United States
Environmental Protection
Agency
Office of Science and Technology
Off ice of Water
Washington, DC 20460
EPA-822-K-94-001
March 2001
www.epa.gov
&EPA
Cryptosporidium:
Human Health
Criteria Document
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
ACKNOWLEDGMENTS
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
OshiroofOST.
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).
-i-
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
EXECUTIVE 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 fora 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 forthis organism in wateris
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.
-11-
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
TABLE 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
-iii-
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
A. 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
VTI. Analysis and Treatment 81
A. Analysis of Water 81
1. 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 Ill
1. Introduction Ill
2. Multibarrier Treatment 112
3. Removal of Cryptosporidium 114
4. Inactivation of Cryptosporidium 120
D. Summary 124
VIII. Research Requirements 127
IX. References 130
-IV-
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (DBF) 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. (Payer et
a/., 1997a). However, the diagnosis of cryptosporidiosis in humans in 1976 and the subsequent connection of
1
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer, 1997). At
the time of this writing, itis 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 H-l, 1994
Cryptosporidium Criteria Document). By 1997, however, interspecies transmission studies, morphological
evaluations and immunological analyses had reduced this number to eight valid species (Payer 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
schneideri), and desert monitors in Australia (Koudela and Modry, 1998). Cryptosporidium andersoni was
recovered from thefeces 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 (Payer, 1997; Payer 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; Xiaoetal., 1999a; Xiaoetal., 1999b).
Table 1. Valid Cryptosporidium Species
CryptosporMum Species
C. andersoni
C. baileyi
C.felis
C. meleagridis
C. muris
C. nasorum
C. parvum
C. saurophilum
C. serpentis
C. wrairi
Initially Described Host Species
Bos taurus (cattle)
Gallus gallus (domestic chicken)
Felis catis (domestic cat)
Meleagris gallopavo (turkey)
Mus musculus (house mouse)
Naso liter atus (fish)
Mus musculus (house mouse)
Eumeces schneideri (skink)
Elaphe guttata (corn snake)
E. subocularis (rat snake)
Sanzinia madagasarensus (Madagascar boa)
Cavia porcellus (guinea pig)
Source: Adapted from Payer et al. (2000) and Payer 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. Payer et al
(2000) documented Cryptosporidium infection in more than 150 mammalian species. Illness in humans,
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer, 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; Spanoetal.,
1998a; Spanoetal., 1998b; Sulaimanet al., 1998; Xiaoetal., 1999a; Xiaoetal., 1999b).
Several studies have suggested the possibility of distinct transmission cycles among different genotypes (Awad-
El-Kariem, 1999; Awad-El-Kariem et al, 1998; Morgan et al., 1998a; Patelef or/., 1998; Pengetal., 1997;
Sulaiman et al, 1998; Widmer et al, 1998c). Alternatively, multiple genotypes maybe 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, isoenzymeelectrophoresis, and
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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-El-
Kariem <^ a/., 1994). Webster (1993) applied a battery of molecular taxonomic methods (flow cytornetry, PCR,
and RFLP) to detect and classify Cryptosporidium oocysts from geographically diverse isolates. The isolates
exhibited genetic homogeneity for the most part, although differences inisoelectric points and restriction maps
indicated genetic differences among C. parvum isolates from humans andbovines.
Isoenzyme electrophoresis studies (O'Donoghue, 1995; Awad-El-Kariem, 1995; Awad-El-Kariem etal., 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. Follow-
up studies using AP-PCR and isoenzyme typing (Carraway et a/., 1994; Awad-El-Kariem et a/., 1996; Awad-
El-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
Analysis of genetic polymorphisms among C. parvum isolates from nine human outbreaks and from several
bovine sources (Peng etal, 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 etal, 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 etal, 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
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 er al, 1999b; Morgan^ al, 1999c; Morgan er al, 1999e; Morgan er al,
1
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 ef or/., 1999e; Morgan ef or/., 1998a; Xiaoetal., 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 ef or/., 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
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 etal., 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
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).
10
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/.,
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 spreadbetween 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 HIV-
positive 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
11
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
12
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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).
Often 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 HIV-
infected 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
13
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer et a/., 1997b) or by intubation (Graczyk et a/., 1996c; Graczyk et a/.,
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 a/., 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.
14
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
House flies (Musca domestica) exposed to bovine feces containing C. parvum oocysts transported oocysts to
other surfaces via fecal deposition (Graczyk et a/., 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-month-
old male child in which Cryptosporidium oocysts were detected in the digestive tract of cockroaches
(Periplaneta americand) 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
15
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 serpentisis
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 (Payer, 1997).
16
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Cryptosporidium infections have been detected in nine species offish. Infections have been
observed in both freshwater and marine species, and in cultured, captive, ornamental, and wild-
caught 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 etal, 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) andLimnodynastes 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). Payer 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 (Payer, 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 (Payer, 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.
17
-------
Drinking Water Criteria Document Addendum: Cryptosporidium
March 2001
Table 2. Geographic Distribution of Human Cryptosporidiosis
North America
Canada
Mexico
United States
Pacific
Australia
Malaysia
New Zealand
Papua-New
Guinea
Philippines
Singapore
Middle East
Egypt
Iran
Israel
Kuwait
Saudi Arabia
Caribbean
Cuba
Haiti
Jamaica
Puerto Rico
St. Lucia
Tobago
Trinidad
Virgin Islands
Asia
Bangladesh
Belarus
Cambodia
China
India
Japan
Korea
Myanmar
Republic
Pakistan
Russia
Sri Lanka
Taiwan
Thailand
Central/ South
America
Argentina
Brazil
Colombia
Chile
Costa Rica
Ecuador
El Salvador
Guatemala
Panama
Peru
Uruguay
Venezuela
Africa
Algeria
Burundi
Cameroon
Ethiopia
Gabon
Ghana
Guinea
Guinea-Bissau
Ivory Coast
Kenya
Liberia
Mauritania
Mauritius
Morocco
Nigeria
Rwanda
South Africa
Sudan
Togo
Tunisia
Uganda
Zaire
Zambia
Zimbabwe
Europe
Austria
Belgium
Czechoslovakia
Denmark
England
Finland
France
Germany
Greece
Hungary
Ireland
Italy
Lithuania
Netherlands
Poland
Portugal
Romania
Serbia
Spain
Sweden
Switzerland
Turkey
Wales
18
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
19
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
20
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
21
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Quinnetal., 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 a/., 1997). These authors speculated
that the decreased pH of the carbonated beverages triggers premature excy station.
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 1991/1992MMWR 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.
22
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 1998), indicating that
cryptosporidiosis was occurring in Milwaukee for more than a year before the outbreak. Hsenberg 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
23
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
24
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 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.
25
-------
Drinking Water Criteria Document Addendum: Cryptosporidium
March 2001
Table 3. Outbreaks of Cryptosporidiosis Associated with Drinking Water in the U.S.
Year
1984
1986
1987
1991
1992
1993
1993
1993
1993
1994
1995
State
Texas
New Mexico
Georgia
Pennsylvania
Oregon
Wisconsin
Washington
Minnesota
Nevada
Washington
Florida
Number of
Cases
2006
78
12,960
551
15,000
403,000
7
27
103
104
72
Source
Groundwater (C)
Surface water (C)
River (C)
Groundwater (C)
Spring/river (C)
Lake (C)
Well (I)
Lake (NC)
Lake (C)
Well (C)
Not applicable
Deficiency
Sewage contamination
Untreated
Treatment deficiency
Treatment deficiency
Treatment deficiency
Treatment deficiency
Surface contamination
Unknown
Inadequate filtration
Sewage contamination
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 (Herwaldtera/., 1991). IntheU.S. surveillance report
from 1991 to 1992 (Moore et a/., 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.
26
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 pool-
related 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
27
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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. a/., 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 areview 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
28
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
(Quinn et a/., 1998; Rose and Slifko, 1999). The Minnesota Department of Health reported cryptosporidiosis in
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. Casern ore 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).
29
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 maybe 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-infectedpeople (Goldstein et al, 1996; Roefer et a/.,
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
30
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 thatC. 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.
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Cryopreservation studies conducted by Payer et al. (1991) and Payer (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, Payer 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) (Payer 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). Payer 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 (Payer et al., 1998b). As water
temperatures increased above 20°C, oocysts retained their infectivity for shorter exposure times (Payer et al,
1998b).
32
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Under conditions of high water temperatures, Payer (1994) indicated that all evidence of C. parvum infectivity
was lost within 60 seconds when temperatures exceeded 72°C or when temperatures of at least 64°C 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
33
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 offish, 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
34
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer, 1997; O'Donoghue, 1995).
In general, the development of cryptosporidiosis depends on the species, age, and immune status of the host
(Payer, 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.
35
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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). Payer (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 (Payer, 1997; O'Donoghue,
1995). Normally, only one condition manifests during an outbreak, and respiratory infections are more common
than enteric or renal infections (Payer, 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 (Payer, 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).
36
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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; Payer, 1997). In these cases,
the kidneys were pale in color (Payer, 1997) and enlarged (O'Donoghue, 1995). Hypertrophic and hyperplastic
epithelial cells were detected throughout the kidney (O'Donoghue, 1995; Payer, 1997). Parasites were observed
in the collecting ducts and convoluted tubules (O'Donoghue, 1995).
Reptiles: C. serpentisis the only valid, named species of Cryptosporidium causing infection in reptiles,
although up to five species may exist based on oocyst morphology (Payer, 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 (Payer, 1997). Cryptosporidiosis in snakes is characterized by
anorexia, regurgitation, lethargy, firm midbody swelling, weight loss, and death (Payer, 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).
37
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 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.
38
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (nentreated) 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 antidiarrhea! drugs (Blagburn and Soave, 1997). In addition, chemotherapy
with anticryptosporidial drugs may be initiated.
C. Epidemiological Data
39
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
The majority of epidemiologjcal 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;
Payer, 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 etal., 1997; Payer, 1997;
O'Donoghue, 1995). Serologic surveys in animals suggest much higher prevalence rates of cryptosporidiosis,
especially in adults (Casemore etal., 1997; Payer, 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 (Payer, 1997).
Additional epidemiological studies reported in the literature, but not mentioned in the review articles, describe
the prevalence of cryptosporidiosis in the following animals:
40
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
cattle/calves (Payer etal, 1998a; Olson ef or/., 1997; Perez et al, 1998; Pena etal, 1997)
horses (Scholes etal, 1998; Bray etal., 1998; Forde etal., 1998; Johnson etal., 1997)
lambs (Bukhari and Smith, 1997)
goats (Koudela and Jiri, 1997; Goyena etal, 1997)
nonhuman primates (Muriukie^al., 1997; Majewska etal., 1997)
rodents (Bajer et al., 1997; Bull et al., 1998)
chickens (Sreter et al., 1996)
ostriches (Jardine and Verwoerd, 1997)
pigeons (Rodriguez etal., 1997)
catfish (Muench and White, 1997)
muskrat (Petri et al., 1997),
African hedgehog (Graczyk et al., 1998a)
dugong (Hill etal, 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
41
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
42
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 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
43
-------
Drinking Water Criteria Document Addendum: Cryptosporidium
March 2001
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
30
100
300
500
lOOOf
Total
No.
Subjects
5
8
3
6
7
29
No. (%)
Infected
1 (20)
3 (37.5)
2 (66.7)
5 (83.3)
7 (100)
18
No. (%) with
Enteric
Symptoms
0
3 (37.5)
0
3 (50)
5 (71.4)
11
No. (%) with
Cryptosporidiosis
0
3 (37.5)
0
2 (33.3)
2 (28.6)
7
* Linear regression analysis of the data yielded an r2 of 0.983 and an ID50 of 132 oocysts.
f The intended dose was 1000 oocysts in two subjects, 10,000 in three, 100,000 in one, and
1 million in one.
Source: DuPont etal. (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 a/., 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.
44
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Juraneket a/., 1995; Addiss et a/., 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 etal, 1997)
• Monitoring logs maintained by health maintenance organizations (HMOs) and hospitals for
complaints of diarrheal illness
45
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
• Monitoring the incidence of diarrhea in nursing homes (also described by Proctor et a/., 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
46
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 1995). This antibody profile suggests that the
infection was of prolonged duration.
47
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
The cryptosporidiosis outbreak in Las Vegas, Nevada (Roefer et a/., 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 ADDS
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 a/., 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 ADDS 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
48
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
be expected to miss a substantial number of Cryptosporidium infections in immunocompromisedand 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
49
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 hadoocysts 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 foroocysts (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
50
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 cryptosporidiosisin 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 co-
infected 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 a/., 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
51
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
52
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Vargases a/., 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 (Vargasesa/., 1993); however, in apilot-scale clinical trial with azithromycin (500mg 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.
53
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 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 a/., 1997). A pilot-scale dinical trial of letrazuril treatment for ADDS
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 a/.,
1997).
lonophores with anticocddial properties have also been evaluated for treatment of cryptosporidiosis in animals
(Mead et a/., 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).
