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Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China
Experimental Parasitology 127 (2011) 42–45
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China Rongjun Wang a, Xiaosan Zhang a, Huili Zhu a, Longxian Zhang a,b,c,*, Yaoyu Feng d, Fuchun Jian a, Changshen Ning a, Meng Qi a, Yang Zhou a, Kanda Fu e, Yaqiang Wang e, Yanru Sun a, Qiang Wang a, Lihua Xiao c,** a
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Atlanta Research and Education Foundation, 1670 Clairmont Road, Decatur, GA 30033, USA Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA d School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China e Inspection Division of Huaihe Hospital, Henan University, Kaifeng 475000, China b c
a r t i c l e
i n f o
Article history: Received 24 March 2010 Received in revised form 10 June 2010 Accepted 28 June 2010 Available online 1 July 2010 Keywords: Cryptosporidium Giardia duodenalis Humans Genotyping Subtyping Cryptosporidium hominis Assemblage A Assemblage B
a b s t r a c t Cryptosporidium and Giardia infections are common causes of diarrhea worldwide. To better understand the transmission of human cryptosporidiosis and giardiasis in Henan, China, 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens were characterized at the species/genotype and subtype levels. Cryptosporidium specimens were analyzed by DNA sequencing of the small subunit rRNA and 60 kDa glycoprotein genes. Among those genotyped, nine belonged to C. hominis and one C. felis, with the former belonging to three subtype families: Ia, Ib, and Id. The three Ib subtypes identified, IbA16G2, IbA19G2, and IbA20G2, were very different from the two common Ib subtypes (IbA9G3 and IbA10G2) found in other areas of the world. The distribution of Giardia duodenalis genotypes and subtypes was assessed by sequence analysis of the triosephosphate isomerase (tpi) gene. The assemblages A (eight belonging to A-I and four A-II) and B (belonging to six new subtypes) were found in 12 and six specimens, respectively. More systematic studies are needed to understand the transmission of Cryptosporidium and G. duodenalis in humans in China. Published by Elsevier Inc.
1. Introduction Cryptosporidium and Giardia infections are significant causes of diarrhea in humans worldwide. The diarrhea caused by cryptosporidiosis may become profuse, chronic, or even life-threatening, particularly in immunocompromised persons. Both parasites have a broad range of hosts and can be transmitted by the fecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao et al., 2004; Smith et al., 2006). Using genotyping tools, it has been shown that eight Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. canis, C. felis, C. suis, C. muris, and C. andersoni) and at least six genotypes (cervine, skunk, chipmunk I, horse, rabbit genotype, and pig genotype II) can infect humans, with C. hominis and C. parvum responsible for
* Corresponding author at: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China. Fax: +86 371 63558180. ** Corresponding author. Fax: +1 (770) 488 4454. E-mail addresses: [email protected] (L. Zhang), [email protected] (L. Xiao). 0014-4894/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.exppara.2010.06.034
most clinical cases. The distribution of C. hominis and C. parvum in humans differs in geographic regions (Xiao, 2010). In addition, DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene has shown the complexity of Cryptosporidium transmission in endemic areas. Among the five common C. hominis subtype families, Ia, Ib, Id, Ie, and If, three to four subtype families were each seen in humans in India, Peru, New Orleans, Malawi, South Africa, Kuwait, and Portugal. In contrast, only one or two C. parvum subtype families (IIa and IIc) were seen in humans in the same areas (Alves et al., 2003, 2006; Peng et al., 2003; Sulaiman et al., 2005; Gatei et al., 2007). In giardiasis, only Giardia duodenalis (synonym G. lamblia or G. intestinalis) is recovered from humans and most other mammals (Adam, 2001). Isolates of G. duodenalis are classified into seven common ‘assemblages’ or genotypes: A–G, based on the characterization of the triosephosphate isomerase (tpi), small subunit (SSU) rRNA, b-giardin, glutamate dehydrogenase (gdh), and other genes (Thompson et al., 2000; Sulaiman et al., 2003; Read et al., 2004). Assemblages A and B infect humans and a broad range of other hosts, including livestock, cats, dogs, and wild mammals. The assemblage A is further divided into two major subassemblages, I
R. Wang et al. / Experimental Parasitology 127 (2011) 42–45
and II, and the less common III. Many subtypes are present in the assemblage B (Feng et al., 2008; Geurden et al., 2009; Mahdy et al., 2009). Assemblages C, D, E, F, and G appear to be mostly restricted to companion animals, livestock, and rodents (Monis et al., 2003; Sulaiman et al., 2003). In China, the number of molecular epidemiological studies of cryptosporidiosis and giardiasis in humans is small. Peng et al. characterized genetically five specimens (all belonged to C. hominis) from children in Tianjing, three specimens were characterized by gp60 sequence analysis and three subtypes (IbA23G2, IdA14, and IeA13G3T3) were identified (Peng et al., 2001). Four Cryptosporidium specimens from HIV-infected patients in Taiwan were identified as C. hominis (two cases), C. felis (one case), and C. meleagridis (one case) (Hung et al., 2007). A report by Yong et al. based on sequence analysis of the SSU rRNA gene of eight G. duodenalis specimens from Anhui Province showed the presence of assemblages A and B in four patients each (Yong et al., 2000). Another study by Lu et al. based on sequence analysis of the tpi gene of three specimens showed the presence of two assemblage B and one A-I mixed with assemblage B (Lu et al., 2002). Chen et al. reported that three G. duodenalis specimens from humans in Hebei Province belonged to subtype A-II (Chen et al., 2001). Therefore, the molecular epidemiology of human cryptosporidiosis and giardiasis in China is still unclear. In this study, to better understand the transmission of cryptosporidiosis and giardiasis, 10 Cryptosporidium specimens and 18 Giardia specimens from humans in Henan were genetically characterized.
