FAO TCP/INT/3502 “Reducing and managing the risk of

Acute Hepatopancreatic Necrosis Disease (AHPND) of Cultured Shrimp”

Review of Vibriosis in Shrimp

Farming prior to EMS and Risk

Management Measures

Dr. Celia R. Lavilla-Pitogo

celia.pitogo@fulbrightmail.org

6/24/2015

International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer Responses” 22-24 June 2015, Tryp Hotel, Panama City

1

What is vibriosis?

Vibriosis is a bacterial disease caused by Vibrio species that are ubiquitous in seawater environments. It affects many aquatic organisms causing lesions, discoloration, or loss of function of affected parts.

Disease Signs and Virulence Factors

• Luminescence

• Discoloration of body parts

• Shell necrosis

• Muscular opacity

• Gill melanization

• Off-feed

• Melanized granulomas

• Whitish muscle

• Adherence, a very important virulence mechanism

• Internalization

• Cytotoxicity

• Production of chitinase and other enzymes

It was observed that survival of V. cholerae and V. parahaemolyticus in water was extended in the presence of copepods, an important factor for ecological viability.

Vibrio harveyi

• Divides once every 30 minutes • Ubiquitous in seawater; < 10

cfu/ml

• Luminescent; Diagnosis is easy.

• The yellow and green colonies on TCBS became the foundation for important disease management strategies.

Luminescent vibriosis in shrimp hatcheries

• The disease started when the hatchery system shifted from community culture to Galveston method and its modifications

• Starting culture with filtered or chlorinated water provided a niche for opportunistic bacteria

• Mass mortality was preceded by appearance of luminescent larvae

• In 1990, we reported the causative agent as luminescent Vibrio harveyi

• Challenge tests showed significant mortality after exposure of larvae to 102 cfu/ml

• V. harveyi came from raw seawater, spawners, contaminated zooplankton

• Algae were not significant sources

Sources of Vibrio harveyi in Penaeus monodon hatcheries: Spawners release fecal material in spawning water; adherence resulted

to biofilm formation on egg surface

Penaeus monodon egg with bacterial plaques on the surface

The bacterial plaques are composed of actively dividing cells similar to Vibrio harveyi

This proves that eggs are colonized by V. harveyi before hatching and this pathogen route can be disrupted by spawner removal and

egg rinsing.

Fate of Penaeus monodon larvae during Vibrio harveyi infection

• Vibrio harveyi in seawater is

<10 cfu/ml

• Contamination at spawning

and feeding

Increase in

number Oral route of

entry

Uninfected

larval surface

Biofilm build up

on feeding

apparatus, mouth

and gills

Biofilm formation

in the mouth and

feeding apparatus

Mass

mortality Systemic

infection Destroys

hepatopancreas

Lavilla-Pitogo et al. 1998

Experiments to Control V. harveyi

Experiments to Control V. harveyi

Lavilla-Pitogo et al. 1998

Management of Risks: Hatchery

Options • Disinfect; hygiene is key #1

• Individual spawning; hygiene is key #2

• Ensure regular molting; remove shells; hygiene is key #3

• Ensure clean natural food; hygiene is key #4

• Cleaning methods and break cycle to dry up facilities should remove biofilms; hygiene is key #5

• Review rearing procedures; no to prophylactic application of antibiotics

• Include bacterial monitoring in PL quality standards

RISKS 1. Vibrios are in seawater

2. Spawners bring in vibrios

3. Vibrios colonize shrimp surfaces

4. Artemia is a crustacean and may be colonized like shrimp larvae

5. Vibrios form biolfilms in rearing facilities

6. Hatchery procedures alter gut flora associated with PLs

7. Quality of PLs affected by hatchery techniques

Vibriosis in shrimp grow-out culture The shrimp pond is like a bank!

A lot is deposited; very little is withdrawn.

Rearing pond Feeds Fertilizers

Shrimp

Algae and zooplankton grow and die Organic matter builds-up

Water quality changes Microorganisms grow

Biofilms form

Feces, metabolites, old exoskeleton, mortalities

Vitamins, additives, binders, uneaten pellets

Probiotics, lime and other chemicals

Thousands of postlarvae with associated bacteria + vibrios

+ vibrios

+ vibrios

+ vibrios ? Urea ✔

+ vibrios

Vibriosis in Grow-out Ponds in the Philippines

• Mass mortality within 60 days of culture (DOC); more often in stocks less than 30 DOC

• A few weak shrimp stayed at the edge of the pond, but dead animals were found on the bottom

