United States

Department of

Agriculture

Natural

Resources

Conservation

Service

Watershed

Science

Institute

Waterborne Pathogens in

Agricultural Watersheds

by Barry H. Rosen

NRCS, Watershed Science Institute

School of Natural Resources

University of Vermont, Burlington

With contributions by

Richard Croft, Natural Resources Conservation Service (retired)

Edward R. Atwill, D.V.M., Ph.D., School of Veterinary Medicine, University of California-Davis,

18830 Road 112, Tulare, California

Susan Stehman, V.M.D., Senior Extension Veterinarian, New York State Diagnostic Laboratory,

College of Veterinary Medicine, Cornell University, Ithaca, New York

Susan Wade, Ph.D., Director Parasitology Laboratory, New York State Diagnostic Laboratory,

College of Veterinary Medicine, Cornell University, Ithaca, New York

Contents

Introduction ..................................................... 1

Pathogens of concern ..................................... 3

Pathogens in the environment ..................... 22

Control methods ............................................ 33

Monitoring and evaluation ........................... 43

Anticipated developments ........................... 47

Summary ........................................................ 48

Glossary .......................................................... 49

References...................................................... 52
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Issued June 2000
WSSI - Waterborne Pathogens—1

This technical note provides an introduction to waterborne pathogens,

the disease-causing organisms that contaminate water. Key organisms of

concern are described in detail, including Escherichia coli 0157:H7,

Cryptosporidium parvum (fig. 1), and Giardia sp. Indicator bacteria

that are normally monitored for water quality are described. Information

on viability of organisms in an agricultural setting is presented, along

with relevant management practices for controlling waterborne pathogens at their source, thereby reducing the overall

pathogen loading within a watershed. The topic of

harmful algal blooms is also addressed, although

these organisms do not fall neatly into the category

of pathogen.

Because the potential does exist for contamination

of water with pathogens from agriculture, this

technical note represents a proactive approach for

reducing this source in watersheds.

Although there are several waterborne pathogens, most disease outbreaks from pathogens

are associated with food contamination. Foodborne pathogens are not specifically described in

this technical note, although those described may

contaminate both food and water. Many other

pathogens affect animal health beyond those described in this technical note, and a professional

animal health advisor should be consulted for these

diseases.

A pathogen is any agent that causes disease in animals or plants. Pathogens may be a bacterium, protozoan, virus, or worm. Waterborne

zoonotic disease is a term used to describe illness that is transmitted

among animals and humans by water. Transmission from livestock to

humans is of particular concern and is emphasized in this technical note.

Most waterborne pathogens are in human and animal feces and enter

water along certain pathways. Important pathways include defecation in

waterbodies, carried by overland flow and/or subsurface waterflow.

Pathogenic bacteria and protozoa are potentially available from many

different animal species in watersheds. Wildlife, pets and companion

animals, agricultural animals, and humans are all possible sources of

pathogens. In addition, urban development is often associated with an

increase in bacteria in runoff (Young and Thackson, 1999).

Human contamination or inadequacies at water treatment plants have

been implicated in the previous large-scale waterborne outbreaks while

most current waterborne outbreaks are associated with swimming pools

and recreational waterbodies (lakes and rivers) (Levy et al., 1998; Upton,

1999).

Introduction to

waterborne

pathogens

Nature of waterborne pathogens

Introduction to waterborne pathogens

Sources of

pathogens

Figure 1 Cryptosporidium parvum (courtesy of USEPA,

1999c)
2—WSSI - Waterborne Pathogens

Agriculture is often attributed as the source of water quality impairments, with both nutrients and bacteria cited as the pollutant. This is

partly attributed to The National Water Quality Inventory Report to

Congress, which is the primary vehicle for informing Congress and the

public about general water quality conditions in the United States. This

report is compiled by the Environmental Protection Agency (EPA) based

on information submitted by the States, tribes, and other jurisdictions in

their water quality assessment reports. These reports are issued every

even-numbered year and are often referred to as State 305(b) reports,

based on the section of the Clean Water Act that describes this reporting

mechanism.

The 305(b) reports include information on the degree to which water

supports the designated uses, such as recreation or shellfish harvesting.

The determination of use support for recreation and shellfish harvesting

is based on monitoring for bacterial indicators. The monitoring results

for the bacterial indicators are compared against acceptable thresholds,

normally the state water quality standards.

The National Water Quality Inventory: 1996 Report to Congress lists

bacteria as the third leading cause of impairment for rivers and streams,

an improvement from being ranked as the leading cause in 1994 (USEPA

1994 305(b) Report). Bacterial impairment of water quality remained

second for estuaries. New State and Federal regulations may have economic implications for farmers if stringent source control measures are

required for agriculture.

Waterborne pathogens are a particular concern at the watershed scale

where they may remain viable and are potentially amplified by agricultural practices. People involved in watershed planning need to understand the basic biological characteristics of these organisms to plan and

implement remedial measures, or to avoid practices that could inadvertently promote pathogen viability.

To ensure a safe drinking water supply, treatment normally involves

filtration and/or disinfection of the water. Chlorination has long been the

preferred method to disinfect drinking water. While effective for bacteria

and viruses, it does not kill protozoan cysts (Erlandsen and Meyer, 1984),

such as those produced by Cryptosporidium.

A collaborative voluntary surveillance system for reporting waterbornedisease outbreaks in humans has been maintained by the Centers for

Disease Control (CDC) and the EPA since 1971. The results are used to

assess water systems and the type of agents associated with waterborne

disease outbreaks, and to improve water quality regulations. The CDC/

EPA survey demonstrates an overall improvement in the performance at

drinking water facilities (Levy et al., 1998). The survey reported drinking

water outbreaks at water treatment plants steadily declined since 1989

(i.e., 72.7% for 1989-1990, 62.5% for 1991-1992, 57.1% for 1993-1994, and

30.0% for 1995-1996).

Introduction to waterborne pathogens

Drinking water
WSSI - Waterborne Pathogens—3

Exposure to pathogens can occur during swimming or other recreational

activities. Exposure is related to ingestion, inhalation, or direct contract

with contaminated water. The most recent waterborne outbreaks are

associated with recreational waterbodies (lakes and rivers) and swimming pools (Levy et al., 1998; Upton, 1999). Lakes and rivers allow direct

exposure of people to water that may contain pathogens. In certain

locations these streams and rivers may link to agriculture. The EPA is

developing policies to ensure that states and tribes strengthen their

water quality standards for recreation (USEPA 1999d).

While a large number of viral, protozoal, and bacterial pathogens are

potentially shed in human and animal feces, relatively few cause waterborne disease outbreaks. Waterborne infections are often, but not exclusively, spread by water contaminated with intestinal organisms. Free

living bacteria and parasites that live in water as their natural environment only sporadically cause skin and respiratory infections in humans.

Table 1 lists pathogens causing gastrointestinal illness. This list is from

Centers for Disease Control/Environmental Protection Agency (CDC/

EPA) report summaries published in the last decade. Pathogens suspended in feces and manure may not survive environmental or treatment

conditions long enough to cause infection. Even if these organisms do

survive to reach waterbodies, they can be diluted to levels below doses

required to infect animals and humans. Because of this dilution effect

and water treatment systems used, the vast majority of human infections

caused by microbial agents commonly associated with waterborne

disease outbreaks are spread by contaminated food or by direct host-tohost contact. For disease outbreaks attributable to waterborne pathogens, the main contributor is water contaminated with human feces

(Levy et al., 1998; Upton, 1999) or sewage rather than agricultural operations or wildlife.

Waterborne pathogens of greatest concern have the following characteristics:

• The organisms are shed into the environment in high numbers, or

they are highly infectious to humans or animals at low doses.

• The organisms can survive and remain infectious in the environment for long periods, or they are highly resistant to water treatment.

• Some types of bacterial pathogens can multiply outside of a host

under favorable environmental conditions.

Principal pathogens of concern

Recreational water

Principal

pathogens

of concern
4—WSSI - Waterborne Pathogens

Table 1 Causes of waterborne disease outbreaks causing gastroenteritis 1989-1996 1/

Type of organism Agent Number of Outbreaks asso- Outbreaks assooutbreaks ciated with ciated with

drinking water recreational water

surface ground natural pool/ park

Protozoa Giardia sp. 27 12 6 4 5

Cryptosporidium parvum 21 4 4 2 11

Bacteria with potential for Escherichia coli O157: H7 11 3 7 1

infecting multiple species Campylobacter jejuni 3 3

Salmonella typhimurium 1 1

Salmonella java 1 1

Leptospira grippotyphosa 1 1

Bacterial infections associated Shigella sonnei 17 7 10

with humans Shigella flexneri 2 11

Human viruses Hepatitis A 3 3

Norwalk virus 1 1

Norwalk like virus 1 1

Small round structured virus 1 1

Acute gastroenteritis Unidentified cause-many 60 8 44 7 1

consistent with viral

epidemiology

Other Cyanobacteria-like bodies 1 1

1/ Summary of results from studies by Herwaldt et al., 1991; Moore et al., 1993; Kramer et al., 1998; and Levy et al., 1998.

Principal pathogens of concern
WSSI - Waterborne Pathogens—5

Protozoa of primary concern in watersheds are Cryptosporidium

parvum and Giardia species (abbreviated as sp. fig. 2). These protozoa

cause mild to severe diarrhea and potentially shorten the lifespan of

immune-compromised individuals, such as those with AIDS. These

protozoa are found throughout the world, can coexist in the same animal, and are commonly transmitted from the feces to the mouth in animals and humans (i.e., diaper changing). Neither Cryptosporidium nor

Giardia can reproduce outside the host. Illness in

humans can be caused by relatively low ingestion

levels for both species, with as few as 10 cysts or

less. They also produce a waterborne cyst (Giardia) or oocyst (Cryptosporidium) that is resistant

to chemical disinfectants (Sterling, 1990).

The CDC/EPA survey for 1995-1996 indicated that

all of the outbreaks of cryptosporidiosis were

associated with recreational water, primarily

swimming pools (Levy et al., 1998). Pool-associated outbreaks were caused by chlorine-resistant

parasites (e.g., Cryptosporidium and to a lesser

extent Giardia). Prevention is particularly difficult because it requires improved filtration methods as well as education of patrons about hazards

associated with fecal accidents, especially in pools

frequented by diaper-aged children. This same

CDC/EPA survey noted an improvement in the

trends from drinking water facilities.

Protozoa

Primary concern

Principal pathogens of concern

What are protozoa?

Protozoa are microscopic, single-celled organisms that belong to the kingdom Protista. Although they are single-celled, their structure is

highly complex. About 66,000 species have been

described, of which 10,000 are parasitic. Only a

few species are important disease-causing parasites in humans. If they cause disease, they are

termed pathogens. While parasites in general

obtain food and shelter from their host, they may

not harm them.

Parasitic protozoa occur in many different species of animals. Many protozoa are host-specific,

but some, such as Cryptosporidium parvum,

can infect many unrelated host species. Protozoa

may complete their lifecycle within one animal

or depend on a change of hosts to complete their

cycle. Some parasitic protozoa are passed from

animal to animal directly, while others form

Figure 2 Giardia and Cryptosporidium micrograph to

illustrate relative size

cysts that are resistant to environmental pressures, such as temperature, moisture, and pH,

and can be passed from host to host via water.

Protozoa are divided into four principal classes

according to their mechanism of locomotion

(table 2).

A protozoan lifecycle represents a series of

stages through which the organism passes in

relation to its environment. In simpler cases an

organism reproduces by simple division when

food supplies and other conditions are favorable.

As conditions change, such as decline of food

supply or an increasingly harsh environment, the

organism stops dividing and encysts by secreting

an outer covering (cyst wall). This cyst wall can

be durable. The encysted stage remains until

changing conditions (varies by species) induce

hatching of the cyst (excystment).
6—WSSI - Waterborne Pathogens

In wilderness settings, water that appears clean is often contaminated

with protozoa, such as Giardia. Wildlife contamination is common, and

water must be treated before consumption.

Cryptosporidium parvum—Cryptosporidium parvum is a protozoan

parasite that infects many humans, agricultural livestock (cattle, sheep,

goats, pigs, and horses), companion animals (i.e., dogs and cats), and

wildlife species, such as mice, voles, and raccoons (Fayer and Ungar,

1986; Fayer, 1997).

The lifecycle of Cryptosporidium parvum may be

completed in 1 to 8 days, but varies with the species.

The small, colorless, ovoid to spherical oocyst, containing four sporozoites (fig. 3), is shed in the feces

and is immediately infective to a susceptible animal.

When the oocyst is ingested, the sporozoites are

released and parasitize the lining cells of the small

intestine.

Two kinds of oocysts—thick walled and thin walled—

are formed during the sexual phase of the lifecycle.

Thick walled oocysts (80% of those formed) are environmentally resistant and are shed in the feces. The

thin walled oocysts (20% of those formed) rupture in

the intestine, and sporozoites invade new intestinal

cells, giving rise to autoinfection. This is one reason

that only a few ingested oocysts can ultimately lead to

a heavy infection. Oocysts can be shed in feces for

several days by an infected animal, with up to 10

million oocysts per gram (about 280 million per ounce) of feces during

the peak of shedding (Blewett, 1989; Goodgame et al., 1993; Xiao and

Herd, 1994a).

Cryptosporidium parvum was described by Tyzzer (1912) from the

small intestine of mice. This species is the one found in most mammals.

Principal pathogens of concern

Figure 3 Cryptosporidium sporozoites (arrows)

released from an oocyst-excystation (courtesy

of Dr. Timothy Flanigan, Brown University)

Table 2 Properties of protozoa groups

Class Description

Mastigophora Flagellated cells for motility; major pathogens are Trypanosoma, Leishmania, Trichomonas, Giardia.

Sarcodina Amoeba-like motility. Major pathogen is Entamoeba hystolytica, the cause of amoebic

dysentery.

Sporozoa All are nonmotile, animal parasites with a complex lifecycle that may require a different

host for sexual and asexual reproduction. Do not engulf particulate matter. Major pathogens are Plasmodium (malaria), Toxoplasma (toxoplasmosis), and Cryptosporidium.

Ciliophora Cells with short, hair-like projections called cilia. Major pathogen is Balantidium coli,

which causes dysentery in humans.
WSSI - Waterborne Pathogens—7

It causes illness in young ruminants, dogs, horses, nonhuman primates,

and humans. Illness occurs in adult humans, which is unusual among

mammals. One strain of Cryptosporidium parvum identified is only

passed among humans (Pieniazek et al., 1999).

Waterborne outbreaks of cryposporidiosis have been reported in the

United States since 1983. The Internet website http://www.ksu.edu/

parasitology/water lists 69 Cryptosporidium outbreaks through September 1999. The data clearly indicate that sewage contamination and

drinking water treatment deficiencies are the major causes of the largescale outbreaks. Farm animal contamination of water was only reported

in three cases.

