International Journal for Parasitology xxx (2014) xxx–xxx
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission q Yuanfei Wang a, Wenli Yang b, Vitaliano Cama c, Lin Wang a, Lilia Cabrera d, Ynes Ortega e, Caryn Bern c, Yaoyu Feng a,⇑, Robert Gilman f, Lihua Xiao b,⇑ a
State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA d Asociación Benéfica PRISMA, Lima, Peru e Center for Food Safety, University of Georgia, Griffin, GA 30223, USA f Department of International Health, Johns Hopkins University, Baltimore, MD 21205, USA b c
a r t i c l e
i n f o
Article history: Received 18 January 2014 Received in revised form 19 March 2014 Accepted 20 March 2014 Available online xxxx Keywords: Cryptosporidium meleagridis Multilocus sequence typing Subtypes Population genetics Zoonosis
a b s t r a c t Population genetic studies have been used to understand the transmission of pathogens in humans and animals, especially the role of zoonotic infections and evolution and dispersal of virulent subtypes. In this study, we analysed the genetic diversity and population structure of Cryptosporidium meleagridis, the only known Cryptosporidium species that infects both avian and mammalian hosts and is responsible for approximately 10% of human cryptosporidiosis in some areas. A total of 62 C. meleagridis specimens from children, AIDS patients, and birds in Lima, Peru were characterised by sequence analysis of the ssrRNA gene and five minisatellite, microsatellite and polymorphic markers in chromosome 6, including the 60 kDa glycoprotein (gp60), 47 kDa glycoprotein (CP47), a serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR) and thrombospondin protein 8 (TSP8). The multilocus sequence analysis identified concurrent infections with Cryptosporidium hominis in four AIDS patients and three children. Unique subtypes of C. meleagridis ranged from eight at the gp60 locus (gene diversity – Hd = 0.651), three at the RPGR (Hd = 0.556), three at the MSC6-5 locus (Hd = 0.242), two at TSP8 (Hd = 0.198), to one at CP47 (monomorphic), much lower than that of C. hominis in the same area. Intragenic linkage disequilibrium was strong and complete at all gene loci. Intergenic linkage disequilibrium was highly significant (P < 0.001) for all pairs of polymorphic loci. Two major groups of subtypes were seen, with most subtypes belonging to group 1. Within group 1, there was no clear population segregation, and two of the 14 multilocus subtypes of C. meleagridis were found in both AIDS patients and birds. We believe that these results provide the first evidence of a clonal population structure of C. meleagridis and the likely occurrence of cross-species transmission of C. meleagridis between birds and humans. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
1. Introduction Cryptosporidiosis is a significant cause of diarrhea and gastroenteritis in humans (Kotloff et al., 2013). Recent molecular epidemiological studies indicate that five species of Cryptosporidium (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis and Cryptosporidium canis) are responsible for most human infections (Xiao and Feng, 2008). Of
q Nucleotide sequence data reported in this paper are available in GenBank under accession numbers KF733815–KF733831. ⇑ Corresponding authors. Tel.: +86 159 2144 6686; fax: +86 21 6425 0664 (Y. Feng). Tel.: +1 404 718 4161; fax: +1 404 718 4197 (L. Xiao). E-mail addresses: [email protected] (Y. Feng), [email protected] (L. Xiao).
these species, C. parvum and C. hominis are the two most common ones. However, there are geographical differences in the distribution of the five species. Some studies have shown a high percentage of C. meleagridis: 75.0% (nine of 12 genotyped) of Cryptosporidiumpositive patients in Cote d’Ivoire (Berrilli et al., 2012), 11.3% in HIV-positive patients in northern India (Sharma et al., 2013), 20% in HIV-positive patients in Thailand (Gatei et al., 2002), and 8.8% in HIV-positive patients and 8.2–8.7% in children in Peru (Xiao et al., 2001; Cama et al., 2007, 2008). Thus, C. meleagridis infection occurs not only in HIV-positive patients but also in immunocompetent persons. In fact, among 16,883 cryptosporidiosis cases investigated between 1985 and 2008 in the United Kingdom, 131 cases of C. meleagridis infection were identified, mostly in immunocompetent persons (Leoni et al., 2006; Chalmers et al., 2009; Elwin et al., 2011).
http://dx.doi.org/10.1016/j.ijpara.2014.03.003 0020-7519/Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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One outbreak of cryptosporidiosis by C. meleagridis has been reported in a high school dormitory in Japan (Asano et al., 2006). In birds, although Cryptosporidium baileyi is the most commonly identified Cryptosporidium sp. in poultry, pet birds and wild birds (Ryan, 2010), a high prevalence of C. meleagridis was recently reported in chickens and turkeys in Algeria (Baroudi et al., 2013). There was a probable case of human C. meleagridis infection on a Swedish farm via direct contact with infected chickens (Silverlas et al., 2012). The use of molecular tools in the detection and characterisation of Cryptosporidium spp. has improved our understanding of the population genetics and transmission of C. hominis in humans and C. parvum in humans and cattle (Xiao, 2010; Widmer and Sullivan, 2012). More recent studies have focused on intra-species substructure of C. parvum and C. hominis in relation to host, temporal, geographical and other factors (Mallon et al., 2003a; Tanriverdi et al., 2006; Gatei et al., 2007; Drumo et al., 2012; Herges et al., 2012; De Waele et al., 2013). Highly polymorphic markers, such as microsatellites and minisatellites, in multilocus typing (MLT) and multilocus sequencing typing (MLST) have been used. A study by Mallon et al. (2003b) provided the first evidence of a panmictic population structure in C. parvum, contradictory to the proposed clonal structure. This has been confirmed by results of other studies (Herges et al., 2012; De Waele et al., 2013). Therefore, recombination takes place in nature, at least in C. parvum. A later study, however, showed a mostly clonal population structure in C. hominis, as significant intra-and inter-genic linkage disequilibrium (LD) was seen with minimum evidence of recombination (Gatei et al., 2007). Currently, there is a lack of understanding of the population genetics and sub-structure of C. meleagridis. Previously, three studies typed 12 isolates from humans and four isolates from birds at three polymorphic loci, including the ssrRNA, 60 kDa glycoprotein (gp60) and 70 kDa heat shock protein (hsp70) genes (Glaberman et al., 2001; Abe, 2010; Abe and Makino, 2010). A high genetic heterogeneity was detected in C. meleagridis but no identical multilocus sequence (MLS) subtypes were found between humans and birds. However, the number of isolates and loci used was insufficient to infer population genetic structure and inter-species transmission. In this study, we used the MLST approach to examine the population genetics of C. meleagridis in humans and birds from the same geographical area.
