Molecular epidemiology of cryptosporidiosis: An update

Experimental Parasitology 124 (2010) 80–89

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Molecular epidemiology of cryptosporidiosis: An update Lihua Xiao * Division of Parasitic Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, Bldg. 22, Rm. 14, 4770 Burford Highway, Atlanta, GA 30341, USA

a r t i c l e

i n f o

Article history: Received 16 October 2008 Received in revised form 17 March 2009 Accepted 27 March 2009 Available online 7 April 2009 Keywords: Cryptosporidium Molecular epidemiology Diagnosis Zoonosis Genotyping Subtyping gp60

a b s t r a c t Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels. These tools have been increasingly used in characterizing the transmission of Cryptosporidium spp. in humans and animals. Results of these molecular epidemiologic studies have led to better appreciation of the public health importance of Cryptosporidium species/genotypes in various animals and improved understanding of infection sources in humans. Geographic, seasonal and socioeconomic differences in the distribution of Cryptosporidium spp. in humans have been identified, and have been attributed to differences in infection sources and transmission routes. The transmission of C. parvum in humans is mostly anthroponotic in developing countries, with zoonotic infections play an important role in developed countries. Species of Cryptosporidium and subtype families of C. hominis have been shown to induce different clinical manifestations and have different potential to cause outbreaks. The wide use of a new generation of genotyping and subtyping tools in well designed epidemiologic studies should lead to a more in-depth understanding of the epidemiology of cryptosporidiosis in humans and animals. Published by Elsevier Inc.

1. Introduction

2. Recent developments in molecular epidemiologic tools

Because of the ability of Cryptosporidium to infect various animals and the ubiquitous presence of Cryptosporidium oocysts in environment, humans can acquire Cryptosporidium infections through several transmission routes, such as direct contact with infected persons (person-to-person transmission) or animals (zoonotic transmission) and ingestion of contaminated food (foodborne transmission) and water (waterborne transmission). The relative importance of these transmission routes in the epidemiology of cryptosporidiosis is not entirely clear, largely due to the fact that traditional diagnostic tools do not have the ability to differentiate sources of parasites, and epidemiologic investigations are expensive to conduct. In the last decade, however, numerous molecular biological techniques have been developed to detect and differentiate Cryptosporidium spp. at species/genotype and subtype levels. These tools are now increasingly used in epidemiological studies of cryptosporidiosis in endemic and epidemic areas, which has improved significantly our understanding of the transmission of cryptosporidiosis in humans and animals. Earlier research data in this area were reviewed previously (Xiao et al., 2004a; Xiao and Ryan, 2004, 2008). This review discusses mostly recent progresses in cryptosporidiosis molecular epidemiology since 2006.

Different types of molecular diagnostic tools have been used in the differentiation of Cryptosporidium species/genotypes and C. parvum and C. hominis subtypes. Small subunit (SSU) rRNAbased tools are now generally used in genotyping Cryptosporidium in humans, animals and water samples. A review of original studies on Cryptosporidium genotyping during the last three years revealed the use of SSU rRNA tools in 100 (86%) of 116 publications. In particular, a PCR-RFLP tool that targets an 830-bp fragment of the gene and uses SspI and VspI restrictions for genotyping (Xiao et al., 1999, 2001) is commonly used, being reported in 70 (60%) publications. The widespread use of the SSR rRNA gene in Cryptosporidium genotyping is largely due to the multi-copy nature of the gene and presence of semi-conserved and hyper-variable regions, which facilitate the design of genus-specific primers. Other molecular diagnostic tools based on other genes were popular previously, but their use in Cryptosporidium genotyping has decreased in recent years. Thus, tools based the oocyst wall protein (COWP) gene were used in only 23 of the 116 original publications (many in combination with SSU rRNA-based tools) and other genes were rarely used. PCR tools based on these genes in general only amplify DNA of C. parvum, C. hominis, C. meleagridis, and species/genotypes closely related to C. parvum. Human, animal and environmental studies that have used these tools have usually showed fewer Cryptosporidium species and genotypes than expected (Gomez-Couso et al., 2007; Meamar et al., 2007; Wolska-Kusnierz et al., 2007;

* Fax: +1 770 488 4454. E-mail address: [email protected] 0014-4894/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.exppara.2009.03.018

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Bajer et al., 2008; Duranti et al., 2008; Giangaspero et al., 2009), and at several occasions the use of COWP tools had led to the identification of the common occurrence of C. parvum in unexpected animal species (Giangaspero et al., 2006; Paziewska et al., 2007; Traversa et al., 2008). COWP-based tools have limited usefulness in genotyping Cryptosporidium spp. of animals because of their narrow specificity. Subtyping tools have been used extensively in studies of the transmission of C. hominis in humans and C. parvum in humans and ruminants. One of the popular subtyping tools is the DNA sequence analysis of the 60 kDa glycoprotein (gp60, also called gp40/ 15). The gp60 gene is similar to a microsatellite sequence by having tandem repeats of the serine-coding trinucleotide TCA, TCG or TCT at the 50 (gp40) end of the gene. However, in addition to variations in the number of trinucleotide repeats, there are extensive sequence differences in the non-repeat regions, which categorize C. parvum and C. hominis each to several subtype families. Within each subtype family, subtypes differ from each other mostly in the number of trinucleotide repeats (TCA, TCG or TCT microsatellite). Representative sequences of some common C. parvum and C. hominis subtype families are shown in Table 1. The name of gp60 subtypes starts with the subtype family designation (Ia, Ib, Id, Ie, If, etc. for C. hominis, and IIa, IIb, IIc, IId, etc. for C. parvum) followed by the number of TCA (represented by the letter A), TCG (represented by the letter G), or TCT (represented by the letter T) repeats (Sulaiman et al., 2005). Thus, the name IbA10G2 indicates that parasite belongs to C. hominis subtype family Ib and has 10 copies of the TCA repeat and two copies of the TCG repeat in the trinucleotide repeat region of the gp60 gene. In the C. parvum IIa subtype family, a few subtypes also have two copies of the ACATCA sequence right after the trinucleotide repeats, which are represented by ‘‘R2” (R1 for most subtypes). Additionally, within the C. hominis subtype family Ia, subtypes are further identified by the copy number of a 15-bp repetitive sequence (represented by the letter R) 50 -AA/GGACGGTGGTAAGG-30 (the last copy is 13-bp: AAA/GACGGTGAAGG) that is located shortly downstream of the trinucleotide repeats. Therefore, the name IaA28R4 indicates that parasite belongs to C. hominis subtype family Ia, and has 28 copies of the TCA repeat in the trinucleotide region and four copies of the 13–15 bp repeat. In the case of C. parvum subtype family IIc, all subtypes had five copies of TCA and three copies of TCG repeats: IIcA5G3. They differed from each other in the nucleotide sequence of the 30 region of the gene. The original gp60 sequence for C. parvum subtype family IIc (GenBank Accession No. AF164491) was assigned as IIcA5G3a. Subtypes that diverged from this sequence were assigned subsequent alphabetical extensions. For example,