Despite using state-of-the-art technology, Cryptosporidium still breaks

through best in water treatment systems (Hayes et al., 1989; Goldstein et

al., 1996). The best way to reduce public exposure to Cryptosporidium

is by optimizing the water treatment process, increasing testing of intake

water for the Cryptosporidium as well as the treated water, and working

closely with public health organizations to analyze risk and quickly

identify outbreaks (Mayo Clinic Health Oasis, 1999).

Studies have shown that Cryptosporidium sp. oocysts were present in

39 to 87 percent of surface water (rivers, lakes) tested throughout the

United States from 1988 to 1993 (Rose et al., 1991; LeChevallier et al.,

1991; LeChevallier and Norton, 1995). The source(s) of the organism is

typically unknown (Juranek, 1995; Atwill, 1996; Sterling, 1990; Smith and

Rose, 1990). In addition, unless species are differentiated, the potential

to cause human infection is not known.

An outbreak of cryptosporidiosis in Milwaukee in 1993 is the largest

waterborne disease outbreak reported in the United States. An estimated

400,000 people were reported ill. High tributary flows into Lake Michigan

because of rain and snow runoff may have transported oocysts great

distances into the lake from its watershed, and from there to the water

plant intake. Although all applicable water quality standards were being

met by the water treatment plant, the facility needed significant upgrades

to reduce the risk of Cryptosporidium in treated water (MacKenzie et

al., 1994; Blair, Internet communications). Outbreaks have been traced to

swimming pools in which the filters were not working properly. Contamination resulted from defecation by one of the swimmers while in the

pool (Porter et al., 1988).

Only two reported waterborne outbreaks have been directly linked to

farms. These two outbreaks occurred in Britain and were traced to

contamination of the drinking water by cow manure. One case was a

result of surface runoff after a heavy rainfall, and the other was caused

by spreading of manure on the watershed of a reservoir (Badenoch,

1990). One study found that after 33 days in river water, an estimated 40

to 45 percent of the oocysts were apparently dead, and after 6 months, 89

to 99 percent were dead (Robertson et al., 1992).

Although severe environmental pressures against oocysts remaining

infective when excreted on land exist, only a few oocysts need to remain

alive to pose a risk to humans. Robertson et al. (1992) postulated that

Principal pathogens of concern
8—WSSI - Waterborne Pathogens

cow feces might provide additional protection by coating the oocysts,

making them less vulnerable to environmental factors.

Experimental studies in healthy humans determined that the infectious

dose at which 50 percent of subjects acquired infection was 132 bovinederived oocysts (DuPont et al., 1995). As few as 30 oocysts were shown

to induce cryptosporidiosis (DuPont et al., 1995), but since a dose of less

than 30 oocysts was not fed to the volunteers, the minimal infectious

dose may be lower.

Production of oocysts generally is limited to livestock that are less than

30 days old (Atwill et al., 1999; Atwill et al., 1998; Xiao and Herd, 1994a;

Xiao and Herd, 1994b; Sanford, 1987; Kirkpatrick, 1985) although fecal

shedding of oocysts around the time of lambing has been documented in

ewes (Xiao et al., 1994). Researchers in Europe have reported evidence

of Cryptosporidium parvum in adult beef cattle (Scott et al., 1995;

Lorenzo et al., 1993), but this has not been confirmed by studies in this

country (Wade et al., 1999; Atwill et al., 1998; Xiao and Herd, 1994a; Xiao

and Herd, 1994b; Sanford, 1987; Kirkpatrick, 1985).

When livestock feces are deposited on dry land, the transport mechanisms for Cryptosporidium oocysts from the manure to a nearby body

of water are not well understood. Soil texture, slope, vegetation, and

other factors affect the process. Research using intact soil columns and

simulated rainfall indicates that Cryptosporidium parvum oocysts are

carried up to 30 centimeters (about 12 inches) through clay loam, silty

loam, and loamy sand. The majority of the oocysts, however, remained

within the upper 2 centimeters (about 0.75 inch) of the soil column

(Mawdsley et al., 1995). Research has also shown that preferential flow

paths formed by soil structure, worms, or roots can move even large

particles through the soil (Mawdsley et al., 1995).

One important finding is that the oocysts die when they dry. About 2

hours of dryness was 100 percent lethal for oocysts in the laboratory

(Robertson et al., 1992) and were not infective after 1 to 4 days in a barn

under summer or winter conditions (Anderson, 1986).

Temperature has also been shown to influence the viability of the oocyst.

Oocysts exposed to environmental conditions in a surface soil under

cold, but above freezing temperatures, and wet conditions did not lose

viability (Ghiorse et al., 1997). On the other hand, freeze-thaw cycles did

reduce viability (Ghiorse et al., 1997) and 10 days of temperature below

zero degrees Celsius (32 °F) reduced viability by 90 percent. Fayer and

Nerad (1996) examined survival in low temperatures and found that

Cryptosporidium parvum remained viable at minus 10 degrees Celsius

(14 °F) and 5 degrees Celsius (41 °F) for 168 hours.

Other species of Cryptosporidium occur in other vertebrate groups.

Birds are infected by two species: Cryptosporidium meleagridis, which

is found mostly in the small intestine, and Cryptosporidium baileyi,

which is found in the respiratory tract and lower intestinal tract. These

can cause illness and death, or be asymptomatic in birds, such as chickens, turkeys, ducks, geese, swans, parrots, and finches. Reptiles have at

least one species, Cryptosporidium serpentis, which can cause clinical

illness, chronic gastric disease, weight loss, and death. Upton et al.

Principal pathogens of concern
WSSI - Waterborne Pathogens—9

(1989) examined 528 individual reptile fecal samples for Cryptosprodium

sp. oocysts and believe more than one species exists for reptiles. Fish

have one species, Cryptosporidium nasorum, which kills fish after

illness and emaciation. Mammals are not infected by Cryptosporidium

from birds, reptiles, or fish.

Mammals contract three species: Cryptosporidium muris, Cryptosporidium felis (Pieniazek et al., 1999) and strains of Cryptosporidium

parvum. Cryptosporidium parvum is the primary organism that causes

infection in humans. Cryptosporidium muris was described by Tyzzer

(1910) from the stomach of mice. It is also found in the stomach lining in

cattle from preweaning through adulthood. It is associated with reduced

weight gain and milk production. Oocysts may be shed for months. This

species has not been reported in humans.

Giardia—Giardia is a protozoa frequently found in rivers and lakes

that infects the intestinal tract of mammals, such as humans, dogs, cats,

bears, muskrats, and beaver, as well as some birds, reptiles, and amphibians (Xiao and Herd, 1994a; Xiao and Herd, 1994b; Xiao et al., 1994;

Erlandsen, 1994; Wallis, 1994). Even more widespread than Cryptosporidium, most surface water contained Giardia cysts (LeChevallier et al.,

1991; Wallis et al., 1996). Which of these source(s), including humans, is

the most important in surface water has not been clearly demonstrated.

The largest outbreak in the 1994-1996 EPA/CDC survey (Levy et al., 1998)

came from a community drinking water distribution system (1,449 infected). The source of infection for sporadic cases of waterborne human

Giardia infection is often not clear (Craun, 1990; Levine et al., 1991;

Thompson and Boreham, 1994).

Giardia is a flagellated protozoa with two forms: a motile form (fig. 4) or

trophozoite, and a cyst. Trophozoite attaches to the surface of the epithelial cells of the upper small intestine near the stomach where

it feeds and reproduces. The cyst is ovoid to ellipsoidal and is

the environmentally resistant stage that is able to infect a new

host. When cysts are ingested by a susceptible host, the

lifecycle averages about a week (7-9 days) to be completed

(fig. 5). Trophozoites leave the cysts and attach to the surface

of the epithelial cells of the small intestine by an adhesive

disc. Here they feed and reproduce asexually. The trophozoites form into cysts in the lower part of the small intestine.

Cysts leave the host in the feces and may or may not be immediately infective to an animal. Cysts are shed in feces intermittently, often in large numbers. Densities of Giardia cysts as

high as 400 per liter in sewage effluent and 5 per liter in river

water have been reported.

Giardia cysts survive for long periods in the environment. They survive

the longest at low water temperatures (0.5 °C or 32.9 °F) (de Regnier et

al., 1989; LeChevallier et al., 1991). Most waterborne outbreaks in North

America occurred in cooler areas during the spring and fall (Wallis et al.,

1996). Cysts are killed by boiling, drying, freeze/thaw cycles, and heating.

Management practices that promote any of these measures are effective.

The three recognized species or morphological groups are based primarily on the appearance of structures, called median bodies (Filice, 1952).

Principal pathogens of concern

Figure 4 Giardia trophozoite (Courtesy of Dr.

James M. Smith, McGill University)

Adhesive disc Median body

Nucleus

Ventral groove Anterior

Posterior Flagella

Caudal
10—WSSI - Waterborne Pathogens

Giardia agilis has a club shaped median body and is found in amphibians. Giardia muris has a round median body and is found in mice and

their relatives, birds, and reptiles. Giardia duodenalis (G. intestinalis

or G. lamblia) has a fang-shaped or claw-shaped median body and is

found in reptiles, such birds as herons and geese, and in man, cats, dogs,

coyotes, ruminants, horses, pigs, and some rodents.

Only the G. duodenalis group appears to be composed of distinct species, strains, or races. Since cysts of the G. duodenalis type cannot be

distinguished morphologically, other methods are needed to determine

the source of cysts found in environmental samples. Methods, such as

DNA probes and genetic typing, are helping to solve this dilemma. Currently, cyst detection in water samples or in soil and any attempted

correlation with infected animals may lead to false conclusions about the

origin of the cysts. Until this issue is resolved, the significance of various

animals in contaminating the environment, including water supplies, is

impossible to determine.

Considerable controversy surrounds the issue of whether Giardia sp.

cysts obtained from livestock can infect humans (Dupont, 1995). Two

reviews (Thompson and Boreham, 1994; Erlandsen, 1994) both concluded that convincing data for the zoonotic potential of Giardia still

does not exist. Additional review articles debating this issue can be

found in an article by Erlandsen (1994). Fayer (1994) also concluded that

"no human infection related to these animals has been reported" with

respect to pigs, cattle, sheep, and goats.

Principal pathogens of concern

Figure 5 Giardia lifecycle—binary fission is

simply the one cell dividing in two

(Courtesy of Dr. James M. Smith, McGill

University)

Oral uptake

Excystment Encystment

Faeces

Binary fission

Attachment

Intestinal villi
WSSI - Waterborne Pathogens—11

Finding clear evidence to link livestock to human infection remains

difficult. Investigators lack the ability to identify which animal sources

Giardia sp. cysts came from (Thompson and Boreham, 1994).

Clinical significance of Giardia sp. infection in domestic ruminants is

currently poorly understood. There are a few reports of diarrhea in

calves caused by Giardia. The age range of infected cattle is 5-day-old

calves to adults, with most of the infected animals being less than 6

months old (Wade et al. 1999). Giardia has been considered nonpathogenic in cattle since it is usually found in animals that have normal feces

and no sign of disease. Recent studies have indicated that the prevalence

of Giardia in cattle ranges from less than 14 percent to 100 percent in

animals less than 6 months old (Buret et al., 1990; Deshpande and

Shastri, 1981; O'Handley et al., 1999; Olson et al., 1997a and b; Pavlasek,

1984; Quilez et al., 1996; Taminelli and Eckert, 1989; Xiao et al., 1993;

Wade, et al., 1999).

Waterborne protozoa of secondary importance are those whose transmission to humans does not appear to have a major livestock component

or where a waterborne route is rarely documented. Cats serve as the

host for Toxoplasma gondii. Livestock are not involved in the contamination of water with this protozoan (Acha and Szyfres, 1987; Fayer,

1994). Balantidium coli, a ciliated protozoan found in the intestines of

humans and pigs, is rarely reported in the United States. Its potential to

be transmitted from pigs to humans remains controversial (Acha and

Szyfres, 1987). One source of the intestinal amoeba, Entamoeba

histolytica, is thought to be humans. Livestock have no clear role in

human infection (Acha and Szyfres, 1987; Fayer, 1994).

Cyclospora cayetanensis and the opportunistic intestinal organism

Enterocytozoon bieneusi and Septata intestinalis are not known to

have a livestock source at this time (Goodgame, 1996).

While this publication focuses on a few bacteria that are harmful to

humans, domestic livestock, or both, most bacteria are beneficial for

decomposing dead material and releasing nutrients back into the environment for sustenance of plants and animals. As an example, cyanobacteria are a source of oxygen, and their nitrogen-fixing ability is used

for rice production in many countries.

The direct identification and counting of bacteria in water are not practical because the cells are not distinguishable from one another under the

microscope. Identification relies on the growth requirements and metabolic functions of cells placed into culture. For water, the procedures for

culturing bacteria allow key organisms associated with fecal material to

be identified and counted. These key organisms are the indicator bacteria that are used to evaluate the potential for water contamination by

pathogens. Although indicator bacteria are not pathogenic in and of

themselves, high numbers may indicate fecal contamination from leaky

septic tanks, animal manure, or faulty wastewater treatment facilities.

Some species also live in soil and on plants and are harmless.

Fecal bacteria have traditionally been used as an indicator of the possible presence of pathogens in surface water and the risk of disease.

Bacteria

Protozoa of

secondary concern

Indicators of bacterial

contamination

Principal pathogens of concern
12—WSSI - Waterborne Pathogens

Both E. coli and enterococci were considered to have a higher degree of

association with outbreaks of gastrointestinal illness than fecal coliforms

and were recommended by the U.S. Environmental Protection Agency

(USEPA, 1986).

Total coliform (table 3) is the

broadest category of indicator

bacteria and was originally

believed to indicate the presence of fecal pollution. Numerous nonfecal sources make this

indicator too generic.

Fecal coliform, a subgroup of

total coliform, originates from

the intestinal tract of warmblooded animals. This subgroup

is the most commonly used

indicator of bacterial pollution

in watersheds. Escherichia coli

(E. coli) is a member of the

fecal coliform subgroup. This

subgroup is used because it

correlates well with illness from

swimming and can cause gastrointestinal problems.

Fecal streptococci, also called

fecal strep, are another grouping of bacteria, similar to the

coliforms that are associated

with feces from warm-blooded

animals. The ratio of fecal

coliform to fecal strep may

indicate something about the

source of the bacteria (table 3).

Enterococci are a subgroup of

fecal strep bacteria. This subgroup is used because it correlates well with human illness in

recreational waterbodies.

Coliphage, a virus that uses

coliform bacteria as a host, can

also be used as an indicator of

fecal contamination.

State and local government

agencies commonly monitor for

coliform bacteria. Data from

monitoring bacterial are reported to the EPA. States and a

local health agency may have

more stringent standards than

the national guidelines. The

What are bacteria?

Bacteria are a group of micro-organisms that lack membrane-bound

organelles, and hence are considered simpler than plant and animal cells.

