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PREVENTIVE / CORRECTIVE STRATEGIES BASED ON HEALTH PROMOTINGFEED ADDITIVES TO REDUCE THE IMPACT OF WHITE FAECES SYNDROME IN
ASIA - LESSONS TO LEARN FOR ECUADOR
Gilberto Hernández, MSc
Waldo Nuez, DVM, PhD
DISEASE
PATHOGENHOST
ENVIRONMENT
HEALTH MANAGEMENT
StressPolluted
environment
Latent
carrier
REDUCE(Functional nutrition)
STRENGTEN(Functional nutrition)
IMPROVE
FEW
PATHOGENS
STRONG
SHRIMP
GOOD ENVIRONMENT
STATE OF HEALTH
APPROACHES TO CONFRONT DISEASE
• Action starts after outbreak
• Cost of treatment
• Cost of mortality
• Many hidden costs
• Action anticipates the problem
• Cost of treatment
• Cost of monitoring
CORRECTIVE PREVENTIVE
DISEASE & LOSSES HEALTH & PROFITABILITY
• Increase HOST resistance to disease• Support of target tissues: intestine, liver, etc.
• Modulation of gut microbiota
• Reinforcement of antioxidant mechanisms
• Modulation of immune and inflammatory response
HEALTH PROMOTING ADDITIVES
• Reduce PATHOGEN virulence/load • QS disruption
• Bacteriostatic/bactericidal activities • Antiviral, antiparasitics, antifungals, etc.
CORRECTION
PREVENTION
CORRECTION PREVENTION
PATHOGENHOST
SYNERGIES BETWEEN COMPOUNDS
SYNERGIES TO BROAD THE ANTIMICROBIAL SPECTRUM
Probiotics
Pathogens0.00 5.00 10.00 15.00 20.00 25.00 30.00
Edwardsiella tarda& E. ictaluri
Flavobacterium columnare
Vibrio anguillarum
Vibrio harveyi
Streptococcus iniae
Aeromonas salmonicida
Photobacterium damselae subsp piscicida
Vibrio parahaemolyticus
Aeromonas hydrophila
Tenacibaculum spp
Vibrio harveyi
Yersinia ruckeri
Streptococcus agalactiae Ia & Ib
Pseudomonas putida
Vibrio alginolyticus
PATHOGENIC BACTERIA
Carnobacterium divergens
Lactococcus lactis
Micrococcus luteus
Bacillus licheniformis
Lactobacillus plantarum
Lactobacillus rhamnosus
Bacillus subtilis
Pediococcus acidilactici
Streptococcus thermophilus
PROBIOTIC BACTERIA
CONCENTRATION OF HEALTH ADDITIVE (mg/ml)
Dietary inclusion
of 2-5 kg/mt
Bactericide/bacteriostatic effect
No effect
QS-inhibition
% of extract
Single
botanicalMIC
Synergistic
blend MIC
0
50
100
-0.00050.00050.00150.00250.0035
0
50
100
-0.00050.00050.00150.00250.0035
QS 50% inhibited at
0.0002 % extract
QS in
hib
ition (%
)Q
S in
hib
ition (%
)
bacteriostatic
...
Strength of QS signal (%)
0 20 40 60 80 100
control
+ component 1
+ component 2
+ component 3
HEALTH ADDITIVE
SYNERGIES TO INCREASE ANTI-QUORUM SENSING (QS) EFFICACY
HEALTH ADDITIVE concentration in the tract
ANTIMICROBIAL EFFICACY AT DIFFERENT CONCENTRATIONS
Bacteri
ostatic
/cidal
Bacteri
ostatic
/cidalQS Inhibition
Feeding
(on pellet)
QS Inhibition
Feed intake
Concentration
HEALTH ADDITIVE
In the tract/HP
Anti-bacterial
action
Feed intake
BROAD SPECTRUM EFFICACY UNDER LABORATORY AND FIELD CONDITIONS
HEPATOPANCREATIC AND GUT DISEASES
From Varela-Mejías, 2018. AquaTIC, 50
• Frequently presented as coinfections
• Acute Hepatopancreatric Necrosis Disease (AHPND)
• Septic hepatopancreatic necrosis (SHPN)
• Necrotizing Hepatopancreatitis (NHP)
• Hepatopancreas Microsporidiosis by Enterocytozoon
hepatopenai (EHP)
• Hepatopancreatic Haplosporidiosis (HPH)
• Gregarines
• White Feces Syndrome (WFS)
SHPN SHPN
NHP NHP
AHPND AHPND
PHYTOBIOTIC-BASED FEED ADDITIVE AS PREVENTIVE AND CORRECTIVE STRATEGY AGAINST WHITE FECES SYNDROME (WFS)
WHITE FECES SYNDROME (WFS)
• Major cause of crop failures in Indonesia, Thailand,
Vietnam and India
• Associated with growth retardation, size disparities
and chronic mortalities
WFS CLINICAL SIGNS
Healthy:
• Full stomach and midgut
• Large and pigmented HP
Infected:
• Loose and soft exoskeleton
• Distended and pale midgut
• Atrophied and pale HP
Aggregated, transformed microvilli (ATM) originated by sloughing from epithelial cells of the
shrimp hepatopancreatic tubules Sriurairatana et al. (2014). PLOSone, 9 (6): e99170
WFS POSSIBLE CAUSATIVE AGENTS
• High stocking densities
• High feeding rates
• Planktoon blooms
• Water temperatureEnterocytozoon hepatopenaei (EHP)
From Flegel 2016, NACA
Vibrio spp.
