Genetic Diversity and Population Structure of Cryptosporidium

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Review

Genetic Diversity and Population Structure of Cryptosporidium Yaoyu Feng,1 Una M. Ryan,2 and Lihua Xiao1,* Cryptosporidium species differ in host range. Parasite–host coevolution, host adaptation, and geographic segregation have led to the formation of subtype families with unique phenotypic traits within the major human-pathogenic species C. parvum and C. hominis. Transmission intensity, genetic diversity, and occurrence of genetic recombination and selective pressure have further shaped their population genetic structures. Panmixia appears to be common within the zoonotic C. parvum, especially its hypertransmissible IIaA15G2R1 subtype. Genetic recombination in C. hominis, in contrast, is more restricted to virulent subtypes, especially IbA10G2. Nonhuman primates and equine animals are commonly infected with genetically divergent C. hominis populations. Systematic studies of these and other host-adapted Cryptosporidium spp. are likely leading to improved understanding of population structures underlying various transmission patterns and intensities of Cryptosporidium. Cryptosporidium Presents a Significant Public Health Problem Cryptosporidiosis is one of the primary causes of diarrhea in both humans and farm animals [1,2]. Children, neonatal animals, and immunocompromised individuals are particularly susceptible [3]. In developing countries, cryptosporidiosis is among the most important causes of diarrhea and diarrhea-associated death in young children [2,4,5]. It remains one of the major causes of waterborne outbreaks of illness in industrialized nations, and can be responsible for a significant number of food-borne and zoonotic outbreaks of illness [6–8].

Highlights Cryptosporidium species and subtypes differ in host range and public health significance. Host-adapted subtype families exist in the human-pathogenic species C. parvum and C. hominis. Genetic recombination is common within the zoonotic C. parvum, especially its hypertransmissible IIaA15G2R1 subtype. Genetic recombination in C. hominis is mostly restricted to its virulent subtypes, especially IbA10G2. Host and geographical segregation, genetic recombination, and selective pressure shape the population structure of C. parvum and C. hominis, leading to the emergence of hostadapted, virulent, and hypertransmissible subtypes with public health significance.

The causative agent of cryptosporidiosis, Cryptosporidium, has thus far 38 recognized species. The description of new Cryptosporidium species has accelerated in recent years, with 12 being established since a recent review of the subject [9–17,128]. There are also at least an equal number of Cryptosporidium genotypes of unknown species status. In this review, we discuss the genetic diversity in Cryptosporidium spp. at various levels and the population genetic structure of major human-pathogenic Cryptosporidium species and subtypes.

Cryptosporidium Species Have Different Public Health Significance Most Cryptosporidium species and genotypes have host specificity. As a result, only between one and four of the named Cryptosporidium species and genotypes are commonly found in each host species (Table 1). For example, humans are mostly infected with C. parvum and C. hominis; cattle with C. parvum, C. bovis, C. ryanae, and C. andersoni; and sheep and goats with C. parvum, C. ubiquitum, and C. xiaoi. However, other Cryptosporidium species and genotypes can be found in these hosts occasionally; thus far, over 20 Cryptosporidium species and genotypes have been identified in humans [18]. The identification of Cryptosporidium spp. at the species or genotype level is essential for the assessment of infection sources in humans and the public health potential of parasites in animals. As some of the occasional detections of unusual Cryptosporidium species and genotypes were based on molecular analysis, it is Trends in Parasitology, Month Year, Vol. xx, No. yy

1 Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China 2 School of Veterinary and Life Sciences, Vector- and Water-Borne Pathogen Research Group, Murdoch University, Murdoch, Western Australia 6150, Australia

*Correspondence: [email protected] (L. Xiao).

https://doi.org/10.1016/j.pt.2018.07.009 © 2018 Elsevier Ltd. All rights reserved.

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Glossary

Table 1. Cryptosporidium spp. in Humans and Domestic Mammals

a

Host

Tier 1 speciesa

Tier 2 speciesb

Tier 3 speciesc

Humans

C. hominis, C. parvum

C. meleagridis, C. felis, C. canis, C. ubiquitum, C. cuniculus, C. viatorum, chipmunk genotype I, C. muris

C. andersoni, C. suis, C. bovis, horse genotype, C. xiaoi, skunk genotype, mink genotype, C. erinacei, C. fayeri, C. scrofarum, C. tyzzeri

Cattle

C. parvum, C. bovis

C. ryanae, C. andersoni

C. occultus, C. ubiquitum, C. xiaoi

Sheep & goats

C. xiaoi, C. ubiquitum

C. parvum

C. andersoni

Pig

C. suis, C. scrofarum

C. muris

–

Camels

C. andersoni, C. muris

C. parvum

–

Horses and donkeys

Horse genotype, C. parvum, C. hominis

C. andersoni

C. muris, C. tyzzeri, C. felis (?), C. proliferans

Dogs

C. canis

C. parvum, C. muris

C. scrofarum, C. ubiquitum, C. hominis

Cat

C. felis

C. muris

C. parvum, C. ryanae, rat genotype III

Rabbit

C. cuniculus

–

–

House mouse

C. tyzzeri, C. muris

C. parvum

–

Tier 1 species: Cryptosporidium spp. that are responsible for over 90% of infections in the specified host. Tier 2 species: Cryptosporidium spp. that have been each reported in at least five cases. Tier 3 species: Cryptosporidium spp. for which confirmed detection has been each reported in fewer than five individuals.

