Toxicity and Related Testing. Possible Etiological Agents

Transcription

1 Toxicity and Related Testing Run to reproduce AHPNS experimentally in the laboratory 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

2 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 Tank Number 1 Treatment Rangen (control) No. Stocked Day 37; No. Collected % Survival % 2 Uni-Pres # % 3 Uni-Pres# % 4 CP feed % 4 2

3 Possible Toxic Agents Toxic agent is possible: Algae (bluegreen, dinoflagellate) ll )in ponds? 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 d sediments & 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 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

4 Toxicity Trials with Cypermethrin in Soil Concentration of Cypermethrin 20 day histological findings* 40 day histological findings* Final Adjusted Survival (%)** 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

5 AHPNS: Study 3 UAZ-APL Cypermethrin AHPNS Pathology Concentration (ppb) 0 Negative 0.01 Negative 0.1 Negative 0.5 Negative AHPNS: Study 4 UAZ-APL Sediment: untreated from Vietnam Length of study: 40 days 5

6 Histological Results: Study 4 Soil origin Vietnam Arizona, USA AHPNS Pathology Negative Negative Infectivity Studies Run to reproduce AHPNS experimentally in the laboratory 6

7 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. 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. 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

8 Summary of Infectivity Study Tank Treatment Number No. Day 36 1 Negative control % Survival % 2 Per os* % 3 Injection % * 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 Oi Origin i of tissue: Vietnam Length of study: 6 days 8

9 Histological Results: Study 1 Per os: No AHPNS Injection: No AHPNS (dose related response caused by a massive systemic bacterial urvivors % Survivors from Injection EMS Study infection) % S 60 Treatment 100 Negative control 1:10 dilution 1:20 dilution 1:40 dilution 1:80 dilution AHPNS: Study 5 UAZ-APL Injection: filtered inoculum Tissue: frozen Origin of tissue: Vietnam Length h of study: 6 days 9

10 Histological Results: Study 5 No AHPNS pathology observed in the filtered inoculum treatments Treatment AHPNS Pathology Negative control Negative 1:10 Negative 1:20 Negative 1:40 Negative Positive control (unfiltered) Negative- massive bacterial infection s % Survivors % Survivors from Injection EMS Study Negative control SPF 1: :10 dilution 60 1:20 dilution :40 dilution 0 Treatment Positive controlunfiltered AHPNS: Study 6 UAZ-APL APL Per os: treated feed Bacterial cultured & filtered Filtrate injected Origin of bacteria: UAZ-APL Study 1 Length of study: 21 days 10

11 Histological Results: Study 6 Bacterial isolate Bacterial ID AHPNS Pathology 1335 Vibrio parahaemolyticus Negative 1336 Bacillus sp. Negative AHPNS: Study 7 Vietnam Reverse gavage Tissue: Fresh P. monodon Origin of tissue: Vietnam Length of study: 7 days Histology: Negative for AHPNS 11

12 AHPNS: Study 8 Vietnam Injection Injection (filtered inoculum) Tissue: Fresh P. monodon Origin of tissue: Vietnam Length of study: 7 days Results: Study 8 Mortalities by injection of non-filtered inoculum: % within 24 hours. No mortalities using filtered inoculum for injection. 12

13 AHPNS: Study 9 Vietnam Perosfedfor5da for 5 days Tissue: Fresh P. monodon Origin of tissue: Vietnam Length of study: 7 days Treatment Results: Study 9 Day Note Case number PO 5/5 3/5 3/5 3/5 3/5 3/5 3/5 2 morts B EMS shrimp for inoculums A 02 PO 5/5 5/5 5/5 5/5 4/5 4/5 4/5 1 mort B EMS shrimp for inoculums A 03 PO 5/5 5/5 5/5 4/5 4/5 4/5 4/5 1 mort B EMS shrimp for inoculums A 13

14 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 AHPNS 3 P. monodon with EMS/AHPNS 6 SPF P. vannamei Origin of shrimp: Vietnam Length of study: 7 days 14

15 Histological Results: Study 10 Treatment 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 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

16 Possible Etiological Agents What We Know to Date: Severe HP dysfunction followed by a terminal Vibrio 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 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