United States Office of Science and Technology EPA-822-K-94-001

Environmental Protection Office of Water March 2001

Agency Washington, DC 20460 www.epa.gov

Cryptosporidium:

Human Health

Criteria Document
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

-iACKNOWLEDGMENTS

This document originally was prepared for the U.S. Environmental Protection Agency, Office of Groundwater

and Drinking Water (OGWDW) by the Office of Science and Technology (OST) under contract with Dr. Jon

Standridge from the University of Wisconsin (Order No. 8W-1644-NASA). The document was updated and

revised by ICF Consulting under the direction of Jennifer Welham (Purchase Order 1C-W010-NALX). Overall

planning and management for the preparation of this document was provided by Lisa Almodovar and Robin

Oshiro of OST. 

EPA acknowledges the valuable contributions of those who wrote and reviewed this document. They include:

Jon Standridge, David Battigelli, Rebecca Hoffman, Amy Mager, and other writers/editors of the University of

Wisconsin; Jennifer Welham, Annabelle Javier, and Kristin Jacobson of ICF Consulting; and Lisa Almodovar,

Robin Oshiro, and Crystal Rodgers of the U.S. EPA. EPA also thanks the following external peer reviewers for

their excellent review and valuable comments on the draft document: Mark Borchardt, Ph.D. (Marshfield

Medical Research and Education foundation); Carrie Hancock (CHDiagnostics and Consulting Service, Inc.);

and Charles Gerba, Ph.D. (University of Arizona).
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

-iiEXECUTIVE SUMMARY

The Safe Drinking Water Act requires the U.S. Environmental Protection Agency (EPA) to publish regulations

to control disease-causing organisms (pathogens) and hazardous chemicals in drinking water. One of the

regulations published by EPA to control pathogens is known as the Surface Water Treatment Rule (54 FR

27486; June 29, 1989). The intent of this rule was to control Giardia, pathogenic viruses, and Legionella, all of

which have caused many outbreaks and cases of waterborne illness.

Another prominent waterborne pathogen is the protozoan Cryptosporidium. This organism has caused a

number of waterborne disease outbreaks in the U.S. and other countries. In 1994, EPA prepared a literature

review of the published data on Cryptosporidium, entitled “Cryptosporidium Criteria Document,” to establish

a basis for a regulation to control this organism. The following document, “Drinking Water Criteria Document

Addendum: Cryptosporidium,” updates the 1994 publication. It includes new information available in the

literature from 1994 to the present and was prepared to support EPA’s Interim Enhanced Surface Water

Treatment Rule, which has as a primary focus the control of Cryptosporidium. The update provides information

on general characteristics of Cryptosporidium, its occurrence in human and animal populations and in water, the

health effects associated with Cryptosporidium infection, outbreak data, and an assessment of risk. The

document also includes information about analytical methods to enumerate Cryptosporidium in water and the

effectiveness of various water treatment practices in its removal. 

The document demonstrates that Cryptosporidium oocysts are common and widespread in ambient water and

can persist for months in this environment. The dose that can infect humans is low, and a number of waterborne

disease outbreaks caused by this protozoan have occurred in the U.S., most notably in Milwaukee, where an

estimated 400,000 people became ill. The document shows that otherwise healthy people recover within several

weeks after becoming ill, but illness may persist and contribute to death in those whose immune systems have

been seriously weakened (e.g., AIDS patients). Drugs effective in preventing or controlling this disease are not

yet available. The public health concern is worsened by the resistance of Cryptosporidium to commonly used

water disinfection practices such as chlorination. However, a well-operated water filtration system is capable of

removing at least 99 of 100 Cryptosporidium oocysts in the water. Monitoring for this organism in water is

currently difficult and expensive. 

EPA believes that the information presented in the 1994 document and in the following update is sufficient to

conclude that Cryptosporidium may cause a health problem and occurs in public water supplies at levels that

may pose a risk to human health.
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

-iiiTABLE OF CONTENTS

I. Introduction ....................... ....................... .................... 1

II. General Information and Properties ....................... ....................... . . 2

A. History and Taxonomy ....................... ....................... ..... 2

1. History ....................... ....................... ............ 2

2. Taxonomy ....................... ....................... ......... 3

B. Life Cycle ....................... ....................... ............... 11

C. Morphological Features ....................... ....................... .... 11

D. Species Transmission ....................... ....................... ...... 12

1. Direct Transmission Between Humans ....................... ......... 12

2. Transmission Between Animals and Humans ....................... .... 14

E. Summary ....................... ....................... ............... 17

III. Occurrence ....................... ....................... .................... 18

A. Worldwide Distribution ....................... ....................... .... 18

1. Distribution in Animal Populations ....................... ............ 18

2. Distribution in Human Populations ....................... ............ 20

B. Occurrence in Water ....................... ....................... ...... 22

1. Surface Water ....................... ....................... ...... 22

2. Groundwater ....................... ....................... ...... 23

C. Occurrence in Soil ....................... ....................... ........ 23

D. Occurrence in Air ....................... ....................... ......... 25

E. Occurrence in Food and Beverages ....................... .................. 25

F. Specific Disease Outbreaks ....................... ....................... . 26

1. Outbreaks Associated with Drinking Water ....................... ..... 26

2. Outbreaks Associated with Recreational Waters ....................... . 31

3. Foodborne Outbreaks ....................... ....................... 32

4. Outbreaks among Travelers ....................... ................. 33

5. Outbreaks at Day Care Centers ....................... ............... 34

6. Outbreaks Among Sensitive (Immunocompromised) Subpopulations ........ 34

G. Environmental Factors ....................... ....................... ..... 35

H. Summary ....................... ....................... ............... 39

IV. Health Effects in Animals ....................... ....................... ........ 40

A. Symptomatology and Clinical Features ....................... ............... 40

B. Therapy ....................... ....................... ................ 44

C. Epidemiological Data ....................... ....................... ...... 45

D. Summary ....................... ....................... ............... 47

V. Health Effects in Humans ....................... ....................... ........ 49
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

-ivA. Symptomatology and Clinical Features ....................... ............... 49

B. Epidemiological Data ....................... ....................... ...... 51

C. Treatment: Clinical Laboratory Findings and Therapeutic Management ............ 58

D. Mechanism of Action ....................... ....................... ...... 62

E. Immunity ....................... ....................... ............... 63

F. Chronic Conditions ....................... ....................... ....... 66

G. Summary ....................... ....................... ............... 66

VI. Risk Assessment ....................... ....................... ............... 67

A. Experimental Human Data ....................... ....................... . . 68

B. Experimental Animal Data ....................... ....................... . 70

C. Environmental Factors ....................... ....................... .... 70

1. Prevalence in Surface Waters ....................... ................ 70

2. Oocyst Survival ....................... ....................... .... 71

3. Cryptosporidium in Drinking Water ....................... ........... 72

D. Epidemiologic Considerations ....................... ...................... 73

E. Risk Assessment Models ....................... ....................... ... 75

F. Federal Regulations ....................... ....................... ....... 78

G. Summary ....................... ....................... ............... 81

VII. Analysis and Treatment ....................... ....................... .......... 81

A. Analysis of Water ....................... ....................... ........ 81

l. Collection of Cryptosporidium from Water ....................... ...... 84

2. Detection of Cryptosporidium in Water ....................... ........ 92

3. Assessment of Laboratory Testing Capabilities ....................... . 103

B. Detection in Biological Samples ....................... ................... 104

C. Water Treatment Practices ....................... ....................... . 111

1. Introduction ....................... ....................... ...... 111

2. Multibarrier Treatment ....................... ..................... 112

3. Removal of Cryptosporidium ....................... ............... 114

4. Inactivation of Cryptosporidium ....................... ............. 120

D. Summary ....................... ....................... .............. 124

VIII. Research Requirements ....................... ....................... ......... 127

IX. References ....................... ....................... ................... 130
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

1

I. Introduction

The United States Environmental Protection Agency (USEPA) Office of Water is preparing and revising the

health criteria documents that will support the Phase I Disinfectant/Disinfectant Byproduct (DBP) Rule, the

Interim Enhanced Surface Water Treatment Rule (IESWTR) and the Groundwater Disinfection Rule (GWDR). 

As part of the rule making process, the USEPA is required to compile a complete and current compendium of

the information used as criteria to support creation of the rules. The first step in this process occurred in June of

1994 with the preparation of the USEPA Draft Drinking Water Criteria Document for Cryptosporidium,

hereafter referred to as the “1994 Cryptosporidium Criteria Document.” This addendum provides an update to

supplement (but not duplicate) the 1994 Cryptosporidium Criteria Document. This addendum uses the same

table of contents formatting as the 1994 document to facilitate cross referencing between the two documents. 

Much of the published research since the 1994 document has focused on the speciation of Cryptosporidium,

better methods to detect Cryptosporidium in the environment, and improvements in water treatment technology. 

Consequently, this addendum includes much new information regarding speciation and improvements in

analyses and treatment. The overall objective is to provide a comprehensive Cryptosporidium information

resource to Federal, State, and local health officials responsible for protecting public health and the

environment.

II. General Information and Properties

A. History and Taxonomy

1. History

Cryptosporidium was described by Tyzzer in 1907 but was considered medically unimportant to humans until

the first cases of cryptosporidiosis in humans were reported in 1976 by Nime et al. and Miesel et al. (Fayer et

al., 1997a). However, the diagnosis of cryptosporidiosis in humans in 1976 and the subsequent connection of
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

2

Cryptosporidium to epidemic waterborne disease have since fostered worldwide interest in the study of this

microorganism. By the time the Centers for Disease Control and Prevention implemented routine reporting of

Cryptosporidium among AIDS patients in 1982, only 13 cases of human cryptosporidiosis had been

documented (Ungar, 1990). Since 1982, more than 1,000 reports of human cryptosporidiosis have been

documented in almost 100 countries, reaching all continents with the exception of Antarctica (Fayer, 1997). At

the time of this writing, it is estimated that the annual number of cryptosporidiosis cases exceeds several million

worldwide (Casemore et al., 1997).

Cryptosporidium was first recognized as a waterborne pathogen during an outbreak in Braun Station, Texas,

where more than 2,000 individuals were afflicted with cryptosporidiosis (D’Antonio et al., 1985; Graczyk et al.,

1998b). Since that time, outbreaks affecting over a million individuals have been documented throughout North

America and Europe, with the single largest epidemic occurring in Milwaukee, Wisconsin, in 1993 (Mackenzie

et al., 1994). A complete history of the waterborne outbreaks of cryptosporidiosis is provided in section III-F.

2. Taxonomy

Cryptosporidium is one of several protozoan genera in the phylum Apicomplexa which develop within the

gastrointestinal tract of vertebrates throughout their entire life cycles. More than 20 species have been described

based upon the hosts from which they were originally isolated (a complete list is included in Table II-1, 1994

Cryptosporidium Criteria Document). By 1997, however, interspecies transmission studies, morphological

evaluations and immunological analyses had reduced this number to eight valid species (Fayer et al., 1997a).

Since 1997, two other species have been identified, brining the total number of valid species to ten.

Cryptosporidium saurophilum was isolated from populations of lizards, Schneider’s skink (Eumeces
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

3

schneideri), and desert monitors in Australia (Koudela and Modry, 1998). Cryptosporidium andersoni was

recovered from the feces of domestic cattle, Bos taurus (Lindsay et al., 2000). Table 1 lists the ten valid

Cryptosporidium species and the host organism(s) in which each parasite was originally found; some of these

species have since been shown to occur in additional hosts (Fayer, 1997; Fayer et al., 2000). Genetic research

has provided support for the species C. felis and C. wrairi, whose distinctness from other Cryptosporidium

species had been previously questioned (Bornay-Llinares et al., 1999; Morgan et al., 1999b; Morgan et al.,

1999c; Morgan et al., 1998b; Sargent et al., 1998; Xiao et al., 1999a; Xiao et al., 1999b). 

Table 1. Valid Cryptosporidium Species

Cryptosporidium Species Initially Described Host Species

C. andersoni Bos taurus (cattle)

C. baileyi Gallus gallus (domestic chicken)

C. felis Felis catis (domestic cat)

C. meleagridis Meleagris gallopavo (turkey)

C. muris Mus musculus (house mouse)

C. nasorum Naso literatus (fish)

C. parvum Mus musculus (house mouse)

C. saurophilum Eumeces schneideri (skink)

C. serpentis Elaphe guttata (corn snake)

E. subocularis (rat snake)

Sanzinia madagasarensus (Madagascar boa)

C. wrairi Cavia porcellus (guinea pig)

Source: Adapted from Fayer et al. (2000) and Fayer et al. (1997a)

O’Donoghue (1995) reported that infection caused by Cryptosporidium had been observed in 79 mammalian

species (including humans) in addition to numerous reptilian, amphibian, avian, and fish hosts. Fayer et al.

(2000) documented Cryptosporidium infection in more than 150 mammalian species. Illness in humans,
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

4

however, is confined primarily to infections associated with C. parvum (O’Donoghue, 1995). A single case of

human cryptosporidiosis in an immunocompromised individual was attributed to C. baileyi (Ditrich, 1991), but

this organism was later shown to be C. parvum (Fayer, pers. comm.). Two recent studies have reported C. felis

infections in HIV-positive patients in the United States (Morgan et al., 2000a; Pieniazek et al. 1999). In

addition, C. meleagridis was detected from an HIV-infected individual in Kenya (Morgan et al., 2000a). 

The taxonomy of Cryptosporidium is in the forefront of current research on the parasite, and changes in

nomenclature may be expected. Molecular studies have found considerable evidence of genetic heterogeneity

among isolates of C. parvum from different vertebrate species, and findings from these studies indicate that a

series of host-adapted genotypes or strains of the parasite exist (Awad-El-Kariem et al., 1998; Morgan et al.,

1999a; Morgan et al., 1999b; Morgan et al., 1999c; Morgan et al., 1999d; Morgan et al., 1998a; Spano et al.,

1998a; Spano et al., 1998b; Sulaiman et al., 1998; Xiao et al., 1999a; Xiao et al., 1999b).

Several studies have suggested the possibility of distinct transmission cycles among different genotypes (AwadEl-Kariem, 1999; Awad-El-Kariem et al., 1998; Morgan et al., 1998a; Patel et al., 1998; Peng et al., 1997;

Sulaiman et al., 1998; Widmer et al., 1998c). Alternatively, multiple genotypes may be able to circulate

among different host species, and mixed infection with genotypically different populations may arise through

selection in different host environments (Widmer et al., 1998b). Nevertheless, some researchers have suggested

that these genotypes should be considered to be separate species (Morgan et al., 1999c; Xiao et al., 1999b).

Until recently, it was not possible to assess genetic variation among Cryptosporidium isolates. Molecular

studies utilizing restriction fragment length polymorphism (RFLP) analysis, isoenzyme electrophoresis, and
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

5

arbitrarily primed polymerase chain reactions (AP-PCR) have helped to characterize these subtle differences

among individual isolates; these recent studies are described below.

One study using PCR and restriction mapping suggested that differences in the genetic sequences within the

18S ribosomal RNA (rRNA) region of Cryptosporidium could be used to distinguish individual species (C.

muris, C. parvum, and C. baileyi) and could assist in the development of taxonomic classification (Awad-ElKariem et al., 1994). Webster (1993) applied a battery of molecular taxonomic methods (flow cytometry, PCR,

and RFLP) to detect and classify Cryptosporidium oocysts from geographically diverse isolates. The isolates

exhibited genetic homogeneity for the most part, although differences in isoelectric points and restriction maps

indicated genetic differences among C. parvum isolates from humans and bovines.

Isoenzyme electrophoresis studies (O’Donoghue, 1995; Awad-El-Kariem, 1995; Awad-El-Kariem et al., 1998)

have been applied to characterize animal and human oocyst isolates of C. parvum from different geographical

locations. The discovery of two unique isoenzyme forms indicates the existence of separate subpopulations

within the C. parvum species, one which infects primarily humans and the other which infects animals. Followup studies using AP-PCR and isoenzyme typing (Carraway et al., 1994; Awad-El-Kariem et al., 1996; AwadEl-Kariem et al., 1998 have confirmed the two unique profiles among C. parvum isolates corresponding to the

animal and human types. Cross-transmission infection studies performed by Awad-El-Kariem et al. (1996,

1998) indicated that most human isolates were not capable of establishing infection in a murine model, whereas

all animal isolates were infectious in mice, supporting the existence of genetically distinct populations of this

strain.
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

6

Analysis of genetic polymorphisms among C. parvum isolates from nine human outbreaks and from several

bovine sources (Peng et al., 1997) indicated the existence of two genotypes with genetic differences among

adhesion proteins. Genotype 1 was observed exclusively in human isolates and has been called the human or H

genotype. Genotype 2 was observed both in calf isolates and in isolates from human patients who reported

direct or indirect exposure to infected cattle, and this genotoype has been called the cattle or C genotype. These

findings support two distinct transmission cycles of C. parvum in humans: (1) human to human, and (2) animal

to human. This hypothesis is supported by the work of Carraway et al. (1997), who conducted RFLP analyses

on C. parvum oocysts isolated from humans and cows. While all calf isolates exhibited genetic homogeneity at

a specific 2.8-kb fragment, human isolates exhibited multiple profiles at this locus: one found exclusively in

humans, and one with a superposition of both profiles, indicative of heterogeneity among parasite populations.

Sequence and/or PCR-RFLP analyses of various loci have confirmed the genetic distinctness of the human and

cattle genotypes. The examined loci include the 18S small subunit (SSU) rRNA gene (Morgan et al., 2000a;

Xiao et al., 1999a; Xiao et al., 1999b), ribosomal ITS1 and ITS2 (internal transcribed spacer) regions (Morgan

et al., 1999a), the acetyl-CoA synthetase gene (Morgan et al., 2000a; Morgan et al., 1998a), the COWP

(Cryptosporidium oocyst wall protein) gene (Patel et al., 1998; Spano et al., 1997), the dhfr (dihydrofolate

reductase) gene (Morgan et al., 1999b), the TRAP-C1 and TRAP-C2 (thrombospondin-related adhesive protein

of Cryptosporidium) genes (Spano et al., 1998b; Sulaiman et al., 1998), and the HSP-70 (heat shock protein)

gene (Morgan et al., 2000a). The genetic distinctness of the two genotypes also was supported by a multilocus

study performed by Spano et al. (1998a) on 28 isolates of C. parvum originating from Europe, North and South

America, and Australia. The study analyzed the poly(T) (polythreonine) gene, COWP gene, TRAP-C1 gene,

and RNR (ribonucleotide reductase) gene by using PCR-RFLP, as well as the ITS1 region using a genotype-
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

7

specific PCR. All isolates clustered into two groups, one comprising isolates of both human and animal origin

and the other comprising only human isolates, and no recombinant genotypes were found. Khramtsov et al.

(2000) demonstrated that two virus-like double-stranded (ds) RNAs are present in C. parvum. Although the

dsRNA sequences were similar in isolates of either human or calf origin, slight but consistent differences in

nucleotide sequences at select sites were noted between the two genotypes.

While researchers have demonstrated substantial genetic differences between the human and cattle genotypes,

some studies also have found variation within these genotypes. Widmer et al. (1998a) reported evidence of

polymorphisms within the human genotype and of recombination between the human and cattle genotypes,

based on sequence and PCR-RFLP analysis of the -tubulin intron. In a separate study (Widmer et al., 1998b),

sequence and PCR-RFLP analyses of the -tubulin intron also revealed polymorphisms within the human and

cattle genotypes, with sequences indicative of interallelic recombination in two isolates. Caccio et al. (2000)

provided further evidence that the human and cattle genotypes are not genetically homogeneous. Sequence

analysis of a locus containing microsatellite repeats in 94 C. parvum isolates demonstrated heterogeneity in both

the human and cattle genotypes. Two subgenotypes of the human genotype and four subgenotypes of the cattle

genotype were identified, but the prevalence and significance of these intragenotype differences are not clear. 

For example, Okhuysen et al. (1999) demonstrated that three different C. parvum isolates of the cattle genotype

differed in their infectivity for humans.

In addition to the human and cattle genotypes, recent characterizations of C. parvum isolates from other

vertebrate species have revealed host-specific genotypes in mice, pigs, marsupials, and dogs (Morgan et al.,

2000b; Morgan et al., 1999a; Morgan et al., 1999b; Morgan et al., 1999c; Morgan et al., 1999e; Morgan et al.,
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

8

1998a; Pereira et al., 1998; Xiao et al., 1999b). A genetically distinct strain referred to as the mouse genotype

has been identified in C. parvum isolates from mice in Australia, the United States, the United Kingdom, and

Spain by using sequence analysis of the 18S SSU rRNA, ITS1 and ITS2, dhfr, HSP-70, COWP, and acetyl CoA

loci, as well as RAPD (random-amplified polymorphic DNA analysis) (Morgan et al., 1999a; Morgan et al.,

1999b; Morgan et al., 1999c; Morgan et al., 1999e; Morgan et al., 1998a; Xiao et al., 1999b).

Another genetically distinct strain referred to as the pig genotype has been identified by sequence analysis of the

18S SSU rRNA, ITS1 and ITS2, dhfr, and acetyl CoA loci, as well as RAPD and AP-PCR, of C. parvum

isolates from pigs in Switzerland, the United States, and Australia (Morgan et al., 1999a; Morgan et al., 1999b;

Morgan et al., 1999c; Morgan et al., 1999f; Morgan et al., 1998a; Pereira et al., 1998; Xiao et al., 1999b). A

marsupial genotype also has been suggested based on genetic analysis of C. parvum isolates from a koala from

South Australia and a red kangaroo from Western Australia (Morgan et al., 1999a; Morgan et al., 1999b;

Morgan et al., 1999c; Xiao et al., 1999b). Sequence analysis of the 18S SSU rRNA, ITS1 and ITS2, and dhfr

loci, as well as RAPD, of these isolates has confirmed their distinctness from all other genotypes of C. parvum.

The dog genotype has been identified from sequence analysis of the 18S SSU rRNA and HSP-70 loci, which

demonstrated genetic distinctness in C. parvum isolates from dogs from Australia and the United States

(Morgan et al., 2000b; Morgan et al., 1999c; Xiao et al., 1999b). The dog genotype also has been isolated from

HIV patients (Pieniazek et al., 1999). Xiao et al. (1999b) found genetically distinct strains in C. parvum

isolates from a ferret and a monkey using sequence analysis of the 18S SSU rRNA gene, suggesting the

possibility of a ferret genotype and a monkey genotype. Although the occurrence of genetically distinct strains
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

9

of C. parvum may warrant a taxonomic revision of the genus, scientists have expressed concern over

designating a new species based on a small number of base pair differences (USEPA, 1999a).

Some researchers have supported the hypothesis that Cryptosporidium may belong to a clonal population

structure, based on correlations between phenotypic and genotypic markers and the widespread occurrence of

identical genotypes (Awad-El-Kariem, 1999; Morgan et al. 1999c). The essential concept of the clonal

hypothesis is that different strains of the same species may clone (or propagate) different forms of the same

gene due to geographic isolation. As a consequence of this isolation, mutated or otherwise modified genetic

loci will become perpetuated in a clonal manner within the species in the absence of sexual reproduction. Other

researchers have questioned the clonal population hypothesis on the basis of mixed infection and apparent

genetic recombination (Patel et al., 1998; Widmer et al., 1998a; Widmer et al., 1998b). Studies are underway to

further elucidate the population structure of Cryptosporidium.

