A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1.

Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA.

A staining method for detection of Enterocytozoon hepatopenaei (EHP) spores with calcofluor white.

A fluorescent stain, calcofluor white (CFW), was used for detection of spores of the microsporidium Enterocytozoon hepatopenaei (EHP). EHP spores in suspension, in feces, or in the infected hepatopancreas of the shrimp Penaeus vannamei, can be easily stained with this chitin and cellulose binding dye to show distinct blue-white fluorescent oval walls. The dye does not stain the host tissues. EHP spores showed orange-red spots by staining with hematoxylin and phloxine (H&P) in the section. CFW staining provides a simple and rapid method for determining the presence of EHP spores in fecal or tissue samples.

Description of a Natural Infection with Decapod Iridescent Virus 1 in Farmed Giant Freshwater Prawn, Macrobrachium rosenbergii.

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called "white head" has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, M. superbum, Penaeus vannamei, and Cladocera from a farm suffering from white head in Jiangsu Province were collected and analyzed in this study. Pathogen detection showed that all samples were positive for Decapod iridescent virus 1 (DIV1). Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue, hepatopancreas, and gills of M. rosenbergii and M. nipponense. Blue signals of in situ digoxigenin-labeled loop-mediated isothermal amplification appeared in hematopoietic tissue, hemocytes, hepatopancreatic sinus, and antennal gland. Transmission electron microscopy of ultrathin sections showed a large number of DIV1 particles with a mean diameter about 157.9 nm. The virogenic stromata and budding virions were observed in hematopoietic cells. Quantitative detection with TaqMan probe based real-time PCR of different tissues in naturally infected M. rosenbergii showed that hematopoietic tissue contained the highest DIV1 load with a relative abundance of 25.4 ± 16.9%. Hepatopancreas and muscle contained the lowest DIV1 loads with relative abundances of 2.44 ± 1.24% and 2.44 ± 2.16%, respectively. The above results verified that DIV1 is the pathogen causing white head in M. rosenbergii. M. nipponense and Pr. clarkii are also species susceptible to DIV1.