Contribution to the OIE standard setting procedure

Dr. Jie HUANGOIE Reference Laboratories for WSD & IHHN

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences

Catchable Resources Lab

Mariculture Ecology Lab

Disease Control Lab

Genetic Breeding Lab

Marine Enzyme Lab

Fisheries Environment Lab

Municipal Key Lab for Marine Enz. Eng.

Ministerial CEM forYellow & Bohai Sea

CIQ for Aquatic Germplasm & Env.

Clinical Test Centerfor Fishery Drugs

Service Organizations

Director

Academic Committee

Appraisal Committee

Institute Committee

Assessment Committee

Research System

Innovation System

Office

R&D System

Administration

National CIQ for Aquatic Products

Fish Farming Tech Lab

Product Quality & Safety LabProvincial Key Lab for Fishery Resource &Ecological Env.

Municipal Key Labfor Marine Fish SeedEng. & Biotech.

CPC Office

Research Division

Personnel Division

Financial Division

R&D Division

Information Center

Municipal Pilot Base for Marine Enz. Eng.

Flat Fish Genetic Breeding Center

Marine Fisheries Res. Center

Investigation Vessel Beidou

Field Exp. Station of Fishery Resource Env. In Yellow & Bohai Sea

Rear Services Center

Aquatic Genetic Breeding Center

Processing & Nutrition Lab

Aquatic Genome Lab

Technological Services

Support Facilities

Main diseases concerned◦ Crustaceans: WSD, IHHN, AHPND, CMD, LvID, YHD,

EHP◦ Fish: TRBD, VNN, Edwardsiellosis, Vibriosis,

parasitosis◦ Mollusk: AVND

Scientific research◦ To exploit microbial resources◦ To study etiology and epidemiology◦ To develop diagnosis, prevention and control

technology and products◦ To establish biosecurity system

Crustacean epidemiology and biosecurity technology◦ Finding new pathogens, reporting emerging

diseases New pathogens: V. parahaemolyticus (2012), CMNV

(2014), V. campbellii (2016), LvIV (2016), YHV-8 Emerging diseases: YHD, AHPND, VCMD, EHP, LvID◦ Developing new detection technology On-site rapid and high sensitive detection kits Biochips detection technology◦ Nationwide surveillance for diseases◦ Proposing biosecurity system Elements of biosecurity plan Setting of four biosecurity grades

Mollusk epidemiology and ecological control technology◦ Nationwide coastal surveillance for mollusk

epidemiology◦ Genome sequencing for OsHV-1 strains◦ Algae-carrying transmission pathway of OsHV-1◦ AVN control by interval culture of scallop and algae

Fish epidemiology and vaccine technology◦ Genome sequencing for TRBIV, NNV, V.

anguillarum, and E. tarda◦ Quorum sensing system of V. anguillarum◦ Inactivated vaccines for V. anguillarum and E. tarda◦ Multigene knock-out strains and attenuated

vaccines for E. tarda◦ Adjuvants for immersion vaccination and

technologies

Collection and utilization technologies of the fishery microbial resources◦ Establishing the collection of fishery microbial

resources with >5000 isolates of viruses, bacteria, fungus, and parasites.◦ Identifying probiotic strains with protection

activities to infections with WSSV or V. parahaemolyticus and the function on inorganic nitrogen.◦ Revealing the establishment of probiotics in shrimp

intestinal tract and its influence on the microflora.◦ Establishing probiotics-enhanced biofloc shrimp

farming technology

20 Staff: ◦ 9 Prof.◦ 4 Associate Prof.

3 Postdoctors 1 Doctoral students 33 Postgraduates

White spot disease (WSD) Infectious hypodermal and hematopoietic

necrosis (IHHN)◦ Designated in May 2011

Since Aug 2011, We started to establish, implement and improve the QMS under the guidance of ISO/IEC 17025 and relevant OIE requirements for diagnosis activities◦ CNAS-CL04: 2010 (ISO/IEC17025: 2009)◦ OIE quality standards and guidelines for veterinary

laboratories: infectious diseases - 2nd Edition:2008 We plan to submit the application of the

certification and on-site evaluation under CNAS in 2017

Disease Diagnostic standard Diagnostic method

WSD OIE Aquatic Manual 2016, Chapter2.2.5

PCR, qPCR, LAMPHistopathology,Bioassay

IHHN OIE Aquatic Manual 2016, Chapter2.2.2

PCR, qPCR, Histopathology, Bioassay

Participating in the Proficiency Testing Programme for Aquatic Animal Disease Diagnostic Laboratoies in Asia-Pacific

Test 2011 2012 2013 2014 2015 2016PCR 51 332+7 283+2 547+11 586+23 528

qPCR 540 30 6

LAMP 230 18 15 0+6 35

Histopathology 6 4 43 476 60

ISH

Bioassay 16 34 6

TEM 31

T-E staining 4 7

Test 2011 2012 2013 2014 2015 2016

PCR 51 194 266+12

435+11

586+23 528

qPCR 20

LAMP 225 16

Histopathology 6 4 40 476

Bioassay 20

A series of field-using high sensitive detection kits were made for detection of WSSV and IHHNV.