They have a cell wall, and some have an outer protective layer. Many

antibiotics act by blocking synthesis of the bacterial cell wall. Most

bacteria are unicellular and may have various shapes: spherical (coccus),

rod-shaped (bacillus), comma-shaped (vibrio), spiral (spirillum), or

corkscrew-shaped (spirochete). Generally, they range from 0.5 to 5.0

micrometers. Motile species (those that can move on their own) bear at

least one fine hair (flagella) arising from their surface. Many possess an

outer, slimy capsule, and some have the ability to produce an encysted or

resting form (endospore). Bacteria reproduce asexually by simple division of cells and rarely by conjugation.

Bacteria are so widespread that only the most general statements can be

made about their life history and ecology. They are everywhere on Earth,

even in the most hostile of environments. Some live in soil, plants, or

water; others are parasites of humans, animals, and plants. Many parasitic bacteria do not harm their hosts; some cause diseases by producing

toxins.

Bacteria have a wide range of environmental and nutritional requirements. They can be classified into three groups based on their need for

oxygen. Aerobic bacteria thrive in the presence of oxygen and require it

for continued growth and existence. Anaerobic bacteria thrive in oxygen-free environments. Facultative anaerobes can survive in either

environment, although they prefer the presence of oxygen.

Cyanobacteria, commonly called blue-green algae, are a separate group

of bacteria that deserves mention here. Cyanobacteria have the ability to

fix atmospheric nitrogen into usable organic molecules. They have chlorophyll and are photosynthetic, and although they are not pathogens,

they can produce potent toxins. Many regulate their buoyancy, often

floating to the surface of a waterbody were livestock have easy accesses

to concentrated populations of organisms. Cattle drinking water contaminated with cyanobacterial toxins can die if sufficient toxin is consumed.

No human fatalities have been reported. Potential toxins from

cyanobacteria have only been a recent concern of water treatment plants.

Drinking water facilities are often faced with trihalomethane problems

associated with the necessity to chlorinate water that has excess algal

growth.

Farm ponds also support a luxuriant growth of cyanobacteria as a result

of nutrient enrichment. Excess algae becomes a major problem when cell

growth stops and the organisms begin to die. As this happens, decomposing bacteria consume oxygen (respiration) as they breakdown the dead

cyanobacteria cells, causing oxygen depletion of the waterbody. This may

lead to fish mortality.

Principal pathogens of concern
WSSI - Waterborne Pathogens—13

Table 3 Comparison of fecal bacteria water quality indicators commonly used (Landry and Wolfe, 1999)

Microbial indicator Properties Federal standard 1/

primary contact 2/

Total coliforms (TC) • Originally believed to indicate the presence 1,000 CFU/100 mL

of fecal pollution

• Widely distributed in nature: soils, water, flora,

fauna

• Contains members of Escherichia, Citrobacyter,

Klebsiella, and Enterobacter identified by

incubation at 35 °C

Fecal coliforms (FC) • Subgroup of TC 200 CFU/100 mL

• Coliforms that originate specifically from intestinal

tracts of warm-blood animals

• Cultured by increasing the incubation temperature

to 44.5 °C

• Remains the predominant indicator used to assess

bacterial pollution in watersheds

Escherichia coli • Member of the FC group 126 CFU/100 mL

• Presence correlates with illness from swimming

in both fresh and marine water

• Has been shown epidemiologically to cause

gastrointestinal symptoms

• O157:H7 is a toxin-producing strain of this common

bacterium

Fecal streptococci (FS) • Differ from FC: FC/FS ratio > 4.0

(1) less dominant in feces (human source)

(2) not known to multiply in the environment

(3) die off more rapidly

• Found in intestinal tracts of warm-blood animals 0.7 < FC/FS ratio < 4.0

and may be found on vegetables and insects (multiple sources)

• FC/FS ratio used to determine fecal source

• Includes Streptococcus faecalis, S. faecium FC/FS ratio < 0.7

S. bovis, S. equinus, and S. avium (animal source)

Enterococci • Subgroup of the FS including Streptococcus 33 CFU/100 mL

faecalis, S. faecium, and S. avium

• Commonly found in intestinal tracts of

humans and other warm-blooded animals

• Presence correlates well with illness from

both fresh and marine water

1/ Individual states may have stricter standards, but not lower. CFU = colony forming units.

2/ Primary contact water includes recreational use, such as swimming and fishing.

Principal pathogens of concern
14—WSSI - Waterborne Pathogens

Federal standards are shown in table 3. The EPA has targeted 2003 for all

states to start using E. coli and enterococci as indicators instead of fecal

and total coliform (USEPA 1999d). The concept is based on information

that these two indicators are better for predicting health risk in primary

contract reaction water.

One important point to note on bacterial indicators is that potential

pathogens, such as Cryptosporidium parvum, Shigella, sp. and E. coli

O157:H7, may be in water that meets all bacterial water quality standards. This clearly indicates that better detection methods are needed

for pathogens in water, such as Method 1623 for Giardia and

Cryptosporidium (USEPA, 1999c).

Escherichia coli O157:H7 (E. coli O157:H7)—The E. coli referred to

during general water quality tests (total coliforms, fecal coliforms, E.

coli) are not necessarily pathogenic strains, but instead are an indicator

of general fecal contamination. When used as an indicator of water

quality, E. coli refers to the harmless strains that help maintain normal

intestinal functions (Draser and Hill, 1974).

E. coli O157:H7, however, is a potentially deadly bacteria that can cause

bloody diarrhea and dehydration. It is an unusually infectious organism

with as few as 10 cells causing illness. Young children, the elderly, and

persons with compromised immune systems are the most susceptible.

The combination of letters and numbers in the name of the bacterium

refers to the specific molecular markers found on its cell surface that

distinguish it from other types of E. coli. Although most strains are

harmless and live in the intestines of healthy humans and animals, the

O157:H7 strain produces a powerful toxin capable of causing severe

kidney failure and can break down the lining of the intestine in humans.

E. coli O157:H7 has been isolated from feces of cattle, white-tailed deer,

sheep, dogs, horses, birds, and flies.

How one is infected with E. coli O157:H7—The infection is usually

acquired by eating food or drinking liquids containing the bacteria. The

bacteria in the guts or hides of livestock can contaminate meat during

the slaughtering process. Eating undercooked meat, especially undercooked ground meat, is the most common way of getting the infection.

Person-to-person transmission can occur if infected persons, especially

food handlers, do not wash their hands. Drinking unpasteurized milk or

fruit juice, or swimming in or drinking sewage-contaminated water can

also cause infection.

The role livestock have with E. coli O157:H7—Although this organism is

not pathogenic to cattle themselves, calf water troughs and moist mixed

cattle rations have been cited as sources of E. coli O157:H7 on farms

(Hancock et al., 1997d). E. coli O157:H7 has been shown to persist in

water trough sediment for at least 4 months and may even grow in the

environment (Hancock et al., 1997d). Two surveys of dairy and beef

cattle (Hancock et al., 1997a and b) showed wide-spread distribution of

E. coli O157:H7 in feedlots, ranging from 63 to 75 percent of the 100

herds sampled. However, the data also showed that the entire herd was

not affected. Only 1 to 2 percent of the cows in any given herd carried E.

coli O157:H7.

Bacteria of

primary concern

Principal pathogens of concern
WSSI - Waterborne Pathogens—15

E. coli O157:H7 appears to be only a transient member of the bacterial

flora of a cow, colonizing cattle from 1 to 2 months, rather than being a

long-term carrier (Besser et al., 1997). One study showed that E. coli

O157:H7 increased with dietary stress (Cray et al., 1995). Other influences on the E. coli O157:H7 are related to season (found more frequently after May 1) and cow age, more common in cows 3 to18 months

old (Hancock et al., 1997c). According to the National Animal Health

Monitoring System (Wells, 1999-Internet communications), herd size also

has an influence, with E. coli O157:H7 more common in larger herds. E.

coli O157:H7 was not associated with manure application to grazing

lands (Hancock, 1997b).

Campylobacter—Several species of Campylobacter can cause infection in humans (e.g., C Campylobacter jejuni, Campylobacter coli,

Campylobacter lardis, Campylobacter fetus subspecies fetus).

Campylobacter jejuni accounts for almost all of the diagnosed cases.

The majority of human campylobacteriosis occurs as sporadic cases as

opposed to outbreaks involving large numbers of people (Tauxe, 1992).

Campylobacter jejuni is common in the environment and is shed in the

feces of humans, livestock, and wildlife, including birds. It is also found

in a variety of surface water, stream sediment, and sewage effluents

(Tauxe, 1992; Stern, 1992; Altekruse et al., 1999). The primary routes of

transmission appear to be ingestion of contaminated foods (primarily

poultry and raw milk), ingestion of untreated surface water, and contact

with pets (primarily dogs) suffering from diarrhea. Direct human-tohuman transmission rarely occurs (Tauxe, 1992; Altekruse et al., 1994;

Franco and Williams, 1994; Adak et al., 1995).

Cattle and poultry feces and effluent from poultry processing facilities

have been shown to contain Campylobacter jejuni that in some cases

are similar to human isolates (Tauxe, 1992; Stern, 1992; Altekruse et al.,

1994; Koenraad et al., 1995).

Campylobacter jejuni can survive for a limited time in stream water

(Terzieva and McFeters, 1991). As few as 500 to 800 organisms appear

sufficient to cause clinical illness in humans (Robinson, 1981; Black et

al., 1988). Campylobacter sp. from water can be detected throughout the

year, with more isolates found during the cold seasons in temperate

climates (Carter et al., 1987; Bolton et al., 1987). Campylobacter can

survive in a nonculturable state in cold water and still be infectious to

livestock (Stern et al., 1994). Unchlorinated drinking water has been

identified as a source of infection to cattle herds (Humphrey, 1987).

Despite these findings, documented cases of human campylobacteriosis

attributable to water contaminated with livestock manure or livestock

effluent are uncommon. Better DNA fingerprinting techniques and more

sensitive and specific methods for identifying Campylobacter jejuni in

water and other environmental sources should help identify the primary

source(s) of this common cause of gastroenteritis in humans.

Salmonella—Salmonella species cause diarrhea and systemic infections. These infections can be fatal in particularly susceptible persons,

such as the immuno-compromised, young children, and the elderly. An

estimated 800,000 to 4 million human infections occur each year in the

Principal pathogens of concern
16—WSSI - Waterborne Pathogens

United States, most of them as individual cases apparently unrelated to

outbreaks. The majority of outbreaks are associated with foodborne

illness. More than 2,200 reported serotypes of Salmonella are found in a

variety of host species.

Animals used for food production are common carriers of Salmonella.

Foods often implicated in outbreaks include poultry and poultry products, meat and meat products, dairy products, egg products, seafood, and

fresh produce. Salmonella enteritidis, now one of the most commonly

isolated serotypes from human cases, is highly associated with ingestion

of contaminated shell eggs (Mishu et al., 1994). Recent papers indicate

that the home environment may be a much more common source then

food.

Whether manure from poultry operations or the effluent from poultry

processing facilities is a significant source of waterborne salmonellosis

in humans is unclear at this time. One outbreak in a large California

poultry flock was associated with the same type of Salmonella enteritidis found in creek water and sewage effluent from a nearby treatment

plant. This indicates that livestock can be the source or recipient of

Salmonella infections (Willoughby et al., 1995).

A concise review of the role of livestock in the annual incidence of

waterborne human salmonellosis is difficult, at best. Only two outbreaks

of waterborne salmonellosis have been reported since 1989. One was

associated with wild bird contamination of water in a storage tank, and

the other was associated with water exposure in a recreational pool. The

most noticeable observation regarding human salmonellosis is the lack

of reported waterborne outbreaks definitively traced to livestock (Acha

and Szyfres, 1987; Ziprin, 1994).

Salmonella newport has been shown to survive for extended periods in

freshwater sediment (Burton et al., 1987). Salmonella typhimurium is

another common isolate from cattle (Graeber et al., 1995). In Germany,

isolates of Salmonella typhimurium that were obtained from nearby

calf-rearing facilities were not the strains contaminating associated

coastal water nor the isolates obtained from nearby human patients

(Graeber et al., 1995). Studies such as this provide no assurance regarding the modes of transmission in the United States.

This group includes waterborne bacteria of secondary importance. In

this group, waterborne transmission to humans does not appear to have

a major livestock component or a waterborne route is rarely documented.

Yersinia—The estimated number of human infections with Yersinia

species (yersiniosis) in the United States is 3,000 to 20,000 per year. This

is far less than the reported number of campylobacteriosis and salmonellosis cases (Roberts, 1989; Todd, 1989). Most Yersinia infections in the

United States result from Yersinia enterocolitica. One of the primary

transmission routes is food (Feng and Weagant, 1994). The proportion of

sporadic cases attributable to food versus water versus other routes of

transmission is not well established. Water is not considered a major

source of infection in North America.

Secondary concern

Principal pathogens of concern
WSSI - Waterborne Pathogens—17

Yersinia species are widespread in water (streams and lakes), foods

(pork products, vegetables, dairy products, tofu, seafood), wild and

domestic animals, and humans, but many of the strains obtained from

these sources were not pathogenic for man. Relative to Campylobacter

jejuni, Yersinia enterocolitica is better able to survive in stream water

(Terzieva and McFeters, 1991). Despite swine being considered as one of

the primary environmental sources of these bacteria, documented waterborne outbreaks of human yersiniosis have not been definitively linked

to livestock (Acha and Szyfres, 1987). Because of the low number of

human yersiniosis cases, this pathogen should be classified as a waterborne disease of secondary importance.

Brucella sp.—The waterborne route of transmission is not known to

play a significant role in human infection with Brucella sp. (Acha and

Szyfres, 1987; Chomel et al., 1994).

Leptospirosis interrogans —Human infections with isolates of Leptospirosis interrogans are rare. They are confined mostly to direct

contact with infected animals, as might occur among veterinarians and

slaughterhouse workers (Acha and Szyfres, 1987; Heath and Johnson,

1994).

Although occasional waterborne outbreaks have been reported, the role

of livestock is unclear (Shaw, 1992; Jackson et al., 1993). Leptospirosis

species are carried by many different mammal hosts.

Principal pathogens of concern
18—WSSI - Waterborne Pathogens

Principal pathogens of concern

Viruses

Table 4 Partial list of types of human virus associated with feces 1/

Group Type 2/ Water relationship

Adenovirus DS DNA Isolated from sewage, rivers, lakes, ground water, drinking water, and

recreational bathing water (swimming pools are a major source)

Astrovirus SS RNA Feces, therefore, inferred in sewage

Caliciviruses SS RNA Feces, therefore, inferred in sewage

Hepatitis SS RNA Sewage and polluted rivers

Norwalk-like SS RNA Municipal drinking water contaminated with sewage, recreational

bathing

Rotavirus DS RNA Sewage, rivers and lakes, estuarine and marine water

Coxsackievirus SS RNA Feces, therefore, inferred in sewage

1/ Adapted from AWWARF Workshop on Emerging Pathogens, 1999.

