Cryptosporidium canis is an important cause of cryptosporidiosis in canines and humans. Studies of the transmission characteristics of C. canis are currently hampered by the lack of suitable subtyping tools.
KEYWORDS: Cryptosporidium canis, whole-genome sequence, 60-kDa glycoprotein gene, subtyping, zoonotic transmission
ABSTRACT
Cryptosporidium canis is an important cause of cryptosporidiosis in canines and humans. Studies of the transmission characteristics of C. canis are currently hampered by the lack of suitable subtyping tools. In this study, we conducted a genomic survey of the pathogen and developed a subtyping tool targeting the partial 60-kDa glycoprotein gene (gp60). Seventy-six isolates previously identified as C. canis were analyzed using the new subtyping tool. Amplicons of the expected size were obtained from 49 isolates, and phylogenetic analysis identified 10 subtypes clustered into five distinct groups (XXa to XXe). The largest group, XXa, contained 43 isolates from four subtypes that differed slightly from each other at the nucleotide level, while groups XXb to XXe contain one to three isolates each. The similar distributions of subtypes in humans and canines suggest that zoonotic transmission might play an important role in the epidemiology of C. canis. In addition, suspected zoonotic transmission of C. canis between dogs and humans in a household was confirmed using the subtyping tool. The subtyping tool and data generated in this study might improve our understanding of the transmission of this zoonotic pathogen.
INTRODUCTION
Cryptosporidium spp. are common enteric pathogens of humans and various animals (1). To date, molecular characterization has identified over 20 Cryptosporidium species and genotypes in humans (2), including Cryptosporidium canis. Over 100 confirmed human C. canis infections have been reported, mostly in people in developing countries or those who have traveled to developing countries (3–9). In addition, C. canis is commonly found in dogs, coyotes, foxes, raccoon dogs, and minks (10–12). Possible direct transmission of C. canis from canines to humans has been described (13).
Subtyping tools targeting the 60-kDa glycoprotein (GP60) gene (gp60) have substantially enhanced our understanding of the transmission of Cryptosporidium hominis and C. parvum (14). Such subtyping tools have been developed for other human-pathogenic Cryptosporidium species (15–18). The application of these subtyping tools for molecular characterization of isolates has revealed the occurrence of host adaptation in some of the zoonotic Cryptosporidium spp. (14).
Due to the lack of genomic data, the gp60 gene of C. canis has not been identified yet, and gp60-based subtyping tools are not available for C. canis. In this study, we conducted a whole-genome survey of C. canis and developed a subtyping tool for this zoonotic pathogen based on the gp60 gene sequence identified.
MATERIALS AND METHODS
DNA preparations.
DNA extracts from 76 C. canis-positive convenience samples were included in this study, including 50 from humans, 17 from dogs, 7 from foxes, 1 from a coyote, and 1 from a beaver. Human samples were collected from the United States, Jamaica, Peru, Kenya, Ethiopia, and China, while animal samples were collected from the United States, Peru, and China (Table 1), all from previous studies (13, 19–23). Among them, there were three samples from a suspected family cluster of C. canis infections, including one sample each from a girl, her sibling, and their dog, in a Peruvian household (samples 10405, 10185, and 10200, respectively). Both children had transient diarrhea, and the dog was asymptomatic (13). These samples were determined to be positive for C. canis by PCR and sequence analysis of the small-subunit (SSU) rRNA gene as previously described (24).
TABLE 1.
