Molecular detection of Cryptosporidium cuniculus in rabbits in Australia

Infection, Genetics and Evolution 10 (2010) 1179–1187

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Molecular detection of Cryptosporidium cuniculus in rabbits in Australia Matthew J. Nolan a,*, Aaron R. Jex a, Shane R. Haydon b, Melita A. Stevens b, Robin B. Gasser a,* a b

Department of Veterinary Science, University of Melbourne, Werribee, Victoria, Australia Melbourne Water Corporation, Victoria, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 May 2010 Received in revised form 26 July 2010 Accepted 26 July 2010 Available online 5 August 2010

In the United Kingdom, rabbits have been reported to harbour genotypes of Cryptosporidium (now recognized as C. cuniculus) identical to those from human patients exhibiting symptoms of cryptosporidiosis. The high density of rabbits in many regions of Australia, including both rural and urban as well as natural water catchments areas, and the absence of any information on Cryptosporidium from lagomorphs in this country stimulated the present study. We undertook an epidemiological study that genetically characterized Cryptosporidium from rabbits from four locations in Victoria by PCRcoupled sequencing and phylogenetic analysis of sequence data for loci within the small subunit of nuclear ribosomal RNA (SSU; for specific identification) and the 60 kDa glycoprotein gene (gp60; for genotypic/subgenotypic identification). Cryptosporidium was detected in 12 (6.8%) of 176 individual faecal samples. For SSU, all 12 sequences were identical to each other and to that of C. cuniculus. For pgp60, all corresponding sequences matched the known genotype Vb, and were classified as subgenotype VbA23R3 (n = 11) and VbA26R4 (n = 1), which are both new records. Present evidence indicates that genotype Vb is limited to rabbits; however, it would be premature to conclude that this genotype is not zoonotic. Future studies should focus on the zoonotic potential of C. cuniculus from rabbits and a wide range of yet unstudied animals. (Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. HM852431–HM852433). ß 2010 Elsevier B.V. All rights reserved.

Keywords: Cryptosporidium Cryptosporidiosis Rabbits Humans Zoonotic potential

1. Introduction Cryptosporidium (Apicomplexa) is a genus of parasitic protistans that mainly infect the gastrointestinal tract and, occasionally, the respiratory system of vertebrates (Xiao et al., 2004). The infection is usually acquired by the direct ingestion of oocysts (containing four infective sporozoites) in contaminated food and/or water or by direct contact. In humans, the disease (cryptosporidiosis) is commonly caused by C. hominis and C. parvum, and less frequently by C. meleagridis, C. canis and C. felis (see Caccio` et al., 2005; Hunter et al., 2004). The clinical manifestation of cryptosporidiosis in immuno-competent individuals includes diarrhoea, vomiting, fever, colic and/or head ache (Chen et al., 2002; Farthing, 2000; Kosek et al., 2001; Tzipori and Ward, 2002), and is usually eliminated after days to weeks, following an effective anticryptosporidial host immune response (Riggs, 2002). However, in high-risk groups (i.e. infants, the elderly, immuno-compromised, or -suppressed individuals), cryptosporidiosis can become a chronic and fatal disease (Caccio` and Pozio, 2006; Chalmers and Davies, 2010; Huang et al., 2004; Hunter and Nichols, 2002).

* Corresponding authors. Tel.: +61 3 97312330; fax: +61 3 97312366. E-mail addresses: [email protected] (M.J. Nolan), [email protected] (R.B. Gasser). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.07.020

Given that there are no highly effective drugs against cryptosporidiosis (Caccio` and Pozio, 2006), the identification and genetic characterization of Cryptosporidium from animals and the environment are central to assessing sources of infection, modes of transmission and the potential risk of infection to humans, and underpin the prevention and control of cryptosporidiosis. As it is not possible to reliably identify or differentiate Cryptosporidium species or genotypes based on host origin or by conventional coproscopic means (including staining and immuno-staining) (Jex and Gasser, 2010; Jex et al., 2008b), tools based on the polymerase chain reaction (PCR) (Saiki et al., 1988) are now widely employed to define species/genotypes and to assess the risk that Cryptosporidium-infected animals pose as reservoirs for human infection (Caccio` and Pozio, 2006; Smith et al., 2006). In particular, PCRcoupled sequencing of genetic loci, including the small subunit (SSU) of the nuclear ribosomal RNA and the 60 kDa glycoprotein (gp60) gene, has been shown to be a practical approach for the genetic identification and characterization of Cryptosporidium (Jex et al., 2008b). Despite the ready availability of molecular tools, there are still substantial gaps in our knowledge of the genetic composition of Cryptosporidium populations in wild and domestic animals globally and of their ability or potential to infect humans (Jex and Gasser, 2010). Nonetheless, some molecular-epidemiological studies have linked waterborne outbreaks of cryptosporidiosis in humans to