B. Life Cycle

A complete description of the life cycle of Cryptosporidium is provided in the 1994 USEPA Cryptosporidium

Criteria Document (Figure II-1, p. II-5). Cryptosporidium is excreted in the feces of an infected host in the form

of an oocyst, which represents the only exogenous stage of the life cycle. The oocyst consists of four

sporozoites housed within a sturdy, multi-layered wall. The thick-walled oocyst is the environmentally resistant

form of the parasite, resulting from the fertilization of macrogametes within the host, and it is appreciably

resistant to natural decay in the environment as well as to most disinfection processes. The life cycle is repeated

when sporulated oocysts excreted by an infected host are ingested by a new host and the sporozoites excyst

within the new host’s gastrointestinal tract.
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

10

C. Morphological Features

A complete description of the morphological features of each life cycle stage (oocyst, sporozoite, trophozoite,

merozoite, microgametocyte, macrogametocyte) of Cryptosporidium is provided in the 1994 Cryptosporidium

Criteria Document (p.II-7–II-9). Robertson et al. (1993, 1994) provided evidence that the suture spanning part

of the circumference of the oocyst inner wall described in ultrastructural studies is not the same structure as the

apparent “fold” in the oocyst wall seen using fluorescence microscopy. Their ability to reversibly induce the

folds suggests that they are probably artifactual. As a result, the researchers suggested that the apparent fold no

longer be considered a diagnostic feature of Cryptosporidium parvum. 

D. Species Transmission

1. Direct Transmission Between Humans

A number of studies show that person-to-person transmission of cryptosporidiosis infection can occur within

families, day care centers, hospitals, and in urban environments where population densities are high (USEPA,

1994). The route of infection follows one of two paths: direct, through fecal-oral contact, or indirect, through

fomites (inanimate objects). Casemore (1990) evaluated cryptosporidiosis associated with nosocomial (hospital

acquired) transmission as well as its association with traveller’s diarrhea. In the hospital setting,

cryptosporidiosis may be spread from one patient to another or from a patient to a staff member. 

Cryptosporidium also is a primary cause of traveller’s diarrhea, typically being transmitted through

contaminated water or food (Casemore, 1990). Transmission is affected by ethnic and dietary differences (e.g.,

Muslims exhibit a lower prevalence than many other ethnic groups).
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

11

After visitors who came to Milwaukee during the 1993 outbreak returned to their homes, 74 members of their

households who had not accompanied them on the visit tested positive for Cryptosporidium (Mackenzie et al.,

1995b). Five percent of these infected household members ultimately developed cryptosporidiosis, thereby

meeting the definition of secondary transmission within the household. Osewe et al. (1996) also evaluated data

following the Milwaukee outbreak and concluded that secondary transmission rates to household members

during the epidemic were comparatively low (3-5%). In addition to the person-to-person transmission, there

appeared to be a recurrence of infections that were acquired by the Milwaukee visitors during the outbreak.

Transmission to susceptible individuals in the Milwaukee area continued after the massive initial outbreak, but

decreased rapidly within the two months that followed.

Cordell and Addiss (1994) tracked cryptosporidiosis in various child care settings and observed a 12 to 22% rate

of secondary spread between children to other household members. Newman et al. (1994) reported household

transmission of C. parvum infection in an urban community in northeast Brazil. In this study, 18 of 31 (58%)

households, whose members ranged in age from 5 months to 47 years, showed at least one secondary case of

cryptosporidiosis (identified either by stool examination or serologic testing). Secondary cases involved a total

of 30 persons, yielding an overall transmission rate of 19%. Of the 202 persons included in this study, 94.6%

had evidence of serum IgG or IgM antibodies to Cryptosporidium, demonstrating that a significant rate of

person-to-person transmission of Cryptosporidium may occur.

The rate of transmission between immunocompromised individuals may be high. In group homes housing HIVpositive individuals, Heald and Bartlett (1994) reported a high (not specified) rate of transmission among

occupants. Lopez-Velez et al. (1995) found an overall prevalence of intestinal cryptosporidiosis of 15.6% in
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

12

AIDS patients. However, the rate among homosexual partners was higher (33.3%) than among intravenous

drug users (10.6%), strongly suggesting person-to-person transmission of cryptosporidiosis. Sears et al. (1994)

concluded that strict infection control measures must be followed especially in crowded living conditions and

where immunocompromised persons reside.

Secondary transmission of cryptosporidiosis has been observed among humans whose occupation places them

near primary cases within a confined space. An outbreak occurred among crew members on a U.S. Coast Guard

cutter that had obtained water from the city of Milwaukee during the 1993 epidemic (Moss et al., 1994). Of 50

crew members, 62% exhibited symptoms of cryptosporidiosis, and oocysts were detected in stool samples of 10

individuals (20%). In such an outbreak, distinguishing between primary infections (i.e., due to ingestion of

contaminated water) and secondary infections (i.e., due to fecal-contaminated fomites, food, or other infected

individuals) is difficult because the individuals involved were obviously in a closed environment where frequent

contact between humans occurs. In some instances it may not be possible to determine whether transmission

between humans is the primary cause of cryptosporidiosis, especially when humans also come into contact with

animals through occupational or recreational activities (Adam et al., 1994).

2. Transmission Between Animals and Humans

The 1994 USEPA Cryptosporidium Criteria Document provided adequate evidence for the transmission of

Cryptosporidium from animals, particularly livestock, to humans. Domestic animals such as calves and lambs

are common zoonotic reservoirs involved in occupational exposure, indirect zoonotic transmission, and

contamination of food (e.g., sausages, offal, and raw milk). Animals may also contribute to environmental

contamination in sources such as watersheds, food crops, and recreational waters. For example, 70 cases of
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13

cryptosporidiosis in the U.K. resulted from exposure to a single swimming pool with water contaminated by

animal excreta. Pets, however, have not often been implicated as a source of infection and are not considered a

major risk factor for acquisition of cryptosporidiosis (Glaser, 1998).

Of ten valid species infecting all vertebrate groups, only one, C. parvum, represents a global public health

problem due to its zoonotic potential (Graczyk, 1998b). Although infections in humans primarily have been

attributed to C. parvum, a single case of C. baileyi infection in an immunosuppressed individual has been

reported (Ditrich, 1991). Two instances of C. felis infections in HIV-positive patients in the United States are

known (Morgan et al., 2000a; Pieniazek et al. 1999). In addition, C. meleagridis was detected from an HIVinfected individual in Kenya (Morgan et al., 2000a). 

A number of studies have been conducted to determine if C. parvum can cross species lines to non-mammalian

species. Graczyk et al. (1996a) attempted to infect a variety of fish, amphibia and reptiles with C. parvum

without success. Although the oocysts were present in the cloaca of two fish and one lizard post-exposure, no

life-cycle stages were detected in histological sections taken from any of the inoculated species. From these

results, the authors concluded that the oocysts were unable to establish an infection in the gastrointestinal tract

of lower vertebrates even though the inoculated C. parvum oocysts were retained by these animals for at least 14

days following ingestion. Although animal excreta have been shown to contaminate watersheds and source

waters linked to outbreaks (see Section III-C), evidence of direct animal transmission of Cryptosporidium to

humans is limited to a few examples (Adam, 1994; Casemore, 1990). The search for the source of

Cryptosporidium in sporadic cases has been just as elusive. To assess infectious risks associated with pet

ownership, Glaser et al. (1998) conducted a case-control study of HIV-infected individuals with and without

cryptosporidiosis. No statistically significant differences were observed in the rate of overall pet ownership, cat
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

14

ownership, or bird ownership between the two groups, and only a slight correlation between dog ownership and

human cryptosporidiosis was noted. The authors concluded that pets do not represent a major risk factor for

acquisition of cryptosporidiosis.

Despite the strict host-specificity of a number of mammalian species of Cryptosporidium, the discovery that a

C. parvum-refractory host (a host which ingests infectious oocysts but is not susceptible to infection) can

excrete intact oocysts has raised the issue that, if oocyst infectivity is retained during travel through the

intestinal tract, the refractory host could serve as a mechanical vector for dissemination of the parasite through

the environment (Graczyk, 1998b). In fact, research with some avian species has proven that oocysts do retain

their infectivity after passing through the intestinal tracts of the animals. Insects have been shown to carry C.

parvum oocysts on their outer surfaces as well as in their intestinal tracts. The following paragraphs briefly

summarize relevant findings about insects and birds as vectors for Cryptosporidium.

The contribution of migratory waterfowl to the overall public health risk of cryptosporidiosis remains unclear. 

Transport of oocysts through migratory waterfowl has been demonstrated. Viable Cryptosporidium oocysts

have been found in fecal samples and cloacal lavages of gulls which fed on sewage or other refuse (Smith et al.,

1993). In experimental studies, C. parvum oocysts retained their infectivity after being excreted in the feces of

ducks and/or geese dosed orally (Fayer et al., 1997b) or by intubation (Graczyk et al., 1996c; Graczyk et al.,

1997a). In another study, C. parvum oocysts which were recovered from goose fecal samples collected in the

Chesapeake Bay successfully infected laboratory mice (Graczyk et al., 1998c). The epidemiological

implications of these findings should be considered, especially in areas near reservoirs or in waters where

shellfish are harvested and may be consumed raw.
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

15

House flies (Musca domestica) exposed to bovine feces containing C. parvum oocysts transported oocysts to

other surfaces via fecal deposition (Graczyk et al., 1999). This study also demonstrated that oocysts were found

on the exoskeletons and in the intestinal tracts of the exposed flies. In a study by Mathison and Ditrich (1999),

oocysts were collected on the external surfaces and in the intestinal tracts of dung beetles exposed to C. parvum

oocyst-supplemented dung. Zerpa and Huicho (1994) reported a case of cryptosporidial diarrhea in a 20-monthold male child in which Cryptosporidium oocysts were detected in the digestive tract of cockroaches

(Periplaneta americana) found in the garden of the child’s home. No other potential sources of infection were

identified.

E. Summary

Cryptosporidium has a complex life cycle that involves numerous developmental stages culminating in the

production of oocysts that are resistant to adverse environmental conditions. Ten species are currently

recognized within the genus; however, recent molecular research may warrant taxonomical revision including

the addition of new species. Cryptosporidium infection has been documented in over 150 mammalian species. 

At least one genotype within C. parvum appears to be transmitted exclusively through humans. There is

sufficient evidence that secondary Cryptosporidium infections can occur in humans following primary infection

from ingestion of contaminated drinking water. Human-to-human transmission occurs through the fecal-oral

route and reinfections are common where human density is high or among people living in close quarters. C.

parvum exhibits specificity with regard to infection in mammals. Lower vertebrates such as fish, frogs, and

lizards are not susceptible to infection. Other than C. parvum, only C. felis and C. meleagridis have been

associated with human cryptosporidiosis. Species that infect other mammals, such as C. wrairi, may be
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

16

genetically linked to C. parvum; however, the public health significance of this relationship concerning potential

transmission of C. wrairi among human populations is not known.

III. Occurrence

A. Worldwide Distribution

1. Distribution in Animal Populations

Although the 1994 Cryptosporidium Criteria Document acknowledged that members of the genus

Cryptosporidium have been identified in many animal species, it focused primarily on the prevalence of

Cryptosporidium in domestic animals (cattle, lambs, pigs, goats, deer, and horses) and house pets (dogs, cats,

hamsters, guinea pigs, and rabbits). Prior to the writing of the 1994 document, and since that time,

Cryptosporidium has been identified in numerous mammalian, avian, reptilian, and piscine hosts worldwide. 

One frequently cited review provided an extensive list of the animals in which Cryptosporidium has been

detected (O’Donoghue, 1995). The findings of this review article are summarized below. 

• Cryptosporidium infections have been recorded in 79 mammalian species (including humans). 

Natural infections (i.e., infections not induced in an experimental setting) have been described in

domestic, wild, and captive mammals with the majority occurring in farm, zoo, pet, and lab

animals. Most infections in mammals have been attributed to C. parvum; however, some natural

infections in mice, rats, cattle, mountain gazelles, and a camel have been attributed to C. muris.

• Cryptosporidium infections have been detected in over 30 species of birds primarily in domestic

flocks or aviary birds, but also in wild bird populations. Two species of Cryptosporidium species

are currently considered as valid pathogens in birds (C. meleagridis and C. baileyi; see Table 1,

section II-A), and infections have been attributed to both, although neither species has

demonstrated host specificity.

• Cryptosporidium infections have been reported in over 57 different reptilian species including 40

species of snakes, 15 species of lizards, and 2 species of tortoises. Cryptosporidium serpentis is

currently the only valid named species of Cryptosporidium known to cause infections in reptiles. 

Previous descriptions of other species causing infections in reptiles (C. crotali, C. lampropeltis,

C. ameivae and C. ctenosauris) are consistent with the morphology of another parasite genus

(Sacrocystis spp.) and therefore are considered invalid (Fayer, 1997).
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• Cryptosporidium infections have been detected in nine species of fish. Infections have been

observed in both freshwater and marine species, and in cultured, captive, ornamental, and wildcaught fish. The only valid named species of Cryptosporidium causing infection in fish is C.

nasorum, named after the first reported infection in a tropical marine fish (Naso lituratus)

(Hoover et al., 1981).

• In addition to the mammalian, avian, reptilian, and piscine hosts mentioned above,

Cryptosporidium infections have been identified in two amphibian species, Ceratophrys ornata

(Bell’s horned frog; Crawshaw and Mehren, 1987) and Limnodynastes tasmaniensis (spotted

grass frog; O’Donoghue and Mirtschin, unpublished), and in one invertebrate species, Ruditapes

decussatus (Portuguese clam; Azevedo, 1989).

Additional hosts reported in the literature, but not mentioned in the O’Donoghue (1995) review article, include

Fell Pony foals (Scholes et al., 1998), muskrat (Petri et al., 1997), African hedgehog (Graczyk et al., 1998a),

dugong (Hill et al., 1997), slow loris, white rhinoceros, Indian elephant, Thorold’s deer (Majewska et al., 1997),

and iguana (Fitzgerald et al., 1998). Fayer et al. (2000) documented Cryptosporidium infection in more than

150 mammalian species.

2. Distribution in Human Populations

The distribution of Cryptosporidium in human populations is worldwide, occurring in both developed and

under-developed countries, urban and rural areas, and in temperate as well as tropical climates (Fayer, 1997;

O’Donoghue, 1995). The 1994 Cryptosporidium Criteria Document described the worldwide distribution of

human cryptosporidiosis in 45 different countries. Since 1982, human cryptosporidiosis has been documented in

95 countries and on every continent except Antarctica (Fayer, 1997). A list of these countries, including the 45

listed in the 1994 document, is given in Table 2.

Worldwide prevalence rates of human cryptosporidiosis from the most recent compilations of coprologic (stool)

and serologic (blood) surveys (through 1991) are included in the 1994 Cryptosporidium Criteria Document.
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Table 2. Geographic Distribution of Human Cryptosporidiosis

North America Caribbean Central/ South

America

Africa Europe

Canada Cuba Argentina Algeria Austria

Mexico Haiti Brazil Burundi Belgium

United States Jamaica Colombia Cameroon Czechoslovakia

Puerto Rico Chile Ethiopia Denmark

Pacific St. Lucia Costa Rica Gabon England

Australia Tobago Ecuador Ghana Finland

Malaysia Trinidad El Salvador Guinea France

New Zealand Virgin Islands Guatemala Guinea-Bissau Germany

Papua-New

Guinea

Panama Ivory Coast Greece

Philippines Asia Peru Kenya Hungary 

Singapore Bangladesh Uruguay Liberia Ireland

Belarus Venezuela Mauritania Italy

Middle East Cambodia Mauritius Lithuania

Egypt China Morocco Netherlands

Iran India Nigeria Poland

Israel Japan Rwanda Portugal

Kuwait Korea South Africa Romania

Saudi Arabia Myanmar

Republic

Sudan Serbia

Pakistan Togo Spain

Russia Tunisia Sweden

Sri Lanka Uganda Switzerland

Taiwan Zaire Turkey

Thailand Zambia Wales

Zimbabwe
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B. Occurrence in Water

1. Surface Water

Since the 1994 Criteria Document was published, dozens of reports have appeared in the literature further

characterizing the occurrence of Cryptosporidium in surface waters. Several of these articles adequately

summarize this large body of work. Lisle and Rose (1995) reviewed the literature for more than 25 studies

involving outbreaks, occurrence, monitoring, and detection, as well as regulatory implications. They reported

that between 5.6 and 87.1% of source waters (i.e., surface, spring, and groundwater samples not impacted by

domestic and/or agricultural waste) tested contained 0.003 to 4.74 oocysts/L. They concluded that better

methods were needed for oocyst recovery, detection, and treatment. In another major study, LeChevallier and

Norton (1995) reported finding oocysts in 60.2% of surface waters tested in the U.S. and Canada. Three

companion articles in the September 1997 issue of the Journal of the American Water Works Association

summarized the current state of knowledge of Cryptosporidium occurrence in watersheds (Crockett and Haas,

1997), rivers (States et al., 1997) and reservoirs (LeChevallier et al., 1997). All three studies concluded that any

surface water is subject to a complex set of watershed characteristics (buffer zones, slope, land use, watershed

management, storm water management, sewage treatment practices, sediment types, soil types, vegetation,

population density, pathogen sources, best management practices, and recreational uses) and watershed

processes (precipitation, snow and ice-melt-derived runoff, sediment resuspension, dumping, spills, wastewater

treatment plant failures, temperature fluctuations, and algal blooms) that may lead to the presence of oocysts.
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2. Groundwater

According to the 1994 Cryptosporidium Criteria Document, oocysts are found less frequently in groundwater

than in surface water and consequently very few cryptosporidiosis outbreaks have been traced to groundwater

contamination. Kramer et al. (1996), in a U.S. national survey for the presence of Cryptosporidium oocysts in

drinking water, showed that, of the five outbreaks recorded from 1993 to 1994, two outbreaks resulted from

untreated well water. Both of the outbreaks occurred in Washington State, one in 1993, which accounted for 7

cases of human cryptosporidiosis, and one in 1994 accounting for 104 cases (Rose et al., 1997). Hancock et al.

(1998) recently reported a study of 199 groundwater samples tested for Cryptosporidium. They found that 5%

of vertical wells, 20% of springs, 50% of infiltration galleries, and 45% of horizontal wells tested were positive

for Cryptosporidium oocysts. This new data will force environmental microbiologists and regulators to reassess

previous assumptions that groundwater is inherently free of parasites.

C. Occurrence in Soil

Most Cryptosporidium research has centered on detecting oocysts in either water or biological samples. 

Limited studies have been performed to ascertain the presence or viability of Cryptosporidium in soil. 

Transport of Cryptosporidium oocysts to water from fecal-contaminated soil within a watershed during weather

events was suggested as the most probable mechanism of source water contamination in several documented

outbreaks (Kramer et al., 1996). Mawdsley et al. (1996a) reported on the vertical movement of

Cryptosporidium oocysts through intact, 30-cm soil cores. Transport of oocysts through the soil cores was

greater in silty loam and clay loam than in loamy sandy soil, which was exactly the opposite of what the

researchers expected. Twenty-one days after inoculation, the majority of oocysts still in the soil remained in the
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21

top 2 cm of the soil cores, but some were found as deep as 30 cm. The number of oocysts recovered decreased

with increasing soil depth. Another study by Mawdsley et al. (1996b) confirmed these results but also implied

that a large proportion of oocysts are retained in the runoff rather than being adsorbed onto the soil surface.

Research funded by the American Water Works Association Research Foundation, aimed at creating a better

understanding of watersheds and soil oocyst interactions, is ongoing. 

A new method was recently reported by Anguish and Ghiorse (1997) for examining Cryptosporidium oocysts in

media other than water. They used a computer-assisted laser-scanning microscope equipped for confocal laser

scanning and color video microscopy to examine two agricultural soils, barnyard sediment and a calf fecal

sample. The authors concluded that this technique provides a better approach for oocyst identification and

enumeration, as well as for in situ assessment of cellular activity and viability. This technology could have

great utility for detecting Cryptosporidium in soil. 

D. Occurrence in Air

The 1994 Cryptosporidium Criteria Document cited no data to show that Cryptosporidium is found in ambient

air. A review of the current literature indicates that this continues to be an unresearched area.

E. Occurrence in Food and Beverages

The occurrence of several foodborne outbreaks in recent years has highlighted the role of Cryptosporidium as a

foodborne pathogen. The presence of Cryptosporidium has been documented in raw milk (Badenoch et al.,

1990), unpasteurized apple cider (Millard et al., 1994), uncooked meat products (Casemore et al., 1997), and
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22

eight types of fresh, Costa Rica-grown produce (Monge et al., 1995). A foodborne outbreak of

cryptosporidiosis associated with eating foods containing uncooked and possibly unwashed green onions in

Spokane, Washington has been reported (Quinn et al., 1998). Refrigeration does not compromise oocyst

viability. The influence of temperature on oocyst survival is discussed in section III-G. Recent studies

evaluating oocyst viability (assessed by vital dye staining) showed greater than 85% of oocysts present in beer

and cola made from intentionally contaminated water lost their viability, while only 35% of oocysts in

reconstituted infant formula and orange juice lost viability (Friedman et al., 1997). These authors speculated

that the decreased pH of the carbonated beverages triggers premature excystation.

F. Specific Disease Outbreaks

1. Outbreaks Associated with Drinking Water

The 1994 Cryptosporidium Criteria Document described a number of waterborne disease outbreaks attributed to

Cryptosporidium. The 1989/1990 and 1991/1992 MMWR papers on waterborne disease outbreaks prepared by

the U.S. Centers for Disease Control and Prevention (Herwaldt et al., 1991 and Moore et al., 1993) were not

included in the 1994 document. Herwaldt et al. (1991) found that no outbreaks in 1989 or 1990 were attributed

to Cryptosporidium. Moore et al. (1993) reported that, for 7 of the 11 outbreaks for which an agent was

determined, a protozoal parasite (Giardia lamblia or Cryptosporidium) was the etiological agent. Since 1994,

numerous papers have appeared in the literature that more completely describe the previous outbreaks and

document new outbreaks; these are described in the remainder of this section.
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Additional research articles have been published specific to the 1993 Milwaukee outbreak. Addiss et al. (1996)

reported on the effectiveness of point-of-use water treatment devices during the Milwaukee outbreak. They

concluded that using sub-micron point-of-use devices may significantly reduce the risk of waterborne

cryptosporidiosis. Rodman et al. (1997) studied the utility of monitoring sales data on nonprescription

antidiarrheal medications to detect enteric disease outbreaks. Although the technique would have been useful in

detecting the Milwaukee outbreak, the information from this study showed that the costs incurred in collecting

the data and the absence of increased sales of antidiarrheal medications during other outbreaks limit the utility

of this type of analysis in other cities. Turbidity spikes at the Milwaukee water treatment plant correlated

strongly with hospital visits for gastrointestinal disease prior to 1993 (Morris et al., 1998), indicating that

cryptosporidiosis was occurring in Milwaukee for more than a year before the outbreak. Eisenberg et al. (1998)

confirmed this finding and further concluded that 85% of the outbreak infections could have been avoided if

Cryptosporidium had been identified as the etiological agent in the smaller outbreak. Researchers concluded,

after studying Milwaukee death certificates from before and after the outbreak, that waterborne outbreaks of

cryptosporidiosis can result in significant mortality, particularly among immunocompromised populations

(Hoxie et al., 1997). Fox and Lytle (1996) published a summary article outlining the results of the investigation

of the Milwaukee outbreak by the USEPA. Moss et al. (1998) reported on an outbreak of cryptosporidiosis

involving more than half of the crew members of a Coast Guard cutter which had filled its water tanks with

water from Milwaukee during March of 1993.

