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Clinical Microbiology Reviews, January 2004, p. 72-97, Vol. 17, No. 1
0893-8512/04/$08.00+0 DOI: 10.1128/CMR.17.1.72-97.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health
Lihua Xiao,1* Ronald Fayer,2 Una Ryan,3 and Steve J. Upton4Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Chamblee, Georgia 30341,1 USDA, ARS, ANRI, Animal Waste Pathogen Laboratory, Beltsville, Maryland 20705,2 Division of Veterinary and Biomedical Sciences, Murdoch University, Western Australia 6150, Australia,3 Division of Biology, Kansas State University Manhattan, Kansas 665064
SUMMARY INTRODUCTION HISTORICAL PERSPECTIVE OF CRYPTOSPORIDIUM TAXONOMY SPECIES CONCEPT IN CRYPTOSPORIDIUM VALID CRYPTOSPORIDIUM SPECIES Cryptosporidium Species of Mammals Cryptosporidium muris Tyzzer, 1907. Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000. Cryptosporidium parvum Tyzzer, 1912. Cryptosporidium canis Fayer, Trout, Xiao, Morgan, Lal, and Dubey, 2001. Cryptosporidium felis Iseki, 1979. Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971. Cryptosporidium hominis Morgan-Ryan, Fall, Ward, Hijjawi, Sulaiman, Fayer, Thompson, Olson, Lal, and Xiao, 2002. Cryptic species. Cryptosporidium Species of Birds Cryptosporidium meleagridis Slavin, 1955. Cryptosporidium baileyi Current, Upton, and Haynes, 1986. Cryptosporidium galli Pavlasek, 1999. Cryptic species. Cryptosporidium Species of Reptiles Cryptosporidium serpentis Levine, 1980. Cryptosporidium saurophilum Koudela and Modry, 1998. Cryptic species. Cryptosporidium Species of Fish Cryptosporidium molnari Alvarez-Pellitero and Sitja-Bobadilla, 2002. Cryptic species. HOST-PARASITE COEVOLUTION AND HOST ADAPTATION IN CRYPTOSPORIDIUM: IMPLICATIONS FOR TAXONOMY Host-Parasite Coevolution Host Adaptation Implications for Taxonomy CRITERIA FOR NAMING CRYPTOSPORIDIUM SPECIES Oocyst Morphology Genetic Characterizations Natural Host Specificity Compliance with ICZN PUBLIC HEALTH IMPORTANCE OF CRYPTOSPORIDIUM TAXONOMY Identity of Cryptosporidium Species in Humans Significance of Cryptosporidium Species in Animals and the Environment Infection and Contamination Sources Implications for the Water Industry CONCLUDING REMARKS ACKNOWLEDGMENTS REFERENCES
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There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades. This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp. A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species. All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN. Altogether, 13 Cryptosporidium spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis, and C. cannis in mammals; C. baïleyi, C. meleagridis, and C. galli in birds; C. serpentis and C. saurophilum in reptiles; and C. molnari in fish. With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium. The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp., assessing the public health significance of Cryptosporidium spp. in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources.
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Cryptosporidium species are apicomplexan parasites that infect the microvillus border of the gastrointestinal epithelium of a wide range of vertebrate hosts, including humans. Infected individuals show a wide spectrum of clinical presentations, but the pathogenicity of Cryptosporidium varies with the species of parasites involved and the type, age, and immune status of the host. In many animals, Cryptosporidium infections are not associated with clinical signs or are associated with only acute, self-limiting illness. In some animals, such as reptiles infected with Cryptosporidium serpentis or individuals who are immunosuppressed, the infection is frequently chronic and can eventually be lethal.
Cryptosporidiosis is a frequent cause of diarrheal disease in humans, and several groups of humans are particularly susceptible to cryptosporidiosis. In developing countries, Cryptosporidium infections occur mostly in children younger than 5 years, with peak occurrence of infections and diarrhea in children younger than 2 years (21, 22, 154). Children can have multiple episodes of cryptosporidiosis, indicating that acquired immunity to Cryptosporidium infection is short-lived or incomplete (154, 248). In industrialized countries, epidemic cryptosporidiosis can occur in adults by the food-borne or waterborne route (117, 124, 191). In immunocompromised persons such as human immunodeficiency virus-positive (HIV+) patients, the incidence and severity of cryptosporidiosis increases as the CD4+ lymphocyte cell count falls, especially when it falls to below 200 cells/µl (152, 188, 198).
Because Cryptosporidium spp. infect humans and a wide variety of animals and because of the ubiquitous presence of Cryptosporidium oocysts in the environment, humans can acquire Cryptosporidium infections through several transmission routes (45, 80). In pediatric and elderly populations, especially in day care centers and nursing homes, person-to-person transmission probably plays a major role in the spread of Cryptosporidium infections (153, 214). In rural areas, zoonotic infections via direct contact with farm animals have been reported many times, but the relative importance of direct zoonotic transmission of cryptosporidiosis is not entirely clear (125). Numerous outbreaks of cryptosporidiosis due to contaminated food or water (drinking or recreational) have been reported in several industrialized nations, and studies have sometimes identified water as a major route of Cryptosporidium transmission in areas where the disease is endemic (67, 116, 156, 239). The sources and human infective potentials of Cryptosporidium oocysts in water, however, are largely unclear.
One major problem in understanding the transmission of Cryptosporidium infection is the lack of morphologic features that clearly differentiate one Cryptosporidium sp. from many others (60) (Fig. 1). Hence, one cannot be sure which Cryptosporidium sp. is involved when one examines oocysts in clinical specimens under a microscope. Another major problem is the inability to grow the organisms in large numbers from contaminated sources. Adding to the diagnosis problem and technical difficulties is the confusion in the taxonomy of Cryptosporidium spp., which is partially caused by the lack of consistency in the classification of protozoan parasites in general.
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FIG. 1. Oocysts of C. parvum and some C. parvum-related species. Modified from reference 247.
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TABLE 1. Similarity in morphometric measurements of oocysts of C. parvum and C. parvum-related Cryptosporidium spp.a
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The first individual to establish the genus Cryptosporidium and to recognize its multispecies nature was Ernest Edward Tyzzer, who described the type species, C. muris, from the gastric glands of laboratory mice (225). He later published a more complete description of the life cycle (226) and subsequently described a second species, also from laboratory mice (227). C. parvum differed from the type species not only by infecting the small intestine rather than the stomach but also because the oocysts were smaller (227, 232).
Following the initial discovery of Cryptosporidium, over 50 years elapsed during which the parasite was commonly confused with other apicomplexan genera, especially members of the coccidian genus Sarcocystis. Because many Sarcocystis spp. have oocysts with thin walls that often rupture, releasing free sporocysts, and because each sporocyst contains four sporozoites like Cryptosporidium oocysts, a variety of named and unnamed species were erroneously assigned to the genus (11, 20, 53, 54, 77, 169, 224, 240). Subsequent ultrastructural studies, however, supported earlier light microscopy studies and reaffirmed endogenous stages of Cryptosporidium spp. to possess a unique attachment organelle (84, 93, 236). This attachment organelle, rather than the oocysts, is the key feature that currently defines the genus and family (230), but it has actually been an integral component of the taxonomic definition of the family since at least 1961 (102, 103, 105).
After the recognition of true differences between Cryptosporidium and Sarcocystis, the erroneous concept of strict host specificity (181) was applied to Cryptosporidium spp. This led to the creation of multiple new species including C. agni in sheep, C. anserinum in geese, C. bovis in calves, C. cuniculus in rabbits, C. garnhami in humans, and C. rhesi in monkeys (18, 23, 89, 104, 190). Subsequent cross-transmission studies demonstrated that Cryptosporidium isolates from different animals can frequently be transmitted from one host species to another, which ended the practice of naming species based on host origin and the synonymization of many of these new Cryptosporidium species as C. parvum. However, for a brief period, these very limited transmission studies were used as evidence for the monospecific nature of the genus Cryptosporidium, resulting in the widespread use of the name C. parvum for Cryptosporidium parasites from all kinds of mammals, including humans. Several Cryptosporidium parasites named during or before the period, such as C. meleagridis in turkeys (197), C. wrairi in guinea pigs (236), and C. felis in cats (90), however, survived because of the demonstrated biological differences from the established species C. parvum and C. muris. More recently, several other Cryptosporidium spp. were also named in a less haphazard fashion, such as C. baileyi in birds (49) and C. saurophilum in lizards (97), all based on biological differences from other established Cryptosporidium species.
In recent years, molecular characterizations of Cryptosporidium have helped to clarify the confusion in Cryptosporidium taxonomy and validate the existence of multiple species in each vertebrate class. As a result, several new species of Cryptosporidium have also been named. Thus, C. andersoni from cattle, C. canis from dogs, C. hominis from humans, and C. molnari from fish were all established by using multiple parameters that included not only morphology but also developmental biology, host specificity, histopathology, and/or molecular biology (4, 63, 111, 146).
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One major reason for the long disputes in Cryptosporidium taxonomy is the difficulty in fulfilling the definition of biological species. The classical definition of species as groups of interbreeding natural populations reproductively isolated from other groups (119) is difficult to apply to many organisms like Cryptosporidium, because it is very difficult to conduct genetic crossing studies with many Cryptosporidium spp. Even though Cryptosporidium has a sexual stage and intraspecies sexual recombination has been demonstrated in C. parvum (65, 118), the huge reproductive potential of the parasite results in vast numbers of genetically similar parasites in localized areas. Therefore, mating in Cryptosporidium normally occurs between siblings. As a result, Cryptosporidium has a large bias toward a clonal population structure, as demonstrated by multilocus analysis (13, 72, 212).
Currently, morphology, especially oocyst measurements, represents the cornerstone of apicomplexan taxonomy. Measurements allow microscopists to identify large numbers of genera and morphologically distinct species, and the importance of a good morphologic description cannot be understated. Therefore, oocyst structure is usually one of the requirements for establishing a new species. However, for Cryptosporidium, morphology is not adequate by itself and should not be the sole criterion for naming a new species. Oocysts of many species are virtually identical in size, and similarities in oocyst structure have even caused confusion about the historical validity of several Cryptosporidium spp.
Because oocyst morphometrics alone is not entirely adequate for descriptions of new species of Cryptosporidium, other characteristics must be included in the taxonomic description. In many cases, experimental transmission followed by light microscopy and sometimes electron microscopy of endogenous stages has proven useful. Cases in point include the paper by Current and Reese (47), who provided an excellent account of the life cycle of C. parvum in experimentally infected mice by using a combination of light and electron microscopy. Current et al. (49) then published an account of the life cycle of C. baileyi in chickens and not only pointed out the morphologic differences in oocyst structure between C. parvum and C. baileyi but also showed that C. baileyi possessed a third type of merogonous stage not seen in C. parvum. More recently, Alvarez-Pellitero and Sitja-Bobadilla (4) utilized light microscopy and ultrastructure to provide an elaborate description of C. molnari in two species of marine teleosts. Nonetheless, a strict requirement for life cycle studies in all taxonomic works seems impractical for two reasons. First, distinct species may have similar endogenous development. Second, and equally important, many host species fail to lend themselves easily to animal experimentation. This latter point is especially true of oocysts derived from rare, exotic, venomous, excessively expensive, or very large hosts, making animal studies prohibitive.
