variation in cryptosporidium: towards a taxonomic revision of the genus

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Invited review Variation in Cryptosporidium: towards a taxonomic revision of the genus Una M. Morgan a, *, Lihua Xiao b , Ronald Fayer c , Altaf A. Lal b , R.C. Andrew Thompson a a World Health Organisation Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, and State Agricultural Biotechnology Centre, Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia b Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA c Immunology and Disease Resistance Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA Received 22 February 1999; received in revised form 23 June 1999; accepted 23 June 1999 Abstract Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the diculty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from dierent species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/ species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cryptosporidium; Genotypes; Species; Taxonomy 1. Introduction Once an emerging pathogen, Cryptosporidium is now firmly established as a serious and wide- spread cause of enteric disease in humans and other animals. Cryptosporidiosis can be severe in the very young and elderly, and life-threatening in individuals with an impaired immune system [1]. Limitations associated with conventional diag- nostic methods for cryptosporidiosis based on morphological features [2, 3], coupled with the diculty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensi- tive molecular approaches has obviated the International Journal for Parasitology 29 (1999) 1733–1751 0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(99)00109-5 * Corresponding author. Fax: 08-9310-4144. E-mail address: [email protected] (U.M. Morgan)

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Page 1: Variation in Cryptosporidium: towards a taxonomic revision of the genus

Invited review

Variation in Cryptosporidium: towards a taxonomic revisionof the genus

Una M. Morgana, *, Lihua Xiaob, Ronald Fayer c, Altaf A. Lal b,R.C. Andrew Thompsona

aWorld Health Organisation Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, and State Agricultural

Biotechnology Centre, Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, AustraliabDivision of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA

cImmunology and Disease Resistance Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA

Received 22 February 1999; received in revised form 23 June 1999; accepted 23 June 1999

Abstract

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associatedwith conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the

di�culty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species.The application of sensitive molecular approaches has obviated the necessity for laboratory ampli®cation. Suchstudies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from di�erent

species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during thelast 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive

value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiologyand transmission and controlling outbreaks of the disease is also discussed. # 1999 Australian Society forParasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Cryptosporidium; Genotypes; Species; Taxonomy

1. Introduction

Once an emerging pathogen, Cryptosporidiumis now ®rmly established as a serious and wide-spread cause of enteric disease in humans andother animals. Cryptosporidiosis can be severe in

the very young and elderly, and life-threatening

in individuals with an impaired immune

system [1].

Limitations associated with conventional diag-

nostic methods for cryptosporidiosis based on

morphological features [2, 3], coupled with the

di�culty of characterising parasites isolated in

the laboratory, have restricted our ability to

clearly identify species. The application of sensi-

tive molecular approaches has obviated the

International Journal for Parasitology 29 (1999) 1733±1751

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(99 )00109-5

* Corresponding author. Fax: 08-9310-4144.

E-mail address: [email protected] (U.M.

Morgan)

Page 2: Variation in Cryptosporidium: towards a taxonomic revision of the genus

necessity for laboratory ampli®cation. Such stu-dies have found considerable evidence of geneticheterogeneity among isolates of Cryptosporidiumfrom di�erent species of vertebrate, and there isnow mounting evidence suggesting that a seriesof host-adapted genotypes/strains/species of theparasite exist. This contrasts with the present tax-onomy of the genus, which presents a rational-istic approach with only eight recognisedspecies [1]. It is only relatively recently that thetaxonomy of the genus was reduced to just eightspecies whereas previously, a much larger num-ber of species had been described [1].

The picture that is emerging as a result of mol-ecular studies clearly indicates that the species-level taxonomy of the genus does not re¯ect mol-ecular phylogenetic analyses or epidemiologicaldata, and warrants reappraisal. Indeed, currentmolecular data provide support for some of theearlier taxonomic separation on the basis of hostoccurrence [4±28].

In this article we review studies on the molecu-lar characterisation of Cryptosporidium duringthe last 5 years, and put this into perspectivewith the past and present taxonomy of the genus.We also cover the predictive value of achieving asound taxonomy for the genus Cryptosporidiumwith respect to understanding its epidemiologyand transmission and controlling outbreaks ofthe disease.

2. Taxonomy of apicomplexansÐhistoricalperspective

Within the phylum Apicomplexa, taxonomicclassi®cation schemes have been proposed to ac-commodate a variety of parasitic eukaryotes pos-sessing an apical complex at some stage in theirlife-cycle. The taxa have been based on combi-nations of morphological features detected bylight microscopy or EM, unique life-cycles andhost speci®city. Morphological features of theoocyst stage at the light-microscope level havebeen used more than any other single character-istic to designate genus and species. The numberof sporocysts within an oocyst and the number ofsporozoites within each sporocyst have provided

patterns useful for designating genera. In turn, thesize and shape of the oocyst, the presence orabsence of a micropyle cap, the size and shape ofsporocysts, the presence or absence of a Stiedabody, and the size of sporozoites have been usefulin great measure for designating species. It hasbeen suggested that, based on oocyst structure,one could expect to identify 2 654 208 specieswithin the genus Eimeria alone [29].

As helpful as these features have been for iden-tifying many species, di�culties began to arisewhen oocysts of a given species were found to bemorphologically indistinguishable from those ofanother species or genus. Three genera, all with afeline or canine ®nal host, have morphologicallyindistinguishable oocysts [30]. Oocysts ofToxoplasma gondii (average 10�11 mm) are dis-poric with tetrazoic sporocysts, and overlap insize and shape with those of Hammondia ham-mondi and Neospora caninum. A similar problemwith identity of the exogenous stage arose withinthe genus Sarcocystis, wherein at the time of ex-cretion from the body, weak-walled, sporulatedoocysts ruptured and released sporocysts thatappeared identical to sporocysts of other species.Although some size di�erences for sporocysts canbe found within the genus, Sarcocystis speciesare di�erentiated from one another primarily onhost speci®city and tissue cyst morphology.Furthermore, sporocysts of some Frenkelia spp.appear identical to some species of Sarcocystis,and the genera are di�erentiated by host and tis-sue cyst characteristics [31]. Because oocysts ofCryptosporidium are among the smallest exogen-ous stages of any apicomplexans, any morpho-logic di�erences may not be discernible at thelight-microscope level. Therefore, it is likely that,within the genus Cryptosporidium, characteristicsother than oocyst morphology will be needed todetermine taxonomic status.

