of
the outbreaks and 41% of the cases (Griffiths, 2000).

Although proper cooking can eliminate most pathogens from meat products, contaminated meat
remains a significant link between humans and pathogenic zoonoses. Outbreaks of
enterohemmhorhagic E. coli in the U.S. between 1982 and 2002 can be attributed primarily to
contaminated meat (41%), followed by produce (21%), person to person contact during illness
(14%), contaminated drinking or recreational water (9%), and directly contacting infected
animals or their wastes (3%) (Rangel et a/., 2005). The emergence of many antimicrobial-
resistant zoonotic pathogens in human populations has been linked to the consumption of food
animals and dairy products.  For instance, Holmberg et a/., (1984) attributed a 6-state outbreak of
multi-drug resistant Salmonella newport to consumption of beef from a feedlot that was using
subtherapeutic doses of chlorotetracycline as a growth promoter. The emergence of multidrug-
resistant Salmonella typhimurium DT 104 in 1988 in cattle in the UK was rapidly followed by its
detection in meat (Threlfall et a/., 1997) and later in humans, presumably via the consumption of
contaminated beef, pork sausages, and chickens. Between 1990 and 1995, human illnesses in
UK attributed to S. typhimurium DT 104 increased from 259 to 3837 (Lee et al, 1994). The
emergence  of fluoroquinolone-resistant pathogens in the Netherlands rapidly followed its
introduction as veterinary drug in chickens and humans. Enrofloxacin-resistant Campylobacter
in poultry increased from 0-14% between 1982 and  1989, while resistant Campylobacter causing
human infections rose from 0-11% (Endtz, 1991). As poultry are a primary reservoir for
Campylobacter spp., the use of fluoroquinol ones in the poultry industry was implicated as the
vehicle for human-acquired enrofloxacin-resistant Campylobacter.

Airborne zoonotic pathogens from animal feeding operations may also infect humans and other
livestock animals. As noted above, pathogens and antimicrobial-resistant bacteria have been
detected at elevated concentrations    	
in confinement house air (Cormier et
al., 1990; Cazwalae^a/., 1990;
Crook et al., 1991; Heederick et al.,
1991; Zahn etal, 2001; Predicala et
al., 2002; Gast et al 2004; Chapin et
al., 2005).  Farm workers exposed to
confinement house air are much
more likely than the general
population to acquire infections of
the lungs and sinuses (Mackiewicz,
1998; Aubry-Damon, 2004;
Armand-LeFevre etal., 2005), and
the potential for secondary infection
of nearby populations is high.
Airborne zoonotic pathogens may
also travel over vast distances
downwind of an infected livestock
source.  Henderson (1969) and
Smith et al. (1993) and Hawker et al. (1998) describe
an outbreak in the West Midlands, UK in which
airborne transmission ofCoxiella burnetii was
identified as the causative agent of 147 illnesses.
Outdoor lambing and calving was performed on
farms south of the urban area. Strong gales blew
towards the urban area on a single day approximately
three weeks prior to the onset of illness.  Coxiella
burnetii is known to multiply to very high
concentrations in the placenta of sheep which,
following deposition on the ground during outdoor
birthing, can dry out allowing bacterial release with
airborne particulates (Welsh et al., 1958; Jones  and
Harrison, 2004). The mean incubation period for
Coxiella burnetii in humans has been shown to  be 20
days, consistent with the period of time between the
day of strong gales and the peak onset of symptoms
in the outbreak (Aitken et al., 1987).

Hughes and Wright (1970) describe a series of airborne picnoravirus outbreaks (foot and mouth
disease) in pigs, cattle, and sheep in Worcester, UK in  1967, in which infected animals at three
pig farms were suspected as the cause.  Casal et a/., (1997) estimated that the airborne dispersion
could have transported an infectious dose of this virus from the three source swine farms to cattle
as far as 7 km away. However, secondary infection from cattle or sheep was unlikely to affect
cattle or sheep more than 200 m away (Donaldson et al., 2002).  It has been estimated that
picnoravirus can be transported in the air over distances as great as 60 km overland and 300 km
over seas (Gloster etal., 1982; OIE, 2005).  Lyytikainen etal., (1998) describe an outbreak of Q-
fever in a small rural community in Germany in which airborne transmission of Coxiella burnetii
from a nearby infected flock of 1,000-2,000 sheep may have caused 45 illnesses over a four
month period. Outdoor calving was performed on the farm, and the wind blew from the farm
towards the town 57% of the time during the course of the outbreak. Both picnoraviruses and
Coxiella burnetii may be shed in animal feces suggesting that these organisms, among others,
could be dispersed over vast distances following spray irrigation of animal manures onto
6.3 Antimicrobial Resistance
Antimicrobial-resistant bacteria and other zoonotic pathogens from CAFOs often infect humans,
many times with serious consequences. Evidence in the reported literature overwhelmingly
supports this conclusion and includes direct epidemiological studies, temporal evidence of the
emergence of resistance in livestock animal populations prior to the emergence in human
populations, trends in resistance among human isolates that mimic the use of antimicrobials in
livestock animals, and studies that show farmers, slaughterers, and their family members are
much more likely than the general population to acquire antimicrobial zoonoses. Antimicrobial
resistance can limit treatment options in sickened individuals and increase the number, severity,
and duration of infections (FAAIR Scientific Advisory Council, 2002).  Varma et al., (2005)
evaluated Salmonella outbreaks in the U.S., and determined that among 32 reported outbreaks
                                                      between 1984 and 2002, 22% of
                                                      13,286 people in ten Salmonella-
                                                      resistant outbreaks were hospitalized
                                                      compared with 8% of 2,194 people
                                                      in 22 outbreaks caused by
                                                      pansusceptible strains.  These
                                                      differences are not only the
                                                      consequence of limited  options for
                                                      antimicrobials, but are also related to
                                                      the increased virulence  often
                                                      associated with antimicrobial-
                                                      resistant organisms. For instance,
                                                      Lee etal., (1994) determined that
                                                      individuals infected with resistant
                                                      organisms were ill 25% longer and
                                                      were significantly more likely to be
                                                      hospitalized than those infected with
                                                      pansusceptible strains.  Those
Bezanson et al (1983) describe the infection of a
newborn child with a multidrug-resistant strain of
Salmonella ser. typhimurium resulting in septicemia
and meningitis. The source of infection was the
child's asymptomatic mother, who acquired the
bacterium through ingestion of unpasteurized milk
and passed it to her child during delivery in the
hospital. Illness in the newborn child manifested
within 24 hours, and within 72-96 hours had spread
to several other infants in the hospital nursery. In
another case described by Lyon et al. (1980), a
multidrug-resistant strain of Salmonella heidelberg
was spread from an asymptomatic mother to
newborn child during delivery via cesarean section.
The mother was a farmer who shortly before delivery
had been working with calves from an infected herd.
Three infants in the hospital nursery were infected
with the organism and developed bloody diarrhea.

infected with resistant strains were hospitalized on average ten days versus eight days for those
infected with susceptible strains, even though most subjects in both cases were treated with an
antimicrobial to which the infectious agent was susceptible. The difference in hospitalization
rates likely reflects the higher virulence of the resistant infectious organism, and, to a much
lesser extent, an inappropriate first choice of antimicrobial for treatment.  Resistance to
antimicrobial agents, resulting from their extensive use in animal agriculture,  may result in tens
of thousands of additional infections by zoonotic pathogens compared to what would be
experienced with pansusceptible strains.  This may result in more than ten thousand additional
days of hospitalization, and hundreds of thousands of excess days of diarrhea  in the U.S. each
year (Barza and Travers, 2002; Travers and Barza, 2002)
6.4 Hydrologic Events
Hydrologic events ranging from mild rainfall to flooding can increase the movement of
pathogens from CAFOs or manure-amended fields to waters that are likely to come into contact
with people. Serious public health consequences of the increased pathogen load, especially
during flooding events, are common (Isaacson et al., 1993; MacKenzie etal., 1994; Health
Canada, 2000; CDC, 1998).  Several studies in low income countries have reported increases in
morbidity and/or mortality following flood events due to cholera, cryptosporidiosis, nonspecific
diarrhea, poliomyelitis, rotavirus, and typhoid and paratyphoid (Fun etal., 1991; van
Middelkoop et al., 1992; Katsumata et al., 1998; Biswas et al., 1999; Sur et al., 2000; Mondal et
al., 2001; Kunji et al., 2002; Kondo et al., 2002; Heller et al., 2003; Vollard et al., 2004). The
increased relative risk (RR) or odds ratio (OR) of contracting disease during flooding in these
cases ranged from 1.39 to 4.52.  Significant increases in vector- and rodent-borne diseases were
also observed (Trevejo etal.,  1998; Han etal., 1999; Sanders etal., 1999; Sarkar etal., 2002;
Leal-Castellanos etal., 2003).

The increased risk of contracting disease post-flood in high income countries such as the U.S.,
UK, and Australia exist, but are less pronounced (Ahern et al., 2005). Bennet et al., (1976)
observed hospital visits by the flooded to more than double in the year following an event in
Bristol, UK, in 1968.  These researchers also observed a 50% increase in mortality among the
flooded, mostly in the elderly. Reacher et al., (2004) interviewed 467 households following a
flood in Lewes, UK, and observed a slight increase in gastrointestinal illness in those whose
homes were flooded.  In Brisbane, Australia (1974), a flood led to increased morbidity, but not
mortality, in the flooded group (Price, 1978; Abrahams et al., 1976). However, Handmer and
Smith (1983) noticed no flood-related increase in hospital admissions during flooding in
Lismore, Australia the same year.  In a cohort study of 1,110 people in a U.S. Midwestern
community, Wade etal., (2004) reported an increase in the incidence of gastrointestinal illness
during a flood event in April and May of 2001.  The increase in gastrointestinal illness was
pronounced in persons with potential sensitivity to infectious gastrointestinal agents and those
who came into contact with the flood water, especially children.  Heather et al., (2004) noted the
significance of heavy rainfall in the Walkerton, Canada outbreak of E. coli O157:H7 and
Campylobacter.  The rainfall was equivalent to a 60-year event,  and it was suggested that this
extreme precipitation may have mobilized animal wastes and led to the outbreak. Curriero et al.,
(2001) studied the link between reported waterborne disease outbreaks in the U.S. between 1948
and 1994 and extreme precipitation events. These authors found a strong correlation between

rainfall and disease. Disease due to surface water contamination primarily occurred during the
month of the precipitation event, whereas disease associated with groundwater contamination
occurred two months following extreme precipitation events.

Serious health outcomes from flooding events can and do occur in the U.S. and may be unfairly
weighted against the underprivileged. The recent flooding of New Orleans, Louisiana following
hurricane Katrina, a category 4 event, resulted in the exposure of tens of thousands of people to
floodwaters  laden not only with chemical wastes, but also decomposing bodies, animal
carcasses, sewage, and animal wastes. E. coli concentrations in these waters reached as high as
42,000 per 100 mL, hundreds of times higher than levels associated with gastrointestinal
illnesses that result from "recreational contact".  Those unable to escape the city prior to the
hurricane were primarily the underprivileged, and illness was exacerbated by the lack of
availability of medical provisions and personnel. The eye of the hurricane traveled through
Mississippi,  the fourth largest poultry-producing state in the U.S., with the highest rainfall
amounts (ranging between 12.5-22.5 centimeters, falling at a rate of 1-2 cm per hour) tracking
over the central and northeastern portions of the state. As can be seen in Figure 4, these regions
are associated  with the bulk of large concentrated swine feeding operations in the state of
Mississippi.  The pollution from these operations has been previously reported to
disproportionately affect impoverished and African-American peoples  (Wilson et a/., 2002).  The
full breadth of public health outcomes from this hurricane, as well as potential environmental
injustice resulting from the flooding, have yet to be fully understood. These events, however,
signify the need for water quality officials to seriously consider precipitation events during
6.5 Economic considerations
Infection by zoonotic pathogens results not only in extensive human suffering, but also
significant economic loss.  For instance, the Milwaukee outbreak of Cryptosporidiosis in 1993
cost the community as much as 96.2 million; 31.7 million in medical costs and 64.6 million in
lost productivity (ASM, 1998; Corso etal., 2003; Water Health Connection, 2005). The
Walkerton, Ontario outbreak of E. coli O157:H7 and Campylobacteriosis in 2002, with 2300
cases and 6 deaths, cost the community an estimated 40 million in lost productivity, medicine,
and hospitalization costs.  The American Society for Microbiology estimates that even a mild
case of diarrhea may cost $330 in lost work productivity and over-the-counter medicines
(adjusted to 2005 dollars).  More severe cases were estimated to cost up to $9,500 per person for
medical diagnosis and treatment. Considering the number of illnesses that may be experienced
in the U.S. each year, foodborne illnesses may amount to three billion dollars per year due to
hospitalization and more than 20 billion in lost work productivity and over-the-counter
medicines, a significant portion of which may be due to transmission of disease from livestock
animals. Similarly, waterborne illnesses may result in a total of two to twenty billion dollars in
costs annually (Garthright etal,  1988; Hardy etal, 1994; Gerba, 1996; Liddle etal, 1997;
Fleisher et a/., 1998; Scott et a/.,  2000; Dwight et aL, 2001; Fruhwirth et a/., 2001; Corso et al.,
2003).  Considering the annual healthcare costs of managing antimicrobial resistance, which may
be in the range of 4-30 billion dollars (Khachatourians, 1998; American Academy of
Microbiology (AAM), 1999; Montague, 2000), the annual costs associated with illness caused by

               Value of Poultry and Eggs as a
               Percent of Total Market Value of
               Agricultural Products Sold, 2002
               Value of Hogs and Pigs as a
               Percent of Total Market Value of
               Agricultural Products Sold, 2002
               Average Number of Cattle and
               Calves per 100 Acres of All Land
               in Farms, 2002
Observed Precipitation,
  August 24-30,2005
                                                  0.0 - 0.9 * 2.0 - 3.9
                                                  1,0-1.9 *a.O-6.9
                                                         • 7.0-9.9
                                                 Maximum Precipitation Rate, August 23-31,2005
                                                August 2&1.
                                                                  Raid Rait (ran/hO

                                                            (i         50   30        50
Figure 4.       Distribution of livestock animals in regions impacted by Hurricane Katrina, August, 2005
               (adapted from USDA, 2002; National Oceanic and Atmospheric Administration, 2005;
               National Aeronautics and Space Administration, 2005).

zoonotic pathogens and antimicrobial resistant bacteria from livestock operations may be

Significant economic losses may also be incurred by the closing of beaches when waters cannot
meet USEPA recreational water guidelines. Of the thousands of beach closings every year, more
than 80 percent are due to excessive levels of bacteria (ASM, 1998).  A beach closing due to
bacteria indicates that levels were excessive the day prior to the closing, during which time
thousands of individuals may have been exposed to contaminated water. Dwight et a/., (2001)
estimated the economic burden from illness associated with recreational coastal water pollution
at Newport and Huntington Beaches, Orange County, California alone to be 3.3 million per year.
Considering the thousands of beaches closed every year, economic losses may be in the billions
of dollars.
6.6 Discussion
Both waterborne outbreaks and those associated with fresh produce have been on the rise in
recent decades and will likely continue to increase as surveillance is improved. Although the
source of contamination in many of these outbreaks remain unreported, poor manure
management in livestock operations most assuredly plays a significant role as alternative sources
of contamination are limited in scale compared to manure applications and typically much less
infectious in character.  The annual costs of infectious zoonotic diseases in the U.S. may reach
into the tens of billions of dollars considering both food and waterborne illnesses.  These
estimates exclude such costs as death, pain and suffering, lost leisure time, financial losses to
food establishments, legal expenses, and long-term health outcomes due to infections that may
result in degenerative diseases or cancer. The economic burden of pathogenic zoonoses has been
shifted from corporate farms who fail to use appropriate manure management at the source of
disease to sickened individuals and businesses that  experience decreased revenues due to beach
closures and lost productivity. Economic burdens of CAFO pollution may be especially
shouldered unfairly by minority groups and the poor, as evidenced by recent works describing
environmental injustice surrounding the swine industries of North Carolina and Mississippi
(Wing et a/., 2000; Wilson et a/., 2002). It is  at present unclear how new molecular
microbiological technologies such as microbial source tracking will affect litigation and potential
liability of CAFO operators in future disease outbreaks.

7.  Emerging Technologies:  Monitoring Pathogens in the
Concentrated animal feeding operations may release pathogens into the environment through a
variety of mechanisms that may result in extensive human suffering and economic loss. Current
surveillance activities may be inadequate to identify fully the scope of problems surrounding the
release of overt pathogens from CAFOs to the environment. For instance, surface water quality
surveillance in the U.S. relies on the quantitative detection of bacterial indicators of fecal
pollution including E. coli and enterococci rather than direct identification of selected etiologic
agents associated with disease in humans.  Although related to gastrointestinal illness following
recreational water contact, these indicators may not be reliable surrogates for all bacterial
pathogens and most parasites and viruses.  Human illness can  occur even when the
concentrations of E. coli and enterococci indicate that bathing waters are safe.  The use of
microbial indicators as surrogates for pathogens continues because infectious concentrations of
pathogens in waters may be low and difficult to detect, and standard methods for analysis do not
exist for many pathogens.