Kits were provided for the shrimp farms, hatcheries, aquaculture disease control stations, some research laboratories.

The total of 3269 and 749 field-using high sensitive detection kits for WSSV and IHHN were distributed from 2011-2016◦ China◦ Southeast Asia countries◦ Egypt

Resulting in 65—95 min◦ Sample preparation: 3—5 min◦ NA extraction: 4—8 min◦ NA denaturation: 4—6 min◦ Amplification: 50—70 min◦ Observation: 4—10 min

2016-11-2Jie Huang - Biosecurity System for

Aquatic Animal Health 17

Comments and suggestions for OIE standards:◦ 2011 WSD: 10 comments IHHN: 4 comments Other Aquatic Manual chapters: 44 comments

◦ 2012 OIE Aquatic Code: 7 comments

◦ 2013 OIE Aquatic Code: 2 comments

◦ 2014 OIE Aquatic Code: 6 comments

◦ 2015 WSD 6 comments IHHN 2 comments OIE Aquatic Code: 3 comments

◦ 2016 IHHN: 3 comments Other Aquatic Manual chapters: 12 comments OIE Aquatic Code: 7 comments

Being invited in the evaluation of OIE registration for diagnostic reagents

Year Type of material Quantity Purpose

2011

WSSV-infected tissues 100g Positive infected shrimp tissues

Purified WSSV preparation 1 tube Positive control

for VP24 and Polyclonal antibodies VP28 of WSSV 2 tube Positive control

2012 WSSV-infected tissues 170 g Infection experiment and positive control

2013 WSSV-infected tissues 30g Infection experiment

2014 WSSV-infected tissues 160g Infection experiment

2015 WSSV-infected tissues 18g Infection experiment and positive control

2016WSSV-infected tissues 100g Infection experiment

WSSV DNA 106 reactions positive control for PT

Year Type Quantity Purpose

2011IHHNV-infected tissues 30g Positive infected shrimp tissues

IHHNV DNA 1 tube Positive control

2012 IHHNV-infected tissues 20 g Positive infected shrimp tissues

2013 IHHNV-infected tissues 1g Positive control

2014IHHNV-infected tissues 2x50g Infection experiment

IHHNV DNA 3x185ul Positive control

2015 IHHNV-infected tissues 6g Positive infected shrimp tissues

2016 IHHNV DNA 106 reactions Positive control

Convenient sampling protocols for aquatic animals

Diagnostic methods for multiple pathogens

Screening of potential infection caused by unknown pathogens

Comprehensive diagnosis

Sample collection and preservation methods for multiple purposes with single animal/tissue◦ Nucleic acid detection: in 3 times of 95% EtOH◦ Histopathology: Davidson’s AFA◦ Bacterial isolation: 2216E broth◦ TEM: TEM fixative◦ Virus isolation: frozen tissues

0

1

2

3

4

5

A05

A06

A07

A35

A36

A37

A55

A56

A57

A75

A76

A77

B05

B06

B07

B35

B36

B37

B55

B56

B57

B75

B76

B77 RL

30%

50%

70%

95%

FMD

‐20°C

‐80°C

RNA h

alf degradation t

ime (d)

Preserving groups

A05~A77: in (NH4)2SO4 w/ 0~70% EtOH at pH5~7; B05~B77: in NH4Ac w/ 0~70% EtOH at pH5~7; RL: in RNAlater; 30%~95%: in 30%~95% EtOH; FMD: in FMD; -20℃ & -80℃: at -20℃ & -80℃.

Technologies◦ Histopathology◦ PCRs for multiple pathogens in single sample◦ LAMPs for multiple pathogens in single sample◦ Multiplex PCR for multiple pathogens◦ Gene chips for multiple pathogens

Evaluation criteria?

V. fiischeri V. splendidus A. hydrophila E. tarda

Gene chips for 7 pathogenic bacteria

V. anguillarum V. alginolyticus V. parahaemolyticus

Reddish body

CMNV

Covert mortality syndrome

Shrimp

Imperfect condition

VPAHPND

TSV

2016-11-8Jie Huang - Shrimp Epidemiology

and Biosecurity Plan

Technologies◦ Histopathology◦ Viral metagenomics◦ Microbial

metagenomics◦ Other high throughput

technologies◦ Bioassay

Technological guideline?