2.2. DNA extraction and C. meleagridis identification Approximately 200 ll of fecal suspension from each specimen was washed three times with distilled water by centrifugation. DNA was extracted using the FastDNA Spin Kit for Soil (MP Biomedicals, Carlsbad, CA, USA). The diagnosis of C. meleagridis in each specimen was confirmed in this study by PCR-RFLP analysis and DNA sequencing of an approximately 830 bp fragment of the ssrRNA gene (Xiao et al., 2001). 2.3. Targets for MLST Five genetic loci were targeted in the subtype analysis of the identified C. meleagridis, including the gp60, 47 kDa glycoprotein (CP47), serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR), and thrombospondin protein 8 (TSP8) genes, all in chromosome 6. These targets were selected due to their polymorphic nature in C. parvum and C. hominis (Gatei et al., 2006, 2007; Xiao and Ryan, 2008) and the ability to detect C. meleagridis by PCR. The targets were amplified by nested PCR using primers based on available C. parvum and C. hominis sequences. For gp60, the primers previously used in the amplification of a 520–620 bp fragment of the gene in C. meleagridis were used due to their greater efficiency than the routine gp60 primers in amplifying the target in this species (Glaberman et al., 2001). 2.4. MLST PCR The total volume of PCR mixture was 100 ll for all PCR analyses and contained 1 ll of the extracted DNA (for primary PCR) or 2 ll of the primary PCR product (for secondary PCR), 0.2 lM (for primary PCR) or 0.4 lM (for secondary PCR) primers, 0.2 mM deoxyribonuleotide triphosphate mix (Promega, Madison, WI, USA), 3 mM MgCl2, 1X GeneAmp PCR buffer (Applied Biosystems, Foster City, CA, USA), and 2.5 U of Taq DNA polymerase (Promega). Primary PCRs also contained 400 ng/ll of non-acetylated BSA (Sigma–Aldrich, St. Louis, MO, USA). PCR amplification consisted of an initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 45 s, the specified annealing temperature (Glaberman et al., 2001; Xiao and Ryan, 2008) for 45 s and 72 °C for 60 s, with a final extension of the PCR products at 72 °C for 10 min. PCR products were visualised under ultraviolet light after 2% agarose gel electrophoresis.
2. Materials and methods 2.5. DNA sequence analysis 2.1. Specimens A total of 62 C. meleagridis-positive stool specimens from Peru (35 from AIDS patients, 20 from children, seven from birds) were used in this study. Fifty-five human specimens were collected in three molecular epidemiological studies of cryptosporidiosis in HIV-positive persons and children in Lima, Peru, two of which were described previously (Xiao et al., 2001; Cama et al., 2007). Seven bird specimens were obtained in the same area in 2006, including five from chickens, one from a pigeon and one from a duck, all from the same small community (Pampas de San Juan de Miraflores, Lima, Peru) where the pediatric specimens came from. All human and bird specimens were chosen based on previous diagnosis of C. meleagridis by PCR-restriction fragment length polymorphism (RFLP) analysis of the ssrRNA gene (Xiao et al., 2001; Cama et al., 2007). Only one specimen from each person or bird was included. Written informed consent was obtained from adult human patients and guardians of study children. The field human and animal studies were approved by the institutional review boards of the Johns Hopkins University, University of Georgia, and the Centers for Diseases Control and Prevention, USA.
PCR products were sequenced in both directions on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). At least two PCR products were sequenced for each specimen at each locus. Nucleotide sequences were read and assembled using the software ChromasPro (http://www.technelysium.com.au/ChromasPro.htm). Alignment of consensus sequences obtained and those from the GenBank database was done using ClustalX (http://www.clustal.org/). Sequence alignments were edited using the BioEdit program version 7.0.4 (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html). Phylogeny of gp60 subtypes was inferred by neighbor-joining (NJ) analysis based on genetic distance calculated by the Kimura 2-parameter model using the program TreeCon (http://www.psb.rug.ac.be/bioinformatics/psb/Userman/treeconw. html). The robustness of the branches was assessed using the bootstrapping method with 1000 replicates. 2.6. Multilocus analysis Genetic diversity included both sequence length polymorphism and single nucleotide polymorphism (SNP), and was used to assess
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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subtype frequencies, gene diversity, pair-wise LD, and recombination rates. Intragenic LD and recombination rates were calculated using DnaSP 5.10.00 (http://www.ub.es/dnasp/). Most multilocus analyses were conducted on allelic data, including pair-wise intergenic LD among polymorphic loci using Arlequin 3.1 (http:// cmpg.unibe.ch/software/arlequin3/), and multilocus LD by calculating the standardised index of association (ISA) using the program LIAN 3.5 (http://pubmlst.org/perl/mlstanalyse/mlstanalyse.pl?site= pubmlst&page=lian&referer=pubmlst.org). Relationships among MLS subytypes were assessed by NJ analysis of concatenated sequences. An eBURST analysis (http://eburst.mlst.net/) of allelic data was used to identify sub-populations present. To assess the robustness of the sub-structuring, the Wright’s fixation index (FST) was calculated between sub-populations using Arlequin.
identified as having C. hominis and C. meleagridis co-infection in ssrRNA PCR (Table 1). These seven specimens were excluded in MLST and population genetic analyses of C. meleagridis. 3.3. Cryptosporidium meleagridis subtypes at gp60 Of the remaining 55 C. meleagridis specimens, 54 (all except for 7345) were amplified in gp60 PCR and all PCR products were sequenced successfully. This locus was highly variable with 76 segregation sites and no singleton mutation. The nucleotide diversity was 0.039 whereas the gene diversity (Hd) was 0.651. Sequence alignment and phylogenetic analysis of the sequences showed the presence of two subtype families (Fig. 1A). There were a total of eight subtypes in two subtype families (Table 2; Fig. 1A). Seven of the subtypes belonged to the IIIb subtype family, including IIIbA13G1 (one specimen), IIIbA15G1 (four specimens), IIIbA24G1 (six specimens), IIIbA26G1 (33 specimens), IIIbA27G1 (four specimens), IIIbA30G1 (one specimen), and IIIbA34G1 (one specimen). The other subtype, IIIcA6, belonged to the subtype family IIIc and was seen in four specimens. The IIIcA6 specimens all generated the type 2 ssrRNA sequence.