The February 28, 2005 ruling by the 2nd U.S. Circuit Court of Appeals required the USEPA to
identify and characterize the performance of animal waste management practices and barrier
technologies that specifically address contamination of the nation's waters by pathogens
emanating from CAFOs. Transport properties and the virulence of various pathogens vary to a
wide degree, and may be poorly represented by the bacterial indicators E. coli and enterococci
(Ferguson et a/., 2003). Thus, this requirement signals the need for new and improved pathogen
detection technologies. New approaches to water quality monitoring and emerging technologies
enabling the identification of overtly pathogenic agents in natural waters and their source will
greatly improve human health and welfare and increase the biosecurity of our natural resources.

Considering the many potential exposure routes following release of pathogenic zoonoses from
CAFO facilities, identification of the risks  associated with pathogens emanating from
concentrated animal feeding operations may require technologies that enable the measurement of
overt pathogens in air, drinking and recreational water, meat and produce,  soil  and sediments,
and feces of various animals among others. For foods, standard methods for analysis for a small
number of zoonotic pathogens already exist (FDA, 2005). For other matrices,  such as soil and
sediments, drinking water, and natural waters, methods are lacking or have not been
standardized.  Methods reported in literature include classical  cultivation approaches, and more
recently, identification and quantification of agents via the detection of surface antigens or
nucleic acids.  In either case, the detection  of zoonotic pathogens with infectious doses as low as
a few ingested or respired particles presents specific challenges including concentration of large
environmental samples, removal of inhibitory compounds from sample concentrates, detection of
viability, detection of multiple agents in a limited sample, and long analysis periods, especially
for cultivation-based approaches.  Considerable advances in emerging nucleic  acids and sensor
technologies are reducing  analysis times from weeks to hours  in some instances, but present
trade-offs in terms of the costs and technical expertise required to apply the technologies.  The
intent of this review is not to provide a complete description of all of the emerging detection
technologies and report their application, but rather to identify their strengths and limitations and
discuss the challenges posed by their use.

7.1 Sample Processing
Appropriate sample processing is critical to detecting pathogenic agents at concentrations
relevant to their infectious dose in environmental matrices. For some media that may contain
high numbers of pathogens but are extremely heterogeneous such as animal manure, obtaining an
appropriate sample may hinge on careful compositing procedures. For instance, Pearce et a/.,
(2004) examined the distribution of E. coli O157 in bovine fecal pats and determined that the
density of O157 in the pats was highly variable, differing by as much as 76,800 CFU/g between
samples of the same fecal pat. These researchers determined that most positive samples
bordered the detection limit, and that testing of only 1 g per pat (as is commonly performed) may
result in as much as 20-50% false-negatives. For other media such as air, sample processing may
need to be particularly careful regarding stressing organisms in the sampling device.

The low infectious dose associated with many etiological agents leads to the need to concentrate
copious quantities of air, food, or water into smaller volumes amenable to detection with
classical cultivation or the newer molecular microbial methods.  Several mechanisms have been
used to concentrate these agents to detectable numbers including filtration, immunocapture, and
enrichment.  However, some of these methods may concentrate  inhibitory and/or interfering
compounds with the agent of interest, while others require additional analysis time or alter the
sample from its initial state. Thus, the use of any of these mechanisms may present tradeoffs in
downstream analysis, potentially affecting analytical detection limits, analysis times, or the
number of agents that can be detected from a single sample.  At present, standard methods for
the concentration of viruses from water samples rely on electrostatic capture from 100 or more
liters of water onto positively-charged filters followed by elution, precipitation, and resuspension
in a small volume of sodium phosphate buffer (USEPA, 1993).  Concentration of the protozoan
parasites Giardia and Cryptosporidium require filtration often or more liters of water through a
depth filter followed by elution, centrifugation, and immunomagnetic separation (USEPA, 2001).
Concentration of bacterial pathogens is usually performed by membrane filtration, although
turbidity of water can severely inhibit the volume of water that can be passed through the  filter.

None of the accepted concentration techniques listed above is applicable to all of the various
groups of etiologic agents (viruses, bacteria, protozoans).  The detection of several agents may
require the collection of multiple large volume samples from a single location and concentration
by a number of techniques. To overcome these limitations, newer methods for sample
concentration applicable to all classes of etiologic agents have been proposed.  Most notably,
hollow-fiber ultrafiltration has been used to simultaneously concentrate viruses, bacteria, and
protozoan parasites from water samples as large as 100 L to volumes as low as 250 mL with
recovery efficiencies on the order of 20-92% (Juliano and Sobsey, 1997;  Kuhn and Oshima,
2001; Olsezewski etal, 2001; Evans-Strickfaden etal, 1996; Simmons etal, 2001; Morales-
Morales etal., 2003; Ferguson etal., 2004). Subsequent analyses with small portions of a single
eluent can lead to detection of several pathogens at environmentally-relevant concentrations
(Olsezewski et a/., 2001; Morales-Morales et a/., 2003). Hollow-fiber ultrafiltration may  also
have the added benefit of allowing small or water soluble inhibitors of nucleic acids techniques
to pass into the permeate, rather than co-concentrate with the pathogens in the retentate, prior to
sample analysis (Wilson, 1997).

7.2 Conventional Cultivation and Nucleic Acids Approaches
Conventional cultivation methods for the detection of bacterial pathogens usually require several
steps including (1) sampling and release of bacteria from the environmental matrix, (2) pre-
enrichment in non-selective broth to allow small numbers of stressed bacterial pathogens to
recover and grow prior to applying further environmental  stress in selective broths, (3) transfer to
selective broth to enrich low numbers of pathogens and reduce competitor bacteria, (4)
inoculation of a selective solid medium to identify presumptive positive colonies, and (5)
biochemical and/or serological confirmation of presumptive-positive colonies.  Most probable
number (MPN) techniques can be used to arrive at a quantitative result (USEPA, 2005).
Depending on the number of steps required; confirmation of the presence of specific pathogens
by conventional methods may take as few as two days to two weeks or more.

Nucleic acid technologies follow the same framework for detection as cultivation methods, but
detection or quantitation can occur prior to or following pre-enrichment in nonselective media or
further enrichment in selective broths. Nucleic acid technologies are also commonly used in lieu
of biochemical and/or serological confirmation for presumptive colonies, or to acquire more
detailed genomic information on bacterial isolates, such as possession of antimicrobial-resistance
or virulence traits. Enrichment broths (both selective and nonselective) for  nucleic acids
techniques are used not only to increase  the numbers of pathogens, thereby improving detection,
but also to dilute potential inhibitors of the polymerase chain reaction (PCR). Immunomagnetic
separations have also been used to separate etiological agents from large volumes and/or samples
with inhibitory agents prior to or following enrichment steps.

The detection limit of nucleic acid assays is usually dependent on the amount of time available
for  analysis. In general, if the etiological agent of interest is in high concentration or the medium
is relatively clean (such as drinking water), short analysis times of less than 6-8 hours can be
realized, as inhibitors may be in low concentration relative to pathogens in the original sample
retentate.  In more turbid samples with low numbers of infectious agents, such as stream waters,
analysis times can extend from a day to as much as four days.  Extensive sample processing and
selective enrichments may be required to achieve detection limits relevant to the infectious dose.
Tables 9 and 10 list several studies that have used nucleic acids techniques for the detection of
etiological agents.

As  can be seen in Table 9, nucleic acids  techniques may provide very sensitive detection of
selected etiological agents in clean samples, such as air or drinking water, even without
enrichment. For turbid environmental samples, such as surface water, feces, or soils, nucleic
acid technologies may have detection limits several orders of magnitude higher than cultivation
techniques (See Table 9).  This is because PCR inhibitors, such as humic substances associated
with many environmental samples, are co-extracted with the etiological agent prior to detection.
Even with inhibitors present, nucleic acid technologies may occasionally yield more sensitive
results than cultivation-based techniques. For instance,  Inglis and Kalischuk (2004) used nested
real-time SYBR Green-PCR to quantify  Campylobacter lanienae in cattle feces without
enrichment. These researchers were able to detect C. lanienae at concentrations as low as 250
CFU/g feces in less than 4 hours, a level more  sensitive than could be achieved with cultivation-
based methods that required 2 days for presumptive results.  However, in most instances,

Table 9.       Sample times and detection limits of several nucleic acids-based techniques for detecting
              pathogens in different matrices without enrichment.
Etiologic Agent
No Enrichment
Anthrax Spores
E. coli
E. coli O157:H7
Hepatitis A
Hepatitis A
Campylobacter spp.
Listeria monocytogenes
E. coli O157:H7
Campylobacter jejuni
Campylobacter lanienae
Clostridium difficile
Salmonella spp.
Staphylococcus aureus
E. coli O157:H7
E. coli O157:H7

Drinking Water
Drinking Water
Ground Water
Sewage effluent
Sewage effluent
Cattle Feces
Cattle Feces
Cattle Feces
Human feces

Detection Limit

1 spore/100 L
20,000 CFU/100 mL
1,000,000 PFU/mL
3,000 PFU/mL
1,000,000 PFU/surface
<100,000 CFU/10 g
100 CFU/g
26,000 CFU/g
3,000 CFU/g
250 CFU/g
50,000 CFU/g
106 CFU/g
106 CFU/g
26,000 CFU/g
35,000 CFU/g

Makino and Chuen (2003)
Abd El-Haleem et al. , (2003)
Abd El-Haleem et al. , (2003)
Vaughn e/ al, (2003)
Jean et al., (2001)
Jean et al., (2002)
Jean et al., (2001)
Uyttendale etal., (1995-1997)
Rodriguez-Lazaro etal.,
Ibekwe and Grieve (2003)
Inglis and Kalischuk (2004)
Inglis and Kalischuk (2004)
Burtscher and Wuertz (2003)
Burtscher and Wuertz (2003)
Ibekwe and Grieve (2003)
Ibekwe et al., (2002)
No Enrichment, Immunomagnetic Separation
Crypto sporidium parvum
Cryptosporidium parvum
Hepatitis A
Enterohemorrhagic E. coli
Campylobacter jejuni
Campylobacter jejuni
Clean Water     <24
Turbid Water     <24
Ground Water      6-12
Chicken Rinsate   <24
Chicken Feces      6
Chicken Ceces      6
  50 Oocysts/100 L  Baeumner et al., (2001)
  50 Oocysts/100 L  Baeumner et al, (2001)
  20 PFU/20 mL   Abd El Galil et al., (2004)
  55 CFU/mL      Call et al., (200Ib)
  230 CFU/g       Rudi et al., (2004)
2,000 CFU/g
Rudi et al, (2004)
       Meat = beef, poultry, or pork
improved sensitivity over cultivation-based techniques will not be realized without further
sample processing.

Immunomagnetic separation methods (IMS) have been used successfully in several studies to
concentrate etiological agents prior to or following sample concentration. These methods use
paramagnetic particles coated with antibodies specific to the pathogen of interest to bind the
agent and remove it from the sample matrix in a concentrated form via magnetic attraction of the
pathogen-paramagnetic particle complex (Campbell and Smith, 1997; Bukhari etal.,  1998;
Rochelle etal., 1999). The separation of the  agent of interest from the environmental sample
reduces the presence of inhibitory substances improving detection by PCR.  For instance, Abd El
Galil et al, (2004) developed a protocol for using combined Immunomagnetic Separation-
Molecular Beacon-Reverse Transcription-PCR to detect Hepatitis A virus in groundwater
samples. These authors  concentrated 100 liters of groundwater using an electropositive

microporous (1-MDS) filter followed by elution in beef extract, centrifugation, and resuspension
of the pellet in 20 mL sodium phosphate buffer (final pH=7.4).  Immunomagnetic separation
with two-hour incubation was used to recover the virus followed by extraction of viral RNA,
reverse transcription, and real-time PCR with a molecular beacon probe. As few as 20 plaque-
forming units (PFU) per 20 mL groundwater concentrate were recovered by these methods.
Immunomagnetic separation methods may be attractive where selective enrichment cannot be
used effectively.

Selected amplification facilitators or specific DNA treatments may also be used to reduce (but
not eliminate) the effects of inhibitory compounds on PCR (Satoh etal., 1998; Abu Al-Soud and
Radstrom, 2000; Boddinghaus et a/., 2001).  Common facilitators  may include bovine serum
albumin (BSA), the single-stranded DNA-binding T4 gene 32 protein (gp32), betaine, and
several proteinase inhibitors, all of which work to a varying degree depending on sample type
(Abu Al-Soud and Radstrom, 2000). Of the facilitors used, BSA is the most common, and tends
to work with samples from a wide variety of origins, including blood, human and animal feces,
surface and ground waters, soils and sediments, and meat. For instance, Rudi et a/., (2004)
applied integrated cell  concentration and DNA purification using immunomagnetic beads with
real-time (TaqMan) PCR to detect and quantify Campylobacter jejuni in chicken fecal samples.
These researchers could detect as few as 1,000-10,000 CFU/g feces in untreated samples, but
with 0.4% BSA in the reaction mix, could reduce PCR inhibition caused by the fecal extract, so
that they could reduce their detection limit to as low as 230-2,300  CFU/g feces. Since the mode
of action of many facilitators is similar (removal of inhibitors), their benefits are not additive
(Abu Al-Soud and Radstrom, 2000).

To improve detection of bacterial pathogens  in difficult matrices, especially for particularly
infectious agents that may be of interest at very low concentrations, enrichments can be used to
revive stressed bacteria and  increase their numbers prior to detection. Enrichments have the
added benefit of diluting PCR inhibitors prior to detection by nucleic acid techniques, resulting
in lower detection limits than can be realized by direct sampling (See Table 10). The enrichment
of bacterial pathogens presents  a trade-off: selective media may enrich one pathogen at the
expense of other agents of concern.  Several  enrichment broths may be required to detect several
agents.  However, in some cases, nonselective broths may improve the detection of many
bacterial pathogens following a single enrichment. For instance, Nam et a/., (2004) evaluated
the  use of universal pre-enrichment broth (UPB) versus selective enrichment broths  [lactose
broth, modified trypticase soy broth (plus novobiocin), and Listeria enrichment broth] for
detection of Salmonella spp., E. coli O157:H7, and Listeria monocytogenes from dairy fecal
slurry, lagoon water, drinking water, silage/feed, trapped rats, bird droppings, calf fecal swabs,
milking parlor floor swabs, bulk tank milk, and in-line milk filters. These researchers observed
no differences between growth in UPB and selective media using pure cultures of the three
pathogens, either individually or mixed.  However, slightly  better recovery of pathogens from
environmental samples was  observed when UPB was used for the  initial enrichment step, and
transfers were made from the single pre-enriched sample to selective media. UPB supported the
growth of all three pathogens to levels detectable by culture techniques within 24 hours from the
different environmental matrices.

