11/3/2012 Toxicity and Related Testing Run to reproduce AHPNS experimentally in the laboratory i t ll i th l b t Possible Etiological Agents • Toxicant – From feed (new ingredient(s) in feed?) – Remember the melamine contamination of “wheat gluten” of 2-3 years ago. – 37 day study using feeds from affected farms gave negative results. 2 1
11/3/2012 Design of Feed Toxicity Study • 3 starter feeds (2 Uni-President & 1 CP) were collected at farms with ongoing EMS. • At UAZ each feed was provided to 30 ~1g SPF P. monodon for 37 days. • Fed at 5% body weight in 2 equal feedings. • Histology of samples at termination (day 37) examined for AHPNS. 3 Feed Toxicity Study Results Day 37; D 37 Tank No. Treatment No. % Survival Number Stocked Collected Rangen 1 30 30 100% (control) 2 Uni-Pres #1 30 29 97% 3 Uni-Pres#2 30 30 100% 4 CP feed 30 29 97% 4 2
11/3/2012 Possible Toxic Agents • Toxic agent is possible: – Algae (bluegreen, dinoflagellate) in ponds? Al (bl di fl ll t ) i d ? Potentially toxic algae not often found in EMS ponds. – Crustacides used in pond preparation prior to stocking? • Hepatotoxic effect of 2 brands of crustacides? – Cypermethrin test results: 5 Toxicity Trials with Cypermethrin • Assumption: Cypermethrin binds to suspended sediments & A ti C th i bi d t d d di t & is available in pond bottom detritus when PLs are stocked. • Commercial grade cypermethrin was purchased in Vietnam. • Pesticide was mixed with soil to give 0 ppb, 50 ppb, 200 ppb & 400 ppb. • A plastic grate with 1 cm 3 cells was added to each p g experimental tank with soil to reduce turbidity. • 40 P. vannamei & 40 P. monodon were used in replicates per dose level of cypermethrin. • Samples for histology were taken at 20 & 40 days post stocking & examined for signs of EMS/AHPNS. 6 3
11/3/2012 Toxicity Trials with Cypermethrin in Soil Concentration 20 day 40 day Final Adjusted of histological histological Survival (%)** Cypermethrin findings* findings* 0 ppb AHPNS N/D AHPNS N/D 100% 50 ppb AHPNS N/D AHPNS N/D 100% 200 ppb AHPNS N/D AHPNS N/D 100% 400 ppb AHPNS N/D AHPNS N/D 100% * AHPNS = acute hepatopancreatic necrosis syndrome. N/D = not detected. ** Survival adjusted for histological samples. 9 AHPNS: Study 3 UAZ-APL � Static renewal bioassay. � artificial seawater renewed daily. � cypermethrin doses renewed daily. � Length of study: 37 days 4
11/3/2012 AHPNS: Study 3 UAZ-APL C Cypermethrin th i AHPNS P th l AHPNS Pathology Concentration (ppb) 0 Negative 0.01 Negative 0.1 Negative 0.5 Negative AHPNS: Study 4 AHPNS: Study 4 UAZ-APL � Sediment: untreated from Vietnam � Length of study: 40 days 5
11/3/2012 Histological Results: Study 4 Soil origin AHPNS Pathology Vietnam Negative Arizona, USA Negative Infectivity Studies Run to reproduce AHPNS experimentally in the laboratory i t ll i th l b t 6
11/3/2012 Possible Etiological Agents • Infectious agent – bacteria ( Vibrio sp.), virus, parasite? • Vibriosis unlikely as agent of EMS/AHPNS: – Bacterial phase of disease occurs after HP begins to degenerate begins to degenerate. – 37-day per os & injection infectivity study gave negative results. 15 Design of Infectivity Study � ~0.5 g P. monodon from affected farms transported frozen to UAZ. � Some used in per os infectivity study with similar size SPF P. monodon . � A second batch was: � homogenized diluted 1:20 in 2% sterile saline � homogenized, diluted 1:20 in 2% sterile saline. � filtered through a 0.45 micron filter to remove bacteria. � 100 ul injected in the 3 rd abdominal segment. � Histology for AHPNS at termination (day 36). 16 7
11/3/2012 Summary of Infectivity Study Tank Treatment Number No. Day % 36 Survival 1 Negative 10 10 100% control 2 Per os* 10 10 100% 3 Injection 10 10 100% * MBV was passed to the SPF P. monodon in the per os group. 17 AHPNS: Study 1 UAZ-APL � Per os � Injection: non-filtered inoculum � Tissue: frozen � Origin of tissue: Vietnam � O i i f i Vi � Length of study: 6 days 8