• Mortality followed periods of high luminous bacteria in the water

• More prevalent in farms with shared water source than those that drew water from open sea or sub-surface wells

Comparison of luminescent bacterial load of 4 ponds

Bacterial load of the hepatopancreas of pond-reared P. monodon juveniles with (A) and without (B)

luminescent vibriosis

1 13 23 33 43 53

Days of Culture

1 13 23 33 43 53

Days of Culture

TPC

PVC

LBC

Log

no

. of

bac

teri

a (c

fu/h

ep

ato

pan

crea

s)

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

A B

Fate of Penaeus monodon juveniles to sub-adults after infection with Vibrio harveyi

Vibrio harveyi in the environment

<10 cfu/ml

Increase in number

Build up in the hepatopancreas

Chronic

Infection

Focal necrosis in the

hepatopancreas

Recovery;

slow growth

Mortality

Severe melanization, fibrosis,

granuloma formation

Acute

Infection

Massive inflammatory

response

Mortality

Were postlarvae a source of infection?

Younger stages were more vulnerable to infection

than older shrimps

Extensive ponds that used wild postlarvae were not

affected.

Differences between 40 batches of wild-caught and 40 batches of hatchery-reared PLs based on 3 features

Criterion Wild-caught PLs Hatchery-reared PLs

Age (based on number of rostral spine)

4-6 dorsal spines 1-3 ventral spines

Depends on age, but usually 3-4 dorsal, 0-1

ventral spines

Infection with monodon baculovirus (MBV)

O out of 40 batches 23 % positive out of 40 batches

Luminous bacterial load

40% are positive counts ranged from

5.0 - 95 cfu/postlarva

79% positive counts range

from 10 - 99,000 cfu/postlarva

Stage

Number of Batches

Examined

Batches Negative for Luminescent Bacteria (%)

Range of Associated Luminescent Bacteria

Hatchery-reared postlarvae = 272 batches PL 12

97

59 (61)

5.0 x 100 - 1.3 x 105

PL 13

36

12 (33.3)

2.5 x 100 - 8.9 x 104

PL 14

25

11 (44)

5.0 x 100 - 2.5 x 104

PL 15

37

11 (30)

7.0 x 101 - 1.7 x 104

PL 16

18

6 (33.3)

5.0 x 100 - 9.9 x 104

PL 17

28

9 (32)

5.0 x 100 - 3.0 x 105

PL 18

31

7 (23)

2.0 x 102 - 4.0 x 104

Wild-caught PLs

31

18 (58)

5.0 x 100 - 3.5 x 102

Comparison of luminescent bacterial load of hatchery-reared and wild-caught postlarvae

Management of Risks: Grow-out OPTIONS

1. Allocation of reservoir for water conditioning

2. Monitoring of water thru bacterial enumeration to ensure that counts are not 102 cfu/ml of luminous bacteria before stocking

3. Use well-water instead of nearshore seawater

4. Thorough drying of earthen pond bottom eliminates vibrios

5. Add niche-filling bacteria like probiotics to exclude or compete with pathogenic vibrios; Use greenwater techniques

6. Selection of postlarvae, including enumeration of associated bacterial flora

7. Provision of treatment pond to clean water before release into environment

8. NEED FOR NEWER CONTROL STRATEGIES

RISKS

1. Water in grow-out ponds lose diversity and favor pathogenic vibrios

2. Shared water sources have higher pathogenic vibrios

3. Wet pond bottom harbors pathogenic vibrios

4. Some hatchery reared postlarvae harbor large numbers of pathogenic vibrios

5. Effluents from ponds with diseased shrimp are discarded to open waters

Probiotic bacterial products are applied to provide diverse and benign bacteria into the

system

The greenwater technique based on SEAFDEC-ASEAN Manual on Best Practices

Pond lay-out of a low-discharge/recirculating shrimp farm at SEAFDEC, Philippines. Blue arrows indicate water flow in a low-discharge system; red arrows indicate water Flow in a closed-recirculation system.

Avoiding too much deposits in the shrimp farm bank through Best Management Practices

Reference: INFOTIP by ASEAN-SEAFDEC

file://localhost/Users/celiapitogo/Desktop/EMS Panama/1Presentation SEAFDEC-ASEAN Publications/SEAFDEC-ASEAN infotip.pdf

Acknowledgments

• All studies on luminescent vibriosis were conducted at SEAFDEC Aquaculture Department, Philippines.

• Studies on the sources of luminescent Vibrio harveyi and microbial manipulation were funded by IDRC of Canada

• I thank FAO for the opportunity to learn with you about how vibrios continue to wreck havoc on the industry we love.

Muchas gracias!