Table 10.      Sample times and detection limits of several nucleic acids-based techniques for detecting
               pathogens in different matrices following enrichment.
Etiologic Agent
Detection Limit
Nonselective Enrichment
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Salmonella spp.
E. coli O157:H7
Salmonella spp.
Salmonella enteritidis
Salmonella typhimurium
Vibrio spp.
Salmonella spp.
E. coli O157:H7
Salmonella spp.
Salmonella spp.
E. coli O157:H7
Salmonella spp.
Staphylococcus aureus
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
Selective Enrichment
Campylobacter jejuni
Campylobacter coli
E. coli O157:H7
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Salmonella spp.
Listeria monocytogenes
Vibrio parahaemolyticus
Campylobacter spp.
Listeria monocytogenes
E. coli O157:H7
Listeria monocytogenes
Listeria monocytogenes
Yersinia enterocolitica
Salmonella spp.
Drinking Water
Surface water
Apple Juice
Chicken Rinsate
Cattle Feces

Surface Water
Surface Water
Surface Water
Dairy Products
Dairy Products
Sewage Sludge

100 CFU/100 mL
600 CFU/100 mL
100 CFU/100 mL
4 CFU/25 g
100 CFU/100 mL
Equal to cultivation §
<10 CFU/25 g
100 CFU/g
100 CFU/g
Equal to cultivation
580 CFU/g
1500 CFU/25 g
Equal to cultivation
1200 CFU/g
10 CFU/g
10 CFU/g
10000 CFU/g
6 CFU/g
2 CFU/g
<10 CFU/g
<10 CFU/g

Equal to cultivation
Equal to cultivation
120 CFU/100 mL
<10 CFU/10 g
<10 CFU/10 g
<10 CFU/60 g
Better than cultivation §
<10 CFU/60 g
Better than cultivation
10 CFU/10 g
<10 CFU/10 g
120 CFU/100 mL
10 CFU/g
10 CFU/g
10 CFU/g
10 CFU/g
Campbells ai, (2001)
Campbells al., (2001)
Porting al, (2001)
Liming and Bhagwat (2004)
Porting/ a/., (2001)
Cooked al., (2002)
Lee et al., (2003)
Lee et al., (2003)
Cheung et al., (2004)
Burtscher and Wuertz (2003)
Burtscher and Wuertz (2003)
Campbells al., (2001)
Campbells al., (2001)
Campbells al., (2001)
Ibekwe and Grieve (2003)

Sails et al., (2002)
Sails et al., (2002)
Uyttendale etal., (1995-1997)
Uyttendale etal, (1995-1997)
Blackstone et al., (2003)
Uyttendale etal, (1995-1997)
Uyttendale etal, (1995-1997)
Burtscher and Wuertz (2003)
Burtscher and Wuertz (2003)
Burtscher and Wuertz (2003)
Burtscher and Wuertz (2003)
        Meat = beef, poultry, or pork
        Nucleic acid techniques provided a result equivalent to or better than cultivation methods based on trials in
        actual samples

Several studies have documented improvements in detection of bacterial pathogens using nucleic
acids techniques that can be realized using enrichments in both non-selective and selective
broths.  Ibekwe and Grieve (2003) used real-time TaqMan PCR to with a detection limit of
26,000 CPU E. coli O157:H7 per gram of soil without enrichment. Using a 16 hour pre-
enrichment in modified Luria-Bertani broth (containing vancomycin, cefoxime, and cefsulodin);
these researchers could reduce their detection limits to less than 10 CPU per gram soil.
Burtscher and Wuertz (2003) evaluated the use of PCR for the detection of Salmonella spp.
Listeria monocytogenes, Yersinia enter ocolitica, and Staphylococcus aureus in biosolids from
anaerobic digesters and aerobic composters following one- and two- step enrichment (24-48
hours) in selective broths.  These researchers were able to detect less than 10 CPU per gram of
waste for each organism following enrichment, versus 106-107 CPU per gram of waste without

Nucleic acids techniques may also be useful as a surrogate for biochemical confirmation or for
genotyping environmental isolates following detection with cultivation techniques, potentially
saving days in analysis time. Miiller et al., (2003) tested sewage sludges and river waters for E.
coli O157:H7 using combined cultivation and nucleic acids techniques.  These researchers
filtered 100 mL river water samples through 0.45  jim nitrocellulose membranes then enriched
the retentate in peptone-saline water (PSW) supplemented with vancomycin-cefixime-cefsulodin
solution for six hours.  Similarly, 100 jiL sewage sludge was enriched for six hours directly in
the antibiotic-PSW solution.  Immunomagnetic  separation was used to isolate E. coli O157:H7
from a small portion of the enrichment media, and the paramagnetic bead-bacteria complexes
were further enriched on selective media for 24  hours. Suspect colonies were investigated with
PCR targeting genes associated with Shiga-like  toxins 1 and 2, attachment and effacement, and
enterohaemolysin. With these methods, the researchers could detect as few as 120 CPU/100 mL
in sewage sludge and river samples in less than 48 hours.

Several researchers have also noted that PCR detection of antibiotic-resistance traits is more
rapid and sensitive, and potentially more cost-effective, than culture or selective media.   This is
attractive for clinical diagnosis and surveillance (Levesque et al., 1995; Briggs et al., 1999; Paule
et al., 2001; White et al., 2001; Paule et al., 2003; Blickwede and Schwartz, 2004; Sundsfjord et
al, 2004;  Shamputa et al., 2004; Jalava and Marttila, 2004). However, in some cases, resistance
to antimicrobials can be phenotypically observed with the lack of detection of antimicrobial
resistance determinants (Patel et al.,  1997). This may occur, as antimicrobial resistance may be
conferred by several different genes, not all of which have been characterized. Because of the
potential risks associated with misdiagnosis of disease, resistance screening by molecular
methods in a clinical setting should be used as a compliment to classical phenotypic approaches.

Other problems may arise when using nucleic acids techniques if researchers are not careful with
their methods. Of particular concern is establishing detection limits for nucleic acid techniques
for pathogens in food, clinical diagnostic samples, and environmental matrices. Detection limits
should be established with samples that closely mimic the matrix of interestenvironmental
conditions. If detection limits are reported that over-estimate the efficacy of the method, their
use for surveillance or diagnosis may put the public at risk.  For instance, Lyon (2001) developed
a real-time (TaqMan) PCR method to detect Vibrio cholerae Ol and 0139 in raw oysters
without enrichment. This researcher spiked 25 g oyster homogenates with a single inocula

(approximately 6.2 x 106 V. cholerae Ol and 6.7 x 106 V. cholerae O139), then serially diluted
with alkaline-peptone water to 6 logio the original concentration.  Because both organisms could
be detected in the most dilute samples, a detection limit of 6-8 CFU/g oyster was reported.
However, by diluting their samples up to six-fold in alkaline-peptone water instead of unspiked
raw oyster homogenate, they may have diluted out a significant amount of PCR inhibitors for
their most dilute samples.  The true detection limits of this assay are unclear.
7.3 Pathogen Viability
For pathogenic zoonoses in different environmental matrices to pose a threat to human health,
they must be in a viable state.  The detection of viability by either cultivation-based techniques
or nucleic acids approaches, however, may not be straight-forward.  Cultivation techniques
require the viability of microorganisms to yield a result. However, viable-but-nonculturable
(VBNC) cells can remain undetected and may complicate interpretation of results. Aside from a
clear definition of what constitutes a VBNC state (Barer and Harwood, 1999; Kell etal., 1998;
Keer and Birch, 2003; Besnard et al., 2000; del Mar Lleo et al., 2000; Grey and Steck, 200Ib;
Nilsson etal.,  1991; Turner etal., 2000: Bogosian etal., 2000), it still remains unclear as to
whether cells in a VBNC  state are pathogenic (Barer et al., 2000; Grimes et al., 1986; Steinert et
al., 1997; Grey and Steck, 200la; Cappelier etal., 1999).  What is known, however, is that
degradation of nucleic acids in VBNC cells may proceed at much slower rates than in killed
cells.  In fact, some studies have indicated that the pool of messenger RNA (mRNA) may
stabilize within VBNC cells rather than continually degrade (Thorne Williams, 1997; Smuelders
et al., 1999). Indeed, VBNC bacterial pathogens may harbor genes encoding antimicrobial
resistance and  other virulence mechanisms for long periods of time after entering a VBNC state
(Chaiyanan et al., 2001), serving as a potential reservoir for virulence determinants in the
environment. It is clear that molecular methods cannot differentiate between viable and VBNC
pathogens when nucleic acids persist in the cells (Thorne and Williams, 1997; Smeulders etal.,
1999; Lazaro etal., 1999; del Mar Lleo etal., 2000; Wei chart etal., 1997).

Nucleic acid techniques may yield more rapid and sensitive results than cultivation-based
techniques for detecting pathogens in different matrices, but there still remains a question as to
what a positive PCR result means. Presence of DNA is not a reliable indicator of bacterial
viability (McCarty and Atlas, 1993; Masters  et al., 1994; Deere et al., 1996; Hellyer et al., 1999).
Ribosomal RNA has been shown to be a better indicator of bacterial viability due to a more rapid
degradation than DNA upon cell death, but may not be reliable in all cases (McKillip et al.,
1999; Villarino et al., 20001 McKillip et al., 1998; Meijer et al., 2000; Tolker-Nielsen et al.,
1997). Because of its extremely short half-life following cell death (seconds), mRNA may be
the most reliable nucleic acid for indicating cell viability (Keer and Birch, 2003). However, it
has been shown that mRNA may still persist.  Therefore, care must be taken to design probes and
primers that target regions of mRNA more susceptible to degradation (Cook, 2003).

Detecting mRNA requires a higher level of technical expertise than standard DNA-based
methods, and does not lend to direct quantitation of pathogens in a sample as multiple and
variable quantities of mRNA may be present in a single cell.  Quantitative results may, however,
be achieved using MPN techniques. Two methods commonly used for detecting  ribonucleic
acids are reverse-transcriptase PCR and nucleic acid sequence-based amplification (NASBA).

Enrichment of environmental or food samples prior to detection may enhance method sensitivity
and aid in the detection of viable versus non-viable cells.  Considering the potential of VBNC
pathogens to act as environmental reservoirs for virulence determinants, as well as the ability of
nucleic acids techniques to detect these cells, molecular methods may offer a distinct advantage
over more classic cultivation-based assays for the protection of human health and the
7.4 Emerging Surveillance Technologies
Considerable advances in nucleic acids and sensor technologies continue at a rapid pace (Walker,
2002; Dunbar et al., 2003; Petrenko and Vodyanoy, 2003; Turnbough, 2003; Olsen et al., 2003;
Greene and Voordouw, 2003; Grow et al., 2003; Unnevehr et al., 2004; Panicker et al., 2004;
Raymond et al., 2005). Of the emerging nucleic acids technologies, perhaps the most promising
for surveillance and biosecurity are microarrays. Call et al., (2003) and Ye et al., (2001)
describe the emerging use of microarrays and their potential for pathogen detection and
genotyping.  Microarrays are essentially a large set of very small southern blots, an array of
many nucleic acid probes complimentary to discrete gene sequences, bound to a solid or semi-
solid matrix, usually a modified glass surface.  Because of the miniscule size of the blots (100-
200 |im diameter "spots" separated from neighbors by typically 200-500  jim), thousands of
sequences can be  screened on a single array of less than four square centimeters. Target nucleic
acids, which are typically, but not necessarily, PCR products, are challenged by the microarray
under stringent hybridization conditions. Targets are usually prepared prior to hybridization with
fluorescent labels or incorporating specific chemistries such as biotin-streptovidin that permit
detection with a secondary fluorescent marker.  Once post-hybridization steps are complete,
arrays are catalogued using high resolution laser- or filter-based scanners and charged-coupled
device (CCD) imaging. Based on hybridization patterns between the spotted arrays and the
nucleic acids targets, the genotype of the original pathogen or the presence of specific pathogens
in complex samples can be identified.

Microarrays are presently limited to endpoint detection, rather than quantification,  of specific
microbial targets.  Although in some instances they can be used to detect nucleic acids isolated
directly from complex matrices, the sensitivity of microarrays severely impedes their use for
pathogen detection at the very low concentrations of interest in environmental samples without a
specific nucleic acid amplification strategy (Call et al, 2003). When coupled with nucleic acid
amplification techniques, microarrays have been used successfully to detect enterohemorrhagic
E. coli in chicken rinsate at concentrations as low as 55 CFU/mL.  Perhaps more promising,
microarrays  can be used to rapidly genotype specific pathogens with greater sensitivity
(Chizhikov etal,  2001; Call etal,  2001b; Johnson and Stell, 2000; Bekal etal, 2003). Whole
and partial genome microarrays for many pathogens are also commercially  available, allowing
for "fingerprinting" of microbial pathogens by establishing patterns unique  to particular species
that may  further enable genotyping studies. By challenging these arrays with nucleic acids of a
wide variety of sources, very small  sets of unique markers for specific pathogens may be
identifiable,  better enabling environmental pathogen detection.

Pathogen detection is performed routinely for meat, produce, seafood, milk, occasionally for
biosolids from human municipal treatment facilities, but rarely for environmental matrices such
as animal manure and their treated residuals, environmental waters, air, soils, and sediments. The
recovery and detection of pathogens in environmental matrices are imperative to identify the
extent to which these agents are removed, inactivated, or persist in livestock animal waste
treatment processes and management systems at CAFOs.  Conventional cultivation-based and
newer nucleic acids-based approaches to detect or enumerate etiological agents in some
environmental matrices are available.  However, these methods are not amenable across different
groups of pathogens or matrices and can complicate environmental sampling.  Very limited
standardization of pathogen detection methods exists, except for in the case of foods
(Association of Analytical Communities (AOAC), 2005; FDA, 2005; USD A, 2005). The
applicability of the standard methods for detecting pathogens in food to other matrices such as
feces, water, and soil has not been established. Standard methods with the required sensitivity
for detecting pathogens at relevant concentrations in environmental milieus are sorely lacking,
especially for hyper endemic or emerging pathogens such asE. coli O157:H7, Salmonella
typhimurium, Yersinia enterocolitica, Campy lobacterjejuni., swine hepatitis E virus and the
protozoan parasites Giardia lamblia and Cryptosporidiumparvum (Sobsey et a/., 2002).  The
efficacy of animal waste management systems for removing zoonotic pathogens and
antimicrobial-resistant bacteria from waste streams at CAFOs remains uncertain.

Aside from assessing the efficacy of livestock animal waste management systems, the recovery
and detection of pathogens in water is imperative to protecting human health and the
environment. The wise old adage of indicator organisms is becoming outmoded, as their
reliability to predict all waterborne outbreaks is uncertain, their results come "a day late and a
dollar short", and newer technologies are becoming available that negate the need to rely solely
on bacterial indicators of pathogenicity. Improving surveillance activities in recreational and
drinking waters will require these new methods for detecting pathogens to be available in near
real time.  However, this may be hindered by specific physical or chemical properties of
environmental waters that, combined with low concentrations of pathogens, may increase sample
processing times. In general, the cleaner the sample, the more methods will be effective for
rapid pathogen detection.  For clean matrices such as drinking water, sample concentration
methods alone may be sufficient to yield results directly usable by both cultivation and direct
molecular detection.  For more challenging matrices, such as ground and surface waters,
significant sample clean-up may be required to remove inhibitors prior to processing with
molecular methods.

As discussed above, the detection of etiologic agents in environmental waters is problematic,  not
only due to very  low (but potentially significant) concentrations, but also due to a lack of
methods with the required sensitivity and competing methods that are not amenable across
different groups of pathogens.  As such, there is a need to develop a unified and automated
system for the detection of all waterborne pathogens (Straub and Chandler, 2003). It has been
suggested that such technologies rely on nucleic acids analyses because they are amenable to
automation and are at present the most promising for rapid and specific quantitation of viable
microbial pathogens (Jothikumar et a/.,  1998; Levin et a/., 2002; Straub and Chandler, 2003).
Hollow-fiber ultrafiltration systems can concentrate all classes of pathogens in a single step and

can be reused (Kuhn and Oshima, 2001; Olszewski etal., 2001). Therefore, these filters may
serve well as a basis for sample concentration in such a system. More recently, renewable
surface technologies for automated sample processing coupled with microarray technologies
have also shown promise as a basis of such a system (Chandler et a/., 2000a,b). Nucleic acids
technologies, however, are still primarily in the hands of researchers and beyond the scope of all
but the most highly trained staff and most affluent utility laboratories (Levin et a/., 2002).
Significant investments need to be made in the development of simple and reliable technologies
that are less technically-demanding.

Several pertinent issues need to be addressed before nucleic acids technologies are exclusively
used as standard methods for surveillance activities, such as monitoring recreational and drinking
water quality.  First and foremost, there remains a need for regulatory establishment of
acceptable concentrations in environmental matrices to determine the relevancy of detection
limits established in these studies. Method development and standardization cannot proceed
until target detection limits that reflect true risks of illness are established. These limits need to
identify target  concentrations based on epidemiological studies of health risk rather than indicate
that pathogens should not be detected in a given sample volume. Regulatory guidelines should
also clearly indicate standards regarding acceptable recoveries from environmental samples
during concentration, analysis sensitivities, and standard errors. This is because sample
concentration methods may present a wide variation in recovery of etiological agents from
environmental waters, and regardless of the sample concentration methods used, PCR-based
detection systems must confront a number of front-end challenges inherent to complex
environmental waters that may reduce the sensitivity of the assay (Chandler, 1998; Loge et a/.,
2002; Call et a/., 2003). As noted by others, a positive detection is relatively simple to interpret.
However, knowledge of assay sensitivity, which varies from sample to sample, is critical to
interpreting negative results (Loge et a/., 2002;  Call et a/., 2003). It is possible that specific
sample properties,  such as the presence of PCR inhibitors, may affect the sensitivity of an assay
thereby  resulting in a false-negative result. The probability of false negative results will
increase with lower numbers of pathogens per sample.  Regulatory guidelines should consider
these limitations in order to reduce  the public health impacts of false-negative results.  Good
sampling designs need to consider how much sample is processed, the efficiency of pathogen
isolation, the efficiency of nucleic acid extraction, and the effect of co-precipitating factors that
inhibit PCR (Loge et al., 2002; Call et a/., 2003).

Considering that acceptable regulatory concentrations of specific pathogens in environmental
matrices will likely be very low, it is unlikely that current technology would be able to detect
pathogens in real-time. Measurement of pathogens to satisfy what would be regulatory levels
may still take 24-48 hours as they will require enrichment to increase pathogen numbers or
reduce co-precipitating factors.  This may also pose challenges as several different enrichment
media may be necessary to detect several different pathogens.  The only true method to alleviate
the need for enrichment to detect low numbers of pathogens in environmental samples would be
intensive sample concentration. Unless significant advances are made in sample concentration,
nucleic acid extraction methods, and nucleic acid clean-up to remove inhibitory compounds,
real-time pathogen detection will remain unrealistic.  This will remain a critical issue for
biodefense applications where near real-time identification of etiological agents may be
imperative to protecting human health.