Some host species harbour numerous species ofApicomplexa, each species of parasite occupying avery speci®c location in the gastrointestinal tractwith developmental stages in speci®c types of cells.For example, chickens, turkeys, cattle, and sheepeach have about seven, seven, 15, and 10 species ofEimeria, respectively [32]. The house mouse, Musmusculus, the type host for Cryptosporidium, is

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511734

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host to about 13 species of Eimeria [32]. For somespecies of Apicomplexa, isolates or strains havebeen recognised that possess unique character-istics. Some are more or less virulent than others,some have a shorter or longer prepatent periodthan others, and some have oocysts that are con-sistently at the larger or smaller end of the sizerange for the species. For isolates within thespecies Cryptosporidium parvum, similar di�er-ences are emerging as more and more biologicaland molecular data accumulate. This diversitybegs the question, `is C. parvum a single specieswith a wide range of biological characteristics or isit a taxon actually consisting of multiple specieswith oocysts that are indistinguishable from oneanother?'.

New knowledge pertaining to a taxon maysuggest the need for change in taxonomic status.In this regard, molecular analysis may help toaddress some taxonomic problems. For example,a new genus Cyclospora was created when an en-teric coccidian parasite in humans was found tohave two sporocysts in each oocyst and two spor-

ozoites in each sporocyst [33]. Like eimerianspecies, Cyclospora sporulates outside the bodyand appears to be very host-speci®c.Furthermore, recent phylogenetic studies haveshown that Cyclospora clusters within variousspecies of Eimeria [34], indicating that oocystmorphology alone may sometimes be misleading.Isospora spp. are traditionally considered as mostclosely related to Eimeria spp. and are placed inthe family Eimeriidae. Unlike Eimeria, however,some Isospora have long been known to formmonozoic cysts in various tissues of intermediatehosts that have ingested oocysts; the cysts com-plete the life-cycle when ingested by the ®nalhost. Indeed, recent molecular characterisationindicates that Isospora are more closely related toToxoplasma and Neospora [35]. Genetic di�er-ences have recently been demonstrated amongsome isolates of Cryptosporidium (see below) andinterpretation of these data, combined with phe-notypic traits of these isolates, may provide acompelling basis for revision of taxa within thegenus Cryptosporidium.

Table 1

Described species of Cryptosporidium

Species of Cryptosporidium Host Reference

C. muris Mus musculus (mouse) [60]

C. parvum Mus musculus (mouse) [134]

C. crotali Crotalus con¯uens (rattlesnake) [135]

C. vulpis Vulpes vulpes (fox) [136]

C. meleagridis Meleagris gallopavo (turkey) [48]

C. tyzzeri Gallus gallus (chicken) [29]

C. lampropeltis Lampropeltis calligaster (king snake) [137]

C. ameivae Ameiva ameiva (lizard) [138]

C. ctenosauris Ctenosaura similis (lizard) [139]

C. wrairi Cavia porcellus (guinea pig) [78]

C. agni Ovis aries (sheep) [140]

C. anserinum Anser anser (goose) [141]

C. bovis Bos taurus (ox) [140]

C. cuniculus Oryctolagus cuniculus (rabbit) [142]

C. felis Felis catis (cat) [85]

C. rhesi Macaca mulatta (rhesus monkey) [143]

C. serpentis Crotalid, colubrid and boid snakes [143]

C. garnhami Homo sapiens (man) [107]

C. nasorum Naso literatus (®sh) [40]

C. baileyi Gallus gallus (chicken) [49]

C. curyi Felis catis (cat) [144]

C. saurophilum Eumeces schneideri (lizard) [72]

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±1751 1735

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3. Inter- and intra-speci®c variation inCryptosporidium

Although 22 di�erent species ofCryptosporidium have been named on the basisof host occurrence (see Table 1), several specieshave been invalidated because oocysts originallydescribed from rattle-snakes, foxes, king snakesand lizards are clearly sporocysts of Sarcocystisspp. [36±38]. There are currently up to eightnamed species of Cryptosporidium which havebeen proposed as valid. These include C. parvumfrom many mammals, Cryptosporidium murisfrom mice and ruminants, Cryptosporidium felisfrom cats, Cryptosporidium wrairi from guineapigs, Cryptosporidium meleagridis andCryptosporidium baileyi from birds,Cryptosporidium serpentis from reptiles andCryptosporidium nasorum from ®sh [1].Comprehensive historical reviews of species inthe genus Cryptosporidium have already beenundertaken (cf. [1, 39]) and therefore the follow-ing is a brief overview of the current status ofspecies and genotypes within the genusCryptosporidium.

3.1. Cryptosporidium in ®shÐC. nasorum

Little is known about the prevalence or geo-graphical distribution of isolates ofCryptosporidium infecting ®sh. The ®rst report ofCryptosporidium sp. in ®sh was in a tropical mar-ine ®sh (Naso lituratus) in 1981 [40] and C.nasorum was subsequently proposed as a speciesin 1984 by Levine [37]. Cryptosporidium has beenreported in a number of species of freshwaterand marine ®sh (cf. [1, 41, 42]). Cryptosporidiumoocysts from humans have been reported to beinfectious for ®sh based on gut histology [43];however, attempts to experimentally infect 1.5-cmguppies [44] and bluegills [45] with C. parvumoocysts from an isolate known to be infective tosuckling mice were unsuccessful. Cryptosporidiumisolates from ®sh have not been genotyped andtherefore the validity of C. nasorum as a species,its relationship to other Cryptosporidium speciesand the presence of multiple species/genotypes ofCryptosporidium infecting ®sh, remain unknown.