Infectivity has sometimes been used to characterize and compare various Cryptosporidium isolates, and many carefully controlled infectivity studies have been published (162-164, 186, 215). Even though infectivity can be used as a good general indicator of host susceptibility and oocyst viability, real quantitative data are limited. Numerous variables affect parasite development, including dosage, oocyst age, oocyst storage conditions, the isolate employed, chemical pretreatments of the oocysts, the age, size, and previous exposure history of the host, whether mixed isolates are represented, and host genetics. For example, Upton and Gillock (233) showed how age and weight alone in ICR outbred suckling mice had dramatic impacts on the numbers of oocysts recovered from experimentally infected suckling mice. Enriquez and Sterling (59) examined C. parvum infections in 19 different strains of adult mice and found that the beige mouse (C57BL/6J-bgJ) harbored the highest levels of infection, with only scant numbers being found in other strains of mice.
In addition to infectivity, host specificity (the broad range of different hosts that can be infected by any one isolate) can prove highly useful when dealing with isolates derived from commonly encountered hosts. For example, one of the earliest ways in which C. andersoni in cattle was distinguished from the morphologically similar C. muris in rodents was by the fact that the former species was never infectious for outbred, inbred, neonatal, or immunocompetent mice (111). Likewise, C. hominis in humans has long been known to have a much narrower host range than the morphologically similar C. parvum, and cross-transmission studies help distinguish between the two (3, 146). Caution should be used when interpreting negative transmission results, however. Even though the lack of ability to infect mice and goats in cross-transmission studies was used as evidence for the separation of C. andersoni from C. muris (111), thus far it has been difficult to infect cattle of different ages and breeds with C. andersoni of bovine origin (9). The earlier conclusion that C. hominis does not infect experimental animals such as mice, calves, lambs, and pigs is apparently premature, since recent studies have clearly shown that calves, lambs, and piglets can be infected with C. hominis (3, 55). "Genotype switching" (a different genotype of oocysts obtained after inoculation with one genotype of oocysts) has also been observed in cross-transmission studies (63, 244). These results are important because they demonstrate that populations of oocysts derived from an individual animal may have low levels of contaminating minor species, which can further compound the interpretation of cross-transmission studies. Nonetheless, determination of at least some aspects of host range can provide highly useful information to support morphologic and genetic data and should be encouraged for as many species accounts as feasible.
Biochemical differences can potentially be used as one criterion in defining Cryptosporidium spp. Early on, restriction fragment length polymorphism analysis (RFLP) of genomic DNA (160), isozyme analysis (14-16, 56, 161), two-dimensional gel electrophoresis (122, 219), and protein or carbohydrate surface labeling of oocysts, sporozoites, or homogenates (112, 115, 155, 157, 158, 217, 218, 220, 222) were all used in an attempt to define both interspecific and intraspecific differences in Cryptosporidium. Differences in protein electrophoretic profiles between C. parvum bovine isolates and C. wrairi lent strong support to the validity of C. wrairi (221). Overall, these methods have proven expensive, technically challenging, and impractical. Not only is there no guarantee that different species would not have identical banding patterns when zymography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis is used, but also relatively large numbers of parasites need to be used in some assays. Typically 107 to 108 highly purified oocysts are used, making repeatability, and sometimes even the initial experiments, impractical without passage and bioamplification in additional hosts.
Recent molecular studies have uncovered an overwhelming amount of genetic diversity within the genus Cryptosporidium (37, 50, 61, 141, 252, 255). In recent years, genetic differences have become a key essential element in defining several new Cryptosporidium spp., such as C. andersoni, C. canis, C. hominis, and C. galli. Thus far, genetic differences identified at the species or genotype level correlate well with other biological characteristics such as the spectrum of natural hosts and infectivity in cross-transmission studies. With further verification, genetic characteristics should play an even greater role in delineating and defining Cryptosporidium spp. Confusion exists, however, about how to distinguish interspecies differences from intraspecific allelic diversity and how much emphasis should be placed on results of molecular analysis.
Because of the uncertainty associated with the extent of intraspecific allelic variation in Cryptosporidium taxonomy, numerous Cryptosporidium genotypes have been described without a designation of species being given or with them all being lumped into C. parvum. Presently, the identification and naming of genotypes is based largely on host origin. When significant or consistent sequence differences from existing genetic data are identified, a new genotype is named after the host from which it was isolated. Although this genotype designation scheme generally reflects significant genetic differences among Cryptosporidium isolates and tends to correlate well with biological differences whenever data are available, not all genotypes differ from each other to the same extent. Thus, some genotypes exhibit extensive nucleotide differences from congenerics whereas others are very similar to each other. The term "subgenotype" is sometimes used to describe relatively minor intragenotypic variations. The use of genotypes and subgenotypes tends to be difficult for researchers in other fields to comprehend. The application of a species designation for some of the well-characterized Cryptosporidium genotypes is useful since it helps relieve much of the confusion.
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Named species of Cryptosporidium that are currently considered valid species now include C. andersoni (cattle), C. baileyi (chicken and some other birds), C. canis (dogs), C. felis (cats), C. galli (birds), C. hominis (humans), C. meleagridis (birds and humans), C. molnari (fish), C. muris (rodents and some other mammals), C. parvum (ruminants and humans), C. wrairi (guinea pigs), C. saurophilum (lizards and snakes), and C. serpentis (snakes and lizards) (Table 2). Other morphologically distinct Cryptosporidium spp. have been found in fish (4), reptiles (234), birds (107), and mammals (61, 255) but have not been named.
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TABLE 2. Valid Cryptosporidium species
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Mammals represent the largest group of animals known to be infected with Cryptosporidium spp., probably due to the greater number of studies as a result of the perceived importance of these animals. The taxonomy of Cryptosporidium in mammals has been the subject of dispute since 1980, and for some time only two species (C. parvum as the intestinal species and C. muris as the gastric species) were recognized (229, 252). We now know that there is enormous biological and genetic diversity in mammalian Cryptosporidium spp., and because of a plethora of molecular studies, multiple new species have been discovered and described.
Cryptosporidium muris Tyzzer, 1907. In 1907, Ernest Edward Tyzzer described a protozoan parasite that he frequently observed in the gastric glands of laboratory mice but not wild mice (225). The asexual meront stage contained six merozoites, each with a distinct nucleus. Sexual stages were observed and measured. All stages were thought to be extracellular, with an unusual knoblike attachment organelle similar to a gregarine epimerite. Spore (oocyst) formation was described, with oocysts measuring about 7 by 5 µm, and fecal-oral transmission was demonstrated. Although Tyzzer thought that the systematic position was uncertain, he nevertheless suggested the name Cryptosporidium muris. Three years later he extended the geographic range of the parasite in Mus musculus from North America to include England. A more detailed description of each life cycle stage (with measurements, drawings, and photographs) was later provided, and all stages were found to localize in the gastric glands of the stomach (226). Sporozoites liberated from oocysts in the gastric glands were thought to be autoinfectious; this has been found to be true for other Cryptosporidium spp. (47, 49). Nonetheless, pathology appeared to be slight. Experimental transmission to other mice was successful, but an attempt to infect a rat was not.
Experimental transmission studies using specific-pathogen-free laboratory rats revealed that a large type of Cryptosporidium oocysts from wild rats trapped in Osaka City developed only in the gastric glands of the stomach. The oocysts measured 8.4 by 6.3 µm and could be transmitted to uninfected rats. Oocysts from this study, identified as C. muris strain RN66, were later used for cross-transmission studies in which mice, guinea pigs, rabbits, dogs, and cats all became infected. Development occurred in the stomach and not the intestine, and oocysts were passed by all hosts (91). Based solely on morphology, C. muris or C. muris-like oocysts have been found in the feces of cattle in the United States (10, 232), Brazil (123, 182), Scotland (30), and Japan, (94) and in cattle and camels in Iran (159). Because species identification was not confirmed genetically or experimentally, many of these authors qualified their findings by calling the organism C. muris-like. Recent molecular characterizations of C. muris and C. muris-like parasites have indicated that all bovine isolates are C. andersoni. Recent studies have shown C. muris to be capable of infecting a wide range of additional hosts including hamsters, squirrels, Siberian chipmunks, wood mice (Apodemus sylvaticus), bank voles (Clethrionomys glareolus), Dolichotis patagonum, rock hyrax, bactrian camels, mountain goats, humans, and cynomolgus monkeys (10, 39, 52, 69, 82, 145, 168, 216, 223, 256; L. Xiao, unpublished data).
Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000. C. andersoni infects the abomasum of cattle and produces oocysts morphologically similar to, but slightly smaller than, those of C. muris (111). It was named after Bruce Anderson, University of Idaho, the original finder of the parasite. Oocysts, passed fully sporulated, were ellipsoid, lacked sporocysts, and measured 7.4 by 5.5 (6.0 to 8.1 by 5.0 to 6.5) µm, with a length/width ratio of 1.35. Unlike those of C. muris, oocysts of C. andersoni were not infectious for outbred, inbred immunocompetent, or immunodeficient mice, nor were they infectious for chickens or goats. C. andersoni was recognized early on to be poorly infective not only to nonbovine hosts but also to cattle. Thus, oocysts derived from cattle, previously identified as C. muris-like, were not infectious for mice or even cattle (10). Similar oocysts from cattle were not transmissible to neonatal or adult BALB/c mice, SCID mice, common voles, bank voles, common field mice, desert gerbils, guinea pigs, rats, rabbits, or goats; only Mongolian gerbils became infected (98). However, successful transmission of large Cryptosporidium oocysts from cattle to mice has been reported by Pavlásek (173) and Kaneta and Nakai (94). Whether these represent isolates of C. andersoni, an isolate of C. muris or C. andersoni with a wide host range, or contamination of the mice with C. muris is unknown. A Danish C. andersoni isolate was found to be infectious to cattle (58), and a so-called novel type of C. andersoni was identified in Japan based on its ability to infect SCID mice (196).
To overcome the conflicting cross-transmission data and the inability to morphologically differentiate oocysts of C. andersoni from those of C. muris, molecular methods have been employed in cross-transmission studies to confirm the species identification. Genetically confirmed C. andersoni infection has thus far been found only in cattle, bactrian camels, and a sheep (145, 256; U. M. Ryan, unpublished data).
