Preservation of necrotizing hepatopancreatitis bacterium (NHPB) by freezing tissue collected from experimentally infected Litopenaeus vannamei

Abstract

Necrotizing hepatopancreatitis (NHP), a severe disease of penaeid shrimp, is caused by bacteria (NHPB) that have previously been demonstrated to reside in tubular epithelial hepatopancreatic (HP) cells of infected shrimp. There has yet to be a successful in vitro culture method to grow the intracellular organism; therefore, it must be propagated in vivo via transmission from NHPB-infected shrimp to healthy individuals. In our studies, NHPB propagation tanks containing infected shrimp were used to maintain a constant supply of organisms for experiments. In order to develop a method for storing infectious NHPB material for future challenge studies, we collected HP tissue containing NHPB by flash freezing whole, fresh HPs at -80°C for up to 80 d and used it to successfully infect specific pathogen-free Litopenaeus vannamei per os in controlled experiments. HP tissue samples were collected from dead shrimp, and PCR was performed to confirm the presence of NHPB. Our results demonstrate that the infectivity of NHPB in tissue is not altered after being frozen at -80°C when compared to NHPB in fresh tissue. Thus, the continual propagation of NHPB in vivo is not required to assure a source of the infectious agent. © Inter-Research 2006.