Recently, a new genus, Piscicryptosporidium, hasbeen proposed for Cryptosporidium species infect-ing ®sh [46]. However, this has not yet been sup-ported by biologic and genetic characterisation.

3.2. Cryptosporidium in birdsÐC. baileyi, C.meleagridis

In 1929, Tyzzer, reported that a species similarto C. parvum was present in the caeca ofchickens [47]. In 1955, Slavin described a similarspecies from turkey poults and named it C.meleagridis [48]. Cryptosporidium baileyi has beendescribed from broiler chickens and is thought tobe a valid species due to di�erences in oocyststructure and to the fact that it develops mainlyin the respiratory tract [49]. Cryptosporidiummeleagridis is thought to be a valid species on thebasis of oocyst and sporozoite morphology [50],and also because C. meleagridis infects the smallintestine and not the respiratory tract [48, 51].Original descriptions of Cryptosporidium fromthe chicken and Cryptosporidium anserinum fromthe goose lacked su�cient detail for subsequentidenti®cation and are considered invalid [1]. Apossible third species from bobwhite quail, withoocysts similar to those of C. meleagridis, di�eredfrom C. meleagridis by infecting the entire smallintestine and by causing severe morbidity andmortality [52±54]. A possible fourth species fromostriches has also been described, which hadoocysts similar in size to C. meleagridis and wasnot infective to freshly hatched chickens, turkeysor quail [55].

Cryptosporidial infections have been reportedin over 30 species of birds (cf. [39]). Only a fewisolates from birds have been analysed geneti-cally, although analysis of oocyst surface antigensand isoenzyme analysis have identi®ed phenoty-pic di�erences between C. baileyi and C.parvum [56±58]. Sequence and phylogeneticanalysis of the 18S rDNA gene has con®rmed thegenetic distinctness and validity of C. baileyi as avalid species [27, 28, 59]. Cryptosporidium melea-gridis has been shown to be genetically distinctbut closely related to C. parvum [28].

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511736

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3.3. Cryptosporidium in rodents and ruminantsÐC. muris

Organisms of Cryptosporidium sp. were ®rstrecognised and described in the peptic glands ofmice by Tyzzer in 1907, who named them as C.muris [60]. Cryptosporidium muris oocysts are lar-ger than C. parvum (7.4�5.6 mm vs 5.0�4.5 mm)and, unlike C. parvum which develops mostly inthe small intestine, C. muris mainly colonises thestomach [1]. Cryptosporidium muris has beendescribed in voles (Microtus sp.), mice (M. mus-culus) and rats (Rattus sp.) (cf. [1]).Cryptosporidium muris-like oocysts have alsobeen reported in cattle (Bos taurus), rock hyrax(Procavia capensis), desert hamsters (Phodopusroborovskii), and camels (Camelus bactrianus) [61±68]. In cattle, C. muris infects only the abomasumand usually causes no overt illness, but retardsacid production. Milk production in cows thatare chronically a�icted with C. muris may bereduced by 13%, and growing calves may also beadversely a�ected [69].

Phylogenetic analysis has shown that C. murisis the most divergent species of Cryptosporidiumand is most closely related to C.serpentis [27, 28, 70]. This is in agreement withbiological data, as C. muris and C. serpentisoocysts are the largest of all the Cryptosporidiumspecies and mainly colonise the stomach, whereasC. baileyi colonises the respiratory tract and thesmall intestine, and most other Cryptosporidiumspecies infect the small intestine [1].

There is some evidence of biological and mol-ecular variation among di�erent isolates of C.muris. Cryptosporidium muris isolates fromrodents, a Bactrian camel and a rock hyrax areinfectious to mice, but C. muris oocysts fromcattle do not seem to infect mice readily [27, 71±73]. Molecular evidence also suggests that thereare distinct genetic di�erences between bovineand other C. muris isolates [27]. Analysis of the18S rRNA gene showed that C. muris Bactriancamel and rock hyrax isolates were identical toeach other, but distinct from C. muris bovine iso-lates. The genetic distance between the bovineand camel/hyrax isolates was 0.0087 nucleotidesubstitutions/site [27]. Future genetic studies are

required in order to determine the extent ofintra-speci®c variation within C. muris.

3.4. Cryptosporidium in reptilesÐC. serpentis, C.saurophilum

Cryptosporidial infections have been reportedin over 57 di�erent reptilian species (cf. [39]).Until recently, however, there have been few mol-ecular characterisation studies on reptilian iso-lates of Cryptosporidium. Tilley et al. [74]di�erentiated reptilian isolates ofCryptosporidium spp. from C. parvum on thebasis of oocyst size (6.2�5.3 mm) and electro-phoretic protein patterns. Phylogenetic analysisof 18S rDNA sequence information has con-®rmed the genetic distinctness of C. serpentis andits validity as a species [27, 28, 70].Cryptosporidium serpentis was also shown to beclosely related to C. muris [27, 28, 70]. The val-idity of C. serpentis as a separate species is sup-ported by its host speci®city and the fact that thesimilarity between two recognised species, C. par-vum and C. wrairi, is 98.87%, which is greaterthan the similarity between C. serpentis and C.muris (97.42%). Data generated by both 18SrDNA sequence analysis and random ampli®edpolymorphic DNA (RAPD) analysis were signi®-cantly correlated (P<0.002), providing ad-ditional evidence to support the clonalpopulation structure hypothesis forCryptosporidium [70].

Morphometric studies on isolates recoveredfrom wild snakes and lizards have indicated theoccurrence of at least ®ve di�erent morpho-types [75]. Some of the morpho-types, however,may represent oocysts of C. parvum and C. murisfrom ingestion of infected preys [70]. The extentof genetic diversities within C. serpentis is notclear. Biologically, C. serpentis infection in lizardsis usually asymptomatic, but the infection insnakes frequently causes clinical diseases [1].Minor genetic di�erences have been observedbetween isolates from snakes and those fromlizards [27]. Recently, a new Cryptosporidiumspecies, Cryptosporidium saurophilum, has beendescribed from lizards, Schneider's skink(Eumeces schneideri) and desert monitors [76].

