Identification of Cryptosporidium parvum and Blastocystis hominis subtype ST3 in Cholga mussel and treated sewage: Preliminary evidence of fecal contamination in harvesting area.

Cryptosporidium parvum and Blastocystis hominis are foodborne parasites known for causing diarrhea. They accumulate in mussels grown on contaminated water bodies, due to the discharge of treated sewage from sewage treatment plants (STP). Despite this, some countries like Chile do not include these parasites in the control or monitoring of sewage water. The objective of this research was to evaluate the contamination of C. parvum. and B. hominis from treated sewage (disinfected by chlorination) and Cholga mussels in a touristic rural cove from the bay of Concepción. Cholga mussels from commercial stores and a treated sewage sample were analyzed. Cryptosporidium spp. was identified by Ziehl-Neelsen-Staining (ZNS) and C. parvum by direct-immunofluorescence assay (IFA) from ZNS-positive samples. Blastocystis hominis was identified by PCR using locus SSU rDNA. C. parvum and B. hominis subtype ST3 were found in 40% and 45% of Cholga mussel samples, respectively, and both parasites were identified in the treated sewage. Blastocystis hominis SSU rDNA gene alignment from Cholga mussels and treated sewage showed 89% of similarity, indicating that could be the same parasite in both samples. We describe the first evidence of possible contamination with these parasites from treated sewage to Cholga mussel suggesting an environmental contamination with high human risk. Based on these results, further studies will consider all the rural coves and STP from the bay to prevent possible contamination of these parasites.

Development of liquid chromatography tandem mass spectrometry method to quantify cyclobutane pyrimidine dimer photolyase activity by detection of 15mer oligonucleotide as reaction product.

Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15 mM diisopropylethylamine/20 mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7 min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was ∼ 94% and the limit of quantification was 13.4 nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.