2/ DS = double-stranded, SS = single-stranded.

Little evidence shows that viruses shed in the excrement of livestock

have posed a waterborne threat to human health in the United States

(Cliver, 1994). Runoff from spreading of municipal sludge and manure

may be a source of viruses to waterbodies. This source is addressed in a

later section. In addition, septic tank effluent may be the most significant

source of pathogenic viruses in the subsurface environment (CAST,

1992). Interspecies transmission of rotaviruses has been demonstrated

experimentally. Livestock-derived rotaviruses are generally not considered a source of human waterborne infection (Acha and Szyfres, 1987).

What are viruses?

Viruses are the smallest known agents of disease

that infect plants, animals, and even bacteria.

They use their host cells for reproduction and

are unable to reproduce outside their host. When

viruses are outside host cells, they exist as DNA

or RNA surrounded by a protein coat or capsid

ranging from 20 to 300 nanometers (1 nanometer

= one billionth of an inch). No other structures

are typically found in cells of other organisms,

such as a nucleus or chloroplast. Because a virus

does not have any of these organelles, it has no

metabolism.

Viruses are not active unless they are within

living cells. When it comes in contact with a host

cell, the virus can inject its genetic material into

the cell, leaving its protein shell behind. Once

within the host cell, the virus uses the cell's own

cellular processes, such as protein synthesis, to

produce more viruses. In essence, the virus

forces the cell to replicate the virus' own genetic

material and protective shell. Once replicated,

the new viruses leave the host cell and are ready

to invade others. Some viruses may remain dormant inside their host cell (lysogenic phase) for

long periods, causing no obvious change in the

host cell. However, when a dormant virus is

stimulated (lytic phase), new viruses are formed

and burst out of the host cell, killing it and going

on to infect other cells.

Hundreds of known viruses cause a wide range of

diseases in humans, other animals, insects, bacteria, and plants. Within a species, 100 or more

viruses may infect that species alone. Most viruses are specific to a single host species. A few

are more general and are capable of infecting one

or more animal species, including humans.

Groups of viruses are classified by the arrangement and type of their genetic material (table 4).

The animal virus group is subdivided into the

following subgroups: double-stranded DNA,

single-stranded DNA, double-stranded RNA,

single-stranded RNA, and retroviruses, a unique

kind of single-stranded RNA virus.
WSSI - Waterborne Pathogens—19

Algae are a natural part of marine and freshwater environments. They

are the base of the food web and provide oxygen to the water. Some

harmful algal blooms, like toxic Pfiesteria outbreaks, cannot be visibly

detected and can be detrimental even at low concentrations. In other

cases, like certain red and brown tides, harmful effects occur when the

algae reach concentrations high enough to discolor the water. However,

not all algal blooms that discolor the water are harmful. Many red tides

have no negative effects on marine life, people, or the environment.

Some kinds of algal blooms, like some kinds of red tides, are harmful

because the algae produce one or more toxins that poison fish or shellfish. They also can pose human health risks when people come in contact with affected water. These toxic algal blooms may also kill seabirds

and other animals indirectly when the toxins are passed up the food

chain. Certain kinds of these toxic algal blooms can cause human health

problems via contaminated seafood. Ciguatara fish poisoning, amnesic

shellfish poisoning, and paralytic shellfish poisoning are examples.

However, there is no evidence that Pfiesteria-related illnesses are associated with eating fish or shellfish. Most algal blooms are not toxic, but

they are still considered harmful if they reduce the amount of light or

oxygen in the water, consequently killing sea grasses, fish, or other

marine life.

Recent attention has be focused on Pfiesteria piscicida, which has been

associated with fish lesions and fish kills in coastal water from Delaware

to Alabama (USEPA, 1999a, Internet communication). These organisms

are believed to be native, not introduced species, and are probably

common inhabitants of estuarine water within their range. These microbes have not been found in freshwater lakes, streams, or other inland

waterbodies.

Pfiesteria identification—Pfiesteria belongs to the dinoflagellates

group of algae. These algae are microscopic, normally free-swimming,

single-celled organisms (fig. 6).

Although many dinoflagellates are plant-like and obtain energy by photosynthesis, others, including Pfiesteria, are more animal-like and acquire

some or all of their energy by eating other algae and often incorporating

their prey’s chloroplast into their own cells. The vast majority of dinoflagellates are not toxic. Pfiesteria, however, is a known toxin producing dinoflagellate.

Discovered in 1988 by researchers at North Carolina State University,

Pfiesteria piscicida is now known to have a highly complex lifecycle

with 24 reported forms, a few of which can produce toxins. Pfiesteria

was named in honor of the late Dr. Lois Pfiester, who contributed much

of what we know today about the complex lifecycles of dinoflagellates.

A few other potentially toxic dinoflagellate species with characteristics

similar to Pfiesteria have been identified, such as Cryptoperidiniopsis

(Steidinger et al., in prep), which also is a species that feeds on

microalgal prey. In North Carolina and Maryland, this species can occur

in brackish systems with Pfiesteria piscicida. These newly-identified

species are referred to as Pfiesteria-like organisms, and they occur from

Delaware to the Gulf of Mexico.

Harmful algal

blooms

Pfiesteria

Figure 6 Pfiesteria scanning

electron micrograph

(Courtesy of Dr. Karen

Steidinger, Florida Marine

Research Institute)

Principal pathogens of concern
20—WSSI - Waterborne Pathogens

Pfiesteria and human health problems—Preliminary evidence

suggests that exposure to water where toxic forms of Pfiesteria are

active may cause memory loss, confusion, and a variety of other symptoms including respiratory, skin, and gastro-intestinal problems. It has

been shown that similar human health effects can be caused by exposure

to Pfiesteria toxins in a laboratory setting. To date, other Pfiesteria-like

organisms have not been shown to cause human illness.

Pfiesteria is not contagious or infectious and cannot be caught like a

cold or flu. No evidence shows that Pfiesteria-related illnesses are associated with the consumption of finfish, shellfish, or crustaceans, such as

crabs, lobsters, and shrimp. Any human health problems associated with

the microbe stem from its release of toxins into river and estuarine water

and human contact with that water, rather than the organism infecting a

person.

The Centers for Disease Control and Prevention, in cooperation with

state health departments in Delaware, Florida, Georgia, Maryland, North

Carolina, South Carolina, and Virginia, have established a surveillance

system to collect reports of human illness thought to be related to exposure to Pfiesteria and Pfiesteria-like organisms in estuarine water. This

and other ongoing research efforts are expected to further delineate the

nature, extent, and duration of any Pfiesteria-related human health

effects.

Nutrients and Pfiesteria—Nutrients, such as nitrogen and phosphorus, are thought to encourage the growth of Pfiesteria populations by

stimulating the growth of algae that Pfiesteria feeds on when in its

nontoxic forms. Some evidence suggests that nutrients may also directly

stimulate the growth of Pfiesteria, but more research is needed to show

this conclusively. At this time the precise role that nutrients and other

factors may play in promoting toxic outbreaks of Pfiesteria is not clear

and is an area of active research.

Excess nutrients are common pollutants in coastal water. Chief sources

of nutrient pollution in coastal areas are sewage treatment plants, septic

tanks, polluted runoff from suburban landscapes and agricultural operations, and air pollutants that settle on the land and water.

Causes of toxic Pfiesteria outbreaks—The exact conditions that

cause toxic outbreaks of Pfiesteria to develop are not fully understood.

Scientists generally agree that a high density of fish must be present to

trigger the shift of Pfiesteria cells into toxic forms. However, other

factors may contribute to toxic Pfiesteria outbreaks by promoting the

growth of Pfiesteria populations in coastal water. These factors include

warm, brackish, poorly flushed water and high levels of nutrients.

What is being done about Pfiesteria—State and Federal agencies are

working closely with local governments and academic institutions to

address the problems posed by Pfiesteria. Federal agencies involved in

the effort include the U.S. Environmental Protection Agency, the National Oceanic and Atmospheric Administration, the Centers for Disease

Control and Prevention, the National Institute of Environmental Health

Sciences, the Food and Drug Administration, the U.S. Geological Survey,

Principal pathogens of concern
WSSI - Waterborne Pathogens—21

and the U.S. Department of Agriculture. Together with state departments

of health and natural resources, these agencies are working to:

• manage the risk of human health effects by monitoring and rapid

response through river closures and public health advisories

• direct funding for technical expertise to do Pfiesteria-related

research and monitoring

• make current and accurate information widely available to public

• understand and address the causes of Pfiesteria outbreaks, especially the possible role of excess nutrients

Helminths are worms that may be free-living or parasitic in plants and

animals. The parasitic worms of greatest concern in water are Platyhelminthes or flatworms (flukes and tapeworms) and Nematoda (roundworms). Most flukes and tapeworms require a minimum of two hosts to

complete their lifecycle.

Infection with one or a few Ascaris sp. may not be noticed. Infection

with numerous worms may result in pneumonia during the migratory

phase when larvae that have hatched from the ingested eggs penetrate

into the lungs. Vague digestive tract discomfort sometimes accompanies

intestinal infection, but small children that have more than a few worms

may have intestinal blockage because of the worms' large size. No

worms stay on the path that is optimal for their development. Those that

wander can penetrate into tissues and locate in various organ systems of

the human body, causing complications.

Ascaris lumbricoides is one of the largest parasitic roundworms and is

the most common parasite found in humans. It is estimated that 20 to 25

percent of the world's population is infected with this nematode (World

Health Organization, 1992). The adult female of this species can measure

up to 18 inches long (males are generally shorter). The adult worms live

in the small intestine and eggs are passed in the feces. A single female

can produce up to 200,000 eggs each day.

Ascarus suum, a parasite common in pigs, has larvae that will migrate to

the lungs and die. This can cause a particularly serious form of pneumonia. Adult worms of this species develop in small children's' intestines.

Nematode eggs, such as those of Ascaris sp., can contaminate crops

when irrigation water has not been adequately disinfected. Sludge may

also be a source of crop contamination if inadequate pretreatment is

used. Humans can be infected if they eat raw produce that is contaminated with live ascaris eggs.

Hookworm is a nematode that is endemic in moist tropical and subtropical regions. When inadequately treated sewage is used on croplands, in

combination with the naturally high soil moisture, one can expect hookworm infection. The infection is usually contracted by persons walking

barefoot over contaminated soil.

Helminths

Principal pathogens of concern
22—WSSI - Waterborne Pathogens

Water plays a major role in mobilizing and transporting micro-organisms.

Rainfall washes organisms from feces or vegetation surfaces and directs

them into the soil or along the land surface and thus into surface water.

Water contaminated by pathogens is often a health risk for swimming

and potentially hazardous for other recreational uses. It must receive

extensive treatment for drinking water use. A major challenge in watersheds is to limit the number of micro-organisms reaching surface and

ground water, thereby avoiding significant contamination. The goal is to

minimize the number of pathogens to a level lower than that needed to

cause infection, thereby protecting water users.

Several potential sources of pathogens are in the environment. Complex

pathways for their distribution are common. Only three waterborne

disease outbreaks have been associated with agriculture. The vast majority are associated with pools, sewage contamination associated with a

drinking water facility or inadequacies or failures at these facilities, or

biofilms in post-treatment distribution systems. In contrast, agriculture

continues to be recognized as a major cause of not attaining water quality based on indicator bacteria standards (USEPA, 1996).

Although sewage and septic systems are the main source of contamination, identifying the potential impacts from agricultural sources is important. Some of the potential pathways for pathogens in the environment

are illustrated in figure 7.

For indicator bacteria, agriculture continues to be recognized as a major

cause of not attaining water quality standards (USEPA, 1996).

Animal feeding operations and grazing lands are all potential sources of

pathogens. How livestock are kept varies, but the trend in modern production agriculture is to confine animals so careful attention can be

given to maximizing meat, fiber, or dairy yields. These are known as

animal feeding operations (AFOs) or concentrated animal feeding operations (CAFOs), based on size and discharge characteristics, and are

described in the EPA-USDA Unified National Strategy for Animal Feeding Operations (USEPA, 1999b, based on 40 CFR 122.23(b) and app. B).

Most poultry, swine, and increasingly, dairy cattle, are confined, creating

a waste stream that may have multiple stages for manure treatment and

disposal. Beef cattle, sheep, and, to a lesser extent, dairy animals and

swine may graze on rangeland or pastureland during part of this production period.

Bare areas, such as open lots with heavy animal traffic, have the greatest

potential for pathogen (and nutrient) runoff into surface water or leaching into ground water. Direct deposit into streams is also an obvious

source. Onfarm recycling of manure has the potential to carry pathogens

into surface water if proper application practices designed to prevent

runoff are not followed.

Biosolids (sewage sludge) are aptly described as a concentrate of pathogens and must require prudent treatment, handling, and land application

and crop utilization techniques to avoid infection of humans and animals.

The class of sludge directly affects pathogen load. Class A sludge is

treated, usually through high temperature, with the goal of reducing

pathogens below detection levels. This sludge can be used without

Pathogens in

the environment

Domestic livestock

Land application of

sewage and sludge

Sources of waterborne pathogens

Pathogens in the environment
WSSI - Waterborne Pathogens—23

Figure 7 Potential pathways for waterborne pathogen movement in watersheds

Pathogens in the environment

Sewage

and sludge

from

municip.

Direct

deposition

of feces

Effluent,

sludge,

sewage,

spills

Direct

deposition

of feces

Livestock

manure

municipal

sludge

Various vegetated buffers

Interaction

Plant, soil, and rock media

Direct

Inputs from

outside of

watershed

Pets and

companion

animals

Wastewater

treatment

plant

Wild animals–

birds and 

mammals

Agricultural

operations

Urban: Lawns, yards,

parks, paved

surfaces, etc.

Non-urban: Forest,

crop lands

grazing lands, etc.

Land uses

Recipient upland features

Waterborne

Affected water

Watershed sources

Surface water Ground water

Recreational

waterbodies

Agricultural

waterbodies

Drinking

water

facilities

Wells
24—WSSI - Waterborne Pathogens

restriction. Class B sludge has received treatment to ensure that pathogens are not likely to pose a threat to public health or the environment,

to reduce, but not eliminate pathogens. When farmers use Class B

sludge, they are advised to avoid direct human contact or inhalation of

dust or spray during and after application. The EPA should be consulted

for a detailed review of regulations and technologies of pathogens in

biosolids (USEPA, 1992).

Many pathogens can survive sewage treatment, although their numbers

are reduced (CAST, 1992; Strauch, 1991). Some pathogens are adsorbed

to particles that remain with the sludge during sedimentation processes.

A number of cases have occurred where animals have become infected

from the spreading of sewage biosolids that were not disinfected. The

salmonellosis in dairy cows from pastures in Switzerland is an example

(Strauch, 1991). The effluent from the sewage treatment process is

generally disinfected by chlorination or other processes and either

directly discharged to surface water or applied to land areas. Certain

organisms, such as Cryptosporidium, are known to survive this disinfection process. For this reason, uses of water downstream of sewage

discharges must be carefully controlled and the water treated

appropriately to avoid infection by such organisms.

