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Cryptosporidium cuniculus and Giardia duodenalis in Rabbits: Genetic Diversity and Possible Zoonotic Transmission

  • Weizhe Zhang ,

    Contributed equally to this work with: Weizhe Zhang, Yujuan Shen

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Yujuan Shen ,

    Contributed equally to this work with: Weizhe Zhang, Yujuan Shen

    Affiliation Key Laboratory of Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Ministry of Health, World Health Organization, Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai, China

  • Rongjun Wang,

    Affiliation College of Animal Science and Veterinary Medicine, Henan Agricultural University Zhengzhou, Henan, China

  • Aiqin Liu , (AL); (LZ)

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Hong Ling,

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Yihong Li,

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Jianping Cao,

    Affiliation Key Laboratory of Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Ministry of Health, World Health Organization, Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai, China

  • Xiaoyun Zhang,

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Jing Shu,

    Affiliation Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China

  • Longxian Zhang (AL); (LZ)

    Affiliations Department of Parasitology, Harbin Medical University, Harbin, Heilongjiang, China, Key Laboratory of Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Ministry of Health, World Health Organization, Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai, China, College of Animal Science and Veterinary Medicine, Henan Agricultural University Zhengzhou, Henan, China



Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China.

Methodology/Principal Findings

378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather's sugar flotation technique and Lugol's iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples.


The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.


Cryptosporidium and Giardia are important intestinal protozoa found in humans and animals worldwide. Both pathogens are responsible for gastroenteritis, chronic diarrhea or even severe diarrhea, depending on the age and health of the infected hosts as well as the genetic background and infective dose of the parasites.

The majority of human Cryptosporidium infections are attributable to C. hominis and C. parvum. However, a number of other Cryptosporidium species and genotypes have also been reported at a lower frequency, including nine Cryptosporidium species (C. meleagridis, C. felis, C. canis, C. muris, C. suis, C. ubiquitum, C. cuniculus, C. fayeri and C. andersoni) and five genotypes (skunk genotype, chipmunk I genotype, horse genotype, monkey genotype and pig genotype II) [1][8]. Although the first report of rabbit Cryptosporidium was noticed in 1912 [9], the concerns regarding Cryptosporidium infection in rabbits have only occurred in recent years due to a few sporadic human cases and a serious waterborne outbreak of cryptosporidiosis caused by C. cuniculus (previously named as Cryptosporidium rabbit genotype) [4], [10], [11]. In addition, although several studies have reported the natural infection of C. cuniculus in rabbits [12], few large-scale studies were available for the prevalence of C. cuniculus in rabbits and only a small number of studies have been conducted on genetic analysis [10], [13][17].

For G. duodenalis, seven G. duodenalis assemblages (A to G) are defined based on genetic analysis and host specificity. More recently, assemblage H has been identified in marine vertebrates [18]. Among which, only assemblages A and B are human pathogens and assemblage A is further classified into two major subtypes, AI and AII. Many subtypes are present in the assemblage B due to its high degree of genetic polymorphism. In contrast, assemblages C to G are mostly found in livestock, companion animals and rodents [5], [19][21]. Previously, most studies focused on the prevalence and molecular identification of G. duodenalis in livestock and wild animals. Thus far, only one G. duodenalis isolate from a rabbit has been identified as assemblage B based on TPI gene [20].

In China, a few studies have reported the prevalence of Cryptosporidium spp. in rabbits (Table 1), and the molecular identification was just seen in a more recent study [17]. In contrast, only one G. duodenalis isolate has been obtained from a rabbit; however, it was not genotyped and subtyped [22]. Thus, the prevalence, distribution and genetic characterization of C. cuniculus and G. duodenalis in rabbits in China are still unclear. In this study, to better understand the prevalence and transmission of cryptosporidiosis and giardiasis, an epidemiologic investigation of two parasites was conducted in rabbits in Heilongjiang province, China; further, the positive isolates of C. cuniculus and G. duodenalis were analyzed for genetic characterization, respectively.

Table 1. Prevalence of Cryptosporidium in rabbits in China.