Cryptosporidium parvum Tyzzer, 1912. The most frequently reported species in mammals, C. parvum, was first found in mice (227). It was differentiated from C. muris based on its smaller oocyst size and its location only in the villi of the small intestine, most frequently near the tips. Transmission experiments from mouse to mouse always resulted in infection of the small intestine as opposed to the stomach. All life cycle stages were described, measurements were provided, and photographs and camera lucida drawings were included. Tyzzer remarked that stages were not strictly extracellular, but he did not consider them intracytoplasmic because they were in contact with the inner or cytoplasmic surface of the cell. Mature oocysts were ovoidal or spheroidal and did not exceed 4.5 µm in greatest diameter. Upton and Current (232) gave measurements of 5.0 by 4.5 (4.5 to 5.4 by 4.2 to 5.0) µm and a length/width ratio of 1.16 for viable oocysts, and Tilley et al. (222) reported that the oocysts measured 5.2 by 4.6 (4.8 to 5.6 by 4.2 by 4.8) µm with a length/width ratio of 1.15 (1.04 to 1.22). Tyzzer (227) also observed similar organisms in the small intestine of a rabbit. Frequently, C. parvum infection involves both the small intestine and the colon (228).
Over 150 species of mammals have been identified as hosts of C. parvum or C. parvum-like parasites. Most descriptions, however, have been based solely on microscopy, with no careful morphometric measurements or genetic or other biological data. Recent molecular characterizations, however, have shown that there is extensive host adaptation in Cryptosporidium evolution, and many mammals or groups of mammals have host-adapted Cryptosporidium genotypes, which differ from each other in both DNA sequences and infectivity. Thus, these genotypes are clearly being delineated as distinct species and include C. hominis (previously termed the human genotype or genotype 1), C. parvum (also termed the bovine genotype or genotype 2), and C. canis (the dog genotype). Other genotypes have been associated with mouse, pig, bear, deer, marsupial, monkey, muskrat, skunk, cattle, and ferret (255). Most of these organisms probably represent individual Cryptosporidium species.
It is possible that the C. parvum isolate originally found in laboratory mice by Tyzzer (227) might be what we now recognize as the Cryptosporidium mouse genotype (132, 134, 136, 138, 257). This is because Tyzzer (225) was able to easily infect adult mice whereas C. parvum senso stricto tends to produce very low-level infections in these hosts. However, because C. parvum is also occasionally found in mice and because no type specimens were originally deposited, we can never be sure what Tyzzer was actually working with. Therefore, when Upton and Current (232) provided a modern morphologic description of the oocysts and Current and Reese (47) provided in-depth life cycle and cross-transmission studies between mice and cattle, they essentially validated the name C. parvum for the bovine genotype. Even if one were to reject this argument and attempt to resurrect C. bovis Barker and Carbonell, 1974, for the bovine genotype in cattle (18), Article 23.9 of the International Code of Zoological Nomenclature (ICZN) specifically addresses reversal of priority in cases that may result in confusion. Specifically, Article 23.9.1.2 states that prevailing usage must be maintained when "the junior synonym or homonym has been used for a particular taxon, as its presumed valid name, in at least 25 works, published by at least 10 authors in the immediately preceding 50 years and encompassing a span of not less than 10 years." Clearly, C. parvum would fall into this category. We recommend the use of C. parvum for the Cryptosporidium parasites previously known as the bovine genotype and avoid the use of C. parvum broadly for Cryptosporidium in mammals.
Thus far, C. parvum is known to infect mainly ruminants (cattle, sheep, goats, and deer) and humans. Earlier reports of naturally occurring C. parvum infections in pigs and mice (129, 138) have yet to be confirmed by other researchers.
Cryptosporidium canis Fayer, Trout, Xiao, Morgan, Lal, and Dubey, 2001. Cryptosporidium oocysts have been observed in the feces of dogs worldwide (reviewed in reference 63). Oocysts from the feces of a naturally infected dog measured 4.95 by 4.71 µm and had a length/width ratio of 1.05 (63). These oocysts were morphologically indistinguishable from those of C. parvum and possessed common surface antigens. Oocysts from the dog were infectious for a calf, but, unlike those of C. parvum, they were not infectious for neonatal BALB/c or for dexamethasone-treated and untreated C57BL6/N mice. Oocysts obtained from a human source were also infectious for a calf, and sequence analysis of the small-subunit (SSU) rRNA and HSP70 showed that these two isolates were identical to the dog genotype previously identified (144, 213, 257). Based on its ability to infect humans and bovines but its inability to infect mice, as well as significant genetic differences from other Cryptosporidium spp., the parasite was named C. canis (63). Confirmed C. canis infections have been found in dogs, coyotes, foxes, and humans (144, 177, 187, 248, 255, 257).
Cryptosporidium felis Iseki, 1979. The first report of Cryptosporidium in cats included a description of the oocyst from the feces, basic observations of endogenous development, and some work on host specificity, and pathogenicity (90). Oocysts measuring 5 by 4.5 µm were fed to four cats, three 7-week-old ICR mice, and three 180- to 200-g guinea pigs. Oocysts were found only in the feces of three cats. Prepatent and patent periods were 5 to 6 and 7 to 10 days postinoculation, respectively. Mtambo et al. (148) obtained oocysts of two sizes from a farm cat and fed these to two lambs, which also shed similar sized oocysts, of 6.0 by 5.0 and 5.0 by 4.5 µm. Oocysts from the lambs were subsequently fed to 20 mice, of which 19 became infected, in contrast to 0 of 10 mice fed oocysts from the cat. Mtambo et al. (148) attributed the lack of infectivity for mice of oocysts from the cat as possibly due to prolonged storage. However, another explanation may be that the lambs acquired an extraneous infection with C. parvum during the time they were being examined for oocyst shedding. Molecular testing would have been necessary to clarify these findings.
Even though the validity of C. felis was in doubt for some years, recent molecular characterizations at the SSU rRNA, ITS-1, HSP70, COWP, and actin loci support the concept of C. felis as a valid species. All Cryptosporidium isolates from cats characterized have thus far shown significant sequence differences from other known Cryptosporidum spp. and genotypes. In addition, all are very similar to each other even though they are from different geographic regions (134, 137, 211, 213, 242, 250). Confirmed C. felis infections have been found in cats, humans, and cattle (27, 33, 127, 137, 177, 187, 248, 257).
Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971. Cryptosporidium wrairi from the guinea pig (Cavia porcellus) was named as an acronym for the Walter Reed Army Institute of Research (236). Only small guinea pigs (weighing 200 to 300 g) were usually found to be infected. Infection was not associated with diarrhea or overt signs of coccidiosis, but only with enteritis (93, 236). Asexual and sexual stages were described, and fresh mucosal scrapings containing these stages were photographed. The times at which developmental stages were observed were recorded, but oocysts were never recognized as such. However, they may have been mistakenly misidentified as second-generation meronts containing four merozoites. Mucosa scraped from the distal ileum was delivered by gastric gavage to 3-week-old rabbits, chickens, and turkeys as well as young guinea pigs. Only the guinea pigs became infected, and this occurred only when scrapings were obtained 6 to 9 days after inoculation. The ultrastructure of all intracellular stages except mature microgametes, zygotes, and oocysts was described (237). Again, a micrograph identified as a second-generation meront containing three or four merozoites might actually be a developing oocyst.
Initially, cross-transmission studies suggested that C. parvum and C. wrairi might actually be the same species. Angus et al. (12) were able to transmit the parasite not only between guinea pigs but also to infant mice and lambs, even though it was not clear that this was the same species as that described by Vetterling et al. (236). Chrisp et al. (44) raised 23 monoclonal antibodies to C. parvum and 12 to C. wrairi, and they all reacted with equal intensity with the heterologous species. However, despite this close antigenic relationship, C. wrairi was not infectious for SCID mice whereas C. parvum was.
When Cryptosporidium from guinea pigs and C. parvum were compared morphologically, oocysts from guinea pigs measured 5.4 by 4.6 (4.8 to 5.6 by 4.0 to 5.0) µm and had a length/width index of 1.17 and those of C. parvum were similar in size and measured 5.2 by 4.6 µm with an index of 1.16 (221). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice inoculated with the guinea pig isolate became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts than did mice infected with C. parvum, and the infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins of C. parvum had a wide molecular size range of labeled bands whereas Cryptosporidium from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with fewer bands greater than 100 kDa (221). Overall, these biological, immunological, and chemical labeling methods were confusing and inconclusive. More recently, molecular characterizations have identified significant differences between C. parvum and C. wrairi at multiple genetic loci (43, 147, 200, 201, 211, 213, 257). These combined data, along with the fact that naturally occurring C. wrairi infections have been found only in guinea pigs, strongly suggest that this organism is a different species from C. parvum.
Cryptosporidium hominis Morgan-Ryan, Fall, Ward, Hijjawi, Sulaiman, Fayer, Thompson, Olson, Lal, and Xiao, 2002. Cryptosporidium parasites infecting humans, previously designated C. parvum human genotype, genotype 1, or genotype H, have been delineated as a separate species, C. hominis, based on molecular and biological differences (146). Numerous studies during the past several years showed not only a plethora of genetic and biological differences but also largely a lack of genetic exchange between this parasite and C. parvum (bovine genotype or genotype 2). C. hominis is morphologically identical to C. parvum, 4.6 to 5.4 by 3.8 to 4.7 µm (mean, 4.2 µm) with a length/width ratio of 1.21 to 1.15 (mean, 1.19). Unlike C. parvum, C. hominis is traditionally considered noninfective for mice, rats, cats, dogs, and cattle (73, 146, 185, 241, 242). However, more recently, C. hominis has been reported from a dugong and a lamb, and calves, lambs, and piglets can also be infected experimentally with at least some C. hominis isolates at high doses (3, 55, 73, 142). Pathogenicity studies with gnotobiotic pigs have shown the prepatent period to be longer than for C. parvum (8.8 and 5.4 days, respectively) and have also shown differences in parasite-associated lesion distribution and intensity of infection (146, 186). C. hominis and C. parvum also have different biological activities in cell culture (85). However, the number of isolates studied in animal and culture models has been small.
There appear to be distinct differences in oocyst shedding patterns between C. hominis and C. parvum in humans. A study in the United Kingdom revealed that C. hominis was detected in a significantly greater proportion of samples with larger numbers of oocysts whereas C. parvum was detected in a significantly greater proportion of the samples with small numbers of oocysts (121). Another study in Lima, Peru, reported that the duration of oocyst shedding in stool was significantly longer and the intensity of infections was significantly higher during C. hominis infections (248). There are also distinct geographical and temporal variations in the distribution of C. parvum and C. hominis infections in humans. In patients in the United Kingdom, C. parvum was more common during spring whereas C. hominis was more common in late summer and autumn in those with a history of foreign travel (120).
Genetic characterization of C. hominis and C. parvum has consistently demonstrated distinct differences between the two species at a wide range of loci (5-7, 15, 16, 25, 26, 31, 32, 35, 37, 68, 72, 76, 82, 83, 120, 121, 127, 130-134, 136, 161, 166, 168, 179, 183, 185, 194, 199-201, 206, 209-213, 235, 241-244, 248, 250, 254, 256, 257). There are also fundamental differences in ribosomal gene expression between C. hominis and C. parvum, since the latter constitutively expresses two types of rRNA genes (type A and type B [101]) whereas more than two transcripts have been detected in C. hominis (251). In addition, despite the large number of isolates examined at multiple unlinked loci from a wide range of geographical locations, putative recombinants between C. hominis and C. parvum have never been explicitly identified (118). Although some interspecific recombination has suggested by several research groups (65, 100, 205, 243), the significance or extent of any recombination is not yet fully clear. If recombination between species does occur, it seems to be very limited.
