This document discusses the development and use of specific pathogen free (SPF), specific pathogen resistant (SPR), and specific pathogen tolerant (SPT) shrimp. It defines SPF shrimp as coming from a population that has tested negative for specific pathogens for at least 2 years through surveillance in a highly biosecure facility. The document outlines FAO guidelines for developing and maintaining SPF status, including facility certification. It describes a project in Ecuador and Nicaragua that selected WSSV tolerant shrimp from exposed populations and tested them over multiple generations to develop WSSV SPT+SPF stocks, finding improved survival and growth compared to local stocks. An external audit approved the SPF status of the selected stocks.

1. SPF, SPR and SPT as part
of a biosecurity strategy
Victoria Alday-Sanz
victoria_alday@yahoo.com

2. WHAT IS SPF?
PCR –ve animals?
Animals that have passed a quarantine and a
sample/various samples are tested?
Are the descendants of SPF still SPF?

3. FAO Expert Workshop
Specific Pathogen-Free (SPF), Specific Pathogen-
Resistant (SPR) and Specific Pathogen-Tolerant (SPT)
Shrimp:
a Need for International Technical Guidelines
26-28 May 2016, Sukosol Hotel, Bangkok, Thailand

4. SPF: SPECIFIC PATHOGEN FREE
• SPF is a animal coming from a population that has tested negative for
SPECIFIC PATHOGENS for at least 2 years (surveillance program in
place), that is raised in highly biosecure facilities (water treatment
and enclosed environment) following biosecure management
measures and have been fed with biosecure feeds.
• Requires transparency: auditing of the facility

5. WHAT PATHOGENS DEFINE SPF?
• No consensus
• Often for OIE listed pathogens (2015)
• DOF only OIE systemic viruses
• India only OIE listed pathogens
• Egypt bacteria (all vibrios) and fungi
No AHPND or EHP –ve requirement!!
• The SPECIFIC PATHOGENS are defined by the supplier and the client
• SPF programs should target all known pathogens (OIE++)

6. SPF
• SPF does not necessary refer to Pathogen Free (PE)
• SPF does not refer to its susceptibility, tolerance or resistance (genetic
traits)
• Genetic characterization:
• Susceptible: sensitive to infection and disease
• SPR: resistant to infection (qualitative)
• SPT: tolerant to disease (quantitive)
• Culture conditions: SPF:
• Susceptible+SPF
• SPR+SPF
• SPT+SPF Common misunderstanding: “either SPF or SPR”

7. FAO Technical Paper and Guidelines
• How to develop SPF
• Criteria for selection
• Genetic variation
• Maintenance of SPF
• Facilities
• Biosecurity measures
• Surveillance program
• Animal health monitoring
• Problem to confirm SPF status
• Sampling
• Standard methods (OIE)
• EVE
• Safeguards for farmers
• Third party validation/evaluation of
SPF (for importers to check facility,
source, quarantine on entry, etc)
• Recommendation for facility
certification by recognized
international standards
• Proper standard diagnostic
procedures
• Significant penalties for fraud
(legislation)
• Facility licence
• Government audit

8. SPF changed shrimp industry
in Asia
SPF=high biosecurity ponds
SPF+SPT/SPR=low biosecurity ponds

9. Development of WSSV SPT+SPF stocks (2010)
Victoria Alday-Sanz

10. Objective
• To identify individuals free of infections (known pathogens) from:
• Exposed populations (APE)
• WSSV tolerant populations

11. • LA: spontaneous development of WSSV tolerance (over 10 years)
• Endemic and widely spread
• Still nowadays:
• Common mortality at 40 days
• Drop in temperature: winter
• WSSV experimentally challenged stocks (over generations) did not show
better tolerance
Thailand: exclusion Ecuador: coped with
Controlled ponds Extensive ponds

12. Criteria
• Selection of animals from farms (no biosecurity)
• Animals between 23-30gr
• From as broad geographic origin as possible (genetic diversity)
• From farms/ponds with good performance

13. ¿Why working with SPF?
• Physiological cost of infection
• Economic cost of disease
• Tolerance to WSSV achieved to an aceptable degree
• To be done before the next epidemic arrived (and got pond broodstock
infected)
• We were shrimp farmers-business is at crop
• Natural selection is too costly for the farmer

14. ¿Where?
• 2 programs:
• Ecuador
• Nicaragua
• Wide genetic diversity
• Sanitary risk dispersion
• Lower risk of closing of borders risk

15. Surveillance nationwide of primary pathogens
• OIE listed pathogens (2010) + EHP + PvNv + Streptococus
• Nested PCR
• Kits IQ2000
• Successful annual participación in Arizona Ring tests
• Consulted with Sanitary Authorities and research centers for endemic
pathogens

16. Transmission Main Shrimp Pathogens
Virus Economic impact Distribution Vertical
transmission
Broodstock
Strategy
WSSV High (highly
prevalent)
systemic Intraovum Eliminate
TSV High (sporadic) systemic Perovum Eliminate
YHV High (sporadic) systemic Perovum Eliminate
IHHNV Medium systemic Intraovum Eliminate
YHV/GAV High (localized) systemic Perovum Eliminate
IMNV High (localized) systemic Perovum Eliminate
Streptococus High (localized) systemic Perovum Management
NHP Medium Digestive track Mouth open Management
BP, MBV, HPV Low Digestive track Mouth open Management
EHP High Digestive track Mouth open Management
AHPND High Internal and external
cuticle
Surface
colonization
Management
Assumption: All pathogens are vertically transmitted

