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Preliminary results of Watercare project

The available classical diagnostic methods, due to many disadvantages, do not allow effective detection of pathogenic enteric viruses in environmental samples. Due to low concentrations of pathogenic viruses in the sea, it is important to develop an effective concentration procedure for their successful detection. In the first part of the doctoral thesis, we focused primarily on the development of a protocol for an effective concentration of pathogenic enteric viruses in coastal water samples. Monolithic chromatographic columns (BIAseparations) were used for the concentration of rotaviruses and noroviruses, prior to the detection with reverse transcription quantitative PCR in real time (RT-qPCR). We tested the efficiency of concentration using columns of various chemical properties and selected pathogenic enteric viruses (rotavirus and norovirus). Among them, hydrophobic interaction monolithic column (CIM® C4) was the most effective. CIM C4 was used to optimize the concentration step and tested in waters with different salinities. The presence of concentrated viruses was confirmed by RT-qPCR and transmission electron microscope. We have developed a protocol that enables rapid concentration of viruses in coastal waters of various salinities and can be used on-site. The presence of RoV and NoV was surveyed, using the developed concentration protocol, prior to one-step RT-qPCR molecular detection, in the inner part of the Bay of Koper, in mussel farming areas and a swimming area. Rotaviruses, noroviruses and fecal indicator bacteria were frequently detected in the inner part of the Bay of Koper. Rotaviruses and noroviruses were detected in the studied area, with higher rates close to the outfall of the wastewater treatment plant in the estuary of river Rižana and were also detected in the middle of the Bay of Koper and in areas used for recreation and mussel farming. The results show that water bodies, which are otherwise defined as suitable for bathing or mussel farming, based on the results of fecal indicator bacteria, still contain low concentrations of pathogenic enteric viruses. In addition to human pathogenic enteric viruses and faecal coliforms, changes in abundance of bacteria and virus particles were studied in relation to temperature, salinity, inorganic and organic nutrient concentrations in the organically polluted Rižana estuary. Preliminary results showed spatially and seasonally changes in bacterial and viral particles abundance, and bacterial composition spatially and seasonally. However, seasonality plays a greater role in bacterial dynamics. Klasične diagnostične metode, zaradi številnih pomanjkljivosti, ne omogočajo učinkovitega odkrivanja, sledenja in določanja patogenih virusov v okoljskih vzorcih. Zaradi nizkih koncentracij patogenih virusov v morju je zato pomembno razviti učinkovit postopek koncentriranja virusov kar bi omogočilo njihovo nadaljnjo uspešno odkrivanje. V prvem delu doktorske naloge smo se osredotočili predvsem na razvoj protokola za koncentriranje patogenih virusov v vzorcih obalne morske vode. Za koncentriranje patogenih rotavirusov in noravirusov smo uporabili monolitske kromatografske kolone (BISseparations), za njihovo detekcijo pa metodo kvantitativnega PCR v realnem času (RT-qPCR). Učinkovitost koncentriranja izbranih modelnih patogenih virusov (rotavirusov in norovirusov) smo optimizirali s uporabo različnih kromatografskih kolon s specifičnimi kemičnimi lastnostmi. Rezultati so pokazali, da je bila hidrofobna interakcijsko-monolitna kolona (CIM® C4) najučinkovitejša. Zato je bila ta kolona izbrana za nadaljnjo optimizacijo procesa koncentriranja virusov in testiranje morske vode različnih slanosti. Prisotnost virusov po koncentriranju smo potrdili z uporabo presevnega elektronskega mikroskopa kot tudi z uporabo RT-qPCR. Razvili smo protokol, ki je uporaben na terenu in ki omogoča hitro koncentriranje virusov v morskih vodah različnih slanosti. V nadaljevanju smo z novim postopkom koncentriranja virusov in z metodo RT-qPCR preverjali prisotnost RoV in NoV, in sicer v vzorcih izoliranih iz notranjega dela Koprskega zaliva, iz področja školjčišč in iz področja kopališč. Ugotovili smo, da so vzorci iz notranjega dela Koprskega zaliva vsebovali rotaviruse in noroviruse in bakterije, ki indicirajo na možnost onesnaženosti morske vode s fekalijami. Onesnaženost z rotavirusi in z norovirusi je bila znatnejša predvsem v področju izliva čistilne naprave v reko Rižano. Ne glede na to pa smo onesnaženost zaznali tudi v sredini Koprskega zaliva in območjih školjčišč in kopališč. Zanimivo je, da so tudi tisti vodni predeli kopališč in školjčišč, ki so glede na teste z indikatorskimi bakterijami uvrščeni kot varni, vsebovali nizke koncentracije patogenih virusov. Poleg samega določanja patogenih virusov in indikatorskih bakterij pa smo testirali tudi kako temperatura, slanost, anorganska in organska hranila v organsko onesnaženem ustju reke Rižane vplivajo na prisotnost celokupnega števila prisotnih bakterij in virusnih delcev. Preliminarni rezultati teh testiranj so pokazali visoke in hitre spremembe v koncentraciji preučevanjih virusov in bakterij še zlasti v odvisnosti od prostorskih in sezonskih vplivov. Letni časi so še posebej vplivali na spremembe v koncentracijah indikatorskih bakterij.