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2.4. Giardia genotyping and subtyping G. duodenalis genotypes and subtypes were determined by DNA sequence analysis of the tpi gene. A nested PCR was used to amplify the partial tpi gene (530-bp) of Giardia specimens (Sulaiman et al., 2003). Sequencing of tpi gene was done as described below. Genotype and subtype identities of G. duodenalis were established by direct comparison of the acquired sequences with reference sequences. 2.5. DNA sequence analysis All PCR products were sequenced on an ABI PRISMTM 3730 XL DNA Analyzer (Applied Biosystems, USA), using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing a new PCR product if necessary. The SSU rRNA and gp60 sequences of Cryptosporidium and tpi sequences of Giardia obtained in this study were aligned with reference sequences downloaded from GenBank using the program ClustalX 1.83. Nucleotide sequences of representative subtypes have been deposited in the GenBank under Accession Nos. FJ707310–FJ707316 (Cryptosporidium) and GU564274–GU564284 (Giardia). 3. Results 3.1. Cryptosporidium genotypes and subtypes
2. Materials and methods 2.1. Stool specimens The 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens from humans were obtained from a survey conducted in hospitals of Zhengzhou, Kaifeng, and Linzhou in Henan Province between June 2007 and December 2008. All specimens were stored at 4 °C in 2.5% (w/v) potassium dichromate solution or frozen at 80 °C without preservatives prior to being used in molecular characterizations.
The 10 Cryptosporidium-positive specimens were successfully amplified at the SSU rRNA locus. RFLP analysis of the 10 PCR products suggested the presence of C. hominis in nine specimens and C. felis in one. This was confirmed by DNA sequence analysis of the PCR products. Cryptosporidium hominis was further subtyped by gp60 gene sequence analysis. Three subtype families were identified in the nine C. hominis-positive specimens: Ia (11.1%), Ib (66.7%), and Id (22.2%). Among subtype family Ib, three subtypes were found: IbA16G2 (in one case), IbA19G2 (in two cases), and IbA20G2 (in three cases). In contrast, only one subtype each was present in subtype families Ia (IaA9R3 in one case) and Id (IdA21 in two cases) (Table 1).
2.2. DNA extraction Genomic DNA was isolated from 200 lL of stool specimens using the QIAampÒ DNA stool kit (QIAGEN Inc., Valencia, CA) and the previously described procedures, after washing of specimens three times with distilled water by centrifugation at 1000g for 10 min, oocyst lysis by alkaline digestion, and crude DNA extraction by the phenol–chloroform method (Xiao et al., 2002). The extracted DNA was eluted in 200 lL of the AE elution buffer and stored at 20 °C. 2.3. Cryptosporidium genotyping and subtyping Cryptosporidium spp. were genotyped by PCR amplification of a 830-bp fragment of the small subunit (SSU) rRNA gene and restriction fragment length polymorphism (RFLP) analysis of the PCR products using restriction enzymes SspI and VspI (Xiao et al., 2001). To confirm the genotype identification, all PCR products were sequenced. Subtyping of C. hominis was done by DNA sequencing of the gp60 gene. A fragment of 855-bp of the gp60 gene was amplified by nested PCR (Alves et al., 2003). The previously established nomenclature system was used to determine subtype families and subtypes within each family (Sulaiman et al., 2005; Xiao, 2010).