subtype IIcA5G3b has a trinucleotide deletion (ACA) shortly after the trinucleotide repeats and 31 nucleotide substitutions whereas subtype IIcA5G3c had 33 nucleotide substitutions. It should be kept in mind that gp60 and other subtyping tools (including the multilocus typing and multilocus sequence typing tools below) do not amplify DNA of C. felis, C. canis, and other species distant from C. parvum and C. hominis. Sequence analysis of gp60 gene is widely used in Cryptosporidium subtyping because of its sequence heterogeneity and relevance to parasite biology. It is the most single polymorphic marker identified so far in the Cryptosporidium genome (Gatei et al., 2006a; Leoni et al., 2007; Wielinga et al., 2008). Unlike other subtyping targets, such as double stranded RNA, internal transcribed spacer-2 and traditional microsatellites and minisatellites, which are generally considered non-functional, gp60 is located on the surface of apical region of invasive stages of the parasite, and is one of the dominant targets for neutralizing antibody responses in humans (O’Connor et al., 2007). Thus, it is possible to link biologic characteristics of the parasites and clinical presentations with the subtype family identity. Some of the C. parvum subtype families, such as IIa and IId, are found in both humans and ruminants, responsible for zoonotic cryptosporidiosis. In areas with both IIa and IId, such as Spain, IIa subtypes preferentially infect calves whereas IId subtypes preferentially infect lambs and goat kids (Quilez et al., 2008a,b). Other C. parvum subtype type families, especially IIc (formerly known as Ic), have been so far only found in humans (Alves et al., 2003; Xiao and Feng, 2008). There are also significant differences in clinical presentations and virulence among some common C. hominis subtype families in cryptosporidiosis-endemic areas (Cama et al., 2007, 2008). The recent whole genome sequencing of C. parvum and C. hominis has allowed the identification of microsatellite and minisatellite sequences in C. parvum and C. hominis genomes and other targets highly polymorphic in C. parvum and C. hominis. They are frequently used in multilocus analysis to increase the subtyping resolution. Two types of techniques are used in the subtyping. In multilocus typing (MLT), variations in microsatellites and minisatellites are assessed on the basis of length variations using polyacrylamide gel electrophoresis or the GeneScan technology (Ngouanesavanh et al., 2006; Tanriverdi et al., 2006; Tanriverdi and Widmer, 2006; Leoni et al., 2007; Morrison et al., 2008). This allows the use of many targets in the MLT techniques economically. The second type of typing techniques, multilocus sequence typing (MLST), relies on the detection of genetic heterogeneity by DNA sequencing of the amplified PCR products (Cama et al., 2006b; Gatei et al., 2006a, 2007, 2008). Comparing to MLT, MLST

Table 1 Major GP60 subtype families and representative sequences. Species

Subtype family

Dominant trinucleotide repeat

Other repeat (R)

GenBank Accession Nos.

C. hominis

Ia Ib Id Ie If Ig

TCA TCA, TCA, TCA, TCA, TCA

AA/GGACGGTGGTAAGG – – – – –

AF164502 (IaA23R4) AY262031 (IbA10G2), DQ665688 (IbA9G3) DQ665692 (IdA16) AY738184 (IeA11G3T3) AF440638 (IfA19G1) EF208067 (IgA24)

IIa IIb IIc

TCA, TCG TCA TCA, TCG

ACATCA – –

IId IIe IIf IIg IIh IIi IIk IIl

TCA, TCG TCA, TCG TCA TCA TCA, TCG TCA TCA TCA

– – – – – – – –

AY262034 (IIaA15G2R1), DQ192501 (IIaA15G2R2) AF402285 (IIbA14) AF164491 (IIcA5G3a), AF164501 (IIcA5G3b), EU095267 (IIcA5G3c), AF440636 (IIcA5G3d) AY738194 (IIdA18G1) AY382675 (IIeA12G1) AY738188 (IIfA6) AY873780 (IIgA9) AY873781 (IIhA7G4) AY873782 (IIiA10) AB237137 (IIkA14) AM937006 (IIlA18)