The Cryptosporidium monkey genotype, which appears to be a variant of C. hominis, has been found in rhesus monkeys (255, 257).
Cryptic species. When Tyzzer initially described C. parvum in the small intestine of mice in 1912, he was working with adult mice (227). Recent research, however, has revealed that mice harbor a genetically distinct form of Cryptosporidium, the mouse genotype, which is different from what we commonly know as C. parvum (this latter species was previously referred to as the C. parvum bovine genotype). Only rarely is C. parvum (bovine genotype) found naturally in mice since it is predominantly a parasite of ruminants and some humans (136, 138, 257). Therefore, as explained above, it is likely that the species described by Tyzzer in 1912 was not C. parvum (bovine genotype) but in fact the mouse genotype. Because the bovine genotype of Cryptosporidium retains the name C. parvum and because the mouse genotype is biologically and genetically distinct, the mouse genotype will almost certainly be named as a new species shortly.
There are probably many other cryptic Cryptosporidium species in mammals, all of which were previously assumed to be C. parvum. Thus far, nearly 20 Cryptosporidium genotypes with uncertain species status have been collectively found in pigs (two genotypes), sheep, horses, cattle, rabbits, marsupials, opossums (two genotypes), ferrets, foxes, deer (two genotypes), muskrats (two genotypes), squirrels, bear, and deer mice (255). The genetic distances among these Cryptosporidium parasites are greater than or comparable to those among established intestinal Cryptosporidium species. Limited cross-transmission studies have shown biological differences among some of the genotypes (57), some of which have even shown oocyst morphology different from that of C. parvum (Table 1; Fig. 1).
Although infections have been found in over 30 species of birds (62, 107, 160, 203), only three avian Cryptosporidium spp. have been named: C. meleagridis, C. baileyi, and C. galli. These three Cryptosporidium spp. can each infect a broad range of birds, but they differ in predilection sites. Even though both C. meleagridis and C. baileyi are found in the small and large intestine and bursa, they differ significantly in oocyst size and only C. baileyi is also found in the respiratory tissues such as the conjunctiva, sinus, and trachea. In contrast, C. galli infects only the proventriculus.
Cryptosporidium meleagridis Slavin, 1955. Developmental stages of a parasite that conformed to those found by Tyzzer (225) from the small intestine of mice were found on the villus epithelium in the terminal one-third of the small intestine of turkeys in Scotland (197). Based on dried smears stained by the MacNeal-modified Romanowsky method, the cytology and measurements of merozoites, trophozoites, meronts, gametes, and oocysts were ascertained and the parasite was named C. meleagridis. Oval oocysts measuring 4.5 by 4.0 µm (197) appeared indistinguishable from those of C. parvum. Lindsay et al. (110) gave measurements of 5.2 by 4.6 (4.5 to 6.0 by 4.2 to 5.3) µm for viable oocysts from turkey feces. Although enormous numbers were observed in smears, sporozoites could not be identified within them (197). Illness with diarrhea and a low death rate in 10- to 14-day-old turkey poults was associated with the parasite, which completed its life cycle on the villus epithelium without appearing to invade host tissues (197). No attempts were made at this time to transmit this parasite to other hosts, although subsequent studies have demonstrated that turkeys and chickens are susceptible to infection after oral inoculation with C. meleagridis oocysts (107).
Subsequent molecular analysis of a turkey isolate in North Carolina and a parrot isolate in Australia at the SSU rRNA, HSP70, COWP, and actin loci demonstrated the genetic uniqueness of C. meleagridis (143, 211, 213, 250, 257). Viable oocysts measured 5.1 by 4.5 µm (143). When the morphology, host specificity, and organ location of C. meleagridis from a turkey in Hungary were compared with those of a C. parvum isolate, phenotypic differences were small but statistically significant (202). Oocysts of C. meleagridis were successfully transmitted from turkeys to immunosuppressed mice and from mice to chickens. Sequence data for the SSU rRNA gene of C. meleagridis isolated from turkeys in Hungary were found to be identical to the sequence of a C. meleagridis isolate from North Carolina. Even though it has been suggested C. meleagridis may be C. parvum (40, 203), results of molecular and biological studies have confirmed that C. meleagridis is a distinct species (135, 143, 202, 211, 213, 250, 255, 257).
C. meleagridis is apparently a misnomer since it infects other avian hosts (for example, parrots), not just turkeys (135, 143). It is also the third most common Cryptosporidium parasite in humans (178, 248). Several subtypes of C. meleagridis have been described based on multilocus analysis (75).
Cryptosporidium baileyi Current, Upton, and Haynes, 1986. A second species of avian Cryptosporidium, originally isolated from commercial broiler chickens, was named C. baileyi based on its life cycle and morphologic features (49). The species was named in honor of the late W. S. Bailey, then President of Auburn University, for his pioneering work on the biology of Spirocerca lupi. The prepatent period was 3 days, and the patent period lasted 20 and 10 days for birds inoculated at 2 days of age and at 1 or 6 months of age, respectively. Developmental stages, found in the microvillus region of enterocytes of the ileum and large intestine, were described, measured, and photographed, and the time of their first appearance was noted. Heavy infection of the bursa of Fabricius (BF) and cloaca did not result in clinical illness. Thin-walled oocysts were observed, but most were thick walled. Viable oocysts measured 6.2 by 4.6 (5.6 to 6.3 by 4.5 to 4.8) µm and thus were much larger and more elongate than those of C. meleagridis. Mice, goats, and quail inoculated with oocysts did not become infected, but limited life cycle stages were observed in some turkey poults, and heavy infections developed only in the BF in 1-day-old ducks and 2-day-old geese (49). Sporozoites excysted in vitro and inoculated intranasally produced upper respiratory infections similar to those reported for naturally infected broilers (49). C. baileyi was found in the colon, cloaca, and BF as well as at several respiratory sites in black-headed gulls (Larus ridibundus) younger 30 days in the Czech Republic (172). Oocysts measuring 6.4 by 4.9 µm were successfully transmitted from gulls to 4-day-old chickens (Gallus gallus domestica).
C. baileyi is probably the most common avian Cryptosporidium sp. and has so far been found in chicken, turkeys, ducks, cockatiels, a brown quail, and an ostrich (108, 135; L. Xiao and U. M. Ryan, unpublished data). Hence, a wide range of birds can be infected with C. baileyi. High morbidity and mortality are often associated with C. baileyi respiratory infections of birds, especially broiler chickens (107).
Cryptosporidium galli Pavlasek, 1999. A third species of avian Cryptosporidium was first found by Pavlásek (174) in hens (Gallus gallus domesticus) on the basis of biological differences. The parasite has recently been redescribed on the basis of both molecular and biological differences (195). C. galli appears to be associated with clinical disease and high mortality (135, 174, 175, 195). Oocysts are larger than those of other avian species of Cryptosporidium and measure 8.25 by 6.3 (8.0 to 8.5 by 6.2 by 6.4) µm, with a length to width ratio of 1.3 (175). Oocysts were infectious for 9-day-old but not 40-day-old chickens (185). Unlike other avian species, life cycle stages of C. galli developed in epithelial cells of the proventriculus and not the respiratory tract or small and large intestines (174). DNA sequence analysis of three different loci confirmed that C. galli was distinctively different from C. baileyi and C. meleagridis and was related to the gastric Cryptosporidium parasites found in reptiles and mammals (C. serpentis, C. muris, and C. andersoni).
Blagburn et al. (24) may also have detected C. galli in birds when they used light and electron microscopy to characterize Cryptosporidium parasites in the proventriculus of an Australian diamond firetail finch that died of acute diarrhea. A subsequent publication also identified a species of Cryptosporidium infecting the proventriculus in finches and inadvertently proposed the name C. blagburni in Table 1 of the paper (135). However, Pavlásek (174, 175) had provided a detailed description of what appeared to be the same parasite and named it C. galli. More recent molecular analyses have revealed C. galli and C. blagburni to be the same species (195).
Confirmed hosts of C. galli include finches (Spermestidae and Fringillidae), domestic chickens (G. gallus), capercaille (Tetrao urogallus), and pine grosbeaks (Pinicola enucleator) (195). Morphologically similar oocysts have been observed in a variety of exotic and wild birds including members of the Phasianidae, Passeriformes, and Icteridae (195). Future studies are required to determine the extent of the host range for C. galli. Genetic heterogeneity probably exists in C. galli, since one finch isolate had significant sequence divergence in the SSU rRNA gene from the other C. galli isolates from finches and other birds (195).
Cryptic species. Based on limited biological and molecular studies, it appears that several other avian Cryptosporidium spp. are distinct species as well (66, 109, 135, 255). It was suggested that bobwhite quails may harbor a different Cryptosporidium species, which had oocysts similar to those of C. meleagridis but differed from C. meleagridis by infecting the entire small intestine and by causing severe morbidity and mortality (81, 86, 193). Another species is possibly present in ostriches, since this parasite has oocysts similar to C. meleagridis but is not infective to freshly hatched chickens, turkeys, or quail (66). These observations have yet to be confirmed by molecular characterizations. More recently, a new genotype of Cryptosporidium parasites has been found in an ostrich, but it is related to C. baileyi rather than C. meleagridis (U. M. Ryan and L. Xiao, unpublished data). Several other new Cryptosporidium spp. have been found in birds by molecular analysis, such as a duck genotype in a black duck and two goose genotypes in Canada geese, all of which are related to intestinal Cryptosporidium species (135, 255). Another new genotype has recently been found in a Eurasian woodcock. Even though it clustered with C. galli in phylogenetic analysis, it may represent a separate species (Ryan and Xiao, unpublished).
Of all the animals, reptiles, especially snakes, are affected most severely by cryptosporidiosis due to the chronic and detrimental nature of the infection in reptiles. Even though a high prevalence of Cryptosporidium infections has sometimes been found in captive reptiles, few studies have attempted to identify the species structure of the parasite in reptiles. For quite some time, one species, C. serpentis, was the only species identified in reptiles. More recently, however, C. saurophilum was described in lizards (97).