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±1751 1737

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The new species di�ers from C. serpentis by hav-ing smaller oocysts, developing in a di�erent lo-cation in the intestine, and by the inability toinfect snakes [76]. A Cryptosporidium isolatefrom a desert monitor has recently been shownto be genetically distinct [28]. It is unclear, how-ever, whether oocysts from the desert monitoralso belong to the proposed new Cryptosporidiumspecies from lizards, and further genetic charac-terisation studies are required to con®rm the val-idity of C. saurophilum as a separate species.

3.5. Cryptosporidium in guinea pigsÐC. wrairi

Cryptosporidium wrairi was ®rst recognised asa cause of chronic enteritis in a colony of guineapigs (Cavia porcellus) at the Walter Reed ArmyInstitute of Research (WRAIR) [77]. It was sub-sequently characterised and named C.wrairi [78, 79]. Genetic characterisation studies ata number of di�erent loci have all con®rmed thegenetic distinctness of C. wrairi [18, 20, 80]. Itsvalidity as a species was supported by its host-speci®city and also by the fact that, unlike C.parvum, C. wrairi is not readily infective formice [78], although it has subsequently beenshown to be moderately infective to mice [81±84],SPF lambs [81] and calves [84]. Cryptosporidiumwrairi also exhibits strikingly di�erent oocystwall proteins than C. parvum [82], although phy-logenetic analysis in the 18S rRNA, heat shockprotein (HSP70) and Cryptosporidium oocystswall protein (COWP) genes indicates that C.wrairi is closely related to C. parvum ([28, 29, 59];Xiao et al., unpublished observations). Becausefew isolates of C. wrairi have been characterised,the level of intra-speci®c variation within thisspecies has yet to be determined.

3.6. Cryptosporidium in catsÐC. felis

There are few reports of Cryptosporidium incats [85±89] but, on the basis of host speci®cityand oocyst morphology, it has been suggestedthat cats may be host to a di�erent species ofCryptosporidium, namely C. felis [1, 85, 90, 91].Recent genetic characterisation studies have con-®rmed the genetic distinctness of feline isolates of

Cryptosporidium [28, 92, 93]. Furthermore,oocysts from cat faeces average 4.6�4.0 mm,whereas oocysts from humans average5.0�4.5 mm [92].

Phylogenetic analysis of cat-derived isolates ofCryptosporidium has also provided strong sup-port for the validity of C. felis as a separatespecies [28, 59]. For example, the levels of simi-larity between C. parvum and C. felis (e.g.97.6%) are within the range of interspeci®c vari-ation observed for avian Eimeria spp. (99±95.3%). To date, over 20 di�erent feline isolatesof Cryptosporidium from di�erent continentshave been characterised using a portion of the18S rRNA gene ([92, 93]; Morgan et al., unpub-lished). All feline isolates so far examined havebeen virtually identical at this locus. The levels ofgenetic divergence between the cat genotype andother C. parvum isolates determined by 18SrRNA and HSP70 nucleotide sequence analysis(in relation to the observed levels of interspeciesdivergence within avian spp. of Eimeria), coupledwith apparent host-speci®city and morphologicaldi�erences [92], con®rm C. felis as a validspecies.

4. Intraspeci®c variation within C. parvum

4.1. Phenotypic variability within C. parvum

The causative agent of cryptosporidiosis inhumans and a range of other mammalian speciesis the species C. parvum. Although few di�erenceshave been found in the morphological character-istics and developmental cycles of parasites cur-rently classi®ed as C. parvum, increasing evidenceof behavioural di�erences has been reportedbetween isolates over the last few years, reinfor-cing the belief that C. parvum is not a uniformspecies. These include di�erences in virulence andpathogenesis, infectivity, and drugsensitivity [56, 94±97]. Antigenic di�erences havealso been detected among di�erent mammalianisolates in Western-blot studies using hostimmune sera, rabbit antisera and mAbantibodies [56, 57, 97, 98]. More recently,Western-blot analysis of oocyst wall antigens has

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511738

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been used to recognise multiple types within C.parvum [99]. Such heterogeneity may explaindi�erences in clinical picture, response to drugsand passive immunotherapy, and could also com-plicate immunological approaches to diagnosis.Since AIDS and immunocompromised patientsare those most at risk from cryptosporidiosis,there is an urgent need to determine the extent ofgenetic diversity within Cryptosporidium isolatesa�ecting humans. This will allow the develop-ment of appropriate molecular characterisationprocedures, so that sources of transmission canbe identi®ed and strain di�erences correlatedwith clinically important parameters such asseverity of infection. Studies in Italy were the®rst to demonstrate behavioural di�erencesbetween isolates of Cryptosporidium from AIDSpatients exhibiting mild and severe cryptospori-diosis, which were thought to re¯ect intrinsiccharacteristics of the parasite isolates or`strains' [100]. Similarly, reported di�erences indisease severity and response to therapeuticagents in another series of AIDS patients [101]may have been due to variability in theparasite rather than infection intensity. Variableresponses to treatment are also considered tobe a factor in the clinical management ofcryptosporidiosis [102].

The genetic basis of such behavioural di�er-ences has still to be demonstrated, but it hasbeen suggested that variability in virulence couldbe correlated with whether or not the source ofinfection was of zoonotic origin [103]. Earlyresults on the molecular characterisation of C.parvum isolates supported the presence of zoono-tic transmission [9] and have been subsequentlycon®rmed by a wealth of data on the geneticvariability of C. parvum (see below). Indeed, untilit was possible to `genotype' isolates of the para-site, it was very di�cult to interpret the results ofcross-infection experiments, particularly thoseusing mice and calves. Results from di�erent lab-oratories using isolates of human origin oftenproduced variable results, which we now know isa re¯ection of the host speci®city of the parasite.Cross-transmission studies of genotyped isolateshas indicated that the `cattle' genotype readilyinfects mice and cattle, while the `human' geno-

type does not [17, 25, 104]. These results thus sup-port those of earlier workers who had founddi�erences in infectivity between isolates ofhuman and cattle origin [94, 100, 105].