b c

difficult to differentiate colonized/replicating parasites from ingested oocysts simply passing through the host [19,20]. The distribution of Cryptosporidium species in humans is different between developing and industrialized countries, with C. hominis as the dominant species in developing countries and both C. hominis and C. parvum as the dominant species in industrialized nations. They are responsible for almost all cryptosporidiosis outbreaks investigated [18]. Nevertheless, they differ from each other in virulence, with C. hominis causing more clinical presentations in most studies [21–25]. Among the less common Cryptosporidium species, C. meleagridis, C. felis, C. canis, C. viatorum, and C. muris are largely seen in developing countries, whereas C. ubiquitum, C. cuniculus, and chipmunk genotype I are mainly seen in industrialized nations. Among the latter, C. ubiquitum and chipmunk genotype I are responsible for substantial numbers of cryptosporidiosis cases in rural states in the USA, while C. cuniculus cases are mostly seen in the UK [26–29]. As these Cryptosporidium species and genotypes are found in various animals, the difference in Cryptosporidium species distribution could be a reflection of the importance of different animals or infection sources in cryptosporidiosis epidemiology [30].

Subtypes and Host Adaptation in C. parvum C. parvum is among the few Cryptosporidium species with a broad host range and is the most important zoonotic species. Subtyping has been used extensively in studies of C. parvum transmission in humans and animals (Figure 1). The most commonly used genetic locus for subtyping Cryptosporidium spp. is the 60 kDa glycoprotein gene (gp60) (see Glossary and Box 1). Thus far, nearly 20 C. parvum subtype families have been described at the locus, some of which (such as IIc) appear to be adapted to humans, and some (such as IIa and IId) adapted to animals (Box 2). Because gp60 is one of the most polymorphic loci in the Cryptosporidium 2

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Clonal population: a population of organisms with a common origin. It is characterized by strong linkage disequilibrium among genetic loci and frequently very little or no genetic diversity among isolates prevalent in the host at any given time. Epidemic population: a population characterized by frequent recombination in which a particular successful clone may be generated, becoming the predominant one for an extended period. gp60: a 60 kDa immunodominant glycoprotein widely believed to be involved in the invasion of Cryptosporidium spp. Its gene is one of the most polymorphic genes in the Cryptosporidium genome because of immune selective pressure, thus it is commonly used in subtyping humanpathogenic Cryptosporidium species. Host adaptation: this refers to the ability of a pathogen to circulate and cause disease in a particular host population. It is an indicator of the pathogen’s fitness or ability to adapt to its host environment, but may lead to reduced occurrence of interspecies transmission of the pathogen. Linkage disequilibrium: the status of a nonrandom association of alleles (sequence types) at different genetic loci. Between two loci with linkage disequilibrium, the allele at one polymorphic locus can predict the allele of an adjacent polymorphic locus. Panmictic population: a population with high frequency of genetic recombination among individuals. As a result, individuals from the population have high genetic diversity and low linkage disequilibrium among genetic loci.

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++++ : ProporƟonal to populaƟon size in humans : GeneƟc diversity : Transmission direcƟon + + : Importance of transmission direcƟon

II a

II c

IIaA15G2

R1

Clonal ? Ɵc mic

Pan

C. parvum

++

+++ ++

II d

Clon

al

++

+ +

Figure 1. Transmission and Population Genetics of Major Host-adapted Cryptosporidium parvum Subtype Families (IIa, IIc, and IId in Humans, Cattle, and Small Ruminants, Respectively). The major directions of transmission are indicated by arrows, with their importance in cryptosporidiosis being indicated by red plus symbols. The size of the pie charts is proportional to the approximate population size of the subtype families in humans, while the relative diversity of the subtype families is indicated by the complexity of the charts. The most common IIa subtype, IIaA15G2R1, as well as the population genetics (clonal or panmictic) of the subtype families is indicated.

Box 1. Cryptosporidium parvum and Cryptosporidium hominis Subtypes
C. hominis and C. parvum are commonly subtyped by DNA sequence analysis of the 60 kDa glycoprotein gene

(gp60) [18].
Each species has multiple subtype families of different host range and/or virulence/transmissibility [18].
These subtype families are traditionally named as Ia, Ib, Id, Ie, If, etc. for C. hominis; IIa, IIb, IIc, IId, etc. for C. parvum;

and IIIa, IIIb, IIIc, IIId, etc. for C. meleagridis.
Within each subtype family, subtypes mainly differ from each other mostly in the trinucleotide repeat (TCA, TCG, or