Cryptosporidium serpentis Levine, 1980. C. serpentis was named by Levine (104) based solely on a clinical report by Brownstein et al. (29) and using the rationale that species of Cryptosporidium had been customarily differentiated by association with their hosts. Based strictly on the ICZN, C. serpentis remained a nomen nudum until it was validated by morphologic and other biological data in a study by Tilley et al. (222). These authors reported oocysts to measure 6.2 by 5.3 (5.6 to 6.6 by 4.8 to 5.6) µm, with a length/width ratio of 1.16 (1.04 to 1.33). Another group (Xiao, unpublished) obtained measurements of 5.9 by 5.1 µm and a length/width ratio of 1.17. Levine (104) noted that cross-transmission among snakes had not been attempted and that therefore the name C. serpentis might encompass more than one species. Indeed, Brownstein et al. (29) reported that 14 snakes of three genera and four species (Elaphe guttata, Elaphe subocularis, Crotalus horridus, and Sansinia madagascarensis) in two zoological parks over a period of 7 years had severe chronic hypertrophic gastritis. Signs included postprandial regurgitation and firm midbody swelling. Gross and histological pathology were described. All developmental forms of Cryptosporidium were identified by ultrastructure in the gastric mucosa. Unlike avian and mammalian cryptosporidiosis, infections occur in mature snakes, the clinical course is usually protracted, and once infected most snakes remained infected (29). Between 1986 and 1988, 528 reptiles from three continents were examined for Cryptosporidium and 14 specimens representing eight genera and 11 species were found infected (234). Although in most cases the investigators were unable to examine the hosts for the site of infection, they presented a morphologic and statistical study of the oocysts of nine isolates and concluded that these isolates could be placed in five separate groups. Without additional isolates to determine the sites of infection and life cycles, the authors were reluctant to name new species.
Thus far, most isolates from snakes characterized by molecular analysis appear to be related to other gastric Cryptosporidium spp. (C. muris, C. andersoni, and C. galli) found in mammals and birds (139, 211, 213, 250, 255, 256). More recently, C. serpentis was found in 25 of 44 isolates from various species of snakes, some of which showed the same clinical signs and pathologic changes described by Brownstein et al. (29). It was also found in gastric washings from three snakes with clinical cryptosporidiosis; based on the collective data, C. serpentis seems to be the most common Cryptosporidium sp. (Xiao, unpublished).
Despite the name, C. serpentis apparently also infects lizards. Two isolates from savannah monitors were shown to be genetically related to C. serpentis based on sequence analysis of the SSU rRNA gene (256). More recently, PCR-RFLP and sequence analysis of the SSU rRNA gene of 24 isolates from various lizards identified 10 C. serpentis infections, and data suggest that C. serpentis is at least as common as C. saurophilum in lizards (Xiao et al., unpublished). There may be some host adaptation, since there are some minor differences between snake and lizard isolates in the sequence of the SSU rRNA and actin genes (211, 256). It is possible, however, to cross-transmit these two C. serpentis subtypes between snakes and lizards (Xiao, unpublished).
Cryptosporidium saurophilum Koudela and Modry, 1998. C. saurophilum was named following an extensive study of the feces or intestinal contents from 220 wild and captive lizards of 67 species (97). Six species of lizards in five genera were found to be passing oocysts, and Schneider's skink (Eumeces schneideri) was designated the type host. The site of infection in the type host was the intestine and cloaca. Oocysts measured 5.0 by 4.7 (4.4 to 5.6 by 4.2 to 5.2) µm, with a length/width ratio of 1.09. No pathological changes were found in the intestine and cloaca of adult lizards, but weight loss, abdominal swelling, and mortality occurred in some colonies of juvenile geckos (Eublepharis macularius). Oocysts from E. schneideri, Varanus prasinus, and Mabuya perrotetii were inoculated into five genera of lizards, and all the animals became infected. Snakes, chickens, BALB/c mice, and SCID mice were also inoculated but did not become infected. Based on oocyst size, site of infection, and cross-transmission studies, the name C. saurophilum was proposed for this species of Cryptosporidium in lizards.
Molecular characterizations support the existence of C. saurophilum. A Cryptosporidium isolate from a desert monitor (Varanus griseus) was shown to be genetically distinct from C. serpentis and to be more closely related to the intestinal Cryptosporidium spp. of mammals and birds (257). More recently, this parasite was found in 9 of 24 Cryptosporidium isolates from monitors, iguanas, and geckoes (Xiao, unpublished). Because of oocyst morphology (4.94 by 4.49 µm, with a length/width ratio of 1.14 [Table 1]), it was concluded that these lizard parasites were C. saurophilum.
Even though C. saurophilum was originally described as a lizard parasite, it has been found recently in two captive snakes in Missouri. Both snakes were heavily infected, and one had clinical signs of diseases (Xiao, unpublished). A group of six snakes housed together with four lizards in the same room in Maryland also had C. saurophilum infections, but with much lower intensity than the infection of the four lizards (Xiao, unpublished). Two corn snakes and two leopard geckos were inoculated with C. saurophilum oocysts isolated from a bull snake. Only the geckos became infected; one had parasites in the intestine only, whereas the other had parasites in both the stomach and intestine (Xiao, unpublished). Therefore, it seems that snakes can also be infected with C. saurophilum and that C. saurophilum infection in snakes is not totally restricted to the intestine. Previously, some snakes were found infected with a Cryptosporidium sp. restricted to the intestine (28).
Cryptic species. More Cryptosporidium species are likely to be present in reptiles, because an earlier study identified at least five morphotypes in wild and captive reptiles (234). Some of these morphotypes might also represent oocysts of C. muris and the Cryptosporidium mouse genotype or other species from ingested prey (pseudoparasites) with cryptosporidiosis. These Cryptosporidium spp. are frequently found in snakes without clinical signs of infection (139, 234; Xiao, unpublished). How many of these represent pseudoparasites and how many represent pathogens is currently unknown. Turtles and tortoises are known to be infected with distinct gastric and intestinal forms of Cryptosporidium (78, 79, 255), and gekkonids harbor a distinct cloacal form (231). Several unknown intestinal Cryptosporidium genotypes have been identified in snakes and lizards by molecular analysis (255; Xiao, unpublished).
Another new species, C. varanii Pavlasek, Lavickova, Horak, Kral, and Kral, 1995, was found in an Emerald monitor (Varanus prasinus) living in the Czech Republic but captured in New Guinea (176). Oocysts of this parasite measured 4.8 by 4.7 (4.8 to 5.1 by 4.4 to 4.8) µm with a length/width ratio of 1.03. Parasite stages were found in the intestine, especially in the caudal section. It remains to be determined whether C. varanii is actually C. saurophilum.
Descriptions of Cryptosporidium-like parasites in fish were reviewed by Alvarez-Pellitero and Sitja-Bobadilla (4). Two named species of Cryptosporidium have been found in fish, C. nasoris Hoover, Hoerr, Carlton, Hinsman, and Ferguson, 1981 (87), and C. molnari Alvarez-Pellitero and Sitja-Bobadilla, 2002 (4).
Cryptosporidium molnari Alvarez-Pellitero and Sitja-Bobadilla, 2002. Cryptosporidium infection in gilthead sea bream and European sea bass from the Atlantic, Cantabric, and Mediterranean coasts of Spain was studied over a period of 3 years (4). Mucosal scrapings, feces, and fixed tissue from gilthead sea bream were examined by light microscopy, and infected stomachs were studied by electron microscopy. Fixed tissues from European sea bass were examined by light microscopy. Developmental stages were described in detail, with numerous photomicrographs and electron micrographs, and oocyst measurements were obtained from fresh and fixed specimens. Most parasite stages were located at the surface of epithelial cells in the stomach but were seldom found in the intestine. Similar to Cryptosporidium in other piscine species, zygotes and oocysts were located mainly in the basal portion of the epithelium. Oocysts, nearly spherical (length/width ratio, 1.05), had a great size range but averaged 4.72 by 4.47 µm. Pathological effects, mostly in fingerlings and juvenile fish, were seen in over 24% of gilthead sea bream versus 4.6% of sea bass. Histological damage was documented. The species was named in honor of the Hungarian parasitologist Kalmar Molnar because of his extensive contribution to fish parasitology.
Unfortunately, no molecular characterization of C. molnari has been conducted thus far, which may cause problems for the identification and naming of other Cryptosporidium spp. in fish. Based on the predilection site, C. molnari may be genetically related to other gastric Cryptosporidium spp. such as C. muris, C. andersoni, and C. galli. However, molecular characterization is needed to determine if this is indeed the case.
Cryptic species. Cryptosporidium nasoris (syn. C. nasorum) should be regarded as a nomen nudum. Only developmental stages of the parasites on the microvillous surface of intestinal epithelial cells were described by light and electron microscopy. No measurements of viable oocysts were provided, and no taxonomically useful diagnostic features were presented. The species was named solely on the basis of the presumed host specificity of Cryptosporidium spp.
Some parasites in fish have been identified only as Cryptosporidium sp. (34, 99, 149). Others have been placed in a genus, Piscicryptosporidium, primarily because oocysts are located deep within epithelial cells and retain residual microvilli (170). These authors described two new species, P. reichenbachklinkei from gourami and P. cichlidaris from cichlids, based solely on transmission electron microscopy. However, additional molecular studies need to be conducted to determine whether the Cryptosporidium spp. of fish warrant generic status, and we regard the parasites as species inquirenda. In any case, it is almost certain that fish harbor more than one species of Cryptosporidium, since Cryptosporidium spp. have also been found in the intestines of various fish (4).
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As with other well-studied parasites, recent studies indicate that both host-parasite coevolution and host adaptation exist in the evolution of Cryptosporidium. Phylogenetic analyses of Cryptosporidium spp. at the SSU rRNA, HSP70, and actin loci all support a general genetic structure of the genus Cryptosporidium, with all gastric and intestinal Cryptosporidium spp. forming their own monophyletic groups. Within each group, parasites of reptiles form the basal branches and most mammalian parasites form later branches (Fig. 2). The placement of C. baileyi is still uncertain; in the SSU rRNA and actin-based phylogenetic trees, it groups with the intestinal parasites, whereas in the HSP70-based tree, it groups with the gastric parasites. The branch orders of individual species or genotypes within the gastric or intestinal species differ somewhat among the three genetic loci (255). These differences may be due to differences in the rate and nature of genetic variations among the three genes. The SSU rRNA gene of Cryptosporidium evolves slowly, with sequence variations limited to several regions of the gene. In contrast, HSP70 and actin are highly polymorphic over the entire length of the genes and the genetic differences between gastric and intestinal Cryptosporidium in the two genes approach those between Cryptosporidium and Plasmodium. Thus, the HSP70 and actin genes are probably very useful to infer the genetic relationship of closely related Cryptosporidium spp. because of the high sequence heterogeneity but less useful to assess the relationship between distant members because of homoplasy.
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FIG. 2. Genetic relationship among named Cryptosporidium species and unnamed genotypes inferred by a neighbor-joining analysis of the partial SSU rRNA gene. Values on branches are percent bootstrapping using 1,000 replicates. Numbers following species or genotypes are isolate identifications used in the construction of the phylogenetic tree, whereas numbers in parentheses are the number of isolates sequenced. Modified from reference 255.