8. Microbial Source Tracking
Microbial source tracking (MST) is a set of methodologies by which the animal or human source
of fecal pollution in a contaminated water body may be identified.  MST technologies rely on
phenotypic and genotypic differences in fecal microorganisms shed in the wastes of animals and
humans that make them unique to a particular animal host (host-specific). These differences
may arise due to variations in growth environments and the selective pressures of various animal
guts such as differences in diet, antimicrobial treatments, temperatures, pH,  and more. Upon
release into the environment, it is assumed that these organisms remain unchanged and migrate
with fecal pollution.  Their detection in concert with indicators of fecal pollution or overt
pathogens is thus assumed to be indicative of the animal source.

Knowledge of the source(s) of microbial contamination in water bodies and their relative load
contribution helps to focus remedial efforts and resources in the right direction at an earlier time.
Source identification may also enable investigation of best management practice (BMP)
effectiveness leading to improvements in total  maximum daily load (TMDL) development and
implementation.  There are many potential sources of bacteria extant in watersheds, and it is
important to be able to sort out the source of observed contamination so that an evaluation of the
effectiveness of control strategies can be made. MST may also improve enforcement activities
when discharges exceed permitted levels.

In MST, the selection of the right indicator is important, since it is the single element which
provides  the measurable parameters to determine the origin of the pollution. Both phenotypic
and genotypic technologies have been developed primarily using fecal coliforms, E. coli, fecal
enterococci, fecal streptococci and viruses. Some of these technologies are library-dependent;
they rely  on comparing fingerprint databases, either phenotypic or genotypic, of microorganisms
from known sources to the fingerprints of unknown samples. These may include antibiotic
resistance analysis (ARA), carbon utilization profiles (CUP), repetitive extragenic palindromic
PCR (rep-PCR) DNA fingerprinting, randomly amplified polymorphic DNA (RAPD) analysis,
amplified fragment length polymorphism (AFLP) analysis, pulse field gel electrophoresis
(PFGE), and ribotyping (Harwood, 2000; McClellan etal, 2001; Hagedorn etal, 2003; Carson
et al, 2003; Ting et al, 2003; Leung et al, 2004;  Scott et al, 2004; Webster et al, 2004). Other
MST technologies are  library-independent; they rely on the conservation of unique genetic
identifiers inherent to a specific fecal  microorganism endemic to the members of a single animal
species (the in-group) that are different from the genetic identifiers of the same or different fecal
microorganisms in other animals or humans (the out-group).  Examples of library-independent
MST technologies include gene-specific PCR, 16S rRNA gene clone libraries, and target-
specific PCR-based methods (Bernhard and Field, 2000; Khatib et al., 2002; Khatib et al., 2003;
Field etal., 2003;  Simpson, etal, 2003; Bonjoch etal, 2004; Scott etal, 2004; Simpson etal,
2004; Suerinck etal, 2005).

At present, MST studies have primarily employed library-dependent methods including ARA,
AFLP,  CUP, and ribotyping (EPA, 2005). Library-independent methods, especially gene-
specific and target-specific PCR, have been the focus of recent literature as extensive libraries
are not needed for their application, and they can be easily  and rapidly applied to source
identification studies.  Potentially, phenotypic  and genotypic methods could complement each
other according to training, equipment, and funding available. This section highlights the more

common methods of analysis that provide information about the source of microbial
contamination and the methods used to analyze the data obtained by these methods. A full
review of all available MST technologies is beyond the scope of this review.  An excellent
resource for further information regarding MST, its application, promises, and limitations is
available in the USEPA Microbial Source Tracking Guide Document (2005).
8.1 Antibiotic Resistance Analysis (ARA)
The antibiotic-resistance analysis (ARA) method has gained popularity over the last decade
because it is readily applicable and simple to use.  However, the classification accuracy is
usually lower than that of the molecular methods at the level of individual species. When animal
species are grouped into larger animal categories like human, livestock, and wildlife, the
accuracy improves notably to values of 95% or more.  This method has been reported to provide
sensible classifications of known and unknown fecal isolates and to resolve MST queries to
satisfaction in various case studies (Parveen etal.,  1997; Hagedorn, 1999; Harwood etal., 2000).

ARA is based on the following two premises (1) the use of antibiotics in humans and animals
can result in antibiotic-resistant bacteria, and (2) differences in the selective pressures resulting
from dosing with different types and concentrations of antimicrobials, as well as different growth
environments in various animal intestines, result in unique patterns of antibiotic resistance
specific to different animal types. The development and study of such specific  patterns are the
basis of this methodology, which uses fecal coliforms, E. coli, enterococci, or fecal streptococci
as indicators. The laboratory procedure requires only conventional microbiology training and
techniques.  Therefore, it is cost-efficient and can be rapid when performed by an experienced
research team. It begins with the recovery of the fecal bacteria from samples, mostly by
membrane filtration and incubation in or on selective media. It continues with the inoculation of
the isolated fecal bacteria onto agar or into broth medium containing a number of antibiotics at
increasing concentrations. Lastly, the results are evaluated by comparing the antibiotic
resistance profiles of the polluted water to the reference source library profiles.

The key to success of this method is having a representative source library with an acceptable
average of correct classification (ARCC). Most important is to perform a cross-validation test
before using the library to classify unknown isolates. The cross-validation test  can be done with
hold-out isolates or with new known isolates that are submitted as unknowns to the statistical
software (Harwood et a/., 2000).  The rate of correct classification from this test should not be
significantly different from the original rates obtained when the library was initially classified.
Similarly, it is important to use the antibiotics and the concentrations that provide the more
accurate classification of the library isolates. For instance (as noted above), several
antimicrobials used in animal agriculture have human-use analogs.  Resistance that develops in
livestock animals may therefore confer resistance to human antimicrobials and vice-versa.
Therefore, preliminary tests are recommended prior to the analysis of the water isolates
(Hagedorn et a/., 1999).  While this method does not classify individual animal species very
accurately, it has been used with satisfaction for human, poultry,  livestock and wildlife
categories (See  examples below). In most cases, this is sufficient and supports the development
of a restoration  strategy.

Several examples of the use of ARA for MST exist.  Parveen, et a/., (1997) used ARA
methodology to differentiate point-source (PS) from nonpoint-source (NPS) E. coli from the
Apalachicola Bay, Florida.  They used 765 isolates and obtained average MAR indexes of 0.25
for PS and 0.13 for NPS.  PS isolates showed higher resistance to single antibiotics and to
combinations of antibiotics than NPS isolates.  Sixty-five resistance patterns were observed for
PS isolates, compared to only 32 patterns for NPS isolates, when cluster analysis was used.
Wiggins (1996) developed a protocol to generate more extensive AR profiles by using various
concentrations for each antibiotic tested. He also introduced the use of a reference source library
and the multivariate analysis of variance discriminant function analysis to the  studies of MST by
ARA.  He studied 193 water fecal streptococci, with a source library of 1,435  fecal streptococci
isolates against a battery of five antibiotics at four concentrations each. An ARCC of 72% was
obtained when the source categories were analyzed at the species level and 82% when some
species were pooled into "poultry" and "beef categories. In general, increasing the number of
antibiotics used in an analysis increased the ARCC that could be achieved.

Others have further validated the ARA method by using additional statistical analysis. Hagedorn
(1999) created separate source databases with 7,058  and 892 isolates with ARCCs of 87% and
88%, respectively, which increased to 97% and 95% after pooling species into poultry and beef.
They used discriminant analysis to classify 4,615 water isolates from Page Brook River and
obtained 82% beef, 7.3% deer, 5.6% waterfowl, and 0% human, and 5.3% unknown.  Cluster
analysis was also used, which generated very good separation between sources with high
antibiotic resistance including chicken, dairy cattle and human clusters.  Among beef cattle and
deer, which had low levels of resistance, there was separation, but the deer tended to sub-cluster
within the larger beef cow isolate cluster. Most of the unknown source isolates were grouped in
beef cow, deer, and waterfowl clusters, whereas none grouped in the human cluster.  Based on
the results of this work, cattle access to the stream was reduced by fencing.

Harwood etal., (2000) obtained ARCCs of 64% and 62 % with a source database  of 6,144 fecal
coliform and 4,619 fecal streptococci isolates.  Similar to Wiggins (1996) and Hagedorn (1999),
the ARCCs improved to 75% and 72%, respectively, after pooling the species into human and
animal groups. Fecal coliforms from cattle were classified correctly at a higher rate than those of
fecal streptococci.  Conversely, fecal streptococci from humans were correctly classified at a
higher rate than those from fecal coliform isolates. Overall, the fecal coliform database had a
significantly greater ARCC  than the fecal streptococci database. Spearman's ranked correlation
using the percentage of correctly classified isolates versus the corresponding number of sampling
events resulted in a significant negative-correlation between sampling events and the percentage
of correctly classified isolates for the fecal coliform database, but not for the fecal streptococci
database. They analyzed 91 fecal coliform isolates from surface water receiving effluent from
faulty septic systems, 81 of which were classified into the human category. Similarly, 38 of 51
fecal streptococci from the same samples were categorized as human.  After the septic systems
were repaired, only 7.8% of fecal coliforms and 1.2% of fecal streptococci were classified as

Graves, etal., (2002) constructed a library of 1,174 enterococci isolates, and with two categories
(human and nonhuman) achieved an ARCC of 96%. By splitting nonhuman sources into
livestock and wildlife, they were able to achieve an ARCC of 92%.  They analyzed 2,012

enterococci isolates from a stream that drains a watershed with large populations of livestock and
wildlife and that passes through a community of 82 homes served by individual septic systems.
The yearly average classification was 10% human, 40% wildlife, and 50% livestock.  Burnes
(2003) analyzed 800 fecal coliform isolates from Big Creek, a mixed-use watershed, against a
source library of 1,125 fecal coliform isolates (human and non-human categories, ARCC=94%).
He found that human sources contributed greater than 50% of the base flow fecal coliforms in
urbanized areas. Chicken and livestock appeared to be responsible for the base flow fecal
coliforms found in rural reaches of the stream.  Hydrologic events changed the contribution of
each source to the stream such that fecal coliform pollution was 16% attributable to domestic
sources, 21% attributable to wildlife, and up to 60% attributable to chickens and other livestock
8.2 Ribotyping
Ribotyping is a DNA fingerprinting method that exploits small differences in 16S and 23 S
rRNA-coding regions of bacterial DNA to identify genetic relationships between unknown
bacteria and a set of known index organisms (Grimont and Grimont, 1986; Stull etal.,  1988;
Graves et a/., 1999). This method works on the premises that (1) multiple copies of the genes
encoding 16S and 23 S rRNA may appear within the bacterial genome with different flanking
restriction site locations, (2) there is variability amongst 16S and 23 S rRNA genes, and (3) there
is variability in the intergenic spacer region between 16S and 23 S rRNA genes.  Ribotyping
involves the culturing of a bacterium followed by DNA extraction and purification.
Subsequently, the DNA is digested with one or more enzymes and the digestion products are
separated by gel electrophoresis.  DNA bands are typically transferred onto a nylon membrane
and challenged by hybridization analysis with a chemically-labeled  nucleic acid probe.  The
probes may be generated from an index bacterium, such as a particular strain of E. coli, by
reverse transcribing the 16S and 23 S rRNA and labeling the cDNA  with a chemical labeling
scheme.  Because of small  differences in the restriction sites of different bacteria, the resulting
band patterns from the hybridization analysis will be distinct.  This pattern is called a ribotype.
By comparing ribotypes of unknown samples to a library of known  samples challenged by
probes from the same index organism, genetic and evolutionary relationships can be discerned.
Ribotypes may be translated to a binary code facilitating a discriminant statistical analysis to aid
in interpretation of results (Grimont and Grimont,  1986; Parveen, 1999; Carson etal., 2001).

In early years, ribotyping was used in epidemiological studies to characterize bacteria such as E.
co//', Salmonella enterica and Vibrio cholerae (Stull  et al., 1988; Olsen etal., 1992; Popovic et
a/.,  1993). Gradually, ribotyping found application in multidisciplinary areas such as plant
pathology, animal science, food technology, and MST (Nassar et a/., 1994; Nagai et a/., 1995;
Kilic etal., 2002; Scott etal., 2004). By comparing libraries of ribotypes grouped by animal
source, some researchers have noted that distinct bands unique to specific sources may emerge
from non-distinct bands, thus facilitating the identification of the source of pollution in unknown
samples. For instance, Parveen (1999) tested the applicability of this methodology to predict the
source of E.  coli pollution in the Apalachicola Bay, Florida. They analyzed a library of 238 E.
coli isolates and found that discriminant analysis of the ribotype profiles showed an ARCC of
82%. A total of 97% of nonhuman and 67% of the human isolates were correctly classified.
Carson etal., (2001) extended the application of ribotyping to distinguishE1.  coli from humans

and seven nonhuman hosts.  When ribotypes from a library of 287 E. coll isolates obtained from
humans, cattle, pigs, horses, chickens, turkeys, migratory geese, and dogs were used, the ARCC
was 73%.  By reducing the discrimination to human and nonhuman sources, these authors were
able to achieve an ARCC of 97%.  Using a library of 160 E. coli isolates, Scott etal., (2004) was
able to employ ribotyping to identify animals as the primary source of pollution in a water way
near Charleston in South Carolina. Prior to this  investigation, a significant human input was
suspected. Kuntz etal., (2003) successfully combined targeted sampling protocols with
ribotyping to identify the source of fecal contamination of Sapelo River in Georgia.  E.faecalis
DNA fingerprints in the river were a 43% match to those in a nearby wastewater lagoon,
suggesting that fecal contamination of the river originated from the wastewater treatment facility.

Other studies have used ribotyping successfully  but have noted caveats in its application.  For
instance, Hartel (2002) studied 568 E. coli isolates from different locations in Georgia and Idaho
to determine the geographic variability of E. coli from different animal species. They found that
the percentage of ribotype sharing within an animal species increased with  decreased distance
between geographic locations for cattle and horses, but not for swine and chicken. The data
suggested that the ability of libraries to classify unknown isolates is good provided both library
and unknown isolates belong to the same geographic area. Wheeler et al., (2002) explored the
potential of E.faecalis as a human fecal  indicator for MST using ribotyping. He analyzed fecal
samples from humans and a variety of livestock, domestic, and wildlife and found that the host
range of E.faecalis was limited to dogs, humans, and chickens and the ribotypes clearly
distinguished between human and chicken hosts. The dog isolates were apparently eliminated
when a protocol to quickly isolate E.faecalis was used.  Hartel et al., (2003) noted that ribotypes
of E. coli isolates from wild deer was significantly affected by their diets. Wild deer exhibited
35 E. coli ribotypes, whereas penned deer generated only 11.  Although issues of geographic
stability, host range of target bacteria, and host stability of ribotypes may be of concern in some
instances, ribotyping remains a preferred and well-accepted method for MST.  From the results
above, it appears to be a reliable tool to discriminate between pollution sources and provide
valuable information for water management purposes.
8.3 Amplified Fragment Length Polymorphisms (AFLP)
AFLP is a DNA-fingerprinting method based on the detection of characteristic differences in the
fingerprints of two genomes resulting from polymorphisms, insertions, and deletions that occur
within or immediately adjacent to restriction sites. In this method, genomic DNA is extracted
from the target cells and digested with a pair of restriction enzymes. Linkers specific to the
restriction enzyme sites chosen are ligated to the DNA fragments providing the sequences for
hybridization of PCR primers in the amplification steps.  As large numbers of different
fragments can be generated during digestion (more than 106 fragments per digestion of a genome
of 109 base pairs (bp)), selective DNA amplification is used to limit the number of amplification
products.  Selective amplification can be achieved through a variety of mechanisms including 3'-
extensions to one or both linkers, "pre-amplification" with only one primer complementary to
one of the restriction enzyme sites, inclusion of 3'-extensions on the "pre-amplification" primer
or one or both AFLP-PCR primers, and labeling only one of the AFLP-PCR primers. Amplified
products are separated on a denaturing polyacrylamide gel in an automated DNA sequencer, and
"fingerprints" are captured by specialized software which can scan the fingerprints for

discriminatory bands. Evaluation of the results by various statistical analyses can be performed
provided the DNA banding patterns are converted to binary form.

AFLP has proved to be highly discriminatory and reproducible when compared to other
molecular typing techniques. During the last decade, it has become a reliable tool to classify
bacteria to the strain level, as well as to perform genetic mapping of higher organisms (Janssen et
al, 1996; Desai etal, 1998; Savelkoul etal, 1999; lyoda etal, 1999; and Zhao etal, 2000).
Two reported evaluations of AFLP as a tool for MST stand out in literature.  Both investigations
compared AFLP to other fingerprinting techniques and utilized E. coli as the indicator organism.
In the first of these studies, Guan etal., (2002) studied a collection of 105 E. coli isolates from
the feces of cattle, poultry, swine, deer, goose, moose, and human samples and compared AFLP
to the ARA and the 16S rDNA methods. The results indicated that AFLP was significantly more
effective than the other two methods. Ninety-four percent of the livestock isolates,  97% of the
wildlife isolates, and 97% of the human isolates were correctly classified by AFLP. In
comparison, 46% of livestock isolates, 95% of wildlife isolates, and 55% of human isolates were
correctly classified by ARA, while 16S rDNA-based techniques resulted in 78%, 74%, and 80%
correct classification, respectively. Although additional isolates in the source library may
improve the ARCCs of the ARA and 16S rDNA methods, the resolution achieved by AFLP with
a small library was impressive in this instance.