TCT) region downstream from the signal peptide sequence of the gene.
Subtypes within each family are named by the number of TCA (represented by the letter A), TCG (represented by the

letter G), or TCT (represented by the letter T) repeats.
In some subtype families, other repetitive sequences (such as ACATCA in the IIa subtype family, AAGACGGTGG-

TAAGG or its variations in the Ia subtype family, and AAGAAGGCAAAGAAG or its variations in the If subtype family) are present, which are designated by R at the end of the subtype names.
The subtype nomenclature of Cryptosporidium spp. has recently been described in detail [18].
The most prevalent subtypes are IbA10G2 and IIaA15G2R1 for C. hominis and C. parvum, respectively [30].
Because of the existence of selective pressure and genetic recombination at the gp60 locus, genetic traits at other genetic loci often do not segregate by gp60 subtype [39].

genome, is under selective pressure, and undergoes through genetic recombination [31–33], its typing results do not always agree with those of other genetic loci for some of the C. parvum subtype families, especially IIa and IId. These two subtype families differ at the whole-genome level mostly in some but not all mucin genes, to which gp60 belongs [34]. Others, such as the newly identified IIo and IIp subtype families, differ further from common C. parvum subtype families at some more conserved genetic loci such as the small subunit (SSU) rRNA, 70 kDa

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Box 2. Cryptosporidium parvum Subtype Families and the Hypertransmissible IIaA15G2R1 Subtype
















Cryptosporidium parvum has broad host specificity and high genetic heterogeneity [16]. Common C. parvum subtype families include IIa, IIc, and IId, all of which have different preferred hosts [18]. In farm animals worldwide, IIa is mostly found in cattle, while IId is mostly found in sheep and goats [18]. In European countries and Australia, IIa subtypes can also be found in lambs and young goats, while in China, dairy calves are mostly infected with IId subtypes [120]. IIc subtypes are mostly found in humans [30]. Comparative genomic analysis has shown high genetic similarity between IIa and IId subtypes of bovine origin, but significant divergence between IIa and IIc subtypes [34,37]. Other C. parvum subtype families have been mostly detected in a few humans or animals, except for IIe and IIm, which appear to be two genetically related human-adapted C. parvum subtype families found in developing countries [30]. A few of the rare C. parvum subtype families, such as IIi (AY873782) in humans and yellow-necked mice and IIu (LC270809 and LC270810) in camels, have gp60 sequences very similar to those of C. hominis (Id and If subtype families, respectively) [63,121]. IIaA15G2R1 is the dominant IIa subtype in calves and lambs as well as humans in most industrialized nations [30,87,122]. There is heterogeneity within IIaA15G2R1, with frequent discordance in subtyping results between gp60 and other genetic markers, due to common occurrence of genetic recombination within this subtype [87]. It has been suggested that the IIaA15G2R1 subtype at the gp60 locus is a fitness marker for C. parvum, with independent sequence characteristics at other genetic loci [87].

heat shock protein (HSP70), actin, and oocyst wall protein (COWP) genes [35]. They could represent more divergent C. parvum. The genetic basis for host adaptation in C. parvum subtypes is not clear. Recently, comparative genomic analysis has been used in addressing this issue. Copy-number variations in two secretory Cryptosporidium-specific protein families (MEDLE proteins and insulinase-like proteases) have been linked to differences in host ranges between C. parvum and C. hominis [36]. These differences have also been observed at the genome level between C. parvum subtype families IIa (preferentially infects cattle) and IId (preferentially infects sheep and goats). Compared with the IIa subtype family, the IId subtype family has lost one of the six genes encoding MEDLE proteins and gained at least one insulinase-like protease gene [34]. In addition to these gene gains and losses, C. parvum IIa, IId, and IIc subtype families have highly divergent subtelomeric genes encoding other families of secretory proteins, such as mucins [34,37]. The distribution of C. parvum subtype families in humans is distinct in different areas and socioeconomic conditions, probably reflecting differences in transmission patterns in cryptosporidiosis epidemiology, especially the importance of zoonotic infection and various farm animals (Figure 1). In most industrialized nations, zoonotic C. parvum infections are caused by IIa subtype families, particularly the dominant IIaA15G2R1 subtype. In contrast, in developing countries, IIa subtypes are rarely seen in humans, where C. parvum infections are mostly caused by the anthroponotic IIc subtypes, together with a few other human-adapted IIe and IIm subtype families. In the Mideast, both IIa and IId subtypes are commonly seen in humans. IId subtypes occur in low frequency in European countries and Australia, but are largely absent in the USA [18].