54
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 protedytic activity
is involved in the infectivity of C. parvum, based on the interaction between human alpha-1-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 1hat Cryptosporidium-mduced 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-mduced diarrhea maybe caused by a toxin (Guarino et al., 1994;
55
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
56
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 HIV-
Cryptosporidium-infected patients. These patients displayed marked elevation in anti-Cryptosporidium IgA and
IgM antibody liters. These high antibody liters 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 a/., 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.
57
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer et a/., 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 maybe treated with macrolides and antimotility agents. A previous Cryptosporidium
58
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 on Cryptosporidium
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
59
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
health officials to communicate with water utilities, interpret water quality surveys, and define the adequacy of
treatment at acceptable public health risks (Rose et a/., 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 a/., 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.
60
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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-poshive stool
samples when only one stool is examined.
Okhuysen etal. (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.
61
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
62
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
63
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
64
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 outbreak-
and 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 (Molbake^a/., 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 (Athertone^a/., 1995). Other groups
at risk for cryptosporidiosis are secondary contacts, farm workers (Lengerich et a/., 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 a/., 1996).
65
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 dose-
response data are required. The human dose-response data currently available are limited to the studies of
DuPontetal. (1995), Chappelletal. (1996), and Okhuysenega/., (1999); however, an exponential dose-
response 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 (P:) as follows:
Equationl. P:=l-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 P: ranged from 0.14 to 0.52.
Exposure values predicted according to this model ranged from a minimum of 0.16 oocysts per liter (P:=0.14) to
a maximum value of 79 (P:=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
66
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 waterbome
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:
\ = 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
67
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
68
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 -
0.0064/1 OOL).
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
69
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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/i cr_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
70
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
71
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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,
72
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 collectit. 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.
73
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
1. 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
74
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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., Envirocheck™ capsules); these filters
contain a pleated polysulfone membrane with a 1- m porosity. Envirocheck™ is a 6-cm-diameterby 21-cm-
long 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 Envirocheck™ 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).
75
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (DIG) 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,
76
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
77
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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-by-
particle basis. It can differentiate and physically separate (sort) particles based on their size, internal
complexity, and fluorescence. Flow cytometry wilh 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-poshive samples than membrane immunofluorescence assay (IFA)
(94.1% vs. 35.3%, respectively) and an equal number of Giardia-positive samples. This technique has been
78
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 anti-
Cryptosporidium 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 bead-
Cryptosporidium 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%, whilethe 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.
79
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
80
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
a/., 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 a/.,
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 (DIG) microscopy (USEPA, 1999b). Well slide staining has been
evaluated as an alternative to membrane IFA staining (Frederickson et a/., 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.
81
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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; Gracyzke^ 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 T™ 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
82
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 aPCR 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,
83
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 transcript!on-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 half-
life 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.
84
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 Iog10
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 a/., 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 peroxidase-
streptavidin conjugate is used for color development. The results of the strand displacement assay correlate well
with evaluation of acid-fast slides of fecal samples.
85
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 F ACS and IMS methods in
samples with colloidal clay (Reynolds etal., 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 a/., 1995), and the inability to perform any light microscopy techniques for visualization of internal
cytoplasm or sporozoites.
Miscellaneous methods
Campbell etal. (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 abiotin-conjugated anti-FITC
antibody and streptavidin-peroxidase. Statistical analysis revealed no difference in results obtained with the
86
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 to400x 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.
87
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
With Method 1622, 10-L sampleis 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
VILA. 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 perL. 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
88
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 Blacker 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
89
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
assessment; however, extraneous factors such as aldehyde fixation may inhibit uptake of PI and falsely elevate
the numbers of viable oocysts (Campbell etal., 1993c).
Various infectivity assays have been described using tissue culture methods to assess the infectivity of C.
parvum (Upton etal, 1994a; Upton ef or/., 1994b; Upton ef or/., 1995; Rochelle etal, 1997a; Slifkoef or/., 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
a/., 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 DIG 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.
90
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Sixteen commercial laboratories were enlisted in a survey (Clancy et a/., 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 thefeces 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
91
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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. Resales 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.
92
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 (Payer, 1997). Kang and Mathan (1996)
compared five staining methods for detection of Cryptosporidium oocysts in fecal smears. The safranin-
methylene 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 Ziehl-
Neelsen 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
93
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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), Grigoriewer 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)
94
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
Mael 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 species-
specific. 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
95
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 DNAfrom 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).
96
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Flow cytometry methods have been described for detection of Cryptosporidium in mice (Arrowood et a/.,
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. Youetal. (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:
97
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 lull-scale water treatment
processes is found in Table 5.
2. Multibairier 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.
98
-------
Drinking Water Criteria Document Addendum: Cryptosporidium
March 2001
Table 5. Cryptosporidium Removal Efficiencies for Selected
Physical and Chemical Processes
Treatment Process Description
Coagulation + Gravity Settling
Coagulation + Filtration
Coagulation + Gravity Settling +
Filtration
Coagulation + Dissolved Air
Flotation
Slow Sand Filtration
Diatomaceous Earth Filtration
Coagulation + Microfiltration
Ultrafiltration
Removal Achieved (log)
Bench Scale
< 1.0a
2.0-2.6a
Pilot Scale
1.4- 1.8b
2.7 - 5.9b
2.5- 3. 8h
2.7-2.91*
4.2- 5. 2b
>5.3f
2.1 -2.81*
>3.7C
>4.0C
>6.0d
>6.0d
Full Scale
0.4- 1.7s
1.6-4.06
1.6-4.06
<0.5-3.0f
1.0-2.5
* 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;8 Kelley et al, 1994; h
Anderson et al, 1996; and' 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.
99
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 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
100
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Cryptosporidium oocysts (Plummer etal., 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 a/., 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.
101
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.I, any watershed, river, or
reservoir is subject to a complex set of watershed characteristics and watershed processes (Crockett and Haas,
1997;LeChevallierera/., 1997; States ef a/., 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
102
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
103
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 etal., 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 etal., 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 etal., 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
104
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
specifications for slow sand filters (Fogel et a/., 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 a/., 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, demand-
free 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
105
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 a/., 1990; Finch et a/., 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 (Finchetal., 1993). CT values fora 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 a/., 1989; Korich et a/., 1990; Parker et
al., 1993; Owensetal., 1994aandb; Qumnetal., 1996; Finch etal., 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 a/., 1990; Finch et a/., 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
106
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
would be necessary. At pH 8, the required CT values appear to be greater than this. Recent results have shown
that chlorine dioxide maybe more effective if ozone is applied upstream of the chlorine dioxide addition point
(Liyanage et a/., 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 (KorichetaL, 1990; Payer, 1995; Pinched a/., 1997; GyureketaL,
1997; Venczel et a/., 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 a/., 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 a/., 1990; Finch et a/., 1997; Gyurek et a/., 1997). Also, preliminary studies have shown that
the ability of monochloramine to inactivate Cryptosporidium may be enhanced by preozonation or by
107
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
prechlorination (Finchetal., 1997; Gyureketal., 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 a/., 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 a/., 1995; Arrowood et a/., 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 a/., 1995). In the other study, up to 6 log inactivation was
observed with an alternative piece of commercial equipment (Arrowood etal., 1996).
D. Summary
108
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Analysis of water samples
109
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
USEPA Method 1622 uses a capsule filter to collect Cryptosporidium oocysts from water samples. Other
collection methods includethe use of filters ofvarying 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.
110
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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 IF A 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
111
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
112
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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
113
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
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.
IX. References
Aarnaes, S.L., Blanding, J., Speier, S., Formal, D., de la Maza, L.M., and Peterson, E.M. 1994. Comparison of
the ProSpectT and Color Vue enzyme-linked immunoassays for the detection of Cryptosporidium in stool
specimens. Diag. Microbiol. Infect. Dis., 19:221-225.
Adam, A.A., Hassan, H.S., Shears, P., and Elshibly, E. 1994. Cryptosporidium in Khartoum, Sudan. J. E.
African Med., 71:11:745-746.
114
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Addiss, D.G., Arrowood, M.J., Bartlett, M.E., Colley,D.G., Juranek, D.D. and Kaplan, I.E. 1995. Assessing the
public health threat associated with waterbome cryptosporidiosis: report of a workshop. MMWR, 44:RR-6.
Addiss, D.G., Pond, R.S., Remshak, M., Juranek, D.D., Stokes, S., and Davis, J.P. 1996. Reduction of risk of
watery diarrhea with point-of-use water filters during a massive outbreak of waterborne Cryptosporidium
infection in Milwaukee, Wisconsin, 1993. Am. J. Clin. Microbiol., 54:6:549-553.
Adegbola, R., Demba, E., De Verr, G., and Todd, J. 1994. Cryptosporidium infection in Gambian children less
than 5 years of age. J. Trop. Med. Hyg., 97:103-107.
Aldom, I.E., and Chagla, A.H. 1995. Recovery of Cryptosporidium oocysts from water by a membrane filter
dissolution method. Lett. Appl. Microbiol., 20:186-187.
Alles, A.J., Waldron, M.A., Sierra, L.S., and Mattia, A.R. 1995. Prospective comparison of direct
immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in
human fecal specimens. J. Clin. Microbiol., 33:6:1632-1634.
Anderson, B.C. 1985. Moist heat inactivation of Cryptosporidium sp. Am. J. Public Health, 75:12:1433-1434.
Anderson, B.C. 1986. Effect of drying on the infectivity of cryptosporidia-laden calf feces for 3- to 7-day-old
mice. Am. J. Vet. Res., 47:10:2272-2273.
Anderson, W.L., Champlin, T.L., Clunie, W.F., Hendricks, D.W., Klein, D.A., Kregrensin, P., and Sturbaum, G.
1996. Biological particle surrogates for filtration performance evaluation. AWWA ACE Proc., Toronto,
Ontario, [as cited in Frey et al. (1998)]
Anguish, L., and Ghiorse, W. 1997. Computer-assisted laser scanning and video microsccopy for analysis of
Cryptosporidiumparvum oocysts in soil, sediment and feces. Appl. Environ. Microbiol., 63:724-733.
Anusz, K.Z., Mason, P.H., Riggs, M.W., and Ferryman, L.E. 1990. Detection of Cryptosporidium parvum
oocysts in bovine feces by monoclonal antibiody capture enzyme-linked immunosorbent assay. Anal. Clin.
Microbiol., 28:2:2770-2774.
Argenzio, R.A., Leece J., and Powell D.W. 1993. Prostanoids inhibit intestinal NaCl absorption in experimental
porcine cryptosporidiosis. Gastroenterol., 104:440^47.
Arrowood, MJ. 1997. Diagnosis. In: Cryptosporidium and Cryptosporidiosis, Payer R (ed), CRC Press, New
York.
Arrowood, M.J., Kurd, M.R., and Mead, J.R. 1995. A new method for evaluating experimental cryptosporidial
parasite loads using immunofluorescent flow cytometry. J. Parasitol., 81:404-409.
Arrowood, M.J., Xie, L.T., Rieger, K., and Dunn, J. 1996. Disinfection of Cryptosporidium parvum oocysts by
pulsed light treatment evaluated in an in vitro cultivation model. J. Eukaryot. Microbiol., 43:5:888.
115
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Atherton, F., Newman, C., and Casemore, D.P. 1995. An outbreak of water-borne cryptosporidiosi s associated
with a public water supply in the UK. Epidemiol. Infect., 115:123-131.
Awad-El-Kariem, F. 1996. Significant parity of different phenotypic andgenotypic markers between human and
animal strains of Cryptosporidiumparvum. J. Eukaryot. Microbiol., 43:5:708.
Awad-El-Kariem, F.M. 1999. Does Cryptosporidium parvum have a clonal population structure? Parasitol.
Today, 15:12:502-504.
Awad-El-Kariem, F.M., Robinson, H.A., Dyson, D.A., Evans, D., Wright, S., Fox, M.T., and McDonald, V.
1995. Differentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing.
Parasitol., 110:129-132.
Awad-El-Kariem, F.M., Robinson, H.A., Petry, F., McDonald, V., Evans, D., and Casemore, D. 1998.
Differentiation between human and animal isolates of Cryptosporidium parvum using molecular and biological
markers. Parasitol. Res., 84:4:297-301.
Awad-El-Kariem, F., Warhurst, D., and McDonald, V. 1994. Detection and species identification of
Cryptosporidium oocysts using a system based on PCR and endonuclease restriction. Parasitol., 109:19-22.
Badenoch, J..,etal. 1990. Cryptosporidium in water supplies. Report of the group of experts. Copyright
controller of HMSO. London, U.K.
Bajer, A., Bednarska, M., and Sinski, E. 1997. Wildlife rodents from different habitats for Cryptosporidium
parvum. Acta Parasitol., 42:4:192-194.
Balatbat, A., Jordan, G., Tang, Y., and Silva, J. 1996. Detection of Cryptosporidium parvum DNA in human
fecesby nested PCR. J. Clin. Microbiol., 34:7:1769-1772.
Baron, E., Schenone, C., and Tanenbaum, B. 1989. Comparison of three methods for detection of
Cryptosporidium oocysts in a low-prevalence population. J. Clin. Microbiol., 27:1:223-224.
