Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and quantitative PCR assays

Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 10(8) copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.