CHASING AHPND: WORKING TOWARD A BETTER

UNDERSTANDING OF ENVIRONMENTAL
CONTRIBUTING FACTORS FOR THE DISEASE

James A. Brock, D.V.M.

brockj001@hawaii.rr.com
 AHPND - Early Clues?
Shrimp Stocked in Containers off the
Bottom, no Die
Stop Feed, Stop Die

Stock in Nursery, Survival Sometimes Better

RESEARCH MILESTONE Larger Shrimp, Less Die

AHPND AGENT AHPND-Vp Challenge % Survival
IDENTIFIED Mixed Culture, TSB 0%
(Tran et al. 2013) Mixed Culture, TSA,
100%
MA or BA
Tran et al. 2013
 AHPND-Vp Toxins Identified!
Han, J.E. et al. 2015. Photorhabdus
insect related (Pir) toxin-like genes
in a plasmid of Vibrio
parahaemolyticus, the causative
agent of acute hepatopancreatic
necrosis disease (AHPND) of shrimp.
Dis Aquat Organ. 113(1):33-40.
Sirikharin, R. et al. 2015. Characterization
and PCR detection of binary, Pir-like toxins
from Vibrio parahaemolyticus isolates that
cause acute hepatopancreatic necrosis
disease (AHPND) in shrimp. www. PlOS
ONE.

We determined that the Pir toxin kills diamondback larvae by destroying

that portion of the digestive system known as the midgut.
Blackburn, M.B. et al. 2006. Remarkable susceptibility of the diamondback
moth (plutella xylostella l.) to ingestion of pir toxins from photorhabdus
luminescens. Entomologia Experimentalis et Applicata 121:31-37.
THE Heterorhabditis/Photorhabdus STORY

Heterorhabditis sp.
Photorhabdus sp.
Pir A/B toxins
AMAZON.com

Pir A/B-like Toxins: the proximate cause of AHPN

 SUMMARY OF IMPORTANT GENETIC FACTORS OF Vibrio
parahaemolyticus STRAINS RELATIVE TO AHPND
Vibrio spp. Virulence
and Pir Toxin
Plasmids
PLASMID
BACTERIA

AHPND
 AHPND PATHOGENESIS: Our Current Model
RECOVERY?

UNLIKELY

High Dose

WSJ
STOMACH
DNA copy/L
4.44E+06
DNA copy/L
5.97E+05
POSSIBLE
6.62E+05 1.88E+05
9.16E+07 2.27E+07
1.27E+07 2.42E+08
3.06E+05 6.07E+07 Low Dose
1.35E+08 7.58E+07
Background: Hypothesis Testing in Bioassays

Laboratory Microcosm Tanks Shrimp Ponds

 Methods: Scale up problematic
Laboratory Most Die Microcosm Most No Die
Means Table for % Survival Interaction Bar Plot for % Survival
Effect: AHPND-Vp Effect: AHPND-Vp
Error Bars: 1 Standard Deviation(s)
Count Mean Std. Dev. Std. Err.
100
5HP 4 83.500 4.435 2.217
none 12 89.500 5.729 1.654
80
P1/1 14 86.714 10.246 2.738

Mean % Survival
60
Fisher's PLSD for % Survival
Effect: AHPND-Vp
Significance Level: 5 % 40
Mean Diff. Crit. Diff. P-Value
5HP, none -6.000 9.632 .2121 20
5HP, P1/1 -3.214 9.458 .4916
none, P1/1 2.786 6.563 .3915
0
5HP none P1/1
AHPND-Vp

DNA copy/L DNA copy/L

2.73E+03 3.96E+03 But AHPND-Vp remained in
6.28E+03 8.36E+03 the stomach
4.27E+03 7.80E+03
 Epidemiological Viewpoint: Risk Factors
HOST AGENT ENVIRONMENT
Species 1. ABUNDANCE Agent present? If Yes..
Age/Size 2. STRAIN
pH
Molt 3. VIRULENCE FACTORS
Salinity
Strain Pir A/B toxins,
Temperature
Defenses Other toxins (?),
Zooplankton/Filter Feeders
Behavior Digestive enzymes,
Nutrients
Quorum sensing,
Biodiversity of pond water
etc.
Bottom sediments
Other Microbial Agents
Agent abundance/Toxin
production
Challenge Density: P1/1 TSB+NaCl Culture
ANOVA COMPARISON FOR P1/1 CFU/ML CHALLENGE DOSAGE AND
MEAN COUNT OF REMAINING SHRIMP ON DOE4, CPF AHPNDS-PLUS
TRIAL, March, 2014
Means Table for DOE4
Effect: P1/1 Conc. Interaction Bar Plot for DOE4
Effect: P1/1 Conc.
Count Mean Std. Dev. Std. Err.
Error Bars: 1 Standard Deviation(s)