Duke et al. (1996) reported an outbreak of cryptosporidiosis in Northumberland, U.K. The source water was a

private untreated water supply that appeared to be contaminated by run-off slurry from surrounding fields and/or

lamb carcasses found in a collection chamber connected to the water supply. Atherton et al. (1995) also
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24

reported an outbreak of cryptosporidiosis in northern England which was attributed to failure of the public

drinking water system to remove oocysts. The outbreak onset was characteristic of a point source infection

occurring during a period of heavy rainfall at the reservoir. Maguire et al. (1995) reported an outbreak in

London, U.K., where 44 individuals were confirmed to have Cryptosporidium infections acquired from drinking

tap water. Bridgman et al. (1995) reported 47 cases of cryptosporidiosis linked to two groundwater sources in

northwestern England. In this case, it was found that one of the water sources could be contaminated with

surface water from a field containing livestock waste in times of heavy rainfall, similar to that experienced

during the time of infection.

Leland et al. (1993) wrote an article not cited in the 1994 Cryptosporidium Criteria Document describing the

Jackson County, Oregon outbreak of 1992 from an engineering perspective. In a Florida outbreak, 77% of the

counselors and campers at a day camp were infected from a municipal drinking water supply (CDC, 1996c). 

Goldstein et al. (1996) reported an outbreak of cryptosporidiosis in Las Vegas, Nevada, within the Lake Mead

watershed area, where 78.2% of the cases occurred in immunocompromised persons and 21.8% of the infected

individuals were immunocompetent. The Nevada treatment system used state-of-the-art technologies and

chemical treatment. Recognition of the outbreak was attributed to surveillance conducted by the State of

Nevada, where cryptosporidiosis is a reportable disease.

A number of review articles concerning Cryptosporidium outbreaks have been published since 1994. Rose et

al. (1997) briefly described a number of outbreaks associated with drinking water, and Solo-Gabriele and

Meumeister (1996) presented an overview of U.S. outbreaks, supporting their view that current practices and
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25

regulations are inadequate to protect consumers from waterborne disease. The MMWR Surveillance for

Waterborne-Disease Outbreaks - United States, 1993-1994 (CDC, 1996b) reported that 10 of 29 total

waterborne disease outbreaks were associated with the protozoans Giardia and Cryptosporidium. A national

survey over a 2-year test period (1993 and 1994) identified five outbreaks resulting in 403,271 cases involving

Cryptosporidium oocysts in drinking water (Kramer et al., 1996). Of this total, 403,000 were from the outbreak

in Milwaukee, Wisconsin, 103 were from Las Vegas, Nevada, and 27 were from an outbreak at a resort in

Minnesota. All three outbreaks were attributed to surface water as the source. The remaining two outbreaks

resulted from contaminated groundwater: one from a private well in Washington State (resulting in seven

cases), and the other from a community well, also in Washington State (resulting in 104 cases). Some notable

outbreaks in the United States from 1984 to 1995 which were associated with drinking water and the

deficiencies which caused them are summarized in Table 3.

In addition to the outbreaks described in the literature, a newsletter called Cryptosporidium Capsule provides

additional, anecdotal information about suspected outbreaks in drinking water in Devon, U.K. in 1995, drinking

water in Maine in 1995, drinking water in Collingwood Ontario in 1996, lakes in Cranbrook and Kelowna,

British Columbia, in 1996, and a groundwater outbreak in the U. K. in 1997.
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Table 3. Outbreaks of Cryptosporidiosis Associated with Drinking Water in the U.S.

Year State

Number of

Cases

Source Deficiency

1984 Texas 2006 Groundwater (C) Sewage contamination

1986 New Mexico 78 Surface water (C) Untreated

1987 Georgia 12,960 River (C) Treatment deficiency

1991 Pennsylvania 551 Groundwater (C) Treatment deficiency

1992 Oregon 15,000 Spring/river (C) Treatment deficiency

1993 Wisconsin 403,000 Lake (C) Treatment deficiency

1993 Washington 7 Well (I) Surface contamination

1993 Minnesota 27 Lake (NC) Unknown

1993 Nevada 103 Lake (C) Inadequate filtration

1994 Washington 104 Well (C) Sewage contamination

1995 Florida 72 Not applicable Cross connection

NC = Non-community; C = Community; I = Individual

No cases of cryptosporidiosis were reported in the waterborne disease outbreak survey published by the CDC

from 1989 to 1990; however, the large number of cases of acute gastrointestinal illness (AGI) of unknown

etiology may have included illness caused by Cryptosporidium. Of the total number of AGI cases reported in

1989 and 1990, 56% (2,402 of 4,288) were unexplained (Herwaldt et al., 1991). In the U.S. surveillance report

from 1991 to 1992 (Moore et al., 1993), approximately 20% of the total enteric illness cases were caused by

Cryptosporidium. Cases of AGI represented 76.5% of the total enteric illnesses reported from 1991 to 1992,

some of which were likely caused by Cryptosporidium. These observations suggest that efforts to identify

Cryptosporidium as the etiological agent during outbreaks frequently fail.
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Two articles have been written about the inadequacies of current surveillance practices in detecting and

preventing cryptosporidiosis from drinking water. Craun et al. (1997) pointed out that the coliform test can no

longer be used as the sole indicator of a water's microbiological safety. Frost et al. (1996) focused on the

importance of epidemiological surveillance and collaboration between water purveyors and community public

health departments to enhance public safety.

2. Outbreaks Associated with Recreational Waters 

Fourteen outbreaks of gastroenteritis related to recreational waters were reported by nine states during 1993 and

1994 (Kramer et al., 1996). Ten of these outbreaks were caused by Cryptosporidium or Giardia, with five

specifically linked to Cryptosporidium. Three of the Cryptosporidium outbreaks were associated with motel

swimming pools; two were associated with community swimming pools. All five pools were filtered or

chlorinated; one had a malfunctioning filter. None of the other pools had identifiable treatment deficiencies.

The inability of chlorine levels normally used in swimming pools to kill Cryptosporidium, coupled with poor

pool filtration equipment maintenance practices, have been suggested as the primary cause of swimming poolrelated cryptosporidiosis.

Outbreaks associated with recreational waters can be difficult to recognize because the individuals using such

facilities may reside in widely separated geographical areas. Forty-four individuals contracted cryptosporidiosis

after swimming in a Los Angeles pool where an accidental fecal release occurred (CDC, 1990; Sorvillo et al.,

1992). McAnulty et al. (1994) reported a community-wide outbreak of cryptosporidiosis in Lane County,

Oregon that was linked to swimming at a wave pool with inadequate sand filtration equipment. MacKenzie et
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28

al. (1995a) reported on a swimming pool-related outbreak involving 51 individuals. The outbreak occurred in a

city 75 miles from Milwaukee about 30 days after the massive Milwaukee outbreak. The authors suggested that

increased attention should be given to preventing swimming pool-related outbreaks following outbreaks of

cryptosporidiosis associated with water supplies. Cryptosporidiosis associated with two pools in Dane County,

Wisconsin was reported during the summer of 1993 (CDC, 1994). Kramer et al. (1998) reported an outbreak

with 38 infected individuals who contracted cryptosporidiosis while swimming in a recreational lake. They

speculated that contamination of the lake came from either infected swimmers or run-off.

3. Foodborne Outbreaks

Foodborne transmission of cryptosporidiosis has only rarely been reported. In October 1993, an outbreak of

cryptosporidiosis occurred among students and staff who consumed contaminated apple cider while attending

an agricultural fair in central Maine. This was the first large outbreak in which foodborne Cryptosporidium

could be identified and documented as the causative agent (Millard et. al., 1994). A survey, completed for 611

(81%) of the estimated 759 attendees at the fair, found 160 (26%) cases of primary cryptosporidiosis.

Cryptosporidium oocysts were detected in the stools of 50 (89%) of the primary and secondary case subjects

tested. Oocysts were detected in the apple cider, on the cider press, and in the stool specimen of a calf on the

farm of the supplier of the apples used to make the cider. Two more foodborne outbreaks, one involving apple

cider and another associated with green onions, are reported in a review by Rose and Slifko (1999). A

community outbreak in New York was associated with a cider mill using apples picked from an orchard located

near livestock. These outbreaks underscore the need for agricultural producers to take precautions to avoid

contamination of foodstuffs by infectious agents commonly present in the farm environment. Another outbreak

was traced back to a dinner banquet in Washington in which unwashed green onions were the suspected cause
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(Quinn et al., 1998; Rose and Slifko, 1999). The Minnesota Department of Health reported cryptosporidiosisin

50 attendees of a social gathering who ate a salad contaminated during preparation by a day care worker (CDC,

1996a). Laberge et al. (1996) prepared a review article in which they list foods associated with

Cryptosporidium infections that include unpasteurized milk, sausage, raw beef, kefir, pelleted feed, silage,

powdered milk, raw tripe, and apple cider. Casemore et al. (1997) referred to sausage, offal, and raw milk

contamination by Cryptosporidium but did not link these foods to specific outbreaks. Harp et al. (1996)

reported that standard commercial pasteurization techniques kill 100% of C. parvum oocysts. Methods to detect

oocysts in food have not been optimized.

4. Outbreaks among Travelers 

The 1994 Cryptosporidium Criteria Document stated that cryptosporidiosis has emerged as an important cause

of traveler’s diarrhea and indicated that illnesses frequently occur in travelers visiting developing countries. 

However, reports since 1994 indicate that travelers within developed countries such as the United States have

also acquired Cryptosporidium infections.

During the Milwaukee outbreak in 1993, visitors became infected with Cryptosporidium as a consequence of

drinking water in that city. In addition, upon returning home, they transmitted the parasite to members of their

households (MacKenzie et al., 1995b). 
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5. Outbreaks at Day Care Centers

Several outbreaks of cryptosporidiosis have occurred in day care centers in the United States; these outbreaks

are summarized in “Cryptosporidium: Risk for Infants and Children” (USEPA, 2001).

6. Outbreaks Among Sensitive (Immunocompromised) Subpopulations

While outbreaks of cryptosporidiosis are rarely limited to immunocompromised subpopulations, cases in

immunocompromised individuals may be detected first because these individuals are more likely to be

diagnosed. For example, an outbreak of cryptosporidiosis in Las Vegas, Nevada (Clark County within the Lake

Mead watershed area) was first recognized among HIV-infected people (Goldstein et al., 1996; Roefer et al.,

1995). Immunocompromised persons accounted for 78.2% of the cases in this outbreak. Although these

individuals had an increased risk of dying by the end of the outbreak (compared to immunocompromised

individuals without cryptosporidiosis), they did not have an increased one-year mortality rate.

G. Environmental Factors

Because Cryptosporidium oocysts are remarkably resistant to inactivation in the environment, the survival of

Cryptosporidium under a variety of environmental conditions has been evaluated by a number of investigators. 

While the majority of these studies have considered the effects of physical antagonism (e.g., freezing, heating,

UV exposure), studies have also been conducted to consider the role of microbial antagonists (microbial

predation), chemical antagonists (such as disinfection) and aging. This section will focus primarily on aspects

of physical antagonists in the environment. The aspects of chemical disinfectants are discussed below in
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Section VII, Inactivation of Cryptosporidium under Water Treatment Practices. Walker et al., (1998) have

reviewed laboratory and field studies on the survival and transport of C. parvum oocysts.

Robertson et al. (1992) evaluated the sensitivity of C. parvum oocysts to a variety of environmental pressures

such as freezing, dessication, and water treatment processes, as well as in physical environments commonly

associated with oocysts. Approximately 97% of the test oocysts were inactivated after 18 days at -22°C,

suggesting that the levels of viable oocysts in surface waters subjected to freezing might be influenced by

seasonal temperature variations. After 2 hours of drying oocysts at room temperature, only 3% of oocysts were

still viable and, after 4 hours, no oocysts were viable. None of the water treatment processes investigated (i.e.,

alum floccing, liming, and ferric sulfate floccing) had any effect on oocyst viability when pH was corrected. 

When stored at 4°C, the percentage of oocysts remaining viable in stool samples decreased steadily with time. 

(In the study, the relationship between oocyst viability and and time varied with individual.) After 176 days in

tap water, river water, or cow feces, there was a statistically significant increase in the proportion of dead

oocysts in test samples. Seawater was even more lethal to oocysts, with a statistically significant increase in

dead oocysts by 35 days of exposure to the test conditions. In summary, the work of Robertson et al. (1992)

demonstrates that C. parvum oocyst viability is sensitive to a wide range of typical environmental conditions

while remaining relatively insensitive to some water treatment processes. Their research also emphasizes that

oocyst viability is also dependent on the amount of time to which oocysts are exposed to environmental

conditions.
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32

Cryopreservation studies conducted by Fayer et al. (1991) and Fayer (1997) indicate that oocyst survival

depends upon the temperature and duration of freezing conditions, implying that C. parvum oocysts are not

necessarily rendered noninfectious by being frozen. In another study, Fayer and Nerad (1996) demonstrated that

the infectivity of C. parvum oocysts after freezing is dependent on the temperature and duration of freezing. In

general, shorter freezing times are required to neutralize infectivity when lower freezing temperatures are

employed (e.g., 1 hour at -70°C vs. 168 hours at -15°C to completely neutralize infectivity) (Fayer and Nerad,

1996). Temperature stability studies were also conducted by Sattar et al. (1999) who evaluated the freeze/thaw

susceptibility of various preparations of oocysts, including highly purified preparations as well as infected calf

feces. This study indicated that oocyst stability under freezing conditions is at least partially dependent upon the

surrounding matrix, with fecal material conferring a cryopreservative effect on oocysts.

In the absence of freezing conditions, colder water temperatures tend to promote the survival of most

microorganisms. C. parvum may survive outside of mammalian hosts for several months or more depending

upon water temperature (Straub et al., 1994). Fayer et al. (1998b) investigated the effect of water temperatures

ranging from -10°C to 35°C on oocyst infectivity. As water temperature increased to a maximum of 20°C,

oocysts remained infectious for longer exposure times. For example, oocysts retained their infectivity for 1

week in -10°C water but remained infectious for up to 24 weeks in 20°C water (Fayer et al., 1998b). As water

temperatures increased above 20°C, oocysts retained their infectivity for shorter exposure times (Fayer et al.,

1998b).
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33

Under conditions of high water temperatures, Fayer (1994) indicated that all evidence of C. parvum infectivity

was lost within 60 seconds when temperatures exceeded 72oC or when temperatures of at least 64oC were

maintained for 2 minutes. It is important to note, however, that such water temperatures are not typical

environmental conditions. Anderson (1985) evaluated the infectivity of Cryptosporidium oocysts following

exposure to a variety of moist heat treatments. Warming oocysts to 45°C for 5 to 20 minutes was effective in

completely neutralizing their infectivity (Anderson, 1985). The efficiency in reducing the infectivity of

C. parvum oocysts through exposure to high temperatures for short time periods, such as the conditions used in

pasteurization processes, was examined by Harp et al. (1996). They demonstrated that oocysts suspended in

water or milk lost infectivity after heating to 71.7°C for 5 to 15 seconds in a laboratory-scale pasteurizer.

Further research on the effects of dessication on C. parvum oocysts demonstrates that typical environmental

conditions are effective in reducing infectivity. Anderson (1986) examined the infectivity of oocysts from calf

fecal samples which had been subjected to drying in either winter or summer months. In summer temperatures

(i.e., 18°C to 29°C) with approximately 60% humidity, oocysts completely lost infectivity in 1 to 4 days. 

Experiments conducted in winter, with temperatures ranging from -1°C to 10°C and humidity of approximately

60%, resulted in a complete loss of infectivity within 2 to 4 days. Control samples kept moist or kept moist and

refrigerated retained infectivity for up to 14 or 21 days, respectively.

Limited studies have been conducted on the effects of physical shear on oocyst viability; these studies have

attempted to assess the potentially abrasive effects of oocyst contact with sand and gravel particles or through

fast-flowing waters. In addition, oocysts could be subject to such shear forces in rapid sand filters. Parker and
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

34

Smith (1993) demonstrated rapid inactivation of oocysts in a mixed sand reactor. Sattar et al. (1999) conducted

studies to evaluate the synergistic effects of mixed sand reactors upon disinfection efficiency with chlorine, and

they observed that shear stress enhanced chlorine inactivation. Sattar et al. (1999) also evaluated the effects of

microbial predation upon oocyst survival and observed that oocysts incubated in dialysis cassettes that were

suspended in natural waters exhibited significantly longer survival times when bacterial populations were

excluded from the suspension water, implying that microbial predation may play an important role in

determining oocyst survival in natural waters.

H. Summary

In summary, cryptosporidiosis is zoonotic, widespread, and often associated with surface water, groundwater,

recreational water, and contaminated food and drink. Outbreaks associated with these sources continue to occur

among travelers, children in day care centers, and immunocompromised as well as healthy individuals

throughout the world. Ten valid species of Cryptosporidium have been described in more than 150 mammalian

species, 30+ avian species, 57 reptilian species, 9 species of fish, and 2 amphibian species. Until more research

is completed, public health workers can do little more than speculate on the human infectivity of all the

Cryptosporidium species. Currently, there is little information published on the cross-reactivity of the nine

species to commercially available antibodies used for detection of the organism. Immunocompromised

individuals such as those with HIV infections or AIDS, very young children, the elderly, and individuals

undergoing therapeutic treatment for cancer are more likely to acquire an infection, develop cryptosporidiosis,

and show more severe clinical symptoms. Deficiencies in water treatment systems are often cited as a major

reason for outbreaks, and even the best of systems can be overwhelmed by a high density of oocysts entering the

source waters over a short period of time. Infected individuals will shed oocysts in their feces and can transmit
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

35

the infection to other family or community members. In addition, day care centers for children, due to their

high density of a sensitive population, are a potential source for secondary spread of cryptosporidiosis from

infected children to others both within and outside of their households. Research on environmental factors has

confirmed previous work showing that oocysts are highly refractory to environmental stressors.

IV. Health Effects in Animals

A. Symptomatology and Clinical Features

The 1994 Cryptosporidium Criteria Document included general information on the symptomatology and

clinical features of cryptosporidiosis in a limited number of domesticated mammals such as calves and lambs. 

Cryptosporidiosis has also been documented in many additional mammals, both domestic and wild, as well as

several species of birds, reptiles, and fish (see section III-A). Two recent review articles contain comprehensive

information on the symptomatology and clinical features of cryptosporidiosis in these different groups of

animals (Fayer, 1997; O’Donoghue, 1995).

In general, the development of cryptosporidiosis depends on the species, age, and immune status of the host

(Fayer, 1997). Younger animals and animals with less developed or compromised immune systems are

generally more susceptible to severe infection than healthy adult animals. In many cases, healthy adult animals

that become infected are asymptomatic or exhibit only mild clinical signs (O’Donoghue, 1995). A summary of

the symptomatology and primary clinical features of infected animals follows.
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36

Mammals: Most clinical cases of cryptosporidiosis in mammals involve infection by C. parvum. The most

common feature of cryptosporidiosis in mammals is profuse, watery diarrhea that may be pale yellow in color

and may have a distinct offensive smell. Other clinical signs of infection include dehydration, fever, anorexia,

weight loss, weakness, and progressive loss of condition (O’Donoghue, 1995). Most animals will recover

spontaneously within 1-2 weeks of infection. Histopathological observations may reveal several lesions in the

small intestine, including mild to moderate villous atrophy and loss of epithelial cells. Developmental stages of

the parasite are often seen within the small intestine and occasionally elsewhere (stomach, colon, liver, lungs)

(O’Donoghue, 1995). Fayer (1997) provided detailed descriptions of the symptomatology and clinical features

of cryptosporidiosis in several species of ruminant and non-ruminant mammals.

Birds: Two Cryptosporidium species, C. meleagridis and C. baileyi, are known to cause infection in birds. 

Avian cryptosporidiosis appears as either a respiratory, enteric, or renal disease (Fayer, 1997; O’Donoghue,

1995). Normally, only one condition manifests during an outbreak, and respiratory infections are more common

than enteric or renal infections (Fayer, 1997; O’Donoghue, 1995). Clinical signs of respiratory infections

include rales, coughing, convulsive sneezing, and dyspnea (O’Donoghue, 1995). Excess mucus may exist in the

trachea, sinuses, and nasal passages, and fluid may be present in the air sac (Fayer, 1997). Histopathological

changes may include hypertrophy and hyperplasia of the respiratory epithelium, with reduced or absent ciliation

(O’Donoghue, 1995). Parasites may be detected throughout the respiratory tract including the nasopharynx,

larynx, trachea, and bronchi (O’Donoghue, 1995).
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37

Clinical signs associated with enteric infections caused by Cryptosporidium in birds include mild to severe

diarrhea, dehydration, malaise, weight loss, and weakness. Histopathology may reveal atrophy and fusion of

villi, along with epithelial hyperplasia and hypertrophy, as well as other malformations (O’Donoghue, 1995). 

Parasites are found primarily in the gastrointestinal tract (O’Donoghue, 1995).

Renal infections in birds have been detected only at necropsy (O’Donoghue, 1995; Fayer, 1997). In these cases,

the kidneys were pale in color (Fayer, 1997) and enlarged (O’Donoghue, 1995). Hypertrophic and hyperplastic

epithelial cells were detected throughout the kidney (O’Donoghue, 1995; Fayer, 1997). Parasites were observed

in the collecting ducts and convoluted tubules (O’Donoghue, 1995).

Reptiles: C. serpentis is the only valid, named species of Cryptosporidium causing infection in reptiles,

although up to five species may exist based on oocyst morphology (Fayer, 1997). Most reports of infections in

reptiles have involved clinical or subclinical infections in captive reptiles (especially snakes); only subclinical

infections have been reported in wild reptiles (Fayer, 1997). Cryptosporidiosis in snakes is characterized by

anorexia, regurgitation, lethargy, firm midbody swelling, weight loss, and death (Fayer, 1997). Interestingly,

most infections in snakes have been detected in mature animals. In addition, most infections have been

associated with chronic gastric disease, as opposed to the acute enteritis which is common in mammals with

cryptosporidiosis. Infections and intermittent oocyst shedding in snakes may last for several months to 2 years

(O’Donoghue, 1995). Histopathological observations have included inflammation, hyperplasia, and

hypertrophy of the gastric glands (O’Donoghue, 1995).
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38

Clinical features of cryptosporidiosis reported in lizards have included primarily subclinical gastric infections,

while cryptosporidiosis in tortoises involves gastritis and regurgitation, or progressive wasting (O’Donoghue,

1995).

Fish: Except for the original report of cryptosporidiosis in fish (Hoover et al., 1981), infections in fish have not

been associated with clinical symptoms. The original report described a progressive illness in a tropical marine

fish (Naso lituratus) which was characterized by anorexia, emaciation, regurgitation, and passage of feces

containing undigested food. Although developmental stages of the parasite were found attached to the intestinal

mucosa, no pathogenic changes were evident in this fish. Since then, parasites have been detected in the

intestines or stomach of other fish, but few histopathological changes have been described (O’Donoghue, 1995).