3.2. Giardia genotypes and subtypes All 18 Giardia-positive specimens produced the expected tpi PCR products. Alignment of the tpi sequences obtained with reference sequences indicated the presence of two genotypes of G. duodenalis; 12 specimens were identified as G. duodenalis assemblage A and six as assemblage B (Table 2). Among the three cities examined in this study, Kaifeng appeared to have more assemblage B infections than Zhengzhou and Linzhou (P < 0.01 between Kaifeng and Linzhou areas by Chi-square analysis, Table 2). Of the 12 assemblage A specimens, eight produced sequences that were identical to the A-I (GenBank Accession No. AF069556), and the remaining four produced sequences identical to the A-II (GenBank Accession No. AF069557). In contrast, the six assemblage B specimens produced sequences that were not identical to any known assemblage B subtypes, thus representing six new subtypes, named B1–B6 for convenience (Table 2). 4. Discussion In this study, 90% of Cryptosporidium specimens were identified as C. hominis. Previously, five human Cryptosporidium isolates from Tianjing also belonged to C. hominis (Peng et al., 2001) and two of four Cryptosporidium-positive specimens from Taiwan had
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R. Wang et al. / Experimental Parasitology 127 (2011) 42–45 Table 1 Cryptosporidium genotypes and subtypes in 10 patients in Henan, China. Area
Sample
Linzhou
146 193
Kaifeng
ZL0821 LB-1 ZZH-1 WJH-35
Zhengzhou
ZYY-3 LFY-6 CHZF1 CHZF2
Age (years)
Sex
Diarrhea
Genotype/subtype
4 5
Female Female
No No
C. hominis/IbA20G2 C. felis
6 1 1 35
Male Female Male Male
No Yes Yes Yes
C. C. C. C.
hominis/IbA20G2 hominis/IaA9R3 hominis/IbA20G2 hominis/IbA19G2
3 6 2 1
Male Male Male Female
Yes Yes Yes Yes
C. C. C. C.
hominis/IbA19G2 hominis/IbA16G2 hominis/IdA21 hominis/IdA21
Table 2 Giardia assemblage/subtype in 18 patients in Henan, China.
a
Area
Sample
Sex
Diarrhea
Assemblage/subtype
Linzhou
LZ1 LZ2 LZ4 LZ5 LZ6 LZ8
Age (year) 5 7 5 5 6 6
Male Female Male Female Female Female
No No No No No No
Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-I A/A-I A/A-I A/A-II A/A-I A/A-I
Kaifeng
KF1 KF2 KF3 KF4 KF5 KF6 KF7
2 26 31 32 9 43 68
Male Female Male Male Female Female Female
No No No No No No No
Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-I A/A-I B/B2 B/B3 B/B4 B/B5 B/B6
Zhengzhou
ZS1 RM1 RM2 RM3 RM4
7 6 78 8 2
Male Male Male Male Male
Yesa No No No Yesa
Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-II B/B1 A/A-II A/A-I A/A-II
The two Giardia-positive cases mixed with Cyclospora infection, P < 0.01 between Kaifeng and Linzhou.
C. hominis (Hung et al., 2007). These data indicate C. hominis is the predominant species in humans in China, an observation consistent with results of previous studies in other developing countries (Xiao, 2010). In contrast, only one case of C. felis was detected in a 5-year-old immunocompetent child in this study. Cryptosporidium felis is one of the five most common Cryptosporidium species that are responsible for human cryptosporidiosis. More than 70 C. felis cases have been reported in immunocompetent patients and HIV-infected patients worldwide (Xiao and Ryan, 2008). Despite of the small number of positive specimens in the present study, three C. hominis gp60 subtype families were detected, including Ia, Ib, and Id. Among the three subtype families, Ib was the predominant and three subtypes, IbA16G2, IbA19G2, and IbA20G2, were observed. Worldwide, IbA9G3 and IbA10G2 are the two common subtypes within the Ib subtype family. IbA9G3 is commonly seen in humans in Malawi, Kenya, and India, and IbA10G2 is commonly seen in South Africa, Botswana, Jamaica, Peru, USA, Canada, Australia, and European countries (Xiao, 2010). In addition, IbA10G2 is responsible for more than half of the waterborne outbreaks in the United States, United Kingdom, Canada, and France (Xiao and Ryan, 2008). The three Ib subtypes found in this study are different from the subtypes reported in other areas of the world. Previously, subtypes IbA19G2 and IbA20G2 were commonly found in wastewater samples in Shanghai (Feng et al., 2009), and another unusual Ib subtype, IbA23G2, was reported in a child in Tianjing, China (Peng et al., 2001). Therefore, the unique Ib subtype populations might indicate the presence of an unique C. hominis transmission in China, as previously suggested (Feng et al., 2009). Although the subtypes in Id and Ia
subtype families in this study also appear to be unusual, there are numerous subtypes within the two subtype families, many which can occur in the same area (Xiao and Ryan, 2008). Thus far, only assemblages A and B of G. duodenalis have been convincingly identified in humans. The proportion of infections attributable to the two genotypes is different among studies and geographical locations (Homan and Mank, 2001; Monis et al., 2003; Sulaiman et al., 2003; Yason and Rivera, 2007; Kohli et al., 2008; Lalle et al., 2009; Mahdy et al., 2009; Singh et al., 2009; Yang et al., 2010). In this small study, assemblage A (66.7%) was the dominant G. duodenalis genotype in the study area. However, there might be differences in the distribution of the two genotypes in humans in Henan, as most assemblage B infections in the study were detected in