MEAN COUNT OF SHRIMP DOE4

10x3 12 9.417 3.753 1.083 20
10x4 12 5.250 1.960 .566
10x5 12 3.083 1.832 .529
17.5
none 8 19.125 .354 .125 15
12.5
Fisher's PLSD for DOE4
Effect: P1/1 Conc. 10
Significance Level: 5 %
7.5
Mean Diff. Crit. Diff. P-Value
10x3, 10x4 4.167 2.000 .0001 S 5
10x3, 10x5 6.333 2.000 <.0001 S
2.5
10x3, none -9.708 2.236 <.0001 S
10x4, 10x5 2.167 2.000 .0344 S 0
10x3 10x4 10x5 none
10x4, none -13.875 2.236 <.0001 S
P1/1 CFU/ML
10x5, none -16.042 2.236 <.0001 S
pH: Challenge via water: P1/1 - TSB+NaCl/5105 cfu/ml

Cell Line Chart

Split By: pH
Error Bars: 1 Standard Deviation(s)
22
20

18
Mean Count of Shrimp

16 7.0
14 7.5
12 8.0

10 8.6

8
6
4
0 hr

12 hrs

24 hrs

36 hrs

48 hrs

60 hrs

72 hrs

84 hrs
Time in the Chalenge
 pH: Pellet Coated with AHPND-Vp, P1/1 1 x 104 cfu
Cell Line Chart
Split By: Treatment Interaction Bar Plot for 120 hrs
Means Table for 120 hrs
Error Bars: 1 Standard Deviation(s) Effect: Treatment
30 Effect: Treatment
Error Bars: 1 Standard Deviation(s)
Count Mean Std. Dev. Std. Err. 10
27

Mean Count of Shrimp at 120 hrs.

pH 7.0 3 8.333 1.155 .667
24 9
Mean Number of Shrimp

pH 7.8 3 5.000 1.000 .577

21 8
pH 8.3 3 4.000 1.732 1.000
18 pH 7.0 7

15 pH 7.8 6
Fisher's PLSD for 120 hrs
12 pH 8.3 Effect: Treatment 5
Significance Level: 5 % 4
9
Mean Diff. Crit. Diff. P-Value
6 3
pH 7.0, pH 7.8 3.333 2.664 .0222 S
3 2
pH 7.0, pH 8.3 4.333 2.664 .0073 S
0 1
pH 7.8, pH 8.3 1.000 2.664 .3938
0 hr

12 hrs

24 hrs

36 hrs

48 hrs

60 hrs

72 hrs

84 hrs

96 hrs

108 hrs

120 hrs

0
pH 7.0 pH 7.8 pH 8.3
Treatment

Time in the Trial

pH 7: Less Die
 Salinity/Temperature:
Shrimp MBW = 0.14 gram
Cell Line Chart
Split By: Salinity, Tem .
Error Bars: 1 Standard Deviation(s) P1/1 TSB+NaCl: 105 cfu/ml
22
20
18
Mean Count of Shrimp

16
14 5, 25
12 5, 30
10 30, 25
8 30, 30
6
4
2
0
1 hr. 6 hr 8 hr 16 hr 24 hr
Time in the Trial
Filter Feeders and Zooplankton

Filter Feeders Zooplankton

High concentration of AHPND-Vp, but the addition of these organisms did not
increase shrimp mortality in bioassay trials
Nutrients: Inoculate P1/1 105 cfu/TSB+NaCl
Feed Pellets Added or Not Added
Cell Line Chart
Split By: Treatm ent
110
100
90
80
Number of Shrimp AHPND-Vp/No Feed
70
AHPND-Vp/Normal Feed
60
No Feed
50
Normal Feed
40
30
20
10
0 hrs.

12 hrs.

24 hrs.

36 hrs.

48 hrs.

60 hrs.

72 hrs.

84 hrs.

96 hrs.

108 hrs.

