Hepatopancreatic Necrosis Disease (AHPND)-like disease in farmed P Hepatopancreatic Necrosis Disease (AHPND)-like disease in farmed P. monodon in Queensland AUSTRALIAN ANIMAL HEALTH LABORATORY (AAHL) Nick Moody | Team Leader, Aquatic Diagnostic Capability Ridley Australian Prawn & Barramundi Farmers Symposium – 2 August 2016

AHPND Also known as Early Mortality Syndrome (EMS) Notifiable to the OIE, from 1 January 2016 Led to significant reductions in prawn production globally Caused by “specific virulent strains of Vibrio parahaemolyticus, namely VPAHPND, which contains one or more extrachromosomal plasmids, including a unique, previously unreported, large, plasmid with a size of ~70 kbp” Australian disease does not meet the current OIE definition of a confirmed case Not in V. parahaemolyticus and in one case not in the plasmid AHPND-like disease in farmed P. monodon in queensland

Molecular tests at AFDL Primer/probe Sequence (5’-3’) Target Amplicon Size (bp) Reference VpPirA F TTGGACTGTCGAACCAAACG PirA gene 135 Han et al (2015) VpPirA R GCACCCCATTGGTATTGAATG VpPirA probe FAM- AGACAGCAAACATACACCTATCATCCCGGA-TAMRA   AP1 F CCTTGGGTGTGCTTAGAGGATG pVA1 plasmid 700 Flegel and Lo (2014) AP1 R GCAAACTATCGCGCAGAACAC C AP2 F TCACCCGAATGCTCGCTTGTGG AP2 R CGTCGCTACTGTCTAGCTGAAG AP3 F ATGAGTAACAATATAAAACATGAAAC 333 Sirikharin et al (2014) AP3 R GTGGTAATAGATTGTACAGAA AP4 F1 PirA + PirB gene 1269 Sritunyalucksana et al (2015) AP4 R1 ACGATTTCGACGTTCCCCAA AP4 F2 TTGAGAATACGGGACGTGGG 230 AP4 R2 GTTAGTCATGTGAGCACCTTC 143F TGCCTTATCAGCTNTCGATTGTAG 18S rRNA gene 848 Lo et al (1996) 143R TTCAGNTTTGCAACCATACTTCCC 16S-27f AGAGTTTGATCMTGGCTCAG 16S rRNA gene 1465 Jiang et al (2006) 16S-1492r AAGGAGGTGATCCAGCCGCA Vpara toxR-F GTCTTCTGACGCAATCGTTG Tox R gene 367 Kim at al (1999) Vpara toxR-R ATACGAGTGGTTGCTGTCATG AHPND-like disease in farmed P. monodon in queensland

FRDC 2016-013: “Aquatic Animal Health and Biosecurity Subprogram: Comparative pathogenicity of exotic AHPND and the presumptive bacterial hepatopancreatitis detected in farmed Penaeus monodon in Queensland AHPND-like disease in farmed P. monodon in queensland

FRDC 2016-013 PI: Nick Moody, AFDL, Geelong CI: Ian Anderson, BSL, Brisbane Start Date: 04-07-16 End Date: 15-12-17 FRDC funds: $131,106 AHPND-like disease in farmed P. monodon in queensland

FRDC 2016-013 Two Australian representative isolates: North Queensland: PirA+PirB toxin gene in the chromosome of Vibrio harveyi (no pVA1 plasmid present) Central Queensland: PirA+PirB toxin gene present in the pVA1 plasmid in something other than Vibrio parahaemolyticus (not V. harveyi) Exotic AHPND strains PirA+PirB toxin gene in the pVA1 plasmid in V. parahaemolyticus AHPND-like disease in farmed P. monodon in queensland

FRDC 2016-013 Infectivity trials will be based on the model of Tran et al (2013) included in the current revision of the OIE Manual for AHPND Use P. monodon as a positive infection control Use P. merguiensis as a negative extraction control Whole genome sequence on the PacBio platform (U Melb) Current MiSeq data generated at AAHL will assist with assembly Develop material for inter-laboratory test evaluation Seeking prawns for bioassay trials in August/September 2016 AHPND-like disease in farmed P. monodon in queensland

Acknowledgements APFA, Helen Jenkins FRDC AAHBS FRDC QRAC Biological Sciences Laboratory CSIRO AAHL AAHL Fish Diseases Laboratory AHPND-like disease in farmed P. monodon in queensland

Thank you Nick Moody Team Leader, Aquatic Diagnostic Capability Senior Research Scientist t +61 3 5227 5749 e nick.moody@csiro.au AUSTRALIAN ANIMAL HEALTH LABORATORY (AAHL)

Toxin gene location in the Plasmid AHPND-like disease in farmed P. monodon in queensland