Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp

[Display omitted] •The EHP histology includes basophilic inclusions within hepatopancreas cells.•We developed a specific EHP in situ hybridization assay using digoxigenin-labeled probe.•An EHP specific PCR was developed targeting its 18S rRNA gene region.•By PCR, EHP was detected in the infected shr...

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Bibliographic Details
Published in:Journal of invertebrate pathology 2015-09, Vol.130, p.37-41
Main Authors: Tang, Kathy F.J., Pantoja, Carlos R., Redman, Rita M., Han, Jee Eun, Tran, Loc H., Lightner, Donald V.
Format: Article
Language:English
Subjects:
Amoeba
Animals
Artemia
Decapoda
EHP
Enterocytozoon
Enterocytozoon - isolation & purification
Enterocytozoon hepatopenaei
Genes, Fungal
In situ hybridization
In Situ Hybridization - methods
Litopenaeus vannamei
Marine
Microsporidium
PCR
Penaeidae - parasitology
Polymerase Chain Reaction - methods
Shrimp disease
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Summary:[Display omitted] •The EHP histology includes basophilic inclusions within hepatopancreas cells.•We developed a specific EHP in situ hybridization assay using digoxigenin-labeled probe.•An EHP specific PCR was developed targeting its 18S rRNA gene region.•By PCR, EHP was detected in the infected shrimp, their feces and water samples.•By PCR, EHP was detected in some Artemia biomass. A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.
ISSN:0022-2011
1096-0805
DOI:10.1016/j.jip.2015.06.009