Monitoring of Hawaiian Invertebrates for White Spot Syndrome Virus. David Albert Dr. Jo-Ann Leong Dr. Teresa Lewis

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1 Monitoring of Hawaiian Invertebrates for White Spot Syndrome Virus David Albert Dr. Jo-Ann Leong Dr. Teresa Lewis

2 What is White Spot Syndrome Virus (WSSV) Enveloped, rod shaped Double stranded DNA virus 70 to 150 nm X 275 to 380 nm 305,107 bp (Yang et. al. J. of Virology, Dec 2001) Viability of free virus in sea water is 3-4 days Mortality in Penaeid shrimp reaches 90% to 100% in 3 to 7 days Images obtained from web site of Dr. Philip C. Loh of the University of Hawaii at Manoa Department of Microbiology

3 What is White Spot Syndrome Virus (WSSV) TIME LINE White Spot was first described in Penaeus monodon in Taiwan This disease caused a 70% reduction in shrimp production in China WSSV quickly spreads to other shrimp growing regions of Asia White spot disease detected in the U.S. at a farm in Texas 1995-Present -- Appeared in every major shrimp producing area of the world

4 Hawaii Dept. of Agriculture April 14, 2004

5 The Problem: Vector for introduction of WSSV into Kauai unknown WSSV may have been introduced into Hawaii by frozen shrimp products WSSV is both detectable and infectious in frozen shrimp imports (Nunan et al., 1998) WSSV also infects a wide variety of crabs, copepods, and other arthropods (Cai et al. 1995; Chang et al. 1998; Lightner et al. 1998; Wang et al. 1998).

6 Natural and Experimental Hosts of WSSV Penaeus monodon Penaeus semisulcatus Penaeus merguiensis Penaeus indicus Penaeus chinensis Penaeus penicillatus Penaeus japonicus Penaeus vannamei Penaeus stylirostris Penaeus setiferus Penaeus occidentalis Penaeus aztecus Penaeus duorarum Penaeus californiensis Metapenaeus ensis Trachypenaeus curviostris Exopalaemon orientalis Macrobrachium rosenbergii Ocronectes punctimanus Procambarus clarkii Charybdis feriatus Charybdis granulata Charybdis annulata Charybdis lucifera Doclea hybrida Grapsus albolineatus Halimede ochtodes Liagore rubronaculata Lithodes maja Matuta miersi Paradorippe granulata Parthenope prensor Philyra syndactyla Podophthalmus vigil Portunus pelagicus Portunus sanguinolentus Scylla serrata Thalamita danae Panulirus versicolor Panulirus penicillatus Panulirus ornatus Panulirus longipes Cherax quadricarinatus Calappa philargius Calappa lophos

7 Research Objectives: 1) Validate a PCR amplification assay for WSSV in the laboratory at HIMB 2) Determine assay sensitivity with known positive samples 3) Run PCR assays for the following samples a) Frozen shrimp products from five stores in Hawaii b) Captured wild crabs/lobster/shrimp from: i) Kauai at Ceatech site ii) Kauai Hanalei Bay iii) Kauai Nawiliwili Bay c) Captured wild crabs/lobsters/shrimp from: i) Kaneohe Bay ii) Waikiki near the aquarium iii) Sand Island near the landfill d) Negative control shrimp from Chen Lu Farms, Kahuku

8 Objective 1: Validate a PCR assay 1. Obtain a positive control WSSV 1 st step PCR on grocery store shrimp WSSV 2 nd step PCR Lane 1: DNA Ladder Lane 2: No template control Lane 3-5: Commodity shrimp from India Lane 6-9: Commodity shrimp from Thailand

9 WSSV Plasmid 4 sequence CTAAAATAGTGCCTAAGAGATTCGACGGAGTTGACCCAGCCTTCCCTGCCGCTCTCTCACCTGCGTCTATCTCACCTCAT GCTTTCTTCCATGGATTCCCATACAAAGTCATCTTTCATGGACAACATCAAATTGCACATGACTGATACTCAATGCTTCT TCAAGAACATTGAACGATTTGAGAAATTCTTGGGAAGATATGGGGACGAATACGCCATGTCCCACAAGCAAAATTGTAAC TGCCCCTTCCATCTCCACCACACTTTTACTCCCTCAGATAACGAGCATCTGGTATCCTCTTTCGCATTCGCCCGCCCAGA AGTCTCCATGGAAGAAATTAGAGCCACACCCTATCAGGCCAACAAGCTTATTAGTGACAAACATTACGTGATGAACATGT CCAAGATCGATTCTAGAGTAACAGGATCTTCCCTCCTTAAGAAGGTTAGCGAATGGACTGAAATGAGAATGAACTCCAAC TTTAATGGAACATTTGAACCATCAAGACTCGCCCTCTCCAACTCTGGCATGACAACGGCAGGAGTCAACCTCGACGTTAT TGTCAAACCAAATAATGCAAGAAGTGTACTAGGAATATTGGAATGTCATCGCCAGCACGTGTGCACCGCCGACGCCAAGG GAACTGTCGCTTCAGCCATGCCAGCCGTCTTCCAGGCAACCGATGGAAACGGTAACGAATCTGAACTGATCCAGAATGCT CTGCCAAGGAACAGATACATCCAAAAGAGCACAATGAACGCTCAAACTGTCGTGTTTGCTAATGTTTTGGAACACTTATC GCCGATCTTGGAAAGGTTATCGTGAACGAACTGGCCGGCACCATCGCTGATCTGTACCAAAAGCGTATATGAAAACACCA AGGAAATGATGATAGACTAGGCTCTGACGACTCTTCAATCTAATAATATGGAGGAGTAGAATCAATGGATTATGGAGATA GCGAACACAATCCAACAATGGTTCGGTCT Distribution of 38 Blast Hits on the Query Sequence Score E Sequences producing significant alignments: (bits) Value gi gb AF Shrimp white spot syndrome virus gi gb AF AF White spot syndrome viru gi gb AF AF White spot syndrome viru gi gb AF AF White spot syndrome viru gi gb U PMU50923 Penaeus monodon nonoccluded gi emb AJ WSP White spot syndrome ba

