Detecção de mixosporídeos por PCR em Tempo Real e por PCR Convencional em amostras de água de pisciculturas

Abstract

Myxosporeans are cnidarians, mandatory cosmopotian, endoparasites, with about 2.600 species listed. They have a complex biological cycle, characterized by transmission via multicellular spores. Myxosporeans infect a variety of tissues and vertebrate organs, mainly fish, but also amphibians, reptiles, birds and mammals. Annelids serve as the invertebrate hosts in the complex life cycle of myxosporeans. The latter are invertebrate hosts. A wide range of myxosporeans species are important fish pathogens, and present a great economic impact in aquaculture and commercial fishing worldwide. In Brazil, 140 myxosporeans species were registered. Some listed in native fish and fish farming have been shown to be pathogenic or in great potential to cause damage to their hosts, causing impacts and economic losses in both fishing and aquaculture. The current methods of detecting these parasites require the capture of hosts to obtain samples, which is toilful and time consuming. Even when there is no visible infection or high fish mortality rates, the detection of parasites can be challenging. This present study aimed to optimize methods to detect the presence of myxosporeans DNA in environmental samples using conventional PCR (cPCR) and Real-Time PCR (qPCR) techniques, offering simple methods of early detection, which can evaluate and quantify the presence of myxosporeans in fish farms water. 48 water samples were collected from two fish farms in the countryside of São Paulo state. They were stored in ice and transported to the laboratory. After, the samples were processed according to the following steps: filtration, vacuum on cellulose nitrate membrane; DNA extraction from the membrane; Reaction of Conventional PCR and Real-Time PCR with primers described in the literature, and primers built for this study. The methodology used for DNA filtration and extraction proved to be efficient for obtaining DNA from myxosporeans present in water and the DNA concentrations varied from 1.6 ng to 53,2 ng. The cPCR was able to detect up to 0.1 x 10-3 ng of DNA and identify 24 positive samples (50%). The qPCR detected concentrations up to 0,1 x 10-4 ng of myxosporeans DNA and 40 samples collected (83%) were positive. The results of this work showing to be more sensitive qPCR than cPCR able to detect the presence of myxosporid SSrDNA in a larger number of samples.