Cryptosporidium canis samples used in this study and their subtype identities
| Sample ID | Host | Source location | Subtypea |
|---|---|---|---|
| 1080 | Human | Peru | XXa4 |
| 3236 | Human | Peru | XXa4 |
| 4495 | Human | Peru | XXa3 |
| 4540 | Human | Peru | XXa4 |
| 4572 | Human | Peru | XXa4 |
| 13023 | Human | Peru | NA |
| 19248 | Human | Peru | XXa4 |
| 35388 | Human | Ethiopia | XXa4 |
| 36061 | Human | Ethiopia | XXa4 |
| 3216 | Human | USA | NA |
| 44220 | Human | USA | NA |
| 25090 | Human | Nigeria | NA |
| 37139 | Human | Kenya | XXc1 |
| 2534 | Human (HIV) | Peru | XXa4 |
| 2847 | Human (HIV) | Peru | XXa4 |
| 3850 | Human (HIV) | Peru | XXa1 |
| 4173 | Human (HIV) | Peru | XXa1 |
| 4192 | Human (HIV) | Peru | NA |
| 6363 | Human (HIV) | Peru | XXa1 |
| 6393 | Human (HIV) | Peru | XXa4 |
| 6401 | Human (HIV) | Peru | XXa4 |
| 6420 | Human (HIV) | Peru | NA |
| 6421 | Human (HIV) | Peru | NA |
| 7348 | Human (HIV) | Peru | XXa4 |
| 7363 | Human (HIV) | Peru | XXa4 |
| 8194 | Human (HIV) | Peru | NA |
| 8297 | Human (HIV) | Peru | NA |
| 8298 | Human (HIV) | Peru | XXa4 |
| 8299 | Human (HIV) | Peru | XXa4 |
| 8300 | Human (HIV) | Peru | XXa4 |
| 8301 | Human (HIV) | Peru | XXa4 |
| 8302 | Human (HIV) | Peru | XXa4 |
| 19247 | Human (HIV) | Peru | NA |
| 19252 | Human (HIV) | Peru | XXa4 |
| 19262 | Human (HIV) | Peru | XXa4 |
| 19267 | Human (HIV) | Peru | XXa4 |
| 19272 | Human (HIV) | Peru | XXa4 |
| 9639 | Human (HIV) | Jamaica | XXa2 |
| 9649 | Human (HIV) | Jamaica | XXa2 |
| 2714 | Human (child) | Peru | XXa4 |
| 9502 | Human (child) | Peru | NA |
| 9624 | Human (child) | Peru | XXa4 |
| 10185 | Human (child) | Peru | XXa4 |
| 10187 | Human (child) | Peru | XXa4 |
| 10191 | Human (child) | Peru | XXa4 |
| 10405 | Human (child) | Peru | XXa4 |
| 9544 | Human (child) | Kenya | NA |
| 9566 | Human (child) | Kenya | XXa3 |
| 10964 | Human (child) | Kenya | XXa3 |
| 26117 | Human (child) | China | XXb2 |
| 105 | Dog | USA | XXa4 |
| 715 | Dog | USA | XXe1 |
| 3217 | Dog | USA | NA |
| 42721 | Dog | USA | NA |
| 45460 | Dog | USA | XXd1 |
| 9875 | Dog | Peru | XXa1 |
| 9876 | Dog | Peru | XXa1 |
| 9883 | Dog | Peru | XXa4 |
| 9884 | Dog | Peru | NA |
| 9942 | Dog | Peru | XXa4 |
| 9943 | Dog | Peru | NA |
| 9944 | Dog | Peru | XXa4 |
| 9945 | Dog | Peru | NA |
| 9947 | Dog | Peru | NA |
| 10200 | Dog | Peru | XXa4 |
| 39706 | Dog | Kenya | NA |
| 45572 | Dog | China | XXb1 |
| 5035 | Fox | USA | NA |
| 5073 | Fox | USA | NA |
| 5074 | Fox | USA | NA |
| 5528 | Fox | USA | NA |
| 5977 | Fox | USA | XXb3 |
| 5978 | Fox | USA | NA |
| 6008 | Fox | USA | NA |
| 2011 | Coyote | USA | NA |
| 6009 | Beaver | USA | NA |
NA, not applicable (PCR negative).
Whole-genome sequencing of C. canis.
To identify the gp60 gene of C. canis, the genome of a canine-derived isolate, 45460, from Tennessee was sequenced. Oocysts were isolated from the stool sample by immunomagnetic separation. Genomic DNA was extracted and amplified as previously described (25). The amplified genomic DNA was sequenced on an Illumina genome HiSeq 2500 instrument (Illumina, San Diego, CA). An estimated 200-fold coverage was achieved using 250-bp paired-end sequencing. The genome was assembled from the raw sequence reads using CLC Genomics Workbench 11.0 (Qiagen) with a word size of 63 and a bubble size of 500, as previously described (25). The contigs generated were aligned with the published genome of C. parvum IOWA using Mauve (http://darlinglab.org/mauve/) to identify the ortholog of the gp60 gene (cgd6_1080) in C. canis.
Subtyping method.