Belosevic M,, Guy R.A., Taghi-Kilani R., Neumann N.F., Gyurek L.L., Liyanage R.J., Millard P.J., and Finch
G.R. 1997. Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability. Int. J. Parasitol.,
27:7:787-798.
Bifulco, J.M. and Schaeffer, F.W. 1993. Antibody-magnetite method for selective concentration ofGiardia
lamblia cysts from water samples. Appl. Environ. Microbiol., 59:3:772-776.
Bissuel, F., Cotte, L., Rabododonirina, M. Rougier, P., Piens, M.-A., and Trepo, C. 1994. Paromomycin: an
effective treatmentfor cryptosporidial diarrhea in patients with AIDS. Clin. Infect. Dis., 18:447-449.
Black, E.K., Finch, G.R., Taghi-Kilani, R., and Belosevic, M. 1996. Comparison of assays for Cryptosporidium
parvum oocysts viability after chemical disinfection. FEMS Microbiol. Letters, 135:187-189.
116
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Blagburn, B.L. and Soave, R. 1997. Prophylaxis and chemotherapy: human and animal. In: Cryptosporidium
and Cryptosporidiosis., Payer R (ed), CRC Press, New York.
Blanshard, C., Shanson, D.C., and Gazzard, E.G. 1997. Pilot studies of azithromycin, letrazuril, and
paromomycin in the treatment of Cryptosporidiosis. Int. J. STD AIDS, 8:124-129.
Blassak, M., Wick, J., and Mueller, R. 1996. Detection of Cryptosporidium DNA in fecal samples. Clin. Chem.,
42:11:189 l(Abstr. 15).
Borchardt M.A. and Spencer S.K. 1996. Recovery of Cryptosporidium and Giardia from environmental
samples using a blood cell separator. In 1997 AWWA WQTC Proceedings, Boston, MA.
Bornay-Llinares, F.J., da Silva, AJ., Moura, IN., Myjak, P., Pietkiewicz, H., Kruminis-Lozowska, W.,
Graczyk, T.K., and Pieniazek, NJ. 1999. Identification of Cryptosporidium feUs in a cow by morphologic and
molecular methods. Appl. Environ. Microbiol., 65:4:1455-1458.
Brandonisio, O., Marangi, A., Panaro, M.A., Marzio, R., Natalicchio, M.I., Zizzadoro, P., and De Santis, U.
1996. Prevalence of Cryptosporidium in children with enteritis in southern Italy. Eur. J. Epidemiol., 12:187-
190.
Brannan, D., Greenfield, R., Owen, W., Welch, D., and Kuhis, T. 1996. Protozoal colonization of the intestinal
tract in institutionalized Romanian children. Clin. Infect. Dis., 22:456-461.
Bray, R.E., Wickler, S.J, Cogger, E.A., Atwill, E.R, London, C., Gallinoa, J.L., and Anderson, T.P. 1998.
Endoparasite infection and Cryptosporidium/Giardia in feral horses on public lands. J. Equine Vet. Sci.,
18:1:41-43.
Bridgman, S. A., Robertson, R.MP., Syed, Q., Speed, N., Andrews, N., and Hunter, P.R. 1995. Outbreak of
Cryptosporidiosis associated with a disinfected groundwater supply. Epidemiol. Infect., 115:555-566.
Bukhari, Z. and Smith, H. 1995. Effect of three concentration techniques on viability of Cryptosporidium
parvum oocyts recovered from bovine feces. J. Clin. Microbiol., 33:10:2592-2595.
Bukhari, Z. and Smith, H.V. 1997. Cryptosporidium parvum-oocyst excretion and viability patterns in
experimentally infected lambs. Epidemiol. Infect, 119:1:105-108.
Bukhari, Z, Smith, H.V, Sykes, N., Humphreys, S.W., Paton, C.A., Girdwood, R.W.A., and Fricker, C.R.
1997. Occurrence of Cryptosporidium spp. oocysts and Giardia spp. cysts in sewage influents and effluents
from treatment plants inEngland. Wat. Sci. Tech, 35:385-390.
Bull, S.A., Chalmers, R.M., Sturdee, A.P., and Healing, T.D. 1998. A survey of Cryptosporidium species in
Skomer Bank voles (Clethrionomys glareolus skoherensis). J. Zool., 244:Part 1:119-122.
117
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Caccio, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T., and Pozio, E. 2000. A microsatellite marker
reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitol.,
120:Pt 3:237-244.
Campbell A.T., Gron, B., Johnsen S.E., and Dynal A.S. 1997. Immunomagnetic separation of Cryptosporidium
oocysts from high turbidity water sample concentrates. In: 1997 Int. Symp. Waterborne Cryptosporidium Proc.,
Flicker et al. (eds), AWWA, Newport Beach, CA.
Campbell, A., Robertson, L., and Smith, H. 1993a. Novel methodology for the detection of Cryptosporidium
parvum: a comparison of cooled charge couple devices (CCD) and flow cytometry. Wat. Sci. Tech., 27:3-4:89-
92.
Campbell, A.T., Robertson, L.J., and Smith, H.V. 1993b. Detection of oocysts of Cryptosporidium by enhanced
chemiluminescence. J. Microbiol. Meth., 17:297-303.
Campbell, A.T., Robertson, L.J., and Smith, H.V. 1993c. Effects of preservatives on viability of
Cryptosporidium parvum oocysts. Appl. Env. Microbiol., 59:12:4361-4362.
Campbell, A.T., Robertson, L.J., Snowball, M.R., and Smith, H.V. 1995. Inactivation of oocysts of
Cryptosporidium parvum by ultraviolet irradiation. Wat. Res., 29:11:2583-2586.
Campbell, A.T. and Smith, H.V. 1997. Immunomagnetic separation of Cryptosporidium oocysts from water
samples: round robin comparison of techniques. Wat. Sci. Tech., 35:11-12:397401.
Carraway, M., Widmer, G., and Tzipori, S. 1994. Genetic markers differentiate C. parvum isolates. J. Eukaryot.
Microbiol.,41:5:268-278.
Casemore D.P. 1987. The antibody response to Cryptosporidium: development of a serdogical test and its use
in a study of immunologically normal persons. J. Infect., 14:125-134.
Casemore, D.P. 1990. Epidemiological aspects of human cryptosporidiosis. Epidemiol. Infect., 104:1-28.
Casemore, D.P. and Roberts, C. 1993. Guidelines for screening for Cryptosporidium in stools: report of a joint
working group. J. Clin. Pathol., 46:2-4.
Casemore, D.P., Wright, S.E, and Coop, R.L. 1997. Cryptosporidiosis - human and animal epidemiology. In:
Cryptospoidium and Cryptosporidiosis., Payer R (ed), CRC Press, New York.
CDC. 1990. Swimming associated cryptosporidiosis — Los Angeles County. MMWR, 39:20:342-345.
CDC. 1994. Cryptosporidium infections associated with swimming pools - Dane County, Wisconsin. 1993.
JAMA, 272:12:914-915.
118
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
CDC. 1996a. Foodborne outbreak of diarrheal illness associated with Cryptosporidium parvum. MMWR,
45:36:783.
CDC. 1996b. Surveillance for Waterborne-Disease Outbreaks - United States, 1993-1994. MMWR, 45:SS-1:1-
33.
CDC. 1996c. Outbreak of cryptosporidiosis at a day camp - Florida, July-August 1995. JAMA, 275:23:1790.
Cevallos, A., Kelly, P., Ngwenya, B., Luo, N., Pobee, J., and Farthing, M. 1995. Antibody response to
Cryptosporidium parvum in patients with HIV and diarrhea. Gasterenterol., 108:4:A794.
Chapman, P. A. and Rush, B.A. 1990. Efficiency of sand filtration for removing Cryptosporidium oocysts from
water. J. Med. Microbiol., 32:243-245.
Chappell, C.L., Okhuysen, P.C., Sterling, C.R., andDuPont, HL. 1996. Cryptosporidium parvum: intensity of
infection and oocyst excretion patterns in healthy volunteers. J. Infect. Dis., 173:232-236.
Clancy, J.L., Golinitz, W.D., and Tabib, Z. 1994. Commercial labs: how accurate are they? J. AWWA, 86:5:89-
97.
Clancy J.L., Hargy T.M., and Schaub S. 1997. Improved sampling methods for the recovery of Giardia and
Cryptosporidium from source and treated water. In 1997 Int. Symp. Waterborne Cryptosporidium Proc., Fricker
etal. (eds), AWWA, Newport Beach, CA.
Clark, D.P. and Sears, C.L. 1996. The pathogenesis of cryptosporidiosis. Parasitol. Today, 12:6:221-225.
Clavel, A., Arnal, A., Sanchez, E., Cuesta, J.,Letona, S., Amiguet, J., Castillo, F., Varea, M., and Gomez-Lus,
R. 1996a. Respiratory cryptosporidiosis: case series and review of the literature. Infect., 24:5:341-346.
Clavel, A., Arnal, A., Sanchez, E., Varea, M., Quilez, J., Ramirez, I, and Castillo, F. 1996b. Comparison of 2
centrifugation procedures in the formalin-ether acetate stool concentration technique for the detection of
Cryptosporidium oocysts. Int. J. Parasitol., 26:6:671-672.
Clayton, F., Heller, T., and Kotler, D. 1994. Variation in the enteric distribution of Cryptosporidia in Acquired
Immunodeficiency Syndrome. Am. J. Clin. Pathol., 102:4:420425.
Compagnon, B., Robert, C., Mennecart, V., de Roubin, M.R., Cervantes, P., and Joret, J.C. 1997. Improved
detection of Giardia cysts and Cryptosporidium oocysts in water by flowcytometry. In 1997 WQTC
Proceedings, Denver,CO.
Connolly, G.M., Dryden, M.S., Shanson, D.C., and Gazzard, E.G. 1988. Cryptosporidial diarrhoea in AIDS
patients and its treatment. Gut, 29:593-597.
119
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Cordell, R.L. and Addiss, D.G. 1994. Cryptosporidiosis in child care settings: a review of the literature and
recommendations for prevention and control. Fed. Infect., 13:310-317.
Crabb, J.H. 1998 Antibody based immunotherapy of Cryptosporidiosis. Adv. Parasitol., 40:121-149.
Craun, G.F., Berger, P.S., and Calderon, R.L. 1997. Coliform bacteria and waterborne disease outbreaks. J.
AWWA., 89:3:96-104.
Crockett, C.S. and Haas, C.N. 1995. Understanding the behavior of Giardia and Cryptosporidium in an urban
watershed: explanation and application of techniques to collect and evaluate monitoring data. Proc. Wat. Qual.
Tech. Conf, New Orleans, pp. 1603-1624.
Crockett, C.S. and Haas, C.N. 1997. Understanding protozoa in your watershed. J. AWWA., 89:9:62-73.
Current, W. 1994. Cryptosporidiumparvum: household transmission. Ann. Int. Med., 120:6:518-519.
D'Antonio, R.G., Winn, R.E., Taylor, J.P., Gustafson, T.L., Current, W.L., Rhodes, M.M., Gary, G.W., and
Zajac, R.A. 1985. A waterborne outbreak of Cryptosporidiosis in normal hosts. Ann. Int. Med., 103: 886.
Dagan, R.,Fraser, D., El-On, J.,Kassis, I, Deckelbaum, R., and Turner, S. 1995. Evaluation of an enzyme
immunoassay for the detection of Cryptosporidium spp. in stool specimens from infants and young children in
field studies. Am. J. Trop.Med. Hyg., 52:2:134-138.
Danielson, R.E., Cooper, R.C., and Riggs, J.L. 1995. Giardia and Cryptosporidium analysis: a comparison of
microscopic and flow cytometric techniques. Proc. Wat. Qual. Tech. Conf, New Orleans, pp. 1673-1685.
de la Cruz, A. A. and Sivaganesan, M. 1994. Detection of Giardia and Cryptosporidium spp. in source water
samples by commercial enyme-immunoassay kits. Proc. 1994 Wat. Qual. Tech. Conf, Nov. 6-10, 1994. San
Francisco, CA.
Ditrich, O., Palkovic, L., Sterba, J., Prokopic, J.,Loudova, J., and Giboda, M. 1991. The first finding of
Cryptosporidium baileyi in man. Parasitol. Res., 77:44.
Drozd, C. and Schwartzbrod, J. 1997. Removal of Cryptosporidium from river water by crossflow
microfiltration: a pilot-scale study. Wat. Sci. Tech., 35:11-12:391-395.
Duke, L.A., Breathnach, A.S., Jenkins, D.R., Harkis, B.A., and Codd, A.W. 1996. A mixed outbreak of
Cryptosporidium and Campylobacter infection associated with a private water supply. Epidemiol. Infect.,
116:303-308.
DuPont, C., Bougnoux, M.E., Turner, L., Rouveix, E., andDorra, M. 1996. Microbiological findings about
pulmonary Cryptosporidiosis in two AIDS patients. J. Clin. Microbiol., 34:227-229.