Some generalities can be made about survival of pathogens that may be

useful in the implementation of conservation practices. For example,

table 5 shows survival times of pathogens in freshwater, sewage, on

crops, and in soils. Septic tank effluent may be the most significant

source of pathogenic bacteria and viruses in the subsurface environment

(CAST, 1992). In the mid 1980s, the overflow or seepage of sewage

primarily from septic tanks and cesspools was responsible for 43 percent

of the reported outbreaks and 63 percent of the reported cases of illness

caused by the use of untreated ground water (Craun, 1985).

Table 5 Survival times of pathogens in various media 1/

Micro-organism - - - - - - - - - - - - - - - - - - - - - - - - Survival (days) - - - - - - - - - - - - - - - - - - - - - - - -

slurry fecal paddies soil water plants

Bacteria

Salmonella sp. 250+ 200+ 150+ 16

E. coli 300+ 200+ 200+ 35

General maxima 2/ 1 year 180

Viruses

General maxima 1 year 60

Protozoan cysts

General maxima 180+ 5 (dry)

Helminth ova

General maxima 7 years ––– 150 (dry)

1/ EPA, 1992; Kelly, 1978.

2/ General maxima is the longest time that the organism can survival.

Pathogens in the environment
WSSI - Waterborne Pathogens—25

Pets and companion animals are often overlooked as a source of water

contamination. Yet their large numbers and the manner in which their

waste is disposed can be of concern. Pets provide a potential reservoir

for a number of pathogens including Giardia sp., Cryptosporidium

parvum, and Salmonella sp. Dogs and cats release waste in yards and

walking areas often adjacent to streams that are subject to direct runoff.

Young horses are also potential carriers of Cryptosporidium parvum

(Coleman, 1989; Johnson et al., 1997) and Giardia (Johnson et al., 1997).

Salt Lake City has enacted strict regulations for companion animals in

the Wasatch and Oquirrh Mountains. These mountains supply drinking

water for the city, and coliform counts indicated a potential pathogen

problem was developing. Management of companion animal waste is

extremely variable by some pet owners, ranging from careful collection

to complete neglect.

Wild mammals and birds also act as pathogen reservoirs. They are dispersed across forest land, idle land, pastureland, cropland, and the urban

landscape. Their wastes most commonly enter surface water, although

leaching to ground water can occur. Wildlife can contribute pathogenic

microbes, such as Salmonella sp. and E. coli (Moorhead and Davis,

1998). Concentrations of wild animals or waterfowl in watersheds can

cause problem levels of indicator bacteria. The high density activities of

these animals close to or in water provide little opportunity for terrestrial die-off of organisms during their lifecycle.

Certain pathogens exit the host in an environmentally resistant form

(e.g., cyst) that can remain viable in the environment for a while. This

allows the organism an opportunity to be ingested by another host and

repeat its lifecycle. Some pathogens are shed in great numbers, with the

random chance that a few organisms will find a new host to repeat the

lifecycle. In either case, the survival of the pathogen outside its host is

dependent on a many variables, including temperature, soil type, and

moisture content. Each pathogen needs to be examined individually for a

complete understanding. The following introduces the survival of pathogens in an agricultural setting.

Enteric organisms differ widely in their ability to survive various environments outside the host. Once outside the host, the organisms are subjected to stresses and processes (fig. 8).

The floors of an animal facility are a major collection point for bacteria

and parasites that leave their animal host. Most facilities have means of

managing wastes from these areas. Manure is stored at various consistencies that are a function of the total solids. The total solid content of

manure depends on the animal species, housing, type of bedding used,

and addition of other waste and water. Generally, the manure is considered to be in a solid form when its solids comprise at least 20 percent of

its mass (see Agricultural Waste Management Field Handbook, figure

9–1). A slurry is defined as a mixture of feces and urine, which may

contain cleaning water, rainwater, and small amounts of feed and

bedding.

Pets and companion

animals

Wildlife—mammals

and birds

Pathogen survival

in the environment

Waste management systems

Pathogens in the environment
26—WSSI - Waterborne Pathogens

Although reports have shown Salmonella reproduction in slurry manure

storage, most pathogens decline in solid manure storage (Jones, 1976;

Jones, 1979). Solids present the greatest opportunity for spontaneous

generation of heat in storage through composting. The resulting high

temperature may be sufficient to kill part of the microbial population

(fig. 9). Some pathogens are destroyed by spontaneous generation of

heat in well-bedded manure packs, a technique for disinfection referred

to as dung packing. A Swedish study using infected cattle examined both

slurry and manure. Salmonella was in 35 percent of the slurry samples

and only in 6 percent of manure with straw bedding samples for similar

herds (Strauch, 1991).

The survival of pathogens in manure, particularly in anaerobic manure, is

of primary concern. Organisms are known to survive longer in the

anaerobic state than in aeration. This is most likely because the generation of heat from bacterial breakdown of organic material in aerated

material is sufficient to shorten bacteria life spans. In a review of several

pathogenic species of bacteria (Salmonella. typhimurium, E. coli,

Pseudomonas aeruginosa, Staphalococcus aureus) in cattle manure,

Strauch (1987) illustrated that organisms survived up to 15 days in aerated liquid manure, but up to 39 days in nonaerated liquid manure. The

inside temperature of the aerated manure ranged from 29 to 34 degrees

Celsius (84-93 °F), while that of the nonaerated ranged from 9 to 23

Figure 8 Factors affecting the viability of pathogens along transport pathways

Precipitation

Waste

storage

Applied

waste

Seepage and filtering

Soil type,

water content

and pH

Soil bacteria

competition

Drying

dilution

Vegetative

cover

Adsorption

Control factors

(metals, disinfectants,

antibiotics, pesticides)

Soil organic matter

soil aeration

Water table 

Runoff:

concentrated

or sheet flow

Slope

Sunlight

ultraviolet

radiation

Air temperature

humidity

freeze-thaw cycle

Barnyard runoff

storage system leakage

Pathogens in the environment
WSSI - Waterborne Pathogens—27

Figure 9 Time and temperature requirements for 100 percent die-off on selected pathogens

from sewage sludge and septic tanks

80

70

60

50

40

30

20

0.2 0.6 1 7 24 100

Time need for die-off (hours)

Temperature (°C)

400 850 1,000 3,500 7,500

Human enteric viruses

Ascaris eggs Salmonella

degrees Celsius (48-73 °F). Other environmental factors remained nearly

the same for both treatments.

Table 6 lists factors that affect the survival of pathogenic micro-organisms during animal waste slurry storage.

Some waste management systems include biogas generation as a component. Thermophilic anaerobic digestion at 49 to 54 degrees Celsius (120–

130 °F) can reduce the number of pathogens. Slurry in mesophilic biogas

generators only attains a temperature of 30 to 38 degrees Celsius (86–100

°F) and will not destroy as many pathogens. Effluents from mesophilic

biogas generators should be considered as having the same hygienic

status as any slurry under normal storage conditions to assure that

species tolerant of higher temperatures are destroyed (Strauch and

Ballarini, 1994).

Pathogens in the environment
28—WSSI - Waterborne Pathogens

Table 6 Factors affecting survival of pathogens during the storage of animal waste as slurry 1/

Factor Considerations

Size of loading dose Percent of herd infected (higher potential for shedding)

Frequency of waste additions (continued supply of organisms)

Amount of dilution (lower concentration, less reproductive success for

bacteria)

Length of storage period Die-off rate

Treatment Anaerobic, aerobic, aeration and composting processes (die-off rate

dependent on temperature – duration)

Disinfectants

Metals and/or pesticide content

Incidental treatment in storage Aerobic digestion

Anaerobic digestion

Drying (die-off from desiccation)

Freeze/thaw cycle

Slurry characteristics Available oxygen (for aeration)

pH

Temperature (see figure 9)

Nutrients (available for sustenance, particularly organic forms)

Total solids content (important to some organisms)

Inhibitors (antibiotics may have minimal effect - organism resistance)

Innate survival of organism Vegetative form vs. spore, endospore, or cyst (high resistance to external

environment)

1/ Adapted from Kelly, 1978.

Pathogens in the environment
WSSI - Waterborne Pathogens—29

Land application areas Vegetation—Manure applied to pasture falls either on the forage or to

the soil surface. Some Salmonella species in droppings from infected

animals grazing in pastures have been known to survive for more than 28

weeks (Jones, 1979). Some Salmonella sp. in digested sewage sludge

spread on grass has been recovered viable up to 5 weeks (Strauch, 1991).

Survival partly depends on the height of grass. For example, Salmonella

dublin survives for 10 days on the upper part of the grass and from 14 to

19 days at the lower part of the stem. This difference in survival rates is

most likely a result of moisture retention (humidity) and shading (Taylor

and Burrows, 1971).

Salmonella longevity also varies by plant species. Those species that

have stems and leaves providing refuge or shading extend the viability of

bacteria. Visible light, ultraviolet radiation, and desiccation (drying) are

the probable factors in destroying the organisms on forage. Rainfall can

extend their life by providing needed moisture and washing the organisms from the plant into the soil environment. Hancock et al. (1997b)

surveyed 36 dairy herds in Idaho, Oregon, and Washington and found

that manure application to forage crops was not associated with the

prevalence of E. coli O157:H7 in cattle on farms.

Soil—Manure and sewage sludge can be applied by incorporation or

injection into cropland soil. Once in the soil subsurface, two major

factors control microbial fate: survival and time of migration. The longer

a micro-organism persists through its soil migration, the greater the

chance it may infect a potential host once it reaches the ground water.

Other than helminths, the survival of various micro-organisms has been

reported to vary from less than 30 days to more than a year in soil. Salmonella after being applied to soil has survived for more than 20 weeks

(Findlay, 1972). Stoddard et al. (1998) found nondetectable levels of fecal

bacteria in leachate 60 days after application of manure.

Pathogen survival in soil is limited by several factors. Entrapment of

micro-organisms by the soil can lead to mortality that is hastened by

environmental factors, such as dryness, pH, predator soil microorganisms, lack of percolation

water, and lack of organic

matter. Figure 10 illustrates the

effect of various soil types on

the recovery of E. coli at

various depths, demonstrating

the greatest potential movement of pathogens to ground

water in sandy soil compared

to clay soil.

Organic and clay particles in

soil effectively trap viruses and

smaller bacteria and protozoa

(Mawdsley et al., 1995). This

occurs as microbes are

adsorbed to clay and some

negatively charged surfaces.

Larger bacteria, protozoa, and

helminths are filtered by

Pathogens in the environment

Figure 10 Effect of selected soil texture types (without preferential flow) and

depth on E. coli numbers in effluent

106

105

104

103

102

10

0

0 0.6 1 2 3 4

Soil depth in cm

E. coli

5 10 15

Arenosa sand

San Angelo sandy clay loam

Houston black clay

Beaumont clay

Adapted from Moore et al., 1998
30—WSSI - Waterborne Pathogens

narrow pore size and through bridging of individuals at pore constrictions. In coarser textured soil, human viruses are more likely to migrate

through pores.

The microbial population of soil is restricted to the aqueous phase and

the solid-liquid interface. Movement of bacteria and viruses increases in

saturated soils. Percolating water provides a mechanism for downward

movement. Saturated water flows through macropores, bypasses the

filtering effect, and transports micro-organisms long distances.

E. coli cells were found to move as much as 1,500 centimeters per hour

through saturated hillside slopes (Rahe et al., 1978). Plant roots tend to

increase the translocation of bacteria through soil (Mawdsley et al.,

1995). Percolating water can accelerate this movement through these

root channels. Soil structure can also provide macropores, as frequently

seen in clay soils.

Once a pathogen leaves the host environment, it must adjust to external

conditions that are different and often stressful. Biological, chemical,

and physical stressors interact in complex ways that have not been well

studied. The survival of most pathogens, once discharged into a waterbody, is highly variable depending upon the quality of the receiving

water, particularly turbidity, oxygen levels, presence of pesticides and

nutrients, temperature, pH, organic matter content, and solar radiation

(Moore et al., 1988). Once in a waterbody, micro-organisms often become adsorbed to organic matter and soil particles. These settle out and

accumulate at the bottom of rivers and lakes, and may become a source

of organism if resuspended. Many small protozoa feed on bacteria,

including pathogens, and many invertebrates feed on both bacteria and

protozoa in a waterbody.

Extensive research on coliforms, such as E. coli, provides the basis for

understanding factors affecting bacterial survival in streams, rivers, and

lakes. Pathogenic bacteria generally are not well suited to aquatic systems, and survival studies indicate that native bacterial flora outcompete

them for nutrients (Korhonen and Martikainen, 1991). In laboratory

studies of lake water, E. coli can survive for prolonged periods (as long

as 260 days) when kept at 4 degrees Celsius (39 °F) (Flint, 1987) when no

other bacteria are present. Low temperatures, which slow metabolism,

generally prolong the survival of pathogens.

Most bacterial pathogens are sensitive to temperatures exceeding 60

degrees Celsius (140 °F). Bacterial pathogens, which produce resistant

endospores or have thick-walled cells, may survive higher temperatures

and only be killed by prolonged heating at temperatures in excess of 100

degrees Celsius (212 °F) (Kelly, 1978). Higher temperatures also kill

protozoan cysts. Freeze-thaw cycles, when the organisms are subjected

to repeated freezing and thawing, also causes pathogen mortality.

The normal pH range for most waterbodies is close to 7 (neutral) and

would not affect bacterial survival. Only at extreme pH (<4.5 and >8.2)

can cell die-off be anticipated.

Survival in water

Pathogens in the environment
WSSI - Waterborne Pathogens—31

Nutrient enrichment of water may play an important role in survival. Lim

and Flint (1989) demonstrated the importance of nitrogen in the survival

of E. coli. They allowed cells to survive the competition from the indigenous bacterial flora. The additional nitrogen appears to be important in

allowing cells to go through a period of progressive cell dormancy that

prolongs their viability.

The effect of ultraviolet radiation on bacterial and protozoan mortality

has long been known. Recent evidence shows visible light as another

inhibitor of cell survival (Barcina et al., 1990). E. coli and Enterococcus

faecalis were significantly reduced when exposed to visible light in both

freshwater and marine systems.

Estimating enteric bacteria die-off—Moore et al. (1988) provided a

way of estimating die-off rates for bacteria. Several algorithms presented

are essentially variations of Chick’s Law, a simulation of a simple firstorder reaction in chemical kinetics given as:

N

N

t kt

0

= 10−

where:

Nt = number of bacteria at time t

N0 = number of bacteria at time 0

t = time in days

k = first order or die-off rate constant

Although several variations of this equation have been used to more

closely portray organism survival under varying environmental conditions, Chick’s Law continues to provide a simple, easy to use approach

for estimating die-off. Example die-off rate constants, k, for various

conditions are provided in table 7. For more detail consult Moore et al.