3. Results 3.1. Confirmation of C. meleagridis identification by PCR-RFLP and sequence analysis of the ssrRNA gene All 62 specimens were re-analysed at the ssrRNA locus by PCRRFLP and DNA sequencing. Fifty-four human specimens and six bird specimens were confirmed as having C. meleagridis, one human specimen had both C. hominis and C. meleagridis, and one bird specimen had both C. baileyi and C. meleagridis. In RFLP analysis, the C. meleagridis specimens produced two VspI banding patterns (type 1 and type 2) identified previously (Glaberman et al., 2001). Within type 2, the copy A gene RFLP product was the dominant in all specimens. DNA sequencing confirmed the identification of two C. meleagridis genotypes, with type 2 differing from type 1 mostly in the presence of a ATT insert (Glaberman et al., 2001). Only five specimens (1021, 1227, 2815, 6536 and 7345) belonged to type 2 C. meleagridis, all from human specimens collected before 2003. One additional human specimen (7342) had both types 1 and 2, with the former dominating. Among the six type 2 ssrRNA sequences obtained, three (specimens 1227, 2815 and 7342) had mixed nucleotides at position 331 (A and G) and 820 (A and G) (using GenBank Accession number AF112574 as the reference) and an ATT insertion, which were previously identified as products of both A and B copies of the type 2 ssrRNA gene (Glaberman et al., 2001). Type 1 was seen in both human and bird specimens.
3.4. Sequence polymorphism at other genetic loci All 55 C. meleagridis specimens produced PCR amplification at the CP47 locus. The sequences obtained were identical to each other. Fifty-three specimens were amplified at the MSC6-5 locus and belonged to three subtypes (Table 2). The dominant subtype (in 46 specimens) had nine TCT repeats and four TCC repeats in the microsatellite region. Another subtype (in four specimens) had nine TCT repeats and three TCC repeats. The third subtype (in three specimens) had 10 TCT repeats and 13 TCC repeats, and was seen only in specimens of the type 2 ssrRNA. There were no segregation sites at this locus. It had a low Hd of 0.242 (Table 3). Fifty specimens were amplified at the RPGR locus. There were three subtypes with four segregating sites. It had a low nucleotide diversity of 0.005 and a modest Hd of 0.556 (Tables 2 and 3). All sequences from three type 2 ssrRNA specimens were identical to each other. In contrast, the two subtypes from type 1 ssrRNA specimens differed from each other by three SNPs. Compared with sequences from type 1 ssrRNA specimens, RPGR sequences from type 2 ssrRNA specimens had a ‘‘GGGAGGTAAAAGAGAAGA’’ insertion, a ‘‘AAGGAGAGAAGAAAGAAG’’ insertion, and an A to T nucleotide substitution. Fifty-five specimens were amplified at the TSP8 locus. Two subtypes were seen at the locus (Table 2), which differed from each other by a single nucleotide: a change of A to G. There was a low nucleotide diversity of 0.001 and a low Hd of 0.198 (Table 3). There was no agreement between ssrRNA sequence types and TSP8 subtypes.
3.2. Identification of co-infections with C. hominis by MLST All 62 specimens containing C. meleagridis were analysed by MLST, including the human specimen with C. hominis and C. meleagridis co-infection and the bird specimen with C. baileyi and C. meleagridis co-infection. Seven human specimens, including four from AIDS patients and three from children, however, produced only C. hominis sequences during MLST, including the specimen
Table 1 Identification of Cryptosporidium hominis co-infection at five genetic loci in seven of 62 Peruvians previously identified as positive for Cryptosporidium meleagridis based on PCRrestriction fragment length polymorphism and DNA sequence analyses of the ssrRNA gene of Cryptosporidium spp. Specimen ID
Patient source
3928 4500 4508 5144 9887 9890 9912
HIV+ adult HIV+ adult HIV+ adult HIV+ adult Child Child Child
Cryptosporidium species identified at the genetic locus ssrRNA
gp60
CP47
MSC6-5
RPGR
TSP8
C. C. C. C. C. C. C.
C. C. C. C. C. C. C.
Negative C. hominis Negative C. hominis C. hominis C. hominis C. hominis
Negative C. hominis C. hominis C. hominis C. hominis C. hominis C. hominis
C. C. C. C. C. C. C.
C. hominis C. hominis Negative C. hominis C. hominis C. hominis C. hominis
meleagridis meleagridis meleagridis meleagridis and C. hominis meleagridis meleagridis meleagridis
hominis hominis hominis hominis hominis hominis hominis
hominis hominis hominis hominis hominis hominis hominis
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Y. Wang et al. / International Journal for Parasitology xxx (2014) xxx–xxx 86
IIIg-JX878614
A
HIV+
80
54 100
0.05 substitutions/site 100
9890-Child-IdA10
100
IIIe-AB539721 IIIa-AF401499 Child
IIIbA24G1
7027, 7047, 7364
IIIbA26G1
2567, 4509, 4517, 4523, 4528, 5714, 5765, 5776, 6484, 6494, 7020, 7043, 7047, 7051, 7053, 7342, 7349, 7424, 7784, 7797, 7801
IIIbA2&G1
5745, 7352, 7355
2713, 2731, 2805, 3252, 9528, 9636, 9917, 10188, 11737, 11749, 11756
Bird 11286, 11308, 11354
11208, 13099
13094
7085-HIV+-IIIbA15G1 9508-Child-IIIbA15G1 7359-HIV+-IIIbA15G1 9633-Child-IIIbA34G1 10173-Child-IIIbA30G1 11371-Bird-IIIbA15G1 2556-HIV+-IIIbA13G1 IIId-DQ067570 100 2815-Child-IIIcA6 100 6536-HIV+-IIIcA6 1021-Child-IIIcA6 1227-Child-IIIcA6 2731-Child-MLG4 2713-Child-MLG4 5776-HIV+-MLG4 7797-HIV+-MLG4 2567-HIV+-MLG4 7053-HIV+-MLG4 10188-Child-MLG4 4523-HIV+-MLG4 6494-HIV+-MLG4 5714-HIV+-MLG4 7784-HIV+-MLG$ 3252-Child-MLG4 9636-Child-MLG4 9528-Child-MLG4 2805-Child-MLG4 4517-HIV+-MLG4 85 7020-HIV+-MLG4 5765-HIV+-MLG4 4528-HIV+-MLG4 84 7342-HIV+-MLG4 7801-HIV+-MLG6 9633-Child-MLG11 57 7364-HIV+-MLG3 57 7355-HIV+-MLG9 58 67 5745-HIV+-MLG8 7352-HIV+-MLG8 13094-Bird-MLG8 7349-HIV+-MLG5 6484-HIV+-MLG5 100 7051-HIV+-MLG5 7043-HIV+-MLG5 11749-Child-MLG5 4509-HIV+-MLG5 7424-HIV+-MLG5 11756-Child-MLG5 11737-Child-MLG5 9917-Child-MLG7 74 10173-Child-MLG10 7359-HIV+-MLG12 9508-Child-MLG12 94 61 7085-HIV+-MLG12 7027-HIV+-MLG1 89 11308-Bird-MLG1 59 11354-Bird-MLG1 7047-HIV+-MLG2 67 2556-HIV+-MLG14 67 11371-Bird-MLG13 1021-Child-MLG15 100 6536-HIV+-MLG16 62 1227-Child-MLG16