Materials and Methods

Ethical Considerations

Before beginning work on the study, we contacted the farm owners and obtained their permission. No specific permits were required for the described field studies. We directly collected the fecal samples instead of operating on the rabbits in this study. Each of the experimental rabbits was fed alone in each cage and the labeled plastic bags were put under each of the cages. One day later, we collected the rabbit feces excreted in the bags. During the procedure, the rabbits were not hurt at all. And the locations where we sampled are not privately-owned or protected in any way. The field studies did not involve endangered or protected species.

Sample Collection

A total of 378 fresh fecal samples were collected from 4-6-month-old experimental rabbits on eight farms in Heilongjiang Province between October 2008 and August 2010. The Sheather's sugar flotation technique and Lugol's iodine stain method were used to detect the Cryptosporidium oocysts and Giardia cysts, respectively. Wet smears were examined using a bright-field microscope with 100× and 400× magnification. Cryptosporidium-positive and G. duodenalis-positive samples were stored in 2.5% potassium dichromate solutions at 4°C prior to DNA extraction, respectively.

DNA extraction

Fecal samples were washed twice with distilled water, and genomic DNA was extracted using a QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted in 200 µL of Buffer AE and stored at −20°C prior to use in PCR analysis.

Cryptosporidium genotyping and subtyping

Cryptosporidium oocysts in the samples were identified to the species/genotype level using nested PCR amplification of an approximately 830 bp fragment of the SSU rRNA gene [23]. Subtyping of Cryptosporidium-positive samples was done by nested PCR amplification of an approximately 800–850 bp fragment of the gp60 gene [24]. All secondary PCR products of both genes were sequenced using the secondary PCR primers.

G. duodenalis genotyping

The identity of G. duodenalis genotypes and subtypes were determined by DNA sequence analysis of the TPI gene. G. duodenalis-positive samples were used to amplify the partial TPI gene of an approximately 530 bp fragment by a nested PCR [20]. Genotype and subtype identities of the G. duodenalis samples were established by direct comparison of the acquired sequences with reference sequences downloaded from GenBank.

Sequence analysis

All secondary PCR products were sequenced in both directions using the secondary PCR primers on an ABI PRISMTM 3730 XL DNA Analyzer (Applied Biosystems, USA) by using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Nucleotide sequences obtained were aligned with Cryptosporidium and G. duodenalis reference sequences from GenBank using ClustalX 1.81. The representative nucleotide sequences obtained in the study were deposited in the GenBank database under the following accession numbers: HQ397716 to HQ397718 (Cryptosporidium) and HQ397719, HQ666892 to HQ666898 (G. duodenalis).


Prevalence of Cryptosporidium and Giardia in rabbits

A total of 378 fecal samples were examined by microscopy. Nine samples were positive for C. cuniculus (2.38%), and 28 samples were positive for G. duodenalis (7.41%). And G. duodenalis was more widespread than C. cuniculus, accounting for C. cuniculus oocysts found in four farms and G. duodenalis cysts found in seven farms. Both Cryptosporidium and Giardia were detected on Farms 2, 3, 4 and 8 (Table 2). Additionally, one mixed infection with both parasites was found on Farm 3.

Table 2. Prevalence and subtype distribution of C. cuniculus and G. duodenalis in rabbits in Heilongjiang Province, China.

Cryptosporidium genotyping and subtyping

DNA sequencing of the SSU rRNA PCR products showed that all the nine sequences were identical to C. cuniculus isolates from China, the UK, the Czech Republic and Australia [4], [10], [13], [14], [16], [17]. Subtyping was further achieved by sequence analysis of the gp60 gene. Two subtypes were identified in the nine C. cuniculus-positive isolates: VbA21 (n = 6) and VbA32 (n = 3).

