Molecular and serological methods for the detection of marine bacteria pathogenic to fish = Métodos serolóxicos e moleculares para a detección de bacterias mariñas patóxenas de peixes

Resumo

Aquaculture industry has developed considerably during the last decades and is, nowadays, one of the fastest-growing food productions sectors in the world. Marine fish culture is dominated by Atlantic salmon (Salmo salar), led by Norway, Chile, United Kingdom, Canada and Ireland. Other economically important marine fish species are gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax), eel (Anguilla anguilla) and turbot (Scophthalmus maximus), which are reared in Mediterranean and southern Atlantic coasts of Europe and yellowtail (Seriola quinqueradiata), ayu (Plecoglossus altivelis), flounder (Paralychthys olivaceous) and seabream (Pagrus major) in Japan. Other high-value flatfish, sole (Solea senegalensis), naturally distributed in Mediterranean and Atlantic waters is also reared successfully in extensive aquaculture productions in Spain and Portugal. As occurs in other areas of animal production, infectious diseases are an ever-present hazard in aquaculture, with the potential to cause heavy stock losses or to reduce the commercial value of the fish as food for humans. Unfortunately, while knowledge of the physiological and nutritional requirements of the cultured species is gradually achieved, the preservation of health status of the new and old species maintained in captivity remains a high priority. The availiability of reliable methods for the detection, identification and epidemiological typing of pathogenic organisms in diseased and carrier fish is crucial for the establishment of effective control measures, in order to eliminate or reduce the impact of the diseases in farmed fish. The main goal of this PhD study was the development and evaluation of molecular and serological methods for the detection of bacteria pathogenic for marine fish. The development of new detection methods will allow a rapid diagnosis of some of the major diseases affecting aquaculture production in Galicia. In the first part of this work the efficacy of two serological commercial methods, AQUAEIA-Va and AQUARAPID-Va (BIONOR), and the Dot blot assay for the detection of Vibrio anguillarum was evaluated. All three serological methods detected the strains of the serotypes O1 and O2 of V. anguillarum, but only the Dot blot assay allows the detection and specific differentiation of the three pathogenic serotypes of this bacterial species. However, the portability and the simplicity of the AQUAEIA-Va and AQUARAPID-Va systems (does not require trained personnel or complex technical requirements for making and interpreting results), could be considered an useful tool for the in situ identification of this fish pathogen, as well as for the diagnostic of the vibriosis caused by the serotypes O1 and O2 of V. anguillarum. In order to develop molecular techniques that do not require prior isolation of the organism, the second part of this study (Chapters 3, 4, 5 and 6) was focused on the design of different diagnostic techniques based on the polymerase chain reaction (PCR). With this aim, we have developed: 1) a single and a multiplex PCR assays for the detection of V. anguillarum and differentiation of the serotype O1 of this bacterial species, respectively, 2) a diagnostic method that combines multiplex PCR and hybridization in DNA microarrays, applicable for the detection of a selected group of bacteria causing mortality in fish (Aeromonas salmonicida, Vibrio anguillarum, Photobacterium damselae subsp. damselae, Vibrio parahaemolyticus and Vibrio vulnificus), and 3) a duplex-Real time PCR method, that allows the simultaneous identification of the fish pathogens A. salmonicida and V. anguillarum, using the intercalating agent SYBR Green for the detection of PCR amplification products, as a less expensive option. The PCR methods developed showed high specificity and high sensitivity when using pure cultures and/or tissue samples of infected fish, allowing detection of pathogens under study, even in asymptomatic carriers.