Molecular diagnosis of Nile tilapia (Oreochromis niloticus, L.) diseases in Egypt

ملخص البحث

The Nile tilapia, Oreochromis niloticus is widely used in aquaculture and is becoming an increasingly popular species for culture in environmentally controlled conditions in temperate countries. Intensification of fish adversely affects fish health and tends to produce a poor environment for fish and increase their susceptibility to infections. Disease outbreaks are encountered in the rapidly developing aquaculture industry and affecting the economic development of this sector Pseudomonas fluorescens and Pseudomonas aeruginosa are widely distributed in the freshwater ecosystem and are recognized as the primary causal agents of bacterial haemorrhagic septicaemia in fish, usually in association with environmentally stressful conditions such as high temperatures or overcrowding. To overcome the problems of traditional methods of diagnosis, molecular diagnostic procedures have been devised. At present, restriction enzyme digestion, probe hybridization and polymerase chain reaction (PCR) are commonly applied in the routine diagnosis of fish diseases. Fish viral diseases have become important, demanding effort in developing cell lines from the tropical cultivable fish species. So, this study aims to develop a molecular diagnosis method for detection of P. fluorescens and P. aeruginosa in tissues of naturally infected O. nilotica and developing a cell culture system from the ovarian tissue of the fish. Therefore the following experiments were performed: - Molecular diagnosis of P. fluorescens and p. aeruginosa by using restriction enzyme digestion (Five Restriction enzymes EcoR1, Tru91, Bam HI, Hae III and Hind III were used), probe hybridization and polymerase chain reaction (simple, and multiplex PCR) by using two sets of specific primers of 16S rRNA gene - Detection of pathogens through purified DNA using different annealing temperature. - Direct detection of pathogens from bacterial colonies and different concentrations of organs homogenates (Six dilutions 1/1, 1/2, 1/3, 1/4, 1/5 and 1/6 w/v). - Random Amplified Polymorphic DNA (RAPD) analysis of P. fluorescens and P. aeruginosa by using 16 random primers. - Developing a cell culture system from the ovarian tissue of O. niloticus through employing three kinds of media (L-15, MEM and RPMI) and deferent incubation temperature (16, 27 and 30oC). The main results could be summarized as follows: - P. fluorescens and P. aeruginosa were pathogens for O. niloticus. - The specific primers of P. fluorescens gave a unique and specific amplification band in an 848 bp length at different annealing temperatures of 50, 53, 55, 57, 59 and 62°C. - The specific primers of P. aeruginosa gave a unique and specific amplification band in a 615 bp length at different annealing temperatures of 55, 59 and 62°C. - Unique and specific amplification band in an 848 bp length were obtained from the all tested dilutions of organs with P. fluorescens and P. aeruginosa with some differences in the density of bands. - Restriction enzyme digestion analysis indicated that there is no difference between inoculated and re-isolated bacteria. - Positive results were obtained with all samples used in DNA hybridization - Among the three media of minimum essential medium MEM, RBMI and L-15, each supplemented with 20% foetal bovine serum, L-15.was found to be the most suitable medium in terms of its efficacy to support rapid cells attachment and cell proliferation. - At different incubation temperature of ovaries cells (16, 27 and 30oC), no results were obtained when cells were incubated at 16 oC compared with 27 and 30oC.

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Molecular diagnosis - Nile tilapia diseases in- Egypt

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