Subtypes and Host Adaptation in C. hominis Over ten subtype families have been recognized in C. hominis, based on sequence analysis of the gp60 gene [18] (Box 3). The lower diversity of gp60 subtypes in C. hominis compared with C. parvum could be due to its narrower host range and lower transmission intensity. Unlike in C. parvum, subtype families in C. hominis at the gp60 locus frequently do not segregate in the same way at other genetic loci [38,39]. Among the common C. hominis subtype families (Ia, Ib, Id, Ie, If, and Ig), there are significant differences in subtype diversity (Figure 2). Infections by the

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Box 3. Cryptosporidium hominis Subtype Families and the Virulent IbA10G2 Subtype









Cryptosporidium hominis has a much narrower host range than C. parvum [30]. Ia, Ib, Id, Ie, If, and Ig are the most common C. hominis subtype families found in humans worldwide [18]. Ii and Ik are nonhuman primate-adapted and equine animal-adapted C. hominis, respectively (see Table 2 in main text). The most dominant C. hominis subtype is IbA10G2, which is widely distributed in both industrialized and developing countries [40,115]. IbA10G2 is responsible for most C. hominis-associated outbreaks in Europe and Australia and numerous outbreaks in the USA [6,18,40,115,123–126]. IbA10G2 is also the predominant subtype among sporadic cases in many countries, and was the main subtype identified during an increase of cryptosporidiosis cases in Europe in late summer 2012 [103,115,127]. It has also been proposed to be more virulent and cause long-term postinfection symptoms [21,40,115]. Genetic recombination has been observed in IbA10G2 from developing countries and the USA [36,40].

Ib : ProporƟonal to populaƟon size in humans : GeneƟc diversity : Transmission direcƟon + + : Importance of transmission direcƟon

IbA10G2

++++

+

++

Epidemic

I a, I d, I e, I f & I g nal

Clo

C. hominis

Clo

nal

Ii&Ik

?

? ++++

+

+

Figure 2. Transmission and Population Genetics of Major Host-adapted Cryptosporidium hominis Subtype Families (Ia–Ig in Humans, Ii in Nonhuman Primates, and Ik in Equine Animals). The major directions of transmission are indicated by arrows, with their importance in cryptosporidiosis being indicated by red plus symbols. The size of the pie charts is proportional to the approximate population size of the subtype families in humans, while the relative diversity of the subtype families is indicated by the complexity of the charts. The most common Ib subtype, IbA10G2, as well as the population genetics (clonal or epidemic) of the subtype families is indicated.

Ib subtype family are mostly caused by IbA10G2 and IbA9G3, with the former being distributed worldwide and the latter mostly seen in some developing countries [18,40–45]. Other subtypes, such as IbA12G3, IbA13G3, IbA16G2, IbA19G2, IbA20G2, and IbA21G2, are restricted to confined areas [43,46–50]. Similarly, there are only two dominant subtypes within the Ie subtype family: IeA11G3T3 and IeA12G3T3. In contrast, there are many subtypes within other common C. hominis subtype families, especially in developing countries [51]. C. hominis subtypes in humans differ in infectivity and virulence, with subtype IbA10G2 dominating in many sporadic and outbreak cases worldwide [21,22,25,50] (Box 3). Although C. hominis is widely considered a human-specific Cryptosporidium species, it is now increasingly reported in animals (Figure 2); C. hominis has been detected in cattle [19,52–56], Trends in Parasitology, Month Year, Vol. xx, No. yy

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T repeat AJ849464_Human_SI1_Slovenia DQ388385_Human_NL1_Netherlands DQ388386_Human_NL2_Netherlands AF093491_Human_HFL2_US AF481962_Human_UG502_Uganda AF108865_Human_H7_Australia DQ286403_Human_Chile01_Chile KM012041_Human_UKH_UK AF112569_Rhesus_CPRM1_US KF679722_Rhesus_CHCM-1_China KP716563_Macaca_ML77_Thailand KP716564_Macaca_ML138_Thailand KX710091_Gibbon_80GYZ_China KX710087_Gibbon_65GYZ_China KX710088_Gibbon_76GYZ_China GU319778_Monkey_China KR296813_Human_SC563_Thailand KR296812_Human_SC476_Thailand KU200954_Donkey_82_China_ KU200955_Horse_Tt_China_ KT948752_Horse_EPC46_Brazil KU892563_Human_Swec466_Sweden KU892567_Human_Swec726_Sweden KX926452_Horse_B12_China KX926453_Horse_B29_China

ATATAAAATA .......... .......... .......... .......... .......... .......... .......... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......T... ......TG.. ......TG..

TTTTGATGAA .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

TATTTATATA .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

ATATTAACAT .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AATTCATATT .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... G......... .......... .......... .......... .......... .......... .......... ..........

ACTATTTTTT .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

TTTTTAGTAT .......... .......... .......... .......... .......... .......... .......... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--..... ...--.....

