Approaches to genotyping individual miracidia of Schistosoma japonicum
- 228 Downloads
Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.
KeywordsSchistosomiasis Adult Worm Praziquantel Whole Genome Amplification Schistosoma Japonicum
The authors wish to thank Kang Jun-xing, former General Director of the Sichuan Center for Disease Control and Prevention (Chengdu, People’s Republic of China) for his continued support and collaboration. The authors also wish to acknowledge Zhong Bo, Lu Ding, Ye Hong, Cui Lina, Chen Lin, Zhang Yi, and Meng Xian-hong of the Institute of Parasitic Diseases, Sichuan Center for Disease Control and Prevention for their support and assistance with the work described herein. We also thank county Anti-Schistosomiasis Control Station leaders and staff from the two counties included in this study for their hard work and hospitality during the fieldwork of sample collection conducted in 2007, 2008, and 2010. This work was supported in part by the NIH/NSF Ecology of Infectious Disease Program (grant no. 0622743), by the National Institute for Allergy and Infectious Disease (grant no. K01AI091864 and R01AI068854), and by the Emory Global Health Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interests
No author has any competing interests to declare.
- Bureau of Disease Control and Prevention, Chinese MOH (2001) Handbook of schistosomiasis control. Shanghai Science & Technology Press, Shanghai, In ChineseGoogle Scholar
- Dean FB, Hosono S, Fang LH, Wu XH, Faruqi AF, Bray-Ward P, Sun ZY, Zong QL, Du YF, Du J, Driscoll M, Song WM, Kingsmore SF, Egholm M, Lasken RS (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 99:5261–5266PubMedCrossRefGoogle Scholar
- Gower CM, Shrivastava J, Lamberton PHL, Tollinson D, Webster BL, Emery A, Kabatereine NB, Webster JP (2007) Development and application of an ethically and epidemiologically advantageous assay for the multi-locus microsatellite analysis of Schistosoma mansoni. Parasitology 134:523–536PubMedCrossRefGoogle Scholar
- Li YL, Guan XH (2010) Human Parasitology, 7th edn. People’s Health Press, Beijing, p 111, In ChineseGoogle Scholar
- Liu Y, Luan RS, Zhong B, Zheng W, Zou J, Qiu DC (2010) Determinants of re-emerging of schistosomiasis epidemic in areas with transmission once controlled in Sichuan Province. J Prev Med Inf 3:165–169, In Chinese, English abstractGoogle Scholar
- Rudge JW, Carabin H, Balolong E, Tallo V, Shrivastava J, Lu DB, Basañez MG, Olveda R, McGarvey ST, Webster JP (2008) Population genetics of Schistosoma japonicum within the Philippines suggest high levels of transmission between humans and dogs. PLoS Negl Trop Dis 2:e340. doi: 10.1371/journal.pntd.0000340 PubMedCrossRefGoogle Scholar
- Stothard JR, Webster BL, Weber T, Nyakaana S, Webster JP, Kazibwe F, Kabatereine NB, Rollinson D (2009) Molecular epidemiology of Schistosoma mansoni in Uganda: DNA barcoding reveals substantial genetic diversity within Lake Albert and Lake Victoria populations. Parasitology 136:1813–1824PubMedCrossRefGoogle Scholar
- WHO (2012) Accelerating work to overcome the global impact of neglected tropical diseases—a roadmap for implementation. WHO, Geneva, www.who.int/neglected_diseases/NTD_RoadMap_2012_Fullversion.pdf Google Scholar