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Genetically related hosts often harbor related species of Cryptosporidium. This is supported by the following observations. (i) All three phylogenetic analyses support a close relatedness of Cryptosporidium parasites from North American and Australian marsupials. (ii) The Cryptosporidium species from coyotes and one of the two genotypes from foxes are related to C. canis from dogs. (iii) C. canis from dogs and the Cryptosporidium genotypes from coyotes and foxes form a cluster with the Cryptosporidium parasite from bears. (iv) The Cryptosporidium deer genotype appears to be related to a newly identified genotype (the bovine genotype B) in cattle. (v) Cryptosporidium parasites from primates (C. hominis and the monkey genotype), lagomorphs (rabbit genotype), and rodents (mouse genotype), which have been shown recently to be genetically related mammals, Euarchonotoglires (150, 151), form a monophyletic group. These observations collectively support a theory of host-parasite coevolution in the genus Cryptosporidium (255).
It is difficult to determine the time of emergence of the genus Cryptosporidium parasites because of the lack of fossil records and controversy in the use of a molecular clock to attempt to time evolutionary events. However, Carreno et al. (36) have shown that Cryptosporidium spp. form a sister clade with the gregarines, parasites of invertebrates, suggesting a very early emergence of the parasite from the rest of the Apicomplexa. However, the two-way split of gastric and intestinal Cryptosporidium spp. probably occurred before the emergence of fish and reptiles, since both types of parasites occur in fish and reptiles. In addition, reptilian Cryptosporidium spp. form a basal position for both groups in all phylogenetic analyses, and the extent of sequence differences between the gastric and intestinal Cryptosporidium spp. is very large (>5.7% in the SSU rRNA gene). This is supported by the small genetic distance (0.38%) between Cryptosporidium species of the American (opossum genotype I) and Australian (marsupial genotype) marsupials, which have been separated from each other for over 50 million years because of continental drift. Further studies of parasites from amphibians and fish would be useful to the understanding of Cryptosporidium evolution.
Two exceptions to the hypothesis of host-parasite coevolution in the genus Cryptosporidium are C. parvum (the bovine genotype) and C. meleagridis. On the parasite side, C. parvum is genetically very similar to the species found in mice (the mouse genotype) and forms a monophyletic group with C. hominis and the monkey and rabbit genotypes (Fig. 2). On the host side, rodents, primates, and lagomorphs originated from a common ancestor different from the ancestor of ruminants (150, 151). It is possible that C. parvum was originally a parasite of rodents that has been recently established in cattle. This host expansion in Cryptosporidium is supported by the ability of C. parvum to infect several types of mammals, including rodents. The genetic diversity of C. parvum is lower than that of C. hominis (205), also supporting the theory of recent introduction of the parasites into ruminants. Cattle also apparently have their own Cryptosporidium genotype, the unnamed Cryptosporidium bovine genotype B, which is genetically related to the Cryptosporidium deer genotype (255). This latter species infects a ruminant related to cattle. It is unclear why this Cryptosporidium genotype is not very prevalent in cattle. It is possible that immunity induced by C. parvum has limited the prevalence of the Cryptosporidium bovine genotype B in cattle. C. parvum predominantly infects neonatal ruminants, including calves, lambs, and kids, at or near the time of birth (62).
Host expansion has also apparently occurred in C. meleagridis. Unlike C. baileyi and Cryptosporidium spp. in ducks and geese, which have near-basal positions in the intestinal Cryptosporidium group in all phylogenetic analyses, C. meleagridis is always placed in a clade containing most mammalian Cryptosporidium spp. and is most closely related to C. parvum, C. hominis, C. wrairi, and monkey, mouse, and rabbit genotypes (Fig. 2), all of which are parasites of the Euarchontoglires (primates, lagomorphs, and rodents). Thus, C. meleagridis originally may have been a mammalian Cryptosporidium sp. that has been subsequently established in birds. This is supported by the recent findings of the ability of C. meleagridis to infect a wide range of mammals, including humans, rodents, gnotobiotic pigs, and calves (2, 178, 202, 248; L. Xiao and L. Ward, unpublished data), which is certainly not the case with C. baileyi.
As expected, as the host evolves, Cryptosporidium spp. gradually become adapted to a particular group of animals and develop some host specificity. This is especially true for the intestinal Cryptosporidium spp. in mammals. In addition to the named species such as C. parvum, C. hominis, C. wrairi, C. canis, and C. felis, there are many Cryptosporidium genotypes with no established species names, such as the mouse, horse, sheep, ferret, cervine, muskrat, fox, skunk, deer, deer mouse, bear, and marsupial genotypes, pig genotypes I and II, opossum genotypes I and II, and bovine genotype B (Fig. 2). All of these parasites are found predominantly in a particular species of mammal or a group of related animals, and many of them were previously considered C. parvum (255). Even within C. canis, there are genetic differences among parasites in dogs, coyotes, and foxes. More host-adapted species and genotypes are likely to be present in mammals, since numerous intestinal Cryptosporidium genotypes in storm water cannot be attributed to specific animals (246).
Host adaptation probably also occurs in Cryptosporidium spp. of reptiles and birds, but to a much less extent. However, this may be due to the paucity of studies. As mentioned, minor genetic differences exist between C. serpentis from snakes and lizards. Unlike species or genotypes in mammals, each of which is usually seen in a very narrow range of animals, each Cryptosporidium species or genotype identified thus far in reptiles apparently can infect a broad range of reptilian hosts. Thus, both C. serpentis and C. saurophilum are seen in both snakes and lizards, and the tortoise genotype infects both tortoises and turtles (255; Xiao, unpublished). This also seems to be true for Cryptosporidium in birds. C. baileyi, C. meleagridis, and C. galli are all found in many different birds, although ducks and geese seem to have host-adapted genotypes (135, 143, 255).
Understanding host adaptation and host-parasite coevolution is important to the revision of Cryptosporidium taxonomy. Recently, the validity of several host-adapted Cryptosporidium spp., such as C. wrairi, C. felis, C. canis, C. hominis, and C. andersoni, has been supported by cross-transmission studies. The former four were originally classified as C. parvum. For example, C. wrairi has been accepted as a valid species because of its difference in host specificity from C. parvum senso stricto. The same is also true for C. andersoni, originally considered to be C. muris but now established as a separate species because of differences in infectivity in cross-transmission studies (111). Prior experience with these newly established species suggest that many of the host-adapted genotypes would exhibit significant differences in infectivity in cross-transmission studies, in addition to the genetic differences greater than or comparable to those between established species. They will probably warrant species designation pending morphologic and biological characterizations. In addition, host adaptation indicates the existence of natural segregation and barriers for genetic recombination.
However, not all host-adapted genotypes should be considered separate species. The Cryptosporidium parasite in coyotes and one of the two genotypes in foxes are genetically very closely related to the C. canis in dogs and should be considered variants of C. canis. The same is probably true of the monkey genotype, which is almost identical to C. hominis. The challenge is in determining which are species and which are variants of the same species. For example, even though the marsupial genotype from Australia and the opossum genotype I in North America have been separated from each other for more than 50 million years and have a genetic distance greater than that between members mentioned above, these two parasites are clearly related. There are even quite big genetic differences among marsupial genotype isolates in Australia. In such cases, biological studies are clearly needed to resolve the taxonomic problem.
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Because of the difficulties associated with the taxonomy of Cryptosporidium spp., general guidelines should be developed as an aid in establishing new species of Cryptosporidium. The fourth edition of ICZN, which governs the taxonomy of protozoan parasites and other animals, took effect on 1 January 2000. Within the introduction, Ride et al. (192) list a series of underlying principles that are pertinent to this discussion. The first principle provides an overall foundation for discussion, and states "The Code refrains from infringing upon taxonomic judgment, which must not be made subject to regulation or restraint." Loosely translated, this means that the criteria used in establishing any new species depend on what experts in each individual field accept as valid criteria. Therefore, there are no set international guidelines for establishing a new species of Cryptosporidium per se, providing that the 90 articles of ICZN are not violated. Therefore, the real question is whether we should, as Cryptosporidium researchers, establish or recommend general guidelines for describing new species of Cryptosporidium. Based on the plethora of synonyms, nomen nuda, and genetically cryptic species that seem to be hidden within the genus, recommendations for naming new species of Cryptosporidium seem to be in order.
At the 6th Meeting on Molecular Epidemiology and Evolutionary Genetics in Infectious Disease at the Pasteur Institute in Paris, France, a session was held entitled "The taxonomy of the genus Cryptosporidium." The objective of this session was to review the current criteria used to name species of Cryptosporidium, to evaluate the merits of these criteria, and to propose the most suitable criteria for future use. The panelists discussed areas of concern and provided recommendations for improvement. It was suggested that when naming new species of Cryptosporidium, four basic requirements should be fulfilled: (i) morphometric studies of oocysts; (ii) genetic characterizations; (iii) demonstration of natural and, whenever feasible, at least some experimental host specificity; and (iv) compliance with ICZN.
Even though oocysts of many Cryptosporidium spp. are morphologically similar, morphometric measurement of oocysts can play a vital role in the differentiation of some Cryptosporidium spp. For example, the established species in birds and reptiles can easily be differentiated on the basis of the size and shape of oocysts. Even among the intestinal species in mammals, there are significant differences in morphometrics (Table 1). Therefore, each species description should be accompanied by a series of morphologic measurements of a population of oocysts, usually 20 to 100 oocysts, complete with the means, ranges, and sometimes standard deviations or confidence limits of the measurements (length, width, and shape index or length/width ratio). Whenever possible, oocysts should be excysted and the size of sporozoites should be measured.
We can no longer describe new Cryptosporidium species solely based on morphologic descriptions or developmental studies. Most animals can be naturally infected with multiple Cryptosporidium spp. (Table 3), which frequently cannot be differentiated from each other on the basis of morphology and development (Table 1; Fig. 1). To avoid confusion in the identity of the parasites involved, defining distinct genetic differences between species will be key when naming new species of Cryptosporidium.
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TABLE 3. Cryptosporidium spp. of humans, domestic animals, and some wildlife
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If nucleotide sequences are to play a pivotal role in differentiating species of Cryptosporidium, two key points should be examined. First, which of the genes and/or noncoding regions should be used in the taxonomic descriptions? Second, how much variability constitutes a separate species? The first query is most easily addressed, although it is not without controversy. In most instances, common nucleotide sequences such as SSU rRNA, actin, and HSP70 have been employed, with SSU rRNA being the most commonly used. These sequences are useful not only because they are universal in distribution but also because generic primers can be employed. Recent examples where common sequences were used include the differentiation of C. andersoni from C. muris, the differentiation of C. canis from C. parvum, and, most recently, the establishment of C. hominis from humans as a species distinct from C. parvum (63, 111, 146). However, other sequences have also been used to great effect in differentiating some species of Cryptosporidium, despite the potential for greater difficulties in primer design. These include genes encoding the oocyst wall protein (COWP [250]), thrombospondin-related adhesive protein 1 (TRAP-C1 [200]), and tubulin (32, 194, 210, 243), to name but a few, as well as noncoding sequences such as the internal transcribed spacer 1 (147) and microsatellites (1, 31, 64). Differences in sequences encoding microneme and rhoptry proteins, such as gp900 (also known as polythreonine protein or poly-T [19, 35]), were identified between C. parvum and C. hominis (35, 212). They may eventually be shown to be pronounced in some isolates and should reflect adaptation of the parasite to specific hosts. Although designing primers against these more variable sequences may prove too difficult (213), they may also, in time, prove to be highly useful in species recognition and pathogenesis studies.
