MOLECULAR PHYLOGENY AND IN-SITU DETECTION OF THE ETIOLOGIC AGENT OF NECROTIZING HEPATOPANCREATITIS IN SHRIMP

Necrotizing hepatopancreatitis (NHP) is a severe disease of farm-raise d Penaeus vannamei that has been associated with mortality losses rang ing from 20 to 95%, NHP was first recognized in Texas in 1985 (S. K. J ohnson, p, 16, in Handbook of Shrimp Diseases, 1989) and is an economi cally important disease that has limited the ability to culture shrimp in Texas, The putative cause of NHP is a gram-negative, pleomorphic, intracellular, rickettsia-like bacterium that remains uncultured in pa rt because of the absence of established shrimp cell lines, The inabil ity to culture the NHP bacterium necessitated the use of molecular met hods for phylogenetic placement of the NHP bacterium, The gene encodin g the 16S rRNA (16S rDNA) of this shrimp pathogen was amplified by PCR , cloned, and sequenced, Sequence analysis of the cloned 16S rDNA indi cates that the NHP bacterium is a member of the a subclass of the Plot eobacteria, Within the alpha subclass, the NHP bacterium is shown to b e most closely related to bacterial endosymbionts of protozoa, Caediba cter caryophila and Holospora obtusa, Also, the NHP bacterium is disti nct from but related to members of the typhus group (Rickettsia typhi and R. prowazekii) and spotted fever group (R. rickettsii) of the fami ly Rickettsiaceae, Fluorescently labeled oligonucleotide DNA probes th at bind to variable regions (V2, V6, and V8) of 16S rRNA of the NHP ba cterium were used to detect the bacterium in infected shrimp by in sit u hybridization. This technique provided direct visual evidence that t he 16S rDNA that was amplified, cloned, and sequenced was derived from the intracellular bacterium that infects the hepatopancreas of farm-r aised P. vannamei shrimp.