Kaifeng, whereas Zhengzhou and Linzhou had mostly assemblage A infections (Table 2). In the present study, A-I was the dominant assemblage A in humans. Previously, two major subassemblages of A, A-I and A-II, were described (Ponce-Macotela et al., 2002; Souza et al., 2007; Fallah et al., 2008; Hussein et al., 2009; Lalle et al., 2009). Results of previous studies in various areas have shown that humans are mostly infected with A-II, although A-I is also seen in some areas or studies. In contrast, animals are mostly infected with A-I, although the A-II is seen occasionally (Cacciò and Ryan, 2008; Xiao and Fayer, 2008). In this study, eight of the 12 assemblage A infections were caused by A-I, and four were caused by A-II. It remains to be determined whether this discordance in subtype distribution of the assemblage A was attributable to the small sample size in this study. In conclusion, C. hominis is the predominant species in Henan, China, and the C. hominis population here appears different from
R. Wang et al. / Experimental Parasitology 127 (2011) 42–45
those in other areas of the world. Moreover, this study provided some preliminary data on genotype and subtype distribution of G. duodenalis in the area. More extensive studies in humans and animals in different areas are needed to better characterize the transmission of cryptosporidiosis and giardiasis in humans and whether the common occurrence of A-I infections in humans is the result of anthroponotic or zoonotic transmission. Acknowledgments This study was supported in part by Key National Science & Technology Special Projects (No. 2008ZX10004–011), the State Key Laboratory of Veterinary Etiological Biology, Ministry of Health Special Funds of Public Sector Research (No. 200808012), and the National Natural Science Foundation of China (No. 30928019). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.exppara.2010.06.034. References Adam, R.D., 2001. Biology of Giardia lamblia. Clinical Microbiology Reviews 14, 447– 475. Alves, M., Xiao, L., Antunes, F., Matos, O., 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitology Research 99, 287–292. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. Journal of Clinical Microbiology 41, 2744–2747. Cacciò, S.M., Ryan, U., 2008. Molecular epidemiology of giardiasis. Molecular Biochemical Parasitology 160, 75–80. Chen, X.N., Lu, S.Q., Li, J.H., Wang, F.Y., Wang, N.X., Wang, F., 2001. Isolation of Hebei isolates of Giardia Lamblia and investigation on their genotypes. Chinese Journal of Parasitic Disease Control 14, 100–102. Fallah, E., Nahavandi, K.H., Rasul, J., Poor, B.M., Mohammad, A., 2008. Molecular identification of Giardia duodenalis isolates from human and animal reservoirs by PCR–RFLP. Journal of Biological Sciences 8, 896–901. Feng, Y., Li, N., Duan, L., Xiao, L., 2009. Cryptosporidium genotype and subtype distribution in raw wastewater in Shanghai, China: evidence for possible unique Cryptosporidium hominis transmission. Journal of Clinical Microbiology 47, 153– 157. Feng, Y., Ortega, Y., Cama, V., Terrell, J., Xiao, L.H., 2008. High intragenotypic diversity of Giardia duodenalis in dairy cattle on three farms. Parasitology Research 103, 87–92. Gatei, W., Das, P., Dutta, P., Sen, A., Cama, V., Lal, A.A., Xiao, L., 2007. Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India. Infection, Genetics and Evolution 7, 197–205. Geurden, T., Levecke, B., Cacció, S.M., Visser, A., De Groote, G., Casaert, S., Vercruysse, J., Claerebout, E., 2009. Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium. Parasitology 136, 1161–1168. Homan, W.L., Mank, T.J., 2001. Human giardiasis: genotype linked differences in clinical symptomatology. International Journal for Parasitology 31, 822–826. Hung, C.C., Tsaihong, J.C., Lee, Y.T., Deng, H.Y., Hsiao, W.H., Chang, S.Y., Chang, S.C., Su, K.E., 2007. Prevalence of intestinal infection due to Cryptosporidium species among Taiwanese patients with human immunodeficiency virus infection. Journal of Formosan Medical Association 106, 31–35. Hussein, A.I., Yamaguchi, T., Nakamoto, K., Iseki, M., Tokoro, M., 2009. Multiplesubgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach. Parasitology International 58, 258–262. Kohli, A., Bushen, O.Y., Pinkerton, R.C., Houpt, E., Newman, R.D., Sears, C.L., Lima, A.A., Guerrant, R.L., 2008. Giardia duodenalis assemblage, clinical presentation
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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China Rongjun Wang a, Xiaosan Zhang a, Huili Zhu a, Longxian Zhang a,b,c,*, Yaoyu Feng d, Fuchun Jian a, Changshen Ning a, Meng Qi a, Yang Zhou a, Kanda Fu e, Yaqiang Wang e, Yanru Sun a, Qiang Wang a, Lihua Xiao c,** a
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Atlanta Research and Education Foundation, 1670 Clairmont Road, Decatur, GA 30033, USA Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA d School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China e Inspection Division of Huaihe Hospital, Henan University, Kaifeng 475000, China b c
a r t i c l e
i n f o
Article history: Received 24 March 2010 Received in revised form 10 June 2010 Accepted 28 June 2010 Available online 1 July 2010 Keywords: Cryptosporidium Giardia duodenalis Humans Genotyping Subtyping Cryptosporidium hominis Assemblage A Assemblage B