4.2. Cryptosporidium in humans and domesticlivestockÐ`human' and `cattle' genotypes

Less than 20 years ago, it was proposed thatCryptosporidium might be a `single speciesgenus' [106] and a rare human infection [107].There is now strong evidence from genetic andbiological characterisation studies indicatingthat there are two distinct genotypes ofCryptosporidium infecting humans: a `human'genotype, which to date has been found only inhumans and possibly in a captive monkey [19],and a `cattle' genotype, which is found in dom-estic livestock such as cattle, sheep, goats, etc., aswell as humans [4±28, 108, 109], and is infectiousfor other animals such as laboratoryrodents [17, 25].

Genetic diversity in C. parvum has been ident-i®ed using isoenzyme analysis which clearly dif-ferentiated between human and animal isolates,with some human isolates exhibiting animal pro-®les as a result of zoonotic transmission [5, 6, 58].The di�erence between human and animal C.parvum isolates was subsequently con®rmed byRAPD analysis [9, 110±112]. However, due to thelow numbers of oocysts frequently recoveredfrom environmental and faecal samples, and thepresence of contaminants, the majority of geneticcharacterisation studies have utilised parasite-speci®c PCR primers to overcome these pro-blems.

Sequence and/or PCR±RFLP analysis of the18S rDNA gene [10, 26±28, 110] and the morevariable internal transcribed rDNA spacers (ITS1and ITS2) [13, 110], the acetyl-CoA synthetasegene [12], the COWP gene ([18], Xiao et al.,unpublished observations), the dihydrofolatere-ductase-thymidylate synthase (dhfr) gene[8, 24, 59], the 70 kDa HSP70 (Sulaiman et al.,unpublished observations), the thrombospondin-related adhesion protein (TRAP-C1 and TRAP-C2) genes [17, 20±22] and an unidenti®ed genomic

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±1751 1739

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fragment [108] have all con®rmed the genetic dis-tinctness of the `human' and `cattle' genotypes.

A recent multilocus approach analysed 28 iso-lates of Cryptosporidium originating fromEurope, North and South America andAustralia [19]. Polymerase chain reaction±RFLPanalysis of the polythreonine [poly(T)] andCOWP gene, TRAP-C1 gene and ribonucleotidereductase gene (RNR), and genotype-speci®cPCR analysis of the rDNA ITS 1 region, clus-tered all the isolates into two groups, one com-prising both human and animal isolates and theother comprising isolates only of humanorigin [19]. Polymerase chain reaction±RFLPanalysis of the poly(T) and COWP gene, RNRand PCR analysis of the 18S rDNA gene wasalso conducted on C. parvum isolates from AIDSpatients [25]. Five of the patients tested exhibitedthe `human' genotype and two exhibited the`cattle' genotype. In both studies, neither recom-binant genotypes nor mixed infections weredetected [25].

Studies to date have indicated substantial gen-etic di�erences between the `human' and `cattle'genotypes, but very little variation within thesegenotypes. Only minor di�erences within thehuman genotype isolates have been identi®ed inthe 18S rRNA [26], TRAP-C2 [17, 22] andpoly(T) genes [25]. The signi®cance and preva-lence of these intragenotype di�erences, however,are not clear. A recent study has reported thatsequence and PCR±RFLP analysis of the b-tubu-lin intron revealed polymorphism within thehuman genotype and evidence of recombinationbetween the `human' and `cattle' genotypes [113].Others have analysed the same region and foundno support for these results [23, 114]. One groupdi�erentiated between `human' and `cattle' geno-types, but found no variation withingenotypes [23]. The region was so conserved thatC. muris and C. serpentis were identical to thebovine genotype at this locus [23]. The samegroup also analysed the Peru isolate of Widmeret al. [113], at the b-tubulin, CP15 and TRAP-C2loci and were unable to detect recombination ormixed infection, after sequencing 10 clones(Sulaiman et al., unpublished observations).Similarly, sequence analysis of the b-tubulin

locus by another group reported a 1.8% diver-gence between `human' and `cattle' genotypes,but no variation within genotypes and no recom-binant genotypes [114]. In the study by Widmeret al. [113], direct extraction of DNA from stoolswas also used, and it is possible that some of thesequences obtained (especially the `new' humangenotype which showed signi®cant divergencefrom the human and bovine genotypes in boththe intron and exon 2), were the result of non-speci®c ampli®cation by the non-Cryptosporidiumspeci®c primers. In light of the parallel resultsobtained from other researchers, the validity ofthe results generated by Widmer et al. [113]remain uncon®rmed.

In foodborne, waterborne and day-care out-breaks of cryptosporidiosis, oocysts of both thehuman and bovine genotypes of Cryptosporidiumhave been identi®ed, with the human genotypeidenti®ed more frequently [17, 22, 26, 115].Outbreaks caused by the bovine genotype haveall been epidemiologically linked to contami-nation from or direct contact with animals, suchas the Maine apple cider outbreak in 1995, theBritish Colombia outbreak in 1996, thePennsylvania rural family outbreak in 1997 andthe Minnesota zoo outbreak in 1997 [22].Results of these studies were also very useful inclarifying the source of contamination in out-breaks, such as the massive one in Milwaukee in1993, which was probably caused byCryptosporidium of human origin contaminatingthe water supply [17, 22].

Phylogenetic analysis of `human' and `cattle'C. parvum genotypes at the 18S rRNA locusrevealed that they exhibit a similarity of 99.7%.The similarity between T. gondii and N. caninumis 99.8% at this locus. For the ITS locus, thesimilarity between `human' and `cattle' genotypesis much less at 82.23%. The similarity betweenT. gondii and N. caninum at this locus is89.49% [13]. There are also biological di�erencesbetween the `human' and `cattle' genotypes, asthe `human' genotype does not readily infect neo-natal mice or cattle [17, 25, 104]. The absence ofrecombinant genotypes using isoenzyme and mul-tilocus analysis at unlinked loci [6, 19, 25] indi-cates reproductive isolation between the `human'

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511740

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and `cattle' genotypes, and further supports theirdistinction as discrete species.