120
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
DuPont, H., Chappell, C., Sterling, C., Okhuysen, P., Rose, 1, and Jakubowski, W. 1995. The infectivity of
Cryptosporidiumparvum in healthy volunteers. New Eng. J. Med., 332:855-859.
Eisenberg, J. N. S., Seto, Y.W., Colford, J.M., Olivieri, A., and Spear, R. 1998. An analysis of the Milwaukee
cryptosporidiosis outbreakbased on a dynamic model of the infection process. Epidemiol., 9:3:255.
Entrala, E., Rueda-Rubio, M., Janssen, D., and Mascaro, C. 1995. Influence of hydrogen peroxide on acid-fast
staining of Cryptosporidium parvum oocysts. Int. J. Parasitol., 25:12:1473-1477.
Farrington, M., Winters, S., Walker, C., Rubenstein, D., and Miller, R. 1994. Cryptosporidium antigen detection
in human feces by reverse passive hemagglutination assay. J. Clin. Microbid., 32:11:2755-2759.
Payer, R. 1994. Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Appl.
Environ. Microbiol., 60:8:2732-2735.
Payer, R. 1995. Effect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for
neonatal BALB/c mice. Appl. Environ. Microbiol., 61:2: 844-846.
Payer, R. (ed). 1997. Cryptosporidium and Cryptosporidiosis. CRC Press, New York.
Payer, R., Gasbarre, L., Pasquali, P., Canals, A., Almeria, S., and Zarlenga, D. 1998a. Cryptosporidium parvum
infection in bovine neonates-dynamic clinical, parasitic, and immunologic patterns. Int. J. Parasitol., 28:1:49-56.
Payer, R., Morgan, U., and Upton, SJ. 2000. Epidemiology of Cryptosporidium: transmission, detection, and
identification. Int. J. Parisitol. 30:1305-1322.
Payer, R. and Nerad, T. 1996. Effects of low temperatures on viability of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol., 62:4:1431-1433.
Payer, R., Nerad, T.,Rall, W., Lindsay, D.S., and Blagburn, B.L. 1991. Studies on the cryopreservation of
Cryptosporidium parvum. J. Parasitol., 77:357-361.
Payer, R., Speer, C.A., and Dubey, J.P. 1997a. The general biology of Cryptosporidium. In: Cryptosporidium
and Cryptosporidiosis, Payer R (ed), CRC Press, New York.
Payer, R., Trout, J.M., Gracyzk, T.K., Farley, C.A., and Lewis, EJ. 1997b. The potential role of oysters and
waterfowl in the complex epidemiology of Cryptosporidium parvum. In: 1997 Int. Symp. Waterborne
Cryptosporidium Proc., AWWA, Newport Beach, CA.
Payer, R., Trout, J.M., and Jenkins, M.C. 1998b. Infectivity of Cryptosporidium parvum oocysts stored in water
at environmental temperatures. J. Parasitol., 84:6:1165-1169.
Filkorn, R., Wiedenmann, A., and Botzenhart, K. 1994. Selective detection of viable Cryptosporidium oocysts
by PCR. Zbl. Hyg., 195:489-494.
121
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Finch, G.R., Black, E.K., Gyurek, L., and Belosevic, M. 1993. Ozone inactivation of Cryptosporidiumparvum
in demand-free phosphate buffer determined by in vitro excystation and animal infectivity. Appl. Environ.
Microbiol., 59:12:4203-4210.
Finch, G.R., Gyurek, L.L., Liyanage, L.R.J., and Belosevic, M. 1997. Effect of various disinfection methods on
the inactivation of Cryptosporidium. Final report. AWWARF, Denver, CO.
Finn, S., et. al. 1996. Letter to the editor. Apr. 15, Clin. Microbiol. Newslett.
Fitzgerald, S.D., Moisan, P.G., and Bennett, R. 1998. Aural polyp associated with cryptosporidiosis in an
iguana (Iguana iguana). J. Vet. Diagn. Invest., 10:2:179-180.
Flanigan, T. 1994. Human Immunodeficiency Virus infection and cryptosporidiosis: protective immune
reactions. Am. J. Trop.Med. Hyg., 50:5:29-35.
Flanigan, T., Whalen, C., Turner, J., Soave, R., Toerner, J., Havlir, D., and Kotler, D.
1992. Cryptosporidium infection and CD4 counts. Ann. Int. Med., 116:840-842.
Fogel, D., Isaac-Renton, J., Guasparini, R., Moorehead, W., and Ongerth, J. 1993. Removing Giardia and
Cryptosporidium by slow sand filtration. J. AWWA, 85:11:77-83.
Forde, K.N., Swinker, A.M., Traubdargatz, J.L., and Cheney, J.M. 1998. The prevalence of
Cryptosporidium'Giardia in the trail horse population utilizing public lands in Colorado. J. Equine Vet. Sci.,
18:1:38-40.
Forney, J., Yang, S., andHealey, M. 1996. Anticryptosporidial potential of alpha-1-antitrypsin. J. Eukaryot.
Microbiol.,45:5:638.
Fout, G.S., etal. 1996. ICRMicrobial Laboratory Manual. EPA 600/R-95/178. USEPA, Office of Research and
Development, Washington, DC.
Fox, K. and Lytle, D. 1996. Milwaukee's Crypto outbreak: investigation and recommendations. J. AWWA,
88:9:87-94.
Fredericksen, D.W., Tabib, Z., Boutros, S., Cullen, W., Dittmer, R., McCarthy, L, and McCuin, R. 1995.
Examination of the slide technique vs. membrane technique to recover Giardia lamblia cysts and
Cryptosporidium parvum oocysts. Proc. Wat. Qual. Tech. Conf, New Orleans, pp. 837-845.
Frey, M.M., Hancock, C., and Logsdon, G.S. 1998. Critical evaluation of Cryptosporidium research and
research needs. AWWARF and AWWA.
Fricker, C.R., Crabb, J.A., Turner, N., and Smith, HV. 1997. The concentration and separation of
Cryptosporidium oocysts and Giardia cysts using vortex flow filtration and immunomagnetic separation. In
1997 Int. Symp. Waterborne Cryptosporidium Proc., Fricker et al. (eds), AWWA, Newport Beach, CA.
122
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Friedman, D.E., Patten, K.A., Rose, J.B., and Barney, M.C. 1997. The potential for Cryptosporidiumparvum
oocyst survival in beverages associated with contaminated tap water. J. Food Safety, 17:125-132.
Frost, F. J., Craun, G.F., and Calderon, R.F. 1996. Water disease surveillance. J. AWWA, 61:66-75.
Garcia, L.S., Brewer, T.C., and Bruckner, D.A. 1987. Fluorescence detection of Cryptosporidium oocysts in
human fecal specimens by using monoclonal antibodies. J. Clin. Microbiol., 25:1:119-121.
Garcia, L.S., Shum, A., and Bruckner, D.A. 1992. Evaluation of a new monoclonal antibody combination
reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal
specimens. J. Clin. Microbiol., 30:12:3255-3257.
Glaser, C.A., Reingold, S.S., and Newman, T.B. 1998. Association between Cryptosporidium infection and
animal exposure in HIV-infected individuals. J. AIDS Human Retrovirol., 171:79-82.
Goldstein, S., Juranek, D., Ravenholt, O., Hightower, A., Martin, D., Mesnik, J., Griffiths, S., Bryant, A., Reich,
R., and Herwaldt, B. 1996. Cryptosporidiosis: an outbreak associated with drinking water despite state-of-the-
art water treatment. Ann. Int. Med., 124:5:459468.
Goyena, M., Ortiz, J.M., and Alonso, F.D. 1997. Influence of different systems of feeding in the appearance of
Cryptosporidiosis in goat kids. J. Parasitol., 83:6:1182-1185.
Graczyk, T.K., Cranfield, M.R., Dunning, C., and Strandberg, J.D. 1998a. Fatal Cryptosporidiosis in ajuvenile
captive African hedgehog (Ateletrixalbiventris). J. Parasitol., 84:1:178-180.
Gracyzk, T.K., Cranfield, M.R., and Payer, R. 1996b. Evaluation of commercial enzyme immunoassay (EIA)
and immunofluorescent antibody (IFA) test kits for detection of Cryptosporidium oocysts of species other than
Cryptosporidium parvum. Am. J. Trop. Med. Hyg., 54:3:274-279.
Graczyk, T.K., Cranfield, M.R., Payer, R., and Anderson, M.S. 1996c. Viability and infectivity of
Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host. Appl.
Envir. Microbiol., 62:9:3234-3437.
Graczyk, T.K., Cranfield, M.R., Payer, R., andBixler, H. 1999. House flies (Musca domestica) as transport
hosts of Cryptosporidium parvum. Am. Journ. Trop. Med. Hyg., 61:3:500-504.
Graczyk, T.K., Cranfield, M.R., Payer, R., Trout, J.M., and Goodale, H.J. 1997a. Infectivity of
Cryptosporidium parvum oocysts is retained upon intestinal passage through a migratory water-fowl species
(Canada goose, Branta canadensis). Trop. Med. Int. Health, 2:4:341-347.
Graczyk, T., Payer, R., and Cranfield, M. 1996a Cryptosporidium parvum is not transmissible to fish, amphibia
or reptiles. J. Eukaryot. Microbiol., 43:5:628.
123
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Graczyk, T.K., Payer, R., and Cranfield, M.R. 1998b. Zoonotic transmission of Cryptosporidiumparvum:
implications for waterbome cryptosporidiosis. Parasitol. Today, 13:9:348-351.
Graczyk, T., Payer, R., Cranfield, M., and Owens, R. 1997b. Cryptosporidium parvum oocysts recovered from
water by the membrane filter dissolution method retain their infectivity. J. Parasitol., 83:1:111-114.
Graczyk, T.K., Payer, R., Trout, J.M., Lewis, E.J., Farley, C.A., Sulaiman, I, and Altaf, A.L. 1998c. Giardia sp.
cysts and infectious Cryptosporidium parvum oocysts in the feces of migratory Canada geese (Branta
canadensis). Appl. Envir. Microbiol., 64:7:2736-2738.
Greenberg, P.D., Koch, J., and Cello, J.P. 1996. Diagnosis of Cryptosporidium parvum in patients with severe
diarrhea and AIDS. Digest. Dis. Sci., 41:11:2286-2290.
Griffiths, J.K. 1998. Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment, and
diagnosis. Adv. Parasitol.,40:37-84.
Grigoriew, G.,Walmsley, S., Law, L. Chee, S.,Yang, J., Keystone, J., andKrajden, M. 1994. Evaluation of the
Merifluor immunofluorescent assay for the detection of Cryptosporidium and Giardia in sodium acetate
formalin-fixed stools. Diagn. Microbiol. Infect. Dis., 19:89-91.
Guarino A., Canani, R.B., Casola, A., Pozio, E., Russo, R., Bruzzese, E., Fontana, M., and Rubino, A. 1995.
Human intestinal cryptosporidiosis: secretory diarrhea and enterotoxic activity in CaCo-2 cells. J. Infect. Dis.,
171:976-83.
Guarino, A., Canani, R.B., Pozio, E., Terraciano, L., Albano, F., and Mazzeo, M. 1994. Enterotoxic effect of
stool supernatent of Cryptosporidium-infected calves on human jejunum. Gastroenterol., 106:28-34.
Gyurek, L.L., Finch, G.R., and Belosevic, M. 1997. Modeling chlorine inactivation requirements of
Cryptosporidium parvum oocysts. J. Env. Eng., 123:9:865-875.
Haas, C.N., Crockett, C.S., Rose, J.B., Gerba, C.P., and Fazil, A.M. 1996. Assessing the risks posed by oocysts
in drinking water. J. AWWA, 88:9:131-136.
Haas, C.N., Hornberger, J., Ammangandla, U., Heath, M., and Jacangelo, J.G. 1994. A volumetric method for
assessing Giardia inactivation. J. AWWA, 86:2:115-120.
Hall, T., Pressdee, J., Gregory, R., and Murray, K. 1995. Cryptosporidium removal during water treatment using
dissolved air floatation. Wat. Sci. Tech., 31:3^:125-135.
Hancock, C. M., Rose, J.B., and Callahan, M. 1998. Crypto and Giardia in U.S. groundwater. J. AWWA,
58:3:58-61.
Harp, J. A., Payer, R.,Pesch, B.A., and Jackson, G.J.. 1996. Effect of pasteurization on infectivity of
Cryptosporidium parvum oocysts in water and milk. Appl. Environ. Microbiol., 62:8:2866-2867.
124
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Harp, J.A. and Moon, H.W. 1991. Susceptibility of mast cell deficient WAV mice to Cryptosporidiumparvum.
Infect. Immun., 59:718-720.
Harris, A.T., Larkin, J., and Blum, K. 1995. The development of a method detection limit (MDL095) for the IFA
analysis ofGiardia and Cryptosporidium. Proc. Wat. Qual. Tech. Conf, New Orleans, pp. 49-55.
Heald, A.E. and Bartlett, J.A. 1994. Cryptosporidium spread in a group residential home. Ann. Int. Med.,
121:467-468.