C. parvum

TCG, TCT TCG TCG, TCT TCG

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allows the detection of length polymorphism and nucleotide substitution in microsatellite and minisatellite markers and the inclusion of other markers with only single nucleotide polymorphisms (SNP). In addition to subtyping, MLT and MLST tools are widely used in population genetic studies and geographic tracking of C. parvum and C. hominis (Ngouanesavanh et al., 2006; Tanriverdi et al., 2006, 2008; Gatei et al., 2007, 2008; Morrison et al., 2008). No matter which PCR tool is used in genotyping or subtyping Cryptosporidium, all broadly specific tools have the problem of detecting only the dominant genotype in the specimen because of the inherent nature of exponential amplification by PCR and the requirement of a substantial amount of PCR product to be visible on an agarose gel. On the other hand, all narrowly specific genotyping and subtyping tools detect only C. hominis, C. parvum and species or genotypes related to them. The former results in the failure in detecting many concurrent infections with mixed Cryptosporidium species/genotypes, whereas the latter leads to the failure in detecting other divergent species/genotypes and the inability to subtype these parasites. Therefore, the type of molecular diagnostic tools used and their order of usage would greatly affect the results of molecular epidemiologic investigations of cryptosporidiosis transmission. In a study reported by Cama et al. (2006a), the use of a SSU rRNA-based PCR-RFLP genotyping tools resulted in the detection of C. canis and C. felis in multiple specimens from 21 HIV+ adults in Peru. Re-analysis of these specimens using COWP- and dihydrofolate reductase-based PCR-RFLP genotyping tools, however, failed to detect C. canis and C. felis in any of the specimens, and identified C. hominis, C. parvum or C. meleagridis in seven of the 21 persons. The species distribution and the number of concurrent infections would have been very different if only one type of genotyping tools or the reverse order of the two types of genotyping tools were used. 3. Cryptosporidium parvum infections in farm animals 3.1. Cryptosporidium parvum in cattle Cattle are commonly infected with three other Cryptosporidium spp. in addition to C. parvum, including C. andersoni, C. bovis, and C. ryanae. Earlier studies in the United States indicated the existence of an age-related occurrence of Cryptosporidium spp. In dairy cattle, C. parvum is mostly found in pre-weaned calves, C. bovis and C. ryanae in weaned calves, and C. andersoni in yearlings and adult cattle (Fayer et al., 2006, 2007; Santin et al., 2008). The development of a MboII RFLP procedure to differentiate the three bovine intestinal species (C. bovis, C. parvum and C. ryanae) (Feng et al., 2007) has facilitated studies on the Cryptosporidium species distribution in cattle. The occurrence of the four species in dairy cattle and their age-related distribution have been confirmed in numerous recent studies in various areas (Geurden et al., 2006, 2007; Starkey et al., 2006; Coklin et al., 2007; Feng et al., 2007; Langkjaer et al., 2007; Plutzer and Karanis, 2007; Thomaz et al., 2007; Thompson et al., 2007; Brook et al., 2009; Halim et al., 2008; Szonyi et al., 2008). The same pattern of infections probably also exist in beef and other breeds of cattle (Geurden et al., 2007; Feltus et al., 2008; Szonyi et al., 2008). Thus, in cattle, only pre-weaned calves are major source of C. parvum. 3.2. Cryptosporidium parvum in sheep and goats An earlier study conducted in Australia suggested that C. parvum was largely absent in sheep. Instead, C. bovis and the Cryptosporidium cervine genotype were the two common parasites in sheep (Ryan et al., 2005b). This has been confirmed in recent studies in the United States, United Kingdom, Belgium, and Tunisia

(Santin et al., 2007; Soltane et al., 2007; Elwin and Chalmers, 2008; Geurden et al., 2008). However, some of the recent studies in UK and Spain have shown exclusive or dominant occurrence of C. parvum in lambs (Pritchard et al., 2007, 2008; Mueller-Doblies et al., 2008; Quilez et al., 2008a). In these studies, the cervine genotype and C. parvum appeared to be more prevalent than C. bovis. Therefore, there appear to be some geographic differences in the occurrence of Cryptosporidium spp. However, results of a recent study in Australia have demonstrated that the failure to detect C. parvum in a previous study in lambs was the preferential detection of the dominant species in samples with mixed parasite populations by a SSU rRNA-based PCR tool (Yang et al., 2009). Whether age differences of the study animals also contributed to the difference in species distribution remains to be decided. Although earlier studies failed to detect any age associated differences in the distribution of Cryptosporidium species/genotypes in sheep (Santin et al., 2007; Mueller-Doblies et al., 2008), results of the recent Australian study seemingly suggest that C. parvum preferentially infects preweaned lambs over old animals (Yang et al., 2009). Few studies have genotyped Cryptosporidium from goats. In one small study in Belgium, C. parvum was the only species found in 11 goat kids (Geurden et al., 2008). Cryptosporidium bovis and a new Cryptosporidium genotype, however, were found in two goats in China (Karanis et al., 2007). Cryptosporidium hominis was the only species identified in three goat, five human, and six cattle samples in South Korea (Park et al., 2006). There is apparently also a Cryptosporidium goat genotype in goats (Ryan et al., 2005a). There are minor genetic differences in the SSU rRNA gene of C. bovis among cattle, sheep, and goats. 3.3. Cryptosporidium parvum in pigs Likewise, an earlier study in Australia identified an absence of C. parvum in pigs and the presence of two major Cryptosporidium spp. in pigs: C. suis and the Cryptosporidium pig genotype II (Ryan et al., 2003b). The occurrence of the two genotypes and absence of C. parvum in pigs have been shown more recently in Australia, Norway, N. Ireland, Denmark and Spain (Xiao et al., 2006; Hamnes et al., 2007; Langkjaer et al., 2007; Suarez-Luengas et al., 2007; Johnson et al., 2008). In two studies, it was shown that C. suis preferentially infected suckling piglets, whereas the pig genotype II was more frequently found in weaners (Langkjaer et al., 2007; Johnson et al., 2008). In two Czech studies, only C. suis was seen in preand post-weaned piglets, both Cryptosporidium pig genotype II and C. suis were found in finishers, and C. parvum was found in two sows (Vitovec et al., 2006; Kvac et al., 2009). Cryptosporidium parvum was also only rarely seen in pigs in a study conducted in Ireland (Zintl et al., 2007). Thus, pigs are not a major source of C. parvum. 3.4. Cryptosporidium parvum in other farm animals Few studies have characterized Cryptosporidium spp. in other farm animals. Cryptosporidium parvum is known to infect horses (Chalmers et al., 2005b; Grinberg et al., 2008a), but the number of specimens characterized is small and horses are known infected with the Cryptosporidium horse genotype (Ryan et al., 2003a). Likewise, C. parvum has also been found in a small number of alpaca fecal specimens genetically characterized (Starkey et al., 2007; Twomey et al., 2008). 4. Cryptosporidium species/genotypes in humans Five Cryptosporidium species/genotypes are responsible for most human cryptosporidiosis cases, including C. hominis, C. parvum, C.

