II 
(Non-legislative acts) 
DECISIONS 
COMMISSION IMPLEMENTING DECISION (EU) 2015/1554 
of 11 September 2015 
laying down rules for the application of Directiv e 2006/88/EC as regards requirements for 
surveillance and diagnostic methods 
(notifi ed under document C(2015) 6188) 
(Text with EEA relevance) 
THE EUR OPEAN COMMISSION, 
Having regar d to the Treaty on the Functioning of the European Union, 
Having regard to Council Directive 2006/88/EC of 24 October 2006 on animal health requirements for aquaculture 
animals and products thereof, and on the prevention and control of certain diseases in aquatic animals (1), and in 
particular Articles 49(3), 50(4), 57(b) and 61(3) thereof, 
Whereas: 
(1)  Directive 2006/88/EC lays down minimum preventive measures for the surveillance and early detection in 
aquatic animals of the listed diseases set out in Annex IV to that Directive (‘the listed diseases’) and the control 
measures to be applied in the event of the suspicion of or an outbreak of the listed diseases. It also lays down the 
requirements for the achi evement of disease-free status for Member State s or for zones or compar tments thereof. 
(2)  The eradication of the listed diseases and the achi evement of disease-free status for a Member State, zone or 
compar tment should be based on the same principles and follow the same scientifi c approach throughout the 
Union. For that reason, it is necessar y to lay down at Union level specifi c requirements for eradication and 
surveillance schemes, and the sampling and diagnostic methods to be used by the Member States to obtain 
disease-free status for the whole of a Member State or a zone or compar tment thereof. 
(3)  The laboratory examinations to be carried out in the event of the suspicion or conf irmation of the presence of 
the listed diseases, should be the same at Union level and should follow the same scientific standards and 
prot ocols. In accordance with Directive 2006/88/EC, it is necessar y to establish specifi c diagnostic methods and 
procedures to be used by the laboratories designat ed for that purpose by the compet ent author ity of the Member 
States. 
(4)  The Aquatic Animal Health Code adopt ed by the World Org anisation for Animal Health (OIE) (the ‘Aquatic 
Code’) sets out standards for improving the health of aquatic animals and the welf are of farmed fish worldwide, 
including standards for safe international trade in aquatic animals and products thereof. A number of chapt ers of 
the Aquatic Code set out recommendations concer ning the use of certain diagnostic tests. Such tests provided for 
by the OIE are laid down in the OIE Manual of Diagnostic Tests for Aquatic Animals (the ‘Aquati c Manual’). In 
order to ensure that Union's requirements with regar d to aquatic animal disease diagnostics are consiste nt with 
international standards, the rules laid down in this Decision should take into account the standards and 
recommendations of the Aquati c Code. 23.9.2015 L 247/1 Official Jour nal of the European Union EN     
(1)OJ L 328, 24.11.2006, p. 14. (5)  In this respect, for many of the liste d diseases, the Aquatic Manual lists several tests and procedures to be used 
for the purpose of laboratory examinations. In order to unif orm the scientific basis for the diagnostic work for 
the listed diseases at Union level, it is necessar y to choose among the diagnostic tests and procedures 
recommended by the OIE and to specify which tests should be mandatory for the purpose of laborat ory 
examination when carrying out surveillance programmes and to rule out the suspicion of or to conf irm the 
presence of the listed diseases. While there also will be a need for having alternative methods and procedures 
available in certain cases, descr iptions and some scientific explanations for when and how the alternati ve 
methods could be applied should be provided. This is in particular necessar y for the more detailed diagnostic 
procedures. 
(6)  In order to produce precise and reproducible diagnostic results, it is important that the detailed procedures and 
prot ocols to be used are validate d in accordance with the relevant quality standards refer red to in Part I of 
Annex VI to Directive 2006/88/EC. For many of the diagnostic methods provided for in this Decision, the use of 
commercial test kits is a necessar y part of the diagnostic prot ocols and those test kits have been validate d in 
accredit ed tests by the European Reference laboratories (EURL) for the respective diseases. In the interest of lega l 
certainty , the commercial names of those validat ed commercial test kits should be referred to in this Decision. 