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livestock, and a range of wild animals, including rabbits (Oryctolagus cuniculus) in Europe and North America (e.g., Chalmers et al., 2009; Hunter, 1999; Ong et al., 1999). The recent discovery that a ‘rabbit genotype’ of Cryptosporidium (now recognized as C. cuniculus; Robinson et al., 2010) has been linked to diarrhoeal disease in humans in the UK (Chalmers et al., 2009) has raised considerable awareness about the importance of investigating rabbits as a source of Cryptosporidium transmissible to humans (Robinson and Chalmers, 2009; Smith et al., 2010). The high density of rabbits in many rural and urban as well as natural water catchment areas of Australia (Clarke et al., 2000), and the absence of any information on Cryptosporidium infecting lagomorphs in this country stimulated the present study. We genetically characterized Cryptosporidium from rabbits from four locations in Victoria by PCR-coupled sequencing of loci in SSU and gp60 and studied the relationships of the species/genotypes defined with known genotypes by phylogenetic inference. 2. Materials and methods

GCCGTTCCACTCAGAGGAAC-30 ) and gp15-15E (reverse: 50 -CCACATTACAAATGAAGTGCCGC-30 ) (Mallon et al., 2003). For the first amplification, the cycling protocol was 94 8C/5 min (initial denaturation), followed by 35 cycles of 94 8C/30 s (denaturation), 55 8C/45 s (annealing) and 72 8C/1 min (extension), with a final extension of 72 8C/10 min. The nested amplification was performed using 94 8C/5 min, followed by 30 cycles of 94 8C/30 s, 55 8C/30 s, and 72 8C/30 s, with a final extension at 72 8C/10 min. For both loci, all secondary amplicons were purified over minicolumns (Wizard1 PCR-Preps DNA Purification System, Promega), according to the manufacturer’s protocol. Amplicons were subjected to bi-directional, automated sequencing (BigDye1 Terminator v.3.1 chemistry, Applied Biosystems, Foster City, California, USA) using (separately) the primers employed for the nested PCR. The quality of each sequence was verified by appraising the corresponding electropherogram using the program FinchTV v1.4.0 (http://www.geospiza.com/Products/finchtv.shtml). Open reading frames (ORFs) were predicted from individual pgp60 sequences using Transeq (http://www.ebi.ac.uk/Tools/emboss/ transeq/index.html).