B. Therapy

Treatment of cryptosporidiosis in animals involves a combination of prophylactic and chemotherapuetic drugs

along with other preventative measures. As stated in the 1994 Cryptosporidium Criteria Document, there is no

approved effective treatment for cryptosporidiosis in animals. However, numerous drugs have been tested in

studies that focused both on the treatment of naturally acquired infections and the treatment or prophylaxis of

experimentally induced infections in animals. The majority of efficacy evaluations of agents tested in animals

have involved prophylactic as opposed to therapeutic drug regimens (Blagburn and Soave, 1997).

A recent review article lists the numerous anticryptosporidial drugs that have been evaluated in animals

(Blagburn and Soave, 1997). The findings of the review article are summarized below.
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39

• Most studies have been conducted in laboratory rodents, including mice, rats, and hamsters. 

Over 30 compounds have proven effective against cryptosporidiosis in rodents, including

maduramicin, alborixin, lasalocid, and salinomycin. In some cases, efficacies of the drugs tested

exceeded 90% compared to control (nontreated) animals.

• Several anticryptosporidial drugs have also been tested in ruminants. Among the drugs

demonstrating activity against C. parvum infections are paromomycin, lasalocid, halofuginone,

and sulfaquinoxaline. 

• Anticryptosporidial drugs have also been tested in several other types of animals including

several species of birds and reptiles, as well as mammals (pigs and cats). There has been little

success in identifying successful drugs in these animals, although some success was achieved in

treating cryptosporidiosis in snakes. 

Prevention of cryptosporidiosis in animals is best achieved by eliminating contact with viable oocysts. This is

particularly difficult in settings with large numbers of animals such as farms or zoos (Blagburn and Soave,

1997). To prevent infections in these types of settings, infected animals should be quarantined in facilities that

can be cleaned and disinfected, contaminated articles and the clothing of animal care workers should be cleaned

thoroughly or discarded, clean food and water should be provided, and access of rodents and other wild animals

should be restricted (Blagburn and Soave, 1997). Prevention can also be enhanced by ensuring that neonatal

mammals receive adequate amounts of colostrum early in life (Blagburn and Soave, 1997).

Treatment of animals suffering from cryptosporidiosis is similar to that in humans, namely, rehydration with

fluids and electrolytes along with antidiarrheal drugs (Blagburn and Soave, 1997). In addition, chemotherapy

with anticryptosporidial drugs may be initiated.

C. Epidemiological Data
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The majority of epidemiological data for cryptosporidiosis in animals is confined to economically important

livestock, especially ruminants (Casemore et al., 1997). Three extensive reviews exist which describe the

epidemiological data available for animals, including both domestic and wild animals (Casemore et al., 1997;

Fayer, 1997; O’Donoghue, 1995). These reviews include information on the prevalence and spread of

cryptosporidiosis in many groups of animals including cattle, sheep, goats, pigs, horses, dogs, cats, deer, mice,

and several other small mammals, as well as many species of birds, reptiles, and fish.

In general, clinical infections are seen primarily in neonates and immunocompromised animals. Age-related

resistance has been documented in several species, and the age of an animal upon infection can greatly alter the

severity of the infection and the prepatent period (Casemore et al., 1997). Adult animals often appear

asymptomatic even when shedding small numbers of oocysts (Casemore et al., 1997; Fayer, 1997;

O’Donoghue, 1995). Serologic surveys in animals suggest much higher prevalence rates of cryptosporidiosis,

especially in adults (Casemore et al., 1997; Fayer, 1997). This high prevalence may be due to cross-reaction of

animal sera infected with coccidial parasites rather than from actual infections (Casemore et al., 1997).

Potential sources of infection in animals include other infected animals of the same or different species (i.e., it is

believed that rodents can infect calves or cattle with C. parvum), mechanical carriers such as insects, birds, and

humans, contaminated feed and water, and other contaminated fomites such as bedding, brushes, shovels, and

feed utensils (Fayer, 1997).

Additional epidemiological studies reported in the literature, but not mentioned in the review articles, describe

the prevalence of cryptosporidiosis in the following animals:
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41

cattle/calves (Fayer et al., 1998a; Olson et al., 1997; Perez et al., 1998; Pena et al., 1997)

horses (Scholes et al., 1998; Bray et al., 1998; Forde et al., 1998; Johnson et al., 1997)

lambs (Bukhari and Smith, 1997)

goats (Koudela and Jiri, 1997; Goyena et al., 1997)

nonhuman primates (Muriuki et al., 1997; Majewska et al., 1997)

rodents (Bajer et al., 1997; Bull et al., 1998)

chickens (Sreter et al., 1996)

ostriches (Jardine and Verwoerd, 1997)

pigeons (Rodriguez et al., 1997)

catfish (Muench and White, 1997)

muskrat (Petri et al., 1997),

African hedgehog (Graczyk et al., 1998a)

dugong (Hill et al., 1997)

deer (Majewska et al., 1997)

slow loris (Majewska et al., 1997)

white rhinoceros (Majewska et al., 1997)

Indian elephant (Majewska et al., 1997)

iguana (Fitzgerald et al., 1998)

D. Summary

Cryptosporidium infections have been documented in many different species of mammals as well as in several

species of birds, reptiles, and fish. In general, the severity of the infection depends on the species, age, and

immune status of the host. Clinical infections are primarily seen in younger animals and animals with

compromised immune systems, while infected healthy adult animals may be asymptomatic or exhibit only mild

clinical signs.

Most clinical cases of cryptosporidiosis in mammals involve infections by C. parvum. The most common

features of cryptosporidiosis in mammals are profuse, watery diarrhea, dehydration, fever, anorexia, and weight

loss. Two species of Cryptosporidium, C. meleagridis and C. baileyi, are known to cause infections in birds. 

Cryptosporidiosis in birds is characterized by respiratory, enteric, or renal infections. Respiratory infections,

which cause rales, coughing, convulsive sneezing, and diarrhea, and enteric infections, which cause
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42

dehydration, weight loss, and weakness, are the most common. The majority of cryptosporidiosis infections in

reptiles have been reported in captive snakes. These infections, caused by C. serpentis, are characterized by

anorexia, postprandial regurgitation, lethargy, and midbody swelling. The only clinical infection described in

fish was caused by C. nasorum and was characterized by anorexia, emaciation, regurgitation, and passage of

feces with undigested food.

Treatment of cryptosporidiosis in animals involves a combination of prophylactic and therapuetic drugs along

with other preventative measures. Although there is no approved, effective treatment for cryptosporidiosis in

animals, several drugs have been tested in rodent and bovine models and have shown substantial success. 

Several drugs have also been tested in reptiles and birds with limited success. Prevention of cryptosporidiosis

in animals is best achieved by eliminating contact with viable oocysts as much as possible. This involves

isolation of infected animals and disinfection of all articles that come into contact with the infected animals.

The majority of epidemiological data for cryptosporidiosis in animals is confined to economically important

livestock, especially cattle. There is also information available on sheep, goats, pigs, horses, dogs, cats, deer,

mice, and several other small mammals. These studies show that the age of an animal can greatly alter the

severity of the infection and the prepatent period and that the primary sources of infection for animals are other

infected animals, mechanical carriers, contaminated feed and water, and other contaminated objects.
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V. Health Effects in Humans

A. Symptomatology and Clinical Features

The clinical manifestations of cryptosporidiosis in humans are directly related to the immunocompetence of the

host and may include profuse, nonbloody, watery diarrhea that generally resolves spontaneously within 48

hours; however, variability in clinical symptoms is appreciable. Diarrheal symptoms are generally not

distinguishable from those caused by other common enteric pathogens. Other symptoms reported by

individuals afflicted with cryptosporidiosis include abdominal cramps, vomiting, lethargy, and general malaise. 

Diarrhea results from a combination of enterocyte damage and physical blockage of the intestinal villi, leading

to a disruption in the normal balance of intestinal absorption and secretion (Clark and Sears, 1996). The

incubation period in humans is estimated to vary between 2 and 10 days (Arrowood, 1997), with a mean

incubation of approximately 7-9 days (Juranek, 1998).

Human volunteer studies have been conducted to assess the infectivity and dose-response of C. parvum in

humans (DuPont et al., 1995). Sixty-two percent of subjects who ingested doses of Cryptosporidium ranging

from 30 oocysts to 1 million oocysts acquired infection. The infectious dose causing disease in 50% of the

population (ID50) for the Iowa strain of C. parvum was 132 oocysts in humans, compared with an ID50 of 60

oocysts in neonatal mice; however, the test strain of C. parvum in this case was adapted to a mouse model prior

to challenge studies, which may account for the disparity in ID50 values. The mean and median incubation

periods for cryptosporidiosis in the study were 9.0 and 6.5 days, respectively. Infected humans developed

clinical enteric symptoms that were associated with excretion of oocysts, although one of the 11 subjects who

did not pass oocysts passed a single soft stool on day 10 and exhibited enteric symptoms on days 23 through 31. 

Symptoms of clinical illness included abdominal pains, cramps, and diarrhea in six subjects; six had nausea; one
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44

reported vomiting; and one had moderate dehydration. Table 4 summarizes the dosing data and related

infection rates from the study. Note that volunteers exhibiting enteric symptoms (e.g., diarrhea, loss of appetite)

did not test positive for cryptosporidiosis in all cases.

Table 4. Rate of Infection and Enteric Symptoms as a Function of Intended Dosage*

Intended

Dose

No.

Subjects

No. (%)

Infected 

No. (%) with

Enteric

Symptoms

No. (%) with

Cryptosporidiosis

30 5 1 (20) 0 0

100 8 3 (37.5) 3 (37.5) 3 (37.5)

300 3 2 (66.7) 0 0

500 6 5 (83.3) 3 (50) 2 (33.3)

1000† 7 7 (100) 5 (71.4) 2 (28.6)

Total 29 18 11 7

* Linear regression analysis of the data yielded an r2

 of 0.983 and an ID50 of 132 oocysts.

† The intended dose was 1000 oocysts in two subjects, 10,000 in three, 100,000 in one, and

1 million in one.

Source: DuPont et al. (1995)

Follow-up studies indicate that the number of excreted oocysts and the pattern and duration of shedding can

vary widely among immunocompetent individuals (Chappell et al., 1996). In the volunteer challenge study,

high variability in shedding patterns was observed, and oocysts were observed intermittently in consecutive

stool samples, implying that production of oocysts is not uniform and may be influenced by unknown factors.

These data may in part account for the observation that fewer than half of the individuals who acquire illness

during an epidemic produce stools positive for Cryptosporidium when single samples are submitted for

diagnostic analysis.
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B. Epidemiological Data

Although only 13 cases of cryptosporidiosis had been documented by the CDC in 1982, human

cryptosporidiosis has been reported in almost 100 countries spanning the globe since that time (Ungar, 1990). 

Because C. parvum is ubiquitous, infects most mammals, and is highly infectious, all human populations are at

risk to some degree (Griffiths, 1998). Screening of select human populations was initiated in the United States

during the 1980s, with special emphasis on children and immune-suppressed individuals. However,

determining the true prevalence of cryptosporidiosis has proven challenging due to the facts that diagnostic

methods have limited sensitivity and the majority of individuals who experience mild to moderate diarrheal

illness do not generally seek the services of a physician (Juranek, 1998).

In 1994, a workshop was organized by the National Center for Infectious Diseases (NCID) and the USEPA to

assist the Centers for Disease Control and Prevention and state public health departments in providing guidance

on public health issues relating to waterborne cryptosporidiosis (Juranek et al., 1995; Addiss et al., 1995). The

workshop, titled “Prevention and Control of Waterborne Cryptosporidiosis: An Emerging Public Health

Threat,” addressed the following topics: surveillance systems and epidemiological study designs, public health

responses, cryptosporidiosis in immunocompromised individuals, water sampling methods, and interpretation of

results. The recommended approaches to surveillance included the following:

• Making cryptosporidiosis incidents or outbreaks reportable to CDC 

• Monitoring sales of antidiarrheal medication through local pharmacies (also described by

Rodman et al., 1997)

• Monitoring logs maintained by health maintenance organizations (HMOs) and hospitals for

complaints of diarrheal illness
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46

• Monitoring the incidence of diarrhea in nursing homes (also described by Proctor et al., 1998)

• Monitoring laboratory data for Cryptosporidium (also described by Proctor et al., 1998) 

• Evaluating water distribution system design in selected cities

• Providing prompt epidemiological assistance during outbreaks

A cohort approach was recommended to facilitate the epidemiological study of outbreak data, with blood tests

performed quickly in order to screen out negative subjects. A cohort analysis would demonstrate whether the

exposure(s) was associated with subsequent infection or disease. Also, since such research requires large

sample sizes, the inclusion of blood tests would make this approach feasible. Strategies recommended for the

improvement of public health responses included the identification of methods for rapid notification of the risks

for waterborne cryptosporidiosis to agencies, advocacy groups, and the public.

The workshop participants emphasized that boiled water advisories are not essential in the absence of

supporting epidemiological information suggesting increases in diarrheal disease in the community. On the

subject of cryptosporidiosis in immunocompromised individuals, the workshop concluded that

immunocompromised individuals are no more likely than immunocompetent individuals to acquire

cryptosporidiosis in an outbreak. However, AIDS patients, patients receiving treatment for cancer, recipients of

organ or bone marrow transplants, and individuals who have congenital immunodeficiencies are at greater risk

than immunocompetent individuals for developing severe, life-threatening cryptosporidiosis if they become

infected.

Additional information on the epidemiologic aspects of cryptosporidiosis in humans is provided in a review by

Casemore (1990). The distribution of Cryptosporidium in humans from several countries was broken down by
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47

age group, non-human reservoirs, and routes of transmission. This study found that when testing was

performed for suspected infections, 60% of the positive findings occurred in children and 30% occurred in

adults less than 45 years old. The disease occurred in single sporadic cases, small cluster cases, and short

clinical series. Cryptosporidium is the third or fourth most commonly identified pathogen in the world, and the

reported rates are higher in underdeveloped countries, especially in children (Casemore, 1990). Seasonal and

temporal trends vary from country to country and occurrence may indirectly reflect rainfall and farming events

such as lambing.

Immunocompromised populations are also at high risk of infection and disease from drinking water

contaminated with Cryptosporidium oocysts. Clayton et al. (1994) studied 41 patients with AIDS who had

become infected with Cryptosporidium, and they found two significant patterns among these individuals. In

61% of these patients, Cryptosporidium was present in the proximal small bowel (i.e., upper small intestine) and

the patients had severe clinical disease characterized by malabsorption of nutrients. In the remaining 39%, who

had less severe disease, Cryptosporidium was seen only in the colon or the stool. An increased susceptibility to

cryptosporidiosis in immunocompromised individuals was reported in Kenya, East Africa, where 42 to 44% of

the HIV-positive patients at the Kenyatta National Hospital tested positive for Cryptosporidium, whereas only

8.6% of the non-AIDS patients carried this pathogen. In Zambia, 25.4% of the HIV-positive patients who were

symptomatic carried oocysts and had increased specific anti-Cryptosporidium IgG and IgA antibodies, but there

was no increase in specific IgM antibodies (Cevallos et al., 1995). This antibody profile suggests that the

infection was of prolonged duration.
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The cryptosporidiosis outbreak in Las Vegas, Nevada (Roefer et al., 1995) was recognized primarily because of

the high incidence among the immunocompromised population and because of state requirements for reporting

of disease from this pathogen. Although the water supply in this city was processed by a state-of-the-art system,

the protection it provided proved inadequate for immunocompromised persons. Research shows HIV-infected

patients who have cleared oocyst infections have much higher levels of specific secretory IgA levels than AIDS

patients with chronic cryptosporidiosis (Flanigan, 1994). This indicates either that secretory IgA is involved in

recovery from infection, or that it is the only marker for an effective immune response at the mucosal surface. 

In a study on asymptomatic carriage of intestinal Cryptosporidium by immunocompetent and immunodeficient

children, the percentage of carriers among immunodeficient children was 22% compared to 6.4% among

immunocompetent children (Pettoello-Mantovani et al., 1995). However, the percentages of symptomatic

children in these groups were similar, with 4.4% of the immunocompetent children and 4.8% of the

immunodeficient children positive for oocysts.

Greenberg et al. (1996) reported on stool sampling from AIDS patients receiving chemotherapy and/or

radiotherapy. The study objective was to determine the yield of Cryptosporidium oocysts in stools versus

biopsies taken from the upper and lower intestines of these patients. Only 53% of 106 patients were positive for

oocysts when a single stool sample was taken, but detection increased to 73% positive when multiple stool

samples were taken (3.3±0.3, mean±SEM). When sampling was by terminal ileum biopsy, the number of

positive samples increased to 91%. From these studies, it appears oocysts may invade the small intestine in

immunocompromised individuals with no oocysts detectable in stool examinations. Thus, stool sampling can
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49

be expected to miss a substantial number of Cryptosporidium infections in immunocompromised and AIDS

patients.

In addition to gastrointestinal disease, AIDS patients can have other complications from Cryptosporidium

infection such as respiratory cryptosporidiosis (Mifsud et al., 1994). Clavel et al. (1996a) reported cases of

intestinal cryptosporidiosis with pulmonary involvement in AIDS patients who had diarrhea and were positive

for Cryptosporidium oocysts. 

Patients with cancer may be immunocompromised as a side effect of their therapeutic treatment. Therefore,

these patients are likely to have an increased susceptibility to cryptosporidiosis. Tanyuksel et al. (1995)

examined 106 cancer patients, all of whom were receiving chemotherapy and/or radiotherapy and surgery, and

found that 17% of the patients who had diarrhea were positive for Cryptosporidium oocysts. They concluded

that individuals who are compromised by such treatments are at high risk for Cryptosporidium infections.

Logar et al. (1996) evaluated the occurrence of C. parvum in Slovenia and reported a higher incidence of

cryptosporidiosis in older patients and young children. The authors believed that the infections in older patients

and young children (median age 3 years) were due to lowered resistance or immune response. In the older

patients, Cryptosporidium infections appeared to be a consequence of other diseases or secondary to irradiation

or other immunocompromising treatments common in this age group. Considerable evidence exists to show

that immunological deficiency is a natural consequence of aging (Miller, 1996). Casemore (1990) observed that

the severity of disease from infection is greatest among children under 5 years of age and among
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

50

immunocompromised patients (e.g., AIDS or cancer patients), and that the impact is greatest in developing

countries.

The 1994 Cryptosporidium Criteria Document discussed the high prevalence of cryptosporidiosis in children

and notes that the evidence comes primarily from reports of diarrhea in day care centers. Cryptosporidiosis is

now recognized as a significant disease in childcare settings (Cordell and Addiss, 1994). Additional data on the

effectiveness of prevention and control strategies, as well as on the economic impact of these outbreaks on the

community, state, and country, should be collected. There is evidence that cryptosporidiosis affects children in

other countries. In China, 42 to 58% of a cross-section of children less than 16 years of age were serologically

positive for Cryptosporidium (Zu et al. 1994). Among a similar group in Virginia, less than 17% tested

positive. The data from the review by Cordell and Addiss (1994) indicate that in impoverished communities,

this parasitic infection is highly endemic and occurs in early childhood. Single stool samples from 1,000

apparently healthy children (ages 6 through 14) in Jordan showed that 4% of the children had oocysts and,

among these children, 37% were symptomatic (Nimri and Batchoun, 1994). Brandonisio et al. (1996) reported

7 (1.9%) of 368 children hospitalized (for unspecified reasons) in Italy tested positive for oocysts (359 of these

children were immunocompetent and 9 were HIV infected). Six of the seven cases were in

immunocompromised children. Brannan et al. (1996) found that 12% of the children in Romania were carrying

C. parvum oocysts, although 73% of these children had either IgA or IgG antibodies to this protozoal parasite.

Adegbola et al. (1994) reported that the occurrence of Cryptosporidium infection in Gambian children has

seasonal peaks associated with rain and high relative humidity. Factors accounting for the seasonal distribution
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

51

may include increased survival of oocysts in a high relative humidity environment and an increased possibility

of dissemination of oocysts to children as a result of the impact of the rainy season on domestic and

environmental hygiene. For additional information regarding cryptosporidiosis in children, refer to

“Cryptosporidium: Risk for Infants and Children” (USEPA, 2001).

C. Treatment: Clinical Laboratory Findings and Therapeutic Management

Cryptosporidiosis is self-limiting in most immunocompetent patients as well as in many immunocompromised

patients. The recommended management of Cryptosporidium-infected patients includes careful monitoring of

hydration and electrolyte balance, with oral or intravenous hydration and nutrition as necessary. Antimotility

agents (i.e., opiates or somatostatin and its analogues) may be helpful to prevent dehydration. Patients coinfected with HIV should continue or begin antiretroviral therapy to suppress viral replication and boost CD4+

cell counts. Patients currently undergoing chemotherapy or immunosuppressive therapy should be removed

from treatment (Griffiths, 1998).

In patients infected with HIV, cryptosporidiosis has been a major cause of morbidity and mortality, resulting

from dehydration and malnutrition (Blanshard et al., 1997). Since the publication of the 1994 Cryptosporidium

Criteria Document, several new treatment strategies have been pursued. The most promising development is

associated with the introduction in 1996 of protease inhibitors for the treatment of HIV infection. Le Moing et

al. (1998) examined data on the prevalence of intestinal cryptosporidiosis in HIV-infected patients for the

period from January, 1995, to December, 1996. They observed a decrease in the prevalence of cryptosporidiosis

at the same time that protease inhibitors first gained widespread use in this population. Although this finding
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

52

does not prove that protease inhibitors were responsible for the decrease in cryptosporidiosis, the study authors

noted that there was no new treatment for cryptosporidiosis during that time and no change in the number of

HIV-infected patients. The study results confirm that protease inhibitors had a beneficial effect on

cryptosporidiosis at the population level.

The results of other studies suggest that combination antiretroviral therapy that incorporates a protease inhibitor

provides HIV-infected patients the best chance for changing the course of cryptosporidiosis. Miao et al. (1999)

examined the effect of three different combinations of antiretroviral therapy with protease inhibitors. After six

months of treatment, two of three patients had fecal smears that were negative for C. parvum. The third patient

stopped treatment after one month due to adverse side effects. While on the treatment, this patient’s fecal

smears were also negative for C. parvum but relapsed within two months after going off antiretroviral therapy. 

The results suggest that the treatment regimen suppresses C. parvum infection when taken for one month but

completely eliminates infection after six months of treatment. Maggi et al. (2000) conducted a retrospective

cohort study to compare the response of HIV-infected patients with cryptosporidiosis to antimicrobial treatment

alone or in conjunction with antiretroviral treatment (up to three drugs). The therapeutic effect of antimicrobial

treatment and combination antiretroviral therapy (either two or three drugs) on cryptosporidiosis was excellent

and was sustained after a lengthy follow-up period of nearly two years. The study authors speculated that the

patients’ responsiveness to combination antiretroviral treatment was due primarily to an increase in CD4+

 cell

count rather than decreased viral load. Antimicrobial treatment alone or in conjunction with a single

antiretroviral drug was not effective in treating cryptosporidiosis in HIV-infected patients.
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

53

To date, no chemotherapeutic agents have been consistently effective in the management of cryptosporidial

infections (O’Donoghue, 1995; Blagburn and Soave, 1997). Although anecdotal success has been reported

following treatment with some compounds, most have proven ineffective in controlled studies. As many as 100

compounds have been shown to be ineffective for the treatment of cryptosporidiosis; some of the many

compounds that have been investigated including spiramycin, azithromycin, clarithromycin, roxithromycin,

diclazuril, letrazuril, paromomycin, nitazoxanide, difluoromethylornithine, and atovaquone (Blagburn and

Soave, 1997). 