In addressing the second point, which attempts to make predictions about the degree of variability to be used to define species, there can be no easy answer. Even with protists such as Cryptosporidium, intraspecific allelic variation occurs and these differences will vary depending on the nucleotide sequence being studied. Recently, over 75,000 common chimpanzee sequences were aligned and mapped to the human genome, resulting in the discovery of 98.77% genetic similarity between the two vertebrates (245). Thus, sometimes a high degree of genetic similarity can exist even when we are dealing with separate genera. To be cautious, one would suspect that a new species is involved if the genetic differences between two isolates are greater than or comparable to those between established Cryptosporidium spp. However, when genetic differences are small, then careful biological studies (developmental biology, host specificity in cross-transmission studies, predilection site, prepatent and patent periods, intensity of oocyst shedding, virulence, etc.) with multiple isolates have to be used to support genetic findings. It cannot be overstated that genetic studies in themselves cannot be used as the sole means of differentiating species of Cryptosporidium. Not only are there no provisions within the ICZN for this, but also newer species may eventually be found to possess identical genetic sequences in some regions, effectively invalidating a genetically defined species.
A mere description of the percent differences or numbers of nucleotide changes in genetic sequences is also not enough when dealing with species description. For comparisons with other species and genotypes, DNA sequence data should be presented in the original paper and/or deposited in public databases (38). Precise areas of nucleotide differences that help define a new species should be clearly defined, and the genetic relationship between the new and existing parasites should be examined by phylogenetic analysis. Therefore, it is important that sizeable DNA fragments of the selected genes should be amplified and informative regions should be targeted. This is especially important for genes with polymorphic regions interspersed between conserved regions, such as the SSU rRNA gene. To avoid PCR contamination and misinterpretation of data, more than one genetic locus should be characterized. Different genes or markers evolve at different rates, and this can produce different results. Genetic diversity exists within an individual Cryptosporidium species or genotype, and different copies of the SSU rRNA gene have minor sequence differences in some intestinal Cryptosporidium spp. (C. parvum, C. hominis, C. felis, marsupial genotype, opossum genotype I, and others). Multilocus characterizations can help to determine whether these observed sequence differences represent genetic differences at the species level. Table 4 lists some of the primers commonly used for the characterization of the SSU rRNA, actin, and HSP70 genes of Cryptosporidium parasites. Occasionally, the genetic differences between two parasites are significant at all genetic loci examined yet the two parasites are clearly related, such as the marsupial genotype and opossum genotype I.
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TABLE 4. Some primers used in the characterization of the SSU rRNA, HSP70, and actin genes of various Cryptosporidium spp.
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Infectivity in cross-transmission studies has often been used as an important criterion in Cryptosporidium taxonomy. Nevertheless, because significant biological variations exist among isolates of the same Cryptosporidium species, various parasite, host, and laboratory factors all help control the susceptibility of a host animal to infection. Only a few species of laboratory animals are in common use, and the usefulness of host specificity as demonstrated in cross-transmission studies in naming new Cryptosporidium species is sometimes limited. There are already conflicting and confusing data regarding host specificity of C. andersoni, C. hominis, C. wrairi, and C. felis. Nevertheless, the extensive data accumulated thus far indicate that in addition to generalist species like C. muris, host-adapted Cryptosporidium spp. exist, which should be useful for studying natural host specificity and barriers to genetic recombination. Therefore, when naming new Cryptosporidium species, it is suggested that researchers should determine the spectrum of host animals within limits of feasibility and compare those isolates with established species. This may seem like a daunting task, but with the ever-increasing amount of molecular characterizations of Cryptosporidium isolates from different animals, it may be as simple as tabulation of the spectrum of host animals infected with an isolate based on previously published literature. Researchers should refrain from naming new Cryptosporidium species based solely on the finding and characterization of one or two isolates.
In recommending general criteria for naming new species of Cryptosporidium, at least two points within the ICZN should be emphasized. First, Articles 8 and 9 of the ICZN spell out clear criteria for publication (192). Most conspicuous is Article 9, which lists formats not constituting valid, published work. These include microfilm, unpublished copies of work even if available in a library or archive, material in the form of electronic signals on the World Wide Web, tape recordings, and abstracts or papers issued primarily to participants at meetings or symposia. Thus, the clear mandate in the ICZN is that the International Committee still prefers taxonomic descriptions to be printed on paper, although publication in the form of CD-ROM appears to be acceptable if the work has "...been deposited in at least 5 major publicly accessible libraries which must be identified by name within the work itself."
The second point pertains to Article 72.3 which states, "Name-bearing types must be fixed originally for nominal species-group taxa established after 1999." Therefore, in order for a species of Cryptosporidium to be valid under the ICZN, the authors must establish a name-bearing type in the form of a holotype or designated syntypes. Because of the difficulty of fixing single (holotype) specimens as name-bearing types for most members of the Apicomplexa, specimens in a type series (syntypes) are almost always used (Article 72.1.1). For coccidia and Cryptosporidium spp., the morphological description itself and the museum photographs of the unpreservable oocysts (sometimes termed phototypes [17]) should be provided. Whenever possible, stained slides and histological sections should be deposited. The museum in North America where the majority of type specimens currently reside is the U.S. National Parasite Collection in Beltsville, Md. It should be noted that photographs and illustrations are not types themselves but, rather, a representative of the name-bearing type (Article 72.5.6).
Currently Cryptosporidium species and genotypes have been named or identified primarily in association with the host from which they were isolated. This can be problematic, since most Cryptosporidium spp. infect more than one species of animals. Therefore, the initial host identified may not be the host the parasite most commonly infects. Also, usually more than one Cryptosporidium species may be commonly found in one host species. For example, if C. meleagridis were first found in a mammal instead of turkeys, it would have been named differently.
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Cryptosporidiosis is a major public health problem in both developing and developed countries. In developing countries, the disease probably exerts most of its impact on pediatric health. In addition to the occurrence of diarrhea, cryptosporidiosis has been attributed to malnutrition and stunted growth (41, 42, 126). In developed countries, waterborne outbreaks of cryptosporidiosis have a significant economic impact. A recent study estimates that the total illness-associated cost of the 1993 Milwaukee waterborne outbreak of cryptosporidiosis is $96.2 million: $31.7 million in medical costs and $64.6 million in productivity losses. The average total costs for a person with mild, moderate, and severe illness during the outbreak are $116, $475, and $7,808, respectively (46). These cost estimates do not include litigations and infrastructure improvements in water treatment facilities after the outbreak. Pretty et al. (189) have estimated that the United Kingdom will expend an average of £23 million/year to meet legal requirements for removal of Cryptosporidium from drinking water supplies. Therefore, it is important for researchers to determine the extent of contamination of surface waters with species of Cryptosporidium infective for humans. Understanding the taxonomy of Cryptosporidium spp. can be also useful in establishment of the identity of the parasites infecting humans, assessment of the public health significance of Cryptosporidium from animals and the environment, and tracking of infection and contamination sources. All these will promote understanding of the transmission and epidemiology of human cryptosporidiosis, development of preventive measures to minimize exposures to infections, accurate risk assessment, and scientific management of the watershed.
For quite some time, it was thought that C. parvum was the species responsible for human cryptosporidiosis. This is largely due to a lack of clear understanding of the species structure of the genus Cryptosporidium and the reliance on morphologic features for differentiation. With the use of genetic analysis in helping define species, we now have a better knowledge of the taxonomy of Cryptosporidium spp. and the molecular tools to differentiate species with similar oocyst morphology. Thus, numerous studies have shown two Cryptosporidium spp., C. parvum and C. hominis, to be responsible for most human Cryptosporidium infections (5-7, 15, 16, 25, 26, 31, 32, 35, 37, 68, 72, 76, 82, 83, 120, 121, 127, 130-134, 136, 161, 166, 168, 179, 183, 185, 194, 199-201, 206, 209-213, 235, 241-244, 248, 250, 254, 256, 257). Subsequently, after the identification of the genetic uniqueness of C. meleagridis, C. felis, and C. canis (137, 256, 257), these three parasites, which are traditionally associated with animals, were found in AIDS patients in the United States, Kenya, and Switzerland (127, 128, 187). More recent studies have confirmed the presence of these parasites in immunocompromised individuals in other parts of the world (5, 6, 71, 82, 216) (Table 5). Other Cryptosporidium spp. found in humans include C. muris, the cervine genotype, and pig genotype I (69, 82, 165, 169, 216, 247). The last three Cryptosporidium spp., however, have a very low prevalence in humans and are unlikely to emerge as major human pathogens. Immunosuppression is apparently not a prerequisite for infections with any of these species since they have been found in immunocompetent persons in Peru, the United Kingdom, and Japan (177, 178, 180, 248, 258). In Peru, where a significant proportion of infections in humans are due to zoonotic Cryptosporidium spp., there is no significant difference between children and HIV+ adults in the distribution of all five common Cryptosporidium parasites (C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis) (249).
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TABLE 5. Prevalence of five common Cryptosporidium spp. in humansa
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The host-adapted nature of most Cryptosporidium spp. indicates that the majority of species probably do not have high infectivity for humans. Thus, Cryptosporidium spp. commonly found in reptiles and most wild mammals have never been detected in humans and probably have no significant public health importance. On the other hand, host adaptation is not strict host specificity. A species or genotype may preferentially infect a species or group of animals, but this does not mean that this parasite cannot infect other animals. As mentioned above, C. parvum, C. meleagridis, C. felis, C. canis, C. muris, the cervine genotype, and pig genotype I have all been found in humans. Others, such as C. andersoni in cattle, pig genotype II, the mouse genotype in rodents, and C. baileyi, C. galli, and goose genotypes I and II, have not been found in humans. Thus, parasites from different animals and different species from the same animal have vastly different zoonotic potentials. More often, a Cryptosporidium species or genotype is found in a few different animals, and one species of animal is usually susceptible to multiple Cryptosporidium spp. (Table 3). Thus, all species of Cryptosporidium infect a limited range of animals (Table 1), and when the host range or infectivity includes humans, the parasite acquires public health significance.
The development of genetic tools as part of the taxonomic characterization of Cryptosporidium spp. makes it possible to directly assess the human infection potential of Cryptosporidium oocysts found in the environment. Currently, the identification of Cryptosporidium oocysts in environmental samples is based largely on the immunofluorescence assay (IFA) after concentration processes (ICR method, method1622/1623, flow cytometric method, solid-phase cytometric method [106]). Because IFA detects oocysts from all species of Cryptosporidium, the species distribution of Cryptosporidium in environmental samples cannot be assessed. Although many surface water samples contain Cryptosporidium oocysts, it is unlikely that all of these oocysts are from human-pathogenic species or genotypes, because only five Cryptosporidium spp. (C. parvum, C. hominis, C. meleagridis, C. canis, and C. felis) are responsible for most human Cryptosporidium infections. Information about the identity of Cryptosporidium oocysts is necessary for accurate risk assessment of contamination in water. Thus, identification of oocysts to species and genotypes is of public health importance.