Primers for a nested PCR assay were designed based on the partial coding region of the gp60 gene derived from the C. canis genome with expected primary and secondary PCR products of approximately 750 bp and 700 bp, respectively. The primers used were GP60-Canis-F1 (5′-ATA CTC TGG TCT CCC GTT T-3′) and GP60-Canis-R1 (5′-GTA CTC GGA AGC GGT GTA-3′) for primary PCR and GP60-Canis-F2 (5′-AAG GCG CCT CAC TCA TT-3′) and GP60-Canis-R2 (5′-TCA GTT AGA TAT CAC CCA TTA A-3′) for secondary PCR. The volume of both PCR mixtures was 50 μl, containing 1 μl of the DNA template (for primary PCR) or 2 μl of the primary PCR product (for secondary PCR), 250 nM primary PCR primers or 500 nM secondary PCR primers, 2.5 mM MgCl2, 200 μM deoxynucleotide triphosphates, 1× PCR buffer (15 mM Tris-HCl, 50 mM KCl, and 2.5 mM MgCl2 [pH 8.0]), and 1.5 U Taq polymerase. To reduce PCR inhibition, 400 ng/μl of nonacetylated bovine serum albumin was used for the primary PCR. Amplification was performed on a GeneAmp PCR 9700 thermocycler (Applied Biosystems, Foster City, CA, USA), consisting of an initial denaturation step at 94°C for 5 min; 35 cycles of 94°C for 45 s, 52°C for 45 s, and 72°C for 80 s; and a final extension step at 72°C for 10 min. Both positive (DNA from a Peruvian C. canis sample) and negative (molecular-grade water) controls were used in each PCR run. The secondary PCR products were visualized under UV light after 1.5% agarose gel electrophoresis.
Sequence analysis.
All positive products of the secondary gp60 PCR were sequenced in both directions on an ABI 3130xl genetic analyzer (Applied Biosystems). The sequences were assembled using ChromasPro 2.1.6 (Technelysium), cleaned using BioEdit 7.0.5.3 (https://bioedit.software.informer.com/7.0/), and aligned using MUSCLE implemented in MEGA 10.0.5 (www.megasoftware.net/). Signal peptides and a glycosylphosphatidylinositol (GPI) anchor in the protein sequences were predicted using GPI-SOM (http://gpi.unibe.ch/). Potential furin cleavage sites in sequences were predicted using the ProP 1.0 server (http://www.cbs.dtu.dk/services/ProP/). N-glycosylated sites and O-glycosylated sites were predicted using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) and YinOYang 1.2 (http://www.cbs.dtu.dk/services/YinOYang/), respectively. To analyze the phylogenetic relationship among various subtype families of C. canis, a maximum likelihood tree was constructed using MEGA 10.0.5 based on substitution rates calculated with the general time-reversible model and gamma distribution with invariant sites (G+I). The reliability of cluster formation was evaluated using the bootstrap method with 1,000 replicates.
Data availability.
Representative nucleotide sequences derived from this study were deposited in GenBank under accession numbers MT954604 to MT954613.
RESULTS
Genomic sequences and identification of the gp60 gene.
After de novo assembly, 11,619,831 bp in 2,446 contigs with an N50 of 76,644 bp and a GC content of 40.43% were obtained from isolate 45460. The larger-than-expected size and poor assembly statistics of this genome were likely caused by contaminating bacteria. The assembled contigs were aligned with the C. parvum IOWA genome using Mauve. A manual inspection of the sequence alignment led to the identification of the ortholog of the partial cgd_1080 (gp60) gene in contig 2334. The partial gp60 gene of C. canis retrieved from this contig contained 945 bp without the 5′-end sequence (Fig. 1).
FIG 1.
Identification of the gp60 (cgd_1080) gene of Cryptosporidium canis in the Mauve alignment of its whole-genome sequence with the assembled and annotated genome of the Cryptosporidium parvum IOWA isolate. A partial gp60 gene of C. canis was identified in contig 2334, with its 5′ end missing.
The C. canis sequences obtained shared ∼32% sequence identity with the gp60 genes of C. hominis, C. parvum, and C. fayeri at the amino acid level and 90 to 97% query coverage. As no start codon and signal peptide could be found at the 5′ end, the full length of the C. canis gp60 gene could not be determined. There were 33 O-linked glycosylation sites in the sequence obtained but no N-linked glycosylation sites. A putative furin cleavage site, “RSRR,” was found in the middle of the C. canis GP60 protein sequence, and a GPI anchor site was found at the C terminus. In addition, insertions of 51 and 33 amino acids were observed upstream and downstream of the furin cleavage site, respectively (Fig. 2).