WFS IS LINKED TO DISTINCT DIGESTIVE MICROBIAL CONFIGURATIONS
Diversity
(Shannon index)
Heterogeneity
(weighted Unifrac distance)
WFS reduces compositional and functional diversity and increases
heterogeneity across individuals
Metagenomic functional predictions16S rRNA gene sequencing profiles
5 13
PREVENTION (+ CORRECTION) STRATEGIES AGAINST WFS
POND MANAGEMENT PHYTOBIOTIC-BASED ADDITIVE
• Chemical treatment before stocking
• Efficient disposal of organic waste
• Use of filtered water
• Water application of probiotics
• Removal of white feces
• Reduction of feeding
• Etc..
• Antibacterial
• Capacity to interrupt the quorum sensing (QS)
communication system of pathogenic bacteria
• Antiparasitic
• Mixed in mash or top coated
MODE OF ACTION OF PHYTOBIOTIC-BASED ADDITIVE
Inhibition of V. parahaemolyticus QS
system
Increased diversity of the digestive bacterial
community
Robles et al. 2016
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Day 0 Day 150 Control Day 150SANACORE
Shannon´s Index H
HEALTH ADDITIVE
0%
20%
40%
60%
80%
100%
NC-20d TR-20d
Bacterial genus (%)
CONTROL SANACORE® GMHEALTH ADDITIVE
NEGATIVE CTRL
POSITIVE CTRL
PHYTOBIOTIC-BASED ADDITIVE
IN VIVO EFFICACY AGAINST WFS-ASSOCIATED BACTERIA (WFD#5)
0
10
20
30
40
50
60
70
Day
0
Day
1
Day
2
Day
3
Day
4
Day
5
Day
6
Day
7
Day
8
Day
9
Day
10
Day
11
Day
12
Day
13
Day
14
(%)
Days post-challenge
CUMULATIVE MORTALITY RATE (%)
NEGATIVE CTRL
POSITIVE CTRL
SANACORE
0
10
20
30
40
50
60
70
Day
0
Day
1
Day
2
Day
3
Day
4
Day
5
Day
6
Day
7
Day
8
Day
9
Day
10
Day
11
Day
12
Day
13
Day
14
(%)
Days post-challenge
PREVALENCE (%)
• Reduced PREVALENCE from 60% to 30% during peak prevalence period
• Reduced MORTALITY from 50% to 30% during peak mortality period
HEALTH ADDITIVE
Aldama-Cano et al. (2018)
Chanthaburi (Batch 1)
Pathum Thani (Batch 2)
CTRL HEALTH ADDITIVE
3 g/L
HEALTH ADDITIVE
6 g/LWFS-INFECTED SHRIMP
120 minutes / ~25°C / n = 3
Phloxin b
IN VITRO EFFICACY AGAINST ENTEROCYTOZOON HEPATOPENAI (EHP)
0
10
20
30
40
50
60
70
80
90
Control SANACORE 3g/L SANACORE 6g/L Control SANACORE 3g/L SANACORE 6g/L
Batch 1 Batch 2
Ger
min
atio
n r
ate
(%)
a
b b
a
b b
0% GERMINATION 0% GERMINATION
Non-germinated
GerminatedBatch of spores
spores/µlGermination
rate (%)
1 1.05 x 106 61.0 ± 7.6
2 3.25 x 106 80 ± 5.0
• Complete inhibition of EHP germination under in vitro conditions
VALIDATION OF SUPPLEMENTATION UNDER FIELD CONDITIONS
#3 Odisha
#2 Subang-Karawang
#1 Penang
VALIDATION OF PREVENTIVE SUPPLEMENTATION UNDER FIELD CONDITIONS
FARM TRIAL #1 : Northern P. Malaysia
• History of area : WSSV, EMS, WFS
• Size of ponds : 0.4 Ha (8 ponds)
• Stocking density : 110pcs/m2
• TREATMENTS :
1) CTRL commercial feed (n=2)
2) PREVENTIVE (n=6) : + top-coating of HEALTH
ADDITIVE
- DOC 0-30 : 3kg/ton
- DOC 30-80 : 2 kg/ton
- DOC 81-129 (harvest) : 1 kg/ton
42
129
0
20
40
60
80
100
120
140
Control Sanacore
days
Emergency
harvest
HEALTH
ADDITIVE
