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Molecular Characterization of Cryptosporidium spp and Giardia spp species in urban water catchment in Campinas, Sao Paulo, Brazil

Grant number: 13/26211-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2014
Effective date (End): June 30, 2014
Field of knowledge:Engineering - Sanitary Engineering - Basic Sanitation
Principal researcher:Regina Maura Bueno Franco
Grantee:Guilherme Batista Felisardo
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated research grant:12/50522-0 - Environmental contamination by protozoa Giardia spp. and Cryptosporidium spp. and Ascaris suum: challenges of detection, removal and inactivation of infective forms, AP.TEM

Abstract

The city of Campinas, SP, is supplied by Atibaia Capivari and rivers, which supply 95 % and 5 % of total water withdrawal for public supply, respectively. Both rivers belong to the Piracicaba Basin, Capivari and Jundiaí, covering one of the most populated and industrialized regions of São Paulo. These water bodies exhibit a high degree of eutrophication due to the release of domestic sewage and industrial effluents. The presence of waterborne pathogenic protozoa, Cryptosporidium spp. and Giardia spp. has been documented in these watersheds. Significant concentrations of microbiological indicators were also detected in the waters of these rivers. Given these findings and in order to identify possible sources of contamination of water sources that supply the city of Campinas, this research aims to investigate the occurrence of different species of Cryptosporidium and Giardia genotypes in samples of raw surface water from rivers Atibaia and Capivari. 8 water samples (10 liters / sample) will be collected for each source. The water samples after the concentration procedure and elution initially using the methodology membrane filtration or Filta -Max system according to the turbidity of the sample, be purified by immunomagnetic separation (IMS). After the acid dissociation fractions resulting from the IMS will be subjected to molecular studies. DNA extraction will be performed with the ZR Fungal / Bacterial DNA miniprep kit - Zymo Research, and then two distinct regions of the " ² - Giardin " gene will be used to molecularly confirm the presence of Giardia in samples (Mahbubani et al, 1992). A fragment of 753pb gene " ² - Giardin " will be sequenced and used to characterize the samples of the seven assemblies described above. The primers G7 and G59 were based on the complete gene sequence and have a resolution not only to determine the genetic assembly of the sample as well as the subgroup to which it belongs (Caccio et al, 2002). Will also sequenced a fragment of 713pb gene " glutamate dehydrogenase " GDH1 and GDH4 using primers (Homan et al, 1998). This region also identifies not only the assemblies as well as existing subgroups. The samples will be sequenced in duplicate, using the direct and reverse primers and employing sequencer ABI 3500 XL (Applied Biosystems). The sequences are edited with the help of the program DNASTAR SeqMan package. All samples sequenced the project will be analyzed together with the reference sequences of seven distinct assemblies available in GenBank (NCBI). Molecular characterization of Cryptosporidium species is performed using the nested PCR technique according to Xiao et al, 2000. The products of the nested PCR will also be sequenced and the sequences obtained are compared with those deposited in GenBank reference through global alignment with the software CLUSTAL W. After alignment with reference sequences, both sequences as Giardia Cryptosporidium will be subjected to different methods of phylogenetic analysis (maximum likelihood, neighbor -joining and Bayesian method) to identify interest groups. The results allow to identify the main sources of contamination of water sources studied, and also help to elucidate the epidemiology and environmental dispersion of these waterborne pathogenic protozoa in the metropolitan region of Campinas, SP.

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