L. Xiao / Experimental Parasitology 124 (2010) 80–89

meleagridis, C. felis, and C. canis (Xiao and Feng, 2008). Among them, C. hominis and C. parvum are the most common, responsible for the majority of human infections, especially in industrialized nations, even though in some areas C. meleagridis infection rates are as high as C. parvum (Cama et al., 2007, 2008). A few other Cryptosporidium species and genotypes are occasionally found in humans, including C. muris, C. suis, C. andersoni and Cryptosporidium cervine, horse, rabbit, skunk and chipmunk I genotypes (Robinson et al., 2008; Xiao and Feng, 2008; Xiao and Ryan, 2008). The distribution of C. parvum and C. hominis in humans differs in geographic regions. In European countries, both C. parvum and C. hominis are common in humans (Leoni et al., 2006; Nichols et al., 2006; Soba et al., 2006; Llorente et al., 2007; Wolska-Kusnierz et al., 2007; Bajer et al., 2008; Savin et al., 2008; Wielinga et al., 2008; Chalmers et al., 2009a; Zintl et al., 2009). In the Middle East, C. parvum is the dominant species in humans (Sulaiman et al., 2005; Meamar et al., 2007; Tamer et al., 2007; Al-Brikan et al., 2008; Pirestani et al., 2008). In the rest of the world, especially developing countries, C. hominis is usually the predominant species in humans (Cordova Paz Soldan et al., 2006; Gatei et al., 2006b, 2007, 2008; Muthusamy et al., 2006; Park et al., 2006; Samie et al., 2006; Ajjampur et al., 2007; Bushen et al., 2007; Cama et al., 2007, 2008; Cheun et al., 2007; Hung et al., 2007; Morse et al., 2007; Araujo et al., 2008; Jex et al., 2008). Geographic variations in the distribution of C. parvum and C. hominis can also occur within a country. For example, C. parvum is more common than C. hominis in rural states in the United States and Ireland (Feltus et al., 2006; Zintl et al., 2009). There are also temporal and age-associated variations in the disease burdens between C. parvum and C. hominis (Chalmers et al., 2009a). Like earlier observations in the United Kingdom and New Zealand, C. hominis was more prevalent in autumn (in the Netherlands), and C. parvum was more prevalent in spring (in Ireland) in some recent studies (Wielinga et al., 2008; Zintl et al., 2009). In the Netherlands, C. hominis was more commonly found in children and C. parvum more in adults (Wielinga et al., 2008). In the United Kingdom, C. hominis was more prevalent in infants less than one year, females aged 15–44 years and international travelers, and there has been a decline in C. parvum cases since 2001 (Chalmers et al., 2008, 2009a). In studies conducted in Peru, there were no significant differences in the distribution of Cryptosporidium species or genotypes between children and HIV+ persons, indicating that there is no preferential infection with zoonotic species/genotype in immunocompromised persons (Cama et al., 2007, 2008). Cryptosporidium parvum and C. hominis are thus far associated with most waterborne, foodborne and direct contact-associated (person-to-person and animal-to-person) outbreaks of cryptosporidiosis (Xiao and Ryan, 2008). One recent drinking waterborne outbreak of cryptosporidiosis in England, however, was caused the Cryptosporidium rabbit genotype (Chalmers et al., 2009b). 5. Cryptosporidium parvum subtypes and zoonotic transmission 5.1. Cryptosporidium parvum subtypes in animals To characterize the transmission dynamics and zoonotic potential of C. parvum, numerous studies have been conducted to subtype C. parvum in farm animals, especially calves. Most of the studies employed gp60 sequence analysis, and were done in industrialized nations. Results of these studies have shown that calves are commonly infected with subtypes in the IIa subtype family (Table 2). One subtype, IIaA15G2R1, is especially common; it is overwhelmingly the dominant subtype in most areas studied. Several other subtypes, however, are more regionally distributed.