In another study, Leung et al, (2004) used Shiga-toxin-producing E. coli (STEC),
enterotoxigenic E. coli (ETEC), and non-pathogenic E. coli from cattle,  swine, and  human
sources from very diverse geographic areas, including the U.S., Canada, Europe,  and Australia,
to construct source libraries.  A multiple-response permutation procedure analysis of the data
obtained with AFLP indicated that the seven groups defined by host-pathogenicity combinations
(bovine STEC, bovine ETEC, bovine non-pathogenic E. coli, human STEC, human ETEC,
human non-pathogenic E. coli and swine non-pathogenic E. coif) were significantly different.
Subsequently, stepwise discriminant function analysis was used to select 39 discriminant DNA
bands distinguishing the host specificity of the E.  coli strains for the analysis. The overall cross-
validation classification efficiency was 93.6% with 91.4% of human, 90.6% of bovine, and
97.7% of swine isolates being classified into their correct host types.

AFLP also distinguished the non-pathogenic E. coli from STEC and ETEC, and was able to
classify the strains based on both host specificity and virulence (Leung et al, 2004). Stepwise
discriminant function analysis selected 41 DNA bands to classify the isolates based on
pathogenicity with an overall cross-validation classification efficiency of 99.1% (100% of non-
pathogenic E. coli, 100% of STEC, and 90.9% of ETEC correctly classified).  Fifty DNA bands
were selected by that same means to differentiate  the seven host-pathogenicity combinations
(bovine VTEC, bovine ETEC, bovine non-pathogenic E. coli, human VTEC, human ETEC,
human non-pathogenic E. coli and swine non-pathogenic E. coli) with an average cross-
validation classification efficiency of 86.4% (individual group classification efficiency ranging
from 50 to 100%).  Despite the wide geographic origin of the E. coli strains in this study, AFLP
was capable of differentiating the E. coli strains with a high rate of correct classification from the
various hosts.

Like most methods used in MST, AFLP needs the development of a reference source library of
the indicator organism. Based on the reports above and the fact that AFLP screens the entire
genome, this tool has great potential for MST.  However, reports of case studies with successful
application of AFLP are necessary to further MST using AFLP.
8.4 Host-specific molecular biomarkers
Host-specific molecular biomarkers for MST studies, target-specific PCR-based methods, are
attractive because they offer rapid analysis (no source library or cultivation are needed) and
greatly reduced cost. They are technically less demanding than most alternative MST
techniques. Several bacterial targets have been proposed in the literature for MST applications
including Bacteroides, Bifidobacterium, Streptococcus Lancefield Group D, and Rhodococcus
coprophilus (Whitehead and Cotta, 2000; Vancanney et al, 2002; Bernhard et al., 2003; Bonjoch
et al., 2004; USEPA, 2005). However, since each marker is specific to a single animal host, a
combination of markers may be required to fully identify potential sources of contamination.
Because no cultivation is required, these methods are applicable to detection of several
biomarkers in a single sample, which may strengthen the argument for source identification
(USEPA, 2005).  For instance,  samples with positive results for several human-specific
biomarkers, such as human-specific Bacteroides spp. (Bernhard et al., 2003), both the human-
specific primer pairs for Bifidobacterium adolescentis and Bifidobacterium dentium (Bonjoch et
al., 2004), and the human-specific Enterococcus spp. esp markers (Scott et al., 2005), but
negative for the cattle-specific Bacteroides (Bernhard et al.,  2003) or E. coli LTIIa toxin (Khatib
et al., 2002), would strongly implicate a human rather than cattle source. At present, Bacteroides
markers are the most commonly used host-specific molecular biomarkers for MST (Bernhard et
al., 2003; Seurinck et al., 2005; USEPA, 2005). However, the use of these markers for resolving
watershed-scale microbial pollution is unknown.

The development of the human- and ruminant-specific Bacteroides biomarkers is described by
Bernard and Field (2000a,b). These researchers created 16S rDNA clone libraries of members of
the Bacteroides-Prevotella group from human and cow fecal samples.  Individual and pooled
clones were examined by length heterogeneity  (LH)-PCR and terminal-restriction-fragment-
length-polymorphism (T-RFLP) techniques. Sequencing and phylogenetic analysis
demonstrated that the human-specific sequences clustered together and were closely related, but
not identical, to sequences of Bacteroides vulgatus, which is commonly found in human feces.
The cattle-specific sequences formed the new gene clusters CF123 and CF151. All human- and
cattle-specific genetic markers were  found in DNA extracted from river and estuary water
contaminated with fecal pollution.

Primer sets were  developed to amplify  specific sequences within the Bacteroides-Prevotella
host-specific gene clusters (Bernhard and Field, 2000a,b): one forward primer specific for the
human-specific gene cluster HF8, two forward primers for the cattle-specific gene clusters
CF123 and CF151, and a general Bacteroides-Prevotella reverse primer for use with all three
forward primers.  These primers were successfully used to amplify 16 human and  19 cow fecal
16S rDNAs (Bernard and Field, 2000b). In subsequent work, Bernhard et al., (2003) used these
primers to test 22 water samples from Tillamook Bay and the results were congruent with land
use. For example, the human specific primer pair, HF183F/Bac708R, amplified only DNA from

waters around urban areas and sewage treatment plants, demonstrating specificity for human
Bacteroides DNA. The cattle specific primers, CF128F/Bac708R and CF193F/Bac708R,
amplified DNA from waters primarily near rural areas.  However, these primers amplified other
ruminant DNA as well. Therefore, positive results with these primers should be scrutinized
against land use and ruminant wild-life populations to prevent misinterpretation of results.  Dick
et al, (2005) have also recently published host-specific primers for swine and horse.

Relatively few other host-specific molecular biomarkers have been reported in literature (Khatib
et al, 2002a,b; Nebra et al, 2003; Bonjoch et al., 2004; Scott et al., 2005).  Most of these
markers target 16S rDNA, but some markers are emerging that detect alternative regions of the
bacterial genome, such as virulence genes (Khatib et al., 2002a,b; Scott et al., 2005).  The
efficacy of these and other biomarkers have not been well established.  Therefore, interpretation
of the presence of these molecular markers in different milieus may be complicated. Because of
the small number of environmental samples studied so far, host-specific molecular biomarker
technology needs further exploration in case studies to show field-applicability.
8.5 Discussion
Several methodologies are available for MST studies.  As shown by the publication evidence,
ARA represents a good tool for MST validated by various applications in real-life case studies.
However, specific considerations may limit its usefulness including human-livestock analogs,
and transfer of resistance traits in different milieus. Ribotyping is probably the most field-tested
method among the molecular methodologies used for MST. From the results above, it appears to
be a reliable tool to discriminate between pollution sources and provides valuable information
for water management purposes.  Although ribotyping may generate high ARCCs, it has also
been associated with a high cost and may be labor intensive, requiring long studies to achieve
results (Hartel et al., 2003). AFLP technologies are emerging as a promising tool for MST.
These methods offer a high degree of specificity, as they can screen the entire genome instead of
selected regions such as the 16S and 23 S rDNA screened by ribotyping. However, considerable
technical expertise and expense may be  needed to fully utilize the technique. Other molecular
methods, although sophisticated and capable of measuring parameters with high resolution, are
in various stages of development.  More research is needed for these methods to become
accessible for a broader population of users. As of today, these techniques have mostly been
used in feasibility studies with a small amount of isolates. Full-scale watershed studies are
needed to assess the potential of these technologies for future use.

Several factors may complicate the use of MST technologies in contaminated watersheds.  Poor
survival of reference organisms in the environment may result in little or no detection, limiting
the ability of the various methods to identify the source of fecal contamination (Simpson et al.,
2003; U.S.EPA, 2005). Even those reference organisms that are reasonably hearty may exhibit
variable survival times for different phenotypes or genotypes dependent on the environmental
milieu. This may lead to changes in genotypic and/or phenotypic signatures of the overall
populations and divergence from fingerprints in source libraries established with the raw fecal
material. For instance, Anderson et al.,  (2005) studied decay rates for fecal bacterial indicator
organisms (fecal coliforms and Enterococcus spp.) originating from dog feces, wastewaters, and
soil in freshwater and  sediment and saltwater and sediment. These researchers observed variable

decay rates based on fecal bacterial source, environment, and even ribotype. These changes may
complicate interpretation of environmental fingerprints and undermine source tracking efforts,
potentially resulting in misinterpretation of environmental data.

Another serious complicating factor is that the transport properties of different bacteria may vary
several-fold depending on the specific microbial agent and the milieu (Ferguson et a/., 2003). If
transport and survival of the index organism(s) used to identify fecal pollution source(s) do not
match that of pathogens emanating from potential sources, MST may yield questionable
information.  For instance, Simpson et a/., (2003) could not establish a relationship between the
molecular fingerprints of 16S rDNA fecal Bacteroides clones in  a large horse manure pile
immediately adjacent  to a receiving stream and downstream water as close as 5 m from the
manure pile.  In contrast, other researchers have noted significant overland and downstream
transport of antibiotic-resistant bacteria and several bacterial, viral, and parasitic pathogens, such
as Cryptosporidiumparvum, Salmonella spp., swine hepatitis E,  and Yersinia enterocolitica, for
several hundred meters from concentrated animal feeding operations (CDC, 1998; Karetnyi et
a/., 1999; Gurdin et a/., 2002). Further, if several different bacterial index organisms are used
for source identification, as suggested by the USEPA  (2005), the transport properties of the
individual agents need to be clearly defined to interpret what differences in the level of detection
for different animal sources mean.  MST studies need to identify the distance at which selected
biomarkers may be detectable from their pollution source(s) and whether or not this may be
indicative of the fecal pathogens emanating from the same source.

9. Treatment Technologies and Management Practices
As animal agriculture has evolved to larger facilities with large numbers of animals in limited spaces,
the problems associated with manure handling have grown. A single swine, beef, dairy, or poultry
facility can produce waste equivalent to a small city. The waste is, for the most part, untreated and
spread into the environment with little control on dissemination of microorganisms in the waste. All
animal manures contain microorganisms, some of which are pathogenic to humans and other animals.
Zhao etal., 1995 surveyed dairy herds in 14 states to determine the prevalence of E. coli O157:H7.
Their results indicated that E. coli O157:H7 was present in about 5% of each herd.

Prior sections of this report have enumerated the pathogenic organisms in manure and detailed the
illnesses associated with them.  Environmental problems originate when the manure containing
pathogens is distributed into the open environment with no effort made to reduce the content of
pathogens or limit their movement in the environment. Wind, surface flow, and subsurface flow can all
carry enough pathogens to receiving waters to exceed water quality standards.  In many cases, streams
and lakes are used for recreation, and the people using them can be exposed to infection without
knowing that they have been exposed.  Knowledge of the survival and transport of potential pathogens
in the environment is critical for implementing corrective actions on the landscape to limit people's
exposure to pathogens.

Microorganisms can move in the environment in several ways. Organisms can move with any dust
produced in animal housing, feedlots, or manure spreading. Other airborne transport can happen as
liquid waste is spread by spraying as an irrigation process, spraying from an application vehicle, or
agitation of lagoons prior to spraying.  After manure has been applied to a field surface, microbes can
move with water when rainfall exceeds the infiltration rate, thereby creating runoff. Rainfall impact
dislodges the organisms from soil or manure  particles, and flowing water transports them to receiving
waters. Another path for movement of organisms is through subsurface drainage. Microorganisms can
enter worm burrows or root channels and move downward in the soil profile as the water flows to
groundwater or to drainage tile. If groundwater is shallow,  it is possible for serious contamination to
occur in wells situated too close to application sites or CAFO installations.

Tile drains can short circuit groundwater recharge by intercepting water and diverting it to streams
before it can percolate through the soil. Water that infiltrates the soil down to tile depth is usually below
the majority of the root zone and thus not available for plant uptake.  Tile drains can accelerate the
movement of nutrients and bacteria  into receiving waters (Joy etal.,  1998, McLellan etal., 1993).
Hunter et a/., 2000 found that sheep grazing in pastures in England could adversely affect water quality
even though the animal population was quite low, one animal per square kilometer basis. Tile drains
and open ditches were conduits for microorganisms from pastures to streams.  Janzen et a/., 1973  found
that water quality in streams near dairy farms frequently exceeded coliform limits due to bacterial
contamination.  About 42 % of the farms were responsible for exceeding standards. Some research has
shown that waste applied to the surface of a tiled field can enter the tile quickly after a rainfall.  The
width of the zone affected by tile at  the soil surface is on the order of a meter.  In fields that have tile
drains, the spacing of tile lines is on the order of 25 or more yards depending on the soil type. Coarser
grained soils will allow wider spacing  of tile  lines than fine grained soils. Each tile line will drain
infiltrating water down to its level after a rain event.  A portion of the infiltrating water will drain below

the tile level and enter groundwater as recharge.  There is a possibility that groundwater can be
contaminated by manure spread on the surface.

As shown earlier in this report, beef, dairy, poultry and swine operations have become fewer in number
and larger in animal populations.  The result is to produce more manure in limited areas with little
opportunity apply it to land at low enough levels to reduce pathogenic organisms to background levels.
Microorganisms in manure produced in large CAFOs pose a serious risk to water quality for recreation,
human health, and possibly to nearby farms by spreading disease.  One of the most feared occurrences in
the agricultural community is an outbreak of disease among farm animals. Recent outbreaks of hoof and
mouth disease in the United Kingdom led to multiple billion dollar losses (Ferguson et a/., 2001).
Reducing the presence of potential pathogens in wastes applied to land will go a long way to improving
the safety of farms from disease. Reduction of the bacterial population in water can also improve
downstream biosecurity of adjacent farm operations.  Manure management practices and potential
treatment technologies can be applied to reduce the number of microorganisms distributed into the open
environment. There is a great need to implement microorganism reduction techniques to animal waste
to prevent detrimental environmental effects.

9.1 Manure  management: active and passive systems
Common practices used for managing manures in the U.S. include passive and active approaches.  The
passive systems include lagoons, storage prior to disposal, vegetated buffer strips, constructed wetlands,
separation of different ages of animals, and land application. The passive systems do involve
manipulation of manure to move it and eventually land apply the materials, but do not require more than
minimal operator input.  Active systems include composting, anaerobic digesters, aerobic digesters, and
actively operated lagoons. Active systems require more operator attention, such as turning  compost
windrows, monitoring digesters, and mixing lagoons.  In both cases, the key factor is operation with
minimum input of labor and capital.

Lagoons are large excavations that may or may not be lined with plastic or clay that receive liquid
wastes from animal confinement buildings. Lagoons can be single or multiple cell designs. A passive
lagoon is effectively anaerobic due to the large load of organic matter flowing into the lagoon and
limited aeration from diffusion and wind.  Multiple cell designs can have anaerobic cells, followed by
increasingly aerobic cells as the organic content of the waste stream decreases by settling and
degradation by microorganisms. Lagoons can also be modified by adding covers to collect methane, or
as a permeable cover to oxidize ammonia to nitrate that can be reduced to nitrogen gas in the
microenvironment of the cover.

In some cases, usually dairy, beef, and poultry operations, manure is simply scraped into piles and held
until a convenient time for disposal by land application.  Separation of animals into age groups has also
been shown to be effective in reducing specific pathogens, especially C. parvum (Atwill et a/.,  1999).
Young animals frequently shed large numbers of oocysts, and older animals do not. Manure from calves
can be collected and treated separately from the larger quantities of manure from older animals
(Hutchinson et a/., 2005). The costs of treatment are lower due to the much smaller mass to treat.  In
some  cases, manure is periodically removed from the animal confinement buildings and directly land
applied with no treatment.  Simple holding of waste for greater than 90 days will achieve bacterial
reductions of >90% (Thayer et a/., 1974).

Vegetated buffer strips (VBS) are placed along the downslope sides of fields where wastes are applied to
intercept runoff water. As the water flows across the buffer strips contaminants are retained in the
vegetated area. The vegetation slows water velocity, allowing settling of particles and infiltration of
water into the soil.  A well-designed VBS will reduce the quantity of microorganisms leaving a field
during runoff events. Many studies have shown greater than 50% reduction of bacterial populations
between water entering a VBS and water leaving a VBS. While this reduction may not fully achieve
primary contact standards, it is a step in the right direction. Key factors in VBS success are the width of
the VBS, slope of the soil, type of soil, and degree of vegetative cover.  Good buffers are usually about
ten meters wide, with slopes less than  8%, and have  about 90% coverage with vegetation. Other
management options can help lower the numbers of organisms being applied to the fields.
Microorganisms can be retained in strips and wetlands to a significant degree; however, reduction of
loads to meet water standards has proven elusive. Many VBS studies show reductions of organisms
reaching streams by as much as 90+% (Coyne et a/., 1998). The difficulty arises in that reducing
populations 90% (e.g., IxlO6 to IxlO5 per  100 mL) can still leave more organisms in the water than
standards allow (Coyne et a/., 1995). Table 11 summarizes research done on the trapping of
microorganisms in VBS systems and wetlands which primarily treat surface water flow and some
shallow ground water.

