TRANSMISSION ELECTRON MICROSCOPIC ANALYSIS OF Vibrio spp.CELLS FROM HEPAROPANCREAS OF WHITE SHRIMP Penaeus vannamei

Shrimp farming has mantained an exponential growth during the last years which also has caused the spread of different viral and bacterial diseases. Vibrio is the most common genus associated with this situation. Acute Hepatopancreatic Necrosis Disease (AHPND) is caused by strains of Vibrio parahaemolyticus (Vp), during infection the bacteria colonize the stomach of the organisms and release a toxin that damages the functionality of the hepatopancreas and causes mortality. In 2013, this disease affected negatively shrimp production in Northwestern Mexico. The aim of this study was to analyze the morphology of Vibrio genus bacteria in white shrimp hepatopancreas Penaeus vannamei using the Transmission Electron Microscopy technique in samples from farms in the State of Sonora, Mexico.

Four samples of hepatopancreas bacteriology were performed on TCBS plates (MCD LAB) in order to obtain green and yellow colonies. Green colonies (presumptive to V. parahaemolyticus) were isolated and enriched in TSB (MCD LAB) (2% NaCl) to obtain DNA by lysis buffer and perform Real Time PCR using the IQ Real AHPND / EMS kit. For the analysis of Transmission Electron Microscopy, the protocol of Segovia and Zavala was used; where the hepatopancreas were fixed in 2% glutaraldehyde (GTA) for 24 h, after that GTA was discarded and 500 µl of sodium cacodylate (two washes) was added, then it was passed to the dehydration process where different washes were made with 70, 90 and 100% alcohol. A mixture of acrylic resin and absolute alcohol (1: 1) was added and maintained for 18 h, then a sample fragment was taken and placed in a 0.2 tube ml where acrylic resin was added, then the tube was placed in a UV lamp for 6 h and passed to the oven at 60 °C for 3 h. After this time the cuts were made in the microtome and the cut was placed on the grids, finally the sample was observed in the Transmission Electron Microscope (MET).

As results the hepatopancreas isolates presented large and green colonies with creamy consistency in TCBS, however, they also observed the presence of yellow colonies. In the Real-Time PCR analysis they were confirmed as positive for AHPND, in the hepatopancreas sections bacteria with a cell size from 0.39 to .90 µm were observed, thus showing very similar characteristics to Vp which have a cell size of between 0.40- 3 µm, however they do not have "waves" on their wall, characteristic of a Vp.