Toxicity and Related Testing Run to reproduce AHPNS experimentally - - PDF document

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Toxicity and Related Testing

Run to reproduce AHPNS i t ll i th l b t 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.

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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.

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Feed Toxicity Study Results

D 37 Tank Number Treatment No. Stocked Day 37; No. Collected % Survival 1 Rangen (control) 30 30 100% 2 Uni-Pres #1 30 29 97% 3 Uni-Pres#2 30 30 100% 4 CP feed 30 29 97%

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Possible Toxic Agents

• Toxic agent is possible:

Al (bl di fl ll t ) i d ? – Algae (bluegreen, dinoflagellate) 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:

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Toxicity Trials with Cypermethrin

A ti C th i bi d t d d di t &

• Assumption: Cypermethrin binds to suspended 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 cm3 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.

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Toxicity Trials with Cypermethrin in Soil

Concentration

• f

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%

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* AHPNS = acute hepatopancreatic necrosis syndrome. N/D = not detected. ** Survival adjusted for histological samples.

AHPNS: Study 3 UAZ-APL

Static renewal bioassay. artificial seawater renewed daily. cypermethrin doses renewed daily. Length of study: 37 days

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C th i AHPNS P th l

AHPNS: Study 3 UAZ-APL

Cypermethrin Concentration (ppb) AHPNS Pathology 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

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Histological Results: Study 4

Soil origin AHPNS Pathology Vietnam Negative Arizona, USA Negative

Infectivity Studies

Run to reproduce AHPNS i t ll i th l b t experimentally in the laboratory

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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.

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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 3rd abdominal segment. Histology for AHPNS at termination (day 36).

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Summary of Infectivity Study

Tank Treatment Number

• No. Day

36 % Survival 1 Negative control 10 10 100% 2 Per os* 10 10 100% 3 Injection 10 10 100%

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* MBV was passed to the SPF P. monodon in the per os group.

AHPNS: Study 1 UAZ-APL

Per os Injection: non-filtered inoculum Tissue: frozen O i i f i Vi Origin of tissue: Vietnam Length of study: 6 days

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Histological Results: Study 1

• Per os: No AHPNS
• Injection:

No AHPNS (dose related response caused by a massive

% Survivors from Injection EMS Study

100 60 100 40 60 80 100 urvivors Negative control 1:10 dilution 1:20 dilution

systemic bacterial infection)

0.8 18 20 40 Treatment % S 1:40 dilution 1:80 dilution

AHPNS: Study 5 UAZ-APL

Injection: filtered inoculum Tissue: frozen Origin of tissue: Vietnam L h f d 6 d Length of study: 6 days

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Histological Results: Study 5

% Survivors from Injection EMS Study

100 100 100 100 100 s Negative control- SPF 1:10 1:10 dilution

No AHPNS pathology observed in the filtered inoculum treatments

Treatment AHPNS Pathology Negative control Negative 1:10 Negative

20 40 60 80 Treatment % Survivors 1:10 dilution 1:20 dilution 1:40 dilution Positive control- unfiltered

filtered inoculum treatments

g 1:20 Negative 1:40 Negative Positive control (unfiltered) Negative- massive bacterial infection

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

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Histological Results: Study 6

Bacterial isolate Bacterial ID AHPNS Pathology 1335 Vibrio parahaemolyticus Negative 1336 Bacillus sp. Negative p 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

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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.

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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

Treat- ment Day Note Case number 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 h i f i l 12 344 A EMS shrimp for inoculums 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

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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 th f t d 7 d Length of study: 7 days

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Histological Results: Study 10

Treatment AHPNS Pathology 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 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.

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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