Active manure management systems involve more operator participation to maintain
functionality. Composting requires attention to carbon to nitrogen ratio, moisture, and periodic
aeration. In composting, the degradable organic matter is consumed by microorganisms
reducing the mass of material. During the process, the temperature of the compost pile will rise
to over 50° C.  Under these conditions, pathogenic organisms cannot survive. Composting is
very effective in reducing pathogenic organism content of wastes (Olson, 2003). Adequate
disinfection requires that conditions of specified time at specified temperatures be met.

Anaerobic digesters can be either plug-flow or mixed reactors.  In both cases, the easily degraded
organic  matter in the waste stream is consumed, reducing the  oxygen content of the reactor to
methanogenic conditions. Generation of methane can partially offset the costs of reactors and
maintenance by providing electricity and / or hot water for the farm.  Anaerobic digesters can be
operated at ambient (20-30°C), mesophilic (30-37°C), or thermophilic (45-55°C) temperatures.
The efficiency of the reaction and reduction of pathogens is different under the different
temperature conditions. The thermophilic reactors are more efficient in production of methane
and in destruction of pathogenic organisms (Sobsey etal., 2002).  However, they are more
susceptible to upsets. Aerobic reactors actively incorporate oxygen into the reactor fluid with the
goal of maintaining aerobic metabolism by the microorganisms. Aerobic reactors can also
operate  at ambient, mesophilic, and thermophilic temperatures.  The benefit of an aerobic reactor
lies in odor reduction and greater carbon mass reduction. Pathogenic organisms are also reduced
in aerobic reactors, with greater reductions occurring at higher temperatures (Hill, 2003).  In an
earlier study, Munch etal., 1987 determined the decimation times for several pathogens in cattle
and swine manure slurries from five herds in the temperature  ranges 18-20°C and 6-9°C. The
results are shown in Table 12. As can be seen in these results  some organisms are relatively poor
in survival. In general, colder environments favor survival, and aeration favors the reduction of
microorganisms.  Other organisms, especially fecal streptococci and E. coli, can have very long
decimation times at cool temperatures. Management practices that address the resistant
organisms should at the same time reduce the less-resistant organisms.

Table 11. Summary of microorganism retention in vegetated buffer strips and wetlands.
Slope Protozoan Parasites Viruses
(Cryptosporidium or Giarda)
Fecal Indicator Bacteria
Coliforms Streptococci
Vegetative Buffer Strips
Grass plus forest
Corn and grass
Grass and barley stubble

2 cell
3 cell
4 cell
5-20 35->99%
5-20 90-99%
10, 20 >99%


10-30 >99%

1.5-4.5 99.4-98.3%

87% and 64% §§


370-600 f
90% >95%
75% 68%
6,000 f
91% 74%

2,900-10,000 f 4,800-17,000 f

69% 70%


McCaskeye/a/., 1971
Coynerf al., 1998
Collins et al., 2005
Busheee/a/., 1998
Coyne et al., 1995
Coyne et al., 1998
Heinonen-Tanski et al., 2001
Entry et al., 2000
Young et al, 1980

Chendoriane/a/., 1998
Ferguson et al., 2003
Sobsey and Hill, 2002
Chendoriane/a/., 1998
Behrendse/a/., 1999
CPU per 100 mL in runoff
under low flow conditions
NR = not reported
in each cell
For 87% for Cryptosporidium, 64% for Giardia

Table 12.
Bacterial decimation times in aerated and non-aerated manure slurries in weeks.
                                   20 °C
                  7 °C            20 °C
 Fecal streptococci

 Escherichia coli

 Salmonella typhimurium

 Staphylococcus aureus

 Yersinia enterocolitica
       NR = Not Reported
The pathogen reduction effectiveness of different manure management practices is shown in
Table 13. In most cases, potential pathogens are reduced in common practices by 2 logic orders
or 99%.  While this is important, it is not enough to achieve acceptable water quality standards in
receiving waters. Lagoon effluent, surface runoff water, tile drain water, or digester effluent may
need to have 4 to 6 logic orders of organism reduction to meet water quality standards.

Olsen and Larsen, 1987, identified bacterial decimation times of several pathogenic organisms in
meso and thermophilic anaerobic digesters.  Their results are shown in Table 14. The important
factors were the species of bacteria and temperature, but not the source of manure, reactor
process (batch or continuous), gas produced, ammonia content, or pH.

Combining management practices has been shown to accomplish greater reductions of
pathogenic organisms than single practices.  Among the practices tested are multicell lagoons
with constructed wetlands (Ibekwe et a/., 2002), multicell lagoons followed by constructed
wetlands (Sobsey and Hill, 2002), solid separation  prior to wetlands (Hill et a/., 1999), lagoons
followed by constructed wetlands followed by infiltration basins (Lorimor etal., 2003), solids
separation followed by composting of solids and treatment of the water, digesters followed by
constructed wetlands (Bicudo and Goyal, 2003), animal diet manipulation to reduce pathogen

          Table 13.
Microorganism inactivation by different management techniques.
Etiologic Agent
Campylobacter spp.
E. coli

Listeria spp.
Salmonella spp.
Yersinia enterocolitica
Protozoan Parasites










Aerobic Solar





Diet Separation Reference

X § Olson, 2003 ; Hutchinson et al. , 2005
X X Olson, 2003; Davies-Colley et al., 1999; Collins et
al., 2005; McCaskey etal., 1998; Martinet al., 2003;
Shaw et al. , 2004; Hutchinson et al. , 2005;
Schamberger et al., 2004; Wright et al., 2003
X X Losinger, 1995

X Olson, 2003 ; Mendez-Hermida, 2005 ; Whitman et al. ,
Olson, 2003
Monteithe/a/., 1986
                  T = Thermophilic process, yields virtually complete reduction of pathogens
                  X = Approximately 90% reduction
                  M= Mesophilic process, yielded approximately 99.9% reduction

Table 14. Bacterial decimation times in anaerobic digesters.
 Etiologic Agent               	Decimation Time

Erysipelothrix rhusiopathiae
Escherichia coli
Salmonella dublin
Salmonella typhimurium
Staphylococcus aureus
Streptococcus faecalis
Group D streptococci
Fecal coliforms
Total coliforms
Days at 35 °C
Hours at 53 °C

 Clostridium perfringens (spores)§        Not inactivated       Not inactivated
 Bacillus cereus (spores)               Not inactivated       Not Inactivated

t      Adapted from Olsen and Larsen, 1987
§      C. perfringens was still present after 300 days at 35°C and 180 days at 53°C

loads, and separation of animals into different buildings at susceptible life stages (Shaw et al.,
2004). North Carolina State University has compared conventional lagoons followed by
sprayfields with solids separation followed by a constructed wetland.  The second treatment
system reduced coliforms and E. coli by 3 to 4 logio, while the first reduced coliforms and E. coli
by 1 to 2 logio. Multicell lagoons have been shown to reduce potential pathogens in wastes by
about 99 % in each cell.  With two or three cells in series, microbial populations can be reduced
to acceptable water quality levels. New York City has published a multiple barrier approach to
protecting source water watersheds (New York City and the Watershed Agricultural Council,
1996). The approach starts with good animal husbandry, including herd health, separation of age
groups, sanitation improvements, and crop system changes. Szostakowska et al., 2004 reported
on the presence of C. parvum and G. lamblia in cattle barn flies and landfill flies. The flies from
the barn had a greater load of infective cysts than the flies from the landfill.  These  studies show
the importance of controlling the spread of pathogenic organisms in the environment by
nonagricultural vectors.  The second stage is improving barnyards, manure handling, application
timing, soil management, and composting. The third stage improves stream corridors, adds
vegetated buffer strips, adds stream crossings for pastured animals, fences animals away from
streams, and adds watering stations remote from streams. Milne (1976) showed that livestock in
proximity to a stream increased the nutrient and organism load in the stream. Fecal coliforms
and fecal streptococci were found in the stream  above water quality standards.  Jellison et al.,
2002 found major sources of Cryptosporidium spp. in a watershed  to be from wildlife and cattle.
C. parvum was found in cattle and deer.  One example of a method of runoff control is simply
fencing livestock away from streams (Line et al., 2000; Owens  et al., 1996).  In both cases,
fencing cattle away from streams led to significant reductions of nutrients and sediments entering
the streams.  Owens et al., 1996 also showed that 1% of storm flow accounted for 27% of the
sediment losses.  Peak losses were in May and June.

9.2 Disussion
Conventional manure management techniques do reduce the populations of pathogenic
microorganisms. The extent of the reduction for most techniques is on the order of 90-99%. The
pathogenic organisms originate in the digestive tracts of warm blooded animals, so it is not
surprising that conditions in the open environment are inimical to their survival. Important
factors in organism survival are nutrient content of the waste, organic content of the waste,
temperature, and the species of microorganism. Organic acids, ammonia, and pH changes can
also act to reduce the survival of microbial populations. Competition and predation can also
affect the population of pathogens in waste materials. Once the manures are spread on fields,
microbes can move with air, surface and subsurface flow. VBSs will retain large fractions of
microbial populations, but not enough to allow discharge into receiving streams.  Solar radiation
and timing of manure application with regard to rainfall have a significant effect on
microorganism survival. Soil management techniques are also important in planning for
reduction of pathogen reduction.  Surface application of manure will take advantage of solar
radiation as a disinfection technique, but ammonia losses to the atmosphere may be increased.
Injection of liquid waste will retain ammonia as a fertilizer, but pathogen survival is enhanced
when organisms are protected from drying and the effects of sunlight. The most effective
methods for manure management that also control pathogenic organisms are composting and
thermophilic digestion. In both cases, the temperature of the process is adequate to destroy many
pathogenic organisms. Composting is probably the least costly process to use.  It does require
attention to solids content, moisture content, and C: N ratios.  Properly done, composting can
yield a value-added product that can be marketed to the public. This does require development
of a marketing plan. High temperature digestion will also destroy pathogens. Under anaerobic
conditions, methane can be recovered and used to offset the cost of building and running the
digester.  High temperature aerobic digesters destroy pathogens and reduce the carbon mass that
must be handled, but operational costs are likely to be high and are hard to justify.

The best methods for reducing pathogen loads in manures will combine more than one
management tool to achieve reduction of microbial populations to levels that will meet water
quality standards. One example of a combined management process is separation of solids from
the  waste stream, composting of the solids, and digestion of the liquid portion followed by a
constructed wetland. This process would reduce pathogen loads in the waste so that land
application of either the solid or liquid phase would pose little or no risk to receiving waters.
Another example of a combined treatment system is an anaerobic digester, followed by solids
separation and constructed wetlands for treatment of the liquid phase of the waste.  Multicell
lagoons, followed by constructed wetlands, are another form of combined systems. Wastes
should not be applied to land unless two or three management/treatment steps are used before
land application. VBS should also be present at the edges of the fields used for application of the
wastes. Waste management will include management of the animals in terms of diet and housing
sanitation.  Animal producers will need to work with agricultural researchers and planners to
reduce the dissemination of pathogenic microorganisms from their facilities.  Achieving these
reductions need not require high cost technologies.  In some cases, the reductions can be
achieved by modifying existing facilities and perhaps adding additional processes, such  as
wetlands.  Adding baffles to lagoons to increase the length of the flow path is one such modest
cost option.

Changing the manure management process at CAFOs will require that each facility examine its
manure handling process and look for ways to incorporate more steps that reduce pathogen
loads.  The USDA Natural Resources Conservation Service publishes conservation standard
practices that can be applied to manure management problems
(http://www.nrcs.usda.gov/technical/Standards/).  Collaboration between producers, conservation
officers, and environmental advisors can lead to great improvements in the handling of animal
manures in the U.S.

10. Ongoing research at the  EPA and Other Federal Agencies
The presence of CAFOs and the associated wastes are a topic of interest for several agencies of
the U.S. government. Natural resources in the U.S. that are potentially affected by the presence
of CAFOs and the attendant manure include, but are not limited to the air, water, and soils. In
particular, the United States Department of Agriculture (USD A) has a significant interest and
commitment to animal issues, both from a production and an environmental perspective. Within
the USD A, two organizations have a dominant interest in manure-related research including the
Agricultural Research Service (ARS) and the Cooperative State Research, Education, and
Extension Service (CSREES).  ARS has a formal research area known as National Program 206
(NP206) that is tasked with manure related research with a wide array of topics of interest to the
EPA.  CSREES  sponsors research by funding of universities and other organizations. Research
goals encompass atmospheric emissions, nutrient management, pathogens, and pharmaceutically
active chemicals, and byproducts. All phases of animal  production will enter into aspects of
manure management from feed formulas to field application of manure. In addition, the Natural
Resources Conservation Service (NRCS) funds implementation of conservation practices
through a variety of programs. NRCS does not conduct research directly.  In the conservation
activities of NRCS, there are several different programs established to help livestock producers
improve the environmental performance of their operations.  These programs are either  cost-
sharing or outright grants to help producers mitigate environmental impacts of livestock
operations. One of the larger programs is the Environmental Quality Incentive Program (EQIP)
that provides low interest loans and cost sharing to producers that install conservation practices
on their property.  The EQIP funding level was about $1.5 billion in Fiscal Year 2005

Another agency that conducts research into microbial problems related to CAFOs is the United
States Geological Survey (USGS). The USGS conducts water quality research across the U.S.
and complements USDA research in several ways.  USGS emphasizes assessment of water
bodies and the impacts of animal waste in karst terrain and ground and surface waters. USGS
tends to place its research in a watershed context while USDA tends to place its research into a
location specific context. Both approaches are needed to fully address the impact of animal waste
on water resources in the U.S.  See Table 15  for a listing of some current microbiological
research carried out by USGS.

The Centers for Disease Control (CDC) has conducted research on  the public health effects of
animal waste in the environment.  The areas of interest to CDC include antibiotic resistance,
bacterial populations, and nitrates in water. The research conducted by CDC has been severely
limited due to budget cutbacks. There is an important need for epidemiological analysis of
microorganisms originating from animal waste to determine if human health is at significant risk.
Waterborne disease is believed to be greatly under-reported (Morris and Levin, 1996; American
Society for Microbiology, 1998). Many cases of gastroenteric illness that could be attributed to
contaminated water are likely to go unreported by people because they simply do not associate
swimming in streams, lakes, and ponds with the onset of symptoms. It is more likely for people
to assume that a gastroenteric illness was associated with foods consumed during recreational

Table 15.


Not identified

Not identified

Not identified


New Mexico



Five States,
eastern US
Not identified


Studies carried out or in progress in the United States Geological Survey
Media f













Nutrients, Fecal Bacteria

Bacteria, Viruses,
Protozoa, Nutrients

Nitrate, Bacteria

E. coli, Salmonella


Nutrients, Bacteria

Nutrients, Bacteria



Viruses, Bacteria


Nitrate, Phosphorus

Nutrients, Bacteria

Bacteria, Nutrients

Waste Type










Wild Birds


Wells showed contamination with
no history of manure in the area

Presence in streams, fate and
transport, methods, isotopes

Effects in karst terrain, spring
resurgences, nitrate from septic
systems, highest bacteria in initial
Resistance development, cost
Survey of waste from hog to stream,
Effect of CAFO on GW,SW
Conforms in Shoal creek watershed
Methods, presence of antibiotics in
Survey of streams for antibiotics
Survey of wells for bacteria and
nitrate, downstream had elevated
levels of both.
Land use effects, seasonal variation,
soil-water effects, BMP effects
94 dairies surveyed, high nitrate
found at many
Survey of wells, few had
contaminants, positives in areas of
high agriculture
Models currently inadequate to
describe transport, no well
Permeability versus runoff

Birds were dominant sources,
antibiotic resistance patterns,
rainfall increased counts in 48-72
hours depending on wind, collection
time affected counts.
Decline of contaminants through
wetlands before a wildlife refuge.
GW = Groundwater; SW = Surface water

The various agencies with interests in microorganisms associated with animal waste have a
broad range of topics that they are pursuing.  A key topic for all agencies is development and
validation of methods for the identification and enumeration of potential pathogens in the
different media associated with manure in the environment.  The methods for total and fecal
coliforms and enterococci are mature, but largely limited to water.  There  are few methods
suitable for a broad range of media such as soil, sediment, lagoons, manure, and water. New
methods need to be developed especially to identify overt pathogens in different media.
Assuming that a given method is credible, the survival and transport of microorganisms in the
environment becomes the next major topic of research.  Currently, there is limited research on
the movement of microorganisms in the environment. Do they move with soil particulates?
What affects movement of organisms in the environment? Are they independent of soil?  How
long do different organisms survive in the environment? What are safe limits of the different
organisms in recreational waters? Beyond the questions raised here are larger questions of how
to control the content of microorganisms in animal waste. What effect do animal  diets have on
microbial populations in the manure? What effect does manure storage have on pathogen
populations?  Is pathogen regrowth a significant problem?