4.3. Cryptosporidium in miceÐthe `mouse'genotype

Wild rodents are thought to provide an im-portant reservoir of infection of C. parvum forfarmed animals, because oocysts of C. parvumhave been detected in wild brown rats (Rattusnorvegicus), wild house mice (Mus domesticus),wild wood mice (Apodemus sylvaticus) and wildbank voles (Clethrionomys glareolus) [116±118] infarm buildings, ®elds and closely associated agri-cultural areas. Recent research, genetically char-acterising isolates of C. parvum from mice (M.musculus) in Australia, the US, the UK andSpain using sequence analysis of the 18S rRNA,ITS, dhfr, HSP70, COWP and acetylCoA loci, aswell as RAPD analysis, has revealed that theseisolates carry a distinct genotype referred to asthe `mouse' genotype ([12, 13, 16, 28, 59]; Xiao etal., unpublished observations). Interestingly,some of the mice were also infected with the`cattle' genotype, indicating that they might serveas reservoirs of infection for humans and otheranimals. Oocysts of the `mouse' genotype werealso identi®ed from a large footed mouse-earedbat (Myotus adversus), extending the host rangeof this genotype [16].

Phylogenetic analysis of this genotype at therDNA ITS1 locus revealed that the `mouse' gen-otype is most closely related to the `cattle'genotype [59]. At this locus, the similaritybetween `mouse' and `cattle' genotypes is93.14%. The similarity between T. gondii and N.caninum at this locus is 89.49%. Similar resultswere also obtained at the 18S rDNA [28].Analysis of murine isolates at the HSP70 andCOWP loci, however, indicated that the distancebetween bovine and murine C. parvum is greaterthan that between bovine and human isolates(Sulaiman et al., unpublished; Xiao et al., unpub-lished).

4.4. Cryptosporidium in pigsÐthe `pig' genotype

Cryptosporidium was ®rst reported in pigs (Susscrofa) in 1977 [119, 120]. Subsequently, porcinecryptosporidiosis has been reported in pigs inAustralia, Europe, Japan, the US and Vietnam(cf. [121]). Recent genetic characterisation studieshave revealed that pigs are infected with a geneti-cally distinct host-speci®c form ofCryptosporidium [12, 13, 15, 28, 122]. Pigs can alsobe infected with the `cattle' strain, indicating thatthey can potentially play a role as reservoirs ofinfection for humans and other animals [15].Phylogenetic analysis has provided support forthe classi®cation of the `pig' genotype as a dis-tinct and valid species. For example, evolutionarydistances between a C. parvum `cattle' genotypeand the `pig' genotype at the 18S rRNA locuswas 0.0121 nucleotide substitutions/site. This isgreater than the observed distance of 0.0051nucleotide substitutions/site between C. parvumand C. wrairi [28, 59]. Similar conclusion wasmade by the analysis of the HSP70 and COWPgenes (Sulaiman et al., unpublished; Xiao et al.,unpublished).

4.5. Cryptosporidium in marsupialsÐthe`marsupial' genotype

Little is known about the prevalence ofCryptosporidium in marsupials. Cryptosporidiuminfections have been reported in southern brownbandicoots (Isoodon obesulus), a hand-reared ju-venile red kangaroo (Macropus rufus) from SouthAustralia and a Tasmanian wallaby (Thylogalebillardierii) [39]. To date, only three marsupialisolates of Cryptosporidium have been genotyped;an isolate from a koala (Phascolarctos cincereus)from South Australia, a second isolate from ared kangaroo from Western Australia and, morerecently, another isolate from a koala fromWestern Australia ( [10, 13, 28, 59]; Morgan et al.,unpublished). However, 18S rDNA, ITS, HSP70,COWP, dhfr sequence analysis and RAPD analy-sis have all con®rmed their genetic identity anddistinctness from other all other genotypes of C.parvum. Indeed, at the ITS locus, the marsupialgenotype exhibit only 64.13% and 58.55% simi-

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±1751 1741

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larity to the `cattle' and `human' C. parvum geno-types, respectively [59].

4.6. New Cryptosporidium genotypesÐdog,ferret, etc.

Genetic analysis of C. parvum-like isolatesfrom two dog (Canis familiaris) isolates from theUSA and a ferret (Mustela furo) at the 18SrDNA and HSP70 loci have also revealed distinctgenotypes ( [28]; Xiao et al., unpublished). Recentgenotyping of a Cryptosporidium dog isolatefrom Australia was identical to the two dog iso-lates from the USA at the 18S rDNA locus(Morgan et al., unpublished). Phylogenetic analy-sis of the entire 18S rDNA gene from these iso-lates revealed the ferret isolate to be most closelyrelated to C. wrairi, although the distancebetween the ferret genotype and C. wrairi(0.0063), and between the ferret genotype and C.parvum bovine genotype (0.0086), was similar tothat between the C. parvum bovine genotype andC. wrairi (0.0058). Among all the C. parvum gen-otypes, the dog genotype was most divergent,with a distance of 0.0228 nucleotide substi-tutions/site between the dog bovinegenotypes [28]. Additional characterisation stu-dies of dog and ferret isolates are required todetermine how widespread and conserved thesegenotypes are in their respective hosts, before anew species designation can be applied tothem. Recently, a monkey genotype has alsobeen identi®ed based on the analysis of the 18SrRNA, HSP70 and COWP genes ( [28], Xiaoet al., unpublished). As expected, this genotypeis most related to the human genotype. Asmore isolates of Cryptosporidium from otheranimal species are analysed genetically, it islikely that new additional genotypes will beidenti®ed.