Ia, Ib, Id & Ie

Ii & Ik

Io

Figure 3. Sequence Differences in the Hypervariable Region of the Small Subunit rRNA Gene among Some Cryptosporidium hominis Subtype Families (Ia–Io). Dots denote nucleotide identical to the reference sequence AJ849464 in the first line, while dashes denote nucleotide deletions. The poly-T region of the gene is indicated as T repeat.

yaks [20], sheep and goats [57–59], kangaroos [55,56,60–62], rodents [14,63], hedgehogs [64], and dogs [65]. As the C. hominis subtype involved in most of the animals (IbA10G2) was also the dominant subtype circulating in humans in these areas, spillover infection of these animals with C. hominis of human origin could be responsible for these C. hominis infections in animals [14,53,54,59–61,63,64] (Figure 2). Persistent circulation of this and other C. hominis subtypes, however, has been reported in cattle, sheep, and nonhuman primates in isolated areas [42,47,53,55,59,66,67]. Contrasting with its occasional detection in other animals, C. hominis is one of the common Cryptosporidium species in nonhuman primates, horses, and donkeys (Table 1), which appear to be normal hosts for C. hominis (Figure 2). Nevertheless, the genetic characteristics of C. hominis in nonhuman primates and equine animals appear to be different from those of C. hominis in humans. At the SSU rRNA locus, C. hominis isolates from some nonhuman primates have nucleotide sequences that are slightly different from human isolates, with T being in nine instead of 11 consecutive places in the hypervariable region of the gene, and an A-to-T nucleotide substitution upstream from the region (Figure 3) [47,68,69]. Minor sequence differences have also been observed at the HSP70, actin, and COWP loci between C. hominis in humans and monkeys (the latter is the monkey genotype) [47,70]. These monkey genotype isolates mostly have an identical gp60 subtype, IiA17, thus representing a unique host-adapted C. hominis subtype family [47,70,71]. A variant of the monkey genotype, which exhibits another unique gp60 subtype that was misnamed as IkA7G4, has also been reported recently [69]. The C. hominis monkey genotype has also been observed in a few human cases [70,72,73]. Other variants of C. hominis related to the monkey genotype are present in horses and donkeys, also representing host-adapted C. hominis subtype families (Figure 2). They exhibit

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Table 2. Distribution of Host-adapted Cryptosporidium hominis Subtypes Subtype family Ii

Ik

Io

Subtype

Host

No. of cases

Location

GenBank accession No.

Refs

IiA17

Rhesus monkey

1

USA

HM234173

[71]

IiA17

Crab-eating macaque

1

China

KF679724

[47]

IiA17

Human

2

Thailand

KR296810, KR296811

[70]

IkA15G1

Horse

1

Algeria

KJ941148

[75]

IkA20G1

Horse

2

Brazil

KT948748

[77]

IkA16

Donkey

2

China

KU200962

[74]

IkA16G1

Horse and donkey

58

China

KU200958, KU200963

[74]

IkA18G1

Human

2

Sweden

KU727290

[70]

IoA15

Horse

2

China

KX926458, KX926459

[76]

SSU rRNA sequences very similar to the C. hominis monkey genotype, with 0–2 nucleotide differences between the two groups (Figure 3) [70,74–76]. This was further supported by sequence analysis of other genetic loci such as the HSP70 and actin genes [70,74–77]. Most of the isolates have been identified as divergent subtypes in the host-adapted Ik subtype family (Table 2). One of the Ik subtypes, IkA18G1, has been found in two human patients in Sweden [70]. Another variant from two horses in China belonged to a new gp60 subtype family related to Id, although it was misdiagnosed as IdA15 [76]. It has now been renamed as IoA15 (Table 2). This subtype family has one additional nucleotide substitution in the hypervariable region of the SSU rRNA gene (Figure 3). It also has HSP70 sequences very similar to those from C. hominis in monkeys in China and Thailand [76]. Comparative genomic analysis of this and other C. hominis subtypes supports the divergent nature of the C. hominis Ik subtype family [78]. It has four times as many single nucleotide polymorphisms (SNPs) as the next most diverse C. hominis isolate.

Subtypes and Host Adaptation in Other Human-Pathogenic Cryptosporidium spp. Host adaptation has also been identified in other Cryptosporidium spp. such as C. ubiquitum and C. tyzzeri [28,79]. Thus far, eight subtype families (XIIa–XIIh) have been identified in C. ubiquitum based on sequence analysis of the gp60 gene [28,80]. Although C. ubiquitum has a broad host range, host adaptation is apparent at the gp60 locus: XIIa is found in ruminants worldwide, XIIb–XIId in rodents in the USA, and XIIe and XIIf in rodents in the Slovak Republic [28]. Humans in the USA are infected with rodent-adapted subtype families XIIb–XIId, whereas those in other areas such as the UK are mainly infected with ruminant-adapted subtype family XIIa [28]. As C. ubiquitum is one of the most common Cryptosporidium species in drinking source water in the USA, and rodent-adapted subtype families are also the dominant C. ubiquitum in drinking source water, consumption of drinking water contaminated by infected wildlife could be the source of human infections [28]. The genetic similarity of Cryptosporidium chipmunk genotype I isolates from humans, wild mammals, and drinking source water also supports the potential role of wildlife and drinking source water in the transmission of emerging Cryptosporidium spp. in the rural USA [27]. Genetic heterogeneity also exists in C. tyzzeri, which is mostly a parasite of small rodents. Two subtype families of C. tyzzeri have been identified in house mice based on sequence analysis of the gp60 gene: IXa and IXb [71]. They differ in the subspecies of house mice they infect: Mus

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musculus musculus (Mmm) and Mus musculus domesticus (Mmd). The IXa subtype family is commonly found in Mmm whereas the IXb subtype family in Mmd. This existence of genetically segregated C. tyzzeri subtype families in the two subspecies of house mice was supported by sequence characterizations of the actin, COWP, and thrombospondin-related adhesive protein 1 genes [79]. Both subtype families have been reported occasionally in humans [81]. Similar host-segregated parasite populations are probably also present among Cryptosporidium genotypes in cricetid rodents [82]. Host adaptation has been further observed in C. canis in canine animals [83] and C. ryanae in bovine animals based on sequence characterization of the SSU rRNA gene [83,84].

