docResults for CAMACO Families Challenge Test with WSSV at UAZ
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Results for CAMACO Families Challenge Test with WSSV at UAZ
Hepatopancreatic Microsporidiosis Enterocytozoon hepatopenaei = a microsporidian infection in the HP affecting cultured Penaeus monodon, P. vannamei & P. stylirostris from East and SE Asia Enterocytozoon hepatopenaei Enterocytozoon hepatopenaei Enterocytozoon hepatopenaei Enterocytozoon hepatopenaei Enterocytozoon hepatopenaei E. hepatopenaei spores in HP squashes Courtesy of Natechanok Somboon, Thailand E. hepatopenaei spores in HP squashes Courtesy of Natechanok Somboon, Thailand E. hepatopenaei spores in HP squashes Courtesy of Natechanok Somboon, Thailand E. hepatopenaei spores in HP squashes Courtesy of Natechanok Somboon, Thailand E. hepatopenaei spores in HP squashes Courtesy of Natechanok Somboon, Thailand Courtesy of Kanokporn Chayaburakul Enterocytozoon hepatopenaei Courtesy of Kanokporn Chayaburakul Enterocytozoon hepatopenaei Photo showing EHP spores; 60x Photo showing EHP spores; 60x Photo showing EHP spores; 60x Photo showing EHP spores; 60x Photo showing EHP spores; 60x Photo showing EHP spores; 60x Photo showing EHP spores & pre-spore stages; 60x Micrograph showing a few spores, but mostly pre-spore stages; 60x Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP) Procedure for development of in situ hybridization EHP probe EHP-infected P. vannamei DNA Extraction from hepatopancreas PCR amplification of the 18S rRNA gene Cloning a 1.1 kb 18S rRNA gene fragment Labeling with digoxigenin-11-dUTP In situ hybridization In situ hybridization EHP ds DNA Adding DIG-EHP probe 90oC, 10 min Adding DIG-AP Ab Colorimetric development H&E staining and ISH detection of EHP in hepatopancreas of penaeid shrimp H&E P. vannamei Vietnam, 2014 P. stylirostris Brunei, 2006 ISH A PCR assay detecting EHP 510F 510R 1.1 kb 18S rRNA gene fragment of EHP 510-bp PCR amplicon EHP-510F: 5’-GCCTGAGAGATGGCTCCCACG EHP-510R: 5’-GCGTACTATCCCCAGAGCCCG PCR detection of EHP in Penaeus vannamei hepatopancreas tissue, feces and tank water HP HP Feces water VN/14 VN/15 Shrimp UAZ quarantined. 6 7 8 9 10 No-template 5 Artemia 4 SPF 3 Ameoba-like 2 Cotton shrimp 1 HP M Summary • ISH results are associated with lesions detected by histology. • EHP positive tissue: hepatopancreas. • EHP probe did not react to a Pleistophora-like microsporidium associated with cotton shrimp disease. • Development of a specific PCR assay detecting EHP. • EHP was detected in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.