120 hrs.
Incubation Time
Nutrients: AHPND-Vp from TSA Plates Diluted in Saline (105 cfu/ml)
Procedure Shrimp Age Shrimp MBW DOE1 DOE2 DOE3 DOE4
20 ppt water PL37 0.78 20 16 16 16
20 ppt water PL37 0.78 20 16 16 15
20 ppt water + 3% TSB media PL37 0.77 20 6 5 5
20 ppt water + 3% TSB media PL37 0.77 20 4 4 4
20 ppt water + 60 ppm
Molass + 3 ppm NH4 + 3 ppm PL37 0.86 20 6 5 5
Fe
20 ppt water + 60 ppm
Molass + 3 ppm NH4 + 3 ppm PL37 0.86 20 5 5 5
Fe
20 ppt water + 60 ppm
PL37 0.77 20 3 2 2
Molass + 3 ppm NH4

20 ppt water + 60 ppm

PL37 0.77 20 3 3 3
Molass + 3 ppm NH4
 Nutrients: Molasses or F2 Fertilizer Added

Molasses or no Molasses Added F2 Fertilizer Added or Not added

Interaction Bar Plot for 120 hrs

Means Table for 120 hrs
Effect: 100 ppm Molasses
Count Mean Std. Dev.
Effect: 100 ppm Molasses
Error Bars: 1 Standard Deviation(s) Interaction Bar Plot for 120 hrs
Means Table for 120 hrs Effect: F/2
Mean Number of Shrimp at 120 Hrs. 24
Effect: F/2 Error Bars: 1 Standard Deviation(s)
22
Std. Err. Count Mean Std. Dev. Std. Err.

Mean Number of Shrimp at 120 Hrs.

20 24
No 9 15.000 7.106 2.369 No 12 16.250 6.468 1.867 22
18
Yes 15 17.133 5.566 1.437 Yes 12 16.417 6.052 1.747 20
16
18
14
16
12 14
Fisher's PLSD for 120 hrs
Fisher's PLSD for 120 hrs 10 Effect: F/2 12
Effect: 100 ppm Molasses 8 Significance Level: 5 % 10
Significance Level: 5 % 6 Mean Diff. Crit. Diff. P-Value 8
Mean Diff. Crit. Diff. P-Value 4 No, Yes -.167 5.303 .9486 6
No, Yes -2.133 5.396 .4210 2 4
0 2
No Yes 0
100 ppm Molasses Added No Yes

F/2 Fertilizer Added

AHPND-Vp Inoculated from TSB

Nutrients: AHPND-Vp Colonization (Experimental) of
Pelleted Feed
7000000
Start CFUs
6000000

5000000
Mean TPC cfu/g

4000000
10
3000000
1000000
2000000

1000000

-1000000
0 hr 1 hr 2 hr 4 hr 8 hr
Incubation Time
Nutrients: Comparison of Challenge Mortality Pattern for
Shrimp Fed Feed Pellets Incubated with AHPND-Vp
Cell Line Chart
Split By: Treatm ent
Error Bars: 1 Standard Deviation(s)
22
20
18
Mean Number of Shrimp

16
Pellet dip 0 hr
14
Pellet dip 2 hrs
12
Pellet dip 4 hrs
10
Pellet dip 8 hrs.
8
Positive Control
6
4
2
0
0 hrs.

24 hrs.

48 hrs.

72 hrs.

96 hrs.

120 hrs.

144 hrs.
Time
Nutrients: Morbid and Dead Shrimp Carcasses

Cohort Shrimp Consume

Water Birds

101 102 103 104 105 NT +C

AP4 1st Step

Water Biodiversity: Floc/Mature/Tilapia/Yellow Vibrio
Interaction Bar Plot for % Survival
Means Table for % Survival Effect: Water Treatm ent
Effect: Water Treatm ent Error Bars: 1 Standard Deviation(s)
120
Count Mean Std. Dev. Std. Err.
Control 9 64.444 27.889 9.296
100

MEAN % SURVIVAL
Floc 9 80.556 14.458 4.819
Mature 9 91.667 15.411 5.137
80
Tilapia 9 93.333 10.308 3.436
Yellow V., 104 9 85.000 22.638 7.546 60

40
Fisher's PLSD for % Survival
Effect: Water Treatm ent 20
Significance Level: 5 %
Mean Dif f . Crit. Dif f . P-Value 0

Tilapia
Mature

Yellow V., 104

Control

Floc
Control, Floc -16.111 18.292 .0827
Control, Mature -27.222 18.292 .0045 S
Control, Tilapia -28.889 18.292 .0028 S
Control, Yellow V., 104 -20.556 18.292 .0286 S WATER TREATMENT
Floc, Mature -11.111 18.292 .2267
Floc, Tilapia -12.778 18.292 .1657
Floc, Yellow V., 104 -4.444 18.292 .6261
Mature, Tilapia
Mature, Yellow V., 104
Tilapia, Yellow V., 104
-1.667
6.667
8.333
18.292
18.292
18.292
.8548
.4657
.3627
Protective
Sediment: Sediment Treatment: Effect on Final Mean Percent Survival
Interaction Bar Plot for % SR
Means Table for % SR Effect: Sedim ent
Effect: Sedim ent Error Bars: 1 Standard Deviation(s)
Count Mean Std. Dev. Std. Err. 100
No 9 86.889 6.972 2.324 90
Yes 21 51.571 18.737 4.089 80