Giardia genotyping and subtyping

Alignment of the TPI sequences indicated that 28 G. duodenalis-positive isolates all belonged to assemblage B and represented eight distinct subtypes, B-I to B-VIII (Table 2). Subtype B-I was identical to a rabbit-derived isolate (AY228639) and it was the most common subtype found in this study, accounting for 64.3% (18/28) of G. duodenalis-positive samples. In contrast, subtypes (B-II to B-VIII) were not identical to any known assemblage B subtypes with each subtype having only one or two cases. By using the GenBank sequence AY368171 as a reference sequence, single nucleotide polymorphisms were present in the eight representative sequences obtained from the 28 G. duodenalis-positive isolates and one to three base variations were found (Table 3).

Table 3. Variation in the TPI nucleotide sequences among subtypes of G. duodenalis assemblage B in rabbits in Heilongjiang Province.


A 2.38% overall infection rate of Cryptosporidium spp. was observed in this study, which was lower than those (2.86% to 33.80%) in rabbits conducted in the other areas in China (Table 1). It was also not as high as those in Japan (19.7%; 13/66) and Australia (6.8%; 12/176) [15], [16]. The differences in prevalence may be related to the factors such as the sensitivity and specificity of detection methods, animal health at the time of sampling, the experimental design and the overall sample size. And health status of animals is closely associated with the animal age. Cryptosporidium is often found in unhealthy juvenile rabbits. A report showed that dead juvenile rabbits had a higher infection rate than healthy rabbits (19.7% vs 3.33%) [15]. In addition, Cryptosporidium infections in neonatal and younger rabbits are associated with high mortality and diarrheic feces. Just like C. parvum in cattle, there appears to be a decline in Cryptosporidium prevalence, symptoms and oocysts shedding as rabbits increase in age [12], [17], [25]. The lower prevalence in this study might result from our fecal sampling of healthy older rabbits ranging from four to six months of age.

Sequence analysis of SSU rRNA gene showed that the nine isolates shared 100% similarity to most of C. cuniculus cases from humans and rabbits although there were two nucleotide differences compared to a New Zealand isolate (AY458612) [4], [10], [11], [13], [14], [16], [17], [26]. Thus, the narrow host spectrum of C. cuniculus further supports previous theory that there is an apparent host adaptation and parasite-host co-evolution in Cryptosporidium [13]. Gp60 gene subtyping revealed the existence of two subtypes in this study, VbA21 and VbA32. To date, at least 20 subtypes from humans and 9 subtypes from rabbits have been identified (Table 4). VbA32 has been isolated from humans in the UK [27]. In contrast, VbA21 was not identical to any known subtypes, thus representing a new subtype. It is unclear about the host specificity of the two subtype families Va and Vb. Va subtype family is mostly found in humans with occasionally seen in rabbits [10], [27], [28]. Vb subtype family is mostly found in rabbits with commonly seen in humans in the UK [10], [16], [17], [27]. This showed that human-derived and rabbit-derived isolates of C. cuniculus do not have strict host specificity and cross transmission may occur between humans and rabbits. The subtypes in this study might have the potential to infect humans, especially for the subtype VbA32 found in humans.

Table 4. Subtypes of C. cuniculus in humans and rabbits in different countries.

For G. duodenalis, only one case of rabbit giardiasis caused by assemblage B has been reported [20]. In this study, a 7.41% infection rate of Giardia was noticed, which was higher than the 2.38% prevalence of Cryptosporidium. Sequence analysis of TPI gene showed that all the isolates belonged to G. duodenalis assemblage B and represented eight different subtypes (B-I to B-VIII) with B-I as the dominant subtype(64.3%, 18/28). Among which, only subtype B-I was identical to a previous rabbit-derived isolate (AY228639), whereas the remaining subtypes have never reported. Previously, the diversity of G. duodenalis assemblage B has also been observed in isolates from humans and other animals [5]. In a recent study in China, the polymorphism of assemblage B is also expected that six human isolates belonged to six distinct subtypes [29]. Unlike assemblage A, the nomenclature of assemblage B subtype was relatively disordered, which might cause trouble in understanding the transmission of G. duodenalis assemblage B. Thus, it is necessary for the researchers to make a uniform rule to name different subtypes of assemblage B in the future.