Population Genetics of C. parvum and the Hypertransmissible IIaA15G2R1 Subtype Multilocus typing tools are increasingly used in advanced typing of C. parvum and C. hominis [73,85,86]. Compared with gp60-based subtyping, the inclusion of other highly polymorphic loci in molecular characterization of clinical isolates allows us to assess the population genetics of Cryptosporidium spp., especially the population structure of different Cryptosporidium species in humans and farm animals; the effect of transmission intensity and patterns, host and geographic segregation, host migration, and agricultural practices; and the mechanism for the emergence of virulent and hypertransmissible C. parvum and C. hominis subtypes. The use of loci beyond the gp60 gene allows not only the measurement of linkage disequilibrium among genetic markers but also comprehensive assessments of the genetic traits of Cryptosporidium spp., as genetic characteristics at other genetic loci do not always segregate by gp60 subtype because of the occurrence of selection at the gp60 locus and genetic recombination or perhaps chromosome missegregation during sexual reproduction [38,87]. Population genetic characterization using multilocus typing tools has mostly identified a panmictic population structure for C. parvum in industrialized nations [88–93]. A panmictic population structure for C. parvum is also seen in both humans and preweaned dairy calves from the same geographic areas [90,93]. This is expected because of the high prevalence of C. parvum in dairy calves due to concentrated animal-feeding operations, the existence of a sexual phase in Cryptosporidium life cycle, and the zoonotic nature of human C. parvum infections in the study areas [94]. Indeed, these studies have identified shared multilocus genotypes (MLGs) between humans and calves from the same areas [90,93]. Results of these population genetic studies have demonstrated the occurrence of geographic segregation in C. parvum subpopulations. Thus, C. parvum isolates formed country-specific clusters in eBURST analysis of MLG data from Uganda, Israel, Serbia, Turkey, and New Zealand [92]. Similarly, a significant geographical segregation was also identified among 692C. parvum isolates originated primarily from cattle and other ruminants from Italy, Ireland, and Scotland. In particular, the genetic distance among C. parvum MLGs was correlated with geographical distance [95]. Population stratifications have also been identified in C. parvum isolates from several other areas [87]. Other studies however, have failed to identify geographical segregation in C. parvum populations [73,90], but those studies were conducted over smaller geographic areas within a country. Data from these studies have also indicated the occurrence of host segregation among C. parvum populations, as previously observed at the gp60 subtype family level [91,93,96]. Thus, C. parvum isolates from humans have two major subpopulations; one is shared with that in calves, whereas the other one appears to be anthroponotic [73,93]. The former appears to represent the gp60 IIa subtype family whereas the latter is probably the IIc subtype family 8

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[38,94]. Host-segregated C. parvum subpopulations have also been reported in ruminants in Northeast Spain, where dairy calves are commonly infected with the IIa subtype family, whereas lambs and young goats are mostly infected with the IId subtype family. In population structure analysis of C. parvum isolates, they formed separate clusters in Bayesian Structure, eBURST, and factorial correspondence analyses [96,97]. In Italy, the subpopulation of C. parvum in goats appears to be different from that in calves and lambs [91]. The occurrence of host-associated and geographically segregated subpopulations appears to shape the population structure of C. parvum through reduced gene flow (Figure 1). In addition to the panmictic population structure discussed above, other genetic modes are present in C. parvum [92]. Thus, several multilocus typing studies have identified clonal populations of C. parvum in dairy cattle and humans in some industrialized nations [87,91,95,98,99]. In Spain, C. parvum in cattle has a panmictic structure while that in sheep has a clonal structure, which is probably a reflection of differences in population genetic structure between gp60 IIa and IId subtype families [88,99]. This was supported by a population genetic study of the C. parvum IId subtype family in China, Egypt, and Sweden, which has mostly a clonal population structure [100]. In Uganda, the genetic structure of C. parvum populations (presumably the anthroponotic IIc subtype family) also appears to be different from the population structures seen in Serbia and New Zealand (presumably the zoonotic IIa), with the former being largely clonal and the latter largely panmictic [92]. However, recombination among host-adapted C. parvum subtype families is possible [87,96], and when the transmission intensity and genetic diversity of C. parvum are high, distinct C. parvum MLGs of the IIa subtype families are frequently confined to individual farms [90,97]. The panmictic population structure of C. parvum described in published studies is probably a reflection of the transmission intensity and nature of reproduction in the IIa subtype family, which is highly prevalent in dairy cattle in industrialized nations. This is further supported by data from population genetic studies conducted with isolates of known gp60 subtype identity [87,89,90,96]. As the hyper-transmissible IIaA15G2R1 subtype is the dominant C. parvum in humans and calves in most of these countries [30], the panmixia reported in C. parvum could be attributed to the reproductive nature of this subtype (Figure 1). Indeed, a comparative study of IIaA15G2R1 and non-IIaA15G2R1 from several geographic areas has shown an epidemic population structure in IIaA15G2R1 and a clonal population structure in non-IIaA15G2R1 [87]. Because of the common occurrence of genetic recombination within IIaA15G2R1, there is frequent discordance in subtype segregation among genetic loci. As a result, there is no clear population differentiation between IIaA15G2R and non-IIaA15G2R. The IIaA15G2R1 subtype at the gp60 locus appears to be a marker for high transmissibility in C. parvum [87]. This suggests that gp60, or other closely linked loci, may play an important role in determining the infectious properties of Cryptosporidium spp., such as virulence or transmissibility [36,39,40,87].