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Thus, IIaA16G1R1 is a major subtype in Hungary, Slovenia and the Belgrade, Serbia and Montenegro area, and IIaA18G3R1 is mostly found in the United Kingdom and Australia (Table 2). On some farms, multiple subtypes are presented in dairy calves, some times concurrently in the same calf (Xiao et al., 2007). All gp60 subtypes described in Europe and Australia have only one copy of the sequence ACATCA (R1) immediately after the trinucleotide repeats, although some subtypes in North American have two copies of such sequence (R2) (Table 2) (Feltus et al., 2006; Trotz-Williams et al., 2006; Xiao et al., 2007). In addition to the IIa subtype family, IId and IIl (misnamed as IIj in some publications) subtypes are also occasionally found in calves in European countries. Thus far, several IId subtypes have been found in dairy calves in Portugal, Spain, Hungary, Germany, Belgium and the Belgrade, Serbia and Montenegro area (Table 2). Likewise, IIl subtypes have also been found in a few calves in the Lithuania, Slovenia and the Belgrade, Serbia and Montenegro area (Table 2). Subtyping of C. parvum has been done in only a few other farm animals. Both IIa and IId subtypes have been found in sheep in Belgium and Spain. However, the prevalence of IId subtypes in these studies are much higher than IIa subtypes (Geurden et al., 2008; Quilez et al., 2008a). IId subtypes are probably especially common in Spain, having been found to far outnumber IIa subtypes in lambs and goat kids, although IIa rather than IId subtypes are commonly seen in calves (Quilez et al., 2008a,b). Like previously seen in calves, IIaA18G3R1 is also common in foals in Australia (Grinberg et al., 2008a; Ng et al., 2008). 5.2. Cryptosporidium parvum subtypes in humans and zoonotic transmission Many of the common bovine IIa subtypes in North America, Europe and Australia are also dominant C. parvum subtypes in humans in these areas (Alves et al., 2006; Feltus et al., 2006; Jex et al., 2007, 2008; Ng et al., 2008; O’Brien et al., 2008; Soba and Logar, 2008; Waldron et al., 2009; Zintl et al., 2009). For example, the predominant C. parvum subtype in calves in Portugal, Slovenia and the Netherlands, IIaA15G2R1, is also the major C. parvum subtype in humans in these countries (Alves et al., 2006; Soba and Logar, 2008; Wielinga et al., 2008). This and other IIa subtypes previously found in calves in Michigan and Ontario also frequently infect humans in Wisconsin and Ontario (Feltus et al., 2006; Trotz-Williams et al., 2006). Likewise, humans in the Republic of Ireland and Northern Ireland are infected with most common IIa subtypes found in calves in Northern Ireland, with the IIaA18G3R1 as the dominant subtype (Thompson et al., 2007; Zintl et al., 2009). This subtype was also the most common subtype in calves and humans in Australia (Jex et al., 2007, 2008; Ng et al., 2008; O’Brien et al., 2008; Waldron et al., 2009). Another less common bovine C. parvum subtype family, IId, may potentially also be responsible for some zoonotic infections in Europe. Four IId subtypes were found in HIV+ persons in Portugal, two of which were previously found in calves and lambs in the same area (Alves et al., 2006). Likewise, a few humans in the Netherlands were also infected with three IId subtypes, although none of them were found in calves in the same study (Wielinga et al., 2008). One person in Ireland and Australia each was also infected with a IId subtype (Waldron et al., 2009; Zintl et al., 2009). IId subtypes of C. parvum, nevertheless, have never been found in humans in the United States and Canada, where they are absent in calves. Another C. parvum subtype family in calves in Europe, IIl, has also been found occasionally in humans in Slovenia (Soba and Logar, 2008). All these are further indicators of differences in the role of zoonotic transmission of C. parvum among geographic areas.

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Table 2 Cryptosporidium parvum gp60 subtypes in cattle in some recent studies.a Country

No. subtyped

No. of subtypes

United States

91

1

United States

175

6

United States

6

1

Canada

36

7

Slovenia

45

5

Belgrade, Serbia and Montenegro England

18

6

51

6

215

16

Hungary

21

5

Germany

53

8

Belgium

90

5

Portugal

72

3

Spain

140

7

The Netherlands

129

17

Italy

62

5

Australia

13

6

Japan

3

1

India

8

4

N. Ireland

a b c

IIa subtypesb

Other subtypesb

Reference

Santin et al. (2008) Xiao et al. (2007) Feng et al. (2007) TrotzWilliams et al. (2006) Soba and Logar (2008) Misic and Abe (2007)

Major subtype

Other subtypes

IIaA15G2R1 (91) IIaA15G2R1 (135) IIaA15G2R1 (6) IIaA15G2R1 (10)

–

–

IIaA15G2R2 (11), IIaA11G2R1 (11), IIaA18G2R1 (10), IIaA17G2R1 (7), IIaA19G2R1 (4) –

–

IIaA16G2R1 (9), IIaA16G3R1 (8), IIaA16G1R1 (4), IIaA13G2R1 (2), IIaA17G2R1 (2), IIaA18G3R1 (1)

–

IIaA15G2R1 (27) IIaA16G1R1 (6)

IIaA16G1R1 (6), IIaA16R1 (3)

IIaA15G2R1 (35) IIaA18G3R1 (120)

IIaA17G1R1 (7), IIaA16G3R1 (4), IIaA18G1R1 (2), IIaA19G1R1 (2), IIaA14G2R1 (1) IIaA15G2R1 (28), IIaA17G2R1 (19), IIaA19G4R1 (15), IIaA20G3R1 (6), IIaA19G3R1 (5), IIaA17G3R1 (5), IIaA20G5R1 (3), IIaA18G2R1 (2), IIaA20G2R1 (2), IIaA16G3R1 (1), IIaA17G1R1 (1), IIaA18R1 (1), IIaA19G2R (1), IIaA20G4R1 (1), IIaA21G2R1 (1), IIa-unknown (5) IIaA17G1R1 (3), IIaA18G1R1 (1)

IIlA16 (2), IIlA18 (2) IIdA18G1b (2), IIlA16 (4), IIlA17 (2) –

IIaA16G1R1 (15) IIaA15G2R1 (43) IIaA15G2R1 (84) IIaA15G2R1 (61) IIaA15G2R1 (106) IIaA15G2R1 (89)

IIaA15G2R1 (34) IIaA18G3R1 (5) IIaA15G2R1 (3) IIaA15G2R1 (5)

IIaA18G1R1 (2), IIaA20G1R1 (2)

IIaA14G2R1 (2), IIaA17G2R1 (2), IIaA18G2R1 (2), IIaA21R1 (1), IIaA22G1R1 (1), IIaA16G1R1 (1) IIaA16G2R1 (3), IIaA13G2R1 (1), IIaA14G2R1 (1)

