Abstract
Cryptosporidium cuniculus was originally detected in rabbits and has been identified as an emerging human pathogen, but the occurrence, prevalence, and epidemiology in human and rabbit populations are poorly understood. As identification of C. cuniculus can be time-consuming and costly using existing molecular assays, a real-time polymerase chain reaction (PCR)-based method targeting specific markers for this species was developed. The assay is based on amplification of the C. cuniculus-specific 60-kDa glycoprotein (GP60) gene using two PCRs targeting subtype families Va and Vb. PCR product formation was monitored by SYBR Green I fluorescence measurement followed by post-amplification melt curve analysis; high resolution melt curve analysis was found to give increased sensitivity over standard melt curve analysis. The real-time PCR correctly identified all 41 C. cuniculus isolates (40 from humans, one from a rabbit) tested, with subtype family in agreement with GP60 gene sequencing. Specificity was demonstrated by lack of detection of nine other Cryptosporidium species and genotypes, including 88 isolates of the closely related species, Cryptosporidium hominis. The PCRs were performed in separate tubes to maximize the possibility of detecting mixed Va–Vb infections; however, none were detected. The potential for multiplexing the reactions was also demonstrated, furthering the utility of the assay for large-scale occurrence and prevalence studies.
References
Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F (2003) Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41:2744–2747
Cama VA, Ross JM, Crawford S, Kawai V, Chavez-Valdez R, Vargas D, Vivar A, Ticona E, Navincopa M, Williamson J, Ortega Y, Gilman RH, Bern C, Xiao L (2007) Differences in clinical manifestations among Cryptosporidium species and subtypes in HIV-infected persons. J Infect Dis 196:684–691
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
Chalmers RM, Robinson G, Elwin K, Hadfield SJ, Xiao L, Ryan U, Modha D, Mallaghan C (2009a) Cryptosporidium rabbit genotype, a newly identified human pathogen. Emerg Infect Dis 15:829–830
Chalmers RM, Elwin K, Thomas AL, Guy EC, Mason B (2009b) Long-term Cryptosporidium typing reveals the aetiology and species-specific epidemiology of human cryptosporidiosis in England and Wales, 2000 to 2003. Euro Surveill 14:73–81
Chalmers RM, Elwin K, Hadfield SJ, Robinson G (2011) Sporadic human cryptosporidiosis caused by Cryptosporidium cuniculus, United Kingdom, 2007–2008. Emerg Infect Dis 17:536–538
Homem CG, Nakamura AA, Silva DC, Teixeira WFP, Coelho WMD, Meireles MV (2011) Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples. Parasitol Res. doi:10.1007/s00436-011-2694-8
Iqbal J, Khalid N, Hira PR (2011) Cryptosporidiosis in Kuwaiti children: association of clinical characteristics with Cryptosporidium species and subtypes. J Med Microbiol 60:647–652
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
Robinson G, Elwin K, Chalmers RM (2008) Unusual Cryptosporidium genotypes in human cases of diarrhea. Emerg Infect Dis 14:1800–1802
Robinson G, Wright S, Elwin K, Hadfield SJ, Katzer F, Bartley PM, Hunter PR, Nath M, Innes EA, Chalmers RM (2010) Redescription of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (Apicomplexa: Cryptosporidiidae): morphology, biology and phylogeny. Int J Parasitol 40:1539–1548
Shi K, Jian F, Lv C, Ning C, Zhang L, Ren X, Dearen TK, Li N, Qi M, Xiao L (2010) Prevalence, genetic characteristics, and zoonotic potential of Cryptosporidium species causing infections in farm rabbits in China. J Clin Microbiol 48:3263–3266
Sulaiman IM, Hira PR, Zhou L, Al-Ali FM, Al-Shelahi FA, Shweiki HM, Iqbal J, Khalid N, Xiao L (2005) Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol 43:2805–2809
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Acknowledgments
We thank the Toxoplasma Reference Unit, Public Health Wales Microbiology and the Haematology Department, Abertawe Bro Morgannwg University Hospital Trust for provision of non-Cryptosporidium DNA and the staff of the UK Cryptosporidium Reference Unit for collection of Cryptosporidium oocysts and preparation of DNA.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hadfield, S.J., Chalmers, R.M. Detection and characterization of Cryptosporidium cuniculus by real-time PCR. Parasitol Res 111, 1385–1390 (2012). https://doi.org/10.1007/s00436-012-2874-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-012-2874-1