Spiramycin, a macrolide antibiotic, was described in the 1994 Cryptosporidium Criteria Document as showing

limited success in the treatment of cryptosporidiosis. Other macrolides that have been evaluated include

erythromycin (Connelly et al., 1988), clarithromycin (Jordan 1996), and azithromycin (Vargas et al., 1993;

DuPont et al., 1996; Hicks et al., 1996). Spiramycin and erythromycin have shown unacceptable side effects

(Connelly et al., 1988). Azithromycin was reported as successful in treating several cases of cryptosporidiosis in

HIV-infected patients (DuPont et al., 1996, and Hicks et al., 1996) and cancer patients undergoing

chemotherapy (Vargas et al., 1993); however, in a pilot-scale clinical trial with azithromycin (500 mg daily), the

compound was ineffective for treating cryptosporidiosis in AIDS patients (Blanshard et al., 1997).

Clarithromycin prophylaxis was considered successful in preventing Cryptosporidium enteritis based on two

retrospective analyses (Jordan, 1996). In the first, a retrospective analysis of 136 AIDS patients revealed that

none of the 63 that received clarithromycin 500 mg twice daily developed Cryptosporidium enteritis, whereas

four patients in the control group developed Cryptosporidium enteritis. In the second, none of 217 AIDS

patients receiving clarithromycin 500 mg twice daily developed Cryptosporidium enteritis over a two-year

period. No other studies on clarithromycin were located.
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54

A one-month course of paromomycin led to the remission of 18 of 24 patients with intestinal cryptosporidiosis

but 10 patients relapsed once treatment was reduced or stopped (Bissuel et al., 1994). In a pilot-scale clinical

trial of AIDS patients with cryptosporidiosis, 60% treated with paromomycin had a complete resolution of

diarrhea and a further 5% had some resolution of symptoms, but paromomycin treatment did not eliminate the

Cryptosporidium infection (Blanshard et al., 1997). A pilot-scale clinical trial of letrazuril treatment for AIDS

patients with cryptosporidiosis resulted in an improvement of symptoms in 40% of the treated patients, and 70%

stopped excreting cryptosporidial oocysts, but biopsies remained positive for Cryptosporidium (Blanshard et al.,

1997).

Ionophores with anticoccidial properties have also been evaluated for treatment of cryptosporidiosis in animals

(Mead et al., 1995). The most promising agents, maduramycin and alborixin, resulted in 96% and 71%

reductions, respectively, in oocysts in immunodeficient mice. Toxicity also was observed in the therapeutic

trials with these compounds in mice, and they are considered to be too toxic for human use in their current

formulations.

The use of hyperimmune bovine colostrum for the treatment of cryptosporidiosis was described by Crabb

(1998). However, considerable variation has been noted in the efficacy of different colostrum preparations

(O’Donoghue, 1995).
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55

D. Mechanism of Action

The 1994 Cryptosporidium Criteria Document reported that the pathogenic mechanisms of Cryptosporidium in

cryptosporidiosis are not known. While the pathogenesis remains unclear, more recent work has helped

elucidate the process. Cryptosporidium sporozoites and merozoites invade the absorptive cells covering small

intestinal villi, damaging and eventually killing enterocytes. Forney et al. (1996) suggested proteolytic activity

is involved in the infectivity of C. parvum, based on the interaction between human alpha-l-antitrypsin (ATT)

and parasite subcellular components. This suggests that the use of serine protease inhibitors may be useful as a

therapeutic strategy. Riggs et al. (1996) identified antigens that may have a critical role in sporozoite infectivity

and therefore may be suitable molecular targets for passive or active immunization against cryptosporidiosis.

Diarrhea occurs when intestinal absorption is impaired or secretion is increased. When killed enterocytes are

extruded from the intestinal epithelium, crypt cells are signaled to repair the damage. Additionally, there is

infiltration of prostaglandin (PGE)-secreting inflammatory cells. Both crypt cells and PGE are known to

stimulate chloride ion secretion; in addition, PGE inhibits NaCl absorption (Clark and Sears, 1996). This

disruption in the absorption/secretion balance can lead to diarrhea. Clinical studies in C. parvum-infected

piglets (Argenzio et al., 1993) have suggested that Cryptosporidium-induced diarrhea is of a secretory nature. 

However, Kelly et al. (1996) conducted perfusion studies to measure water and electrolyte transport in vivo in

five HIV-cryptosporidiosis patients and nine healthy volunteers. There were no differences in net water,

sodium, and chloride movement in the jejunum of the two groups. In addition, there was no evidence

demonstrating that cryptosporidial diarrhea was due to a secretory state in the proximal small intestine. Other

studies have suggested that Cryptosporidium-induced diarrhea may be caused by a toxin (Guarino et al., 1994;
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

56

Guarino et al., 1995). A thorough review of cryptosporidial pathogenesis can be found in Clark and Sears

(1996).

E. Immunity

The importance of cellular immunity in resolving Cryptosporidium infection is highlighted by the contrasting

ability of immunocompetent and immunocompromised individuals to resolve infections. While depletion of

CD8+

 cells (Ungar, 1990), NK cells (Rasmussen and Healy, 1992), mast cells (Harp and Moon, 1991), tumor

necrosis factor (McDonald et al., 1992), or interleukin-2 (Ungar et al., 1991) did not result in enhanced

infection in mice, removal of CD4+

 cells and/or gamma interferon caused severe chronic infections (Ungar et

al., 1991). Additionally, Cryptosporidium-infected immunodeficient mice that were reconstituted with spleen

or lymph node cells from immunocompetent Balb/c mice were able to recover from infection, but upon

depletion of the CD4+

 T cells from the donor, the curative effects were abrogated (Kuhls et al., 1996). In

humans, HIV-infected patients with CD4+ counts of 180 cells/mm3 cleared the infection in 4 weeks, while of

those with lower counts, 87% developed chronic disease (Flanigan et al., 1992). Little is known about the gut

mucosal response to the parasite. Wyatt et al. (1996) used a bovine animal model to examine mucosal

immunity during cryptosporidiosis and showed that ileal intraepithelial T lymphocytes are activated coincident

with enteric disease. This suggests the importance of cell-mediated activity during Cryptosporidium infection.

Specific IgG, IgM, IgA, and IgE antibodies have been detected in patients with confirmed Cryptosporidium

infection (Ungar et al., 1986; Casemore, 1987; Laxer et al., 1990; Kassa et al., 1991). The presence of local and

secretory antibodies has also been confirmed (Laxer et al., 1990); however, the role of these antibodies in
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

57

combating infection is unclear (O’Donoghue, 1995). Kapel et al. (1993) used a time-resolved

immunofluorometric assay to determine the presence of Cryptosporidium antibodies in 12 HIVCryptosporidium-infected patients. These patients displayed marked elevation in anti-Cryptosporidium IgA and

IgM antibody titers. These high antibody titers were not correlated with the gravity of infection in terms of

oocyst shedding. Also, there was no evidence of protection even though there was a mucosal immune response. 

Studies comparing C. parvum infections in B cell-depleted mice showed that infections were similar to those in

normal mice (Taghi-Kilani et al., 1990).

There is evidence for protective immunity to cryptosporidial infection. Repeat infections in dairy cattle workers

occur but are generally much milder than the first infection (Reese et al., 1982). Permanent residents in areas

where cryptospordiosis is common often acquire mild or asymptomatic infections; however, visitors may

become very ill (Current, 1994). 

Okhuysen et al. (1998) reported on the rechallenge of human volunteers previously infected with

Cryptosporidium. Nineteen healthy, immunocompetent adults were challenged with approximately 500

oocysts, 1 year following primary infection. Fewer subjects shed oocysts after the second exposure (16% vs.

63%). Although the percentage of subjects with diarrhea was similar, the clinical severity of infection, as

determined by the number of unformed stools passed, was less following rechallenge compared to the primary

challenge response. The number of IgG and IgA seroconversions increased, but the antibody response did not

correlate to the presence or absence of infection.
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58

F. Chronic Conditions

Duration of illness in cryptosporidiosis patients is influenced primarily by the immune response of the

individual, with most immunocompetent individuals overcoming the acute enteritis stage within two weeks. 

Immunocompromised individuals generally present with chronic enteritis which may last as long as the immune

impairment. Immunocompromised populations include patients undergoing chemotherapy for treatment of

neoplasms, persons undergoing immune suppression treatment to prevent rejection of skin or organ transplants,

malnourished individuals, patients who present with concurrent infectious diseases such as measles, the elderly,

and AIDS patients. Chronic illness may manifest itself as a series of intermittent episodes or may be persistent. 

A functional threshold has been established using CD4+ cells to define the probability that infection will

resolve; patients presenting with CD4+

 counts exceeding 200/ L can generally expect to clear the infection,

while those with CD4+ counts falling below this level may suffer chronic infection (Fayer et al., 1997a).

G. Summary

The primary symptom of cryptosporidiosis in humans is fulminant watery diarrhea. The limited data available

from human volunteer feeding studies indicate a mean ID50 of 132 oocysts. Recent research suggests that the

pathological response to Cryptosporidium is initiated when the sporozoites and merozoites invade and kill the

intestinal epithelial cells (enterocytes). The enterocytes are extruded from the intestinal epithelium, triggering

epithelial repair and infiltration of inflammatory cells. The host responds with the production of antibodies as

well as intraepitheleal T lymphocytes. The infection may cause malabsorptive and secretory diarrhea. 

Management of infected patients includes maintenance of fluid and electrolyte balance. Patients with

unresolved infection may be treated with macrolides and antimotility agents. A previous Cryptosporidium
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

59

infection does not confer resistance to reinfection although reinfection will result in fewer episodes of diarrhea. 

A CDC workshop panel focusing on the application of epidemiologic information onCryptosporidium

recommended making surveillance information available to the appropriate federal agencies, HMOs, hospitals,

and others who play roles in maintaining the public health. Panel recommendations also included performing

cohort analyses of outbreaks using information such as blood tests in populations where exposure to

Cryptosporidium is likely. In addition, workshop participants suggested strategies to improve public health that

included identifying methods for informing agencies, advocacy groups, and the public about risks for

waterborne Cryptosporidium transmission and providing the public with information on dealing with a known

or suspected contamination of a drinking water source.

VI. Risk Assessment

The International Life Sciences Institute (ILSI) Risk Science Institute (RSI) Pathogen Assessment Working

Group (1996) defined pathogen risk assessment as a process that evaluates the likelihood of adverse human

health effects following exposure to pathogenic microorganisms in a medium such as water. Until recently,

most formal risk assessments on pathogenic microorganisms such as Giardia and Cryptosporidium have utilized

a conceptual framework that was developed to assess risks due to chemical exposures; however, it is notable

that the framework for assessing chemical exposures does not account for a number of microbial considerations,

including pathogen-host interactions, secondary spread of microorganisms, short- and long-term immunity, the

carrier state, host animal reservoirs, animal-to-human transmission, human-to-human transmission, and

conditions that lead to propagation/multiplication of microorganisms. Although significant data gaps exist in

the complete characterization of the pathogenesis of Cryptosporidium, risk assessment approaches will enable
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60

health officials to communicate with water utilities, interpret water quality surveys, and define the adequacy of

treatment at acceptable public health risks (Rose et al., 1997).

A. Experimental Human Data

The 1994 Cryptosporidium Criteria Document referred to human volunteer studies by DuPont and colleagues

that were in progress at the time of manuscript preparation. The aim of this study was to determine the

infectivity of C. parvum in healthy adults, in order to predict the likelihood of enteric infection following

exposure to contaminated drinking water. The results of this study have since been published (DuPont et al.,

1995) and are presented in Table 4 of Section V-A. Among 29 subjects who were inoculated with 30 or more

oocysts, 62% (18 subjects) became infected. Of those inoculated with 30 oocysts, 20% became infected,

whereas of those inoculated with >1000 oocysts, 100% became infected. Illness lasted 58 to 87 hours, with 4 to

11 loose stools produced per day, suggesting that human-to-human transmission of C. parvum is more likely to

occur 2.5 to 3.5 days following infection in the primary case. Linear regression of the dose-response data

indicated a human ID50 of 132 oocysts. The research team concluded that a low dose of C. parvum oocysts was

sufficient to cause infection in healthy adults with no serologic evidence of past infection by this parasite.

Follow-up analysis of the 18 individuals who presented with cryptosporidiosis infections indicated appreciable

variation in the numbers of oocysts excreted and in the duration of excretion (Chappell et al., 1996). Only 1 of

the 7 volunteers who exhibited diarrhea had oocysts in every stool during the shedding period, and oocyst

numbers in consecutive stool samples collected from individual volunteers varied by as much as 30 fold. 
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61

Hence, production of oocysts during infection may be intermittent, which may help to explain the observation

that fewer than 

50% of the individuals acquiring illness during waterborne outbreaks produce Cryptosporidium-positive stool

samples when only one stool is examined.

Okhuysen et al. (1999) investigated the infectivity of three geographically diverse isolates (IOWA, UCP, and

TAMU) of C. parvum genotype C in healthy adult volunteers. The TAMU isolate had significantly higher

virulence, based on ID50 (9, 87, and 1042 oocysts for the TAMU, IOWA, and UCP isolates, respectively) and

attack rate (86, 59, and 52% for TAMU, UCP, and IOWA, respectively). In addition, the mean time to onset of

illness was shorter for the TAMU isolate (5 days, versus 9 to 11 days with the other two isolates), and a trend

toward longer duration of diarrhea was observed in subjects infected with the TAMU isolate (94.5 hours,

compared to 81.6 and 64.2 hours for the UCP and IOWA isolates, respectively).

B. Experimental Animal Data

A number of dose-response studies using monkeys, gnotobiotic lambs and several strains of mice were

presented in the 1994 Cryptosporidium Criteria Document. Casemore (1990) reported a 2-to-5-day incubation

period for C. parvum and an excretion period of about 8 to 14 days in animals (species not identified). DuPont

et al. (1995) reported that the ID50 for the Iowa strain of C. parvum oocysts necessary to infect the neonatal

mouse was 60, which is approximately half of the ID50 required to produce infection in humans (132 oocysts). 

The relative similarity among infectious doses in mice and humans suggests that the mouse model is potentially

useful in defining risks associated with human cryptosporidiosis.
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62

C. Environmental Factors 

1. Prevalence in Surface Waters

Cryptosporidium oocysts are more likely to occur in surface waters than in groundwater, as described in Section

III of this document and in the 1994 Cryptosporidium Criteria document. Since the majority of source waters

used for production of drinking water are surface supplies, and because these waters are more vulnerable to

direct contamination from sewage discharges and runoff, the presumption has been that Cryptosporidium will

likely be more common in these supplies. Wallis et al. (1996) found Cryptosporidium oocysts in 6.1% of raw

sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples in Canada. Analyses of raw 

sewage samples indicated that Cryptosporidium was present in more than 50% of samples where one or more

liters was examined (Bukhari et al, 1997; Zuckerman et al., 1997). Ong et al. (1996 a and b) studied the source

of parasite contamination in different watersheds to assess the potential impact upon drinking water sources and

found that water from rivers flowing through cattle pastures in British Columbia exhibited higher

Cryptosporidium counts than did water from a protected watershed. Lisle and Rose (1995) reviewed 25

monitoring studies and found reports of Cryptosporidium in as much as 87.1% of the source waters (i.e.,

surface, spring, and groundwater samples not impacted by domestic and/or agricultural waste), with levels of

oocysts as high as 4.7 per liter. LeChevallier et al. (1995) observed oocysts in 60.2% of surface waters tested in

North America. Crockett and Haas (1995) investigated three water treatment plants located in a major

metropolitan area where watershed monitoring was conducted over one year. They found that creeks which

empty into a river were a primary source of parasites in the urban river-derived water. Each creek’s parasite

density was greatly influenced by suburban wastewater discharges, although the authors did not rule out other

sources which might have influenced the microbial density in the river. In determining the implications of these
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63

studies, however, it is important to note that erratic and insensitive oocyst detection methods may contribute to

an underestimation of oocyst contamination levels in surface waters.

2. Oocyst Survival

The resistance of oocysts to inactivation is an important factor in determining the extent to which humans or

reservoir/host animals can become infected; however, most detection methods for Cryptosporidium cannot

distinguish between viable and nonviable oocysts. Furthermore, some detection methods may render oocysts

nonviable due to chemical antagonism during sample processing. Additionally, the following practices and

issues must be characterized more completely in order to more accurately evaluate the risk of contracting

cryptosporidiosis: sewage discharges, watershed protection, agricultural practices, wildlife management, strain

specificity (animal or human), and oocyst survival under various environmental conditions. The survival of

oocysts under various environmental pressures has been evaluated by several groups and is described in Section

III-G “Environmental Factors.” The majority of these survival studies have relied upon animal infectivity or in

vitro excystation to assess changes in oocyst viability in natural waters. A complete discussion on methods for

the assessment of oocyst viability is also provided in Section VII-A- “Detection of Cryptosporidium in Water.”

3. Cryptosporidium in Drinking Water 

Identification of the specific pathogen and route of infection is an early step in the risk assessment process. The

primary route of human infection by C. parvum involves ingestion of contaminated drinking water and food

(Casemore, 1990); other routes of transmission are described in Section II-D. One of the difficulties in

conducting a risk assessment of Cryptosporidium lies in the uncertainty associated with the level of infectious
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

64

oocysts in drinking water supplies. There are also viability, infectivity, and specific epithet issues. Surveys

such as those described in Section III-B indicate the numbers of oocysts that may occur in drinking water;

however, the impact of these oocyst levels may be underestimated, given the number of gastrointestinal

illnesses that occur each year for which the etiology is undetermined.

Nahrstedt and Gimbel (1996) examined the influence of various factors contributing to the uncertainty

associated with the estimation of Cryptosporidium and Giardia concentrations in water samples. A statistical

model was designed using experimental data. The model provides reliable estimates of the oocyst/cyst

concentration in a given water body from which a representative sample has been taken and analyzed. Their

discussion of the effects of errors in detection methods may lead to improved analytical methods and a better

understanding of results obtained from current detection methods.

D. Epidemiologic Considerations

The USEPA estimated in 1993 that approximately 155 million people may be exposed to Cryptosporidium in

contaminated water every year; however, the estimated population at risk cannot be reconciled with the reported

numbers of infected individuals, even when correction factors for asymptomatic infections and underestimated

environmental levels are included (USEPA, 1994). Factors contributing to the disparity between the

environmental occurrence data and the clinical data are outlined in the 1994 document; additional factors are

described in Section V-B of this document. It remains quite problematic to generate accurate estimates of the

risk of acquiring cryptosporidiosis.
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65

Despite the limitations, the incidence of cryptosporidiosis in the United States is typically assessed through

surveillance reports and the documentation of outbreaks in the published literature. The CDC currently

maintains a surveillance system for cryptosporidiosis that is aimed at collecting information on both outbreakand non-outbreak-related cases. Cases are reported using standard forms which originate from state and local

health departments, but the agency also receives updates from federal agencies and occasionally from private

physicians. While cryptosporidiosis is not a reportable disease in all states (CDC, 1994), it was designated as

notifiable at the national level as of January 1, 1997. It is important to note, however, that the CDC’s

surveillance of cryptosporidiosis is passive, in that the system is dependent upon a physician ordering a

diagnostic test for Cryptosporidium. Most of this testing is done on adults who have AIDS and, as such, these

surveillance data are not an adequate basis for determining the true incidence of cryptosporidiosis in the U.S.

A number of reports describe the severe effects of cryptosporidiosis in children, particularly in malnourished

infants (Molbak et al., 1994; Griffiths, 1998). However, it is generally difficult to determine if malnourished

children are at higher risk of chronic cryptosporidiosis due to immune suppression, or if cryptosporidiosis is an

independent risk factor for becoming malnourished (Griffiths, 1998). Reports from the U.K. show the

occurrence of cases to be highest among children less than five years old (Atherton et al., 1995). Other groups

at risk for cryptosporidiosis are secondary contacts, farm workers (Lengerich et al., 1993), immune-suppressed

individuals, those living in institutional settings such as group homes and orphanages (Heald and Bartlett,

1994), and international travelers who visit regions where cryptosporidiosis is endemic. There is little evidence

that risks differ between genders (Meinhardt et al., 1996).
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

66

E. Risk Assessment Models

Since Cryptosporidium monitoring does not presently provide a true picture of the number of infectious

particles and the efficacy of oocyst removal from treated drinking water, risk calculations involve many

uncertainties. In order to develop risk estimates for specific pathogens such as Cryptosporidium, reliable doseresponse data are required. The human dose-response data currently available are limited to the studies of

DuPont et al. (1995), Chappell et al. (1996), and Okhuysen et al., (1999); however, an exponential doseresponse model has been developed, based upon the data set and a number of assumptions governing the

Milwaukee epidemic of cryptosporidiosis (Haas, 1994). This model describes the probability of infection given

exposure (PI) as follows:

Equation 1. PI

 = 1-e-rN

The values of r and N represent the fraction of ingested oocysts which must survive to establish infection and

the daily exposure, respectively. A value of r specific for Cryptosporidium has been derived (r=0.0047). 

Oocyst concentrations were derived for exposures ranging from 1 to 30 days and PI

 ranged from 0.14 to 0.52. 

Exposure values predicted according to this model ranged from a minimum of 0.16 oocysts per liter (PI=0.14) to

a maximum value of 79 (PI=0.52).

Cryptosporidium concentrations in the Milwaukee water supply were estimated by considering the numbers of

oocysts present in ice produced during the epidemic and correcting for losses associated with poor analytic

recovery efficiencies. According to the exponential model, Cryptosporidium exposure during the epidemic

ranged from 0.6 to 1.3 oocysts per liter. Haas (1994) also applied the risk assessment model to consider data
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67

from previous water monitoring studies and calculated that the annual risk of contracting cryptosporidiosis in

the United States may range from 1 in 100,000 to 4 in 1,000.

Perz et al. (1998) applied a risk assessment approach to examine the role of tap water in waterborne

cryptosporidiosis. This model was based upon the assumption that clinical infection results from exposure to a

single oocyst, and it utilized a theoretical C. parvum density in drinking water of 1 oocyst per 1,000 liters. 

Uncertainties in the model were analyzed by considering ranges and distributions among the input variables. 

The number of annual Cryptosporidium infections (Ij

) was estimated according to the following relationship:

Ij

 = C • POPj

 • Qj

 • rj

where C = concentration of C. parvum per liter of water

j = population subgroup (categorized by age and AIDS status)

POPj = number of persons in the exposed subgroup

Q = annual water intake (liters per year)

r = single organism infectivity (infection/organism/person)

The model was applied to derive the median annual risk of infection among immunocompetent individuals (1 in

1,000 probability, using the assumed exposure level of 1 oocyst per 1,000 liters). The dominant parameter

contributing to uncertainties in the risk assessment was oocyst concentration (e.g., a 10-liter sample volume for

monitoring is too small to detect concentrations of 1 oocyst per 1,000 liters). Therefore, improvements in

Cryptosporidium monitoring techniques for drinking water will improve future risk assessment efforts.

The ILSI workgroup (1996) has developed guidance for an infectivity model that may also be useful when

sufficiently sensitive detection methods become available. Since exposure analysis for humans and animals is
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an essential step in risk assessment, developing animal systems that can be used to estimate infectivity will be

essential for modeling, because detection of oocysts without knowledge about their infectivity is inadequate. 