2.1. Samples and isolation of genomic DNA 2.3. Phylogenetic analysis of sequence datasets One-hundred and seventy-six fresh faecal deposits from rabbits were collected from four locations in Victoria, Australia: (1) 378400 S 1458550 E [n = 80]; (2) 378400 S 1458180 E [n = 33]; (3) 378330 S 1458080 E [n = 46]; and (4) 378370 S 1448540 E [n = 17]. These localities are north-east of Melbourne and <90 km apart. The samples were frozen at 20 8C until further investigation. Genomic DNA was extracted directly from 0.2 to 0.4 g of faeces using the PowerSoilTM DNA Isolation Kit (MoBio, USA), according to the manufacturer’s instructions. 2.2. PCR-coupled sequencing Genomic DNA was subjected to nested PCR-coupled sequencing of two independent loci. For the specific identification of Cryptosporidium, part of the small subunit (SSU) of the nuclear ribosomal RNA gene (designated pSSU; 240 bp) was utilized. For genotypic classification, a part of the 60 kDa glycoprotein (gp60) gene (designated pgp60; 250–350 bp) was amplified and sequenced. PCR was carried out in a 50 ml volume containing 10 mM Tris–HCl (pH 8.4), 50 mM KCl (Promega, Madison, USA), 2.0– 3.0 mM of MgCl2, 200 mM of each deoxynucleotide triphosphate, 100 pmol of each primer and 1.25 U of either GoTaq polymerase (Promega) or MangoTaqTM DNA polymerase (Bioline, USA). The SSU gene was first amplified using primers XF2 (forward: 50 -GGAAGGGTTGTATTTATTAGATAAAG-30 ) and XR2 (reverse: 50 AAGGAGTAAGGAACAACCTCCA-30 ) (Xiao et al., 1999c), followed by the nested amplification of pSSU from 1 ml of primary amplicon using the internal primers pSSUf (forward: 50 -AAAGCTCGTAGTTGGATTTCTGTT-30 ) and pSSUr (reverse: 50 -ACCTCTGACTGTTAAATACRAATGC-30 ); primer pSSUf is located between nucleotide positions 600 and 624, and pSSUr is between positions 837 and 861 (linked to the reference sequence with GenBank accession number AF093492; Xiao et al., 1999a). For the first amplification, the cycling protocol was 94 8C/5 min (initial denaturation), followed by 30 cycles of 94 8C/45 s (denaturation), 45 8C/2 min (annealing) and 72 8C/1.5 min (extension), with a final extension of 72 8C/10 min. The nested amplification was performed employing 94 8C/5 min, followed by 35 cycles of 94 8C/30 s, 55 8C/30 s, and 72 8C/30 s, with a final extension of 72 8C/10 min. The gp60 gene (1 kb) was first amplified using primers gp15ATG (forward: 50 -ATGAGATTGTCGCCTCATTATC-30 ) and gp15STOP (reverse: 50 -TTACAACACGAATAAGGCTGC-30 ) (Strong et al., 2000), followed by a nested amplification of pgp60 from 1 ml of primary amplicon using primers gp15-15A (forward: 50 -

Sequences were aligned using the program Clustal X (Thompson et al., 1997), and alignments adjusted by eye. Pairwise comparisons of nucleotide differences were calculated using the program BioEdit (Hall, 1999). Phylogenetic analysis of sequence data was conducted by Bayesian inference (BI) using Monte Carlo Markov Chain (MCMC) analysis in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). The likelihood parameters set for BI were based on the Akaike Information Criteria (AIC) test in Modeltest version 3.7 (Posada and Crandall, 1998). The general time-reversible model of evolution with gamma distribution and a proportion of invariable sites (GTR + G + I) was employed for the analysis of the pSSU sequence dataset, whereas GTR + G was used for pgp60. Estimates of the base frequencies, the substitution rate model matrix and the proportion of invariable sites determined using AIC were fixed for each analysis (data not shown). Posterior probabilities (pp) were calculated via 2,000,000 generations (pSSU) or 1,000,000 generations (pgp60), utilizing four simultaneous tree-building chains, with every 100th tree being saved. At this point, the standard deviation of split frequencies was <0.01, and the potential scale reduction factor (PSRF) approached one. A 50% majority rule consensus tree for each analysis was constructed based on the final 75% of trees generated by BI. The outgroups used in the analyses were seven species of Eimeria (GenBank accession nos. U67115–U67121; Barta et al., 1997) for pSSU, and Cryptosporidium hominis Ib (AB237130; Abe et al., 2006) for pgp60. 3. Results All 176 genomic DNA samples representing rabbit faecal deposits from four locations in Victoria, Australia, were tested using the PCR for pSSU. Twelve amplicons (6.8%) were produced and then sequenced. All 12 pSSU sequences determined (242 nucleotides) were identical and had mean nucleotide frequencies of 0.351 (A), 0.087 (C), 0.157 (G) and 0.405 (T). These sequences were also identical to previously published reference sequences for C. cuniculus (see Table 1). To provide statistical support for this classification, a representative pSSU sequence (accession no. HM852431) determined here was aligned with selected representative reference sequences obtained from GenBank (see Table 1; alignment available from primary author upon request). The reference sequences represented 21 Cryptosporidium species and 33 Cryptosporidium genotypes from 22 different host species as