a b s t r a c t Cryptosporidium and Giardia infections are common causes of diarrhea worldwide. To better understand the transmission of human cryptosporidiosis and giardiasis in Henan, China, 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens were characterized at the species/genotype and subtype levels. Cryptosporidium specimens were analyzed by DNA sequencing of the small subunit rRNA and 60 kDa glycoprotein genes. Among those genotyped, nine belonged to C. hominis and one C. felis, with the former belonging to three subtype families: Ia, Ib, and Id. The three Ib subtypes identified, IbA16G2, IbA19G2, and IbA20G2, were very different from the two common Ib subtypes (IbA9G3 and IbA10G2) found in other areas of the world. The distribution of Giardia duodenalis genotypes and subtypes was assessed by sequence analysis of the triosephosphate isomerase (tpi) gene. The assemblages A (eight belonging to A-I and four A-II) and B (belonging to six new subtypes) were found in 12 and six specimens, respectively. More systematic studies are needed to understand the transmission of Cryptosporidium and G. duodenalis in humans in China. Published by Elsevier Inc.
1. Introduction Cryptosporidium and Giardia infections are significant causes of diarrhea in humans worldwide. The diarrhea caused by cryptosporidiosis may become profuse, chronic, or even life-threatening, particularly in immunocompromised persons. Both parasites have a broad range of hosts and can be transmitted by the fecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao et al., 2004; Smith et al., 2006). Using genotyping tools, it has been shown that eight Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. canis, C. felis, C. suis, C. muris, and C. andersoni) and at least six genotypes (cervine, skunk, chipmunk I, horse, rabbit genotype, and pig genotype II) can infect humans, with C. hominis and C. parvum responsible for
* Corresponding author at: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China. Fax: +86 371 63558180. ** Corresponding author. Fax: +1 (770) 488 4454. E-mail addresses: [email protected] (L. Zhang), [email protected] (L. Xiao). 0014-4894/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.exppara.2010.06.034
most clinical cases. The distribution of C. hominis and C. parvum in humans differs in geographic regions (Xiao, 2010). In addition, DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene has shown the complexity of Cryptosporidium transmission in endemic areas. Among the five common C. hominis subtype families, Ia, Ib, Id, Ie, and If, three to four subtype families were each seen in humans in India, Peru, New Orleans, Malawi, South Africa, Kuwait, and Portugal. In contrast, only one or two C. parvum subtype families (IIa and IIc) were seen in humans in the same areas (Alves et al., 2003, 2006; Peng et al., 2003; Sulaiman et al., 2005; Gatei et al., 2007). In giardiasis, only Giardia duodenalis (synonym G. lamblia or G. intestinalis) is recovered from humans and most other mammals (Adam, 2001). Isolates of G. duodenalis are classified into seven common ‘assemblages’ or genotypes: A–G, based on the characterization of the triosephosphate isomerase (tpi), small subunit (SSU) rRNA, b-giardin, glutamate dehydrogenase (gdh), and other genes (Thompson et al., 2000; Sulaiman et al., 2003; Read et al., 2004). Assemblages A and B infect humans and a broad range of other hosts, including livestock, cats, dogs, and wild mammals. The assemblage A is further divided into two major subassemblages, I
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and II, and the less common III. Many subtypes are present in the assemblage B (Feng et al., 2008; Geurden et al., 2009; Mahdy et al., 2009). Assemblages C, D, E, F, and G appear to be mostly restricted to companion animals, livestock, and rodents (Monis et al., 2003; Sulaiman et al., 2003). In China, the number of molecular epidemiological studies of cryptosporidiosis and giardiasis in humans is small. Peng et al. characterized genetically five specimens (all belonged to C. hominis) from children in Tianjing, three specimens were characterized by gp60 sequence analysis and three subtypes (IbA23G2, IdA14, and IeA13G3T3) were identified (Peng et al., 2001). Four Cryptosporidium specimens from HIV-infected patients in Taiwan were identified as C. hominis (two cases), C. felis (one case), and C. meleagridis (one case) (Hung et al., 2007). A report by Yong et al. based on sequence analysis of the SSU rRNA gene of eight G. duodenalis specimens from Anhui Province showed the presence of assemblages A and B in four patients each (Yong et al., 2000). Another study by Lu et al. based on sequence analysis of the tpi gene of three specimens showed the presence of two assemblage B and one A-I mixed with assemblage B (Lu et al., 2002). Chen et al. reported that three G. duodenalis specimens from humans in Hebei Province belonged to subtype A-II (Chen et al., 2001). Therefore, the molecular epidemiology of human cryptosporidiosis and giardiasis in China is still unclear. In this study, to better understand the transmission of cryptosporidiosis and giardiasis, 10 Cryptosporidium specimens and 18 Giardia specimens from humans in Henan were genetically characterized.