(1988).

Pathogens in the environment
32—WSSI - Waterborne Pathogens

Table 7 Die-off rate constants for various wastes and conditions 1/

Material/study Organism pH Season or Type of study Die-off rate, k

temperature (°C) (days–1)

Dairy manure

 Pile Fecal coliform --- Oct–Feb Field 0.066

 Pile - covered Fecal coliform --- Oct–Feb Field 0.028

 Stored slurry Salmonella dublin --- Feb Lab 0.107–0.428

 (anaerobic/ E. coli 0.102–0.287

 inoculated)

Swine manure E. coli 7.0 4 Lab 0.686

 (inoculated) 8.0 4 0.867

 (slurry storage) 9.0 4 0.931

7.0 20 0.588

8.0 20 0.816

9.0 20 1.079

Swine manure Fecal coliform 6.4 0–25 Soil: fsl 0.47

 applied to grass 0–25

 field plots 0–25

Poultry manure

 applied to bare Fecal coliform 4.5–6.5 25 Soil: cl 0.342

soil plots Fecal strep 0.093

Stream water E. coli 8.4 4–6 Field 1.970

8.1 4–6 3.140

E. coli 8.1 5 Lab 0.151

10 0.231

15 0.495

20 0.990

1/ Moore et al. (1988).

Pathogens in the environment
WSSI - Waterborne Pathogens—33

Measures for

the control of

pathogens from

agricultural

sources

It is beyond the focus of this publication to describe all the potential

points of control for waterborne pathogens. Herd health issues, including

the clinical diseases associated with pathogens, require the expertise of a

veterinarian or an animal healthcare provider. This section focuses on

the points of control that have an application in agricultural settings.

A multiple-barrier approach can help control pathogen transport and

proliferation. Four control points are illustrated in figure 11. They are:

• Pathogen import to the farm, which is intended to prevent the

initial infection by these organisms

• The cycle of pathogen amplification or proliferation in the animal

operation

• Waste management

• Pathogen export or transport from the farm

These control points should not be treated separately. For example,

waste management is an important part of the amplification/proliferation

control point when feed becomes contaminated with waste. Waste

management also is an important part of the export control point; adequate treatment, such as composting, may have killed the pathogens

before they leave the farm.

Several conservation practices have a role in reducing pathogen load in a

watershed (table 8). Details on each conservation practice are available

from the NRCS National Handbook of Conservation Practices (NRCS,

1999) [http://www.ncg.nrcs.usda.gov/nhcp_2.html]. From a watershed

perspective, any practice that reduces runoff and erosion will reduce the

transport of pathogen directly to surface water.

Table 8 Multiple-barrier approach and selected conservation practices that can reduce pathogen loading to watersheds

Practice 1/ Import Amplification/ Waste Export

control proliferation management control

(source) (transport)

Composting facility (317) x xxx x

Constructed wetland (656) x xxx

Filter Strip (393) and grassed waterway (412) xxx

Residue management (329 & 344) x xxx

Riparian buffers (390 & 391) xxx

Nutrient management (590) xxx

Sediment basin (350) xxx

Waste management system (312) [including: xxx xxx

Waste storage facility (313) Waste treatment

lagoon (359) Waste utilization (633)]

Irrigation water management (449) xxx

Prescribed grazing including use exclusion (472) xxx xxx

 and Fence (382)

1/ Example practices selected from the NRCS National Handbook of Conservation Practices (number indicated-NRCS, 1999). A single x

indicates an indirect relation to pathogen control, while xxx indicates a direct relationship between a practice and pathogen control.

Additional practices may also be appropriate.

Measures for the control of pathogens from agricultural sources
34—WSSI - Waterborne Pathogens

Figure 11 Multiple-barrier points of pathogen control in agricultural operations

Measures for the control of pathogens from agricultural sources

Export from farms

2.

Control Point

Animal operation

amplification/

proliferation

1.

Control

Point

4 .

Control

Point

3.

Control

Point

Conservation practices

management actions

Waste stream

Feeds

Waste management

Import to farm

Contamination

Drinking

water

New animals

Feeds

Wildlife

Rodents

People and 

implements

Drinking water
WSSI - Waterborne Pathogens—35

Drinking water

New livestock

People and implements

A primary avenue for pathogens to access animals is through the feeding

process. Animal feed brought into the farm needs to be free of pathogens, especially for the susceptible, young animals. Wild mice have been

demonstrated to have a high prevalence of Cryptosporidium and Salmonella infection to calves (Klesius et al., 1986), which can easily contaminate grain. Rodents are also a source of Salmonella contamination of

feed on farms.

Salmonella contamination of feed is recognized by the Food and Drug

Administration, and is addressed in their Analysis Critical Control Point

Program for Food Safety (FDA, 1994). Any feed contaminated with

rodent feces should be suspect, and feed imports as well as onfarm

handling of feeds are important.

Some farms purchase hay and green fodder from outside the farm, or

they rent pasture. These feed sources can become contaminated from

the spreading or deposition of wastes. Appropriate waste handling practices need to be followed before this forage is used. Areas receiving

waste applications should not be used for at least 30 days following

waste application (based on Salmonella - Strauch and Ballarini, 1994;

Jones, 1979).

Litter from poultry has been investigated as cattle feed from the perspective of pathogens. Dried and composted poultry litter was not a significant source of Salmonella, Campylobacter or E. coli O157:H7 (Jeffrey et

al., 1998).

Contaminated drinking water may be a source of pathogens. Keeping

livestock and manure out of drinking water sources, such as streams and

ponds, reduces pathogen contamination by direct deposit. Prescribed

grazing, which includes exclusion fencing and alternative watering

facilities (spring development, troughs, water rams, solar and cattle

operated pumps), is an important measure to consider. Onfarm handling

of the waste stream must prevent waste from accidentally mixing with a

clean drinking water source. Surface water sources can also be contaminated by improper practices in the watershed. Contamination of ground

water that comes into a farm and then is used as drinking water is less

frequent. Monitoring of this water source for pathogens should be considered if unexplained disease outbreaks occur.

When an animal operation acquires new stock from outside the farm,

separation or quarantine of the new animals is a practice that may prevent pathogen transmission to healthy animals. The veterinarian or

animal health care provider can be consulted for additional measures

that may decrease the risk of pathogen introduction from new animals.

Some animals that are carriers of pathogens are of special concern

because they shed pathogens, yet do not develop illness.

Anyone who visits the farm may be a carrier of pathogenic organisms.

This is a special concern for those who visit or make deliveries to multiple farms on a regular basis. Farm-to-farm transmission can be prevented by common sense biosecurity measures, such as thorough cleaning of boots, gloves, and any animal handling equipment including vehicle tires.

Control point:

Import to farms

Feeds

Measures for the control of pathogens from agricultural sources
36—WSSI - Waterborne Pathogens

A major goal of any animal operation is the prevention of pathogen

spreading within the farm. Animal health is an essential part of that

control and may include immunization, treatment, culling, and other

measures needed to maintain animal health and reduce the growth and

spread (amplification) of an infection on the farm.

Several common sense practices reduce amplification of infection cycles

in an animal operation. One major source of pathogens is feed and drinking water contamination by manure in the barn or feeding areas. This

point of control is critical, and appropriate actions include:

• frequent cleaning of housing units

• use of separate implements for feeding and cleaning, including

shovels, brooms, or hoses

• frequent cleaning of equipment, such as tractor tires and blades

• prevention of foot traffic contamination of feed with manure

• prevention of contamination with fecal material from other animals, such as other agricultural animals, humans, birds, pets, and

wildlife

• no feeding of potentially contaminated leftover adult feed to young

stock

Some bacteria are unique in their ability to rapidly proliferate under the

right conditions outside the host. Viruses, helminths, and most protozoa,

on the other hand, cannot multiply outside their specific hosts. Once

reduced by treatment or attrition, their populations stay reduced

(USEPA, 1992). To reduce the potential for contamination, manure solids

known to contain pathogens can be spread in areas of low hydrologic

sensitivity. Hydrologically sensitive areas are those areas were water

moves. These areas are time-sensitive. For example, during the dry

season, some parts of a flood plain may not be considered hydrologically

sensitive.

Composting temperatures for solid manure is known to eliminate the

targeted organisms and is another control method.

Several options are available for treatment of manure transferred from

animal operations. These options include aerobic lagoons, anaerobic

lagoons, controlled anaerobic digestion for methane, composting, and

constructed wetlands.

Lagoons are used to reduce pollution potential of the waste through

biological, physical, and chemical processes. Composting solid manure,

if properly managed, provides an opportunity to attain elevated temperatures needed to destroy various organisms including pathogens and plant

seeds. Constructed wetlands are used to process effluent that, for various reasons, cannot be land applied. All of these components reduce

pathogen populations; some more effectively than others.

Anaerobic lagoons reduce the organic content of wastes primarily in the

absence of oxygen. Farm lagoons are generally low rate systems and

receive no supplemental heat. Thus, temperatures in these lagoons

normally do not elevate to more than a few degrees above ambient.

Studies on municipal sludge processed anaerobically indicated this

Control point:

Amplification and

proliferation

Waste management

Storage and treatment

Measures for the control of pathogens from agricultural sources
WSSI - Waterborne Pathogens—37

process may reduce viruses and coliform bacteria to a greater extent

compared to aerobic sludge (Farrah et. al, 1981).

Aerobic lagoons reduce the organic concentration of manure where

minimizing odors is critical. Temperatures in these lagoons also remain

within a few degrees above ambient. In each of these methods, pathogens are reduced by being deprived of organic material that is needed for

survival, and also as a result of the length of storage.

Composting is an effective means of reducing pathogen concentrations.

Temperatures over 55 degrees Celsius (131 °F) are easily attained and

maintained for sufficient duration. All but the most persistent organisms

(e.g., certain viruses and worm eggs) can survive. The main concern in

composting is to assure that the entire waste mass is uniformly treated

and there are no hot or cold spots. Therefore, a high degree of management is required to completely mix the composted material.

Constructed wetlands can be an important tool in the management of

liquid animal waste, including swine, dairy and poultry (CH2M Hill and

Payne, 1997). Constructed wetlands can have an important role in confined animal operations, providing a cost-effective treatment system that

improves water quality.

Constructed wetlands effectively reduce bacterial pathogen numbers

(Watson et al., 1989). Monitoring of constructed wetlands at several

small municipal systems in various climates showed removal efficiencies

from 82 to 100 percent for fecal coliforms (Hammer, 1989). Gravelbed

constructed wetlands have been demonstrated to decrease fecal coliform

by 90 to 99.9 percent (Tanner et al., 1998). Constructed wetlands remove

organisms as follows:

• Viruses may be adsorbed by soil and organic particles.

• Bacteria are removed by sedimentation, ultraviolet radiation,

chemical reactions, natural die-off, and predation by zooplankton.

The control of Cryptosporidium and Giardia through constructed

wetlands is unknown. In some instances, the wildlife that wetlands

attract may actually cause a net increase in pathogens.

The main strategy for managing the waste stream following storage and/

or treatment is to control the release of pathogens from the waste application areas. Climatic conditions, application technique, timing, and

where applications are made are the major considerations for minimizing

the loss of micro-organisms in runoff and leaching. Agricultural Waste

Management Field Handbook (NRCS, 1992), chapter 5, table 5–3 should

be consulted for a detailed review of restricting features that need to be

considered during waste application.

On cropland, two approaches to application of animal waste should be

considered depending on the field conditions that will most likely follow

application.

The first approach, incorporating the waste into the soil, is used when

storm events are anticipated. This allows the reduction of potentially

harmful organisms through adsorption, filtration, and attack from

Measures for the control of pathogens from agricultural sources

Application

Application techniques
38—WSSI - Waterborne Pathogens

predator organisms. Direct incorporation also reduces the potential for

surface applied waste to be carried away by surface runoff. Incorporating surface-applied waste and injection of waste are effective practices

to move organisms into the soil profile. In a study of sod-covered versus

tilled soil, McMurry et al. (1998) found greater infiltration of fecal bacteria in sod-covered soil blocks compared to the tilled soils. The soil was

well-structured. They also concluded that infiltration in these soils contributed to fecal coliform getting into shallow ground water. Irrigation

soon after manure application moved fecal coliform into the drainage

effluent in less than an hour (Goehring et al., 1999) and should be

avoided.

The second approach to waste application, surface application without

incorporation, can be taken when the soil is dry and at summer temperature. This approach allows significant pathogen die-off (Vendrell et al.,

1997) by exposure to UV light and desiccation.

Timing of waste applications is also important especially on established

grasslands where incorporation into the soil is not feasible. If waste is

applied at the beginning of a dry weather period, the number of organisms can be reduced. On the other hand, waste applied just before a

rainfall period will most likely move into runoff. An exception may be in

karst soil where there is little runoff. Spring no-tillage manure application on this soil showed no greater fecal bacteria in leachate compared

to fall applications (Stoddard et al., 1998).

If waste reaches a waterbody in spring or summer, bacterial growth is

stimulated by moderate water temperatures. Proliferation may be the

most important factor responsible for the observed seasonal increase in

indicator bacteria (Edwards et al., 1997).

If the grassland area is to be grazed, waste that has been stored at least

60 days should be applied at least 30 days before the scheduled grazing

period. This may need to be modified if there is evidence to support a

longer wait (Strauch, 1991). Use of these areas for grazing should be

limited to mature animals.

Application of waste to forage vegetation should take place when the

minimal amount of forage biomass is present. This allows sunlight and

desiccation to destroy the greatest amount of pathogens and reduces the

chances of pathogen adherence to the forage. The minimal forage biomass is present just after harvest or grazing.

Winter application should be avoided in hydrologically sensitive areas

because most pathogens survive longer at cold temperatures although

they do not proliferate, and the potential for runoff is high from frozen or

saturated soil.

Hydrology of the waste application area deserves special attention.

Waste placement should be avoided in areas of major flow paths. The

major drainage areas of the waste application area can be determined

and avoided, when possible.

Measures for the control of pathogens from agricultural sources

Timing

Hydrologic considerations
WSSI - Waterborne Pathogens—39

Although some research indicates that runoff volume does not influence

indicator bacterial counts (Edwards et al., 1997), one part of certain

watersheds may be the most significant source of surface runoff and its

associated pathogen. Watershed runoff processes are affected by soil

permeability, soil profile storage, slope steepness, soil complexity, flood

plains, frozen soils, surface and subsurface flow paths, antecedent moisture, and saturated conditions.

Saturated areas, also known as critical source areas (Gburek and Pionke,

1995), expand and contract rapidly into adjacent areas during storms in

response to precipitation, soils, topography, ground water level, and

moisture status. These areas are dominated by saturated overland flow

and rapidly respond to subsurface flow. They often form in areas where

subsurface lateral flow converges, depth to an impervious layer decreases, or an abrupt change in slope occurs. Thus, a strategy in humid

areas may involve identifying the boundaries of critical source areas and

applying waste outside those boundaries to minimize pathogen runoff.

