Necrotizing hepatopancreatitis: Validation of an environmental PCR-based detection assay for use in descriptive epidemiologic studies

Necrotizing Hepatopancreatitis (NHP) is a poorly-understood bacterial disease of shrimp that has been responsible for devastating economic losses to the commercial shrimp industry over recent years. The etiologic agent for this disease is a nonculturable dimorphic rickettsia-like organism (RLO) that targets the shrimp hepatopancreas. When conducting NHP surveillance studies of certain shrimp farms, onset of clinical signs of disease by shrimp generally coincided with histologic detection of NHP in the hepatopancreatic tissues. Clinical response to administration of oxytetracycline-medicated feeds has been demonstrated to be more efficacious if given during the initial stages of an outbreak. Development of a screening assay for early detection of the NHP agent in shrimp tissue and/or environmental samples may facilitate determination of preclinical NHP infection in shrimp populations to enhance the administration of therapeutic measures for better disease control, as well as help define basic NHP spatial and temporal descriptors.;In this study, methodologies for the detection of the NHP-specific 16S rRNA biomarker, used for diagnostic verification in animal tissues, were modified for environmental screening purposes. The relative assay performance values for recently developed NHP-specific genetic-based assays were estimated in comparison to the histologic gold standard for NHP diagnosis on shrimp hepatopancreatic tissues. Matched pair analysis of assay results demonstrated moderate agreement between in situ hybridization and histologic examination results for duplicate samples, therefore, ISH was selected as the "genetic" gold standard for assay comparison with the NHP-specific environmental PCR assay. Since NHP is non-culturable in vitro, the clinical sensitivity limits of the NHP-specific assay on environmental samples was determined indirectly by conducting various dilution studies on NHP-seeded pond water and sediment samples. In a field-based prospective transitional study, the detection of NHP in the environment was compared with detection in shrimp tissues on four Texas shrimp farms with and without a clinical history of NHP. Screening shrimp postlarval tissues was determined to be a more reliable indicator of NHP in a shrimp pond than monitoring the environment. An environmental source/reservoir of NHP was not identified in this study, but incoming water appears to be the probable vehicle for NHP introduction as a point and/or vector-mediated source. Exploration of possible bacterial-protozoal or invertebrate associations is a potential focus for future epidemiologic studies.