The use of beta-tubulin gene for phylogenetic analysis of the microsporidian parasite Enterocytozoon hepatopenaei (EHP) and in the development of a nested PCR as its diagnostic tool

Abstract

Tubulins are major components of microtubules, structures that are involved in many cellular processes. The gene β-tubulin, which encodes a β-tubulin protein, is conserved, and yet with sufficient variability to be useful for phylogenetic studies and for use in molecular diagnostic protocols. We sequenced and analyzed a fragment (870-bp) of β-tubulin gene of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp and associated with growth retardation. The sequence was then used to conduct a phylogenetic study to determine the position of EHP within the Microsporidia and to develop primers for an EHP nested PCR method. Blast analysis revealed a 75% nucleotide identity of EHP β-tubulin to that of E. bieneusi. Similar to other studies based on the small subunit rRNA sequence, the phylogenetic analysis using β-tubulin sequences clustered EHP with E. bieneusi and two other crustacean microsporidia, Agmasoma penaeii and Hepatospora eriocheir. As for the diagnostic use, a nested PCR method was developed that successfully detected EHP in infected Penaeus vannamei from Indonesia, Thailand, Vietnam, and India and in some cases of Artemia biomass. There were no cross-reactions to the shrimp and other crustacean genomes. This nested PCR is 100-fold more sensitive than the one-step PCR and thus suitable for detecting a low level of EHP infection.