FIG 2.
Alignment of the partial GP60 protein sequence of Cryptosporidium canis with those of C. parvum (GenBank accession no. AFF78281), C. hominis (GenBank accession no. ACQ82748), C. meleagridis (GenBank accession no. AHW45799), Cryptosporidium chipmunk genotype I (GenBank accession no. AJW72317), and C. ubiquitum (GenBank accession no. ALH06585). The signal peptides in amino acid sequences are shaded in green, the GPI anchor is in red, and the putative furin cleavage sites are in yellow. Potential N-linked and O-linked glycosylation sites are indicated in boldface type with italics and underlining, respectively. Unique sequence insertions in C. canis are highlighted in red. Dashes denote amino acid deletions.
Efficiency of amplification of the gp60 gene.
PCR products of the expected size were obtained from 49 of the 76 C. canis DNA preparations, including 38 from humans, 10 from dogs, and 1 from a fox. Among the subtyped isolates, 36 were from Peru, 4 were from the United States, 3 were from Kenya, and 2 each were from Jamaica, Ethiopia, and China (Table 1). With the exception of a beaver sample, PCR failures mainly occurred in canine samples, especially those from foxes.
Sequence characteristics of the C. canis gp60 gene.
Ten types of sequences were obtained from the PCR products. Multiple-sequence alignment showed the presence of five major groups of sequences that differed from each other by length and single nucleotide substitutions (single nucleotide polymorphisms [SNPs]). Within the largest sequence group, 43 isolates had nearly identical sequences. As the PCR targeted only the partial gene without the 5′ end, the typical trinucleotide repeats TCA/TCG/TGT seen in the gp60 gene of many Cryptosporidium spp. were not observed in the obtained sequences.
Phylogenetic relationship among C. canis subtypes.
In the maximum likelihood tree, the 49 nucleotide sequences formed five clusters, as expected. They were named the XXa, XXb, XXc, XXd, and XXe subtype families in concordance with the established nomenclature for Cryptosporidium subtype families (14). Within major subtype family XXa, the 43 sequences generated 4 subtypes, named XXa1 (n = 5), XXa2 (n = 2), XXa3 (n = 3), and XXa4 (n = 33). Similarly, subtype family XXb, which was genetically related to subtype family XXa, contained 3 subtypes, XXb1 (n = 1), XXb2 (n = 1), and XXb3 (n = 1) (Fig. 3). In contrast, subtype families XXc, XXd, and XXe contained only one sequence each. Phylogenetically, subtype families XXd and XXe were distant from the other three subtype families, with numerous SNPs.
FIG 3.
Phylogenetic relationship among five Cryptosporidium canis subtype families (XXa to XXe) revealed by maximum likelihood analysis of the partial gp60 gene. Substitution rates were calculated by using the general time-reversible model. Numbers on branches are percent bootstrapping values over 50% using 1,000 replicates. Round and square labels indicate samples from humans and animals, respectively.
Distribution of C. canis subtypes by host and country.
Based on the phylogenetic analysis, human samples were observed in subtype families XXa (n = 38), XXb (n = 1), and XXc (n = 1), while canine samples were observed in subtype families XXa (n = 7), XXb (n = 2), XXd (n = 1), and XXe (n = 1). The one sequence obtained from a fox was identified as a unique subtype, XXb3 (Table 2).
TABLE 2.