–

–

IIdA19G1 (1), IIdA22G1 (1) IIdA22G1 (1) IIdA22G1 (1)

IIaA16G2R1 (7)

IIdA17G1 (4)

IIaA16G3R1 (14), IIaA18G3R1 (8), IIaA16G2R1 (4), IIaA17G2R1 (4), IIaA19G3R1 (2) IIaA17G1R1 (14), IIaA16G3R1 (6), IIaA13G2R1 (2), IIaA14G2R1 (2), IIaA17G2R1 (2), IIaA18G4R1 (2), IIaA18R1 (2), IIaA19G2R1 (2), IIaA11G2R1 (1), IIaA12G2R1 (1), IIaA16G1R1 (1), IIaA16G2R1 (1), IIaA18G3R1 (1), IIaA19G1R1 (1), IIaA21G3R1 (1) IIA18G2R1 (10), IIaA17G2R1 (9), IIaA14 (5), IIaA13 (4)

IIdA23G1 (2)

–

IIaA16G3R1 (3), IIaA19G4R1 (2), IIaA17G2R1 (1), IIaA20G3R1 (1), IIaA21G3R1 (1) –

–

IIaA13G2R2 (1), IIaA14G2R1a (1), IIaA14G2R1b (1)

–

IIlA24 (1)c

Brook et al. (2009) Thompson et al. (2007)

Plutzer and Karanis (2007) Broglia et al. (2008) Geurden et al. (2007) Alves et al. (2006) Quilez et al. (2008b) Wielinga et al. (2008)

Duranti et al. (2008) Ng et al. (2008) Abe et al. (2006) Feng et al. (2007)

See Xiao et al. (2007) for a summary of results of older studies. Numbers in parentheses are number of samples with the subtype. Found in a calf from Lithuania.

What proportion of human infections with C. parvum subtypes in industrialized nations are attributable to zoonotic transmission remains unclear, as the source of C. parvum in humans can be of bovine or human origin. Thus, not all C. parvum subtypes in humans result from zoonotic infections. Studies in Portugal and Slovenia showed that the genetic diversity of C. parvum was much higher in humans than in calves, and of the three C. parvum subtype families, one (IIc) was not found in animals (Alves et al., 2006; Soba and Logar, 2008). The anthroponotic nature of the IIc subtype family has been subsequently demonstrated in comparative subtyping studies of human and bovine cryptosporidiosis in United States, Canada, Portugal, Slovenia, United Kingdom, Ireland, The Netherlands and Australia, where IIc subtypes have only been found in humans (Tables 2 and 3). In urban areas in the United States, IIa subtypes are rarely seen in humans. Instead, the anthroponotic IIc subtype family is responsible for most human C. parvum infections in these areas (Xiao et al., 2004b). In urban areas in European countries, such as Lisbon, Portugal, both IIa and IIc are fairly common in humans (Alves et al., 2006). Children in the Kuwait City are almost exclusively infected with IIa and IId subtypes, although

they have little contact with farm animals, the city uses desalinized sea water as drinking water, and the transmission appears to be anthroponotic in origin (Sulaiman et al., 2005). IId subtypes are also common in children in Saudi Arabia (Al-Brikan et al., 2008). Results of MLT studies support the occurrence of anthroponotic C. parvum. MLT analysis has identified human-adapted C. parvum populations in the United Kingdom and the Netherlands (Leoni et al., 2007; Grinberg et al., 2008b; Morrison et al., 2008; Wielinga et al., 2008). Like the previous observation of greater C. parvum genetic diversity in humans than bovines in the gp60 gene in Portugal, humans in Scotland were infected with significantly wider spectra of C. parvum multilocus types than cattle. Thus, a significant fraction of human C. parvum infections may not have originated from bovine reservoirs (Grinberg et al., 2008b). All these MLT studies included the gp60 locus, which is the most polymorphic marker among the loci targeted. In contrast, lower MLT diversity of C. parvum was observed in France in humans than in animals and none of the two populations of C. parvum seen in France were restricted to humans (Ngouanesavanh et al., 2006). However, gp60 was not a marker included in the French study.

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L. Xiao / Experimental Parasitology 124 (2010) 80–89 Table 3 Distribution of C. parvum gp60 subtype families in humans in developing and industrialized countries. Location

Patient type

Developed countries Ireland Lisbon, Portugal Slovenia Netherlands Australia Australia Australia Australia Australia Australia Japan Kuwait Canada Wisconsin, USA New Orleans, USA

Children and adults HIV+ adults Children and adults Children and adults Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified Children Adults Unspecified HIV+ adults

Developing countries India Indiaa Malawi South Africa Uganda Madagascar Jamaica Guatemala Peru Peru

Children HIV+ adults Children HIV+ children Children Children HIV+ adults Children HIV+ adults Children

a

N

Subtype family IIa

IIc

79 25 31 13 6 24 23 4 9 32 2 59 4 30 6

78 9 29 9 6 23 18 4 8 30 1 28 4 30 1

7 9 2 5 15 1 7 1 22 15

0 0 0 0 0 0 0 0 0 0

Reference IIb

IId

IIe

Other

0 7 1 1 0 1 5 0 1 1 1 2 0 0 5

0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

1 8 0 3 0 0 0 0 0 1 0 29 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 0 0 0 0 0 1 0 0 0

Zintl et al. (2009) Alves et al. (2006) Soba and Logar (2008) Wielinga et al. (2008) Chalmers et al. (2005a) Jex et al. (2008) Jex et al. (2007) Ng et al. (2008) O’Brien et al. (2008) Waldron et al. (2009) Abe et al. (2006) Sulaiman et al. (2005) Trotz-Williams et al. (2006) Feltus et al. (2006) Xiao et al. (2004b)

7 3 1 5 10 1 7 1 22 15

0 5 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 0 0 0 0

0 1 0 0 5 0 0 0 0 0

Ajjampur et al. (2007) Muthusamy et al. (2006) Peng et al. (2003) Leav et al. (2002) Akiyoshi et al. (2006) Areeshi et al. (2008) Gatei et al. (2008) Xiao et al. (2004a) Cama et al. (2007) Cama et al. (2008)

Based on RFLP analysis.