Varga et al. (1995) reported on a model using C. baileyi infection in chickens. The authors developed a

quantitative method to assess oocyst shedding which was based on the rather slow sedimentation of oocysts. A

threshold of sensitivity of between 5,000 and 10,000 oocysts per gram of feces was reported for the technique. 

Triplicate assays over a wide range of oocyst concentrations (i.e., 2,500 to 1.25 x 106

) were in good agreement. 

Considering the level of oocysts excreted by infected humans, this model system appears to have adequate

sensitivity for demonstrating infection. This model’s sensitivity threshold may be adequate for assessing stool

samples of infected individuals, but it is inadequate for monitoring oocysts in water samples for potential

infectivity; thus, further development of sensitive infectivity models is needed.

The usefulness of the ILSI Framework for microbial risk assessment was tested by Teunis and Havelaar (1999). 

They used the Framework to determine the human health risk of C. parvum in an urban population obtaining

drinking water from a river. In the model, agricultural run-off and a sewage plant were contaminating sources

and the water was treated conventionally (i.e., coagulation/flotation, and filtration and ozonation). In order to

assess exposure, the progression of the pathogen from the river water to the tap water was broken down into the

following stages: oocyst counts in source water (corrected for detection method), source water concentration,

removal by storage, and removal by treatment. Each stage was analyzed successively by means of statistical

models. The daily ingested dose, which was calculated by means of a Monte Carlo simulation, was a single

distribution for the population as a whole because data for various subpopulations were not available. A Beta

Poisson model was developed for the dose-response assessment using experimental data from infection of

human volunteers with C. parvum. Based on the model assumptions and data used, the median yearly
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individual risk of infection resulting from a well performing water treatment process was calculated as

approximately 10-6. The authors concluded that the ILSI Framework was a useful tool for defining information

needs and organizing available information in a consistent manner. Future research needs and suggestions for

improving the framework were also discussed.

Haas et al. (1996) used dose-response data on Cryptosporidium to establish waterborne concentrations of

pathogen that led to various levels of risk. The concentration of oocysts in finished water for daily risks

identical to a 1 in 10,000 annual risk of infection is 0.003/100L (95% confidence interval 0.0018 -

0.0064/100L).

F. Federal Regulations

Since the 1994 Cryptosporidium Criteria Document was published, Cryptosporidium is now specifically

regulated by the federal government as a primary drinking water contaminant. The federal regulatory activity

associated with Cryptosporidium in drinking water and its threat of waterborne disease was prompted primarily

by the 1996 Amendments to the Safe Drinking Water Act. The most significant promulgated and proposed

rules addressing Cryptosporidium since 1994 are the Information Collection Rule, the Interim Enhanced

Surface Water Treatment Rule, and the Long Term I Enhanced Surface Water Treatment and Filter Backwash

Rule.

On May 14, 1996 the USEPA promulgated the Information Collection Rule (USEPA, 1996a). The rule required

those water utilities serving more than 10,000 people to test source water and finished water for an 18-month
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period (from July, 1997, to December, 1998). The monthly testing included a variety of analytes such as

coliforms, turbidity, and Cryptosporidium. The rule was primarily a research effort and the USEPA is using the

information collected during the testing period for the development of future rules. The data generated from the

Information Collection Rule is now available to the public through Envirofacts (http://www.epa.gov/enviro/

html/icr/icr_query.html).

The Interim Enhanced Surface Water Treatment Rule was promulgated on December 16, 1998 (USEPA, 1998). 

The rule applies to water utilities using surface water, or groundwater under the direct influence of surface

water, and serving more than 10,000 people. The rule set a maximum contaminant level goal (MCLG) of zero

for Cryptosporidium. For systems that filter water during the treatment process, the rule requires a minimum 2-

log Cryptosporidium removal efficiency. In addition, the Interim Enhanced Surface Water Treatment Rule

includes Cryptosporidium in the watershed control requirement for unfiltered public water systems. This rule

was designed to establish physical removal efficiencies and to minimize Cryptosporidium levels in finished

water. The Agency estimates that as a result of the implementation of this rule, the likelihood of endemic

illness from Cryptosporidium will decrease by 110,000 to 463,000 cases annually. The Agency believes that

the rule also will reduce the likelihood of the occurrence of outbreaks of cryptosporidiosis by providing a larger

margin of safety against such outbreaks for some systems.

The Long Term I Enhanced Surface Water Treatment and Filter Backwash Rule was proposed April 10, 2000

(USEPA, 2000) and should be finalized in late Spring 2001. The Long Term I Enhanced Surface Water

Treatment provisions apply to smaller water systems (i.e., those serving less than 10,000 people) using surface
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water or groundwater under the direct influence of surface water systems. The requirements for the control of

Cryptosporidium are similar to those of the Interim Enhanced Surface Water Treatment Rule. The Long Term I

Enhanced Surface Water Treatment provisions make Cryptosporidium a pathogen of concern for unfiltered

systems, and such systems must comply with updated watershed control requirements. The Filter Backwash

provisions will reduce the potential risks associated with recycling of contaminants removed during the

filtration process. These provisions apply to all water systems that recycle water, regardless of population

served. Physical removal is critical to the control of Cryptosporidium because it is highly resistant to standard

disinfection practices.

G. Summary

Environmental risk assessments have historically relied upon a conceptual framework based upon exposure to

chemical pollutants and are generally considered inadequate for pathogen risk assessment. Although most

human populations are assumed to be at risk for cryptosporidiosis at least to some degree, it has been difficult to

collect accurate figures describing the prevalence of infection in humans due to limitations in public health

reporting systems and due to incomplete characterizations of oocyst speciation and survival under various

environmental conditions. Dose-response data obtained from human volunteer challenge studies contribute to

the ability to quantify the risks associated with Cryptosporidium exposure. Data from animal infectivity studies

suggest that infectious doses may be lower (e.g., ID50 of ~60 oocysts). Risk models have been developed to

assess the probability of cryptosporidiosis infection based upon assumptions governing the levels of infectious

oocysts in drinking water and upon the data generated from volunteer challenge studies. The estimated annual

risk of waterborne cryptosporidiosis based upon these models ranges from 1 in 1,000 to 1 in 1,000,000.
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VII. Analysis and Treatment

A. Analysis of Water

The Information Collection Rule-modified American Society of Testing and Materials method (ASTM ICR)

was described in the 1994 Cryptosporidium Criteria Document. The method, which detects both

Cryptosporidium and Giardia, is tedious and requires high levels of technical expertise. While reproducible and

sensitive for Giardia detection, the method is not reproducible between laboratories and suffers from low

sensitivity for Cryptosporidium detection (Clancy et al., 1994; USEPA, 1996b). The current standard method

for monitoring Cryptosporidium in water is EPA’s Method 1622 (USEPA, 1999b).

The accuracy and reproducibility of method development and comparison studies were questioned by Klonicki

et al. (1997). The authors noted that vital information regarding oocyst source, purification method, age,

storage conditions, and enumeration method was inadequately cited in most publications. Further studies

compared three enumeration methods, hemacytometer, membrane, and well slide, and showed statistically

significant differences among the methods. The authors demonstrated the effect of counting method variability

on recovery values and stressed the importance of standardizing method comparisons and providing adequate

information in publications to allow valid comparison studies.

LeChevallier et al. (1995) investigated numerous methodological variations in the collection, elution, and

concentration portions of the assay to determine their influence on recovery of Cryptosporidium in seeded tap

water and Mississippi River water samples. The authors tested multiple filter types, centrifugation speeds,
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density gradient modifications, elution buffer pH values, and centrifuge bottle types. Although they identified

several sources of oocyst loss, their modifications resulted in variable recovery (7-129%).

Before detection data can be applied in a meaningful way, it must be adjusted to reflect the limits of the

methodology used to collect it. For example, if the method can detect 1,000 oocysts in 100 L of water and a

sample contains only 50 oocysts, a result showing 0 oocysts could be obtained. The results, therefore, must be

reported as “less than 1,000 oocysts.” Harris (1995) developed a method detection limit for indirect

immunofluorescence assay (IFA) analysis of Cryptosporidium and Giardia. The MDL0.95 in this study is

defined as the minimum concentration that the procedure can analyze with 95% confidence that the organism

will be detected. 

Each water type presents unique problems in collection, concentration, isolation, and staining techniques. 

Organic and inorganic particulates present in the water can clog filters or interfere with other portions of the

analysis such as clarification or antibody staining. Additionally, the ASTM ICR immunofluorescent staining

methods do not give information regarding viability, infectivity, and speciation which is essential to assessing

the threat to public health. The reader is advised to review Table 3.19 in Frey et al. (1998) for a complete

synthesis of the research status of Cryptosporidium detection methods. Current efforts to develop improved

Cryptosporidium collection, concentration, and detection methods are described below.
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l. Collection of Cryptosporidium from Water

Filtration-based concentration methods

The 1994 Cryptosporidium Criteria Document described a filtration method using polypropylene wound yarn

filter with a 1- m porosity. This collection method can be used for large volume samples with varying

turbidity. LeChevallier et al. (1995) tested 10 cartridge filters varying in composition (polypropylene, nylon,

rayon, and cotton) and porosity (0.5 and 1.0 m) for removal of Cryptosporidium- and Giardia-sized particles. 

Although retention of 3- and 7- m particles was greater using filters with a 0.5 m porosity, they tended to

clog, limiting the amount of water that could be filtered. The use of cotton, nylon, and rayon filters led to the

most efficient removal of Cryptosporidium and Giardia-sized particles. The authors tested the filters using 1

gallon (3.78 L) volumes of water. Wound fiber filters may not necessarily be superior to wound filters for

samples greater than 1 gallon in volume. To further minimize losses during filtration, the filter housing was

matched with the filter, and a screw press was used to wring the filters. Concentration of the eluate was best

performed at centrifuge speeds of 6,700 to 10,000 x g.

Also described in the 1994 Cryptosporidium Criteria Document was the use of cellulose acetate membrane

(CAM) filters. Nieminski et al. (1995) compared recovery rates of a method using CAM filters to the ASTM

ICR method using wound yarn filters. Prior to filtration by either method, Cryptosporidium and Giardia were

spiked into environmental water samples varying in quality and turbidity. Cyst and oocyst recoveries decreased

with increasing water turbidity, regardless of the filter type. Overall, the cellulose acetate method gave higher

recoveries; however, because the parasites were stained on polycarbonate filters, microscopic confirmation was

not possible. Therefore, the authors recommended the use of the ASTM ICR method for environmental sample
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analysis and the cellulose acetate method for spiking studies. Adlom and Chagla (1995) modified the CAM

method by including an acid dissolution step following filtration. This modification resulted in a 70.5%

recovery of oocysts spiked into 3 liters of treated municipal water. Graczyk et al. (1997b) used cellulose acetate

filters, followed by filter dissolution and ASTM ICR method processing to test recovery of Cryptosporidium

from spiked drinking water. The overall mean recovery rate was reported as 77.7%. Further studies by Graczyk

et al. (1997b) indicate that the acetone dissolution step does not compromise viability or infectivity. 

EPA’s Method 1622 requires a capsule filter (USEPA, 1999b) (e.g., EnvirocheckTM capsules); these filters

contain a pleated polysulfone membrane with a 1- m porosity. EnvirocheckTM is a 6-cm-diameter by 21-cmlong capsule with a surface area of 1,300 cm2

. Clancy (1997) compared throughput and recovery rates of this

capsule filter with those of polycarbonate membrane filters, vortex flow filtration, and cellulose acetate

membrane filters which were dissolved post-filtration. All four filters were challenged with 10 liters of

municipal raw and finished waters. The cellulose acetate membranes and polycarbonate membranes were

blocked at 8 and 2.5 liters, respectively, at a raw nephlometric turbidity unit (NTU) of 5. The polymer vortex

flow and EnvirocheckTM capsule filters were able to process the entire 10 liters of raw water and gave recovery

rates of 11-57% and 8-78%, respectively. In finished waters from five utilities, the vortex flow recovered 18-

69% of the seeded oocysts, while the capsule filter recovered 45-117%. The researchers concluded that the

capsule filter performed best with the various water matrix conditions tested. Other membrane filters composed

of glass fiber have been evaluated but shown to be of poor integrity (Whitmore and Carrington, 1993).
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Centrifugation-based concentration methods

Vortex flow filtration (VFF) is a centrifugation-based filtration method in which a water sample is passed

through a cylindrical membrane filter rotating at high speed within a second cylinder. The rotation sets up forces

that scrub the membrane surface and prevent blockage by particulates. The liquid phase (permeate) crosses the

filter while the particulate-containing phase (retentate) is continuously recirculated and concentrated. Whitmore

and Carrington (1993) evaluated the ability of a VFF apparatus with a 0.45- m polysulfone membrane cartridge

filter, followed by a clarification step using density gradient centrifugation, to concentrate Cryptosporidium

from spiked bore hole or river water samples. The VFF device recovered between 30 and 40% of the seeded

oocysts. Fricker (1997) reported greater than 60% recovery using VFF in seeded river water samples.

Whitmore and Carrington (1993) evaluated cross-flow filtration for recovery of oocysts in clean water. This

apparatus pumps water across a set of alumina filters in a parallel series. In this study, the retentate, typically

150-200 ml, was collected, centrifuged, resuspended in phosphate-buffered saline (PBS), and counted using a

hemacytometer under Nomarski differential interference contrast (DIC) illumination. The authors noted that

this method is rapid and recovers 70% of the seeded oocysts in small-volume samples. Studies using larger

volume samples recovered 40%. The authors suggested that more effective cleaning or replacement of the

filters between runs may result in higher recovery rates. The device produces small volumes of retentate, which

facilitate further concentration, is compact, and can be sterilized. 

Researchers at Marshfield Clinic in Marshfield, Wisconsin have developed a continuous centrifugation method

to concentrate parasites from water (Borchardt and Spencer, 1996). This method uses a blood cell separator,
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operating on the principle of channel-type centrifugation, to concentrate oocysts from water samples. 

Recoveries over a range of water turbidities, oocyst concentrations, and water volumes were reported to be 78-

101% .

Concentration using flocculation

Calcium carbonate flocculation has also been used to concentrate Cryptosporidium oocysts in up to 10 L of

water. The method, developed by Vesey et al. (1993b), uses calcium chloride and sodium bicarbonate in a high

pH solution to crystallize organic particles. The crystals are allowed to settle, the supernatant is discarded, and

the calcium carbonate precipitate is dissolved with sulfamic acid. Vesey et al. (1993b) reported recoveries

greater than 68% with this method. Subsequent analyses of this method by Shepherd and Wyn-Jones (1995 and

1996) were in agreement and reported that calcium carbonate flocculation consistently gave higher recoveries

than cellulose acetate membrane and cartridge filters. However, calcium carbonate flocculation is not

recommended for drinking water analysis when viability is important. Studies by Robertson et al. (1994)

showed significant viability reduction, as determined by vital dye staining (see Viability, VII-A-2) following a

4-hour exposure to a pH of 10.

Clarification by density gradient centrifugation

Density gradient flotation methods are commonly used to clarify samples and concentrate oocysts prior to

detection. Centrifuge speed and time and the density of the solution vary among laboratories using this method. 

LeChevallier et al. (1995) reported that a Percoll sucrose gradient with a specific gravity of 1.15 was 67% better

than a gradient with a specific gravity of 1.0 for recovery and concentration of oocysts. A gradient with a
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specific gravity of 1.3 did not clarify the sample and interfered with microscopic analyses. This study also

indicated that flotation selects for empty oocysts while live, intact oocysts sink to the bottom of the gradient

rather than floating to the upper fraction (LeChevallier et al., 1995). Shepherd and Wyn-Jones (1995) reported

significant decreases in recovery (>50%) when sucrose flotation techniques were included in detection methods.

Flow cytometry

A flow cytometer is a laser-based instrument that analyzes particles in a liquid suspension on a particle-byparticle basis. It can differentiate and physically separate (sort) particles based on their size, internal

complexity, and fluorescence. Flow cytometry with cell sorting (FACS) is used routinely in the U.K. and

Australia for detection of Cryptosporidium and Giardia in environmental samples (Vesey 1993a,1994). Briefly,

a concentrated portion of the sample is incubated with a fluorochrome-conjugated monoclonal antibody that

binds a portion of the oocyst wall, causing the organism to fluoresce. The instrument analyzes the particles and

electrically charges those selected by the operator based on a signal (e.g., fluorescence). The charged particles

are pulled out of the sample stream using oppositely charged electrical plates and deflected onto a microscope

slide. The slides, free of the debris which can obscure oocysts on a membrane filter slide, can be read in 5 to 10

minutes as opposed to the 90 to 120 minutes typically cited for membrane analysis. In studies published by

Vesey et al. (1994), FACS detected greater than 92% of the Cryptosporidium and Giardia in seeded river and

reservoir samples. Additional work incorporating a calcium carbonate flocculation concentration step prior to

FACS reported 64.1% and 62.7% recovery of the oocysts seeded in filtered and raw waters, respectively (Veal,

1997). Studies by Hoffman et al. (1997) with a variety of environmental samples reported that FACS detected

almost three times more Cryptosporidium-positive samples than membrane immunofluorescence assay (IFA)

(94.1% vs. 35.3%, respectively) and an equal number of Giardia-positive samples. This technique has been
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repeatedly shown to be superior to traditional membrane IFA analysis (Danielson, 1995; Compagnon, 1997). 

Researchers at Macquarie University in Australia, working with a flow cytometer manufacturer, have modified

the traditional flow cytometer to optimize it for water quality analysis. This type of flow cytometer is not

currently available in the U.S. The FACS method provides increased sensitivity and requires less time, expense,

and experience than the ASTM IFA method. Additionally, this method can, with no extra effort,

simultaneously detect Giardia. Disadvantages of this method include the initial expense of the instrument

($150,000-200,000) and the level of flow cytometry expertise required with the non-optimized models.

Immunomagnetic separation

Method 1622 uses immunomagnetic separation (IMS) to separate oocysts following a filtration step (USEPA,

1999b). IMS concentrates Cryptosporidium oocysts by using magnetic beads coated with an antiCryptosporidium antibody. Following elution, the sample is incubated with magnetic beads that bind the

oocysts. The solution is inserted into a magnetic particle concentrator that binds the magnetic beadCryptosporidium complex. After the supernatant is decanted, the beads are released from the magnet. Oocysts

are dissociated from the magnetic particles using an acid wash, neutralized with base, and subjected to analysis.

This method was evaluated for Cryptosporidium by Robertson and Smith (1992). Later efforts to develop a

Giardia detection system were pursued by Bifulco and Schaeffer (1993) who reported an 82% recovery rate for

Giardia in waters of varying turbidities. Campbell et al. (1997) spiked laboratory-grade and turbid water

samples with Cryptosporidium and concentrated them using either the ASTM IFA, the U.K. Standing

Committee of Analysts method (SCA), or IMS. IMS gave the highest recoveries from either water type. Clean

water recoveries using IMS were 97.4%, while the ASTM IFA and SCA methods recovered 26.9% and 19.3%,

respectively. Turbid water recoveries were 65.8% using IMS, 5.4% with the ASTM IFA, and 11.7% with SCA. 
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A five-site evaluation comparing IMS to FACS and a modified SCA reported the IMS method consistently

resulted in higher oocyst recoveries than FACS and SCA methods in low turbidity waters (Campbell and Smith,

1997). However, efficiency of the IMS method was decreased in high turbidity water samples. Flow cytometry

showed the greatest recoveries in higher turbidity waters. When Fricker et al. (1997) evaluated the IMS

procedure in water samples seeded with 100 oocysts, recovery rates ranged from 71-120%.

Panning

Panning is a technique originally developed to isolate specific cell types from a mixed cell population. An

antibody raised to the target is attached to a solid substrate and incubated with the target cell-containing

suspension. During the incubation period, the target cell or organism is bound by the antibody and the

remaining cells or debris can be washed off. Direct and indirect panning methods were tested by Stone (1997)

to concentrate Cryptosporidium oocysts during the clarification stage. The authors recovered 50% of the

oocysts seeded into Hank’s balanced salt solution by direct panning and 20% of the seeded oocysts using

indirect panning.
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2. Detection of Cryptosporidium in Water

IFA

Direct and indirect immunofluorescent antibody detection methods facilitate the visualization of oocysts which

may be obscured by debris in environmental samples. The indirect immunofluorescence assay (IFA) (Fout et

al., 1996), described in the 1994 Cryptosporidium Criteria document, remains the most widely used detection

method. The test, however, does not provide information regarding viability, infectivity, or species. The

HydroFluor Combo staining reagents have been shown to cross-react with various algal species (Rodgers et al.,

1996), and an experienced microscopist is essential for accurate and reliable examination. 

Well slide staining

To determine oocyst concentrations, Method 1622 requires well slide staining using fluorescently labeled

monoclonal antibodies and 4',6-diamidino-2-phenylindole (DAPI), and the cells are visualized by fluorescence

and differential interference contrast (DIC) microscopy (USEPA, 1999b). Well slide staining has been

evaluated as an alternative to membrane IFA staining (Frederickson et al., 1995). In a five-site side-by-side

comparison, Cryptosporidium recoveries using the well slide method were in excess of 50% higher than those

obtained using traditional membrane staining. Additionally, the well staining procedure took 50% less time to

perform. The authors suggested the physical forces of the vacuum used in membrane IFA staining may result in

destruction of cysts and oocysts. Microscopic examination of the contents of the apparatus used for membrane

IFA staining revealed a 78% loss of Cryptosporidium, with 2% remaining intact. Cryptosporidium oocysts have

been shown to be compressible (Li et al., 1995) and may slip through filters with pore sizes smaller than their 4-

6 m diameter.
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Enzyme immunoassays

Traditional enzyme immunoassays (EIA) can provide rapid detection of oocysts with little tedium. While these

assays have been used clinically, their use in environmental analyses is not common. Several papers have

shown that EIA can be used to analyze environmental samples (Siddons, 1991; Chapman, 1990; Gracyzk et al.,

1996b). In one instance, Siddons (1991) reported a positive EIA detection of one oocyst. Chapman and Rush

(1990) reported EIA sensitivity equal to microscopic examination in both environmental and human samples. 

De la Cruz and Sivagansen (1994) tested two C. parvum EIA kits for detection of oocysts in buffered saline and

river water. The authors found the kits were capable of detecting <10 oocysts; however, results were variable

with fixed and unfixed organisms. Both EIA kits cross-reacted with algae. Gracyzk et al. (1996b) compared

the specificity of the Prospect TTM enzyme-linked immunosorbent assay (ELISA) to that of the HydroFluor

Combo antibody assay used in the ASTM ICR method and the Merifluor direct stool kit antibody. Their results

showed the ELISA gave a positive reaction with only 6 of 25 non-C. parvum isolates tested, while the

HydroFluor Combo and Merifluor direct antibodies each cross-reacted with 19 of such isolates.