G. duodenalis assemblage B has a broad range of host. Except for having been found in humans, it has also been detected in cattle sheep, horses, dogs, and cats [30][35]. So far, although no strong evidence has supported the zoonotic transmission of G. duodenalis between humans and animals, case control studies showed that contacting with farm animals was related to the increasing infection rates of giardiasis [36], [37].

In conclusion, the present study provided useful data for further studying the C. cuniculus and G. duodenalis infections in rabbits at prevalence rates and molecular levels. Based on the fact that both parasites all belonged to zoonotic pathogens, more extensive studies in rabbits in different areas are needed to better characterize the transmission of cryptosporidiosis and giardiasis and to assess the public health significance of these parasites.

Author Contributions

Conceived and designed the experiments: AL LZ WZ. Performed the experiments: WZ YS XZ. Analyzed the data: YL RW AL. Contributed reagents/materials/analysis tools: LZ HL JC YL JS. Wrote the paper: AL WZ LZ.


  1. 1. Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, et al. (2003) Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. Eukaryot Microbiol 50: Suppl531–533.
  2. 2. Sulaiman IM, Hira PR, Zhou L, Al-Ali FM, Al-Shelahi FA, et al. (2005) Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol 43: 2805–2809.
  3. 3. Feng Y, Alderisio KA, Yang W, Blancero LA, Kuhne WG, et al. (2007) Cryptosporidium genotypes in wildlife from a New York watershed. Appl Environ Microbiol 73: 6475–6483.
  4. 4. Robinson G, Elwin K, Chalmers RM (2008) Unusual Cryptosporidium genotypes in human cases of diarrhea. Emerg Infect Dis 14: 1800–1802.
  5. 5. Xiao L, Fayer R (2008) Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol 38: 1239–1255.
  6. 6. Fayer R (2010) Taxonomy and species delimitation in Cryptosporidium. Exp Parasitol 124: 90–97.
  7. 7. Waldron LS, Cheung-Kwok-Sang C, Power ML (2010) Wildlife-associated Cryptosporidium fayeri in human, Australia. Emerg Infect Dis 16: 2006–2007.
  8. 8. Xiao L (2010) Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124: 80–89.
  9. 9. Tyzzer EE (1912) Cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse. Arch Protistenkunde 26: 394–412.
  10. 10. Chalmers RM, Robinson G, Elwin K, Hadfield SJ, Xiao L, et al. (2009) Cryptosporidium sp. rabbit genotype, a newly identified human pathogen. Emerg Infect Dis 15: 829–830.
  11. 11. Molloy SF, Smith HV, Kirwan P, Nichols RA, Asaolu SO, et al. (2010) Identification of a high diversity of Cryptosporidium species genotypes and subtypes in a pediatric population in Nigeria. Am J Trop Med Hyg 82: 608–613.
  12. 12. Robinson G, Chalmers RM (2010) The European rabbit (Oryctolagus cuniculus), a source of zoonotic cryptosporidiosis. Zoonoses Public Health 57: e1–13.
  13. 13. Xiao L, Sulaiman IM, Ryan UM, Zhou L, Atwill ER, et al. (2002) Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. Int J Parasitol 32: 1773–1785.
  14. 14. Ryan U, Xiao L, Read C, Zhou L, Lal AA, et al. (2003) Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl Environ Microbiol 69: 4302–4307.
  15. 15. Shiibashi T, Imai T, Sato Y, Abe N, Yukawa M, et al. (2006) Cryptosporidium infection in juvenile pet rabbits. J Vet Med Sci 68: 281–282.
  16. 16. Nolan MJ, Jex AR, Haydon SR, Stevens MA, Gasser RB (2010) Molecular detection of Cryptosporidium cuniculus in rabbits in Australia. Infect Genet Evol 10: 1179–1187.
  17. 17. Shi K, Jian F, Lv C, Ning C, Zhang L, et al. (2010) Prevalence, genetic characteristics, and zoonotic potential of Cryptosporidium species causing infections in farm rabbits in China. J Clin Microbiol 48: 3263–3266.