B
0.02 substitutions/site
2
C 13
1 12
3 8 5
9
4 11
7 6 10 Fig. 1. Genetic relationship of Cryptosporidium meleagridis isolates. (A) Relationship among subtypes at the 60 kDa glycoprotein (gp60) locus as inferred by a neighbor-joining analysis of nucleotide sequences. (B) Relationship among multilocus sequence types as inferred by neighbor-joining analysis. (C) Patterns of evolutionary descents among multilocus sequence subtypes based on eBURST analysis of allelic data. The primary founder is shown as the smaller open circle and subgroup founders are shown as the larger open circle. The sizes of dots and circles are proportional to the number of specimens of the sequence type. Two multilocus sequence types of the gp60 subtype IIIcA6 and one of the gp60 IIIbA13G1 subtype did not cluster within this diagram.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Y. Wang et al. / International Journal for Parasitology xxx (2014) xxx–xxx Table 2 Frequency and allele composition of multilocus sequence types of Cryptosporidium meleagridis in 50 human and bird specimens with sequence data at all five genetic loci. MLST
Distribution
Allele composition
ID
Count
Frequency
Children
HIV+ adult
Birds
gp60
CP47
MSC6-5
RPGR
TSP8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3 1 1 20 9 1 1 3 1 1 1 3 1 1 1 2
0.06 0.02 0.02 0.40 0.18 0.02 0.02 0.06 0.02 0.02 0.02 0.06 0.02 0.02 0.02 0.04
0 0 0 7 3 0 1 0 0 1 1 1 0 0 1 1
1 1 1 13 6 1 0 2 1 0 0 2 0 1 0 1
2 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0
1 1 1 2 2 2 2 3 3 4 5 6 6 7 8 8
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 2 1 1 1 2 1 1 1 1 1 1 2 2 3 3
1 1 2 2 1 2 1 2 2 1 2 1 1 1 3 3
1 1 1 1 1 1 2 1 2 1 1 1 1 2 1 2
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Table 3 Gene diversity in 55 Cryptosporidium meleagridis specimens based on intragenic analysis of five polymorphic loci and concatenated multilocus gene sequences. Locus
No. of Haplotype Intralocus genetic 95% CI (ZnS) No. of No. of No. of polymorphic segregating haplotype diversity (Hd) association (ZnS) site site site (bp)
Intragenic linkage disequilibrium (LD, |D0 |)
Recombination events (Rms)
CP47 gp60 MSC6-5 RPGR TSP8 Concatenated sequence including gp60a Concatenated sequence excluding gp60a
392 721 425 380 327 1853
0 323 33 40 1 397
0 76 0 4 1 81
1 8 3 3 2 18
0 0.651 0.242 0.556 0.198 0.829
– 1 – 0.454 – 0.913
– 0.05557–0.40285 – 0.00188–0.40285 – 0.05599–0.42813
– Y = 1.0000 + 0.0000X – Y = 1.0000 + 0.0000X – Y = 1.0178–0.1110X
– 0 –
1132
74
5
9
0.656
0.301
0.00292–0.62149 Y = 1.0515–1.4573X
– 1 1
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8; CI, confidence interval; |D0 |, linkage disequilibrium (LD), where Y is the LD value, and X is the nucleotide distance in kb. a From 50 specimens with sequence data at all five loci.
3.5. MLS subtypes The 50 C. meleagridis specimens with sequence information available at all loci produced a total of 16 MLS subtypes (Table 2). One dominant subtype consisted of 40.0% of the typed specimens. A second subtype was found in 18.0%. Three other subtypes were found in 6.0% and one subtype in 4.0%. The remaining 10 multilocus subtypes were each found in one specimen (2.0%). The frequency and allelic composition of the multilocus subtypes are shown in Table 2. Among the 16 MLS subtypes, 14 belonged to ssrRNA type 1 and two belonged to ssrRNA type 2. There were no significant changes in the distribution of MLS subtypes within type 1 C. meleagridis during the study period.
with 81 polymorphic sites. As expected, results of analysis showed strong LD among all polymorphic sites. A recombination analysis observed only one minimum Rm and an estimate of R per gene of 0.001 (Table 3). In analysis of allelic data, highly significant (Markov chain parameters significance, P < 0.05) intergenic LD was seen in all pairs among gp60, MSC6–5, RPGR and TSP8 (Table 4). Multilocus LD was further assessed by calculating ISA between alleles for all pair-wise combinations of all polymorphic loci, the variance of pair-wise differences (VD), and the 95% critical value for VD (L). In this analysis of allelic data of 55 specimens at all genetic loci, the value of ISA was positive (0.2390) and VD (1.4154) was more than L (0.9505), both indicative of the existence of LD and a non-panmictic structure. A Monte Carlo analysis was further used to test the
3.6. LD and population structure Population structure was assessed by LD among polymorphic sites within each locus and between pairs of polymorphic loci. Intragenic LD based on nucleotide sequences was measured as the non-random association of adjacent nucleotides sites along the DNA sequences. Intragenic LD was strong and complete at all genetic loci with three or more segregation sites such as gp60 and RPGR (Table 3). Recombination analysis using DnaSP programs revealed no recombination events (Rm) at these loci. We also analysed LD in the concatenated multilocus sequences of 1853 bp long
Table 4 Intergenic linkage disequilibrium among genetic loci for Cryptosporidium meleagridis (Markov chain parameters significance, P < 0.05).