Molecular methods: polymerase chain reaction assays

Several reports describe various applications of the polymerase chain reaction (PCR) for the detection of

Cryptosporidium in drinking water (Rochelle et al., 1997a and b; Johnson et al., 1995; Wagner-Wiening and

Kimmig, 1995; Filkorn et al., 1994; Johnson et al., 1993). These methods rely upon in vitro enzyme-mediated

amplification of Cryptosporidium-specific nucleic acids in order to facilitate identification in water samples. In

theory, this technique should offer unmatched endpoint sensitivity as well as the possibility of distinguishing

subtle differences among discrete strains of parasites. A number of techniques aimed at distinguishing viable
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and nonviable oocyst populations are also reported. These applications are summarized below. It is important

to emphasize that, due to the enzymatic nature of the PCR process, these assays may fail due to inhibition of

enzyme activity caused by compounds commonly found in natural waters. Additionally, the majority of the

documented studies have been limited to evaluations of seeded water samples rather than actual comparative

field trials. Hence, the application of these techniques toward the detection of Cryptosporidium in

environmental water samples should be considered developmental.

Johnson et al. (1995) reported a PCR protocol to detect Cryptosporidium in environmental samples, based upon

oligonucleotide primers specific to a portion of the small 18S ribosomal RNA. Detection sensitivities of 1 to 10

oocysts were achieved in purified oocyst preparations; however, the detection sensitivity in seeded

environmental samples was up to 1,000-fold lower. Poor endpoint sensitivity was at least partially offset by a

concentration step using either flow cytometry or immunomagnetic capture and by oligoprobe hybridization

using a chemiluminescent technique. These methods were applied to confirm the presence of oocysts in water

samples from the Milwaukee outbreak of cryptosporidiosis, and the results of the PCR assays were comparable

to those observed when immunofluorescence methods were used.

Rochelle (1997b) evaluated four pairs of previously published primers aimed at the specific detection of C.

parvum. Detection sensitivities ranged from 5 to 50 oocysts in seeded environmental samples when PCR was

followed by oligoprobe hybridization, with some primer pairs offering species specificity while others were

only genus-specific. Successful multiplex reactions aimed at the simultaneous detection of G. lamblia and C.

parvum were evaluated and demonstrated the utility of PCR for the detection of waterborne parasites. However,
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no primer combinations were identified which exhibited the ideal combination of sensitivity, specificity, and

compatibility with multiplex reactions, and several primer sequences previously reported failed to amplify their

targets.

Since PCR is capable of detecting the genetic material of both live and dead microorganisms, a number of

studies have targeted unique sequences to distinguish viable from nonviable oocysts. Wagner-Wiening and

Kimmig (1995) applied PCR to detect Cryptosporidium by targeting a large DNA fragment specific to C.

parvum. In order to differentiate between live and dead oocysts, this group reported the practice of applying an

excystation protocol prior to PCR and targeting sporozoite DNA to ensure that amplified material was

associated only with viable oocysts. Endpoint sensitivity as low as 100 sporozoites was observed and was

reduced to 10 sporozoites when nested PCR was practiced. Filkhorn et al. (1994) also evaluated RNA-based

measurement of viable oocysts by practicing excystation prior to PCR. To preclude spurious contamination

caused by the presence of DNA in nonviable parasites, a DNase enzyme was applied to digest free DNA,

leaving only free RNA from viable oocysts.

Stinnear et al. (1996) described a reverse transcription-PCR (RT-PCR) detection method specific for C. parvum

which can detect single viable oocysts and is based upon the assumption that only viable oocysts are

metabolically active and will produce messenger RNA (mRNA). Since mRNA exhibits an extremely short halflife in living cells (and perhaps even shorter outside of the protective environment of the oocyst wall), only the

mRNA from viable oocysts will be captured and amplified.
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PCR has also been used to track Cryptosporidium reductions during water treatment. Mayer and Palmer (1996)

evaluated several methods to identify Cryptosporidium oocysts in sewage and to determine reductions during

wastewater treatment. The nested PCR technique described above was compared to a modified ASTM method

to track reductions during treatment, and strong correlations were observed, with approximately 2 log10

Cryptosporidium reductions observed. The authors concluded that the PCR method was preferable due to a

substantial reduction in sample collection and processing during analysis.

Molecular methods: cell culture-PCR

At least one integrated approach has been reported that utilizes tissue culture, PCR, and in situ PCR (IS-PCR) to

assess seeded natural water concentrates for the presence of infectious oocysts (Rochelle, 1997a). This

technique offers the possibility of screening out noninfectious oocysts, since only the infectious oocysts will

develop during the initial phase of amplification in human adenocarcinoma cells. Preliminary experiments

suggest that IS-PCR may offer quantitative detection of infectious oocysts in natural water concentrates.

Molecular methods: strand displacement amplification

A strand displacement assay for the detection of Cryptosporidium in natural waters samples has been reported to

overcome the comparatively long cycle times associated with PCR (Blassak et al., 1996). This method is based

upon the selective replication of a single DNA strand while the other parental strand is displaced from the

template, with colorimetric detection of oocyst DNA under a microplate format. A horseradish peroxidasestreptavidin conjugate is used for color development. The results of the strand displacement assay correlate well

with evaluation of acid-fast slides of fecal samples.
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Laser scanning microscopy

The ChemScan RDI is a laser scanning device linked to an epifluorescent microscope with a motorized stage. 

Sample analysis includes a filter staining procedure, followed by automated scanning with the ChemScan

instrument. The instrument digitizes the location of fluorescent objects, allowing for quick confirmation. This

instrument has the advantage of being highly automated and more sensitive than FACS and IMS methods in

samples with colloidal clay (Reynolds et al., 1997). The recovery rate, minimal detection limit, and oocyst

spike concentration were not specified. Disadvantages of this method include the cost of the instrument

($200,000-300,000), use of membrane filters which have been shown to contribute to oocyst loss (Frederickson

et al., 1995), and the inability to perform any light microscopy techniques for visualization of internal

cytoplasm or sporozoites.

Miscellaneous methods

Campbell et al. (1993a) contrasted flow cytometry and a slow scan cooled-charge couple device (CCD) for

detection of Cryptosporidium. The authors discussed the advantages of the CCD, including its ability to

simultaneously assess viability by DAPI staining; however, a comparison of results was not provided. The need

for sophisticated and currently unavailable software was noted by the authors.

Campbell et al. (1993b) also evaluated enhanced chemiluminescence for detection of Cryptosporidium in 21

environmental samples previously assayed by microscopy. The oocysts were labeled with a fluorescein

isothiocyanate (FITC)-conjugated anti-Cryptosporidium antibody, followed by a biotin-conjugated anti-FITC

antibody and streptavidin-peroxidase. Statistical analysis revealed no difference in results obtained with the
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enhanced chemiluminescence assay and microscopy. The authors reported that the chemiluminescence method

is faster than the currently used microscopic method; however, visual confirmation was still necessary.

The electrorotation assay (ERA) relies on the principle that small particles can be induced to rotate in the

presence of a rotating electric field. The rate of rotation is due to the field and surface charge of the particles. 

Jakubowski et al. (1996) described a proprietary electrorotation assay in which oocysts are attached to an

antibody-coated magnetic bead and placed in a filter electrode assembly. The assembly is placed under a

microscope, connected to an electric field generator, and the number of rotating oocysts are enumerated. This

method may differentiate between viable and non-viable oocysts, based on differences in their rotation rates. 

Oocyst recovery rates have been reported to range from 30-95%; however, the efficiency of this method is

dependent on the type and characteristics of the water. A major disadvantage to ERA is that magnification can

only be performed up to 400× due to the thickness of the ERA unit.

Method 1622

Recognizing the need for an improved Cryptosporidium detection method, the USEPA initiated an effort to

identify new and innovative technologies for protozoan monitoring and analysis. Following a comprehensive

evaluation of existing and emerging technologies, the USEPA Office of Water developed an initial draft of

Method 1622 in December 1996 (EPA-821-R-97-023). This method has been validated and is now used as a

standard procedure (EPA-821-R-99-001) for collection and quantification of Cryptosporidium oocysts from

water samples.
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With Method 1622, 10-L sample is shipped to the laboratory, where it is filtered through a polysulfone capsule

filter. Following filtration, the capsule is filled with eluting solution and shaken on a wrist action shaker for

approximately 5 minutes. The capsule is drained and the elution procedure is repeated. The combined eluate is

concentrated by centrifugation, reconstituted to 10 ml, and subject to IMS as described previously in Section

VII.A.1. The solution is stained with anti-Cryptosporidium antibodies using the well slide method described

previously in this section.

Viability determinations

The public health significance of Cryptosporidium relates primarily to the ability of this parasite to initiate

infection in humans and animals. Although the gold standard among infectivity assays remains the animal

model, the high costs and ethical considerations associated with assays using animals preclude their routine use. 

Additionally, these methods do not offer adequate sensitivity for testing of environmental samples when the

level of oocyst contamination may be on the order of 1 or 2 oocysts per L. Differences in pathogenesis among

humans and animals have also called into question the applicability of animals in providing an accurate

reflection of the number of Cryptosporidium required to cause cryptosporidiosis in humans. Nonetheless,

innovative viability assessment methods for Cryptosporidium oocysts are inevitably compared to animal

models, primarily using mice, and these methods are described below.

The in vitro excystation (IVE) method estimates infectivity by determining the number of potentially infectious

oocysts based on simulating the conditions of the mammalian gastrointestinal tract. Oocysts exposed to an acid

pretreatment and bile salts at elevated temperatures will release progeny sporozoites (excyst), whereas
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metabolically inactive (nonviable) oocysts will fail to excyst under such conditions. Excystation is tracked by

direct microscopic visualization of treated samples and their comparison to the initial untreated population. 

Black et al. (1996) and Belosevic et al. (1997) indicate that the IVE assay may significantly overestimate the

true infectivity of oocysts treated with chemical antagonists, compared to the results of animal infectivity

studies. Although the IVE assay is relatively time-consuming and is generally not applicable toward tracking

reductions during water treatment (which may exceed several orders of magnitude), Vesey et al. (1997) have

reported an adaptation of this method that uses flow cytometry to increase the sensitivity and throughput, and

they have observed good agreement with the microscopic method.

The Cryptosporidium Criteria Document (1994) described the early work of Campbell et al. (1993b), which

focused on vital dye staining. This group evaluated the application of dyes which are preferentially absorbed by

viable oocysts, compared to the technique with IVE, and strong correlations were observed. This work was

confirmed by Bukhari (1995) who compared vital dye staining and IVE of oocysts concentrated using several

different methods. Subsequent studies by Black et al. (1996) indicated that vital dye staining correlated well

with IVE but had a tendency to overestimate infectivity of ozone-treated oocysts when compared to animal

infectivity. Jenkins et al. (1997) confirmed that vital dye staining tends to overestimate infectivity relative to

animal infectivity, but they supported its use as an economical, user-friendly method that provides information

on the effects of stresses on the surface of oocysts over time. Belosevic et al. (1997) evaluated 14 nucleic acid

stains as indicators of Cryptosporidium viability and compared the staining to both animal infectivity and IVE

assays. Both heat-treated and chemically inactivated oocysts were consistently stained with novel stains

(SYTO-9, SYTO-59, and hexadium). This staining correlated well with animal infectivity but not the IVE

assay. The authors also developed an IFA viability assay that relies only on propidium iodide (PI) for viability
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assessment; however, extraneous factors such as aldehyde fixation may inhibit uptake of PI and falsely elevate

the numbers of viable oocysts (Campbell et al., 1993c).

Various infectivity assays have been described using tissue culture methods to assess the infectivity of C.

parvum (Upton et al., 1994a; Upton et al., 1994b; Upton et al., 1995; Rochelle et al., 1997a; Slifko et al., 1997). 

These methods track the development of progressive Cryptosporidium infections in cell cultures. Evaluation of

infected cultures can be facilitated by conducting ELISAs following a 1-2 day incubation, and then scoring the

extent of infection (and hence number of viable oocysts present in the original inoculum) by spectrophotometry

in an automatic plate reader. A detection sensitivity of approximately 100 oocysts has been described (Upton et

al., 1994a and b). Slifko et al. (1997) describes a semi-quantitative method which relies upon staining infected

tissue cultures with fluorescent antibody and then tracking the numbers of infectious foci using epifluorescence

and DIC microscopy. An adaptation of the tissue culture method using PCR (Rochelle et al., 1997a) is

described above in Section VII-A-2 “Molecular methods: polymerase chain reaction assays.”

3. Assessment of Laboratory Testing Capabilities

Detection of Cryptosporidium and Giardia and the ability to distinguish them from other organisms of

comparable size and appearance is a major problem that presents most commercial, state, and local laboratories

with a difficult challenge. However, it is important to establish whether these laboratories can follow a standard

procedure and be successful in recovering and detecting these pathogenic protozoans in water samples.
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Sixteen commercial laboratories were enlisted in a survey (Clancy et al., 1994) to assess the ability of the

laboratories to recover and detect Giardia and Cryptosporidium using the ASTM method. Filters spiked with

either Giardia (approximately 740 cysts) and Cryptosporidium (approximately 500 oocysts) or with

approximately 500 cells of Oocystis minuta (algal cells measuring 8-18 m by 5-15 m) were sent to the

laboratories for analysis. Of the 11 laboratories that provided results of their analyses, four reported O. minuta

samples as positive for either Giardia or Cryptosporidium, while four others failed to recover Giardia from the

cyst-spiked filter and six laboratories failed to recover Cryptosporidium from the oocyst-spiked filter. Giardia

cyst recovery ranged from 0.8 to 22.3% (with an average of 9.1%), while Cryptosporidium cyst recovery ranged

from 1.3 to 5.5% (with an average of 2.8%). It was concluded that not all laboratories strictly followed the

ASTM methods and that the majority of laboratories need to improve in one or more of the following areas:

client response, quality of sampling equipment and directions for use, analytical methods, data accuracy, and

reporting format. Expertise in microbiological identification also appeared to be lacking, as indicated by the

relatively high numbers of false positives. The USEPA established an approval process for laboratories that

analyzed samples for the Information Collection Rule for Cryptosporidium and Giardia. The USEPA required

approved laboratories to have trained and experienced personnel performing Cryptosporidium and Giardia

testing, in addition to the necessary processing equipment. Initial and continuous passing of performance

evaluation samples and passing of the on-site inspection were also required.

B. Detection in Biological Samples

Diagnosis of Cryptosporidium infection is typically performed by examining the feces of an infected individual. 

Feces of patients with active cryptosporidiosis normally do not require concentration since oocysts are shed in

great numbers; however, the number of oocysts can fluctuate during the course of infection (Casemore and
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Roberts, 1993). This emphasizes the importance of making diagnoses using multiple specimens. Concentration

methods are employed when trying to assess infection in immunocompromised patients with a history of

unexplained diarrhea or in asymptomatic patients. Concentration methods including formalin-ether and

formalin-ethyl acetate sedimentation are commonly used in clinical laboratories. Sheather’s sucrose flotation,

zinc sulfate flotation, saturated sodium chloride flotation, discontinuous Percoll gradients, and cesium chloride

gradient centrifugation are methods more common in research laboratories. Evaluations of various

concentration techniques have been published and the results are summarized below.

Concentration methods

A study by Bukhari and Smith (1995) comparing water-ether, sucrose density gradient, and zinc sulfate

concentration methods showed significantly higher numbers of oocysts were recovered from bovine feces using

water-ether concentration. Rosales et al. (1994) used concentration by Sheather’s solution to obtain greater

numbers of oocysts than by discontinuous Percoll gradients and a commercially manufactured parasite

concentrator device. Concentration of oocysts from cat feces (Mtambo, 1992) was best accomplished using

formalin-ether sedimentation, which recovered 37% of the original oocysts compared to 11% and 33% for zinc

sulfate and sucrose flotation, respectively. Clavel et al. (1996b) showed that simply increasing centrifugation

times augmented oocyst recovery when using the standard formalin-ether acetate concentration method. Finn et

al. (1996) demonstrated that straining the feces contributed to the loss of oocysts. This study showed a nearly

four-fold overall reduction in the number of oocysts detected using a wash procedure.
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Traditional staining methods

Traditional staining methods were described in the 1994 Cryptosporidium Criteria Document. Numerous new

staining methods and variations of traditional methods have also been employed. A review is included in

Chapter 2 of the book, Cryptosporidium and Cryptosporidiosis (Fayer, 1997). Kang and Mathan (1996)

compared five staining methods for detection of Cryptosporidium oocysts in fecal smears. The safraninmethylene blue technique, a modified Ziehl-Neelsen method, was used as the “gold standard” and compared to

two methods each using auramine and mepacrine stains with potassium permanganate and carbol fuschin as

counterstains. The authors concluded that mepacrine and auramine staining procedures were both easily

performed. However, they preferred mepacrine to auramine because it is less toxic and can be used with carbol

fuschin without a decolorization step. Work by Ungureanu and Pontu (1992) supported these results. In the

Cryptosporidium Screening Guidelines established by a joint working group, Casemore and Roberts (1993)

recommended an auramine method to be used as a screening method with confirmation using a modified ZiehlNeelsen method. Acid-fast staining methods do not stain all oocysts. Entrala et al. (1995) showed hydrogen

peroxide treatment increased the percentage of oocysts displaying acid-fast characteristics. The authors

speculated that treating oocysts with hydrogen peroxide may have affected a component of the oocyst wall and

composition of the oocyst contents or granules.

Immunofluorescence methods

The use of monoclonal antibody detection assays increases the sensitivity of Cryptosporidium detection

compared to various acid-fast methods and auramine-rhodamine staining methods, as described in the following

studies. Garcia et al. (1987) tested 297 human fecal samples using a modified acid-fast method and monoclonal
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antibody staining. Sensitivity and specificity using the monoclonal antibody were 100%, while the acid-fast

method failed to detect 7 of 99 positive samples. Subsequent testing by Baron et al. (1989), Rusnak et al.

(1989), Garcia et al. (1992), Tee et al. (1993), Grigoriew et al. (1994), Alles et al. (1995) and Roberts et al.

(1996) confirmed these results. Studies using monoclonal antibodies for detection of Cryptosporidium in

animals have also shown increased sensitivity (Wee et al., 1995; Mtambo et al., 1992; Xiao and Herd, 1993).

EIAs

Commercial EIAs have been developed to replace time-consuming microscopic methods. Most studies have

shown EIA methods perform better than (Siddons et al., 1991; Dagan et al., 1995) or equal to (Chapman and

Rush, 1990; Rosenblatt and Sloan, 1993; Parisi and Tierno 1994) conventional microscopic methods; however,

work by Newman et al. (1993) indicated that EIA methods were not sensitive enough to be used for patients

without diarrhea. EIAs have been shown to be equal in sensitivity to the IFA (Siddons et al., 1991; Rosenblatt

and Sloan, 1993) and inferior using an experimental EIA (Anusz et al., 1990) and a commercially available EIA

(Ignatius et al., 1997). Two commercial EIAs were compared to a direct immunofluorescence assay, the

ProSpecT and the ColorVue (Aarnes et al., 1994) and found to have differences in performance, i.e., the

sensitivities were 96% and 72-76%, respectively. Specificities were 97.6-99.5% using the ProSpecT and 100%

using ColorVue.

Molecular methods: PCR assays

PCR-based assays are becoming more common and may provide a useful alternative for detecting and

quantifying Cryptosporidium in water, stools, and tissue/organ samples. Awad-El-Kariem et al. (1994)
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described a PCR method that identifies Cryptosporidium at the species level and requires no DNA probes. The

procedure is based on use of the published 18S rRNA genes of C. parvum and C. muris. One of the sequence 3

MaeI endonuclease restriction sites is present only on the C. parvum gene, while others are specific for C. muris

and C. baileyi, which allows screening for human, mouse, and avian species, respectively. The authors

suggested that the protocol they developed is adaptable to detection of small numbers of C. parvum oocysts in

environmental samples. Morgan et al. (1996) noted the importance of PCR in processing clinical as well as

environmental specimens suspected of being contaminated with Cryptosporidium. PCR primers specific for

Cryptosporidium have been developed, and random amplified polymorphic DNA (RAPD) is a simpler approach

for developing diagnostic primers, since many of the products generated by RAPD-PCR are frequently speciesspecific. Leng et al. (1996) developed an assay to identify Cryptosporidium DNA in bovine feces involving

standardization of sample preparation and simplification of the DNA recovery process for PCR amplification

and DNA-hybrid detection. The DNA recovery/PCR detection procedure can recover DNA suitable for PCR

amplification and can detect 103 to 104 fewer oocysts diluted in water or buffered saline and 102 fewer oocysts

from diarrheic fecal samples than the commercial ELISA Color-Vue-Cryptosporidium kit. 

Amplification methods have also been described which may assist in future efforts to define the role of

livestock in waterborne outbreaks of cryptosporidiosis. Blassak et al. (1996) described a rapid assay kit

designed to test fecal samples for live Cryptosporidium using a gene probe method. Oocyst DNA is released by

cyclical freeze/thaw and is then amplified by isothermal strand displacement using biotinylated primers. The

amplification product is detected colorimetrically in a microwell system in which a complementary capture

probe binds to oocyst DNA and then reacts with a horseradish peroxidase-streptavidin conjugate. This complex

emits a color that can be readily detected with qualitative or semi-quantitative results. The authors suggest that
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this method could be applied toward the analysis of genetic similarity among Cryptosporidium strains isolated

from livestock and humans.

Balatbat et al. (1996) developed a nested PCR assay for detection of C. parvum directly from stool specimens. 

After extracting DNA from a formaldehyde-treated stool, a 400-bp fragment of DNA was amplified with two

26-mer primers. The amplicon from the first reaction was then subjected to a second round of amplification

using a second set of primers. With these nested primers, a 194-bp fragment of DNA was amplified and

confirmed as C. parvum DNA by internal probing with an enzyme-linked chemiluminescence system. The test

can detect as few as 500 oocysts per gram of stool and has the potential to detect asymptomatic infections,

monitor response to therapy, or monitor environmental samples. The preliminary results indicate a significantly

enhanced sensitivity compared with traditional assays.

Kelly et al. (1995) developed a sensitive and specific PCR test to confirm Cryptosporidium infections. The test,

which uses previously published primers to detect Cryptosporidium in distal duodenal biopsies, was used to

identify infections in HIV-positive patients in Zambia and proved especially useful in identifying those

infections that were limited to the distal small intestine.

Other methods

Serological methods have been used to monitor exposure to Cryptosporidium. There is limited information

regarding the seroprevalence of Cryptosporidium-infected individuals. The serologic response to

Cryptosporidium is discussed in section V-B of this report and by Lengerich et al. (1993).
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 Flow cytometry methods have been described for detection of Cryptosporidium in mice (Arrowood et al.,

1995). More recent studies using seeded human feces showed a four-fold increase in detection over direct

immunofluorescence methods (Valdez et al., 1997). Chemiluminescence assays have been used by Clavel et al.

(1996b) to detect cultured oocysts and other fecal parasites. You et al. (1996a, b) showed positive detection of

Cryptosporidium in MDCK cell cultures and the potential of this cell culture system for testing

chemotherapeutic agents is promising. A reverse passive hemagglutination (RPH) assay (Farrington et al.,

1994) measuring agglutination of anti-oocyst antibody-coated sheep erythrocytes with oocysts in diluted fecal

suspensions was compared to auramine phenol staining for detection of Cryptosporidium and showed equal

sensitivity. 

C. Water Treatment Practices

1. Introduction

Multiple barriers are used in most surface water treatment plants in an effort to prevent public exposure to

waterborne pathogens like Cryptosporidium. These barriers include removal of pathogens from water by

processes like clarification and filtration, which are generally preceded by coagulation and flocculation

processes. Another type of barrier is inactivation by disinfectants like ozone and chlorine. The purpose of this

section is to summarize the removal and/or inactivation of Cryptosporidium through multibarrier systems and

through individual treatment processes. The reader is strongly encouraged to look at the referenced studies to

gain detailed information regarding site-specific raw water quality and treatment conditions.