The combination of subtyping and conventional epidemiological tools can improve the assessment of the disease burden attributable to zoonotic transmission. The use of MLT in a case control study in Wales and northwest England suggested the occurrence of zoonotic transmission (Hunter et al., 2007). At the ML1 locus, significantly more persons with C. parvum subtype ML1–242 had touched or handled farm animals than those with ML1–227. Similarly, at ML2, significantly more specimens with alleles between 223 and 237 were from patients who had touched or handled farm animals than were specimens with alleles 193 and 197. At the gp60 locus, patients who had contact with farm animals yielded significantly greater product sizes than those who reported no animal contact before onset of illness (Hunter et al., 2007). Still, quantitation of zoonotic transmission of C. parvum is impossible in this type of studies. Cryptosporidium parvum transmission in humans in developing countries appears largely anthroponotic. Unlike European countries, Australia, Canada and the United States, the zoonotic IIa subtypes are rarely seen in humans in developing countries. Instead, the anthroponotic IIc subtype family is responsible for most human C. parvum infections in these areas (Table 3). In some regions such as Lima, Peru and Kingston, Jamaica, the IIc subtype family is the only C. parvum in humans, whereas in other developing countries such as India, Malawi, Uganda and Kenya, some unusual C. parvum subtype families such as IIb and IIe, are also seen in humans, which have never been seen in animals anywhere (Table 3)(Cama et al., 2003, 2008; Akiyoshi et al., 2006; Muthusamy et al., 2006; Gatei et al., 2008). The anthroponotic nature of IIc in developing countries still needs support of results from animal studies, as gp60 subtyping has been done on only a few C. parvum isolates from calves in these areas (Feng et al., 2007). Nevertheless, the anthroponotic transmission of C. parvum IIc in developing countries is also in agreement with the predominance of C. hominis in these areas and the absence of IIc in ruminants in industrialized nations, both of which are transmitted through anthroponotic routes. The existence of human-adapted C. parvum was also confirmed by MLST in a study conducted in Jamaica (Gatei et al., 2008).

6. Cryptosporidium hominis infections As mentioned above, developing and developed countries differ in the proportion of human infections attributable to C. hominis, probably as a result of differences in the disease burden attributable to zoonotic transmission. In addition to this, there are also some differences in molecular epidemiology of C. hominis between the developing and developed countries. In developing countries, the complexity of C. hominis infections in humans is reflected by the occurrence of multiple subtype families and multiple subtypes within families Ia and Id. Thus, 3–4 C. hominis subtype families were seen in humans in India, Peru, Kenya, Malawi and South Africa, and there were many subtypes within C. hominis subtype families Ia and Id in one endemic area (Leav et al., 2002; Peng et al., 2003; Muthusamy et al., 2006; Ajjampur et al., 2007; Cama et al., 2007, 2008; Gatei et al., 2007). The high C. hominis heterogeneity in developing countries is likely an indicator of intensive and stable cryptosporidiosis transmission in these areas. Four common C. hominis subtype families, Ia, Ib, Id and Ie, are usually seen in humans in developing countries. However, there are geographic differences in the distribution of them. For example, all the four common subtype families were seen in children and HIV+ adults in Peru, Malawi, Madagascar and India (Peng et al., 2003; Ajjampur et al., 2007; Cama et al., 2007, 2008; Gatei et al., 2007; Areeshi et al., 2008). In contrast, subtype family If instead of Ie was seen in some South African children (Leav et al., 2002). In Jamaica, Ib subtype family was the major C. hominis seen in HIV+ persons, with only a few other cases caused by other subtype families (Gatei et al., 2008). Within each subtype family, one subtype is frequently seen in certain areas but not in others. For example, there are only two common subtypes within the C. hominis subtype family Ib: IbA9G3 and IbA10G2. The former is commonly seen in Malawi, Kenya and India, whereas the latter is commonly seen in South Africa, Botswana, Jamaica and Peru (Leav et al., 2002; Peng et al., 2003; Cama et al., 2007, 2008; Gatei et al., 2007, 2008). One unusual Ib subtype,

