Lethal toxicity of Vibrio harveyi to cultivated Penaeus monodon induced by a bacteriophage

In Southern Thailand in 1996, intense luminescence in many shrimp rearing ponds was accompanied by massive mortality resulting in total crop loss within 3 or 4 d. Mortality was correlated with gross signs which shrimp farmers called (English translation) tea-brown gill syndrome (TBGS). Histological examination of moribund shrimp revealed massive lesions of the hepatopancreas characterized by hemocytic infiltration and the presence of bacterial cells. Bacterial isolation yielded several strains tentatively identified as Vibrio harveyi on the basis of luminescence and growth on BTB Teepol agar. Representative isolate VH1039 was selected and identified by biochemical tests as V. harveyi. When strain VH1039 and the other luminescent isolates were injected into normal shrimp (1 x 10(7) cells shrimp(-1)), no significant mortality was observed in comparison with control shrimp injected without bacteria. Nor was any significant mortality observed after injection of supernatant fluids from normal or sonicated bacterial cultures of VH1039 (1 x 10(8) cells ml(-1)). Transmission electron microscopy (TEM) of hepatopancreatic tissue from farmed TBGS shrimp revealed bacterial cells of Vibrio morphology together with large numbers of bacteriophage particles that had round to hexagonal heads of approximately 60 nm diameter and tails of approximately 100 nm length. These were either free, attached to cell walls of intact bacteria or in various stages of replication within bacterial cells. Gills of farmed TBGS shrimp were subsequently homogenized in lobster haemolymph buffer (LHB) and membrane filtered (0.22 mu m) Compared to control shrimp injected with LHB, shrimp injected with the 1000x diluted gill filtrate (DGF) showed no significant mortality. However, when DGF was injected together with 1 x 107 cells of strain VH1039, there was total mortality within 48 h. High and rapid mortality concurrent with brown gills was seen only in the mixed injection group. TEM of the artificially infected shrimp tissues did show the presence of bacterial cells, but no mature bacteriophage particles or lysed bacterial cells were found similar to those seen in farmed TBGS shrimp. In further tests, addition of DGF to cultures of VH1039 induced extreme but transitory toxicity of culture filtrates from 24 to 36 h. Treatment of DGF with a germicidal lamp (UV) for 30 min or with heat at 100 degrees C for 15 min failed to stop shrimp mortality from mixed DGF/VH1039 injections. However, mortality was stopped if the heated DGF was treated with DNase. The results suggested that a bacteriophage may sometimes mediate the toxicity of V. harveyi in Penaeus monodon by the transfer of a toxin gene(s) or a gene(s) controlling toxin production.