Distribution of Cryptosporidium canis subtypes by country and host
| Subtype | No. of isolates | Country(ies) (no. of isolates) | Host(s) (no. of isolates) |
|---|---|---|---|
| XXa1 | 5 | Peru (5) | Human (3), dog (2) |
| XXa2 | 2 | Jamaica (2) | Human (2) |
| XXa3 | 3 | Peru (1), Kenya (2) | Human (3) |
| XXa4 | 33 | Peru (30), Ethiopia (2), USA (1) | Human (28), dog (5) |
| XXb1 | 1 | China (1) | Dog (1) |
| XXb2 | 1 | China (1) | Human (1) |
| XXb3 | 1 | USA (1) | Fox (1) |
| XXc1 | 1 | Kenya (1) | Human (1) |
| XXd1 | 1 | USA (1) | Dog (1) |
| XXe1 | 1 | USA (1) | Dog (1) |
Among the 10 C. canis subtypes, 4 were detected in the United States, including XXa4 (n = 1), XXb3 (n = 1), XXd1 (n = 1), and XXe1 (n = 1); 3 were detected in Peru, including XXa1 (n = 5), XXa3 (n = 1), and XXa4 (n = 30); 2 were detected in Kenya, including XXa4 (n = 2) and XXc1 (n = 1); 2 were detected in China, including XXb1 (n = 1) and XXb2 (n = 1); and 1 each was detected in Jamaica (XXa2) (n = 2) and Ethiopia (XXa4) (n = 2) (Table 2).
Household transmission of a C. canis subtype.
The possible transmission of C. canis among humans and a dog in a household previously described in Peru was confirmed by subtyping. Sequences generated from isolates 10405, 10185, and 10200, which were from the index case, her sibling, and the household dog, were identical to each other and identified as subtype XXa4.
DISCUSSION
Although C. canis has been widely reported in humans and canines, its genetic diversity and transmission dynamics remain largely unclear due to the lack of subtyping tools (26, 27). In the present study, we conducted a genomic survey of C. canis to identify its gp60 gene. Based on the partial gene sequence obtained, we developed a subtyping tool to assess its genetic diversity. The application of this tool in the analyses of isolates from humans and animals confirmed the occurrence of zoonotic transmission of this pathogen.
In this study, the gp60 gene of C. canis was identified for the first time. Although only a partial sequence was obtained, results of the amino acid sequence analysis indicate that the GP60 protein of C. canis shares some of the typical sequence characteristics of GP60 proteins of other Cryptosporidium spp., with numerous O-linked glycosylation sites, a furin cleavage site, and a C-terminal GPI anchor. As the 5′ region of the C. canis gp60 gene was absent in our genomic data, the signal peptide and serine repeat region were absent in the sequence that we obtained.
The GP60 sequences of C. canis, however, differ from those of other Cryptosporidium spp., notably in length. Although all GP60 sequences are highly conserved in the GP15 and furin cleavage regions, there are long amino acid inserts in the sequence from C. canis upstream and downstream of the furin cleavage site, which contains most of the O-linked glycosylation sites of the protein. As the GP60 protein is involved in the invasion and attachment phases of the Cryptosporidium life cycle (28, 29), such long sequence insertions and major sequence differences could contribute to the canine-adapted nature of C. canis.
Based on the identified gp60 gene, we developed a subtyping tool for C. canis for the first time. Subtyping data generated from 49 PCR-positive samples identified 10 subtypes clustered into five subtype families, indicating that C. canis has abundant genetic diversity, as with other Cryptosporidium spp. Likely due to the genetic diversity in the primer region, PCR failures in this study mainly occurred in the analysis of canine samples, especially those from foxes. This supports the existence of genetic differences among C. canis isolates from different hosts. Therefore, PCR primers targeting a more conserved region are needed to increase the amplification efficiency and to better understand the genetic diversity and subtypes of C. canis. As the majority of isolates (43 of 49) analyzed in this study belong to subtype family XXa, humans and dogs could be preferentially infected with this group of C. canis isolates. We found this subtype family in extracts of isolates from five countries on three continents. In contrast, subtype families XXb to XXe had only 1 to 3 isolates, indicating that they might be biologically or epidemiologically different.
The inclusion of both human and canine isolates in the largest subtype family, XXa, reinforces the possibility of zoonotic transmission of C. canis, which is further supported by the fact that both subtypes XXa1 and XXa4 have been found in dogs as well as humans. Additionally, our data confirmed the occurrence of zoonotic transmission of C. canis in a household (13). The gp60 sequences obtained from the index case, her sibling, and their dog all belong to subtype XXa4.
In conclusion, we conducted a genomic survey of C. canis and developed a gp60-based subtyping tool for the first time. The genetic similarity among 49 human and canine isolates at the subtype level suggests that zoonotic transmission plays an important role in the epidemiology of C. canis. The representativeness of the findings of this study elsewhere, however, cannot be inferred from this study. In addition, more sequence data are needed to substantiate the limited distribution of subtype families XXc to XXe and demonstrate their biological uniqueness.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (31820103014) and the 111 Project (D20008).