In this section, log removal is defined by the following equation:
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Equation 2. log removal = -log(N/N0

)

where N is the concentration of Cryptosporidium oocysts remaining after treatment and N0

 is the concentration

of Cryptosporidium oocysts prior to treatment. Log inactivation is given by a similar equation, but N and N0

refer to the concentration of infectious Cryptosporidium oocysts in treated water and in untreated water,

respectively. A comparison of removal efficiencies of some bench-, pilot-, and full-scale water treatment

processes is found in Table 5.

2. Multibarrier Treatment

Several studies have evaluated the occurrence of Cryptosporidium in raw and finished waters from multibarrier

treatment facilities (LeChevallier and Norton, 1995). In a survey of 72 North American drinking water plants,

Cryptosporidium was present in 51.5% of raw water samples and in 13.4% of finished water samples. In an

earlier survey of 66 drinking water plants, Cryptosporidium was observed in 87% and 27% of raw and finished

waters, respectively. The authors attributed the different occurrence levels between studies to normal variations

in raw water quality and treatment performance. The authors claimed that microscopic analyses of

Cryptosporidium oocysts in the finished waters suggested that most of the oocysts were nonviable. However,

no attempts were made to specifically assess oocyst viability in this study. 

These findings suggest that a significant percentage of Cryptosporidium oocysts are removed by current

drinking water treatment practices.
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Table 5. Cryptosporidium Removal Efficiencies for Selected 

Physical and Chemical Processes

Treatment Process Description

Removal Achieved (log)

Bench Scale Pilot Scale Full Scale

Coagulation + Gravity Settling < 1.0a1.4 - 1.8b0.4 - 1.7g

Coagulation + Filtration 2.7 - 5.9b

1.6 - 4.0e

2.5 - 3.8h

2.7 - 2.9i

*

Coagulation + Gravity Settling +

Filtration

4.2 - 5.2b

1.6 - 4.0e

> 5.3f

< 0.5 - 3.0f

2.1 - 2.8i

* 1.0 - 2.5

Coagulation + Dissolved Air 

Flotation

2.0 - 2.6a

Slow Sand Filtration > 3.7c

Diatomaceous Earth Filtration > 4.0c

Coagulation + Microfiltration > 6.0d

Ultrafiltration > 6.0d

* Range of average removal efficiencies based on reservoir and river water sources.

Source: Adapted from Frey et al. (1998)

References (cited in Frey et al., 1998): a

 Plummer et al., 1995; b

 Patania et al., 1995; c

 Schuler et al., 1988; d

Jacangelo et al., 1995b; e Nieminski and Ongerth, 1995; f LeChavallier et al., 1991; g Kelley et al., 1994; h

Anderson et al., 1996; and i

 Nieminski, 1995.

Other studies have reported removal of Cryptosporidium oocysts through conventional filter treatments using

chlorine as the primary disinfectant. The combination of coagulation, flocculation, and sedimentation achieved

3.8 log removal of oocysts in a treatment plant near Montreal, Canada (Payment and Franco, 1993). In the same

treatment plant, a 4.6 log removal of oocysts was observed through coagulation, flocculation, sedimentation,

and granular media filtration (also known as conventional treatment). No attempts were made to assess oocyst

viability in this study.
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In a more comprehensive study of a conventional treatment plant near Pittsburgh, Pennsylvania,

Cryptosporidium oocysts were detected in 63% of raw water samples, 29% of settled water samples, and 13% of

filtered water samples (States et al., 1997). For those cases where oocysts were detected in both raw and settled

waters, the treatment plant achieved 0.8 to 1.3 log removal of oocysts prior to filtration. For those cases where

oocysts were detected in both raw and filtered waters, the treatment plant achieved 1.7 to 3.6 log removal of

oocysts through filtration. Oocyst viability was not measured in this study.

3. Removal of Cryptosporidium

Introduction

As noted in the previous section, detectable Cryptosporidium concentrations occur infrequently in treated

waters. Because of this, the effectiveness of treatment processes has been evaluated in challenge studies where

oocysts are spiked into raw water at a concentration high enough for oocysts to be detected in treated water. 

This section summarizes challenge study results for those processes that remove Cryptosporidium oocysts;

challenge study results for those processes that inactivate Cryptosporidium oocysts are summarized in the next

section.

Coagulation, flocculation, and clarification

Several clarification methods are available for drinking water treatment, and these methods are usually preceded

by coagulant addition and flocculation. Currently, sedimentation is the most commonly practiced method of

clarification in the United States. Other options include dissolved air flotation and sludge blanket clarification. 

One bench-scale study showed that dissolved air flotation was superior to sedimentation for removal of
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Cryptosporidium oocysts (Plummer et al., 1995). With proper coagulation and flocculation conditions, the

combination of coagulation, flocculation, and dissolved air flotation could achieve 2.5 to 3.5 log removal of

Cryptosporidium. Under similar conditions, however, the combination of coagulation, flocculation, and

sedimentation could achieve no more than 1.0 log removal of oocysts. Similar bench-scale studies showed that

a 1.3 to 2.8 log removal of Cryptosporidium could be achieved by the combination of coagulation, flocculation,

and dissolved air flotation (Hall et al., 1995).

Plummer et al. (1995) observed relatively weak correlations between the log removal of Cryptosporidium and

removal of turbidity (r2

 = 0.53), UV absorbance (r2

 = 0.52), or dissolved organic carbon (r2

 = 0.50).

Coagulation, flocculation, sedimentation, and filtration (conventional treatment)

Pilot-scale treatment studies with two water supplies in the western United States showed that conventional

treatment could obtain 3.0 to 6.2 log removal of Cryptosporidium, with a median of approximately 4.6 log

removal (Patania et al., 1995). An average of 3.0 log removal was observed in another pilot-scale study in

Utah, with a range of 1.9 to 4.0 log removal (Nieminski and Ongerth, 1995). Another Utah study was designed

to evaluate Cryptosporidium removal in a full-scale treatment plant that was not delivering water to customers. 

In this case, removal efficiencies ranged from 1.9 to 2.8 log removal with an average of 2.3 log (Nieminski and

Ongerth, 1995). Actual performance depends on numerous factors including source water quality, chemical

pretreatment conditions (e.g., coagulant dose and pH), filtration rate, bed depth, and filter media types. As

noted earlier, the reader is advised to read the referenced reports for more detailed information regarding the

results summarized here.
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The results to date suggest that the 2.0 log removal credit proposed for Cryptosporidium removal by

conventional treatment is reasonable and, as shown by the above data, may be conservative. However, studies

have only been published for waters from the western United States and need to be confirmed with results from

other types of water supplies from other geographic areas. As noted in Section III.B.1, any watershed, river, or

reservoir is subject to a complex set of watershed characteristics and watershed processes (Crockett and Haas,

1997; LeChevallier et al., 1997; States et al., 1997).

After combining data from the two Utah studies, Nieminski and Ongerth (1995) reported an r2

 of 0.79 for the

relationship between log removal of Cryptosporidium and log removal of 4-7 m sized particles. The

correlation between log removal of Cryptosporidium and log removal of turbidity was weaker, with an r2

 of

0.55. Patania et al. (1995) reported no significant correlations between log removal of Cryptosporidium and log

removals of turbidity, 1-2 m sized particles, 2-5 m sized particles, 5-15 m sized particles. These latter

results were obtained from a broader range of source waters and suggest that correlations between

Cryptosporidium removal and removal of surrogate indicators may be site specific. Further work is necessary

before definitive conclusions can be reached. These two studies also based their conclusions on data obtained

from different types of filtration practices (e.g., conventional treatment, direct filtration, in-line filtration).

Further analysis is needed to determine whether their observations were dependent on the type of filtration

practice.

Although no significant correlations were obtained between Cryptosporidium removal and turbidity removal,

Patania et al. (1995) observed that filter effluent turbidities less than or equal to 0.1 NTU were required to
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obtain a 5 log Cryptosporidium removal on a reliable basis. Filter effluent turbidities of 0.2 NTU or less were

needed to reliably maintain a 4 log Cryptosporidium removal.

Coagulation, flocculation, flotation, and filtration

The effect of substituting sedimentation with dissolved air flotation on filtered water Cryptosporidium

concentrations has not been directly compared in literature reports. However, pilot-scale studies in the United

Kingdom have shown that the combination of coagulation, flocculation, flotation, and filtration can achieve 2.9

to 4.4 log removal of oocysts (Hall et al., 1995). These results are consistent with those described above.

Coagulation, flocculation, and filtration (direct filtration)

The performance of direct filtration was assessed for the same Utah waters described in the conventional

treatment section (Nieminski and Ongerth, 1995). For the pilot-scale system, Cryptosporidium removal

efficiencies ranged from 1.3 to 3.6 log with an average of 3.0 log. The full-scale system achieved an average

oocyst removal of 2.8 log with a range of 2.6 to 2.9 log. Comparable removals were observed in pilot-scale

studies with water from Seattle (Ongerth and Pecoraro, 1995). Results in this study showed 2.7 to 3.1 log

removal of Cryptosporidium.

Direct comparisons between direct and conventional filtration were only obtained with the pilot-scale system in

Utah. Results indicate that there was no statistically significant difference between the two types of filtration. 

At this time, this conclusion can only be applied to this case and further studies are necessary to determine those

situations in which direct filtration achieves performance comparable to conventional treatment.
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Coagulation and filtration (in-line filtration)

Using water from the Seattle water supply, pilot-scale treatment studies showed that in-line filtration could

obtain 1.6 to 4.2 log removal of Cryptosporidium. The median value was approximately 2.8 log removal

(Patania et al., 1995). Unfortunately, a direct comparison between in-line filtration and conventional treatment

has not been made for the same water supply.

Diatomaceous earth filtration

A recent study with water from Sydney, Australia, concluded that diatomaceous earth filtration could perform

significantly better than conventional treatment for removal of Cryptosporidium oocysts (Ongerth and Hutton,

1997). In this bench-scale study, removal efficiencies ranged from 3.6 to 6.7 log. Although these results appear

to show superior performance of diatomaceous earth filtration, they were performed at filtration rates lower than

those commonly used in conventional treatment facilities. Another study has shown comparable

Cryptosporidium oocyst removal by pilot-scale diatomaceous earth filtration with a Pennsylvania water sample,

in which removal efficiencies ranged from 4.6 to 5.9 log (Schuler et al., 1991).

Slow sand filtration

Several studies have been performed to evaluate Cryptosporidium oocyst removal by slow sand filtration. 

Removal efficiencies ranging from 3.9 to 7.1 log were observed in pilot-scale studies with water from

Pennsylvania (Schuler et al., 1991). Another study in the United Kingdom showed that oocyst removal

exceeded 4.5 log removal (Timms et al., 1995). Only 0.3 log oocyst removal was obtained in a slow sand filter

located in British Columbia, Canada. However, the filter media in this filter did not meet standard
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specifications for slow sand filters (Fogel et al., 1993). Therefore, the overall results obtained to date suggest

that slow sand filtration is an effective barrier to oocyst passage when the process is properly designed.

Membrane processes

In several microfiltration and ultrafiltration experiments, Cryptosporidium oocysts were not detected in treated

waters when the membranes were intact (Jacangelo et al., 1995a). This result was observed with feed oocyst

concentrations as high as 9.1 x 104

 oocysts/L, suggesting a removal capability of more than 7.1 log. This result

is expected because the pores within the membrane skin are smaller than Cryptosporidium oocysts. More recent

studies have shown that Cryptosporidium oocysts could pass through a ceramic microfiltration membrane

having a nominal porosity of 0.2 m (Drozd and Schwartzbrod, 1997). In this case, oocyst removals ranged

from 4.3 to 5.5 log. However, no attempts were made to assess the integrity of the membrane and its associated

equipment in this study.

4. Inactivation of Cryptosporidium

Introduction

The majority of studies performed to date have evaluated Cryptosporidium inactivation in buffered, demandfree waters and in small, batch reactors. Very little is known about the ability of alternative disinfectants to

inactivate Cryptosporidium oocysts in natural waters or in flow-through treatment systems. Results depend on

the method used to quantify inactivation and, if mouse infectivity is used to quantify inactivation, results may

depend on the strain of mouse and on the strain of Cryptosporidium used in the study. For these reasons, the

reader is cautioned against extrapolating information presented in this section to inactivation of
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Cryptosporidium in natural waters and to infectivity in humans. The purpose of this section is to summarize the

inactivation work performed to date for four disinfectant chemicals – ozone, chlorine dioxide, chlorine, and

monochloramine.

Ozone

Cryptosporidium inactivation by ozone has received a significant amount of attention. Ozone clearly achieves

the most significant levels of Cryptosporidium inactivation when compared to the other three disinfectants listed

above (Korich et al., 1990; Finch et al., 1997). In order to achieve 3 log inactivation of Cryptosporidium at pH

7, the product of contact time and ozone concentration (CT) needs to be in the range of 8 to 16 mg•min/L at 7°C

and in the range of 3 to 15 mg•min/L at 22°C (Finch et al., 1993). CT values for a 2 log inactivation of

Cryptosporidium by ozone at pH 7 are in the 5 to 10 mg•min/L range at 7°C and in the 2 to 8 mg•min/L range at

22°C. It is important to stress that these values are based on mean performance and do not account for the wide

variability observed in test results. Overall results observed by other investigators appear to be in general

agreement, when the variability in data is taken into account (Peeters et al., 1989; Korich et al., 1990; Parker et

al., 1993; Owens et al., 1994a and b; Quinn et al., 1996; Finch et al., 1997).

Chlorine dioxide

Results obtained to date suggest that chlorine dioxide is the second most effective disinfectant on the above list

(Peeters et al., 1989; Korich et al., 1990; Finch et al., 1997, Liyanage et al., 1997a). CT values needed for

Cryptosporidium inactivation by chlorine dioxide are considerably higher than those needed for ozone. To

achieve a 1 log inactivation of Cryptosporidium at pH 7 and 25°C, CT values of approximately 60 mg•min/L
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001

107

would be necessary. At pH 8, the required CT values appear to be greater than this. Recent results have shown

that chlorine dioxide may be more effective if ozone is applied upstream of the chlorine dioxide addition point

(Liyanage et al., 1997b). However, this practice is not likely to become common in the United States due to the

implementation of the Stage I Disinfectants and Disinfection Byproducts Rule, which set a maximum residual

disinfectant level goal (MRDLG) for chlorine dioxide of 0.8 mg/L.

Chlorine

At CT values commonly used in drinking water treatment, chlorine does not achieve more than a 1 log

inactivation of Cryptosporidium oocysts (Korich et al., 1990; Fayer, 1995; Finch et al., 1997; Gyurek et al.,

1997; Venczel et al., 1997). This is true even in demand-free water at pH 6 and 22°C, conditions under which

chlorine can be expected to achieve the best level of performance. Recent screening studies have suggested that

the ability of chlorine to inactivate Cryptosporidium may be enhanced by preozonation (Finch et al., 1997). At

this time, published reports are very preliminary and further studies will be needed to determine if this

phenomenon is observed in natural waters and in other laboratories.

Monochloramine

Ever since the passage of the Surface Water Treatment Rule, monochloramine has not been used very frequently

as a primary disinfectant in drinking water treatment. Like chlorine, monochloramine is not capable of

achieving more than a 1 log inactivation of Cryptosporidium at CT values commonly encountered in treatment

practice (Korich et al., 1990; Finch et al., 1997; Gyurek et al., 1997). Also, preliminary studies have shown that

the ability of monochloramine to inactivate Cryptosporidium may be enhanced by preozonation or by
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108

prechlorination (Finch et al., 1997; Gyurek et al., 1997). Again, published reports are very preliminary and

further studies will be needed to determine if this phenomenon is observed in natural waters and in other

laboratories.

Mixed oxidants

A proprietary system that electrochemically produces a mixed oxidant solution was recently evaluated for its

ability to inactivate Cryptosporidium oocysts (Venczel et al., 1997). This system was observed to achieve more

than a 3 log inactivation of Cryptosporidium oocysts with a 5 mg/L oxidant dose and a 4-hour contact time.

UV irradiation

Recent studies have demonstrated that some ultraviolet irradiation technologies may be promising for

Cryptosporidium inactivation (Campbell et al., 1995; Arrowood et al., 1996). Because the technologies

employed in these two studies are not comparable, their results cannot be compared. In one case, ultraviolet

irradiation produced a 2 to 3 log inactivation of Cryptosporidium oocysts at ultraviolet doses and contact times

achievable by commercial equipment (Campbell et al., 1995). In the other study, up to 6 log inactivation was

observed with an alternative piece of commercial equipment (Arrowood et al., 1996).

D. Summary
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109

Analysis of water samples
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110

USEPA Method 1622 uses a capsule filter to collect Cryptosporidium oocysts from water samples. Other

collection methods include the use of filters of varying compositions (e.g., wound yarn, cellulose acetate). 

Capsule filters and cellulose acetate membrane filters appear to have better performance than wound yarn filters. 

Calcium carbonate flocculation methods, which can concentrate up to 10 L of water, have also been shown to be

superior to wound yarn filters but may interfere with viability determinations. Centrifugation-based

concentration technologies such as vortex flow filtration, cross-flow filtration, and continuous centrifugation

could potentially recover greater numbers of oocysts than the currently used ASTM ICR methods; however, the

methods still require interlaboratory validation. Immunomagnetic capture and flow cytometry also show

considerable recovery increases using either seeded or environmental samples. Immunomagnetic capture is the

currently recommended method for recovering oocysts from water samples, as described in Method 1622. 

Laser scanning devices have also performed well in early studies, but more research is required. Several

applications of PCR for the detection of Cryptosporidium have been described in the literature, some of which

may be able to distinguish viable from nonviable oocysts; however, enzymatic inhibition in PCR assays remains

problematic.

Since the determination of Cryptosporidium viability is critical in assessing the public health threat of

cryptosporidiosis, a number of viability assays have been described and compared to animal infectivity models. 

Some viability assays have produced conservative estimates of oocyst viability compared to animal modeling

data; however, limitations in viability assays have precluded their routine use in environmental samples. The

USEPA has established an approval process for laboratories performing detection of Cryptosporidium and

Giardia in water.
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Analysis of biological samples

The 1994 Cryptosporidium Criteria Document described the increased sensitivity of IFA-based procedures. 

Traditional staining methods such as the Ziehl-Neelsen stain, however, are still widely used. EIA methods are

fast, inexpensive, easily performed, and show sensitivity approaching that of IFA methods. However, a lack of

confirmatory analyses may preclude the routine use of EIA methods. Enzyme immunoassays may be useful for

busy hospital laboratories or large-scale screening surveys. Several PCR-based methods capable of

distinguishing differences among specific strains have been described in the literature. As with testing the

efficacy of different water analysis methods, interlaboratory comparisons require strict adherence to oocyst

quality and rigorous enumeration procedures. Recommendations by Klonicki et al. (1997) should be observed

in future studies.

Summary of removal studies

Of the technologies available to the drinking water industry, membrane processes appear to provide the most

significant levels of Cryptosporidium removal. However, full-scale testing of membrane processes has not yet

been conducted. Conventional treatment practices appear capable of meeting at least 2 log removal in most of

the cases studied to date. Although direct filtration and in-line filtration appear to be less effective than

conventional treatment, this has not been demonstrated in a conclusive manner for full-scale treatment systems. 

In bench- and pilot-scale studies, alternative technologies like diatomaceous earth filtration and slow sand

filtration appear capable of achieving comparable, if not better, levels of Cryptosporidium removal than

conventional treatment.

Summary of inactivation studies
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Ozone appears to be the best chemical disinfectant for Cryptosporidium inactivation at this time. The mixed

oxidant and ultraviolet light systems appear to be promising but have only been tested in minimal fashion

compared with ozone. Also holding some promise are the sequential disinfection systems of ozone followed by

the combination of chlorine and ozone, followed by monochloramine. Very few studies have evaluated

Cryptosporidium inactivation in natural waters.

VIII. Research Requirements

Frey et al. (1998) evaluated the current state of Cryptosporidium research, determined the gaps in the data, and

assessed future research needs. This section presents some of the existing needs for research.

Many of the data gaps in our knowledge regarding Cryptosporidium previously identified in the 1994

Cryptosporidium Criteria Document have been filled, and an enormous amount of information has become

available from research conducted in association with the Information Collection Rule. Data gaps that persist in

the areas of source water occurrence, health effects, risk assessment, analysis, and treatment are described

below.

Source Water Occurrence: The source and occurrence of Cryptosporidium in watersheds has been

characterized, although continued improvements in monitoring methods and analytical techniques would

increase our understanding of these issues. Research to discover specific contamination sources also would

contribute to public health protection.
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113

Health Effects: Progress has been made in identifying compounds that can be used for human and animal

therapy/treatment, although evaluation, validation, and clinical trials will be required, and these drugs will be

subject to FDA approval after such research and clinical trials are completed. However, studies to develop new

drugs should be continued. Information about the mechanism of pathogenicity might explain strain differences

in the production of diarrhea. There has been very little progress in elucidating the pathogenic mechanisms

involved in cryptosporidiosis, but USEPA-sponsored human infectivity studies should provide useful

information. 

Risk Assessment: More information is needed to better identify and characterize outbreaks, to assess the risks

to susceptible populations, and to determine the infectious dose and virulence of Cryptosporidium across

different populations. In addition, better diagnostic serological methods need to be developed, validated, and

more serology-based epidemiology studies need to be completed. Risk assessment also would be improved by

calibration of risk assessment models to make them more precise, such as the work done by Nahrstedt and

Gimbel (1996) and Teunis and Havelaar (1999) described in Section VI.

Analysis: Research efforts for recovery of Cryptosporidium oocysts from water samples as well as from

clinical samples have been improved and many of the steps in these processes that historically have been

responsible for oocyst loss have been identified. Studies comparing methods have been conducted, and the

advantages and disadvantages of various approaches have been elucidated. New detection methods are being

developed, especially those using molecular biology approaches (e.g., PCR/gene probe procedures), laser-based

technologies, and computer-assisted microscopy. Using these approaches, methods for determination of oocyst

survivability in the environment and infectivity should improve significantly. Detection methods continue to be
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114

quite variable and the need for a standard method that is accurate, precise, quick, and affordable still exists. 

Many of the newer technologies are yet unproven with real-world samples, and validation testing must be

completed. The analysis of large sample volumes still presents a challenge for detection of Cryptosporidium

using routine collection methods. In addition, not enough is known about the basic cell biology of

Cryptosporidium. Greater knowledge in this area will not only help in the development of an accurate detection

method, but it will also advance the improvement of viability, infectivity, and speciation assays for

environmental Cryptosporidium. Finally, researchers are still faced with the challenge of overcoming

interferences posed by environmental samples for molecular-based techniques.

Treatment: There is a great need to develop, identify, and evaluate new methods for disinfection and removal

of Cryptosporidium (e.g., ozonation, UV, improved filtration). In addition, due to concerns associated with

chlorination byproducts, compounds other than chlorine should be sought as residual disinfectants in finished

drinking water supplies. Complete evaluation of treatment for oocyst removal is dependent on better detection

methods and more rigorous enumeration practices. Other gaps in the data regarding treatment of drinking water

include the usefulness and efficiency of surrogates to determine success of treatment, the impact of the

treatment process on oocyst viability and survival at the molecular level, and guidelines or a decision matrix to

assist in treatment selection.

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