11/3/2012 Histological Results: Study 1 • Per os: No AHPNS % Survivors from Injection EMS Study • Injection: 100 100 100 No AHPNS (dose urvivors Negative control 80 60 related response 1:10 dilution 60 caused by a massive 1:20 dilution 40 40 systemic bacterial % S 18 1:40 dilution 20 infection) 0.8 1:80 dilution 0 Treatment AHPNS: Study 5 UAZ-APL � Injection: filtered inoculum � Tissue: frozen � Origin of tissue: Vietnam � L � Length of study: 6 days h f d 6 d 9
11/3/2012 Histological Results: Study 5 % Survivors from Injection EMS Study No AHPNS pathology observed in the Negative control- 100 100 100 100 SPF 1:10 filtered inoculum treatments filtered inoculum treatments 100 1:10 dilution 1:10 dilution s % Survivors 80 60 1:20 dilution 40 20 1:40 dilution 0 0 Positive control- Treatment unfiltered Treatment AHPNS Pathology Negative control Negative 1:10 Negative g 1:20 Negative 1:40 Negative Positive control Negative- massive bacterial infection (unfiltered) AHPNS: Study 6 UAZ-APL UAZ APL � Per os: treated feed � Bacterial cultured & filtered � Filtrate injected � Origin of bacteria: UAZ-APL Study 1 � Length of study: 21 days 10
11/3/2012 Histological Results: Study 6 Bacterial isolate Bacterial ID AHPNS Pathology 1335 Vibrio Negative parahaemolyticus 1336 Bacillus sp. p Negative g AHPNS: Study 7 Vietnam � Reverse gavage � Tissue: Fresh P. monodon � Origin of tissue: Vietnam � Length of study: 7 days � Length of study: 7 days � Histology: Negative for AHPNS 11
11/3/2012 AHPNS: Study 8 Vietnam � Injection � Injection (filtered inoculum) � Tissue: Fresh P. monodon � Origin of tissue: Vietnam � Origin of tissue: Vietnam � Length of study: 7 days Results: Study 8 � Mortalities by injection of non-filtered inoculum: 60-100% within 24 hours. � No mortalities using filtered inoculum for injection injection. 12
11/3/2012 AHPNS: Study 9 Vietnam � Per os fed for 5 da s � Per os fed for 5 days � Tissue: Fresh P. monodon � Origin of tissue: Vietnam � Length of study: 7 days Results: Study 9 Note Case Day Treat- number ment 01 02 03 04 05 06 07 01 PO 5/5 3/5 3/5 3/5 3/5 3/5 3/5 2 morts 12-343 B EMS shrimp for inoculums 12-342 A 02 PO 5/5 5/5 5/5 5/5 4/5 4/5 4/5 1 mort 12-344 B EMS EMS shrimp for inoculums h i f i l 12 344 A 12-344 A 03 PO 5/5 5/5 5/5 4/5 4/5 4/5 4/5 1 mort 12-345 B EMS shrimp for inoculums 12-345 A 13
11/3/2012 Histological Results: Study 9 Treatment AHPNS Pathology Negative control Negative PO 1 Positive G1-2 PO 2 Positive G1-2 PO 3 Negative Positive control Positive G4 AHPNS: Study 10 Vietnam � Cohabitation � Cohabitation � AHPNS 3 P. monodon with EMS/AHPNS � 6 SPF P. vannamei � Origin of shrimp: Vietnam � L � Length of study: 7 days th f t d 7 d 14
11/3/2012 Histological Results: Study 10 Treatment Treatment AHPNS Pathology AHPNS Pathology Negative control Negative Cohabitation Positive G2-3 Positive control Positive G4 � 50% mortality of P. vannamei � 33% mortality of P. monodon Comments and Caution in Interpretation of Results � The Penaeus vannamei used in the � The Penaeus vannamei used in the cohabitation studies were not from SPF broodstock. � The P. vannamei stock may have gone into the study with developing EMS/AHPNS. � Likewise, the source shrimp for these EMS/AHPNS tests were Penaeus monodon and these were collected & used because they had signs of EMS/AHPNS. 15
11/3/2012 Possible Etiological Agents – What We Know to Date: • Severe HP dysfunction followed by a terminal Vibrio infection of the HP infection of the HP. • Vibrio sp. may not be the agent of AHPNS because terminal phase of disease may be opportunistic. • Feeds (e.g. a new ingredient) tested do not cause AHPNS. • Cypermethrin in static renewal bioassays or when • Cypermethrin in static renewal bioassays or when added to soil does not cause AHPNS in lab trials. • Except for promising cohabitation studies, all tests for an infectious agent (viral, parasitic, bacterial) have been negative to date. Thank you for your attention! Reference Lab for Shrimp Diseases 16
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