5. Population genetics and mode of reproduction

Understanding the extent and nature of geneticvariation in Cryptosporidium is an essential pre-requisite in determining the epidemiology andtransmission of this pathogen. It is especially

important to know whether genotypes of

Cryptosporidium are stable (indicative of clonal

propagation) or unstable (due to frequent genetic

recombination). Although the population struc-

ture of Cryptosporidium has not been conclusively

determined, it appears likely to be clonal, based

on several criteria that have been used as a basis

for a clonal population structure in the related

parasite Toxoplasma [123].

The widespread occurrence of identical geno-

types is an important criterion supporting this

concept of a clonal population structure in

Cryptosporidium [11]. The broad distribution of

the cattle, human, pig and mouse genotypes

supports this concept, as does the correlation

between phenotypic and genotypic markers

[6, 124] and, more recently, evidence of parity

between unlinked genetic loci [19, 25, 59]. Parity

between two sets of independent genetic mar-

kers suggests that recombination might be bio-

logically restricted. This is one of a number of

criteria claimed to provide evidence of clonal

population structure [123]. Conversely, it has

been argued that for regions under selection,

linkage disequilibrium can be maintained

despite su�cient recombination and that selec-

tion, for example as a result of host immunity,

can cause discrete `strains' or genotypes to

persist [125].

To test adequately the clonal model theory

for Cryptosporidium and determine population

structure, a population genetic study is

required, in which data are analysed in terms

of allelic and genotypic frequencies, rather than

simply assessing distances as done in the past.

This would require examining a much larger

number of isolates from localised endemic foci.

This study is now in progress, in collaboration

with colleagues in Australia, North America

and Europe. Evaluation of intragenotype diver-

sity will also be helpful for the elucidation of

Cryptosporidium population structure in view of

the proposed species status of some of the cur-

rent genotypes. Future research should provide

a clearer understanding of the population struc-

ture and transmission characteristics of this ubi-

quitous parasite [109].

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511742

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6. Phylogeny

Phylogenetic analysis of Cryptosporidium iso-lates, using distance-based and parsimony analy-sis at di�erent loci, has provided strong evidencethat this genus is composed of several distinctand valid species [27, 28, 59, 70]. These ®ndingscontrast with those in which a pairwise compari-son of 18S rDNA sequence data obtained fromGenBank for C. wrairi, C. baileyi, C. murisand several bovine C. parvum isolates suggestedthat interspecies and intraspecies values didnot appear appreciably di�erent [126]. Thissuggestion was developed by comparingCryptosporidium species variation to the consider-able degree of variation within Plasmodiumspecies [126]. This comparison betweenCryptosporidium and Plasmodium may be inap-propriate for several reasons: (1) levels of inter-species divergence within Plasmodium is verylarge, and greater in some cases than theintra-genera di�erences between other apicom-plexans (e.g. Cryptosporidium vs Eimeria,Cryptosporidium vs Toxoplasma); (2) the origin ofthe Plasmodium sequences indicating speci®crRNA loci that are expressed at di�erent stagesof the Plasmodium life-cycle was not considered;and (3) the levels of genetic divergence (based onsequences from GenBank) between some 18SrRNA loci within Plasmodium vivax are largerthan divergences between P. vivax and otherPlasmodium species when compared at the same18S rRNA locus (data not shown).

In other studies, pairwise comparison of 18SrDNA sequence data for some Cryptosporidiumisolates revealed interspecies values signi®cantlyhigher than intraspecies values [27, 28, 59, 70].For example, the similarity between two bovineisolates of C. parvum (AUCP and KSU-1) is99.94%, whereas the similarity between C. par-vum (AUCP) and C. baileyi is 96.26% [59]. Theseresults compare well with the level of similaritybetween avian Eimeria species whose phyloge-netic relationships have been wellcharacterised [127]. For example, similaritieswithin avian Eimeria range from 99 to 95.3%.Cryptosporidium parvum and C. muris exhibit asimilarity of 93.27%, which is considerably less

than the similarity between E. tenella andCyclospora spp. at 96.68% [59]. Analysis byother workers has also con®rmed the validity ofspecies within the genus Cryptosporidium [26±28].In addition, Cryptosporidium parasites are not re-lated to other coccidians and the recognisedspecies in Cryptosporidium separate into twobroad groups, with C. muris and C. serpentisforming one group and C. parvum, C. felis, C.wrairi, C. meleagridis, and C. baileyi forming asecond broad group (see Fig. 1).

7. Proposals for a revised taxonomy

7.1. The importance of species discrimination

One of the fundamental issues for researchersin infectious diseases is to determine the taxo-nomic status of the pathogen. Therefore, mean-ingful genetic characterisation of Crypto-sporidium should also address the species natureof the organisms that various researchers arestudying. However, with Cryptosporidium, aswith many other parasitic organisms, determin-ing exactly what constitutes a species is notstraightforward.

The most commonly used species concept,the `biological species concept', has severaldrawbacks, particularly for protozoan parasites.Interbreeding is often di�cult to test directly,as groups may be geographically separated andlaboratory crosses may not re¯ect naturaloccurrence; most importantly, it is applicableonly to sexually reproducing, outcrossingorganisms [128]. Recent studies, especially withbacteria, have shown that low levels of recom-bination occur in many species, allowing thebiological species concept to be extended togroups previously thought to be strictly clonal.There is a continuum from panmictic (strictlysexual, outcrossing) to strictly clonal breedingsystems, and in some species, this may varygeographically, between ecological niches oreven over time [129]. In the case ofCryptosporidium, more comprehensive popu-lation genetic studies are required to determine

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±1751 1743

Page 12: Variation in Cryptosporidium: towards a taxonomic revision of the genus

whether it is strictly clonal or whether there is

any evidence of recombination between clonal

lineages.