Several species-differentiating and genotyping tools have been used to determine whether Cryptosporidium oocysts found in water are from human-infective species (Table 6). An SSU rRNA-based nested PCR-RFLP method has been successfully used for the detection and differentiation of Cryptosporidium oocysts present in storm water, raw surface water, and wastewater (246, 253). In one study, 29 storm water samples were collected from a stream that contributes to the New York City water supply system and were analyzed for Cryptosporidium oocysts. Twelve wildlife genotypes of Cryptosporidium were detected in 27 of 29 positive samples. None of the genotypes have ever been found in humans and therefore probably do not infect humans. Of the 27 PCR-positive samples, 12 contained multiple genotypes (246).
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TABLE 6. Detection of Cryptosporidium in natural water by PCR-based molecular techniques
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Promising results in genotyping Cryptosporidium spp. in water samples have also been generated in recent studies using other techniques (Table 6). HSP70 sequence analysis of PCR-amplified cell culture products revealed the presence of six sequence types of C. parvum in raw surface water samples and filter backwash water samples (51). Comparison of these sequences with the HSP70 sequences collected from various Cryptosporidium spp. indicates that these sequences were from C. parvum, C. hominis, and the mouse genotype (213). Analysis of six river water samples by an HSP70-based reverse transcription-PCR technique also showed the presence of C. parvum and C. meleagridis in two samples (95), even though the primers used in the study were previously shown to have poor specificity (96).
Two SSU rRNA-based PCR sequencing tools have also been successfully used in the differentiation of Cryptosporidium oocysts in surface water and wastewater samples (92, 238). Sequences of C. andersoni and presumably C. parvum and C. baileyi were obtained from seven samples of surface water from a watershed in Massachusetts (82). Analysis of 17 positive surface water samples and 6 wastewater samples from Germany and Switzerland showed the presence of eight Cryptosporidium genotypes, with C. parvum, C. hominis, C. muris, and C. andersoni being the most prevalent parasites, and showed that 4 samples contained three unidentified wildlife genotypes and C. baileyi (238). Using sequencing analysis of TRAP-C2, C. parvum was also found in 11 of 214 surface and finished water samples in Northern Ireland in one study and in 2 of 10 river water and sewage effluent samples in another study (113, 114). Results of these recent studies support the conclusion that many Cryptosporidium spp. found in water do not have high potential for infecting humans.
The host-adapted nature of most Cryptosporidium spp. makes it possible to track the source of Cryptosporidium infection in humans and oocyst contamination in the environment. The high prevalence of C. hominis in humans indicates that humans are a major source of infection for human cryptosporidiosis. The finding of C. parvum, C. meleagridis, C. felis, C. canis, C. muris, pig genotype I, and cervine genotype in humans suggests that farm animals, domestic pets, and some wildlife can be potential sources. Indeed, direct transmission of C. parvum from animals to humans is well documented (48, 124, 125, 185, 204). Many food-borne and waterborne outbreaks of cryptosporidiosis have been caused by C. parvum in North America and Europe (120, 166, 185, 209), even though it is frequently unclear whether humans or animals are the source of contaminations.
Caution should be used in interpreting the significance of finding parasites traditionally associated with animals in humans. It is always tempting to attribute the source of C. parvum and other zoonotic Cryptosporidium spp. found in humans and the environment solely to animals (166, 204). Contradictory to what may be intuitive, many human C. parvum infections in localized areas may indeed have originated from humans themselves. This is supported by sequence analysis of the GP60 gene, a sporozoite surface glycoprotein of C. parvum and C. hominis. As shown in Fig. 3, five of the six C. parvum isolates from AIDS patients in New Orleans possessed the allele family Ic (249). Likewise, all C. parvum isolates (>50 isolates) from children and AIDS patients in Peru also exhibited this allele family (Xiao, unpublished). This allelic type was apparently also the dominant type in South Africa (100), and 5 of 16 C. parvum isolates from Portuguese AIDS patients had this allele (8). In contrast, allele family Ic has never been found among over 500 bovine C. parvum isolates analyzed thus far in the United States, Portugal, and the United Kingdom (8, 74, 184; L. Xiao and J. E. Moore, unpublished data). It was previously suggested that GP60 allele family Ic resulted as a recombination between the C. parvum allele families IIa and C. hominis allele Ib (100). However, among the GP60 allele families of C. parvum and C. hominis, C. parvum allele family Ic appears phylogenetically related to C. hominis allele families Ib and Ie whereas C. parvum allele family IIa is related to C. hominis allele families Ia and Id (Fig. 3). The pattern of sequence diversity in allele family is also different from that in all other C. parvum and C. hominis allele families. It remains to be determined whether the genetic and biological uniqueness of allele Ic is truly due to sexual recombination. In any case, the previously identified two transmission cycles (the anthroponotic transmission cycle with C. hominis and the zoonotic transmission cycle with C. parvum) of cryptosporidiosis in humans is apparently a simplistic view of the complexity of Cryptosporidium infection.
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FIG. 3. Genetic diversity in C. parvum and C. hominis from AIDS patients in New Orleans, based on neighbor-joining analysis of the partial GP60 gene. Values on branches are percent bootstrapping using 1,000 replicates. Five allele families of parasite are seen: Ia, Ib, and Ie are C. hominis allele families, and Ic and IIa are C. parvum allele families. Modified from reference 249.
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C. hominis is also frequently seen in surface water in some areas and is generally considered a relatively strict human pathogen. However, most Cryptosporidium spp. identified in mammals have preferred host taxa but also infect multiple hosts to lesser degrees. Indeed, C. hominis has been identified recently in a few animals, such as a dugong and a sheep (73, 142). Lambs, calves, and gnotobiotic pigs have also been successfully infected with some isolates of C. hominis (3, 55, 73, 186). Recently, some wetland mammals such as beavers and muskrats have been identified as reservoir hosts for Giardia intestinalis and Enterocytozoon bieneusi infections in humans by genetic analysis (207, 208). Similarly, an undiscovered zoonotic reservoir for C. hominis may also exist.
The water industry should take comfort in knowing that even though oocysts of all Cryptosporidium spp. can potentially appear in water, only a few of them are known human pathogens. Even among the latter, not all have the same infectivity for humans. Very limited studies have been conducted to determine the identity of Cryptosporidium oocysts in water, but the results generated thus far indicate that a large proportion of Cryptosporidium oocysts in water are not from species harmful to humans. Current detection methods for Cryptosporidium oocysts in water do not include a species differentiation process. Therefore, the human health impact could be overestimated if the risk assessment models do not take this into consideration. More studies involving systematic sampling of different types of water are clearly needed to develop a better picture of the extent of contamination of water with human-infective Cryptosporidium spp. in different environmental settings.
Currently, surface water in the United States is frequently used as drinking water after conventional treatment, which includes coagulation, flocculation, sedimentation, filtration, and chlorination. When working properly, this process is very effective in removing Cryptosporidium oocysts (88). Therefore, determining the species nature and human-infective potentials of Cryptosporidium oocysts in the source water has not been a high-level concern, especially when the treatment plant also incorporates ozone or uv light treatment near the end of the treatment process. In some areas, however, surface water is treated only by chlorination or is processed by partial treatment without filtration. Under such circumstances, it is critical to minimize the presence of human-pathogenic Cryptosporidium spp. in source water. Thus, the use of surface water potentially contaminated by human and agricultural activities as source water is problematic because humans and farm animals are major sources of contamination by oocysts of human-pathogenic Cryptosporidium spp. In contrast, for water utilities that use pristine water as the source water, partial treatment of surface water may indeed be enough, since Cryptosporidium oocysts in these watersheds are from species originating from wildlife, which are mostly not human pathogens. Thus, periodic determination of the species of Cryptosporidium oocysts in any watershed or source water may be very helpful in the development of strategies for the scientific management and protection of source water.
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The taxonomy of Cryptosporidium, like the taxonomy of most organisms, remains fluid. Even though new criteria may be introduced in future, the taxonomic framework discussed above should be helpful in guiding us to minimize the creation of invalid names for species. As with the revision of the taxonomy of any pathogen, the above suggestions will not be without controversy. However, the goal of this paper is to put forth a scheme that incorporates taxonomic characteristics based on classical methods with newly emerging techniques. When combined, these data should prove useful in understanding the biology, epidemiology, and public health importance of various Cryptosporidium spp. With the separation of C. hominis and, shortly, the mouse genotype from C. parvum, it is expected that some of the previous confusion will be eliminated.
In the meantime, we suggest that researchers follow the proposed guidelines in naming Cryptosporidium species. Thus, a description of new species should provide easily identifiable characteristics that enable other researchers to clearly differentiate this parasite from known Cryptosporidium species, and from other yet unnamed Cryptosporidium spp. that may be found in the same or related hosts. This, at minimum, should include detailed morphometric descriptions of the oocysts, comparative data about the natural host specificity or spectrum of known hosts, and results of genetic characterizations and should comply with all 90 articles of the ICZN. Past experience in the area indicates that excessive reliance on any single criterion or results of cross-transmission is risky and frequently results in the later invalidation of the described species.
Researchers should carefully use Cryptosporidium nomenclature to avoid unnecessary confusion. The name C. parvum should be used only for parasites previously known as the bovine genotype or genotype 2. Even though we have known for years that there is extensive genetic and biologic heterogeneity in mammalian Cryptosporidium isolates and that multiple Cryptosporidium spp. infect humans, too often researchers habitually refer to the parasites in humans or mammals as C. parvum without providing any evidence to support this identification. Therefore, unless the nature of these species is resolved by biological or molecular characterizations, it is scientifically incorrect to assign species names. In most such cases, the use of the term Cryptosporidium sp. or merely Cryptosporidium should be enough.
With the establishment of a framework for naming Cryptosporidium species and the availability of new genetic tools, there should be reduced confusion associated with the taxonomy of the genus. We also hope that the more detailed suggestions for naming new Cryptosporidium spp. will reflect the evolutionary relationships within the genus as well as providing a rational basis for delineating species. This, in turn, will promote the assessment of the public health importance of various species and isolates of Cryptosporidium and allow researchers to better understand the transmission dynamics, to identify risk factors and reservoir hosts, and to establish preventive measures.
We thank researchers who have been working on Cryptosporidium taxonomy in our laboratories over the years. We thank Caryn Bern, Anne Moore, John E. Moore, Michael Arrowood, and Lucy Ward for sharing some unpublished data.
These studies were supported by interagency agreements between the U.S. Environmental Protection Agency and CDC and the Opportunistic Infectious Diseases Program of CDC.
* Corresponding author. Mailing address: Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, GA 30341. Phone: (770) 488-4840. Fax: (770) 488-4454. E-mail: lxiao{at}cdc.gov.
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Clinical Microbiology Reviews, January 2004, p. 72-97, Vol. 17, No. 1
0893-8512/04/$08.00+0 DOI: 10.1128/CMR.17.1.72-97.2004
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