M.J. Nolan et al. / Infection, Genetics and Evolution 10 (2010) 1179–1187

well as seven species of avian Eimeria (outgroups). All 65 sequences were aligned over 279 positions. Phylogenetic analysis by BI grouped the pSSU sequence determined here (HM852431) and five publicly available sequences representing C. cuniculus (see Fig. 1 and Table 1) with strong support (pp = 0.76), to the exclusion of sequences representing all other Cryptosporidium species and/or genotypes. On the basis of a distance matrix (data not shown) derived from the alignment utilized herein to construct the phylogeny, the rabbit genotype was genetically closest to C. parvum (98% similar; six nucleotide differences over 279 positions), C. hominis (98%) and the Cryptosporidium sp. ‘minkgenotype’ (98%). In order to further characterize the samples from rabbit faeces, pgp60 was amplified from each of the 12 samples from which pSSU was derived and sequenced. Two sequence types were determined and are represented by GenBank accession numbers HM852432 (n = 11) and HM852433 (n = 1). These two sequence types had lengths of 274 and 286 bp, respectively, and mean nucleotide frequencies of 0.329 (A), 0.268 (C), 0.180 (G) and 0.223 (T). Sequence variation between these two sequence types was 4.6% (13 nt over 286 positions), with the majority of variation being restricted to alignment positions 76–87 (a region associated with variation in the number of TCA/ACA trinucleotide repeats; n = 0–4) and position 125 (A $ G). Within the entire variable microsatellite region (alignment positions 7–84), the number of trinucleotide repeats ranged from 26 to 30. Prior to phylogenetic analysis, the two pgp60 sequence types defined were compared with publicly available sequences. They matched closely (91.3– 95.6% over 300 nucleotide positions) with published pgp60 reference sequences (accession nos. FJ262733 and FJ262734; Chalmers et al., 2009) representing C. cuniculus (i.e. genotype Vb) (cf. Table 2). Using the system of nomenclature proposed previously (Chalmers et al., 2009) and based on a comparison with the reference sequences, the two sequence types represented genotype Vb, subgenotype VbA23R3 (accession no. HM852432) and VbA26R4 (HM852433). To provide unequivocal support for the genotypic and subgenotypic identities, both pgp60 sequences were aligned with 48 reference sequences obtained from GenBank (see Table 2; alignment available from primary author upon request). These reference sequences represented 10 of 11 currently recognised C. parvum genotypes (IIa–IIh, IIj and IIk), and six pgp60 reference sequences representing C. cuniculus (i.e. genotypes Va and Vb). All 50 sequences were aligned over 337 positions. Phylogenetic analysis grouped the four pgp60 sequences representing genotype Vb from rabbit (see Table 2 and Fig. 2), with strong statistical support (pp = 1.00), to the exclusion of sequences representing genotype Va (which formed a monophyletic group; pp = 1.00) and all other C. parvum sequence types. The C. parvum genotypes IIa–IIe

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also resolved as separate groups, to the exclusion of each other and all other genotypes, with strong support (pp = 0.84–1.00). The analysis also showed that the C. parvum genotypes analyzed herein did not form a monophyletic group to the exclusion of sequences representing either genotype Va or Vb; i.e. genotype IIa grouped with IIg; genotype IIc grouped with IIh, which, in turn, grouped with Va; IIc/IIh/Va grouped with IIk which, in turn, grouped with IIa/IIg; Vb grouped with IIb which, in turn, formed a polytomy with IIf and the group formed by IIc/IIh/Va/IIg/IIa; genotypes IIe and IId formed separate groups to that formed by genotypes Vb/IIb/IIf/IIc/ IIh/Va/IIg/IIa (see Fig. 2). 4. Discussion The present study represents the first detailed molecular epidemiological investigation of Cryptosporidium in wild rabbits outside of Europe. Most previous reports described Cryptosporidium from incidental findings in domestic and/or wild rabbits (Araya et al., 1987; Cox et al., 2005; Feng et al., 2007; Marounek et al., 1985; Medema, 1999; Nagy, 1995; Pavla´sek et al., 1996; Peeters et al., 1986; Rehg et al., 1979; Ryan et al., 1986; Skrivanova et al., 1999, 1997; Soltane et al., 2007; Sturdee et al., 1999; Tian et al., 2002, 2001; Tyzzer, 1912); although previous epidemiological studies had been conducted to estimate the prevalence of infection (Chalmers, 1996; Chalmers et al., 1995; Epe et al., 2004), samples were not characterized genetically. Herein, Cryptosporidium was detected in 6.8% of 176 individual samples; this percentage is higher than prevalences determined in previous studies in the United Kingdom (0.9% of 109 samples) (Chalmers, 1996; Chalmers et al., 1995) and Germany (0% of 232) (Epe et al., 2004). However, in these studies, conventional microscopic and immunological methods (including formol-ether sedimentation, immunofluorescence microscopy and modified Ziehl Neelsen staining) were employed for the detection of oocysts. The limited sensitivity of these conventional methods (Jex et al., 2008b) relative to the nested PCRs used here (cf. Pangasa et al., 2010) may explain the low prevalences recorded in previous studies. In the present study, the pgp60 sequence type representing genotype VbA23R3 (accession no. HM852432) was most prevalent (11 samples from locations 1 and 2); the second pgp60 sequence type representing genotype VbA26R4 (HM852433) was limited to a single test-positive sample from location 2. Both pgp60 sequence types are new records, likely reflecting the paucity of information for Cryptosporidium in wild rabbit populations worldwide. Indeed, previous reports of pgp60 sequence data for Cryptosporidium from rabbits are limited to two published studies (Chalmers et al., 2009; Robinson et al., 2010), in which two genotypes (Va and Vb) and 20 subgenotypes (Va = seven and Vb = 13) were defined from humans, rabbits and treated drinking water in England, China and the Czech