Although there are a large number of species

concepts and guidelines for designating species,

they share common attributes that to be a

Fig. 1. (a) Phylogram depecting relationships inferred by NJ analysis of evolutionary distances inferred from 18S rRNA gene

sequences. Bootstrap values (distance-based, parsimony analysis) of >50% are indicated at each node. (Reproduced with per-

mission from [57].) (b) Phylogenetic relationships among Cryptosporidium species and genotypes inferred by NJ analysis of 18S

rRNA gene sequences. Bootstrap values (Kimura two-parameter distance-based) of >70% are indicated at each node. (Modi®ed

with permission from [28].)

U.M. Morgan et al. / International Journal for Parasitology 29 (1999) 1733±17511744

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species, a group must be cohesive (monophy-letic) and distinct from other groups. A com-prehensive taxonomic study of a group thusinvolves studying numerous individuals/isolatesfrom the entire geographic range and individ-uals from similar species, looking for distinctclusters. This must be done without makingassumptions about what constitutes a popu-lation, as there may be invisible ecologicalsubdivisions [129]. As with studies on a numberof other parasite groups, the molecular charac-terisation of Cryptosporidium has revealed sub-divisions not apparent using morphology. Thesesubdivisions appear to represent host-adaptedgenotypes between which there may be very lit-tle gene¯ow and thus may represent distinctspecies. However, for Cryptosporidium, as forother protozoan groups with a clonal popu-lation structure, the question of an appropriatespecies concept has hardly been addressed,although Tibayrenc et al. [130] suggested thatgenetically distinct groups of clonal parasitesshould be labelled as di�erent species onlywhen the genetic discontinuities correspondwith di�erences in characters of medical signi®-cance. Pathogenic species should thus bede®ned by both biological and genetic criteria,with only those forms exhibiting speci®c bio-logical and/or medical speci®cities deserving thestatus of species [131, 132]. In this respect, theidenti®cation of di�erent genotypes of di�erentCryptosporidium species may have importantepidemiological consequences in terms of under-standing transmission cycles and sources ofinfection in outbreak situations.

Tibayrenc [133] has recently provided a usefulinterpretation of genetic data of infectious agentsby referring to `discrete typing units' (DTUs)which have been characterised genetically andcorrespond to discrete, stable evolutionary units.These units, which may have been di�cult toidentify in the past, may or may not representdistinct species. In such determinations it is use-ful to determine the stability of such DTUs [133]and, in this respect, knowledge of their clonalityis very useful. As such, DTUs may correspond toeither clonal lineages or to cryptic species, andcan be identi®ed by speci®c genetic markers [132].

7.2. A revised taxonomy of the genusCryptosporidium

We believe that limiting species in the genusCryptosporidium to eight is an oversimpli®cationin light of earlier reports of unique oocyst mor-phology and/or host speci®city and recent dataobtained from a comprehensive genetic character-isation of isolates from di�erent species of hosts.As we have detailed above, a range of host-adapted, geographically conserved, genotypeshave been identi®ed that appear likely to re¯ecttaxonomic discrimination as well as providing avaluable basis for the predictive epidemiology ofcryptosporidial infections.

In summary, phylogenetic analysis has indi-cated that C. parvum is not monophyletic, as C.wrairi is clearly placed within C. parvum [28, 59](see Fig. 1a, b). Acceptance of C. wrairi as avalid species (see above) provides strong supportfor accepting the pig, marsupial, and dog geno-types as separate cryptic species. The evolution-ary distances between the `cattle' genotype of C.parvum (KSU-1) and the pig, marsupial, and doggenotypes at 0.0121, 0.015, and 0.0228 nucleotidesubstitutions/site, respectively, are considerablygreater than the evolutionary distances betweenC. parvum (KSU-1) and C. wrairi at 0.0051nucleotide substitutions/site. All these genotypesappear to be host-speci®c and, in the case of thepig, marsupial and human genotypes, there areother biological di�erences, i.e. they do notreadily infect mice ( [15, 17, 25, 104]; Morgan etal., unpublished). Whereas the ferret isolate isalso genetically distinct, to date only one isolatehas been genotyped and, therefore, more isolatesneed to be genotyped from a large geographicarea to conclusively determine the validity of theferret genotype as a separate species. Similarly,more isolates from dogs also need to be geno-typed. Indeed, a wider range of isolates from allgenotypes needs to be examined in the future ata number of di�erent loci. In the case of the mar-supial genotype, isolates of Cryptosporidium frommarsupials from the American continent shouldalso be examined.

The `human' and `cattle' genotypes may be dis-tinct species. At the rDNA ITS locus, the simi-

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Page 14: Variation in Cryptosporidium: towards a taxonomic revision of the genus

larity between `human' and `cattle' genotypes was82.23%, which is less than the similarity betweenT. gondii and N. caninum at this locus at89.49% [59]. There are also biological di�erencesbetween the `human' and `cattle' genotypes. The`human' genotype does not readily infect neo-natal mice or cattle [17, 25, 104]. The absence ofrecombinant genotypes using isoenzyme and mul-tilocus analysis at unlinked loci [6, 19, 25] indi-cates reproductive isolation between the `human'and `cattle' genotypes, and further supports theirdistinction as discrete species. Furthermore, theirgenetic identity is maintained in endemic areaswhere both occur and are transmitted.

The `mouse' genotype may also be a separatespecies (see above). Although the mouse geno-type is related to the cattle genotype, and thegenetic distance between the cattle genotype andthe mouse genotype is less than the distancebetween C. wrairi and the cattle genotype at the18S rRNA and ITS loci, at the HSP70 andCOWP loci the distance between the mouse andcattle genotypes is greater than the distancebetween C. wrairi and the cattle genotype. In ad-dition, biologically, the mouse genotype is com-monly found in adult mice but never in cattle,whereas the cattle genotype is infectious for onlyneonatal mice. As more polymorphic loci becomeavailable, the presence or absence of hybridsbetween di�erent genotypes infecting the samehosts, such as the `human' and `cattle' genotypes,and the `cattle' and `mouse' genotypes, can bemore accurately determined. This will providenot only important information on the popu-lation structure of Cryptosporidium, but also in-formation as to whether these genotypes arereally reproductively isolated and thus representdistinct species.

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