(7)  It may be difficult for certain Member State s to achi eve disease-free status for the whole of the Member State or 
for a zone or compar tment thereof with regar d to one or more of the listed diseases. In such situations the 
Member State may wish not to obtain or regain disease-free status for those listed diseases. The minimum control 
measures to be applied in cases where the Member State concer ned does not wish to obtain or regain disease-free 
status, should be the same at Union level and should follow the same criteria. It is theref ore necessar y in 
accordance with Directive 2006/88/EC to lay down detailed rules for the containment of those liste d diseases and 
the minimum requirements for the lifting of those containment measures. 
(8)  Commission Decision 2001/183/EC (1) lays down the requirements concer ning samp ling plans and diagnostic 
methods for the detection and conf irmation of the listed diseases infec tious haemato poietic necrosis and viral 
haemor rhagic septicaemia. Commission Decision 2003/466/EC (2) lays down requirements concer ning sam pling 
plans and diagnostic methods for the detec tion of infectious salmon anaemia, as well as criteria for zoning and 
official surveillance following suspicion and conf irmations of the presence of that disease. Commission Decision 
2002/878/EC (3) lays down the requirements concer ning the samp ling plans and the diagnostic methods for the 
detection and conf irmation of the mollusc diseases Bonamiosis and Mar teiliosis. In order to updat e the 
requirements, those three Decisions should be replaced by this Decision. According ly, Decision 2001/183/EC, 
Decision 2002/878/EC and Decision 2003/466/EC should be repealed. 
(9)  As certain Member States require time to update their national reference laboratori es in order to compl y with the 
requirements laid down in this Decision, it should apply from 1 Apr il 2016. 
(10)  The measures laid down in this Decision are in accordance with the opinion of the Standing Committ ee on 
Plants, Animals, Food and Feed, 
HAS ADOPTED THIS DECISION: 
Article 1 
Subject matter 
This Decision lays down rules for the following: 
(a)  the surveillance, buff er zones, sampling and diagnostic methods to be used by Member States in connection with the 
disease status of the Member State s or zones or compar tments thereof for the non-ex otic aquatic animal diseases 
liste d in Part II to Annex IV to Directive 2006/88/EC (the ‘listed diseases’) ; 23.9.2015 L 247/2 Official Jour nal of the European Union EN     
(1)Commission Decision 2001/183/EC of 22 Febr uary 2001 laying down the samplin g plans and diagnostic methods for the detect ion and 
confir mation of certain fish diseases and repealing Decision 92/532/EEC (OJ L 67, 9.3.2001, p. 65). 
(2)Commission Decision 2003/466/EC of 13 June 2003 establishing criteria for zoning and official surveillance following suspicion or 
confir mation of the presence of infectious salmon anaemia (ISA) (OJ L 156, 25.6.2003, p. 61). 
(3)Commission Decision 2002/878/EC of 6 November 2002 establishing the samplin g plans and diagnostic methods for the detection and 
confir mation of the presence of the mollusc diseases Bonamiosis (Bonamia ostreae) and Mar teiliosis (Mar teilia refringens) (OJ L 305, 
7.11.2002, p. 57). (b)  the diagnostic methods to be used for laborat ory examinations in the case of the suspicion or conf irmation of the 
presence of listed diseases; and 
(c)  the minimum control measures to be applied in the event of the suspicion or conf irmation of a listed disease in a 
Member State, zone or compar tment not declared free of that listed disease. 