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2.4. Giardia genotyping and subtyping G. duodenalis genotypes and subtypes were determined by DNA sequence analysis of the tpi gene. A nested PCR was used to amplify the partial tpi gene (530-bp) of Giardia specimens (Sulaiman et al., 2003). Sequencing of tpi gene was done as described below. Genotype and subtype identities of G. duodenalis were established by direct comparison of the acquired sequences with reference sequences. 2.5. DNA sequence analysis All PCR products were sequenced on an ABI PRISMTM 3730 XL DNA Analyzer (Applied Biosystems, USA), using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing a new PCR product if necessary. The SSU rRNA and gp60 sequences of Cryptosporidium and tpi sequences of Giardia obtained in this study were aligned with reference sequences downloaded from GenBank using the program ClustalX 1.83. Nucleotide sequences of representative subtypes have been deposited in the GenBank under Accession Nos. FJ707310–FJ707316 (Cryptosporidium) and GU564274–GU564284 (Giardia). 3. Results 3.1. Cryptosporidium genotypes and subtypes
2. Materials and methods 2.1. Stool specimens The 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens from humans were obtained from a survey conducted in hospitals of Zhengzhou, Kaifeng, and Linzhou in Henan Province between June 2007 and December 2008. All specimens were stored at 4 °C in 2.5% (w/v) potassium dichromate solution or frozen at 80 °C without preservatives prior to being used in molecular characterizations.
The 10 Cryptosporidium-positive specimens were successfully amplified at the SSU rRNA locus. RFLP analysis of the 10 PCR products suggested the presence of C. hominis in nine specimens and C. felis in one. This was confirmed by DNA sequence analysis of the PCR products. Cryptosporidium hominis was further subtyped by gp60 gene sequence analysis. Three subtype families were identified in the nine C. hominis-positive specimens: Ia (11.1%), Ib (66.7%), and Id (22.2%). Among subtype family Ib, three subtypes were found: IbA16G2 (in one case), IbA19G2 (in two cases), and IbA20G2 (in three cases). In contrast, only one subtype each was present in subtype families Ia (IaA9R3 in one case) and Id (IdA21 in two cases) (Table 1).
2.2. DNA extraction Genomic DNA was isolated from 200 lL of stool specimens using the QIAampÒ DNA stool kit (QIAGEN Inc., Valencia, CA) and the previously described procedures, after washing of specimens three times with distilled water by centrifugation at 1000g for 10 min, oocyst lysis by alkaline digestion, and crude DNA extraction by the phenol–chloroform method (Xiao et al., 2002). The extracted DNA was eluted in 200 lL of the AE elution buffer and stored at 20 °C. 2.3. Cryptosporidium genotyping and subtyping Cryptosporidium spp. were genotyped by PCR amplification of a 830-bp fragment of the small subunit (SSU) rRNA gene and restriction fragment length polymorphism (RFLP) analysis of the PCR products using restriction enzymes SspI and VspI (Xiao et al., 2001). To confirm the genotype identification, all PCR products were sequenced. Subtyping of C. hominis was done by DNA sequencing of the gp60 gene. A fragment of 855-bp of the gp60 gene was amplified by nested PCR (Alves et al., 2003). The previously established nomenclature system was used to determine subtype families and subtypes within each family (Sulaiman et al., 2005; Xiao, 2010).