GP60 MSC6-5 RPGR TSP8
GP60
MSC6–5
RPGR
TSP8
– 0.00001 0.00000 0.00001
+ 0.00000 0.00012
+ + 0.00177
+ + +
GP60, 60 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Table 5 Linkage disequilibrium analysis based on allelic profile data for Cryptosporidium meleagridis. Population group
Number
H
ISA
PMC
VD
L
VD > L
LD or LE
gp60 gp60 gp60 gp60
55 23 55 23
0.4463 ± 0.1170 0.4463 ± 0.1170 0.3737 ± 0.1297 0.5468 ± 0.1084
0.2390 0.0609 0.2646 0.0964
Contents lists available at ScienceDirect
International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission q Yuanfei Wang a, Wenli Yang b, Vitaliano Cama c, Lin Wang a, Lilia Cabrera d, Ynes Ortega e, Caryn Bern c, Yaoyu Feng a,⇑, Robert Gilman f, Lihua Xiao b,⇑ a
State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA d Asociación Benéfica PRISMA, Lima, Peru e Center for Food Safety, University of Georgia, Griffin, GA 30223, USA f Department of International Health, Johns Hopkins University, Baltimore, MD 21205, USA b c
a r t i c l e
i n f o
Article history: Received 18 January 2014 Received in revised form 19 March 2014 Accepted 20 March 2014 Available online xxxx Keywords: Cryptosporidium meleagridis Multilocus sequence typing Subtypes Population genetics Zoonosis
a b s t r a c t Population genetic studies have been used to understand the transmission of pathogens in humans and animals, especially the role of zoonotic infections and evolution and dispersal of virulent subtypes. In this study, we analysed the genetic diversity and population structure of Cryptosporidium meleagridis, the only known Cryptosporidium species that infects both avian and mammalian hosts and is responsible for approximately 10% of human cryptosporidiosis in some areas. A total of 62 C. meleagridis specimens from children, AIDS patients, and birds in Lima, Peru were characterised by sequence analysis of the ssrRNA gene and five minisatellite, microsatellite and polymorphic markers in chromosome 6, including the 60 kDa glycoprotein (gp60), 47 kDa glycoprotein (CP47), a serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR) and thrombospondin protein 8 (TSP8). The multilocus sequence analysis identified concurrent infections with Cryptosporidium hominis in four AIDS patients and three children. Unique subtypes of C. meleagridis ranged from eight at the gp60 locus (gene diversity – Hd = 0.651), three at the RPGR (Hd = 0.556), three at the MSC6-5 locus (Hd = 0.242), two at TSP8 (Hd = 0.198), to one at CP47 (monomorphic), much lower than that of C. hominis in the same area. Intragenic linkage disequilibrium was strong and complete at all gene loci. Intergenic linkage disequilibrium was highly significant (P < 0.001) for all pairs of polymorphic loci. Two major groups of subtypes were seen, with most subtypes belonging to group 1. Within group 1, there was no clear population segregation, and two of the 14 multilocus subtypes of C. meleagridis were found in both AIDS patients and birds. We believe that these results provide the first evidence of a clonal population structure of C. meleagridis and the likely occurrence of cross-species transmission of C. meleagridis between birds and humans. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
1. Introduction Cryptosporidiosis is a significant cause of diarrhea and gastroenteritis in humans (Kotloff et al., 2013). Recent molecular epidemiological studies indicate that five species of Cryptosporidium (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis and Cryptosporidium canis) are responsible for most human infections (Xiao and Feng, 2008). Of
q Nucleotide sequence data reported in this paper are available in GenBank under accession numbers KF733815–KF733831. ⇑ Corresponding authors. Tel.: +86 159 2144 6686; fax: +86 21 6425 0664 (Y. Feng). Tel.: +1 404 718 4161; fax: +1 404 718 4197 (L. Xiao). E-mail addresses: [email protected] (Y. Feng), [email protected] (L. Xiao).
these species, C. parvum and C. hominis are the two most common ones. However, there are geographical differences in the distribution of the five species. Some studies have shown a high percentage of C. meleagridis: 75.0% (nine of 12 genotyped) of Cryptosporidiumpositive patients in Cote d’Ivoire (Berrilli et al., 2012), 11.3% in HIV-positive patients in northern India (Sharma et al., 2013), 20% in HIV-positive patients in Thailand (Gatei et al., 2002), and 8.8% in HIV-positive patients and 8.2–8.7% in children in Peru (Xiao et al., 2001; Cama et al., 2007, 2008). Thus, C. meleagridis infection occurs not only in HIV-positive patients but also in immunocompetent persons. In fact, among 16,883 cryptosporidiosis cases investigated between 1985 and 2008 in the United Kingdom, 131 cases of C. meleagridis infection were identified, mostly in immunocompetent persons (Leoni et al., 2006; Chalmers et al., 2009; Elwin et al., 2011).
http://dx.doi.org/10.1016/j.ijpara.2014.03.003 0020-7519/Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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One outbreak of cryptosporidiosis by C. meleagridis has been reported in a high school dormitory in Japan (Asano et al., 2006). In birds, although Cryptosporidium baileyi is the most commonly identified Cryptosporidium sp. in poultry, pet birds and wild birds (Ryan, 2010), a high prevalence of C. meleagridis was recently reported in chickens and turkeys in Algeria (Baroudi et al., 2013). There was a probable case of human C. meleagridis infection on a Swedish farm via direct contact with infected chickens (Silverlas et al., 2012). The use of molecular tools in the detection and characterisation of Cryptosporidium spp. has improved our understanding of the population genetics and transmission of C. hominis in humans and C. parvum in humans and cattle (Xiao, 2010; Widmer and Sullivan, 2012). More recent studies have focused on intra-species substructure of C. parvum and C. hominis in relation to host, temporal, geographical and other factors (Mallon et al., 2003a; Tanriverdi et al., 2006; Gatei et al., 2007; Drumo et al., 2012; Herges et al., 2012; De Waele et al., 2013). Highly polymorphic markers, such as microsatellites and minisatellites, in multilocus typing (MLT) and multilocus sequencing typing (MLST) have been used. A study by Mallon et al. (2003b) provided the first evidence of a panmictic population structure in C. parvum, contradictory to the proposed clonal structure. This has been confirmed by results of other studies (Herges et al., 2012; De Waele et al., 2013). Therefore, recombination takes place in nature, at least in C. parvum. A later study, however, showed a mostly clonal population structure in C. hominis, as significant intra-and inter-genic linkage disequilibrium (LD) was seen with minimum evidence of recombination (Gatei et al., 2007). Currently, there is a lack of understanding of the population genetics and sub-structure of C. meleagridis. Previously, three studies typed 12 isolates from humans and four isolates from birds at three polymorphic loci, including the ssrRNA, 60 kDa glycoprotein (gp60) and 70 kDa heat shock protein (hsp70) genes (Glaberman et al., 2001; Abe, 2010; Abe and Makino, 2010). A high genetic heterogeneity was detected in C. meleagridis but no identical multilocus sequence (MLS) subtypes were found between humans and birds. However, the number of isolates and loci used was insufficient to infer population genetic structure and inter-species transmission. In this study, we used the MLST approach to examine the population genetics of C. meleagridis in humans and birds from the same geographical area.