Herwaldt, B.L., Craun, G.F., Stokes, S.L., and Juranek, D.D. 1991. Waterborne-disease outbreaks, 1989-1990.
MMWR, 40:SS-3:1-21.
Hicks, P., Zweiner, R.J., Squires, J., and Savell, V. 1996. Azithromycin therapy for Cryptosporidium parvum
infection in four children infected with human immunodeficiency virus. J. Fed., 129:297-300.
Hill, B.D., Fraser, I.R., and Prior, H.C. 1997. Cryptosporidium infection in a dugong (Dugongdugon). Austral.
Vet. I, 75:9:670-671.
Hoffman, R.M., Standridge, J.H., Prieve, A.F., Cucunato, J.C., and Bernhardt, M. 1997. Using flow cytometry
to detect protozoa. J. AWWA, 89:104-111.
Hoover, D.M., Hoerr, F.J., Carlton, W.W., Hinseman, E.J., and Ferguson, H.W. 1981. Enteric cryptosporidiosis
in a nasotang, Naso liturata. J. Fish Dis., 4:425-428.
Hoxie, N. J., Davis, J.P., Vergeront, J.M., Nashold, R.D., and Blair, K.A. 1997. Cryptosporidiosis-associated
mortality following a massive waterborne outbreak in Milwaukee, Wisconsin. Am. J. Public Health,
87:12:2032-2035.
ILSI Risk Science Institute Pathogen Risk Assessment Working Group. 1996. A conceptual framework to
assess the risks of human disease following exposure to pathogens. Risk Anal., 16:6:841-847.
Ignatius, R., Eisenblatter, M., Regnath, T., Mansmann, U., Futh, U., Hahn, H., and Wagner, J. 1997. Efficacy
of different methods for detection of low Cryptosporidium parvum oocyst numbers or antigen concentration in
stool specimens. Eur. J. Clin. Microbiol. Infect Dis., 16:10:732-736.
Jacangelo, J.G., Adham, S.S., and Laine, J.M. 1995a. Mechanism of Cryptosporidium, Giardia, and MS2 virus
removal by MF and UF. J. AWWA, 87:9:107-121.
Jacangelo, J.G., Adham, S.S., and Laine, J-M. 1995b. Mechanisms of Cryptosporidium, Giardia and MS2 virus
removal by MF and UF. J. AWWA, 87:9:107-121. [as cited inFrey et al. (1998)]
Jakubowski, W., Boutros, S., Faber, W., Faver, R., Ghiorse, W., LeChevallier, M., Rose, J., Schaub, S, Singh,
A., and Stewart, M. 1996. Environmental methods for Cryptosporidium. J. AWWA, 88:9:107-121.
125
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Jardine, J. E. and Verwoerd, DJ. 1997. Pancreatic cryptosporidiosis in ostriches. Avian Pathol., 26:3:665-670.
Jenkins, M.B., Anguish, L.J., Bowman, D.D., Walker, M.J., Ghiorse, W.C. 1997. Assessment of a dye
permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts. Appl. Env.
Microbiol., 63:10:3844-3850.
Johnson, E., Atwill, E.R., Filkins, M.E., and Kalash, J. 1997. The prevalence of shedding of Cryptosporidium
and Giardia spp. based on a single fecal sample collection from each of 91 horses used for backcountry
recreation. J. Vet. Diagn. Invest., 9:1:56-60.
Johnson, D.W., Pieniazek, N.J., Griffin, D.W, Misener,L., and Rose, J.B. 1995. Development of aPCR
protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl. Env. Microbiol., 61:3849-
3855.
Johnson, D.W., Pieniazek, N.J., and Rose, J.B. 1993. DNA probe hybridization and PCR detection of
Cryptosporidium compared to immunoflurescence assay. Wat. Sci. Tech.., 27:77-84.
Jordan, E.G. 1996. Clarithromycin prophylaxis against Cryptosporidium enteritis in patients with AIDS. J. Nat.
Med. Assoc., 88:425-427.
Juranek, D.D., Addiss, D.G., Bartlett, M.E., Arrowood, M.J., Colley D.G., Kaplan J.E., Perciasepe, R., Elder,
J.R., Regli, S.E., and Berger, P.S. 1995. Cryptosporidiosis and public health: workshop report. J. AWWA,
87:9:69-80.
Juranek, D.D. and MacKenzie, W.R. 1998. Drinking water turbidity and gastrointestinal illness. Epidemiol.,
9:3:228-230.
Kang, G. and Mathan, M.M. 1996. A comparison of five staining methods for detection of Cryptosporidium
oocysts in faecal specimens from the field. Indian J. Med. Res., 103:264-266.
Kapel, N., Meillet, D., Buaud, M., Favennec, L., Magne, D., and Gobert J.G. 1993. Determination of anti-
Cryptosporidium coproantibodies by time-resolved immunofluorometric assay. Trans. Roy. Soc. Trop. Med.
and Hyg., 87:330-332.
Kassa, M., Comby, E., Lemeteil, D., Brasseur, P., and Ballet, J.J. 1991. Characterization of anti-
Cryptosporidium IgA antibodies in sera from immunocompetent individuals and HIV-infected patients. J.
Protozool.,38:Suppl:179S-180S.
Kelley, M.B., Brokaw, J.K., Edzwald, J.K., Fredericksen, D.W., and Warrier, P.K. 1994. A survey of eastern
U.S. Army installation drinking water sources and treatment systems for Giardia and Cryptosporidium. Proc.
AWWA Wat. Qual. Tech. Conf, Denver, CO. [as cited in Frey et al. (1998)]
Kelly, P., Carnaby, S., Ngwenyha, B., Luo, N., Pobee, J., and Farthing, M. 1995. Cryptosporidiosis in small
intestine-detection by polymerase chain reaction. Gastroenterol., 108:4:A847.
126
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Kelly, P., Thillainayagam, A., Smithson, J., Junt, J., Forbes, A., Gazzard, B., and Farthing, M. 1996. Jejunal
water and electrolyte transport in human cryptosporidiosis. Digest. Dis. Sci., 41:10:2095-2099.
Khramtsov, N.V., Chung, P.A., Dykstra, C.C., Griffiths, J.K., Morgan, U.M, Arrowood, M.J., and Upton, S J.
2000. Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum. J. Parasitol.,
86:2:275-282.
Klonicki, P.T., Hancock, C.M., Straub, T.M., Harris, S.I., Hancock, K.W., Alyaseri, A.N., Meyer, C.J., and
Sturbaum, G.D. 1997. Crypto research: are fundamental data missing? J. AWWA, 89:97-103.
Korich, D.G., Mead, J.R, Madore, M.S., Sinclair, N. A., and Sterling, C.R. 1990. Effects of ozone, chlorine
dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability. Appl. Env. Microbiol.,
56:5:1423-1428.
Koudela, B. and Jiri, V. 1997. Experimental cryptosporidiosis in kids. Vet. Parasitol., 71:4:273-281.
Koudela, B. and Modry, D. 1998. New species of Cryptosporidium (Apicomplexa, Cryptosporidiidae) from
lizards. Folia Parasitol., 45:93-100.
Kramer, M.H., Herwaldt, B.L., Craun, G.F., Calderon, R.L., and Juranek, D.D. 1996. Waterborne disease: 1993
and 1994. J. AWWA, 88:3:66-80.
Kramer, M.H., Sorhage,F.E., Goldstein, S.T., Dalley, E., Wahlquist, S.P., andHerwaldt, B.L. 1998. First
reported outbreak in the United States of cryptosporidiosis associated with a recreational lake. Clin. Infect. Dis.,
26:1:27-33.
Kuhls, T., Mosier, D., Crawford, D., Abrams, V., and Greenfield, R. 1996. Improved survival of severe
combined immunodeficiency (scid) mice with cryptosporidiosis by adoptively transferring CD4+ and CD4-CD8_
B220-BALB/C splenocytes(Spis). J. Eukaryot. Microbiol., 43:5:718.
Laberge, I, Griffiths, M.W., and Griffiths, M.W. 1996. Prevalence, detection and control of Cryptosporidium
parvum in food. Int. J. Food Microbiol., 31:1-26.
Laxer, M.A., Alcantara, A.K., Javato-Laxer, M., Merorca, D.M., Fernando, M.T., and Roaoa, C.P. 1990.
Immune response to cryptosporidiosis in Philippine children. Am. J. Trop. Med. Hyg., 42:131-139.
LeChevallier, M.W. and Norton, W.D. 1995. Giardia and Cryptosporidium in raw and finished water. J.
AWWA, 87:9:54-68.
LeChevallier, M. W., Norton, W., and Atherholt, T. 1997. Protozoa in open reservoirs. J. AWWA, 899:84-96.
LeChevallier, M.W., Norton, W.D., Lee, R.G., and Rose, J.B. 1991. Giardia and Cryptosporidium in water
supplies. AWWARF and AWWA, Denver, CO. [as cited in Frey et al. (1998)]
127
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
LeChevallier, M.W., Norton, W.D., Siegal, I.E., and Abbaszadegan, M. 1995. Evaluation of the
immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water. Appl.
Env. Microbiol., 61:690-697.
Leland, D., McAnulty, J., Keene, W., and Stevens, G. 1993. A cryptosporidiosis outbreak in a filtered-water
supply. J. AWWA, 85:6:34-42.
LeMoing V., Bissuel, G., Costagliola, D., Eid, Z., Chapuis, F., Molina, J.-M., Salmon-Ceron, D., Brasseur, P.,
and Leport, C. 1998. Decreased prevalence of intestinal cryptosporidiosis in HIV-infected patients concomitant
to the widespread use of protease inhibitors. ADDS, 12:11:1395-1397.
Leng, X., Mosier, D., and Oberst, R. 1996. Simplified method for recovery and PCR detection of
Cryptosporidium DNA from bovine feces. Appl. Env. Microbiol., 62:2:643-647.
Lengerich, E.J., Addiss, D.G., Marx, J.J., Ungar, B.L., and Juranek, DD. 1993. Increased exposure to
Cryptosporidia among dairy farmers in Wisconsin. J. Infect. Dis., 167:1252-1255.
Li, S., Goodrich, J., Owens, J., Clark, R., Willeke, G., and Schaefer, F. 1995. Potential Cryptosporidium
surrogates and evaluation of compressible oocysts. 21st Ann. RREL Research Symp. Abstr. Proc., Cincinnati,
OH.
Lindsay, D.S., Upton, S.J., Owens, D.S., Morgan, U.M., Mead, J.R., Blagburn, B.L. 2000. Cryptosporidium
andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus. J Eukaryot. Microbiol., 47:1:91-95.
Lisle, J. and Rose, J. 1995. Cryptosporidium contamination of water in the USA and UK: a mini-review. J. Wat.
SRT-Aqua, 44:3:103-117.
Liyanage, L.R.J., Finch, G.R., and Belosevic, M. 1997a Effect of aqueous chlorine and oxychlorine compounds
on Cryptosporidiumparvum oocysts. Env. Sci. Tech., 31:7:1992-1994.
Liyanage, L.R.J., Finch, G.R., and Belosevic, M. 1997b. Sequential disinfection of Cryptosporidium parvum by
ozone and chlorine dioxide. Ozone Sci. Eng., 19:409-423.
Logar, J., Poljsak-Prijatelj, M., and Andlovic, A. 1996. Incidence of Cryptosporidium parvum in patients with
diarrhea. J. Eukaryot. Microbiol., 43:5:678.
Lopez-Velez, R., Tarazona, R., Camacho, A., Gomez-Mampaso, E., Guerrero, A., Moreira, V., and Villanueva.,
R. 1995. Intestinal and extraintestinal cryptosporidiosis in AIDS patients. Eur. J. Clin. Microbiol. Infect. Dis.,
14:8:677-681.
MacKenzie, W., Hoxie,N., Proctor, M., Gradus, M., Blari, K., Peterson, D., Kazmierczak, J., and Davis, J.
1994. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water
supply. New Eng. J.Med., 331:3:161-167.
128
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
MacKenzie, W. R., Kazmierczak, J.J., and Davis, J.P. 1995a. An outbreak of cryptosporidiosis associated with a
resort swimming pool. Epidemiol. Infect., 115:545-553.
MacKenzie, W., Schell, W., Blair, K., Addiss, D., Peterson, D., Hoxie, N, Kazmierczak, J., and Davis, J.
1995b. Massive outbreak ofwaterborne cryptosporidiosis infection in Milwaukee, Wisconsin: recurrence of
illness and risk of secondary transmission. Clin. Infect. Dis., 21:57-62.
Maggi, P., Larocca, A.M.V., Quarto, M., Serio, G., Brandonisio, O., Angarano, G., and Pastore, G. 2000. Effect
of antiretroviral therapy on cryptosporidiosis and microsporidiosis in patients infected with human
immunodeficiency virus type 1. Eur. J. Clin. Microbiol. Infect. Dis. 19:3:213-217.