Mean Percent Survival

70
60
Fisher's PLSD for % SR
Effect: Sedim ent 50
Significance Level: 5 % 40
Mean Diff. Crit. Diff. P-Value 30
No, Yes 35.317 13.276 <.0001 S
20
10
0
No Yes
Pond Sediment Added

Danger!
Summary: AHPND Environmental Risk Factors

Factor Risk Avoidance

pH Lower is better
Salinity <6 ppt
Temperature Avoid Hot Water
Filter Feeders, Zooplankton
Manage Densities
Vectors
Uneaten Feed Accumulation Avoid
Microbial Biodiversity Promote
Sediments Remove Promptly
Conclusions
AHPND is an infectious, foodborne disease.

AHPND epidemics in farmed shrimp are strongly influenced by

environmental determinants.

Environmental conditions can be manipulated to reduce the proliferation

of the AHPND-Vp.

Management strategies that result in the reduction of the density of

AHPND-Vp in ponds will result in reduction of mortality from AHPND-Vp.
 GREATER CONTROL OF ENVIRONMENTAL FACTORS ON
FARMS: (Post July 2015 FAO AHPND Meeting in Panama)

THAILAND: GENERALLY RESULTS HAVE BEEN FAVORABLE (FEWER

LOST PONDS AND IMPROVED POND SURVIVALS WITH AN INCREASE
IN THE NUMBER OF FARMERS EMPLOYING, MODIFYING AND
IMPROVING THE APPLICATION TECHNIQUES

R&D FOLLOW-UP: ENCOURAGE BROADER ADOPTION AND

DEVELOPMENT OF CREATIVE IMPROVEMENTS SHIFT FOCUS OF
R&D LABORATORY WORK TO OTHER PRIORTIES
TRIALS TESTING CARBOHYDRATE MANIPULATION/SUCROSE VS. GLUCOSE
UPDATE OF FINDINGS SUBSEQUENT TO JULY 2015
(FAO AHPND Meeting in Panama)

PARAMETER RESULT BENCH TOP RESULT TRIALS IN

TRIALS IN LABORTORY COMMERCIAL PONDS
GLUCOSE VS. SUCROSE PROMISING OUTCOME TOTAL DISASTER
INTERPRETATION: BAD CONCEPT, DISCONTINUE FURTHER WORK, RETHINK
HYPOTHESES
 GENETIC SELECTION
UPDATE ON STATUS SINCE JULY 2015 (FAO AHPND Meeting in Panama)

SELECTION APPROACH RESULT

CHALLENGE WITH LIVE AHPND WEAK SELECTION PRESSURE/VARIABLE
BACTERIA RESULTS
CHALLENGE WITH PIR TOXIN ROBUST SELECTION PRESSURE/RESULTS
PROMISING
SHRIMP SAMPLE COLLECTION STRATEGY TO IMPROVE THE PCR TEST
DETECTION SENSITIVITY FOR APHND WHEN SHRIMP ARE NOT DYING
UPDATE ON STATUS SINCE JULY 2015 (FAO AHPND Meeting in Panama)

Table 3 PCR Primers/% PCR Positive

Status
Case Sample ~ Wt.
Species at Vpldh AP4 VpPirAB
Group Count (gms)
fixation
O1 Pv 18 live 0.3 16.7% 0% 0%
O2 Pv 8 deada 0.3 100% 37.5% 12.5%
P1 Pm 21 live 1.5 14.3% 0% 0%
P2 Pm 8 deada 1.5 100% 100% 100%
Q1 Pm 30 live 2 16.7% 0% 0%
Q2 Pm 8 deada 2 87.5% 0% 0%
R1 Pv 15 live 4.5 3.3% 0% 0%
R2 Pv 8 deada 4.5 25% 0% 0%
 WATERBIRD TRANSMISSION OF AHPND-Vp
UPDATE SINCE JULY 2015 (FAO AHPND Meeting in Panama)

BIRD FECES CULTURES:

FARM PONDS WITH
ONLY NON-AHPND Vp
NO ACTIVE AHPND
ISOLATED
ACKNOWLEDGEMENTS

Ms. Nathene Antonio, BS Hank Bauman, Belize

Mr. Robins McIntosh Ms. Mineyo Shimojo, BA
Mr. Sudharma Choosuk
Mr. Punya Makawan
Mr. Pakpoom Nunjuy
Mr. Apiwat Rattanawongchiya
Mr. Pongsak Kanson
Charoen Pokphand Foods,
Bangkok Thailand
 THANK YOU FOR YOUR KIND ATTENTION!

Something new (?), autosomal homozygous color variant monodon