Fecal samples as DNA source for the diagnosis of Necrotizing Hepatopancreatitis (NHP) in Penaeus vannamei broodstock

Abstract

Necrotizing Hepatopancreatitis (NHP) is a severe disease of cultivated penaeid shrimp caused by a pleomorphic, gram-negative, intracellular rickettsia-like bacterium. Current diagnostic methods for this disease are invasive, requiring dissection of the animal to perform histopathological analysis. In Colombia, NHP affects mainly broodstock, being a major cause of mortalities in maturation laboratories. In order to identify the presence of NHP without having to dissect the animal, we developed a PCR-based method using fecal samples as the DNA source. The DNA was extracted using a quick isolation protocol followed by amplification with primers specific for 16S ribosomal RNA gene sequences. To verify the sensitivity and specificity we analyzed samples from the same animal by PCR and in situ hybridization, and found 100% agreement. In addition, we amplified DNA extracted form paraffin blocks to confirm NHP diagnosis. PCR amplification from fecal samples and paraffin blocks yielded the expected 440 bp fragment. We conclude that PCR amplification from fecal samples is a valuable tool for the diagnosis of NHP in broodstock organisms, and that paraffin-fixed tissues can be used as a source of DNA for PCR amplification of NHP.