Maguire, H.C., Holmes, E.,Hollyer, J., Strangeways, J.E.M., Foster, P., Holliman, R.E., and Stanwell-Smith,
R. 1995. An outbreak of cryptosporidiosis in South London: what values the/? value. Epidemiol. Infect.,
115:279-287.
Majewska, A.C., Kasprzak, W., and Werner, A. 1997. Prevalence of Cryptosporidium in mammals housed in
Poznan Zoological Garden, Poland. ActaParasitol., 4:24:195-198.
Mathison, B.A. and Ditrich, O. 1999. The fate of Cryptosporidiumparvum oocysts ingested by dung beetles and
their possible role in the dissemination of cryptosporidiosis. J. Parasitol., 85:4:678-681.
Mawdsley, J.L, Brooks, A.W., and Merry, RJ. 1996a. Movement of the protozoan pathogen Cryptosporidium
parvum through three contrasting soil types. Biol. Fertil. Soils, 21:1-2:30-36.
Mawdsley, J.L., Brooks, A.E, Merry, R.J., and Pain, B.F. 1996b. Use of a novel soil tilting table apparatus to
demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil.
Biol. Fertil. Soils, 21:1-2:215-220.
Mayer, C.L. and Palmer, CJ. 1996. Evaluation of PCR, nested PCR, and flourescent antibodies for detection of
Giardia and Cryptosporidium species in wastewater. Appl. Env. Microbiol., 62:2081-2085.
McAnulty, J., Fleming, D., and Gonzalez, A. 1994. A community-wide outbreak of cryptosporidiosis associated
with swimming at a wave pool. JAMA, 272:20:1597-1600.
McDonald, V., Deer, R.,Uni, S., Eseki, M., and Bancroft, GJ. 1992. Immune responses to Cryptosporidium
muris and Cryptosporidium parvum in adult immunocompetent orimmunocompromised (nude and SCID)
mice. Infect. Immun., 59:3325-3331.
Mead, J.R., You, X., Pharr, I.E., Belenkaya, Y., Arrowood, M.J., Fallen, M.T., and Schinazi, R.F. 1995.
Evaluation of maduramicin and alborixin in a SCID mouse model of chronic cryptosporidiosis. Antimicrob.
Agents Chemo., 39:854-858.
Meinhardt, P. L., Casemore, D. P., and Miller, K. B. 1996. Epidemiologic aspects of human cryptosporidiosis
and the role ofwaterborne transmission. Epidemiol. Rev., 18:2:118-136.
129
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Miao, Y.M., Awad-El-Kariem, P.M., Gibbons, C.L., and Gazzard, E.G. 1999. Cryptosporidiosis: eradication or
suppression with combination antiretriviral therapy? AIDS, 13:6:734-735.
Mifsud, A., Bell, A., and Shafi, M. 1994. Respiratory cryptosporidiosis as a presenting feature of AIDS. J.
Infect., 28:2:227-229.
Millard, P., Gensheimer, K, Addiss, D., Sosin, D., Beckett, G., Houck-Jankoski, A., and Hudson, A. 1994. An
outbreak of cryptosporidiosis from fresh-pressed apple cider. JAMA, 272:20:1592-1596.
Miller, R.A. 1996. The aging immune system: primer and prospectus.Science, 273:70-74.
Molbak, K., Aaby, P., Hojlyng, N., and Da Silva, A.P.J. 1994. Risk factors for Cryptosporidium diarrhea in
early childhood: a case-control study from Guinea-Bissau, West Africa. Am. J. Epidemiol., 139:7:734-740.
Monge, R. and Chinchilla, M. 1995. Presence of Cryptosporidium oocysts in fresh vegetables. J. Food Protect.,
59:2:202-203.
Moore, A.C., Herwaldt, BL., Craun, G.F., Calderon,R.L., Highsmith, A.K., and Juranek, D.D. 1993.
Surveillance for waterborne disease outbreaks - United States, 1991-1992. MMWR, 42:SS-5:l-22.
Morgan, U.M., Buddie, J.R., Armson, A., Elliot, A., and Thompson, R.C. 1999f Molecular and biological
characterisation of Cryptosporidium in pigs. Austral. Vet. J., 77:1:44-47.
Morgan, U.M., Deplazes, P., Forbes, D.A., Spano, F., Hertzberg, H., Sargent, K.D., Elliot, A., and Thompson,
R.C. 1999a. Sequence andPCR-RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in
isolates of Cryptosporidium from different hosts. Parasitol., 118:Pt 1:49-58.
Morgan, U.M., Monis, P.T., Payer, R., Deplazes, P., and Thompson, R.C. 1999b. Phylogenetic relationships
among isolates of Cryptosporidium: evidence for several new species. J. Parasitol., 85:6:1126-1133.
Morgan, U.M., O'Brien, P., and Thompson, A. 1996. The development of diagnostic PCR primers for
Cryptosporidium using RAPD-PCR. Mol. Biochem. Parasitol., 77:103-108.
Morgan, U.M., Sargent, K.D., Deplazes, P., Forbes, D.A., Spano, F., Hertzberg, H., Elliot, A., and Thompson,
R.C. 1998a. Molecular characterization of Cryptosporidium from various hosts. Parasitol., 117: Pt 1:31-37.
Morgan, U.M., Sargent, K.D., Elliot, A., and Thompson, R.C. 1998b. Cryptosporidium in cats—additional
evidence for C.felis. Vet. I, 156:2:159-161.
Morgan, U.M., Sturdee, A.P., Singleton, G., Gomez, M.S., Gracenea, M., Torres, J., Hamilton, S.G., Woodside,
D.P., and Thompson, R.C. 1999e. The Cryptosporidium "mouse" genotype is conserved across geographic
areas. J. Clin. Microbiol., 37:5:1302-1305.
130
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Morgan, U.M., Weber, R., Xiao, L., Sulaiman, I, Thompson, R.C., Ndiritu, W., Lai, A., Moore, A., and
Deplazes, P. 2000a. Molecular characterization of Cryptosporidium isolates obtained from human
immunodeficiency virus-infected individuals living in Switzerland, Kenya, and the United States. J. Clin.
Microbiol. 38:3:1180-1183.
Morgan, U.M., Xiao, L, Payer, R.,Lal, A. A., and Thompson, R.C. 1999c. Variation in Cryptosporidium:
towards ataxonomic revision of the genus. Int. J. Parasitol., 29:11:1733-1751.
Morgan, U.M., Xiao, L, Monis, P., Fall, A., Irwin, P.J., Payer, R., Denholm, K.M., Limor, J., Lai, A., and
Thompson, R.C. 2000b. Cryptosporidium spp. in domestic dogs: the "dog" genotype. Appl. Env. Microbiol.,
66:5:2220-2223.
Morgan, U.M., Xiao, L., Sulaiman, L, Weber, R., Lai, A.A., Thompson, R.C., and Deplazes, P. 1999d. Which
genotypes/species of Cryptosporidium are humans susceptible to? J. Eukaryot. Microbiol., 46:5:42S43S.
Morris, R. D., Naumova, E.N., and Griffiths, J.K. 1998. Did Milwaukee experience waterborne cryptospoidiosis
before the large documented outbreak in 1993?Epidemiol., 9:3:264-270.
Moss, D., Bennett, S., Arrowood, M.J., Kurd, M., Lammie, P., Wahlquist, S., and Addiss, D. 1994. Kinetic and
isotypic analysis of specific immunoglobulins from crew members with cryptosporidiosis on a U.S. Coast
Guard cutter. J. Eukaryot. Microbiol., 41:5:528.
Moss, D.M., Bennet, S.N., Arrowood, M.J., Wahlquist, S.P., and Lammie, PJ. 1998. Enzyme linked
immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter. Am.
J. Trop. Med. Hyg., 58:1:110-118.
Mtambo, M.M.A., Nash, A.S., Blewett, D.A., and Wright, S. 1992. Comparison of staining and concentration
techniques for detection of Cryptosporidium oocysts in cat faecal specimens. Vet. Parasitol., 45:49-57.
Muench, T.R. and White, M.R. 1997. Cryptosporidiosis in tropical freshwater catfish (Plecostomus spp.). J. Vet.
Diagn. Invest, 9:1:87-90.
Muriuki, S.M.K., Farah, I.O., Kagwiria, R.M., Njamunge, G., Suleman, M., and Olobo, J.O. 1997. The presence
of Cryptosporidium oocysts in stools of clinically diarrhoeic and normal nonhuman primates in Kenya. Vet.
Parasitol., 72:2:141-147.
Nahrstedt, A. and Gimbel, R. 1996. A statistical method for determining the reliability of the analytical results
in the detection of Cryptosporidium and Giardia in water. J. Wat. SRT-Aqua, 45:3:101-111.
Newman, R.D., Jaeger, K.L., Wuhib, T., Lima, A.A.M., Guerrant, R.L., and Sears, C.L. 1993. Evaluation of an
antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts. J. Clin.
Microbiol., 31:2080-2084.
131
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Newman, R.D., Zu, S.-X., Wuhib, T., Lima, A., Guerrant, R., and Sears, C. 1994. Household epidemiology of
Cryptosporidiumparvum infection in an urban community in northeast Brazil. Ann. Int. Med., 120:6:500-505.
Nieminski, E.G. 1995. Giardia and Cryptosporidium cysts removal through direct filtration and conventional
treatment. Paper presented at Ann. AWWA Conf, New York, NY, June 19-23, 1994. [as cited in Frey et al.
(1998)]
Nieminski, E.G. and Ongerth, I.E. 1995. Removing Giardia and Cryptosporidium by conventional treatment
and direct filtration. J. AWWA, 87:9:96-106. [as cited in Frey et al. (1998)]
Nieminski, E.G., Schaefer, F.W.I., and Ongerth, I.E. 1995. Comparison of two methods for detection of Giardia
cysts and Cryptosporidium oocysts in water. Appl. Env. Microbiol., 61:1714-1719.
Nimri, L.F. and Batchoun. R. 1994. Prevalence of Cryptosporidum species in elementary school children. J.
Clin. Microbiol., 32:4:1040-1042.
O'Donoghue, P. 1995. Cryptosporidium and cryptosporidiosis in man and animals. Int. J. Parasitol., 25:2:139-
195.
Okhuysen, P.C., Chappell, C.L., Crabb, J.H., Sterling, C.R., andDuPont, H.L. 1999. Virulence of three distinct
Cryptosporidium parvum isolates for healthy adults. J. Infect. Dis., 180:4:1275-1281.
Okhuysen, P.C., Chappell, C.L, Sterling, C.R., Jakubowski, W., and DuPont, H.L. 1998. Susceptibility and
serologic response of healthy adults to reinfection with Cryptosporidium parvum. Infect. Immun., 66:2:441-443.
Olson, M.E., Guselle, N.J., Ohandley, R.M., Swift, M.L.,Mcallister, T.A., Jelinski, M.D., and Morck, D.W.
1997. Giardia and Cryptosporidium in dairy calves in British Columbia. Canad. Vet. J.-Rev. Vet. Canad.,
38:11:703-706.
Ong, C., Moorehead, W.,Ross, A., and Isaac-Renton, J. 1996a. Giardia spp. and Cryptosporidium spp. in
British Columbia watersheds. J. Eukaryot. Microbiol., 43:5:658.
Ong, C., Moorehead, W., Ross, A., and Isaac-Renton., J.L. 1996b. Studies of Giardia spp. and Cryptosporidium
spp. in two adjacent watersheds. Appl. Env. Microbiol., 62:8:2798-2805.
Ongerth, I.E. and Hutton, P.E. 1997. DE filtration to remove Cryptosporidium. J. AWWA, 89:12:39-46.
Ongerth, I.E. and Pecoraro, J.R. 1995. Removing Cryptosporidium using multimedia filters. J. AWWA,
87:12:83-89.
Osewe, P., Addiss, D., Blair, K., Hightower, A., Kamb, M., and Davis, J. 1996. Cryptosporidiosis in Wisconsin:
a case-control study of post-outbreak transmission. Epidemiol. Infect., 117:297-304.
132
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Owens, J.H., Miltner, R.J., Schaefer, F.W.I., and Rice, E.W. 1994a. Pilot-scale ozone inactivation of
Cryptosporidium. J. Eukaryot. Microbiol., 41:5:568-57S.
Owens, J.H., Miltner, R.J., Schaefer, F.W.I., and Rice, E.W. 1994b. Pilot-scale ozone inactivation of
Cryptosporidium and Giardia. Proc.Wat. Qual. Tech. Conf, San Francisco, pp. 1319-1324.
Parisi, M.T. and Tierno, P.M. 1995. Evaluation of new rapid commercial enzyme immunoassay for detection of
Cryptosporidium oocysts in untreated stool specimens. J. Clin. Microbiol., 33:7:1963-1965.
Parker, J.F.W., Greaves, G.F., and Smith, H.V. 1993. The effect of ozone on the viability of Cryptosporidium
parvum oocysts and a comparison of experimental methods. Wat. Sci. Tech., 27:34:93-96.
Parker, J.F.W. and Smith, H.V. 1993. Destruction of oocysts of Cryptosporidium parvum by sand and chlorine.