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IbA13G3, was seen in Peru (Cama et al., 2007), and several otherwise rare subtypes, IbA19G2, IbA20G2, IbA21G2 and IbA23G2, were the most common C. hominis subtypes in Shanghai and Tianjing, China (Peng et al., 2001; Feng et al., 2009). Likewise, in most developing countries, humans with the subtype family Ie are mostly infected with IeA11G3T3, with the exception of Kingston, Jamaica and Shanghai, China, where IeA12G3T3 is seen (Gatei et al., 2008; Feng et al., 2009). This geographic segregation of C. hominis subtypes became much more obvious when specimens from different countries were genetically compared using MST and MLST tools (Gatei et al., 2006a, 2008; Tanriverdi et al., 2008). Much less genetic diversity is seen in C. hominis in industrialized nations. In European countries and Australia, most C. hominis infections are caused by the Ib subtype family (Alves et al., 2006; Jex et al., 2007, 2008; Chalmers et al., 2008; O’Brien et al., 2008; Wielinga et al., 2008; Waldron et al., 2009; Zintl et al., 2009). The majority of these cases had the IbA10G2 subtype, although in some areas of Australia, both IbA10G2 and IbA9G3 were common (Jex et al., 2008), and several unusual Ib subtypes (IbA18G1, IbA5G2T3 and IbA9G2T1) were also occasionally seen (Jex et al., 2007; O’Brien et al., 2008). In the United Kingdom, having a non-IbA10G2 subtype was significantly linked to recent travel outside Europe (Chalmers et al., 2008). In New Orleans, the United States, results of gp60 and MLST studies indicated that HIV+ adults were infected with geographically diverse C. hominis populations (Gatei et al., 2006a, 2008). The clinical significance of various C. hominis subtypes in humans is not yet clear. In a case control study of HIV+ patients in Lima, Peru, infections with C. hominis Id were significantly associated with diarrhea in general and chronic diarrhea in particular. The association between diarrhea and Ib infections was also marginally significant. In contrast, infections with the Ia and Ie subtype families were more likely asymptomatic (Cama et al., 2007). Very different associations between C. hominis subtype families and clinical presentations, however, were seen in a longitudinal cohort study of cryptosporidiosis in children in the same area (Cama et al., 2008). Although all subtype families were associated with diarrhea, the Ib subtype family appeared to be much more virulent, being significantly associated with diarrhea, nausea, vomiting and general malaise during both first episode of infection or all infections. The Ia subtype family was also very pathogenic in children, being significantly associated with diarrhea, nausea and vomiting during the first episode of cryptosporidiosis (Cama et al., 2008). These results demonstrate that C. hominis gp60 subtype families are linked to different clinical manifestations, and children and HIV+ adults may have different clinical responses to infections with different C. hominis subtype families. The high virulence of the Ib subtype family in immunocompetent persons is also reflected in the number of outbreaks caused by the parasite. Almost half of the C. hominis outbreaks in the United States, especially the major ones, were caused by the Ib subtype family, particularly the IbA10G2 subtype (Xiao and Ryan, 2008). IbA10G2 is apparently also a major C. hominis subtype responsible for cryptosporidiosis outbreaks in Europe (Glaberman et al., 2002; Cohen et al., 2006; Leoni et al., 2007). 7. Human infections with other Cryptosporidium spp In most countries studied, C. parvum and C. hominis are responsible for greater than 90% of human cases of cryptosporidiosis, with the balance attributable to C. meleagridis, C. canis and C. felis. Some areas, especially developing countries, however, have high prevalence of these unusual species (see recent review by (Xiao and Feng, 2008). In Lima, Peru and Bangkok, Thailand, C. meleagridis is as prevalent in humans as C. parvum, responsible for 10–20% of human cryptosporidiosis cases (Gatei et al., 2002; Cama et al.,

2007, 2008). Most human C. canis infections were reported in persons in developing countries, whereas most human infections with the Cryptosporidium cervine genotype were reported in industrialized nations (Xiao and Feng, 2008). Subtyping tools are largely unavailable for these rare species because of the inability of PCR amplification using C. parvumand C. hominis-based primers. Like C. parvum, some human infections with the unusual species are probably the result of anthroponotic transmission, especially in developing countries. Using C. hominis and C. parvum-specific genotyping tools, the analysis of C. canis- and C. felis-infected specimens from HIV+ persons in Lima, Peru revealed concurrent presence of C. hominis and C. parvum IIc subtype family in 6 of 21 patients. The concurrent presence of the human-specific C. hominis and C. parvum suggests that some of the C. canis and C. felis infections in humans were probably transmitted through the anthroponotic rather than zoonotic pathway (Cama et al., 2006a). Nevertheless, possible transmission of C. canis between two siblings and a dog occurred in a household in a slum in Lima, Peru, although the direction of the transmission was not clear (Xiao et al., 2007). In developing countries, children frequently have multiple episodes of cryptosporidiosis, although the likelihood of clinical illness decreases with increased infection episodes (Cama et al., 2008). Molecular analysis of longitudinal specimens from Peruvian children with multiple cryptosporidiosis episodes indicated that immunity against both homologous and heterologous Cryptosporidium species/genotypes was short-lived, with a median interval between infections of 10 months. As might be expected, sequential infections with heterologous Cryptosporidium species were more common than sequential infections with homologous Cryptosporidium species (Cama et al., 2008). Even though some children had the same species (C. hominis) in sequential infections, these were in fact due to heterogeneous subtype families (Cama et al., 2008). In Lima, Peru, HIV+ patients infected with C. canis and C. felis were more likely to have diarrhea in general, those infected with C. parvum, C. canis and C. felis were more likely to have chronic diarrhea, and those with C. parvum were also likely to have vomiting. In contrast, infections with C. meleagridis were more likely asymptomatic (Cama et al., 2007). In the same area, however, children infected with C. parvum, C. meleagridis, C. canis and C. felis were less likely to have non-diarrheal symptoms such as general malaise, nausea and vomiting; only C. hominis infections were significantly associated with these clinical symptoms (Cama et al., 2008). These results demonstrate that different Cryptosporidium spp. are linked to different clinical manifestations in different populations of humans. 8. Conclusion Results of recent molecular epidemiological studies have significantly improved our knowledge of human cryptosporidiosis. We now have a much better understanding of the complexity of Cryptosporidium infection in humans at the species and subtype levels, transmission routes in developing and developed countries, parasite population structure and host-parasite interactions. We are also beginning to use the second generation molecular diagnostic tools in conjunction with traditional epidemiologic investigations to answer some epidemiological questions that are difficult to address by traditional methods, such as the disease burdens attributable to zoonotic transmission, frequency of mixed infections, maintenance of immunity and cross protection, transmission dynamics in different populations and settings, temporal and geographic variations in Cryptosporidium transmission, and the role of parasite factors in transmission, outbreak occurrence and clinical spectra of cryptosporidiosis. These should lead to a more in-depth

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