Table 1 Cryptosporidium pSSU sequence data determined in the present study, together with reference sequences (see accession numbers) for comparison, with epidemiological information. Species/type

Host or environmental source

Locality

GenBank accession nos.

References

Cryptosporidium C. andersonia C. baileyi C. bovis C. canis C. canisb C. canis C. cuniculusc C. cuniculusc C. cuniculus C. cuniculusd C. cuniculusc C. cuniculusc C. fayerie

Cattle (calf) (Bos taurus) Chicken (Gallus domesticus) Cattle Coyote (Canis latrans) Dog (Canis familiaris) Fox (Vulpes vulpes) Human Human Rabbit (Oryctolagus cuniculus) Rabbit Rabbit Treated drinking water Red kangaroo (Macropus rufus)

USA USA USA USA USA USA England England Australia China England England Australia

AF093496 AF093495 AY741305 DQ385546 AF112576 AY120908 EU437413 FJ262726 HM852431 AY120901 FJ262725 FJ262724 AF159112

Xiao et al. (1999a) Xiao et al. (1999a) Fayer et al. (2006) Trout et al. (2006) Xiao et al. (1999c) Xiao et al. (2002) Robinson et al. (2008) Chalmers et al. (2009) Present study Xiao et al. (2002) Chalmers et al. (2009) Chalmers et al. (2009) Xiao et al. (1999b)

M.J. Nolan et al. / Infection, Genetics and Evolution 10 (2010) 1179–1187

1182 Table 1 (Continued ) Species/type

Host or environmental source

Locality

GenBank accession nos.

References

C. felis C. fragile (syn. C. fragilis) C. galli C. hominis C. macropodumf C. meleagridis C. cf. molnari C. muris C. parvum C. ryanae C. varanii (syn. C. saurophilum)g C. varanii (syn. C. saurophilum)h C. serpentis C. serpentis C. suisi C. wrairi C. xiaoi Cryptosporidium ‘genotypes’ Beaver Bearj Chipmunk Coyotek Deer mouse I Deer mouse II Deer mouse III Deer mouse IV Foxl Goose Im Goose II Horse Koalan Lizardo Mink Muskrat I Muskrat II Opossum Ip Ostrichq Pig II Seal Ir Seal IIs Shrew

Cat (Felis catus) Black-spined toad (Duttaphrynus melanostictus) Capercaille (Tetrao urogallus) Human (Homo sapiens) Eastern grey kangaroo (Macropos giganteus) Turkey (Meleagris gallopavo) Guppy (Poecilia reticulata) Rock hyrax (Procavia capensis) Cattle (calf) Cattle Desert monitor (Varanus griseus) Corn snake (Elaphe guttata) Corn snake Savannah monitor (Varanus exanthematicus) Pig (Sus domestica) Guinea pig (Cavia porcellus) Sheep (Ovis aries)

Australia Malaysia Czech Republic Slovenia Australia USA Australia USA USA USA USA Germany USA USA Switzerland USA USA

AF112575 EU162751 AY168848 AJ849464 AY237630 AF112574 AY524773 AF093498 AF093490 EU410344 AF112573 EF502042 AF151376 AF093500 AF108861 AF115378 FJ896046