2.2. DNA extraction and C. meleagridis identification Approximately 200 ll of fecal suspension from each specimen was washed three times with distilled water by centrifugation. DNA was extracted using the FastDNA Spin Kit for Soil (MP Biomedicals, Carlsbad, CA, USA). The diagnosis of C. meleagridis in each specimen was confirmed in this study by PCR-RFLP analysis and DNA sequencing of an approximately 830 bp fragment of the ssrRNA gene (Xiao et al., 2001). 2.3. Targets for MLST Five genetic loci were targeted in the subtype analysis of the identified C. meleagridis, including the gp60, 47 kDa glycoprotein (CP47), serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR), and thrombospondin protein 8 (TSP8) genes, all in chromosome 6. These targets were selected due to their polymorphic nature in C. parvum and C. hominis (Gatei et al., 2006, 2007; Xiao and Ryan, 2008) and the ability to detect C. meleagridis by PCR. The targets were amplified by nested PCR using primers based on available C. parvum and C. hominis sequences. For gp60, the primers previously used in the amplification of a 520–620 bp fragment of the gene in C. meleagridis were used due to their greater efficiency than the routine gp60 primers in amplifying the target in this species (Glaberman et al., 2001). 2.4. MLST PCR The total volume of PCR mixture was 100 ll for all PCR analyses and contained 1 ll of the extracted DNA (for primary PCR) or 2 ll of the primary PCR product (for secondary PCR), 0.2 lM (for primary PCR) or 0.4 lM (for secondary PCR) primers, 0.2 mM deoxyribonuleotide triphosphate mix (Promega, Madison, WI, USA), 3 mM MgCl2, 1X GeneAmp PCR buffer (Applied Biosystems, Foster City, CA, USA), and 2.5 U of Taq DNA polymerase (Promega). Primary PCRs also contained 400 ng/ll of non-acetylated BSA (Sigma–Aldrich, St. Louis, MO, USA). PCR amplification consisted of an initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 45 s, the specified annealing temperature (Glaberman et al., 2001; Xiao and Ryan, 2008) for 45 s and 72 °C for 60 s, with a final extension of the PCR products at 72 °C for 10 min. PCR products were visualised under ultraviolet light after 2% agarose gel electrophoresis.
2. Materials and methods 2.5. DNA sequence analysis 2.1. Specimens A total of 62 C. meleagridis-positive stool specimens from Peru (35 from AIDS patients, 20 from children, seven from birds) were used in this study. Fifty-five human specimens were collected in three molecular epidemiological studies of cryptosporidiosis in HIV-positive persons and children in Lima, Peru, two of which were described previously (Xiao et al., 2001; Cama et al., 2007). Seven bird specimens were obtained in the same area in 2006, including five from chickens, one from a pigeon and one from a duck, all from the same small community (Pampas de San Juan de Miraflores, Lima, Peru) where the pediatric specimens came from. All human and bird specimens were chosen based on previous diagnosis of C. meleagridis by PCR-restriction fragment length polymorphism (RFLP) analysis of the ssrRNA gene (Xiao et al., 2001; Cama et al., 2007). Only one specimen from each person or bird was included. Written informed consent was obtained from adult human patients and guardians of study children. The field human and animal studies were approved by the institutional review boards of the Johns Hopkins University, University of Georgia, and the Centers for Diseases Control and Prevention, USA.
PCR products were sequenced in both directions on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). At least two PCR products were sequenced for each specimen at each locus. Nucleotide sequences were read and assembled using the software ChromasPro (http://www.technelysium.com.au/ChromasPro.htm). Alignment of consensus sequences obtained and those from the GenBank database was done using ClustalX (http://www.clustal.org/). Sequence alignments were edited using the BioEdit program version 7.0.4 (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html). Phylogeny of gp60 subtypes was inferred by neighbor-joining (NJ) analysis based on genetic distance calculated by the Kimura 2-parameter model using the program TreeCon (http://www.psb.rug.ac.be/bioinformatics/psb/Userman/treeconw. html). The robustness of the branches was assessed using the bootstrapping method with 1000 replicates. 2.6. Multilocus analysis Genetic diversity included both sequence length polymorphism and single nucleotide polymorphism (SNP), and was used to assess
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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subtype frequencies, gene diversity, pair-wise LD, and recombination rates. Intragenic LD and recombination rates were calculated using DnaSP 5.10.00 (http://www.ub.es/dnasp/). Most multilocus analyses were conducted on allelic data, including pair-wise intergenic LD among polymorphic loci using Arlequin 3.1 (http:// cmpg.unibe.ch/software/arlequin3/), and multilocus LD by calculating the standardised index of association (ISA) using the program LIAN 3.5 (http://pubmlst.org/perl/mlstanalyse/mlstanalyse.pl?site= pubmlst&page=lian&referer=pubmlst.org). Relationships among MLS subytypes were assessed by NJ analysis of concatenated sequences. An eBURST analysis (http://eburst.mlst.net/) of allelic data was used to identify sub-populations present. To assess the robustness of the sub-structuring, the Wright’s fixation index (FST) was calculated between sub-populations using Arlequin.
identified as having C. hominis and C. meleagridis co-infection in ssrRNA PCR (Table 1). These seven specimens were excluded in MLST and population genetic analyses of C. meleagridis. 3.3. Cryptosporidium meleagridis subtypes at gp60 Of the remaining 55 C. meleagridis specimens, 54 (all except for 7345) were amplified in gp60 PCR and all PCR products were sequenced successfully. This locus was highly variable with 76 segregation sites and no singleton mutation. The nucleotide diversity was 0.039 whereas the gene diversity (Hd) was 0.651. Sequence alignment and phylogenetic analysis of the sequences showed the presence of two subtype families (Fig. 1A). There were a total of eight subtypes in two subtype families (Table 2; Fig. 1A). Seven of the subtypes belonged to the IIIb subtype family, including IIIbA13G1 (one specimen), IIIbA15G1 (four specimens), IIIbA24G1 (six specimens), IIIbA26G1 (33 specimens), IIIbA27G1 (four specimens), IIIbA30G1 (one specimen), and IIIbA34G1 (one specimen). The other subtype, IIIcA6, belonged to the subtype family IIIc and was seen in four specimens. The IIIcA6 specimens all generated the type 2 ssrRNA sequence.