Wat. Res., 27:4:729-731.
Patania, N.L., Jacangdo, J.G., Cummings, L., Wilczak, A., Riley, K., and Oppenheimer, J. 1995. Optimization
of filtration for cyst removal. Final report. AWWARF, Denver, CO. [as cited in Frey et al. (1998)]
Patel, S., Pedraza-Diaz, S.,McLauchlin, J., andCasemore, D.P. 1998. Molecular characterisation of
Cryptosporidium parvum from two large suspected waterborne outbreaks. Outbreak Control Team South and
West Devon 1995, Incident Management Team and Further Epidemiological and Microbiological Studies
Subgroup North Thames 1997. Commun. Dis. Public Health, 1:4:231-233.
Payment, P. and Franco. E. 1993. Clostridium perfringens and somatic coliphages as indicators of the efficiency
of drinking water treatment for viruses and protozoan cysts. Appl. Env. Microbiol., 59:8:2418 -2424.
Peeters, I.E., Ares-Mazas, M.E., Masschelein, W.J., Villacorta-Martinez de Maturana, I, and Debacker, E.
1989. Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium
parvum oocysts. Appl. Env. Microbiol., 55:6:1519-1522.
Pena, H.F.D., Kasai, N., and Gennari, S.M. 1997. Cryptosporidium in dairy cattle in Brazil. Vet. Parasitol.,
73:3-4:353-355.
Peng, M.M., Xiao, L., Freeman, A.R., Arrowood, MJ.,Escalante, A.A., Weltman, A.C., Ong, C.S.L.,
MacKenzie, W.R., Lai, A. A., and Beard, C.B. 1997. Genetic polymorphism among Cryptosporidium parvum
isolates: evidence of two distinct human transmission cycles. J. Emerg. Infect. Dis., 3:4:567-573.
Pereira, M., Atwill, E.R., Crawford, M.R., and Lefebvre, R.B. 1998. DNA sequence similarity between
California isolates of Cryptosporidium parvum. Appl. Env. Microbiol., 64:4:1584-1586.
Perez, E., Kummeling, A. Janssen, M., Jimenez, C., Alvarado, R. Caballero, M., Donado, P., and Dwinger, R.H.
1998. Infectious agents associated with diarrhoea of calves in the Canton of Tilaran, Costa Rica. Prevent. Vet.
Med., 33:1-4:195-205.
133
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Perz, J.F., Ennever, F.K., and LeBlancq, S.M. 1998. Cryptosporidium in tap water: comparison of predicted
risks with observed levels of disease. Am. J. Epidemiol., 147:3:289-301.
Petri, C., Karanis, P., and Renoth, S. 1997. Cryptosporidium infections in muskrat (Ondatra zibethica). Paras.-
J. Soc. Franc. Parasitol, 4:4:369-371.
Pettoello-Mantovani, M., Martino, L., Dettori, G., Vajro, P., Scotti, S., Ditullio, M., and Guandalini, S. 1995.
Asymptomatic carriage of intestinal Cryptosporidium in immunocompetent and immunodeficient children: a
prospective study. Fed. Infect. Dis. J., 14:12:1042-1047.
Pieniazek, N.J., Bornay-Llinares, F.J., Slemenda, S.B., da Silva, A.J., Moura, IN., Arrowood, M.J., Ditrich, O.,
and Addiss, D.G. 1999. New Cryptosporidium genotypes inHIV-infected persons. Emerg. Infect. Dis., 5:3:444-
449.
Plummer, J.D., Edzwald, J.K., and Kelley, M.B. 1995. Removing Cryptosporidium by dissolved-air flotation. J.
AWWA, 87:9:85-95. [as cited in Frey et al. (1998)]
Proctor, M.E., Blair, K.A., and Davis, J.P. 1998. Surveillance data for waterborne illness detection - an
assessment following a massive waterborne outbreak of Cryptosporidium infection. Epidemiol. Infect.,
120:1:43-54.
Quinn, C.M., Archer, G.P., Berts, W.B., and O'Neill, J.G. 1996. Dose-dependent dielectrophoretic response of
Cryptosporidium oocysts treated with ozone. Lett. Appl. Microbiol., 22:224-228.
Quinn, K., Balwin, G., Stepak, P., Thorburn, K., Bartleson, C., Goldoft, M., Kobayashi, J., and Stehr-Green, P.
1998. Foodborne outbreak of cryptosporidiosis-Spokane, Washington, 1997. MMWR, 47:27:565-567.
Rasmussen, K.R. and Healy, M.C. 1992. Experimental Cryptosporidiumparvum infections in
immunosuppressed adult mice. Infect. Immun., 60:1648-1652.
Reese, N.C., Current, W.L., Ernst, J.V., and Bailey, W.S. 1982. Cryptosporidiosis of man and calf: a case report
and results of experimental infections in mice and rats. Am. J. Trop. Med. Hyg., 31:226-229.
Reynolds, D.T., Slade, J.S., and Fricker, C.R. 1997. Laser scanning device for detecting Cryptosporidium. In:
1997 AWWA WQTC Proc., Denver, CO.
Riggs, M., Yount, P., Stone, A., and Langer, R. 1996. Protective monoclonal antibodies define a distinct,
conserved epitope on an apical complex exoantigen of Cryptosporidium parvum sporozoites. J. Eukaryot.
Microbiol.,43:5:748-758.
Roberts, C.L., Morin C., Addiss, D., Wahlquist, S., Mshar, P., and Hadler, J. 1996. Factors influencing
Cryptosporidium testing in Connecticut. J. Clin. Microbiol., 34:9:2292-2293.
134
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Robertson, L.J., Campbell, A.T., and Smith, H.V. 1992. Survival of Cryptosporidium parvum oocysts under
various environmental pressures. Appl. Env. Microbiol., 58:11:3494-3500.
Robertson, L.J., Campbell, AT., and Smith, H.V. 1993. Induction of folds or sutures on the walls of
Cryptosporidium parvum oocysts and their importance as a diagnostic feature. Appl. Env. Microbiol.,
59(8):2638-2641.
Robertson, L.J., Campbell, AT., and Smith, H.V. 1994. Is the 'fold/suture line' of diagnostic significance in the
identification of waterborne Cryptosporidium oocysts? Royal Soc. Trop. Med. and Hyg., 88:25.
Robertson, LJ. and Smith, H.V. 1992. Cryptosporidium and cryptosporidiosis, part 1: current perspective and
present technologies. Eur. Microbiol., 1:20-29.
Rochelle, P., DeLeon, R., Ferguson, D.M., Stewart, M.H., and R.L. Wolfe. 1997a. Optimization of an
infectivity assay, combining cell culture and PCR for waterborne Cryptosporidium parvum. In: 1997 Int. Symp.
Waterborne Cryptosporidium Proc., AWWA, Denver, CO.
Rochelle, P., DeLeon, R, Stewart, M., and Wolf, R. 1997b. Comparison of primers and optimization of PCR
conditions for detection of Cryptosporidium parvum and Giardia lamblia in water. Appl. Env. Microbiol.,
63:1:106-114.
Rodgers, M.R., Flanigan, D.J., and Jakubowski, W. 1995. Identification of algae which interfere with detection
of Giardia cysts and Cryptosporidium oocysts and a method for alleviating this interference. Appl. Env.
Microbiol., 61:10:3759-3763.
Rodman, J.S., Frost F., Davisburchat L., Fraser D., Langer J., and Jakubowski, W. 1997. Pharmaceutical sales -
a method of disease surveillance. J. Env. Health, 60:4:8-14.
Rodriguez, F., Oros, J., Rodriguez, J.L., Gonzalez, J., Castro, P., and Fernandez, A. 1997. Intestinal
cryptosporidiosis in pigeons (Columba livid). Avian Dis., 41:3:748-750.
Roefer, P., Monscvitz, J.T., and Rexing, DJ. 1995. The Las Vegas Cryptosporidium experience. Proc. Wat.
Qual. Tech. Conf, New Orleans, pp. 2243-2262.
Resales, M., Lazcano, C., Arnedo, T., and Castilla, J. 1994. Isolation and identification of Cryptosporidium
parvum oocysts with continuous percoll gradients and combined alcian blue-giemsa staining. Acta Trop.,
56:371-373.
Rose, J., Griffin, D., and Friedman, D. 1994. What is Cryptosporidium? 20th Ann. Conv. Exhib., Wat. Qual.
Assoc., March 15-20, Phoenix, AZ, 11 pp.
Rose, J.B., Lisle, J.T., and LeChevallier, M. Waterborne cryptosporidiosis: incidence, outbreaks, and treatment
strategies. In: Cryptosporidium and Cryptosporidiosis. Payer R (ed), CRC Press, New York, 1997, p. 100.
135
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Rose, J.B. and Slifko, T.R. 1999. Giardia, Cryptosporidium, and Cyclospora and their impact on foods: a
review. J. Food Protect, 62:9:1059-1070.
Rosenblatt, I.E. and Sloan, L.M. 1993. Evaluation of an enzyme-linked immunosorbent assay for detect!on of
Cryptosporidium spp. in stool specimens. J. Clin. Microbiol., 31:1468-1471.
Rusnak, J., Hadfield, T., Rhodes, M., and Gaines, J. 1989. Detection of Cryptosporidium oocysts in human fecal
specimens by an indirect immunofluorescence assay with monoclonal antibodies. J. Clin. Microbiol.,
27:5:1135-1136.
Sargent, K.D., Morgan, U.M., Elliot, A., and Thompson, R.C. 1998. Morphological and genetic characterisation
of Cryptosporidium oocysts from domestic cats. Vet. Parasitol., 77:4:221-227.
Sattar, S.A., Chauret, C., Springthorpe, V.S., Battigelli, D.A., Abbaszadegan, M., and LeChevallier, M. 1999.
Giardia cyst and Cryptosporidium oocyst survival in watersheds and factors affecting inactivation. AWWARF,
Denver, CO.
Scholes, S.F.E., Holliman, A., May, P.D.F., and Holmes, M.A. 1998. A syndrome of anaemia,
immunodeficiency, and peripheral ganglionpathy in Fell pony foals. Vet. Record, 142:6:128-134.
Schuler, P.F., Ghosh, M.M., and Boutros, S.N. 1988. Comparing the removal of Giardia and Cryptosporidium
using slow sand and diatomaceous earth filtration. Proc. AWWA. [as cited in Frey et al. (1998)]
Schuler, P.F., Ghosh, M.M., and Gopalan, P. 1991. Slow sand and diatomaceous earth filtration of cysts and
other particulates. Wat. Res., 25:8:995-1005.
Sears, C.L., Newman, R.D., and Guerrant. R.L. 1994. Cryptosporidium spread in a group residential home.
Ann. Int. Med., 121:6:468.
Shepherd, K.M. and Wyn-Jones, A.P. 1995. Evaluation of different filtration techniques for the concentration of
Cryptosporidium oocysts from water. Wat. Sci. Tech., 31:5-6:425^29.
Shepherd, K.M. and Wyn-Jones, A.P. 1996. An evaluation of methods for the simultaneous detection of
Cryptosporidium and Giardia cysts from water. Appl. Env. Microbiol., 62:1317-1322.
Siddons, C.A., Chapman, P. A., and Rush, B.A. 1991. Evaluation of an enzyme immunoassay kit for detection
Cryptosporidium in faeces and environmental samples. J. Clin. Palhol., 45:6:479-482.
Slifko, R.R., Friedman, D., Rose, J.B., and Jakubowski, W. 1997. An in vitro method for detecting infectious
Cryptosporidium oocysts with cell culture. Appl. Env. Microbiol., 63:9:3669-3675.
Smith, H.V., Brown, J., Coulson, J.C., Morris, G.P., and Girdwood, R.W.A. 1993. Occurrence of oocysts of
Cryptosporidium spp. inLarus spp. gulls. Epidemiol. Infect., 110:135-143.
136
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Solo-Gabriele, H. and Meumeister, S. 1996. US outbreaks of cryptosporidiosis. J. AWWA, 61:76-86.
Sorvillo, F. J., Fujioka, K., Nahlen, B., Tormey, M.P., Kebabjian, R., and Mascola, L. 1992. Swimming-
associated cryptospoidiosis. Am. J. Public Health, 82:5:742-745.
Spano, F., Putignani, L, Crisanti, A., Sallicandro, P., Morgan, U.M., Le Blancq, S.M., Tchack, L., Tzipori, S.,
and Widmer, G. 1998a. Multilocus genotypic analysis of Cryptosporidium parvum isolates from different hosts
and geographical origins. J. Clin. Microbiol., 36:11:3255-3259.
Spano, F., Putignani, L, Guida, S., and Crisanti, A. 1998b. Cryptosporidium parvum: PCR-RFLP analysis of
the TRAP-C1 (thrombospondin-related adhesive protein of Cryptosporidium-1) gene discriminates between two
alleles differentially associated with parasite isolates of animal and human origin Exp. Parasitol., 90:2:195-198.