Xiao et al. (1999c) Jirku et al. (2008) Ryan et al. (2003b) Soba et al. (2006) Power et al. (2004) Xiao et al. (1999c) Ryan et al. (2004) Xiao et al. (1999a) Xiao et al. (1999a) Fayer et al. (2008) Xiao et al. (1999c) Plutzer and Karanis (2007b) Kimbell et al. (1999) Xiao et al. (1999a) Morgan et al. (1999) Xiao et al. (1999c) Fayer and Santı´n (2009)

Beaver (Castor canadensis) Black bear (Ursus americanus) Eastern chipmunk (Tamias striatus) Coyote Deer mouse (Peromyscus sp.) Deer mouse Deer mouse Deer mouse Fox Canada goose (Branta canadensis) Canada goose Human Koala (Phascolarctos cinereus) Leopard gecko (Eublepharis macularius) Mink (Mustela vison) Muskrat (Ondatrini zibethicus) Muskrat Opossum (Didelphis virginiana) Ostrich (Struthio camelus) Human Ringed seal (Phoca hispida) Ringed seal Northern short-tailed shrew (Blarina brevicauda) Human New Guinea boa (Candoia aspera) Star tortoise (Geochelone sp.) Meadow vole (Microtus pennsylvanicus) Black wildebeest (Connochaetes gnou)

USA USA USA USA USA USA USA USA USA USA USA England Australia Czech Republic USA USA USA USA Brazil Czech Republic Canada Canada USA

EF641022 AF247535 EF641026 DQ385545 EF641028 EF641027 EF641014 EF641019 AY120907 AY120912 EF641009 EU437418 AF108860 AY120915 EF641015 EF641013 EF641021 AY120902 DQ002931 EU331243 AY731234 AY731235 EF641010

Feng et al. (2007) Xiao et al. (2000) Feng et al. (2007) Trout et al. (2006) Feng et al. (2007) Feng et al. (2007) Feng et al. (2007) Feng et al. (2007) Xiao et al. (2002) Xiao et al. (2002) Feng et al. (2007) Robinson et al. (2008) Morgan et al. (1999) Xiao et al. (2002) Feng et al. (2007) Feng et al. (2007) Feng et al. (2007) Xiao et al. (2002) Meireles et al. (2006) Kvac et al. (2009) Santı´n et al. (2005) Santı´n et al. (2005) Feng et al. (2007)

England USA USA USA Portugal

EU437415 AY120913 AY120914 EF641020 AY883022

Robinson et al. (2008) Xiao et al. (2002) Xiao et al. (2002) Feng et al. (2007) Alves et al. (2005)

Chicken Chicken Chicken Chicken Chicken Chicken Chicken

USA USA USA USA USA USA USA

U67115 U67116 U67117 U67118 U67119 U67120 U67121

Barta Barta Barta Barta Barta Barta Barta

Skunk Snaket Tortoiseu Vole Wildebeestv Eimeria E. acervulina E. brunetti E. maxima E. mitis E. necatrix E. praecox E. tenella a

Originally lodged on the GenBank database as Cryptosporidium muris Calf genotype (IDVS-811). Cryptosporidium parvum strain CPD1. Cryptosporidium sp. rabbit genotype. d Cryptosporidium parvum isolate 2246. e Cryptosporidium parvum isolate K2. f Cryptosporidium sp. EGK1. g Cryptosporidium sp. CSP06. h Cryptosporidium saurophilum. i Cryptosporidium Pig 1. j Cryptosporidium sp. k Cryptosporidium sp. ex Canis latrans. l Cryptosporidium sp. 2041. m Cryptosporidium sp. 886. n Cryptosporidium K1. o Cryptosporidium sp. 1665. p Cryptosporidium sp. 1041. q Cryptosporidium sp. ex Struthio camelus 2005. r Cryptosporidium sp. ex Phoca hispida genotype seal 1. s Cryptosporidium sp. ex Phoca hispida genotype seal 2. t Cryptosporidium sp. 938. u Cryptosporidium sp. 750. v Cryptosporidium sp. zoo 195. b c

et et et et et et et

al. al. al. al. al. al. al.

(1997) (1997) (1997) (1997) (1997) (1997) (1997)

[(Fig._1)TD$IG]

M.J. Nolan et al. / Infection, Genetics and Evolution 10 (2010) 1179–1187

1183

Fig. 1. The relationships between 58 species and genotypes of Cryptosporidium inferred from pSSU sequence data following analysis by Bayesian inference (BI). Posterior probabilities are indicated at all major nodes. Refer to Table 1 for epidemiological information linked to GenBank accession numbers (in parentheses).