3. Results 3.1. Confirmation of C. meleagridis identification by PCR-RFLP and sequence analysis of the ssrRNA gene All 62 specimens were re-analysed at the ssrRNA locus by PCRRFLP and DNA sequencing. Fifty-four human specimens and six bird specimens were confirmed as having C. meleagridis, one human specimen had both C. hominis and C. meleagridis, and one bird specimen had both C. baileyi and C. meleagridis. In RFLP analysis, the C. meleagridis specimens produced two VspI banding patterns (type 1 and type 2) identified previously (Glaberman et al., 2001). Within type 2, the copy A gene RFLP product was the dominant in all specimens. DNA sequencing confirmed the identification of two C. meleagridis genotypes, with type 2 differing from type 1 mostly in the presence of a ATT insert (Glaberman et al., 2001). Only five specimens (1021, 1227, 2815, 6536 and 7345) belonged to type 2 C. meleagridis, all from human specimens collected before 2003. One additional human specimen (7342) had both types 1 and 2, with the former dominating. Among the six type 2 ssrRNA sequences obtained, three (specimens 1227, 2815 and 7342) had mixed nucleotides at position 331 (A and G) and 820 (A and G) (using GenBank Accession number AF112574 as the reference) and an ATT insertion, which were previously identified as products of both A and B copies of the type 2 ssrRNA gene (Glaberman et al., 2001). Type 1 was seen in both human and bird specimens.
3.4. Sequence polymorphism at other genetic loci All 55 C. meleagridis specimens produced PCR amplification at the CP47 locus. The sequences obtained were identical to each other. Fifty-three specimens were amplified at the MSC6-5 locus and belonged to three subtypes (Table 2). The dominant subtype (in 46 specimens) had nine TCT repeats and four TCC repeats in the microsatellite region. Another subtype (in four specimens) had nine TCT repeats and three TCC repeats. The third subtype (in three specimens) had 10 TCT repeats and 13 TCC repeats, and was seen only in specimens of the type 2 ssrRNA. There were no segregation sites at this locus. It had a low Hd of 0.242 (Table 3). Fifty specimens were amplified at the RPGR locus. There were three subtypes with four segregating sites. It had a low nucleotide diversity of 0.005 and a modest Hd of 0.556 (Tables 2 and 3). All sequences from three type 2 ssrRNA specimens were identical to each other. In contrast, the two subtypes from type 1 ssrRNA specimens differed from each other by three SNPs. Compared with sequences from type 1 ssrRNA specimens, RPGR sequences from type 2 ssrRNA specimens had a ‘‘GGGAGGTAAAAGAGAAGA’’ insertion, a ‘‘AAGGAGAGAAGAAAGAAG’’ insertion, and an A to T nucleotide substitution. Fifty-five specimens were amplified at the TSP8 locus. Two subtypes were seen at the locus (Table 2), which differed from each other by a single nucleotide: a change of A to G. There was a low nucleotide diversity of 0.001 and a low Hd of 0.198 (Table 3). There was no agreement between ssrRNA sequence types and TSP8 subtypes.
3.2. Identification of co-infections with C. hominis by MLST All 62 specimens containing C. meleagridis were analysed by MLST, including the human specimen with C. hominis and C. meleagridis co-infection and the bird specimen with C. baileyi and C. meleagridis co-infection. Seven human specimens, including four from AIDS patients and three from children, however, produced only C. hominis sequences during MLST, including the specimen
Table 1 Identification of Cryptosporidium hominis co-infection at five genetic loci in seven of 62 Peruvians previously identified as positive for Cryptosporidium meleagridis based on PCRrestriction fragment length polymorphism and DNA sequence analyses of the ssrRNA gene of Cryptosporidium spp. Specimen ID
Patient source
3928 4500 4508 5144 9887 9890 9912
HIV+ adult HIV+ adult HIV+ adult HIV+ adult Child Child Child
Cryptosporidium species identified at the genetic locus ssrRNA
gp60
CP47
MSC6-5
RPGR
TSP8
C. C. C. C. C. C. C.
C. C. C. C. C. C. C.
Negative C. hominis Negative C. hominis C. hominis C. hominis C. hominis
Negative C. hominis C. hominis C. hominis C. hominis C. hominis C. hominis
C. C. C. C. C. C. C.
C. hominis C. hominis Negative C. hominis C. hominis C. hominis C. hominis
meleagridis meleagridis meleagridis meleagridis and C. hominis meleagridis meleagridis meleagridis
hominis hominis hominis hominis hominis hominis hominis
hominis hominis hominis hominis hominis hominis hominis
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Y. Wang et al. / International Journal for Parasitology xxx (2014) xxx–xxx 86
IIIg-JX878614
A
HIV+
80
54 100
0.05 substitutions/site 100
9890-Child-IdA10
100
IIIe-AB539721 IIIa-AF401499 Child
IIIbA24G1
7027, 7047, 7364
IIIbA26G1
2567, 4509, 4517, 4523, 4528, 5714, 5765, 5776, 6484, 6494, 7020, 7043, 7047, 7051, 7053, 7342, 7349, 7424, 7784, 7797, 7801
IIIbA2&G1
5745, 7352, 7355
2713, 2731, 2805, 3252, 9528, 9636, 9917, 10188, 11737, 11749, 11756
Bird 11286, 11308, 11354
11208, 13099
13094
7085-HIV+-IIIbA15G1 9508-Child-IIIbA15G1 7359-HIV+-IIIbA15G1 9633-Child-IIIbA34G1 10173-Child-IIIbA30G1 11371-Bird-IIIbA15G1 2556-HIV+-IIIbA13G1 IIId-DQ067570 100 2815-Child-IIIcA6 100 6536-HIV+-IIIcA6 1021-Child-IIIcA6 1227-Child-IIIcA6 2731-Child-MLG4 2713-Child-MLG4 5776-HIV+-MLG4 7797-HIV+-MLG4 2567-HIV+-MLG4 7053-HIV+-MLG4 10188-Child-MLG4 4523-HIV+-MLG4 6494-HIV+-MLG4 5714-HIV+-MLG4 7784-HIV+-MLG$ 3252-Child-MLG4 9636-Child-MLG4 9528-Child-MLG4 2805-Child-MLG4 4517-HIV+-MLG4 85 7020-HIV+-MLG4 5765-HIV+-MLG4 4528-HIV+-MLG4 84 7342-HIV+-MLG4 7801-HIV+-MLG6 9633-Child-MLG11 57 7364-HIV+-MLG3 57 7355-HIV+-MLG9 58 67 5745-HIV+-MLG8 7352-HIV+-MLG8 13094-Bird-MLG8 7349-HIV+-MLG5 6484-HIV+-MLG5 100 7051-HIV+-MLG5 7043-HIV+-MLG5 11749-Child-MLG5 4509-HIV+-MLG5 7424-HIV+-MLG5 11756-Child-MLG5 11737-Child-MLG5 9917-Child-MLG7 74 10173-Child-MLG10 7359-HIV+-MLG12 9508-Child-MLG12 94 61 7085-HIV+-MLG12 7027-HIV+-MLG1 89 11308-Bird-MLG1 59 11354-Bird-MLG1 7047-HIV+-MLG2 67 2556-HIV+-MLG14 67 11371-Bird-MLG13 1021-Child-MLG15 100 6536-HIV+-MLG16 62 1227-Child-MLG16
