DAO 67:163-169 (2005)  -  doi:10.3354/dao067163

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Amanda G. Vincent, Jeffrey M. Lotz*

Department of Coastal Sciences, The University of Southern Mississippi, Gulf Coast Research Laboratory, PO Box 7000, Ocean Springs, Mississippi 39566-7000, USA
*Corresponding author. Email:

ABSTRACT: Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular α-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 4 l of aerated artificial seawater at salinity 30 ± 1 ppt and maintained in a water bath at 30 ± 1°C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 103 to 107 copies mg–1 of hepatopancreas and 101 to 105 copies mg–1 of feces. Lethal infections contained 106 to 107 copies mg–1 of hepatopancreas and 103 to 106 copies mg–1 of feces.

KEY WORDS: Necrotizing hepatopancreatitis · Litopenaeus vannamei · Real-time PCR · Shrimp diseases · Aquaculture

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