Spano, F., Putignani, L, McLauchlin, J., Casemore, D.P., and Crisanti, A 1997. PCR-RFLP analysis of the
Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum., and
between C. parvum isolates of human and animal origin. FEMS Microbiol. Lett., 150:2:209-217.
Sreter, T., Varga, I, and Bekesi, L. 1996. Effects of bursectomy and thymectomy on the development of
resistance to Cryptosporidium baileyi'm chickens. Parasitol. Res., 82:174-177.
States, S., Stadterman, K., Ammon, L., Vogel, P., Bladizar, J., Wright, D., Conley, L., and Sykora, J. 1997.
Protozoa in river water: sources, occurrence, and treatment. J. AWWA, 89:9:74-83.
Stinear, T., Matusan, A., Hines, K., and Sandery, M. 1996. Detection of a single viable Cryptosporidium
parvum oocyst in environmental water concentrates by reverse transcription-PCR. Appl. Env. Microbiol.,
62:3385-3390.
Stone, D.L., Dickson, K., Goven, A., and Cairns, S. 1997. Comparison of direct and indirect 'panning'
techniques for clarification of Cryptosporidium parvum from aqueous samples. Lett. Appl. Microbiol., 25:415-
418.
Straub, T., Mena, H., and Gerba, C. 1994. Viability of Giardia muris and Cryptosporidium parvum oocysts after
aging, pressure, pH manipulations, and disinfection in mountain reservoir water. Proc. 94th Am. Soc.
Microbiol. Gen. Meeting, Las Vegas, NV.
Sulaiman, I.M., Xiao, L., Yang, C., Escalante, L., Moore, A., Beard, C.B., Arrowood, M.J., and Lai, A. A. 1998.
Differentiating human from animal isolates of Cryptosporidium parvum. Emerg. Infect. Dis., 4:4:681-685.
Taghi-Kilani, R., Sekla, L, and Hayglass, K.T. 1990. The role of humoral immunity in Cryptosporidium spp.
infection: studies with B cell-depleted mice. J. Immunol., 145:1571-1576.
Tanyuksel, M., Gun, H., and Doganci, L. 1995. Prevalence of Cryptosporidium spp. in patients with neoplasia
and diarrhea. Scand. J. Infect. Dis., 27:69-70.
137
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Tee, G.H, Moody, A.H., Cooke, A.H., and Chiodini, P.L. 1993. Comparison of techniques for detecting
antigens ofGiardia lamblia and Cryptosporidiumparvum in faeces. J. Clin. Pathol., 46:555-558.
Teunis, P.F.M. and Havelaar, A.H. Cryptosporidium in drinking water: evaluation of the ILSI/RSI quantitative
risk assessment framework. RIVM Report no. 284 550 006. National Institute of Public Health and the
Environment (RIVM), The Netherlands, 1999.
Timms, S., Slade, J.S., and Fricker, C.R. 1995. Removal of Cryptosporidium by slow sand filtration. Wat. Sci.
Tech., 31:5-6:81-84.
Ungar, B.L.P. 1990. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal
specimens. J. Clin. Microbiol., 28:11:2491-2495.
Ungar, B.L.P., Burris, J.A., Quinn, C.A., and Finkelman, F.D. 1990. New mouse modelsfor chronic
Cryptosporidium infection in immunodeficient hosts. Infect. Immun., 58:961-969.
Ungar, B.L.P., Kao, T.-C., Burris, J.A., and Finkelman, F.D. 1991. Cryptosporidium infection in an adult mouse
model: independent roles for IFN-alpha and CD4 T lymphocytes in protective immunity. J. Immunol.,
147:1014-1022.
Ungar, B.L.P., Soave, R., Payer, R., and Nash, T.E. 1986. Enzyme immunoassay detection of immunoglobulin
M and G antibodies to Cryptosporidium in immunocompetent and immunocompromised persons. J. Infect. Dis.,
153:570-578.
Ungareau, E.M. and Dontu G.E. 1992. A new staining technique for the identification of Cryptosporidium
oocysts in faecal smears. Trans. Royal Soc. Trop. Med. Hyg., 86:638.
Upton, S.J., Tilley, M., and Brillhart, D.B. 1994a. Comparative development of Cryptosporidium parvum
(Apicomplexa) in 11 continuous host cell lines. FEMS Microbiol. Lett., 118:233-236.
Upton, S.J., Tilley, M., and Brillhart, D.B. 1995. Effects of select medium supplements on in vitro development
of Cryptosporidium parvum in HCT-^ cells. J. Clin. Microbiol., 33:2:371-375.
Upton, S.J., Tilley, M., Nesterenko, M.V., and Brillhart, D.B. 1994b. A simple and reliable method of
producing in vitro infections of Cryptosporidium parvum (Apicomplexa). FEMS Microbiol. Lett., 118:45-50.
USEPA. 1994. Draft Drinking Water Criteria Document for Cryptosporidium. Prepared by Clement
International Corporation. Prepared for EPA Office of Water, Office of Science and Technology, Washington,
D.C. June 1994.
USEPA. 1996a. Monitoring Requirements for public drinking water supplies. Final rule. Fed. Reg.,
61:94:24354.
138
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
USEPA. 1996b. Field Recovery of Giardia cysts and Cryptosporidium oocysts Draft Report. EPA Contract
68-C5-3909. Work AssignmentB-03. Office of Science and Technology.
USEPA. 1998. Interim Enhanced Surface Water Treatment Rule. Proposed Rule. Fed. Reg.,
63:241:69477-69521.
USEPA. 1999a. Cryptosporidium spp. Systematics and Waterborne Challenges in Public Health: Summary
Report. United States Environmental Protection Agency, Office of Water, Office of Science and Technology,
Health and Ecological Criteria Division.
USEPA. 1999b. Method 1622: Cryptosporidium in water by filtration/IMS/FA. United States Environmental
Protection Agency, Office of Water. EPA-821-R-99-001.
USEPA. 2000. Long Term 1 Enhanced Surface Water Treatment and Filter Backwash Rule. Proposed Rule.
Fed. Reg. 65:69:19045-19094.
USEPA. 2001. Cryptosporidium: Risk for Infants and Children. United States Environmental Protection
Agency, Office of Water, Washington, DC.
Valdez, L.M., Dang, H., Okhuysen, P.C., and Chappell, C.L. 1997. Flow cytometric detection of
Cryptosporidium oocysts in human stool samples. J. Clin. Microbiol., 35:8:2013-2017.
Varga, I, Sreter, T., and Bekesi, L. 1995. Quantitative method to assess Cryptosporidium oocyst shedding in the
chicken model. Parasitol.Res., 81:262-264.
Vargas, S.L., Shenep, JL., Flynn, P.M., Pui, C.H., Santina, V.M., and Hughes, W.T. 1993. Azithromycin for
treatment of severe Cryptosporidium diarrhea in two children with cancer. J. Fed., 123:154-156.
Veal, D., Vesey, G., Fricker, C., Ongerth, J., LeMoenic, S., Champion, A., Rossington, G., and Faulkner, B.
Routine flow cytometric detection of Cryptosporidium and Giardia recovery rates and quality control. In: 1997
Int. Symp. Waterborne Cryptosporidium Proc., Fricker et al. (eds), AWWA, Newport Beach, CA, 1997.
Venczel, L.V., Arrowood, M., Kurd, M., and Sobsey, M.D. 1997. Inactivation of Cryptosporidiumparvum
oocysts and Clostridiumperfringens spores by a mixed-oxidant disinfectant and by free chlorine. Appl. Env.
Microbiol., 63:4:1598-1601.
Vesey, G., Griffiths, K.R., Gauci, M.R., Deere, D., Williams, K.L., and Veal, D.A. 1997. Simple and rapid
measurement of Cryptosporidium excystation using flow cytometry. Int. J. Parasitol., 27:11:1353-1359.
Vesey, G., Hutton, P., Champion, A., Ashbolt, N., Williams, K., Warton, A., and Veal, D. 1994. Application of
flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry, 16:1-6.
Vesey, G., Slade, J., Byrne, M., Shepherd, K., Dennis, P., and Fricker, C. 1993a. Routine monitoring of
Cryptosporidium oocysts in water using flow cytometry. J. Appl. Bacteriol., 75:87-90.
139
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Vesey, G., Slade, J., Byrne, M., Shepherd, K., and Fricker, C. 1993b. A new method for the concentration of
Cryptosporidium oocysts from water. J. Appl. Bacteriol., 75:82-86.
Wagner-Wiening, C. and Kimmig, P. 1995. Detection of viable Cryptosporidium parvum oocysts by PCR.
Appl. Env. Microbiol., 61:4514-4516.
Walker, M.J., Montemagno, C.D., and Jenkins, M.B. 1998. Source water assessment and nonpoint sources of
acutely toxic contaminants: a review of research related to survival and transport of Cryptosporidium parvum.
Wat. Resour. Res., 34:12:3383-3392.
Wallis, P.M., Erlandsen, S.L., Isaac-Renton, J.L., Olson, M.E., Robertson, W.J., and Van Keulen, H. 1996.
Prevalence ofGiardia cysts and Cryptosporidium oocysts and characterization ofGiardia spp. isolated from
drinking water in Canada Appl. Env. Microbiol., 62:8:2789-2797.
Webster, K.A. 1993. Molecular methods for the detection and classification of Cryptosporidium. Parasitol.
Today, 9:7:263-266.
Wee, S., Lee, C., and Joo, H. 1995. Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence
assay using monoclonal antibody. Kor. J. Parasitol., 33:2:107-115.
Whitmore, T.N. and Carrington, E.G. 1993. Comparison of methods for recovery of Cryptosporidium from
water. Wat. Sci .Tech., 27:3-4:69-76.
Widmer, G., Tchack, L, Chappell, C.L., and Tzipori, S. 1998a. Sequence polymorphism in thebeta-tubulin
gene reveals heterogeneous and variable population structures in Cryptosporidium parvum. Appl. Env.
Microbiol., 64:11:4477-4481.
Widmer, G., Tchack, L., Spano, F., and Tzipori, S. 1998b. A study of Cryptosporidium parvum genotypes and
population structure. Mem. Inst. Oswaldo Cruz, 93:5:685-686.
Widmer, G., Tzipori, S., Fichtenbaum, C.J., and Griffiths, J.K. 1998c. Genotypic and phenotypic
characterization of Cryptosporidium parvum isolates from people with AIDS. J. Infect. Dis., 178:3:834-840.
Woods, K., Nesterenko, M., and Upton, S. 1995. Development of microtitre ELISAto quantify development of
Cryptosporidium parvum in vitro. FEMS Microbiol. Lett., 128:89-94.
Wyatt, C., Brackett, E., and Ferryman, L. 1996. Characterization of small intestine mucosal lymphocytes during
cryptosporidiosis. J. Eukaryot. Microbiol., 43:5:668.
Xiao, L., Escalante, L., Yang, C., Sulaiman, L, Escalante, A.A., Montali, R.J., Payer, R., and Lai, A.A. 1999a.
Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl. Env.
Microbiol., 65:4:1578-1583.
140
-------
Drinking Water Criteria Document Addendum: Cryptosporidium March 2001
Xiao, L. and Herd, R.P. 1993. Quantitation ofGiardia cysts and Cryptosporidium oocysts in fecal samples by
direct immunofluorescence assay. J. Clin. Microbiol., 31:2944-2946.
Xiao, L., Morgan, U.M., Limor, J., Escalante, A., Arrowood, M., Shulaw, W., Thompson, R.C., Payer, R., and
Lai, A.A. 1999b. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl.
Env. Microbiol., 65:8:3386-3391.
You, X., Arrowood, MJ.,Lejkowski, M., Xie, L., Schinazi, R.F., and Mead, J.R. 1996a. In vitro evaluation of
anticryptosporidial agents using MDCK cell culture and chemiluminescence immunoassay. J. Eukaryot.
Microbiol.,43:5:878.
You, X., Arrowood, M.J., Lejkowski, M., Xie, L., Schinazi, R.F., and Mead, J.R. 1996b. A chemiluminescence
immunoassay for evaluation of Cryptosporidium parvum growth in vitro. FEMS Microbiol. Lett., 136:251-256.
Zerpa, R. and Huicho, L. 1994. Childhood cryptosporidial diarrhea associated with identification of
Cryptosporidium sp. in the cockroachPeriplaneta americana. Fed. Infect. Dis. J. 13:6:546-548.
Zu, S.-X., Li, J.-F., Barrett, L.J., Payer, R., Shu, S.-Y., McAuliffe, J.F., Roche, J.K., and Guerrant, R.L. 1994.
Seroepidemiologic study of Cryptosporidium infection in children from rural communities of Anhui, China and
Fortaleza, Brazil. Am. J. Trop. Med. Hyg., 51:1:1-10.
Zuckerman, U., Gold, D., Ghelef, G., Yuditsky, A., and Arm on, R. Microbial degradation of Cryptosporidium
parvum by Serratia marcescens with high chitinolytic activity. In: Proc. 1997 Int. Symp. Waterborne
Cryptosporidium. Flicker et al. (eds), Newport Beach, CA, 1997.
141
------- |