B
0.02 substitutions/site
2
C 13
1 12
3 8 5
9
4 11
7 6 10 Fig. 1. Genetic relationship of Cryptosporidium meleagridis isolates. (A) Relationship among subtypes at the 60 kDa glycoprotein (gp60) locus as inferred by a neighbor-joining analysis of nucleotide sequences. (B) Relationship among multilocus sequence types as inferred by neighbor-joining analysis. (C) Patterns of evolutionary descents among multilocus sequence subtypes based on eBURST analysis of allelic data. The primary founder is shown as the smaller open circle and subgroup founders are shown as the larger open circle. The sizes of dots and circles are proportional to the number of specimens of the sequence type. Two multilocus sequence types of the gp60 subtype IIIcA6 and one of the gp60 IIIbA13G1 subtype did not cluster within this diagram.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Y. Wang et al. / International Journal for Parasitology xxx (2014) xxx–xxx Table 2 Frequency and allele composition of multilocus sequence types of Cryptosporidium meleagridis in 50 human and bird specimens with sequence data at all five genetic loci. MLST
Distribution
Allele composition
ID
Count
Frequency
Children
HIV+ adult
Birds
gp60
CP47
MSC6-5
RPGR
TSP8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3 1 1 20 9 1 1 3 1 1 1 3 1 1 1 2
0.06 0.02 0.02 0.40 0.18 0.02 0.02 0.06 0.02 0.02 0.02 0.06 0.02 0.02 0.02 0.04
0 0 0 7 3 0 1 0 0 1 1 1 0 0 1 1
1 1 1 13 6 1 0 2 1 0 0 2 0 1 0 1
2 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0
1 1 1 2 2 2 2 3 3 4 5 6 6 7 8 8
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 2 1 1 1 2 1 1 1 1 1 1 2 2 3 3
1 1 2 2 1 2 1 2 2 1 2 1 1 1 3 3
1 1 1 1 1 1 2 1 2 1 1 1 1 2 1 2
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Table 3 Gene diversity in 55 Cryptosporidium meleagridis specimens based on intragenic analysis of five polymorphic loci and concatenated multilocus gene sequences. Locus
No. of Haplotype Intralocus genetic 95% CI (ZnS) No. of No. of No. of polymorphic segregating haplotype diversity (Hd) association (ZnS) site site site (bp)
Intragenic linkage disequilibrium (LD, |D0 |)
Recombination events (Rms)
CP47 gp60 MSC6-5 RPGR TSP8 Concatenated sequence including gp60a Concatenated sequence excluding gp60a
392 721 425 380 327 1853
0 323 33 40 1 397
0 76 0 4 1 81
1 8 3 3 2 18
0 0.651 0.242 0.556 0.198 0.829
– 1 – 0.454 – 0.913
– 0.05557–0.40285 – 0.00188–0.40285 – 0.05599–0.42813
– Y = 1.0000 + 0.0000X – Y = 1.0000 + 0.0000X – Y = 1.0178–0.1110X
– 0 –
1132
74
5
9
0.656
0.301
0.00292–0.62149 Y = 1.0515–1.4573X
– 1 1
gp60, 60 kDa glycoprotein; CP47, 47 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8; CI, confidence interval; |D0 |, linkage disequilibrium (LD), where Y is the LD value, and X is the nucleotide distance in kb. a From 50 specimens with sequence data at all five loci.
3.5. MLS subtypes The 50 C. meleagridis specimens with sequence information available at all loci produced a total of 16 MLS subtypes (Table 2). One dominant subtype consisted of 40.0% of the typed specimens. A second subtype was found in 18.0%. Three other subtypes were found in 6.0% and one subtype in 4.0%. The remaining 10 multilocus subtypes were each found in one specimen (2.0%). The frequency and allelic composition of the multilocus subtypes are shown in Table 2. Among the 16 MLS subtypes, 14 belonged to ssrRNA type 1 and two belonged to ssrRNA type 2. There were no significant changes in the distribution of MLS subtypes within type 1 C. meleagridis during the study period.
with 81 polymorphic sites. As expected, results of analysis showed strong LD among all polymorphic sites. A recombination analysis observed only one minimum Rm and an estimate of R per gene of 0.001 (Table 3). In analysis of allelic data, highly significant (Markov chain parameters significance, P < 0.05) intergenic LD was seen in all pairs among gp60, MSC6–5, RPGR and TSP8 (Table 4). Multilocus LD was further assessed by calculating ISA between alleles for all pair-wise combinations of all polymorphic loci, the variance of pair-wise differences (VD), and the 95% critical value for VD (L). In this analysis of allelic data of 55 specimens at all genetic loci, the value of ISA was positive (0.2390) and VD (1.4154) was more than L (0.9505), both indicative of the existence of LD and a non-panmictic structure. A Monte Carlo analysis was further used to test the
3.6. LD and population structure Population structure was assessed by LD among polymorphic sites within each locus and between pairs of polymorphic loci. Intragenic LD based on nucleotide sequences was measured as the non-random association of adjacent nucleotides sites along the DNA sequences. Intragenic LD was strong and complete at all genetic loci with three or more segregation sites such as gp60 and RPGR (Table 3). Recombination analysis using DnaSP programs revealed no recombination events (Rm) at these loci. We also analysed LD in the concatenated multilocus sequences of 1853 bp long
Table 4 Intergenic linkage disequilibrium among genetic loci for Cryptosporidium meleagridis (Markov chain parameters significance, P < 0.05).
GP60 MSC6-5 RPGR TSP8
GP60
MSC6–5
RPGR
TSP8
– 0.00001 0.00000 0.00001
+ 0.00000 0.00012
+ + 0.00177
+ + +
GP60, 60 kDa glycoprotein; MSC6-5, serine repeat antigen; RPGR retinitis pigmentosa GTPase regulator; TSP8, thrombospondin protein 8.
Please cite this article in press as: Wang, Y., et al. Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.03.003
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Table 5 Linkage disequilibrium analysis based on allelic profile data for Cryptosporidium meleagridis. Population group
Number
H
ISA
PMC
VD
L
VD > L
LD or LE
gp60 gp60 gp60 gp60
55 23 55 23
0.4463 ± 0.1170 0.4463 ± 0.1170 0.3737 ± 0.1297 0.5468 ± 0.1084
0.2390 0.0609 0.2646 0.0964