Some tools have been or are being developed to delineate critical source

areas (e.g., Cornell Soil Moisture Routing Model-Frankenberger, 1999).

However, more general delineations can be made using the following

factors:

• areas of concentrated flow

• soil drainage class

• flooding frequency

• wetland mapping

• aerial photo interpretation

The final barrier to pathogen movement into the watershed includes the

control of diseased animals or contaminated animal products, grazing

land management, and physical strategies, such as buffers and filter

strips. The containment of pathogens from contaminated animal products and diseased animals is not addressed in this publication. In practical terms, the nutrient management component of the overall conservation plan includes conservation and erosion prevention measures designed to reduce runoff from areas that have manure applications or

intensive animal use. These practices may have a similar effect on reducing the transport of pathogens off the farm and into the watershed.

Although conclusive evidence that one specific practice is more effective

than another is lacking, general trends are apparent (Meals, 1989). As

pathogens move off the farm, any pathogens present will most likely

move with them.

An important control measure is appropriate grazing land management

techniques. Along with waste application precautions to grazing lands,

these techniques include proper grazing use (wastes distributed more

evenly), avoidance of overgrazing to reduce runoff, and keeping animals

out of waterbodies to avoid direct deposits of pathogens. For the latter,

the most commonly used approaches include alternative watering facilities, salt/molasses, and feed and shade away from open water. Fencing

can keep livestock out of streams and direct them to crossings.

Control point:

Export from farms

Grazing land management

Measures for the control of pathogens from agricultural sources
40—WSSI - Waterborne Pathogens

Overall, management strategies designed to minimize direct livestock

contamination of surface water with Cryptosporidium should focus on

the young animals (less than 3 months old). These efforts should focus

on cow herds when calves are present (Ongert and Stibbs, 1989; Atwill et

al., 1998; Atwill et al., 1999). Handling of calves and their manure is the

critical point of control with Cryptosporidium and other pathogens.

Those operations that normally separate young animals include feedlots,

some slaughterhouses, dairies who do not raise their own calves, and

backgrounder/stocker beef cattle operations (calves 6 to 14 months old).

The effectiveness of riparian buffers and filter strips in trapping sediment

and associated contaminates is well documented. The primary mechanism of filter strips involves the infiltration of runoff. When the volume

of runoff exceeds the infiltration capacity of a riparian buffer, then

movement of water and associated pathogens into the receiving waterbody should anticipated.

Although buffers and filter strips can also trap fecal waste (Dickey and

Vanderholm, 1981; Doyle et al., 1977; Young et al., 1980), their effectiveness in reducing pathogen transport is not readily apparent (Coyne et al.,

1995). Their removal rate is often as high as 95 percent (Coyne and

Blevins, 1995); however, the number of indicator bacteria in runoff is

initially in the 106 cells per 100 milliliter range, leaving about 50,000 cells

per 100-milliliter organisms that may still reach the waterbody. This

amount of bacteria still greatly exceeds the standards (table 3).

Flow length is an important consideration. Buffers and filter strips are

often wider than practical for most farmers. Coyne and Blevins (1995)

used grass filter strips that were 4.5 meters wide and concluded that they

deter fecal contamination of water from manured fields on most occasions. Only significant runoff-producing rainfall was a problem. Hubbard

et al. (1999) recommend a 10- to 20-meter-wide vegetated filter strip

between animal confinement areas and watercourses. They also recommend that wastewater from animal operations not be applied to areas of

land that are subject to surface runoff into a watercourse.

Moore et al. (1988) summarized the work of several investigators and

suggested that vegetative filters are most reliable in the removal of

bacteria at high concentrations (at least 105 organisms per 100 milliliters)

from waste effluent. The bacterial populations in runoff from these

buffer areas seem to equilibrate at about 104 to 105 organisms per 100

milliliters regardless of the experimental conditions. For this reason,

buffers and filter strips should be considered as secondary practices in

control and should only be used in conjunction with a full suite of

source, proliferation, and treatment control measures (composting,

lagoon processing, and constructed wetlands) as a part of the overall

waste management system. These buffers become the final polishing

effort before runoff entering waterbodies. All feasible practices along the

pathway from source to the edge of the farm should be implemented

first.

Grass, shrubs, and trees along the riparian interface with cropland and

pasture may be effective treatment of overland and shallow subsurface

flows for nitrogen and particulate phosphorus removal under the correct

Buffers and filter strips

Measures for the control of pathogens from agricultural sources

Specific watershed

management strategies

for Cryptosporidium
WSSI - Waterborne Pathogens—41

Watershed-scale efforts

Figure 12 Mean annual fecal coliform (FC) and fecal streptococcus counts in relation to animal

units under BMPs for the St. Albans watershed

conditions. Moore et al. (1988) and Palone and Todd (1997) identified the

important mechanisms involved in this reduction as:

• a reduction in the volume of runoff from increased infiltration

• a decrease in runoff velocity caused by the vegetative cover with a

resultant increase in sedimentation of pollutants that are adsorbed

to particulate matter

• increased adsorption of pollutants by soil particles under a lower

ionic concentration regime than found on the waste application site

• dilution by incoming rainfall

• physical adhesion to vegetation and soil particles

Watershed-scale research has been conducted to evaluate the collective

effectiveness when multiple practices and Best Management Practices

(BMPs), including buffers and filter strips, are implemented. The following examples illustrate the potential reduction in fecal bacterial contamination without specifically targeting the results of an individual practice.

One study, the St. Albans Bay Rural Clean Water Project (RCWP),

showed a fecal bacterial reduction trend when all watershed waste

management efforts were collectively examined (fig. 12). Monitoring of

fecal coliform and fecal streptococcus in the tributaries during the implementation period showed a close association with increasing proportions

of watershed animals under BMPs.

In Oregon, the Tillamook Bay RCWP directly addressed water quality

impairment resulting from high fecal coliform levels. The problem was

caused primarily by manure in runoff from the dairy farms. High fecal

coliform levels were causing potential health hazards and negatively

affecting commercial oyster industry, recreational clam digging, fishing,

boating, and other tourist activities. Many of the oyster beds were closed

periodically because of excessive coliform levels. The primary objective

of the project was to reduce fecal coliform levels by 70 percent so the

deleterious effects being caused to the oyster and tourism industries

Measures for the control of pathogens from agricultural sources

0

10

100

1,000

10,000

10 20 30 40 50

Percent animal units under BMPs

Mean annual fecal coliform (FC) Fecal strep

(FS) Counts/100 mL

60 70 80 90 100

FC

FS
42—WSSI - Waterborne Pathogens

would also be reduced. The influence of BMPs on the water quality of

Tillamook Bay is inconclusive at this time (Tillamook Bay Internet communication) although water quality improvements have most likely

occurred.

In a study of three Utah watersheds, Glenne (1984) found that percent

reduction of bacteria could be correlated to the buffer strip length and

slope (Moore et al., 1988). Figure 13 provides a plot of some of these

results. It provides only an indication of potential reduction in bacterial

numbers in the Utah watersheds where concentrations were high.

In summary, further investigation is needed into the effectiveness of

buffers and filter strips for removal of pathogens. It appears they can be

effective in removing bacteria from effluent with higher concentrations

(at least 105 per 100 milliliters), but that reductions below 104 per 100

milliliters cannot be expected. Application of percent removal rates to

concentrations below this level is misleading. It should be emphasized

that buffers should be used as a final polish in the context of an overall,

integrated treatment effort on the farm and in the watershed.

Figure 13 Effectiveness of filter strips for bacterial removal (from Utah watershed study, Glenne, 1984)

20

30

40

50

60

70

80

10

0

10 15 20 25 30 35 45 40

Filter strip width, feet

Percent bacteria removed

50 55 60

1 % Slope

5% Slope

10% Slope

Measures for the control of pathogens from agricultural sources
WSSI - Waterborne Pathogens—43

The NRCS National Handbook of Water Quality Monitoring describes

methods for monitoring the water quality response to land use and land

management activities and conservation practices. This handbook

should be consulted for designing a water quality monitoring project and

analyzing the monitoring results.

This technical note is intended to present an overview of selected, specific evaluation methods related to particular pathogens.

Identification and enumeration techniques for coliform bacteria are

inexpensive and simple to perform and some have been developed into

field kits (Pedley and Bartram, 1996).

Fecal bacterial counts in rivers and lakes around the world that have

little human impact vary from less than 1 to as many as 3,000 organisms

per 100 milliliters. Remote mountainous areas may have up to 100 organisms per 100 milliliters; however, they generally are below 30 organisms

per 100 milliliters. Waterbodies that receive waste from around high

population densities can have counts of up to 10 million organisms per

100 milliliters (Chapman, 1996). Ground water not directly connected to

surface water should contain no fecal bacteria.

Coliforms themselves are not pathogenic and only act as an indicator of

fecal contamination. They can come from multiple sources including

wildlife, livestock, companion animals, sewage, and soil. The presence of

Cryptosporidium oocysts and Giardia cysts is not correlated with fecal

coliform counts in water (Fricker and Crabb, 1998). Some data indicate

that fecal bacterial counts derived from a water sampling program do not

correlate with important pathogen sources, such as diapered recreational users (Keene et al., 1994). An outbreak of the bacterium E. coli

O157:H7 in Vancouver, Washington, was traced to a recreation lake

where the fecal coliform counts indicated it was safe. E.coli O157:H7 is

not detected by the normal culturing technique used to enumerate E. coli

in water samples.

Sewage is a major factor in waterborne outbreaks, and coliforms are

useful as a first warning. If used properly, a site that has high coliform

counts should start the process of a more detailed investigation, usually

by public health officials. If the site has unusually high numbers and is

used as a recreational site (i.e., swimming), it may be closed immediately.

Fingerprinting or ribotyping (see sidebar on page 44) is a new method

that is used to identify the sources of bacterial contamination in some

watersheds (Samadpour and Chechowitz, 1995). This method, also called

multiple source tracking, is an expensive technique.

Several evaluation methods are used in testing for the presence of enteric bacteria in water and solids. Positive identification for specific

organisms may at times be necessary and is often complex for pathogenic bacteria, such as Salmonella or Campylobacter sp. These organisms usually occur in low numbers in water samples and may need to be

concentrated through filtration and enrichment techniques. Two of the

Detection methods

Monitoring and

evaluation of

pathogens

Coliform bacteria

Monitoring and evaluation of pathogens

Are coliforms good and

accurate indicator

organisms?
44—WSSI - Waterborne Pathogens

more common counting and identification methods use the multiple tube

and the membrane filter techniques. In the multiple tube method, a

sample is processed through a prescribed series of fermentation tube

tests, which involve several sequences of carefully controlled incubations and observations for presence of organisms. A statistical analysis

of the results in terms of the number of fermentation tubes processed

provides the estimated density of the fecal bacteria expressed as most

probable number (MPN) per 100 milliliters. The membrane filter or

colony count technique involves filtration of the sample, controlled

incubation including the appropriate medium, and a colony count of the

bacteria. The density of the organisms is then

computed and is normally expressed as number per

100 milliliters. This number is actually in colony

forming units (CFU) and may underrepresent the

actual individuals because a colony might have

originated from a clump of bacteria. Tests for

bacteria conducted on semi-solids, such as manure,

are normally expressed in number of micro-organisms per unit mass (dry weight basis unless noted

otherwise).

A test for the presence of viable helminth ova is

available. The test monitors for Ascaris ova, which

serve as an indicator of several helminth species

(USEPA, 1992). Viable helminth ova are observed

under a microscope and counted as individuals.

Methodologies for the detection of viruses are

constantly being improved, but they require expensive laboratory facilities.

The samples may need to be prepared for transport

to highly specialized laboratories. A test is available

that simultaneously monitors for several enteric

virus species that are presumed to be good indicators for other types of enteric viruses. Viruses are

usually counted as plaque forming units (PFU).

Each PFU represents an infection zone where a

single infectious virus has invaded and infected a

layer of animal cells in culture.

Cryptosporidium and Giardia were traditionally

observed microscopically from fecal material or a

concentrated water sample. Recent improvements

include the use of fluorescent dyes coupled with

antibodies that specifically adhere to these organisms including the

ELISA technique. The main difficulty is determining if the observed

organisms are alive or not.

Frequently problems associated with waterborne pathogens involve

multiple sources within a watershed, such as wildlife, domestic animals,

and humans, so that sources may be difficult to isolate. Monitoring

strategies generally include a reach-by-reach analysis of the main river

Watershed evaluation of pathogens

Fingerprinting to identify

sources

Two unrelated watersheds in the Mid-Atlantic Region have high fecal coliform levels, and managers

have been perplexed as to the source. The Philadelphia Suburban Water Company and Montgomery

County, Pennsylvania, were faced with finding

more sources to treat so that Deep Creek Lake

could be reopened to swimmers. Many of the usually suspected sources had already been treated. A

small Chesapeake Bay watershed on Virginia’s

Eastern Shore was overloading a shellfish area

with fecal coliforms and threatened to close down

the clam business in the area. Again, it seemed that

traditional sources (leaky onsite wastewater systems and farms) were exclusively the source.

Scientists tracked fecal coliform levels in the field

to generally identify source areas. In the Deep

Creek Lake Watershed, two microbiologists applied different genetic identification techniques,

called microbial source tracking (MST), to arrive at

similar conclusions: resident geese, ducks, and

other wildlife accounted for more than 70 percent

of the E. coli samples taken in the lake (CTIC,

1997). In the Chesapeake watershed, microbiologists developed a library of more than 200 DNA

patterns to which numerous samples were compared. They concluded that deer and raccoon were

primary sources (Simmons, 1997).

Monitoring and evaluation of pathogens
WSSI - Waterborne Pathogens—45

stem and tributary inputs to identify locations of high pathogen concentration in an effort to isolate the source(s). The evaluation may become

quite complex to determine the principal sources as in the Deep Creek

Lake Watershed, Pennsylvania, or a tributary of southern Chesapeake

Bay, Virginia.

In watershed evaluations, monitoring of indicator bacteria provides the

baseline data needed to identify segments of water bodies with limited

uses. Source(s) may be difficult to isolate using monitoring techniques

without the expenditure of considerable resources, so information is

gained from the ratio of fecal coliform to fecal strep (see table 3).

Modeling is an alternative to intensive monitoring. Models are useful

tools not only to help identify sources, but to also estimate the relative

loading from various management scenarios.

Models of indicator bacteria for surface runoff are available for site and

watershed scales (table 9). Generally, the logic in these models is to

generate organism numbers at the source, assume there is no regrowth,

and using literature or observed coefficients or algorithms for die-off,

route the populations through various pathways to the impaired water

body or threatened location.