Biosecurity —— the OIE definition◦ Objective To reduce the risk of introduction, establishment and

spread of pathogenic agents to, from and within an aquatic animal population

◦ Approach A set of management and physical measures designed

The key of biosecurity definition◦ Risk analysis based Hazard identification Risk assessment Risk management Risk communication

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GAPDisease

resistance breeding

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Diseasesurveillance

Environment management

Microbiological control technologies

Ecological management

ImmunologicalControl

technologies

Farm construction and planning Training program for personals Traceability system Disease surveillance project Biological risk assessment Disinfection management for farm and ponds Water treatments for inlet and outlet Management of seed introduction Management of feeds and inputs Farming management Disease diagnosis and control Contingency plan

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Biosecurity grade 1: Diagnosis based treatment◦ Temporary control according to diagnosis or contingency

plan only, without surveillance and risk assessment Biosecurity grade 2: Surveillance based prevention◦ Planed control with improved facilities and managements

according to partial population based surveillance, inadequate risk assessment

Biosecurity grade 3: Risk analysis based control◦ Integral control under guidance of risk analysis, inadequate

traceability system Biosecurity grade 4: Systemic disease freedom◦ Comprehensive biosecurity, disease free status auditable

and sustainable Biosecurity grade 5: Official certification◦ Official veterinary certification for free compartment

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BSG1 farm with treatment based on diagnosis◦ Cleaning of disinfection◦ Ecological control◦ Disease diagnosis◦ Contingency plan

BSG2 farm with prevention based on surveillance◦ Population based

surveillance◦ Cleaning of disinfection◦ Water barrier◦ Ecological barrier◦ Targeted prevention◦ Contingency plan

BSG3 farm with control based on risk analysis◦ Disease-free larva◦ Systemic surveillance◦ Critical control points◦ Risk management measures◦ Contingency plan

BSG4 hatchery with disease free status◦ Disease-free screening for all

families◦ Systemic surveillance◦ Critical control points◦ Risk management measures◦ Comprehensive audit system of

implementation of biosecurity plan

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Selection of location and construction of hatchery Standard operating procedures Documentation and traceability system Team building and training of personals Evaluation of pathogen risk levels Establishment of independent maternal families Determination of pathogen detection technologies Determination of breeding strategies Quarantine for broodstock candidates◦ Preliminary detection (2—5 months)◦ Secondary screening (5—12 months)

Quarantine in cultivation of generations◦ For each batch of broodstocks◦ For each batch of larva◦ For growth-out stage

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Risk-free feed◦ Complete formula feed For maturation of broodstocks For larvae rearing

◦ Live feed SPF live feed Strict quarantine / feeding tests

Disinfection system◦ Farm/tools disinfection measures◦ Water disinfection measures◦ Disinfection measures for broodstocks and eggs

Management of inlet and waste water Third-party quarantine Official certification

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Selection of location and construction of hatchery

Standard operating procedures Documentation and traceability system Team building and training of personals Management of inlet and waste water Population based surveillance plan◦ Determine technological protocols◦ Determine targeted populations◦ Determine targeted life stages

Risk assessment Third part quarantine

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Quarantine for broodstocks or postlarva Production management Low risk feed◦ Live feed Quarantine Disinfection Quality control

◦ Formula feed For maturation of broodstocks For larvae rearing

Disinfection system◦ Farm/tools disinfection measures◦ Water disinfection measures◦ Disinfection measures for broodstocks and eggs

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布局建场，风险预防Build a farm with well layout for prevention of bio-risk

生产规划，安保先抓Prepare the farming with efforts on biosecurity plan

池塘整备，清消到位Recondition ponds by well dredging and disinfection

早纳先净，培水充分Flood ponds and reservoirs for early disinfection and fertilization

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检疫测毒，精选苗源Select postlarva by pathogen detection to avoid infection

标粗转料，平稳过渡Use nursery culture for stocking and quarantine

分级定密，合理放养Determine stocking density according to stages and health conditions

虾鱼搭配，生态防病Use polyculture with fish for ecological prevention

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精细投喂，少吃多餐Feed Precisely with little feed and many meals

验水刮底，勤测勤调Monitoring and control water and bottom quality regularly

查生测长，依诊防控Examine survival and growth, treat according to diagnosis

培菌投糖，絮团加强Ferment probiotics and use sucrose with enhancement of biofloc

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Thank You！