Republic. The limited information for Cryptosporidium from rabbits is in stark contrast to the number of reports of gp60 sequence data from Cryptosporidium infecting other animals [e.g., cattle (n = 1128), sheep (n = 83), goats (n = 26) and pigs (n = 38)] reviewed by Jex and Gasser (2010). In addition, to date, published data for SSU (partial or complete) are limited to seven sequences from studies of humans and rabbits in England, China, the Czech Republic and New Zealand (Chalmers et al., 2009; Learmonth et al., 2004; Robinson et al., 2008; Ryan et al., 2003a; Xiao et al., 2002). This lack of data for the rabbit genotype of Cryptosporidium impairs the critical assessment of the potential for these genotypes to infect humans. The host specificity and zoonotic potential of species and genotypes of Cryptosporidium have been areas of significant

interest and research (Xiao et al., 2002, 2004). Currently, it is hypothesized that C. parvum represents the species of greatest zoonotic importance (Caccio`, 2005; Xiao and Feng, 2008). However, the zoonotic potential of a number of other species and ‘unusual’ genotypes is unclear (Chalmers and Davies, 2010; Robinson et al., 2008). Recently, C. cuniculus, specifically genotype Va (based on pgp60), was implicated in an outbreak of cryptosporidiosis in humans in England in 2008 (Anon., 2008; Chalmers et al., 2009; Robinson et al., 2008). More recently, genotype Vb has also been characterized from humans (Robinson et al., 2010). However, only a small number of studies has characterized Cryptosporidium from humans in Australia (Jex et al., 2008a; Jex et al., 2007; O’Brien et al., 2008; Waldron et al., 2009),

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1184

Table 2 Cryptosporidium cuniculus defined on the basis of partial 60 kDa glycoprotein (pgp60) gene sequence data determined in the present study, together with reference sequences (see accession numbers) for comparison, with epidemiological information. Species Genotype Cryptosporidium cuniculus Va

Vb

C. parvum IIa

IIb IIc

IId

IIe IIf IIg IIh IIj IIk C. hominis Ia

Host species

Locality

GenBank accession nos.

References

Human Human Rabbit (Oryctolagus cuniculus) Treated drinking water Rabbit Rabbit Rabbit

England England England England Australia China Czech Republic

FJ262732 EU437420 FJ262731 FJ262730 HM852432, HM852433 FJ262734 FJ262733

Chalmers et al. (2009) Chalmers et al.a Chalmers et al. (2009) Chalmers et al. (2009) Present study Chalmers et al. (2009) Chalmers et al. (2009)

Cattle (calf) (Bos taurus)

Australia

Nolan et al. (2009)

Cattle Cattle (calf) Horse (foal) (Equus ferus) Human (Homo sapiens) Human Human Human Human Human Human Human Cattle (calf) Goat (kid) (Capra hircus) Sheep (lamb) (Ovis aries) Human Human Human Human Human Human Human Cattle (calf) Raccoon dog (Nyctereutes procyonoides)

Netherlands USA New Zealand Australia Australia Australia China; Kenya; Portugalb Portugal Australia Australia Kuwait Hungry Spain Spain Australia Portugal Malawi Uganda Kuwait Uganda Uganda Northern Ireland Japan

FJ825018, FJ825019, FJ825022, FJ825023, FJ825026–FJ825028 EF576961, EF576964 DQ630515, DQ630516, DQ630519 EU483077, EU483080 EU164810, EU164811 EU379548, EU379550 FJ861283, FJ861293, FJ861302, FJ861304 AF402285 AY166805 EU164812 EF025581 AY738195 EF073048, EF073049 EU549712, EU549713 EU549718 FJ839877 AY166806 AY382675 EU877260 AY738188 AY873780 AY873781 DQ648547 AB237137

Wielinga et al. (2008) Xiao et al. (2007) Grinberg et al. (2008) Jex et al. (2007) Jex et al. (2008a) Waldron et al. (2009) Peng et al. (2001) Alves et al. (2003) Jex et al. (2007) O’Brien et al. (2008) Sulaiman et al. (2005) Plutzer and Karanis (2007a) Quı´lez et al. (2008) Quı´lez et al. (2008) Waldron et al. (2009) Alves et al. (2003) Peng et al. (2003) Jex and Gasser (2008) Sulaiman et al. (2005) Akiyoshi et al. (2006) Akiyoshi et al. (2006) Thompson et al. (2007) Abe et al. (2006)