  18. 18. Lasek-Nesselquist E, Welch DM, Sogin ML (2010) The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol 40: 1063–1074.
  19. 19. Monis PT, Andrews RH, Mayrhofer G, Ey PL (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect Gene Evol 3: 29–38.
  20. 20. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, et al. (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 9: 1444–1452.
  21. 21. Sprong H, Cacciò SM, van der Giessen JW, ZOOPNET network and partners (2009) Identification of zoonotic genotypes of Giardia duodenalis. PLoS Negl Trop Dis 3: e558.
  22. 22. Lu SQ, Wang ZY, Yan G, Chen PH, Zhu H, et al. (1996) Four isolates of Giardia lamblia cultivated axenically in China and the restriction endonuclease analysis of their DNA. J Parasitol 82: 659–661.
  23. 23. Xiao L, Morgan UM, Limor J, Escalante A, Arrowood M, et al. (1999) Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl Environ Microbiol 65: 3386–3391.
  24. 24. Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, et al. (2003) Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41: 2744–2747.
  25. 25. Fayer R, Santin M, Trout JM (2007) Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet Parasitol 145: 260–266.
  26. 26. Learmonth JJ, Ionas G, Ebbett KA, Kwan ES (2004) Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand. Appl Environ Microbiol 70: 3973–3978.
  27. 27. Robinson G, Wright S, Elwin K, Hadfield SJ, Katzer F, et al. (2010) Re-description of Cryptosporidium cuniculus (Apicomplexa: Cryptosporidiidae): Morphology, biology and phylogeny. Int J Parasitol 40: 1539–1548.
  28. 28. Bouzid M, Tyler KM, Christen R, Chalmers RM, Elwin K, et al. (2010) Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci. BMC Microbiology 10: 213.
  29. 29. Wang R, Zhang X, Zhu H, Zhang L, Feng Y, et al. (2011) Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China. Exp Parasitol 127: 42–45.
  30. 30. Learmonth JJ, Ionas G, Pita AB, Cowie RS (2003) Identification and genetic characterisation of Giardia and Cryptosporidium strains in humans and dairy cattle in the Waikato Region of New Zealand. Water Sci Technol 47: 21–26.
  31. 31. Read CM, Monis PT, Thompson RC (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Genet Evol 4: 125–130.
  32. 32. Traub RJ, Monis PT, Robertson I, Irwin P, Mencke N, et al. (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128: 253–262.
  33. 33. Traub R, Wade S, Read C, Thompson A, Mohammed H (2005) Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses. Vet Parasitol 130: 317–321.
  34. 34. Castro-Hermida JA, Almeida A, González-Warleta M, Correia da Costa JM, Rumbo-Lorenzo C, et al. (2007) Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitol Res 101: 1443–1448.
  35. 35. Coklin T, Farber J, Parrington L, Dixon B (2007) Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada. Vet Parasitol 150: 297–305.
  36. 36. Hoque ME, Hope VT, Kjellström T, Scragg R, Lay-Yee R (2002) Risk of giardiasis in Aucklanders: a case-control study. Int J Infect Dis 6: 191–197.
  37. 37. Hoque ME, Hope VT, Scragg R, Kjellström T (2003) Children at risk of giardiasis in Auckland: a case-control analysis. Epidemiol Infect 131: 655–662.
  38. 38. Men J, Zang X, Yu S, Li J, Gong P, et al. (2009) Cryptosporidiosis molecular epidemiology of sheep and rabbits in Changchun area. J Jilin Agric Univ 31: 447–451. (in Chinese).
  39. 39. Tian ZC, Zhang XC, Li JH, Yin JG, Yang J, et al. (2002) Cloning of a species-specific gene fragment from Cryptosporidium parvum and the development of diagnostic PCR primers. Chin J Parasitol Parasitic Dis 20: 72–75. (in Chinese).
  40. 40. Ni H, Gong Y, Han J, Zhang Z, Yang Y (2008) An investigation of Cryptosporidium in mammals in Qingdao region. Acta Acad Med Qingdao Univ 42: 158–161. (in Chinese).