Findings and conclusions are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention.
REFERENCES
- 1.Checkley W, White AC, Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen X-M, Fayer R, Griffiths JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA, Jr, Priest JW, Roos DS, Striepen B, Thompson RCA, Ward HD, Van Voorhis WA, Xiao L, Zhu G, Houpt ER. 2015. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis 15:85–94. doi: 10.1016/S1473-3099(14)70772-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Feng Y, Ryan U, Xiao L. 2018. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol 34:997–1011. doi: 10.1016/j.pt.2018.07.009. [DOI] [PubMed] [Google Scholar]
- 3.Hijjawi N, Ng J, Yang R, Atoum MF, Ryan U. 2010. Identification of rare and novel Cryptosporidium GP60 subtypes in human isolates from Jordan. Exp Parasitol 125:161–164. doi: 10.1016/j.exppara.2010.01.011. [DOI] [PubMed] [Google Scholar]
- 4.Xiao L. 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124:80–89. doi: 10.1016/j.exppara.2009.03.018. [DOI] [PubMed] [Google Scholar]
- 5.Lucio-Forster A, Griffiths JK, Cama VA, Xiao L, Bowman DD. 2010. Minimal zoonotic risk of cryptosporidiosis from pet dogs and cats. Trends Parasitol 26:174–179. doi: 10.1016/j.pt.2010.01.004. [DOI] [PubMed] [Google Scholar]
- 6.Xiao L, Feng Y. 2008. Zoonotic cryptosporidiosis. FEMS Immunol Med Microbiol 52:309–323. doi: 10.1111/j.1574-695X.2008.00377.x. [DOI] [PubMed] [Google Scholar]
- 7.Chalmers RM, Elwin K, Thomas AL, Guy EC, Mason B. 2009. Long-term Cryptosporidium typing reveals the aetiology and species-specific epidemiology of human cryptosporidiosis in England and Wales, 2000 to 2003. Euro Surveill 14:19086. doi: 10.2807/ese.14.02.19086-en. [DOI] [PubMed] [Google Scholar]
- 8.Elwin K, Hadfield SJ, Robinson G, Chalmers RM. 2012. The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000–2008. Epidemiol Infect 140:673–683. doi: 10.1017/S0950268811000860. [DOI] [PubMed] [Google Scholar]
- 9.Gonzalez-Diaz M, Urrea-Quezada A, Villegas-Gomez I, Durazo M, Garibay-Escobar A, Hernandez J, Xiao L, Valenzuela O. 2016. Cryptosporidium canis in two Mexican toddlers. Pediatr Infect Dis J 35:1265–1266. doi: 10.1097/INF.0000000000001287. [DOI] [PubMed] [Google Scholar]
- 10.Zhang X-X, Cong W, Ma J-G, Lou Z-L, Zheng W-B, Zhao Q, Zhu X-Q. 2016. First report of Cryptosporidium canis in farmed Arctic foxes (Vulpes lagopus) in China. Parasit Vectors 9:126. doi: 10.1186/s13071-016-1396-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Zhang S, Tao W, Liu C, Jiang Y, Wan Q, Li Q, Yang H, Lin Y, Li W. 2016. First report of Cryptosporidium canis in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) and identification of several novel subtype families for Cryptosporidium mink genotype in minks (Mustela vison) in China. Infect Genet Evol 41:21–25. doi: 10.1016/j.meegid.2016.03.016. [DOI] [PubMed] [Google Scholar]
- 12.Trout JM, Santin M, Fayer R. 2006. Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans). J Zoo Wildl Med 37:141–144. doi: 10.1638/05-06TYM-123005.1. [DOI] [PubMed] [Google Scholar]
- 13.Xiao L, Cama VA, Cabrera L, Ortega Y, Pearson J, Gilman RH. 2007. Possible transmission of Cryptosporidium canis among children and a dog in a household. J Clin Microbiol 45:2014–2016. doi: 10.1128/JCM.00503-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Xiao L, Feng Y. 2017. Molecular epidemiologic tools for waterborne pathogens Cryptosporidium spp. and Giardia duodenalis. Food Waterborne Parasitol 8–9:14–32. doi: 10.1016/j.fawpar.2017.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Guo Y, Cebelinski E, Matusevich C, Alderisio KA, Lebbad M, McEvoy J, Roellig DM, Yang C, Feng Y, Xiao L. 2015. Subtyping novel zoonotic pathogen Cryptosporidium chipmunk genotype I. J Clin Microbiol 53:1648–1654. doi: 10.1128/JCM.03436-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, Santin M, Fayer R, Kvac M, Ryan U, Sak B, Stanko M, Guo Y, Wang L, Zhang L, Cai J, Roellig D, Feng Y. 2014. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg Infect Dis 20:217–224. doi: 10.3201/eid2002.121797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Rojas-Lopez L, Elwin K, Chalmers RM, Enemark HL, Beser J, Troell K. 2020. Development of a gp60-subtyping method for Cryptosporidium felis. Parasit Vectors 13:39. doi: 10.1186/s13071-020-3906-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Stensvold CR, Beser J, Axen C, Lebbad M. 2014. High applicability of a novel method for gp60-based subtyping of Cryptosporidium meleagridis. J Clin Microbiol 52:2311–2319. doi: 10.1128/JCM.00598-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Adamu H, Petros B, Zhang G, Kassa H, Amer S, Ye J, Feng Y, Xiao L. 2014. Distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Ethiopia. PLoS Negl Trop Dis 8:e2831. doi: 10.1371/journal.pntd.0002831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Cama VA, Bern C, Roberts J, Cabrera L, Sterling CR, Ortega Y, Gilman RH, Xiao L. 2008. Cryptosporidium species and subtypes and clinical manifestations in children, Peru. Emerg Infect Dis 14:1567–1574. doi: 10.3201/eid1410.071273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, Vivar A, Kawai V, Vargas D, Zhou L, Xiao L. 2003. Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. J Eukaryot Microbiol 50:531–533. doi: 10.1111/j.1550-7408.2003.tb00620.x. [DOI] [PubMed] [Google Scholar]
- 22.Gatei W, Wamae CN, Mbae C, Waruru A, Mulinge E, Waithera T, Gatika SM, Kamwati SK, Revathi G, Hart CA. 2006. Cryptosporidiosis: prevalence, genotype analysis, and symptoms associated with infections in children in Kenya. Am J Trop Med Hyg 75:78–82. doi: 10.4269/ajtmh.2006.75.78. [DOI] [PubMed] [Google Scholar]
- 23.Xu H, Jin Y, Wu W, Li P, Wang L, Li N, Feng Y, Xiao L. 2016. Genotypes of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in dogs and cats in Shanghai, China. Parasit Vectors 9:121. doi: 10.1186/s13071-016-1409-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Xiao L, Sulaiman IM, Ryan UM, Zhou L, Atwill ER, Tischler ML, Zhang X, Fayer R, Lal AA. 2002. Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. Int J Parasitol 32:1773–1785. doi: 10.1016/S0020-7519(02)00197-2. [DOI] [PubMed] [Google Scholar]
- 25.Guo Y, Li N, Lysen C, Frace M, Tang K, Sammons S, Roellig DM, Feng Y, Xiao L. 2015. Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing. J Clin Microbiol 53:641–647. doi: 10.1128/JCM.02962-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Taghipour A, Olfatifar M, Bahadory S, Godfrey SS, Abdoli A, Khatami A, Javanmard E, Shahrivar F. 2020. The global prevalence of Cryptosporidium infection in dogs: a systematic review and meta-analysis. Vet Parasitol 281:109093. doi: 10.1016/j.vetpar.2020.109093. [DOI] [PubMed] [Google Scholar]
- 27.Ryan U, Fayer R, Xiao L. 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141:1667–1685. doi: 10.1017/S0031182014001085. [DOI] [PubMed] [Google Scholar]
- 28.Boulter-Bitzer JI, Lee H, Trevors JT. 2007. Molecular targets for detection and immunotherapy in Cryptosporidium parvum. Biotechnol Adv 25:13–44. doi: 10.1016/j.biotechadv.2006.08.003. [DOI] [PubMed] [Google Scholar]
- 29.Cevallos AM, Zhang X, Waldor MK, Jaison S, Zhou X, Tzipori S, Neutra MR, Ward HD. 2000. Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infect Immun 68:4108–4116. doi: 10.1128/iai.68.7.4108-4116.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Representative nucleotide sequences derived from this study were deposited in GenBank under accession numbers MT954604 to MT954613.