10 Objective 2: Determine sensitivity Serial dilutions of cloned positive control WSSV 1 st Step WSSV 2 nd Step Lane 1: DNA Ladder Lane 2-9: serial dilution 10 6 through 10-1

11 Research Objective Results 3a: Perform PCR assay on frozen commodity shrimp Stores: Foodland, Safeway, Star Market, and Nanko s Country of origin: Thailand Vietnam India Mexico Viral presence: POSITIVE POSITIVE POSITIVE Not Detected

12 Objective 3b: Ceatech Ceatech quarantined by Hawaii Department of Agriculture Estimated $2 million in lost revenue 20 million shrimp buried 48 ponds drained into Kinikini Ditch No clear indication of how this virus was introduced into the farm Multiple sampling trips were taken by members of the Department of Agriculture (DOA) and Department of Land and Natural Resources (DLNR) P. vannamei were found outside the confines of the farm No samples have tested positive for WSSV

13 Objective 3b: Ceatech Location Name Across the street from CEATECH Hatchery PMRF culvert PMRF mouth of Kinikini Ditch Kinikini Ditch near intersection of ditches Kinikini Ditch Kinikini Ditch near farm drain Number of samples Species collected Palaemon debilis Litopenaeus vannemei Litopenaeus vannemei Ghost Crab sp? No samples found No samples found No samples found

14 Objective 3c: Kaneohe Bay Collection sites: Coconut Island and Heeia State park Heeia State Park Coconut Island

15 Objective 3c: Kaneohe Bay Animals Collected and Tested: Thalamita crenata Blue crab 2 nd step positive Portunus sanguinolentus Blood-spotted crab Not detected Pachygrapsus sp. rock crabs 2 nd step positive Palaemon debilis O pae 2 nd step positive

16 Objective 3c: Waikiki Aquarium Following the recommendations of Dr. Jim Brock, purchase of SPF shrimp for use as feed began several years ago. No testing for viral diseases was done on shrimp prior to Dr. Brock s recommendation Recent testing of shrimp feed from Kauai was negative for WSSV Prior to the release of several slipper lobster back into the wild a specimen was tested for WSSV No significant loses of animals ever recorded Slipper, Spiny and Reef lobsters Various endemic crabs Opa e Cleaner shrimp Harlequin shrimp

17 Research Objectives: Future Plans Work with Tony Montgomery to sample Sand Island sites Return to Kauai to continue monitoring around CEATECH Continue sampling of Kaneohe Bay Develop a bioassay to study the susceptibility of local crustaceans to WSSV Perform transmission trials per os under quarantine conditions

18 Notifiable Diseases of Crustaceans: Taura syndrome (viral/penaeid shrimp) White spot disease (viral/penaeid shrimp and other decapod crustaceans) Yellowhead disease (viral/penaeid shrimp) Other Significant Diseases of Crustaceans: Tetrahedral baculovirosis (Baculovirus penaei) (viral/penaeid shrimp) Spherical baculovirosis (Penaeus monodon-type baculovirus) Infectious hypodermal and haematopoietic necrosis (viral/penaeid shrimp) Crayfish plague (Aphanomyces astaci) (fungal/freshwater crayfish) Spawner-isolated mortality virus disease (viral/penaeid shrimp)

19 Conclusions: We now have a valid PCR amplification assay for WSSV at HIMB Sensitivity of this 2-step assay is on the order of 1 viral particle/µl Commodity shrimp sold in grocery stores and bait shops contain WSSV Samples taken of local crustaceans in Kaneohe Bay have tested POSITIVE for WSSV The impact on local crustacea in Kauai remains undetermined Much more work needs to be done

20 Anuenue Fisheries Research Center Dee Montgomery-Brock Department of Land and Natural Resources Tony Montgomery Waikiki Aquarium Jerry Crow Dr. Andrew Rossiter