In addition to development of methods for enumerating pathogenic organisms in environmental
samples, there are several other common topics of interest to USD A, USGS, CDC, and EPA.
These topics include survival and transport of organisms in the environment, source
identification and tracking, and antibiotic resistance characteristics of the microorganisms.  Do
tile drain lines enable transport of bacteria into receiving waters? What effects do different soil
types have on microorganism survival?  What effect does timing of manure application to soil
have on microbial populations? What is the effect of solar radiation on bacterial survival? What
effect does rainfall have on transport of microorganisms from application  sites to nearby
streams? What effects do different Best Management Practices (BMPs) have on the movement
of microorganisms in the environment?  BMPs commonly include vegetated buffer strips,
constructed wetlands, runoff retention basins, infiltration basins, terracing, injection of waste into
the soil rather than broadcast application, and more. The development of models of microbial
behavior in the environment is a topic of interest to the different Agencies because good models
can help to conserve resources and assist in planning for Total Daily Maximum Load (TMDL)
implementation and assessing plans for placement of new animal operations on the landscape.

Research carried out by the different agencies is performed on many different scales.  Laboratory
scale studies are performed to develop new detection and enumeration methods, measure
movement of microorganisms through small  soil columns under controlled conditions, develop
source-tracking techniques, and evaluate small model digester performance.  The work done to
develop new methods for detecting and estimating microbial populations uses several
approaches:  1) Culture-based methods are being refined to be more selective and to reduce the
number of steps or time required to provide data for analysis; 2) antimicrobial compound
resistance patterns are used as one approach to identify organisms originating either from
humans or animals;  3) genetic analysis techniques are also being developed to discriminate
human from nonhuman isolates to help identify sources of organisms in water.  Another goal is
to develop robust methods that can detect and estimate the population of specific organisms in
the environment and track them to their source.  The benefit of source identification is that
corrective actions can be focused  more effectively on specific problems rather than being applied

to a broad area. Laboratory-scale work is also done to conduct analyses of samples from
different environments using legally standard methods. Beyond the laboratory, field experiments
are conducted at plot-scale, field-scale and watershed-scale. The plot and field-scale
experiments usually evaluate the effects of application rate, application timing, rainfall effects on
transport, and survival of microorganisms in the field.  Also included at this scale are effects of
vegetated buffers, wetlands, and other field management practices that can impact the transport
and fate of microorganisms.  Watershed-scale examination of effects of CAFOs on microbial
populations in waters is perhaps the most difficult to carry out.  Examination of waters for
populations of total or fecal coliforms only reveals if the waters meet standards or not.  The
current methods simply do not allow for estimation of the contribution of human versus other
animal inputs. Similarly, the inputs of wildlife cannot be separated from domestic animals or
humans using total or fecal coliform methods.  Consequently, installation of management
practices at one CAFO may reduce that facility's input to the stream, but have little effect on the
total load of microorganisms. Much work needs to be done to adequately model microorganism
behavior in the environment and to identify critical control points. A means for separating the
total microbial load into its important  components needs to be developed and validated to enable
estimation of the maximum load for individual water bodies. Associated with this work is a need
to estimate or identify the health risk of fecal organisms in water.

The majority of the research conducted by USD A has dealt with the control and retention of
plant nutrients in manures. The nitrogen and phosphorus content of manure represents a
valuable resource for fertilization of agricultural land.  Similarly, the organic matter content of
manure is a valuable soil conditioner.  In recent years, the ARS has added a significant amount
of research on the microbial content of manures with regard to the presence of pathogenic
microorganisms, the transport of organisms in the environment, and the survival of organisms in
the environment (Table 16).  When farms were smaller and had small numbers of livestock
present, the manure produced was largely used as a soil amendment and fertilizer. In most cases,
the small farm manure load would have been indistinguishable from the background of wildlife
sources. The advent of large animal production units has altered the quantity of manure
generated in small land areas. It is common now to have poultry houses with over 100,000 birds
and swine houses with more than 1,000 animals in a building. Beef feedlots and dairy facilities
can also have very large numbers of animals present. The amount of land available within
economical transport distances for application of manure is also limited. The result is that too
much manure is applied to too little land, leading to the possibility for serious runoff losses of
nutrients and potential pathogens.

The importance of understanding microbial behavior in the environment cannot be
overestimated. The health of humans  and animals can be seriously affected by microorganisms
that are commonly present in manures. If one farm has animals that are shedding pathogens in
their manure, that manure can be a source of infection for other farms, recreational water users,
and possibly municipal water supplies downstream of the farm. The true risks remain largely
unknown because there is little information on the presence and survival of pathogens in animal
waste after it enters the environment.  USDA research  on the microorganisms in manure is
addressing this concern in studies from laboratory to entire watershed studies. The indicator
organisms (coliforms and Enterococci) are useful for screening of waters for the presence of
fecal contamination, but are limited in revealing the presence of pathogens.

Table 16.      Studies carried out or in progress by the United States Department of Agriculture,
              National Program 206t

North Carolina








Media §
SW, Soil









Nutrients, Bacteria


Cryptosporidium parvum
Bacteria, Protozoa
Nutrients, Bacteria
Nitrate, Bacteria
Nutrients, Bacteria

Bacteria, Protozoa

Nutrients, Bacteria
Nutrients, Bacteria


Nutrients, Bacteria
BMP Effectiveness
Nutrients, Bacteria
Nutrients, Bacteria
Nutrients, Bacteria

Nutrients, Bacteria

Waste Type




Poultry, Fish

Poultry, Swine



Survival and transport of
Advanced waste treatment system
Runoff content of bacteria,
vegetative treatment system

Modeling, runoff, transport

Integrated waste systems

Dairy lagoon water site
Gases, PM2.5, management
effects, percolation
Best Management Practice effects,
method recovery efficiency
Commercial additive effects
Transport in soil columns
Survival of pathogens
Runoff, antibiotics, treatment
Transport, riparian buffer effects,
at different scales
BMP effectiveness
BMP placement, stream processes
BMP effectiveness, crop effects
Tile water, soil effects, survival

Pond effects on removal
Pollutant removal at edge of field

Methods, source ID, E. coli O157
       There are also several projects not associated with a specific state that are examining fate
       and transport of microorganisms in the environment. These projects also examine the
       factors affecting microorganisms and their movement. Pathogen identification, antibiotic
       resistance, modeling, composting, wetlands, management practices, and animal diet
       effects are among the research topics.
       GW = Groundwater; SW = Surface water

Table 17.       Studies carried out or in progress by USDA or cooperating Universities listed in the CRIS
               database t

Media §
North Carolina SW,













t Some
§ GW =


Air, SW





SW, soil


of the entries may
GrniinHwatpr S^
E. coli, Salmonella





E. coli O157:H7
Protozoa, Bacteria


Bacteria, Antibiotic


Bacteria, nutrients


Bacteria, Hormones,

Bacteria, Hormones


duplicate entries in Table 2.
/ = Siirfarp watpr
Waste Type


Swine, Poultry




Horse, cattle,

Dairy, swine,

Dairy, Swine,






5 to 7 types of E. coli dominate
each group.

Buffers can be effective in
removing bacteria

E. coli declines with time after
E. coli can survive 45 days after
Pathogen transport
Protozoans increased, bacteria
decreased after application
Method development to measure

Buffers are effective with swine
waste, upland cropping was
effective with poultry and dairy
Buffers alone are not adequate,
multiple cropping and forest help
limit loads.
Composting, UV, Chemical
treatment effects on survival
E. coli not best source tracking
organism, protozoa can penetrate
soil to depth, small ponds can
reduce organism load, tillage,
temperature, texture were
Watershed, landscape scale,
methods, filtering by plants
Compost has to be well managed
to reduce pathogen levels
Multiple scales, bacterial reduction
Use of flocculants as a treatment
Diet modification effects
Landuse and conform levels
Feed and odor, antibiotic resistance


Table 17.       Studies carried out or in progress by USDA or cooperating Universities listed in the CRIS
                database (continued)t
 Location	Media§     Analytes
                                      Waste Type   Observations










 New York
Tile drain   Bacteria



Water, air,





 North Carolina   SW

 North Carolina   Wetlands

Bacteria, Viruses


E. coli O157, C. parvum

Bacteria, Nutrients


Bacteria, Antibiotics



Bacteria, protozoa, Phage

C. parvum


Nutrients, Metals,
Various         DOC and pathogen transport,
                Effect of manure on bacterial
Swine           Bacteria at different places in
                waste  streams, diet effects
Swine           Control strategies, native
                community effects on manure
Various         Waste management in karst areas
Dairy           Differentiation of sources
Dairy, swine     Multiple research areas to reduce
                bacteria and recover value from
Dairy           3 to 8  % of milk tanks had
Dairy, beef      Land use and buffers affect
                organisms, methods, source ID
Dairy, beef      0157 is more diverse than
                previously  known, DOC enables
                percolation of pathogens, urban
                water has greater E. coli, oysters
                can be 90% contaminated
Dairy           Algal treatment of dairy waste
                retained nutrients
                Manure particles reduce
                attachment and enable percolation
                of bacteria
Beef, swine,     Tillage, soil type had large effects
turkey           on resistance, transport
Poultry, swine   Feeding study, methods, survival,
                phage  control
Beef            Runoff control, compost,
                vegetative treatment area
Beef            Survival, wetland, runoff, methods

Various         Transport models, vegetation, soil
                type, slope, management practices

Swine, poultry   Diet, new waste systems, survival
                after treatment and application
Swine           Continuous marsh reduced
                nutrients better than other patterns,
                water depth was important, solid
                liquid  separation
        Some of the entries may duplicate entries in Table 2.
        GW = Groundwater; SW = Surface water

Table 17.      Studies carried out or in progress by USDA or cooperating Universities listed in the CRIS
              database (continued)t
 Location	Media§     Analytes
                                  Waste Type  Observations
 North Carolina  Various

 Oklahoma      Soil
 Pennsylvania    SW, soil
            Nutrients, Bacteria

            Bacteria, Metals
 South Carolina  Wastewater   Nutrients
 South Carolina  Wastewater,   Nutrients, Bacteria


Bacteria, Protozoa,
Nitrification, denitrification,
phosphorus recovery

Management practice effects,
wetlands, hydrogen production
Nitrification denitrification
Waste treated with different
materials and practices for the
recovery of nutrients and reduction
of pathogens.
Develop phage as a bacterial
control technology for waste
Multiple aspect study examining
many aspects of animal waste in
the environment.
       Some of the entries may duplicate entries in Table 2.
       GW = Groundwater; SW = Surface water
Considering that microorganisms originating in animal waste represent a significant risk to
people and animals, methods to reduce the microbial load of waste are important.  There is a
great need to develop manure management procedures that will reduce the load of
microorganisms before waste is allowed to enter the open environment.  Anaerobic digestion is
one technique with promise to be cost neutral or beneficial due to the use of generated methane
as a fuel source. Aerobic digestion is a net cost, but reduces odors  and microorganisms.
Composting reduces odors and microorganisms and produces a potentially salable product.
Composting may be practical if markets can be developed.  There are other approaches that
generate activated carbon, pelletized fertilizers and other products.  Combinations of waste
management methods may also be used to reduce microorganism loads before waste disposal.
Storage of wastes for six months has shown  reduction of bacterial populations. Storage in
concert with another management practice may be able to reduce loads of organisms to the point
where application to land would pose little fecal load runoff potential.   The important factor is
that any treatment approach has to be economically feasible in comparison to existing manure
management practices.

A key task to be completed is integration of the various government agency research activities.
The benefit of integration will be  to maximize efficiency of planned research by expanding the
scope of work, avoiding duplication of effort, and sharing of information across interest groups.
EPA is establishing a scientist to scientist level series of workgroups with the goal of integrating
work across agencies. Other goals include enabling scientists to participate in larger projects
than any individual could manage alone and prepare documents that are useful to producers at
the farm level for implementation of environmentally sound practices. Collaboration with the
USDA and Extension services will facilitate these goals.

11. Summary and Outstanding  Issues
Bacteria, viruses, and parasites that can cause disease in humans are endemic in livestock
animals.  The confinement of animals into densely-populated feeding operations exacerbates the
spread of disease and encourages the use of antimicrobial agents for both prophylaxis and to
increase animal growth rates, resulting in the emergence of antimicrobial-resistant bacteria.
These zoonotic pathogens may proliferate in  confinement houses and are shed in animal wastes
that, in most cases, are stored and eventually  spread onto land.  Exposure to antimicrobial-
resistant bacteria and other zoonotic pathogens may occur through direct contact with livestock
animals, breathing confinement house air, contact with insect and animal vectors, recreational or
drinking waters contaminated with manure runoff or leaking manure storage pits, eating produce
from manure fertilized fields, and secondary  infection from exposed individuals. Several
mechanisms are in place to prevent the spread of disease from livestock animals to humans and
may include animal stocking techniques, animal waste treatment practices to destroy pathogens
(such as composting and thermophilic anaerobic digestion), storage of animal manure to reduce
pathogen concentrations prior to spreading, barriers (such as wetlands and buffer strips) to
control runoff from manured fields, and surveillance of our nation's food and waters for
pathogenic organisms. However,  from reported literature, it is clear that exposure to zoonotic
pathogens cause significant human suffering  and economic losses in the billions of dollars
annually  due to lost productivity, treatment of disease, and beach closures. Because of the
continuing human disease caused by zoonoses contaminating food and water resources in the
U.S., we  believe that the current environmental regulations and conventional animal manure
management practices are inadequate for protection of human health and the environment.

The USEPA and other governmental entities  including the USD A, USGS, and the CDC are
actively working towards resolving the threat to human health and welfare posed by
antimicrobial-resistant bacteria and other zoonotic pathogens that may be released into the
environment from CAFOs. As can be seen in this review, the outstanding issues regarding the
fate and transport of zoonotic pathogens are vast; addressing these issues will require the
expertise of all of these agencies and the many disciplines they represent.  Of particular concern
is the synthesis of information generated in these studies into a comprehensive and usable
package,  so that resources can be pooled to arrive at a more complete and usable plan.

Much work is still needed to fully address issues surrounding the contamination of our
environment and with antimicrobial-resistant bacteria and zoonotic pathogens originating from
livestock animals. Based on our review, we recommend that the pathway forward involve not
only value-added research, but also policy changes that are consistent with current limitations on
the use of human waste biosolids as fertilizers.

11.1 General recommendations
Animal agriculture produces copious amounts of manure, most of which is stored untreated and
spread onto land. Based on available manure management technologies, ensuring the safety of
food crops and water resources will require active treatment practices that greatly reduce or
eliminate pathogens in manures and other CAFO wastes prior to land application or discharge to
natural waters. At present, animal manures applied to land as a fertilizer are not regulated in
terms of pathogen reduction.  This lack of regulation is at odds with requirements for the
application of biosolids originating from human septage (USEPA, 2003). Consider:

    *  Even moderately-sized concentrated animal feed operations, such as a 2,500 dairy cattle
       operation, may produce as much manure as a city of 61,000 people.  Serious fines for
       environmental pollution and lawsuits would result if a city of that size spread all of its
       sewage onto land without treatment.
    *  Animal manures and other animal wastes may  contain high concentrations of pathogens,
       hormones, antimicrobials and other pharmaceutically active compounds, metals,
       nutrients, and other chemicals, similar to human sewage.
    *  Animal manures can be as much as 100 times more concentrated than human sewage, as
       human wastes are diluted with other domestic wastewaters prior to treatment.
    *  Because of their concentrated form, animal manures have a higher demand for oxygen,
       higher nutrient content, and higher concentration of pathogens than human septage on a
       per weight basis.
    *  Every year animals raised in CAFOs produce three times as much manure as humans in
       the U.S.

Regulatory bodies should carefully weigh the full costs associated with zoonotic disease, which
are estimated to reach into the billions annually, when considering difficult decisions regarding
the regulation of livestock animal wastes. Several cost effective options for animal waste
treatment can be implemented at CAFO facilities that would reduce pathogens to safe levels
prior to application as a fertilizer.  The most effective and cost-efficient methods for achieving
these ends may be composting or thermophilic anaerobic digestion with recovery of methane that
can be used as a fuel. However, circumstances specific to each animal  confinement facility
would need to be considered when  choosing appropriate manure treatment systems.  These active
treatment systems should be used in concert with management practices to reduce pollution of
water bodies by treated manure fertilizers, such as vegetative filter strips, terraced landscapes,
and constructed wetlands.