DOC
FARM TRIAL #1 : Northern P. Malaysia
1.5
1.33
1.25
1.3
1.35
1.4
1.45
1.5
1.55
Control Sanacore
FCR
Outbreak
Detection
(DOC 60)
Additive supplemented shrimp at
HARVEST
38
66
0
10
20
30
40
50
60
70
Control Sanacore
%
Survival rate
HEALTH
ADDITIVE
4
30
0
5
10
15
20
25
30
35
Control Sanacore
g
Mean Body Weight
HEALTH
ADDITIVE
HEALTH
ADDITIVE
640
5541
0
1000
2000
3000
4000
5000
6000
Control Sanacore
Harvest per acre pond (kg/ acre)
HEALTH
ADDITIVE
FARM TRIAL #2 : Northern Jakarta, Indonesia• History of area: WSSV, WFS
• Size of ponds: 3000 m2 (6 ponds)
• Stocking density : 100 pcs/m2
• TREATMENTS:
1) CORRECTIVE (n=3) :
- DOC 28-35 (7d) : 5 kg/ton (top-coated)
2) PREVENTIVE + CORRECTIVE (n=3) :
- DOC 20-100 (harvest) : 3 kg/ton (preventive)
- DOC 23-30 (7d) : preventive + 5kg/ton (corrective, top-coated)
VALIDATION OF PREVENTIVE AND CORRECTIVE SUPPLEMENTATION UNDER FIELD CONDITIONS
2
4
6
8
10
12
14
16
39 49 59 69 79 89 99
g
DOC
Average Body Weight (g)
*
**
*
24.5%
Harvest at DOC 85
Harvest at DOC 100
FARM TRIAL #2 : Northern Jakarta, Indonesia
Outbreak detected at DOC 23
20
25
30
35
40
45
50
55
Control (DOC 85) anacore (DOC 100)
Survival (%)
Control (DOC 85) Sanacore (DOC 100)HEALTH ADDITIVE
DOC 100
0
1
2
3
4
5
Control (DOC 85) Sanacore GM (DOC 100)
FCR
*
Control (DOC 85) Sanacore (DOC 100)HEALTH ADDITIVE
DOC 100
FARM TRIAL #3 : Odisha, India (September 2018)• History of area: WSSV, WFS and others
• Size of ponds: 0.75 Ha (2 ponds)
• Stocking density : 65 pcs/m2
• TREATMENTS:
1) CTRL commercial feed (n=1)
2) PREVENTIVE + CORRECTIVE (n=1) :
- DOC 1-51 : 4 kg/ton (preventive) / 3 out of 4 meals
- DOC 52-58 (7d) : preventive + 4 kg/ton (corrective)
- DOC 59-99 : 5 kg/ton (preventive)
VALIDATION OF PREVENTIVE AND CORRECTIVE SUPPLEMENTATION UNDER FIELD CONDITIONS
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94
gAverage body weight
0
20
40
60
80
%
Survival rate
Control TreatmentControl HEALTH
ADDITIVE
+44%
0
2000
4000
6000
8000
Kg/
0.75
Ha
Total biomass
Control TreatmentControl HEALTH
ADDITIVE
+50%
1
1.2
1.4
1.6
1.8
2FCR
Control HEALTH
ADDITIVE
-25%
0
5
10
15
20
g
Average body weight (at harvest)
Control Treatment
+35%
HEALTH
ADDITIVE
Control
FARM TRIAL #3 : Odisha, India
ControlHEALTH ADDITIVE
0.2
Control HEALTH ADDITIVE
CONCLUSIONS
• Health promoting additives aim to reinforce the prevention and correction strategies against
hepatopancreatic diseases (FEED MILL OR TOP DRESSING APPLICATION)
• Synergetic combination of phytobiotics has proved to inhibit Vibrio spp. and EHP isolated from WFS
infected shrimp (BROAD SPECTRUM ACTIVITY)
• Under field conditions, corrective dose mitigates the gross signs of infection but only the combined
preventive and corrective strategy can maintain or bring up growth rates and survival to pre-WFS
levels
• The combined preventive and corrective application strategy shows potential against other
hepatopancreatic and gut diseases