3.2. Giardia genotypes and subtypes All 18 Giardia-positive specimens produced the expected tpi PCR products. Alignment of the tpi sequences obtained with reference sequences indicated the presence of two genotypes of G. duodenalis; 12 specimens were identified as G. duodenalis assemblage A and six as assemblage B (Table 2). Among the three cities examined in this study, Kaifeng appeared to have more assemblage B infections than Zhengzhou and Linzhou (P < 0.01 between Kaifeng and Linzhou areas by Chi-square analysis, Table 2). Of the 12 assemblage A specimens, eight produced sequences that were identical to the A-I (GenBank Accession No. AF069556), and the remaining four produced sequences identical to the A-II (GenBank Accession No. AF069557). In contrast, the six assemblage B specimens produced sequences that were not identical to any known assemblage B subtypes, thus representing six new subtypes, named B1–B6 for convenience (Table 2). 4. Discussion In this study, 90% of Cryptosporidium specimens were identified as C. hominis. Previously, five human Cryptosporidium isolates from Tianjing also belonged to C. hominis (Peng et al., 2001) and two of four Cryptosporidium-positive specimens from Taiwan had
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R. Wang et al. / Experimental Parasitology 127 (2011) 42–45 Table 1 Cryptosporidium genotypes and subtypes in 10 patients in Henan, China. Area
Sample
Linzhou
146 193
Kaifeng
ZL0821 LB-1 ZZH-1 WJH-35
Zhengzhou
ZYY-3 LFY-6 CHZF1 CHZF2
Age (years)
Sex
Diarrhea
Genotype/subtype
4 5
Female Female
No No
C. hominis/IbA20G2 C. felis
6 1 1 35
Male Female Male Male
No Yes Yes Yes
C. C. C. C.
hominis/IbA20G2 hominis/IaA9R3 hominis/IbA20G2 hominis/IbA19G2
3 6 2 1
Male Male Male Female
Yes Yes Yes Yes
C. C. C. C.
hominis/IbA19G2 hominis/IbA16G2 hominis/IdA21 hominis/IdA21
Table 2 Giardia assemblage/subtype in 18 patients in Henan, China.
a
Area
Sample
Sex
Diarrhea
Assemblage/subtype
Linzhou
LZ1 LZ2 LZ4 LZ5 LZ6 LZ8
Age (year) 5 7 5 5 6 6
Male Female Male Female Female Female
No No No No No No
Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-I A/A-I A/A-I A/A-II A/A-I A/A-I
Kaifeng
KF1 KF2 KF3 KF4 KF5 KF6 KF7
2 26 31 32 9 43 68
Male Female Male Male Female Female Female
No No No No No No No
Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-I A/A-I B/B2 B/B3 B/B4 B/B5 B/B6
Zhengzhou
ZS1 RM1 RM2 RM3 RM4
7 6 78 8 2
Male Male Male Male Male
Yesa No No No Yesa
Assemblage Assemblage Assemblage Assemblage Assemblage
A/A-II B/B1 A/A-II A/A-I A/A-II
The two Giardia-positive cases mixed with Cyclospora infection, P < 0.01 between Kaifeng and Linzhou.
C. hominis (Hung et al., 2007). These data indicate C. hominis is the predominant species in humans in China, an observation consistent with results of previous studies in other developing countries (Xiao, 2010). In contrast, only one case of C. felis was detected in a 5-year-old immunocompetent child in this study. Cryptosporidium felis is one of the five most common Cryptosporidium species that are responsible for human cryptosporidiosis. More than 70 C. felis cases have been reported in immunocompetent patients and HIV-infected patients worldwide (Xiao and Ryan, 2008). Despite of the small number of positive specimens in the present study, three C. hominis gp60 subtype families were detected, including Ia, Ib, and Id. Among the three subtype families, Ib was the predominant and three subtypes, IbA16G2, IbA19G2, and IbA20G2, were observed. Worldwide, IbA9G3 and IbA10G2 are the two common subtypes within the Ib subtype family. IbA9G3 is commonly seen in humans in Malawi, Kenya, and India, and IbA10G2 is commonly seen in South Africa, Botswana, Jamaica, Peru, USA, Canada, Australia, and European countries (Xiao, 2010). In addition, IbA10G2 is responsible for more than half of the waterborne outbreaks in the United States, United Kingdom, Canada, and France (Xiao and Ryan, 2008). The three Ib subtypes found in this study are different from the subtypes reported in other areas of the world. Previously, subtypes IbA19G2 and IbA20G2 were commonly found in wastewater samples in Shanghai (Feng et al., 2009), and another unusual Ib subtype, IbA23G2, was reported in a child in Tianjing, China (Peng et al., 2001). Therefore, the unique Ib subtype populations might indicate the presence of an unique C. hominis transmission in China, as previously suggested (Feng et al., 2009). Although the subtypes in Id and Ia
