Assessing the viability of necrotizing hepatopancreatitis bacterium (NHPB) stored at − 20 °C for use in forced-feeding infection of Penaeus (Litopenaeus) vannamei

Hepatopancreatitis is a severe disease of cultivated penaeid shrimp caused by a bacterium called necrotizing hepatopancreatitis bacterium (NHPB). There are currently no techniques available to recover and isolate NHPB from cell lines or artificial culture medium. The aim of this study was to develop a method for collection and long-term storage of NHP-infected material without ultra freezing processes. Our method involved collection and freezing of NHPB-infected hepatopancreas tissue (HP) at − 20 °C, using glycerol as a cryoprotectant. This infective material was later used to orally infect shrimp. The viability of the NHPB extracts stored for 14 months in cryoprotectant at − 20 °C was tested by infecting healthy shrimp. Infection progress was monitored by PCR analysis of feces and by scanning electron microscopy. Additionally, we developed a forced-feeding infection procedure to transmit the disease. This type of infection method should allow for greater homogeneity and reproducibility in experimental procedures involving transmission of NHPB to Penaeus (Litopenaeus) vannamei shrimp. Forced-feeding procedure provided more efficient transmission than other previously reported methods. Our findings indicate that in vivo continuous propagation is not required to have a source of viable NHPB. The approach provides an easier, more economically advantageous and more feasible method for experimental infection for NHPB studies.