Of great concern is the continued use of antimicrobials in animal agriculture for growth
promotion and prophylaxis. Many  of the drugs used to promote growth in animal agriculture are
the same as or very similar to human medicines, and result in the shedding of high
concentrations of antimicrobial resistant bacteria that may infect humans and other animals.
Antimicrobial resistant zoonotic pathogens are a serious threat to human health (Ghidan et a/.,
2000; Cheng etal., 2002; Travers and Barza, 2002), and billions of dollars are spent in the U.S.
every year treating diseases resistant to antimicrobials and managing the spread of resistance in
hospital environments.  The benefits of growth promotion in livestock animals are certain, and at

present, difficult to offset completely with market alternatives (Harper, 2004; Gill, 2005).
However, a combination of education of owner/operators, alternative feed additives, and
improved and more sanitary animal husbandry practices are promising for achieving this end
(Gill, 2005). Regulatory agencies should fully weigh the costs and benefits of continued use of
antimicrobial compounds in animal agriculture for growth promotion and consider the phased
removal of these feed additives from the market in favor of alternative technologies.  Tighter
regulation of the use of antimicrobial compounds for prophylaxis should also be considered.
11.2 Recommendations for Future Research
Significant progress has been made to date to address the release and movement of
microorganisms from CAFOs and fields fertilized with their manure byproducts. Research has
ranged from bench studies on pathogen survival to investigations of specific management
practices for impeding the movement of fecal indicator bacteria to receiving waters and specific
surveys of pathogens and antimicrobial-resistant bacteria near CAFO facilities. Current research
is exploring new and innovative ways to detect and quantify pathogens in soils, manures, and
natural waters that are enabling more specific characterization of animal waste management
practices and technologies performance. These techniques have also opened the door for
development of improved monitoring and surveillance systems that may revolutionize the way
we look at water quality.  Some of these new technologies are progressing rapidly towards the
end of being able to identify with great accuracy the source of pathogenic agents in recreational
and drinking water resources that may cause disease. Other advances are being made in the
development of cost efficient and reliable livestock animal waste treatment technologies that
may ultimately reduce the burden of zoonotic disease in the U.S.

Although advances are being made, significant amounts of work are still required to fully
address the issues surrounding antimicrobial resistant bacteria and other zoonotic pathogens from
CAFO facilities.  There is a need for fundamental information on specific etiological agents
pertinent to their movement and inactivation in manures, soils and sediments, and natural waters.
There remain questions as to what levels of these agents are acceptable in natural systems such
that the risk of contracting disease upon accidental exposure is low.  New models that can predict
with accuracy the fate  and transport of pathogens in the environment following the application of
manure fertilizers to land are needed to identify potential control points to locate new operations
safely and in a sustainable manner.  In addition, integrated systems that can monitor our nation's
water resources in real-time for threats that may be posed by zoonoses and other biological
agents are needed to improve biosecurity. All of these research needs are integral to improving
human health and welfare in the U.S., especially in areas of intensive livestock farming. The
following is a top ten list of research needs to address the pathogen issue and reach this goal.

1.  There is a need for standardized methods of analysis for zoonotic pathogens in animal
   manure, soil and sediments, wastewater, recreational water, and drinking water.

       Standard methods with the required  sensitivity for recovering and enumerating pathogens
       at environmentally relevant concentrations in animal manures, soils, wastewaters,
       recreational water, and drinking water are sorely lacking, especially for hyper-endemic or
       emerging pathogens. These methods are needed to (a) identify the extent to which these
       agents are removed, inactivated, or persist in animal waste treatment processes and

       management systems at livestock operations, (b) determine the survival of these agents in
       manures, soils, sediments, and natural waters to improve our ability to predict their fate
       and transport in the environment, and (c) improve surveillance and biosecurity of our
       nation's recreational and drinking water resources.

2.   There is a need for rapid methods of analysis for pathogens in recreational and drinking
    water to improve surveillance and biosecurity of our nation's water resources.

       Microbiological water quality surveillance in the U.S. relies on the detection of bacterial
       indicators of fecal pollution.  Although epidemiologically related to gastrointestinal
       illness, these indicators do not fully describe the risks associated with recreational or
       drinking waters contaminated with some bacteria and most viruses and parasites.
       Furthermore, since conventional cultivation methods take 18-24 hours to yield a
       presumptive-positive result, a positive result today means that everyone drinking the
       water or swimming in it yesterday was exposed to unacceptable levels of fecal pollution.

       As such, there is a need to develop rapid and reliable methods for the detection of fecal
       bacterial indicators and overt pathogens in recreational and drinking waters. A tiered
       approach to rapid monitoring methods may be the most reasonable, starting with
       indicators and then adding pathogens as methods become available.  It has been
       suggested that such technologies rely on nucleic acids analysis because the tests lend
       themselves to automation and are at present the most promising for rapid and specific
       quantitation of both fecal  indicator organisms and viable microbial pathogens
       (Jothikumar et aL, 1998; Levin et a/., 2002; Straub and Chandler, 2003).

       Current technologies for rapid detection of the fecal bacterial indicators are being field
       tested against proven cultivation methods to develop guidelines for improving
       recreational water quality monitoring by the USEPA and CDC (USEPA, 2005).
       However, the near real-time detection of overt pathogens with very low infective doses,
       such asE1. coli O157:H7,  Campy'lobacter jejuni, and Cryptosporidium will require
       significant advances in technologies to concentrate these agents from large volumes of
       potentially dirty water. In particular, there is a need for effective and reliable sample
       concentration technologies capable of co-concentrating viruses,  bacteria, and parasites
       into clean samples amenable to detection with nucleic acids technologies. At present,
       hollow fiber ultrafiltration systems may be the most promising to this end. However, the
       retention of a variety of pathogens from different waters by these filters needs testing and

       Ultimately, the development of a unified and automated system  for the detection of all
       waterborne pathogens is needed (Straub and Chandler, 2003). Hollow fiber ultrafiltration
       devices, renewable surface technologies for automated sample processing, and
       microarray  technologies have shown promise as a basis of such  a system (Chandler et a/.,
       2000a,b). However, such a system should be constructed in a way that it is simple,
       reliable, and technically less demanding than current nucleic acids technologies. The
       need for rapid pathogen detection technologies will remain a critical issue for biodefense
       where real-time identification of etiological agents may be imperative to protecting
       human health.

3.   There is a need for epidemiological data to establish regulatory guidelines for pathogens in
    manure, wastewater, recreational water, and drinking water.

       Regulatory guidelines on the concentrations of pathogens in the manure, wastewater,
       recreational water, and drinking water protective of human health do not exist because (a)
       there is a lack epidemiological data to ascertain the risks of illness associated with
       exposure, and (b) there remain questions as to what level of risk is acceptable.  There
       remains a need for epidemiological data to enable the identification of appropriate and
       safe limits of pathogens in manure, drinking water, recreational water, and in food.
       Based on surveillance of water and foodborne outbreaks in the U.S., priority should be
       given to Salmonella spp., Campylobacterjejuni, E. coll O157:H7, Cryptosporidium.,
       Giardia, and viral agents such as swine hepatitis E virus.

4.   There is a need to  identify inactivation kinetics of zoonotic pathogens in manures, soils, and
    environmental waters.

       Relatively few studies are available describing the survival of zoonotic pathogens in
       environmental matrices, especially considering the broad range of properties of soils,
       manures, and waters that may potentially be contaminated. A significant limitation is the
       lack of information regarding the survival of antimicrobial-resistant bacteria in various
       milieus, including the persistence of phenotypic and genotypic antimicrobial-resistance
       traits. Most studies on the survival of pathogens have been carried out in the laboratory
       instead ofin-situ, and only a few examined more than one environmental stressor
       simultaneously. Accurate information regarding the survival of pathogenic  zoonoses and
       antimicrobial resistant bacteria is necessary for modeling their fate and transport from

       Based on these limitations, the following needs have been identified:
           *  Comprehensive studies that examine the combined effect of several  stressors
              simultaneously on the survival of zoonotic pathogens and antimicrobial-resistant
              bacteria in manures, soils, and surface water sediments, and natural waters are
              needed. Stressors that should be considered include:
                     o   Biological factors, such as antagonism,  competition, and  predation;
                     o   Physical factors, such as temperature, soils and sediment properties,
                        and solar radiation;
                     o   Growth factors, such as  pH and availability of nutrients.
           *  There is a need to identify the effect of the retention of some pathogens on soils
              and sediments on survival in various matrices.
           *  There is a need for small-scale studies to determine the concentration of
              antimicrobial compounds needed for an organism to maintain antibiotic resistance
              and the number of growth cycles that lead to the loss of the resistance trait.

5.  There is a need for fundamental research to characterize the transport of zoonotic pathogens
   over land, through soils and ground water, and in surface water bodies.

       The movement of antimicrobial-resistant bacteria and other zoonotic pathogens from
       animal wastes through the environment is a complex issue. Research is needed to
       address significant data gaps regarding the properties of etiological agents that may affect
       their retention or mobilization in soils and stream bed sediments.  In order to better
       address the transport of pathogens in the environment, several needs must be met,

          * Characterization of the properties of zoonotic pathogens that may affect their fate
             and transport in the environment, which, if understood, would allow them to be
             incorporated into existing  hydrologic and geogaphical information systems (GIS)-
             based transport models.
          * Identification of the particle sizes with which zoonotic pathogens may be
             transported in the environment.
          * Identification of the potential effects of soil and  sediment retention of some
             pathogens on overland transport and resuspension in stream bed sediments.
          * Verification that batch and column studies performed in the laboratory to
             determine pathogen fate and transport properties accurately describe field

6.  There is a need for research to characterize the movement of antimicrobial-resistant bacteria
   and other pathogenic zoonoses into the environment following land application of animal
   manures with particular attention paid to the effects of hydrologic (rainfall) events.

       Information is lacking regarding the concentrations of antimicrobial-resistant bacteria and
       zoonotic pathogens in the environments proximal to CAFOs and fields where their
       manures are applied. However, on a larger scale, significant microbial contamination in
       agricultural watersheds has been observed by the USEPA.  Rainfall has been noted to
       increase concentrations of fecal indicator bacteria in agricultural watersheds, and much  of
       the outbreaks of waterborne disease in the U.S. and Canada have been linked to heavy
       rainfall events.

       Surveillance of pathogens and antimicrobial resistant bacteria  near several CAFOs with
       different confinement animals and manure management practices is needed to ascertain
       the potential  pathways for pathogen transport from manured fields. Monitoring plans
       should also consider sampling at the sub-watershed and watershed scales. Field studies
       are needed to identify the role of drainage tiles and overland transport of pathogens to
       receiving waters during rainfall events. Continuous or event-triggered sampling devices
       should be used so that events are not missed.  Samples should  be taken following manure
       application and 24 to 48 hours after a substantial rainfall. Future studies should also
       consider management records on the use of antimicrobials on each specific farm that may
       be helpful to correlate farm practices with findings  obtained through the studies.

7.  There is a need for continued research into methods for tracking fecal pollution in natural
   waters to its source.

       Much of our nation's water resources are impaired due to high concentrations of fecal
       microorganisms. In many instances, the source(s) of fecal contamination are unclear.
       MST is an emerging technology that identifies the animal origin of fecal bacterial
       pollution.  However, many caveats to the use of MST still exist, and much work is
       needed to improve these technologies for more widespread application. Some of the data
       gaps for the various methods include:

          * Poor survival of MST reference organisms in the environment may result in little
             or no detection, limiting the ability to identify the source of fecal contamination.
             Reference organisms need to be chosen so that they are useful at a distance from
             the potential source.
          * Variability in the survival of different phenotypes or genotypes of MST  reference
             organisms in environmental matrices that may lead to divergence from host-
             specific fingerprints in source libraries  need to be clearly defined. Limitations to
             interpretation of MST results dependent on these findings need to be documented.
          * Variability in transport and survival of the index organism(s) used to identify
             fecal pollution source(s) and pathogens of the same source that may lead to
             misidentification of the source of disease.  Studies are needed to ascertain whether
             or not reference organisms are reliable  indicators of pathogen transport.  It may be
             that several reference organisms are needed to describe the full suite of pathogens
             that may contaminate water bodies.
          * Variability in the transport properties of different index organisms needs to be
             clearly defined to enable interpretation of MST results.
          * Advances in MST technology need to be made to reduce the time of analysis, the
             level of expertise required, and the cost. Host-specific molecular biomarkers
             offer the most promise for achieving this end, but significant advances in their
             development must be achieved  before they  are off-the shelf ready.
          * MST techniques need more field-scale  testing to prove their utility in varied
             circumstances. Full-scale watershed type studies are needed to assess the
             potential of these technologies for future use.
          * There is a need for different levels of analytical methods to address
             microorganism tracking from simple indicators to methods for exact pathogen and
             source identification.
          * Additional research is needed in the area of spatial and temporal variability for
             library-independent MST methods.

Fundamental studies on the efficacy of various manure management practices including
uncertainty in their performance are needed.

   Many management practices have been proven effective for reducing the discharge of
   stressors such as nutrients and sediment runoff to surface waters. However, the efficacy
   of different management practices for impeding the movement of zoonotic pathogens and
   antimicrobial-resistant bacteria to receiving waters following land application of animal
   manures remains uncertain.  Based on studies using fecal indicator organisms, these
   practices may reduce the discharge of pathogenic microorganisms. However, the
   reductions associated with most practices are only on the order of 90-99%, a scant
   number considering that animal manures may contain billions to trillions of bacteria,
   viruses, and parasites per gram. Therefore, although specific management practices such
   as vegetative buffer strips will retain large fractions of microbial populations, they will
   not retain them well enough to protect receiving streams from contamination.

   There is a need to identify the performance of common barrier technologies such as
   infiltration basins, wetlands, and buffer strips for the retention and inactivation of
   pathogenic organisms. Studies should address retention  in the context of factors related to
   the design of the systems such as size, slope, solids or hydraulic residence time,
   vegetation, undercutting by tile drainage, etc.  Studies are also needed to address the
   impacts of rainfall on management practice performance.  Of particular interest is
   exploration of a multibarrier approach versus single barrier BMPs.

   Aside from barrier technologies, there is a need to verify and field test manure treatment
   technologies like anaerobic digestion, not only for pathogen reduction, but also to
   identify the potential for fuel recovery.  These technologies should be compared and
   contrasted to conventional manure  storage technologies in terms of stressor reduction and
   cost. Vector attraction reduction and pathogen regrowth in treated materials should also
   be explored.

   Many of these questions are being addressed in the areas of public wastewater treatment
   and biosolids from public treatment works. Analogies for manure treatment and runoff
   barrier technologies for  pathogens, as well as vector attraction reduction, may be drawn
   from the extensive pool  of research available within the biosolids community. However,
   livestock animal wastes tend to be more concentrated than human sewage; thus, treatment
   solutions for human wastes need to be feild-tested for application at CAFOs. The most
   readily applicable technologies may be those for pathogen reduction in biosolids, but
   liquid separation and treatment may need to be performed prior to application of solids
   treatment technologies.

9.  Models are needed to better predict site-specific optimal manure treatment technologies and
   runoff management practices for pathogen and other stressor reductions

       Better models of microbial behavior in the environment are needed to assist in planning
       for TMDL implementations and assessing plans for placement of new animal operations
       on the landscape.  Of particular interest would be lifecycle assessment models capable of
       analyzing the effects of different treatment technologies and management practices.
       Models should be capable of predicting potential outcomes regarding not only pathogens
       but other stressors such as nutrients and pharmaceutically active compounds.  Best
       possible treatment and management practice combinations, as well as sustainable
       livestock populations based on environmental and human health outcomes, should be
       predicted considering uncertainty in the performance of the various treatment
       technologies and management practices.  Models that integrate the fate and transport of
       antimicrobial-resistant bacteria and zoonotic pathogens may be different from present
       models in many ways. The issues of multi-drug resistance, microbial  reservoirs,
       horizontal gene transfer of resistance determinants, and the ranges of infectious doses
       resulting from various host characteristics are not part of current models for chemical risk
       assessment.  These factors need to be integrated into CAFO models.  Further, particular
       attention should be given to the relationship between pathogens and organic matter,
       sediments, and nutrients, particularly in terms of survival and facilitated transport during
       hydrologic (rainfall) events. These models will ultimately need to be proven at the sub-
       watershed and watershed scales.

10. There is a need to improve the coordination of research activities and dissemination of
   technical information, methodologies, and new technologies between research scientists of
   the various agencies and to a vast array of end users such as educators, regulators, and
   CAFO owners and operators.

       There is a confounding level of technical literature relevant to pathogens  and livestock
       animals dating back more than 100 years, and literature propagates at  an astounding rate.
       Researchers in the fields of engineering, microbiology, agronomy, epidemiology and
       infectious diseases, as well as the geological sciences and others, are conducting a wide
       variety of studies on pathogens and/or fecal bacteria relevant to CAFO issues.
       Interpreting the literature is difficult not only due to the massive amounts of technical
       information available, but also due to the diverse nature of these disciplines. As such,
       there remains a need for better integration of the various government research activities.
       The benefit of integration would include (a) pooling of resources, (b) broadening of
       technical expertise, (c) maximizing efficiency, (d) expanding the scope of work that can
       be performed, (e) avoiding duplication of effort, and (f) sharing information across
       interest groups.  Without significant interdisciplinary integration and cooperation, the
       assimilation of available information into a comprehensive and meaningful form for the
       waste managers, educators, and regulators is unlikely.

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