Population Genetics of C. hominis and the Virulent IbA10G2 Subtype The population genetic structure of C. hominis appears to be significantly different from that of C. parvum in the same areas [93]. In contrast to the common occurrence of panmixia observed in the C. parvum IIa subtype family, most studies have shown a clonal population structure for C. hominis in industrialized nations [93,98,101,102] (Figure 2). This is expected considering the narrow host range and lower transmission intensity of C. hominis. This is especially the case for European countries, where almost all autochthonous C. hominis infections are caused by the IbA10G2 subtype. Thus, population genetic characterizations of isolates from the UK, The Netherlands, and Spain have shown very limited genetic diversity of C. hominis as a result of clonality [92,93,98,103]. In fact, the presence of one or two dominant MLGs in these areas Trends in Parasitology, Month Year, Vol. xx, No. yy

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essentially demonstrates the epidemic nature of the clonal C. hominis population. In Spain, C. hominis subpopulations are segregated by gp60 subtype family, with non-IbA10G2 subtypes being more divergent due to their nature of being imported from developing countries [98]. Sequence conservation in IbA10G2 isolates in European countries has been confirmed by comparative genomic analysis, which has identified fewer than 50 SNPs at the whole-genome level among most IbA10G2 isolates from Europe [44,78]. The clonal expansion of IbA10G2 in European countries presents a significant challenge in epidemiologic tracking of this subtype, which is a major outbreak subtype there [86]. Recently, based on WGS data from 17 isolates, a panel of nine loci with ten SNPs was selected for the development of a PCR next-generation sequencing (NGS) technique, which identified ten sequence types among 44 clinical isolates in Sweden [104]. Conventional MLST tools would be of limited utility in epidemiological tracking of this hypertransmissible C. hominis subtype [18]. The population structure of C. hominis in developing countries, where transmission intensity and the genetic diversity of C. hominis isolates are much higher, appears to be more complicated. Although a study of isolates from India has shown a mostly clonal population genetic structure, the occurrence of genetic recombination among some of the subtypes could not be excluded, as the linkage disequilibrium was incomplete [102]. Data from an earlier study of isolates from Uganda have indicated frequent genetic exchange among C. hominis isolates [92]. Incomplete linkage disequilibrium was also found in the overall C. hominis population from a small community in Lima, Peru, although genetic recombination was largely restricted to the virulent IbA10G2 subtype [40]. Decay of linkage disequilibrium in the genome has also been observed recently in C. hominis in a slum in Dhaka, Bangladesh, probably due to frequent genetic recombination among diverse subtypes in the community. The genetic structure of IbA10G2 in Jamaica, however, appears to be similar to the one in European countries, with minimum genetic heterogeneity [101]. The genetically recombining nature of IbA10G2 in non-European countries has been supported by comparative genomics. In the USA there are two types of IbA10G2 genomes among the few isolates sequenced, one of which is similar to the one in European countries, whereas the other one has sequences of IaA28R4, another hypertransmissible subtype that has emerged in the USA in recent years, at several genetic loci [36]. In fact, these two C. hominis subtypes have very similar genomes, with major sequence differences concentrated at the 50 (around cgd6_60), 30 (around cgd6_5270), and gp60 (cgd6_1080) regions of chromosome 6. In these regions, C. hominis genomes in the USA have biallelic sequences, with a mosaic pattern in sequence types among isolates. Thus, genetic recombination could play a potential role in the emergence of hypertransmissible C. hominis subtypes, as previously indicated by MLST characterization of IbA10G2 and IaA28R4 [39,40]. This has been facilitated by the common presence of multiple C. hominis subtype families within the USA. Results of the population genetic studies and comparative genomics have also indicated the presence of geographical segregation within C. hominis, especially among developing countries. Earlier MLST studies had shown significant population differentiation in C. hominis isolates from Jamaica, Peru, India, and Kenya [85,101]. This was also supported by a comparison of population genetic structure of C. hominis isolates from Uganda and the UK [92]. In those studies, IbA10G2 isolates from different areas formed different clusters in the phylogenetic analysis of MLST data, further supporting the genetically recombinant nature of this virulent subtype [101]. Comparative genomics analysis of IbA10G2 isolates confirmed that the genome of IbA10G2 from Guatemala was very divergent compared to that from Europe [78]. 10

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Population Genetics of Other Cryptosporidium spp.