subtype families in this study also appear to be unusual, there are numerous subtypes within the two subtype families, many which can occur in the same area (Xiao and Ryan, 2008). Thus far, only assemblages A and B of G. duodenalis have been convincingly identified in humans. The proportion of infections attributable to the two genotypes is different among studies and geographical locations (Homan and Mank, 2001; Monis et al., 2003; Sulaiman et al., 2003; Yason and Rivera, 2007; Kohli et al., 2008; Lalle et al., 2009; Mahdy et al., 2009; Singh et al., 2009; Yang et al., 2010). In this small study, assemblage A (66.7%) was the dominant G. duodenalis genotype in the study area. However, there might be differences in the distribution of the two genotypes in humans in Henan, as most assemblage B infections in the study were detected in Kaifeng, whereas Zhengzhou and Linzhou had mostly assemblage A infections (Table 2). In the present study, A-I was the dominant assemblage A in humans. Previously, two major subassemblages of A, A-I and A-II, were described (Ponce-Macotela et al., 2002; Souza et al., 2007; Fallah et al., 2008; Hussein et al., 2009; Lalle et al., 2009). Results of previous studies in various areas have shown that humans are mostly infected with A-II, although A-I is also seen in some areas or studies. In contrast, animals are mostly infected with A-I, although the A-II is seen occasionally (Cacciò and Ryan, 2008; Xiao and Fayer, 2008). In this study, eight of the 12 assemblage A infections were caused by A-I, and four were caused by A-II. It remains to be determined whether this discordance in subtype distribution of the assemblage A was attributable to the small sample size in this study. In conclusion, C. hominis is the predominant species in Henan, China, and the C. hominis population here appears different from
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those in other areas of the world. Moreover, this study provided some preliminary data on genotype and subtype distribution of G. duodenalis in the area. More extensive studies in humans and animals in different areas are needed to better characterize the transmission of cryptosporidiosis and giardiasis in humans and whether the common occurrence of A-I infections in humans is the result of anthroponotic or zoonotic transmission. Acknowledgments This study was supported in part by Key National Science & Technology Special Projects (No. 2008ZX10004–011), the State Key Laboratory of Veterinary Etiological Biology, Ministry of Health Special Funds of Public Sector Research (No. 200808012), and the National Natural Science Foundation of China (No. 30928019). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.exppara.2010.06.034. References Adam, R.D., 2001. Biology of Giardia lamblia. Clinical Microbiology Reviews 14, 447– 475. Alves, M., Xiao, L., Antunes, F., Matos, O., 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitology Research 99, 287–292. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. Journal of Clinical Microbiology 41, 2744–2747. Cacciò, S.M., Ryan, U., 2008. Molecular epidemiology of giardiasis. Molecular Biochemical Parasitology 160, 75–80. Chen, X.N., Lu, S.Q., Li, J.H., Wang, F.Y., Wang, N.X., Wang, F., 2001. Isolation of Hebei isolates of Giardia Lamblia and investigation on their genotypes. Chinese Journal of Parasitic Disease Control 14, 100–102. Fallah, E., Nahavandi, K.H., Rasul, J., Poor, B.M., Mohammad, A., 2008. Molecular identification of Giardia duodenalis isolates from human and animal reservoirs by PCR–RFLP. Journal of Biological Sciences 8, 896–901. Feng, Y., Li, N., Duan, L., Xiao, L., 2009. Cryptosporidium genotype and subtype distribution in raw wastewater in Shanghai, China: evidence for possible unique Cryptosporidium hominis transmission. Journal of Clinical Microbiology 47, 153– 157. Feng, Y., Ortega, Y., Cama, V., Terrell, J., Xiao, L.H., 2008. High intragenotypic diversity of Giardia duodenalis in dairy cattle on three farms. Parasitology Research 103, 87–92. Gatei, W., Das, P., Dutta, P., Sen, A., Cama, V., Lal, A.A., Xiao, L., 2007. Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India. Infection, Genetics and Evolution 7, 197–205. Geurden, T., Levecke, B., Cacció, S.M., Visser, A., De Groote, G., Casaert, S., Vercruysse, J., Claerebout, E., 2009. Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium. Parasitology 136, 1161–1168. Homan, W.L., Mank, T.J., 2001. Human giardiasis: genotype linked differences in clinical symptomatology. International Journal for Parasitology 31, 822–826. Hung, C.C., Tsaihong, J.C., Lee, Y.T., Deng, H.Y., Hsiao, W.H., Chang, S.Y., Chang, S.C., Su, K.E., 2007. Prevalence of intestinal infection due to Cryptosporidium species among Taiwanese patients with human immunodeficiency virus infection. Journal of Formosan Medical Association 106, 31–35. Hussein, A.I., Yamaguchi, T., Nakamoto, K., Iseki, M., Tokoro, M., 2009. Multiplesubgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach. Parasitology International 58, 258–262. Kohli, A., Bushen, O.Y., Pinkerton, R.C., Houpt, E., Newman, R.D., Sears, C.L., Lima, A.A., Guerrant, R.L., 2008. Giardia duodenalis assemblage, clinical presentation
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