Immunofluorescence detection of Ecytonucleospora hepatopenaei (EHP) in Penaeus vannamei

Abstract

Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum, a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.