NESTED POLYMERASE CHAIN-REACTION FOR DETECTION OF ENTEROCYTOZOON SALMONIS GENOMIC DNA IN CHINOOK SALMON ONCORHYNCHUS-TSHAWYTSCHA

A nested polymerase chain reaction (PCR) was developed for detection o f the microsporidian parasite Enterocytozoon salmonis in biological sa mples (blood buffy-coat cells, feces, tissues, lymphocyte cultures) of chinook salmon Oncorhynchus tshawytscha. A major second-round PCR pro duct of 407 bp was readily identifiable in ethidium bromide-stained ag arose minigels. An internal probe was used to verify the identity of t he amplified product by non-radioactive (digoxigenin-based) Southern b lotting; final confirmation was made by DNA sequence analysis. A dilut ion study using infected lymphocytes from in vitro cultures indicated that a single sound of PCR (35 cycles) was able to detect E. salmonis DNA from approximately 1000 infected cells. Sensitivity was increased with the full nested PCR (35 additional cycles), which detected parasi te DNA from less than or equal to 10 infected lymphocytes. The specifi city of the PCR was assessed with a panel of microsporidian and myxosp orean DNAs. In an experimental infection study, E. salmonis DNA was de tected in blood, feces, and tissues of infected chinook salmon but not in uninfected control fish.