Outstanding Questions

The population genetics of other human-pathogenic Cryptosporidium spp. has attracted some attention. A comparison of C. meleagridis isolates from humans and birds using an MLST tool has shown a clonal population structure of the pathogen and likely occurrence of cross-species transmission of infections in Lima, Peru [105]. A similar population structural was also seen in C. cuniculus in rabbits in Heilongjiang, China, using the same tool [106]. Another MLST characterization of C. ubiquitum from humans and various animals has confirmed the existence of host-adapted parasite populations in ruminants and rodents and the presence of genetic recombination among rodent-adapted gp60 subtype families [107]. Population genetic analysis has been widely used in the characterization of bovine C. andersoni in China, which has mostly identified a clonal or epidemic population structure [108–112].

To what extent does genetic diversity at the gp60 locus correlate with genome diversity among Cryptosporidium species and subtype families?

Concluding Remarks Clearly, genetic diversity exists in Cryptosporidium spp. at both species and subtype levels, although it is lower than that commonly observed in other apicomplexans such as Plasmodium spp. and Toxoplasma gondii because of differences in life cycle strategies (monoxenous versus heteroxenous, gastrointestinal versus blood- or tissue-dwelling) and transmission intensity [113,114]. The coevolution, genetic recombination, adaptive selection, and geographical segregation within these species all help in shaping the population genetics of Cryptosporidium spp. These processes have led to the association of some population structures with specific transmission patterns, and the emergence of host-adapted, virulent, and hypertransmissible populations with different genetic structures and public health potential. The global expansion of some virulent C. hominis and C. parvum subtypes, such as IbA10G2 and IIaA15G1R1, presents major challenges to the identification and investigation of cryptosporidiosis outbreaks. This is especially problematic in Europe, where most autochthonous C. hominis infections are caused by genetically conserved IbA10G2 [115]. Knowledge of the mechanisms behind the emergence of successful subtypes with diverse phenotypic traits is critical to the improved understanding of cryptosporidiosis epidemiology in both industrialized and developing nations (see Outstanding Questions) and the development of advanced molecular detection tools for the investigation of cryptosporidiosis outbreaks [18].

What is the mechanism for the emergence of host-adapted C. parvum and C. hominis populations? What are genetic determinants for virulent, and hypertransmissible C. parvum and C. hominis subtypes. What is the mechanism for the C. hominis subtype drift in the USA? What are the population genetic structures of C. parvum and C. hominis in developing countries? What are major genetic differences in C. parvum IId subtypes between China and European countries? Can advanced typing tools be developed for clonally expanded subtype IbA10G2 and newly emerged subtype IfA12G1R5?

Recent technological advances present us with some opportunities to further understand the genetic diversity and population structure of Cryptosporidium spp. NGS and comparative genomics are now increasingly used in the characterization of Cryptosporidium spp. in humans and animals [18,116]. NGS allowed advanced typing of IbA10G2 in Europe [78,104] and may provide new tools in the characterization of emerging C. hominis subtypes in the USA, where IbA10G2 was the dominant C. hominis subtype for cryptosporidiosis outbreaks in previous years [36]. In 2005, however, a new subtype, IaA28R4, appeared and soon became the dominant C. hominis subtype for sporadic cases and outbreaks in the USA [39,117]. In recent years, this subtype has virtually disappeared, being replaced by IfA12G1R5 as the dominant C. hominis subtype in sporadic and outbreak cases [118]. The mechanism involved in C. hominis subtype drift is not well understood. In addition, whole-genome sequencing and comparative genomics are yet to be used in advanced characterization of the hypertransmissible C. parvum subtype IIaA15G2R1 in humans and cattle in industrialized nations. Population genetics and comparative genomics are also needed in advanced molecular characterization of human-pathogenic Cryptosporidium spp. in developing countries. The high prevalence and high genetic diversity of C. hominis and C. parvum in humans in developing countries indicate that genetic recombination among diverse subtypes could occur more frequently there. Thus far, only limited numbers of population genetic studies and only one comparative genomic Trends in Parasitology, Month Year, Vol. xx, No. yy

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study of C. hominis have been conducted in this setting [40,92,101,102,119]. Additional data from developing countries should be valuable in understanding the role of genetic recombination and selective pressure in virulence and hypertransmissibility of some Cryptosporidium subtypes and the mechanism for host adaptation and cross-species transmission of others. They will also be useful in understanding the significance of the unique distribution of C. hominis and C. parvum subtypes in some geographic areas, such as the dominance of C. parvum IId subtypes in humans, farm animals, and rodents in China. Acknowledgments This work was supported by the National Natural Science Foundation of China (31630078 and 31425025).

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