Table 9 Selected models that simulate indicator bacteria

Model name Scale Main land use Hydrology Time scale Main input data Available from

Simple methods:

EPA screening Watershed Mixed N/A Mean Watershed NTIS PB86122496

procedures watershed annual data, land (EPA/600/6-85/

use, loading 002a),

factors (703) 487-4650

(default values)

Mid-range:

Auto-QI Watershed Urban Water Storm Hourly, daily Robert A. Sinclair,

balance event, rainfall; water- IL Water Survey,

continuous shed data, land 2204 Griffith Dr.,

use, BMP re- Champaign, IL

moval rates 61820 ($50)

(217) 333-4592

MWASTE/ Farm Feedlot/ ARS- Storm Animal waste Environmental

MSPRED field/ CREAMS event, system; hydrolo- engineer, National

pasture annual gy; field and Water & Climate

buffer strip Ctr, USDA-NRCS,

parameters; 101 SW Main St.,

coliform conc. Ste 1600,

in runoff from Portland , OR

various waste 97204-3224

management

systems

Monitoring and evaluation of pathogens
46—WSSI - Waterborne Pathogens

Table 9 Selected models that simulate indicator bacteria—Continued

Model name Scale Main land use Hydrology Time scale Main input data Available from

SLAMM Watershed Urban Small Storm Hourly rainfall, Dr. Robert Pitt,

storm- event, characteristics Dept. Civil/Envibased continuous of pollution ronmental Engicoefficient sources, areas, neering, UAL,

soil type, imper- 1150 Tenth Ave. S

viousness traffic Rm 257,

structure Birmingham, AL

35204-4401

(205) 934-8430

Detailed:

BASINS Watershed Mixed land HSPF— Storm Meteorologic & <http://www.epa.

uses water event, hydrologic, land gov/ost/BASINS/>

balance of continuous use distribution & BASINS 3.0

land surface characteristics, scheduled for

and soil loading factors & release in fall 2000

processes washoff parame- will incorporate

ters, receiving ArcView-SWAT

water characteris- with the capabiltics, decay coeffi- ity to simulate

cients, GIS & bacteria; current

national data version <http://

bases www.brc.

tamus.edu/swat/>

SWMM Watershed Urban Nonlinear Storm Meterologic & <http://www.

reservoir event, hydrologic data, ccee.orst.edu/

continuous land use distribu- swmm/>

tion, decay rates;

accumulation

& washoff

parameters

Monitoring and evaluation of pathogens

Ground water can also be affected by pathogens in watersheds in which

the soils are permeable, prone to macropore development, or they are

underlain by fractured bedrock. The potential movement of pathogens in

ground water including viruses, bacteria, Cryptosporidium and Giardia

was reviewed by Robertson and Edberg (1997). Fecal bacteria and other

organisms can contaminate springs and wells where waste is spread on

permeable soil, where waste is not properly contained in storage, or

where animals are concentrated near the water supply. The potential for

contamination of ground water from fecal organisms is normally less

than that of surface water because of the soil's filtering action and the

antagonistic competition with other soil organisms. Short circuits to

natural filtering, such as uncapped or improperly capped wells, are a

major source of fecal bacterial contamination in deeper ground water

systems. Because most individual ground water supplies are not treated

and have zero tolerance (EPA acceptable limit) as it relates to potable

water, protection of ground water supplies is a critical consideration.

Ground water
WSSI - Waterborne Pathogens—47

Anticipated

developments

from ongoing

pathogen

research

Detection of

protozoan cysts

in environmental

samples

Risk analysis

Cysts in water and soil are hard to detect. Membrane filters have been

introduced that may require less water. An immunofluorescent antibody

test, which stains cysts after they have been recovered from the filters,

helps detect the protozoan cysts. Research is currently in progress to

produce more efficient and effective methods for isolating cysts from

water. Methods proposed to date increase efficiency in sampling or

sample processing and analysis by using filters or other analytical methods (Krir and Genthe, 1993; Nieminski et al., 1995; Fricker and Crabb,

1998). The EPA recently released a methodology (Method 1623) for

detecting Giardia cysts and Cryptoporidium oocysts (USEPA, 1999c).

Easily used tests are needed to determine the species and host of origin

for Giardia cysts and Cryptoporidium oocysts, especially in environmental samples (water and soil). These tests would make it possible to

determine whether cysts are of public health concern; i.e., infective to

humans.

As part of its role of ensuring that major regulations proposed by USDA

are based on sound scientific and economic analysis, the Office of Risk

Assessment and Cost Benefit Analysis (ORACBA) is undertaking a series

of case studies to apply a multiple criteria decisionmaking technique

called Adaptive Risk Analysis (ARA) to USDA's Resource Conservation

Programs (Sadeghi et al., 1998; Tumeo et al., 1998 - Internet communications). In the case studies, ecological evaluation is an essential part of

the overall ARA method. Ecological endpoints, such as nutrients from

fertilizers and sediment from soil erosion, have been estimated using

environmental fate models. However, with the increased use of manure

in various agricultural production systems and heightened concern over

potential ecological and human health impacts, the fate and transport of

micro-organisms resulting from manure application need to be better

understood.

Microbial scenarios for various pathogens associated with different types

of manure in terms of their persistence, die-off, and regrowth rates have

been completed and are incorporated into the SWAT (Soil and Water

Assessment Tool) model. Work is underway to use several existing

microbial transport data bases on the fate and transport of pathogens

from manure for initial testing and validation of the microbial component

of the model. However, a more precise calibration and validation of the

model is expected to be done after more quantitative time zero microbial

population and transport data are obtained from the controlled field

lysimeters. These lysimeters are designed specifically to evaluate the

transport capability of Cryptosporidium oocysts in overland waterflow

conditions.

The incorporation of the newly developed SWAT model in the ARA

method provides a more comprehensive tool to examine the success of

USDA’s Environmental Quality Incentives Program (EQIP). This newly

developed process-oriented tool allows a look at specific watersheds and

determines the impact of a range of management practices, such as

manure management strategies, on multiple environmental and human

health objectives. This information provides the basis for comparison of

risk to the costs associated with a set of potential alternative management strategies.

Initial findings

Application of results

Anticipated developments from ongoing pathogen research
48—WSSI - Waterborne Pathogens

The transport of Cryptosporidium at the watershed level is also being

modeled in Canada (Park and Huck, 1999). They have developed a framework for estimating the relative contributions of cattle farms and wastewater treatment plants to Cryptosporidium oocysts in watersheds

(Zhang et al., 1999). The relationship between waterborne parasites

(Cryptosporidium and Giardia) and beef production in the North

Saskatchewan River is also being examined (Cooke, 1999 - Internet

communications).

An ARS-designed rainfall simulator has been setting the stage for what

will probably be the largest outdoor experimental study of the movement

of coliform bacteria and Cryptosporidium parvum in rain runoff and

soil water. The simulator has a boom that flexes as the land's slope

changes, keeping the rain the same distance from the soil up and down

the slope. The simulator will help scientists trace water movement to

track possible routes for bacterial pathogens and for Cryptosporidium

parvum (Shelton, 1999).

Studies are underway to identify flushwater systems that minimize the

risk of environmental contamination and inter-cattle transmission of

Cryptosporidium and Giardia and at the same time conserve water

usage through flushwater recycling (Atwill, 1999-Internet communications). Other studies have shown or are being conducted to examine the

prevalence of and risk factors for the shedding of Cryptosporidium and

Giardia in livestock populations, llamas, wild pigs, and horses (Atwill et

al., 1997; Cole et al., 1998; Wade et al., 1999; Mohammed et al., 1999).

The vast majority of waterborne pathogens affecting people do not

originate from agricultural sources. Human contamination or inadequacies at water treatment plants have been implicated in almost all largescale waterborne outbreaks. The potential does exist for contamination

of water with pathogens from agriculture, warranting a proactive approach for reducing this source in watersheds. Because of the large

amount of fecal material produced and used on the farm, as well as the

spreading of human waste (sewage and sludge), this potential exists. At

this point in time, scientific literature indicates that sound agricultural

practices currently in place significantly reduce the opportunity for the

introduction of pathogens to the watershed.

This publication introduces the subject of pathogenic organisms and

measures used to reduce their transport into the environment. A multiple-barrier approach is recommended. This approach includes prevention of pathogen imports to the farm, breaking the amplification cycle on

the farm, proper handling of the waste stream, and control of transport

from the farm. On-farm measures need to include a veterinarian or other

health care provider who can target control points for pathogen introduction, amplification, and export from the farm before they get into the

environment. Although pathogen-specific practices have not been developed, those practices used for waste management and to control runoff

help control pathogen transport from the farm to the watershed.

Summary

Pathogen transport

Conceptual modeling

Distribution, dissemination, and fate of Cryptosporidium oocysts

Anticipated developments from ongoing pathogen research
WSSI - Waterborne Pathogens—49

Glossary

Aerobic A habitat or organism that relies on the presence of oxygen for continued growth and existence.

Amnesic shellfish poisoning An impairment or lack of memory caused by the ingestion of shellfish

(ASP) contaminated with the diatom Pseudo-nitzschia sp. that has produced

the toxin domoic acid. ASP can be a life-threatening syndrome.

Amoeba A single-celled (protozoan) organism that constantly changes shape.

Anaerobe Used to describe a biological habitat or an organism that exists and

grows with oxygen.

Asymptomatic Carrying a particular disease, but not showing any symptoms.

Autoinfection When a parasite reproduces in a host that then re-infects itself.

Biofilm A thin layer of organisms that colonize and coat the surface in a water

environment.

Campylobacteriosis An infection of the intestines caused by bacteria of the Campylobacter

genus. Symptoms of campylobacteriosis include mild to severe diarrhea

(often bloody), stomach pain, fever, nausea, and vomiting.

Capsid A protein coat that covers the nucleoprotein core or nucleic acid of a

virus.

Ciguatara fish poisoning Gastrointestinal, neurological, and cardiovascular symptoms associated

 (CFP) the ingestion of fish contaminated with toxic dinoflagellates (any of the

following: Gambierdiscus toxicus, Prorocentrum sp., Ostreopsis sp.,

Coolia monotis, Thecadinium sp. and Amphidinium carterae). Paralysis and death have been documented, but symptoms are usually less

severe although debilitating.

Ciliate(d) A grouping of microscopic single-celled organisms that have hair-like

projections on their surface used for locomotion.

Coccus A bacterial cell that has the shape of a sphere.

Coliphage A bacterial virus (phage) that uses coliform bacteria as a host.

Cryptosporidiosis A diarrheal illness of varying severity caused by a microscopic intestinal

parasitic protozoan, Cryptosporidium. It is a common cause of diarrhea

worldwide and is common in AIDS patients.

Cyst A resting stage of an organism that has a tough outer coating.

Dinoflagellate Photosynthetic organisms of the order Dinoflagellida (for botanists

Dinophyceae). They are aquatic and have two flagella that allow the

organism to move.

DNA (deoxyribonucleic acid) The genetic material of all cells and many viruses.

Glossary
50—WSSI - Waterborne Pathogens

E. coli Short for Escherichia coli, the colon bacillus, a bacterium that normally

resides in the intestine of many animals.

ELISA (enzyme linked An assay designed for detection of many different organisms, such as

immuno sorbent assay) certain Salmonella sp., E. coli, and Cryptosporidium (many organismspecific test kits available) http://www.bioline.dk/index.html, http://

www.maff.gov.uk/animalh/bse/animal-health/feedban-elisatest.html.

Encyst The process a cell undergoes to produce a cyst.

Endospore An asexual spore formed within a bacterial cell.

Enteric Of or relating to the small intestine.

Epidemiology The study of populations to determine the frequency and distribution of

disease and to measure risks.

Facultative anaerobe An organism that normally lives in an oxygen-free environment, but can

survive oxygen conditions.

Fecal coliform A grouping of coliform bacteria that lives in the intestines of warmblooded animals. Elevated measurements of these bacteria in surface

water may indicate the presence of human and/or animal waste. Health

advisories may be posted when measurements indicate an increased risk

to humans from exposure.

Flagellum A long, thin, hair-like projection from a cell used in movement.

Gastroenteritis Inflammation of the mucous membrane of the stomach and intestine.

Harmful algal blooms Elevated growth of one or more species of algae, which may result from

excessive nutrient loading in combination with adequate light, temperature, and other environmental factors.

Immunofluorescent antibody A technique developed to specifically identify an organism. Initially, a

probe is developed from the surface properties (antigens) of an organism. These are used to stimulate the production of an antibody that is

specific to that species. The antibody is conjugated (bonded) with a

fluorescent dye that allows it to be seen.

Inoculated The process of adding the material containing seed organisms to start

growth.

Isolate(s) An organism that is isolated from a single source, usually by culturing.

Lysogenic phase The ability of some bacterial viruses to survive in a bacterium as a result

of the integration of their DNA into the host chromosome.

Lytic phase The normal cycle of infection of a cell by a virus in which a mature virus

is produced and the cell is then ruptured.

Mesophilic Requiring a warm temperature in which to develop.

Nonculturable Not being able to grow an organism in laboratory culture on media.

Glossary
WSSI - Waterborne Pathogens—51

Oocyst The environmentally resistant stages of protozoan, such as

Cryptosporidium.

Organelle A structurally discrete component of a cell.

Paralytic shellfish poisoning A life-threatening syndrome caused by consumption of contaminated

 (PSP) shellfish. The causative organisms are the dinoflagellates: Alexandrium

sp., Gymnodinium catenatum, Pyrodinium bahamense that can be

accumulated by shellfish. The toxins produced are called saxitoxins.

Parasite An organism that lives in or on an animal or plant, called a host, and

takes its nourishment to the host's detriment.

Pathogen An agent of disease; it can be a parasite.

RiboNucleic acid (RNA) A chemical similar to DNA. The several classes of RNA molecules play

important roles in protein synthesis and other cell activities.

Ribotype The RNA signature or fingerprint of a cell used to identify the source of

the organism.

Rotavirus A virus that is the leading cause of severe winter diarrhea in young

children.

Salmonellosis An infection of the intestines caused by the Salmonella bacteria, which

causes severe diarrhea and death in some cases.

Serotype A technique used to determine different stains of bacteria.

Shedding The releasing of organisms from the host, usually in feces.

Shigella A group of bacteria that normally inhabits the intestinal tract and causes

infantile gastroenteritis, summer diarrhea of childhood, and various

forms of dysentery.

Spirillum A fairly rigid, helically twisted bacterial cell often, but not necessarily, a

member of the genus Spirillum.

Spirochete Bacteria that appear worm-like, spiral-shaped, and wiggle vigorously

when viewed under a microscope.

Sporozoites A stage in the development oocyst of Cryptosporidium that infects

intestinal cells.

Thermophilic Requiring high temperature in which to develop.

Trophozoite The feeding stage of a protozoan (as distinct from reproductive or encysted stages).

Zoonotic A disease caused by pathogens that are transmitted among animals and

humans.

Zooplankton The floating, often microscopic animals that live in aquatic environments.

Glossary
52—WSSI - Waterborne Pathogens

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