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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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 26
GAPDisease
resistance breeding
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 27
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 28
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 29
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 30
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 31
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 32
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 33
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 34
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 35
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 36
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
2016-11-2, Xi'an, China
Jie Huang - Annual Workshop of Chinese Expert Commission for Aquatic Animal Disease Control 37
Thank You!
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