17. Monitoring results:
Found: WSSV, IHHNV and NHP
Not found: TSV, IMNV, YHV/GAV, BP, PvNv or EHP
• Transmission
• WSSV and IHHNV:
• Transmisión vertical intra ovum: discharged
• HPV:
• ¨Vertical transmission¨ by contamination of the eggs: antibiotic treatment

18. Other tools
• Several parallel facilities (quarantine 1 and 2, broodstock multiplication center,
larvicultures)
• Additional water treatments
• Biosecurity protocols
• Indoor biofloc culture
• Fresh feed testing for pathogen (viruses, parasite and bacteria)
• Staff training

19. Selection of ponds
• Criteria:
• Ponds with good productivity
• Ponds with low prevalence
• Preliminary collection of 75 shrimp/pond (IHHNV, WSSV and NHP)
• Animals with phenotipic characteristics of good health

20. Selection procedure
• Individual reception and maintenance of animals
• PCR after temperature stress (22-24oC for 48h)
• Individual testing: WSSV, IHHNV y NHP
• Individual testing of each female after ablation and spawning
• Pools (10): TSV, IMNV, YGV/GAV, PvNv, BP, EHP (every single animal included in
the reaction
• Pools de PLs (120): WSSV and IHHNV
• Histology of broodstock
• NHP was erradicated with antibiotic treatment of broodstock upon
arrival and egg washing

22. • Each animal was tested individually 2 times (males) or 3 times (females)
and as a pools at least twice
• The process was repeated for 3 generations with consecutive negative
results.
• International standard: 2 years free
Additional testing
• Different tissues (hemolymph, gills and lymphoid organ), different type
of stress (pH, salinity and alcalinity)
• Not detected

24. Year Ecuador Nicaragua
2010 7.000 26.500
2011 18.176 62.000
2012 39.434 13.831
2013
Total 64.610 102.331
Number of PCR reactions de PCR
47%
53%
Descartados Negativos
30,000 broodstock
discharged
Ecuador:
74% broodstock
discharged: 7,435

25. External audit
• Dr. Ignacio de Blas et al.
• DG SANCO European expert
• Departament of Infectious Diseases, Veterinary Faculty, University of
Zaragoza, Spain
• Pathology and Epidemiology
• Oficially recognized lab to declare an area free from disease.

26. Process audited and aproved-annual
revision
Not simply PCR but SPF

27. Improvements in maturation facilities-Nicaragua
27%
25% 24%
18%
0.9% 0.50%
0%
5%
10%
15%
20%
25%
30%
2008 2009 2010 2011 2012 2013
% de Mortalidad
Mortality in reception rooms
18%
17%
15%
13%
1% 0.30%0%
2%
4%
6%
8%
10%
12%
14%
16%
18%
20%
2008 2009 2010 2011 2012 2013
% de…
Mortality post-ablation
5%
4%
3%
2%
0.4% 0.1%
3% 3%
2%
1%
0.1% 0%0%
1%
2%
3%
4%
5%
6%
2008 2009 2010 2011 2012 2013
HEMBRAS
Mortality during
production

28. Improvements in larviculture-Ecuador
No SPF SPF
Days to PL12 20-21 17-18
PLs/gr 350 200
Survival 45-50% 70-75%
Coeficient of variation >15% 10-12%

29. 29
Crecimientos semanales

30. 303
304
305
302
306
311 AVG CAM
AVG SIS
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
%SV
Ponds
% SV 91 Days culture - CAMANICA vs controls
74 % versus 82%
Comparison between SPF
and tolerant+SPF
0
2
4
6
8
10
12
14
16
0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105
Grams
Days
GROWTH - CAMANICA vs Control 80PL/m
303
304
305
302
306
311
PONDS

31. 0
0.5
1
1.5
2
2.5
0%
10%
20%
30%
40%
50%
60%
70%
80%
Larva SPF selcc. Cto Larvanica
FCA
Supervivencia
0.0
1,000.0
2,000.0
3,000.0
4,000.0
5,000.0
6,000.0
7,000.0
8,000.0
0%
10%
20%
30%
40%
50%
60%
70%
80%
Larva SPF selcc. Cto Larvanica
Rendimiento (Kilos/ha)
Supervivencia
Comparison
SPF versus
SPF+tolerance
under WSSV
conditions in
intensive system

32. WSSV SPT SPF shrimp
• Reverse SPF ?
• Broodstock exported to Saudi Arabia (risk analysis)
• PL exported to Perú, Honduras and Guatemala

33. Exported to Saudi Arabia
2 detections (2015 and 2016): no massive mortalities (>70% survival)
20590.6
10466.9
4932
652.9
12980
22350
29055
0
5000
10000
15000
20000
25000
30000
35000
2010 2011 2012 2013 2014 2015 2016
Production
year
P. vannameiP. indicus