Human

Japan

AB237130

Abe et al. (2006)

a

For details, refer to the GenBank database. b Table 4 in Peng et al. (2001) states that the genotype ‘IIb’ was isolated from stool samples from patients from China (Xiao et al., 2004) and Kenya (Okhuysen and Chappell, 2002) only; in contradiction, Fig. 1 indicates that isolate ‘1946’ represents ‘IIb’ and is from Portugal.

and it would be premature to conclude that the subgenotypes VbA23R3 and VbA26R4, characterised herein, are not zoonotic. Investigating the presence and/or distribution of genotypes in wild rabbits in Australia and globally is imperative, not only to determine the significance of this host group as reservoirs of Cryptosporidium for transmission to humans, but also to establish the mode(s) of transmission and ‘true’ host range. Furthermore, the results of the present study emphasize the need for increased investigation into the true host ranges of all Cryptosporidium species infecting other domesticated and non-domesticated animals not studied to date. Cervids, including Alces alces, Capreolus capreolus, Ca. elaphus, Cervus canadensis, Ce. nippon, Odocoileus columbianus, O. virginianus, Rangifer tarandus and Rusa unicolour, are host records for Cryptosporidium spp. (e.g., Alves et al., 2006; Cinque et al., 2008; Deng and Cliver, 1999; Fayer et al., 1996; Feng et al., 2007; Hajdusˇek and Ditrich, 2004; Hamnes et al., 2006; Paziewska et al., 2007; Perz and Le Blancq, 2001; Pople et al., 2001; Siefker et al., 2002; Skerrett and Holland, 2001; Wang et al., 2008) in a range of countries. Some of these host species have been shown, using molecular tools, to harbour Cryptosporidium genotypes that are also known to occur in humans (Alves et al., 2006; Perz and Le Blancq, 2001); yet, there is a paucity of information on the genetic structures of Cryptosporidium populations (particularly in relation to gp60) in cervid populations world-wide (Alves et al., 2006). Clearly, further molecular studies of species, genotypes and subgenotypes of Cryptosporidium infecting hosts other than

humans are required to assess their zoonotic potential (Jex and Gasser, 2010). A number of authors (Alves et al., 2001; Jex et al., 2008b) have recommended that multiple genetic loci should be employed for assessing the population genetic richness and diversity within Cryptosporidium and the epidemiology of cryptosporidiosis. Phylogenetic analysis of sequence data from both loci employed in the present study (cf. Figs. 1 and 2) supported the detection of C. cuniculus in south-eastern Australia. The analyses also indicated a close relationship of this genotype to C. parvum for pgp60, and C. parvum, C. hominis (i.e. the two most common species of Cryptosporidium infecting humans) and Cryptosporidium sp. mink-genotype (98% sequence similarity) for pSSU. Given the substantial differences in gp60 sequences types among Cryptosporidium species (Jex et al., 2008b), the magnitude of sequence similarity (up to 75%) and genetic relationship between the sequences of C. parvum and C. cuniculus suggests that the genetics/ genomics of this and other new species and genotypes should be explored further. However, presently, only a small number of molecular loci is available to allow comparisons among the many species and genotypes recognized within Cryptosporidium. As molecular technologies become more efficient, affordable and accessible, whole genomic sequencing using next-generation platforms (Mardis, 2008) will provide a wealth of new genetic markers for systematic studies and unique prospects for large and detailed molecular epidemiological and population genetic

[(Fig._2)TD$IG]

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Fig. 2. The relationships of genotypes/subgenotypes of Cryptosporidium cuniculus and C. parvum inferred from pgp60 sequence data following Bayesian inference (BI) analysis. Posterior probabilities are indicated at all major nodes. Refer to Table 2 for epidemiological information for each sequence, linked to GenBank accession numbers.

explorations of Cryptosporidium (Jex et al., 2008b). Combined with enhanced bioinformatic annotation and analysis pipelines, these advances will provide unique opportunities for genetic investigations into both recognized and yet undescribed genotypes of Cryptosporidium.

Acknowledgements Funding from Melbourne Water Corporation and the Australian Research Council is gratefully acknowledged. ARJ holds a Career Development Award (CDA) from the National Health and Medical Research Council (NHMRC) of Australia. Thanks to Kathy Cinque for technical assistance.

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