Article 2 
Def initions 
For the purpose of this Decision, the following definitions shall apply: 
(a)  ‘viral haemor rhagic septicaemia’ (‘VHS’) means a disease caused by the viral haemor rhagic sept icaemia virus (VHSV), 
also kno wn as the Egtved virus, a virus belonging to the genus Novirhabdovir us, within the family Rhabdo viridae; 
(b)  ‘infectious haemato poietic necrosis’ (‘IHN’) means a disease caused by infectious hemat opoietic necrosis virus (IHNV), 
a virus belonging to the genus Novirhabdo virus, within the family Rhabdovir idae; 
(c)  ‘koi herpesvir us disease’ (‘KHVD’) means a disease caused by koi herpesvir us (KHV), which belongs to the family of 
Alloher pesvir idae. The scientific name is cypr inid herpesvir us 3 (CyHV -3); 
(d)  ‘infectious salmon anaemia’ (‘ISA ’) means a disease caused by infection with highly polymor phic region (HPR)-delete d 
salmon anaemia virus (ISAV), a virus belonging to the genus Isavir us, within the family Orthomyxovir idae; 
(e)  ‘infection with Mar teilia refrin gens’ means a disease caused by an infection with the param yxean protozoan Mar teilia 
refrin gens; 
(f)  ‘infection with Bonamia ostreae’ means a disease caused by an infection with the haplospor idian protozoan Bonamia 
ostreae; 
(g)  ‘white spot disease’ (‘WSD’) means a disease caused by whit e spot syndrome virus (WSSV), which is a double 
stranded DNA virus of the genus Whispov irus, in the family Nimavir idae. 
Article 3 
Minimum requirements for eradication and surveillance prog rammes 
Member State s shall ensure that the rules on surveillance and eradication programmes, buffe r zones, sam pling and 
diagnostic methods set out in Annex I and the specific methods and detailed procedures set out in Annex II are 
compl ied with, when disease-free status is to be granted, withdrawn or restored for a Member State or for a zone or 
compar tment thereof for one or more of the liste d diseases. 
Article 4 
Minimum requirements for diagnostic methods and specif ic procedures 
Member State s shall ensure that the control methods set out in Annex I and the specif ic diagnostic methods and detailed 
procedures set out in Annex II are compl ied with, when laboratory examinations are carried out in order to conf irm or 
rule out the presence of a listed disease. 
Article 5 
Minimum control measures for the containment of listed diseases and minimum requirements for the lifting of 
cont ainment measures in Member States, zones or compar tments not declared free of listed diseases 
Member States shall ensure that the minimum control measures and minimum requirements for the lifting of 
containment measures set out in Annex I are compl ied with, when carrying out control measures and the lifting of 
containment measures for one or more of the listed diseases in a Member State or in a zone or compar tment thereof 
not declared free of those liste d diseases. 23.9.2015 L 247/3 Official Jour nal of the European Union EN     Article 6 
Repeals 
Decisions 2001/183/EC, 2002/878/EC and 2003/466/EC are repealed. 
Article 7 
Date of application 
This Decision shall apply from 1 Apr il 2016. 
Article 8 
Addresses 
This Decision is addressed to the Member States. 
Done at Brussels, 11 Sept ember 2015. 
For the Commission 
Vytenis ANDRIUKAITIS 
Member of the Commission  23.9.2015 L 247/4 Official Jour nal of the European Union EN     ANNEX I 
SUR VEILL ANCE AND CONTR OL METHODS 
I.  Introduction 
This Annex sets out: 
(a)  requirements for eradication and surveillance programmes, as provided for in Article 44 of Directive 
2006/88/EC, and the sampli ng and diagnostic methods to be used to declare disease-free status for Member 
States or zones or compar tments thereof as provided for in Chapt er VII of that Directive; 
(b)  sampling and diagnostic methods to be used for laboratory examinations in the case of the suspicion of and to 
conf irm the presence of the non-ex otic diseases listed in Part II of Annex IV to Directive 2006/88/EC (the ‘liste d 
diseases’) as provided for in Articles 28(a) and 57(b) of that Directive; 
(c)  the containment measures to be taken in the case of conf irmation of the presence of a liste d disease, as provided 
for in Article 39 of Directive 2006/88/EC, and the measures to be taken in order to obtain Categor y III health 
status for a Member State, zone or compar tment that has Categor y V health status. 
The requirements set out in this Annex cover the following listed diseases: 
1. Viral haemor rhagic septicaemia (VHS) Part 1 
2. Infectious haemato poietic necrosis (IHN) Part 1 
3. Koi herpesvir us (KHV) disease Part 2 
4. Infectious salmon anaemia (ISA) Part 3 
5. Infection with Mar teilia refrin gens Part 4 
6. Infection with Bonamia ostreae Part 5 
7. White spot disease (WSD) Part 6  
II.  Def initions 
For the purpose of Annex I and II, the following definitions shall apply: 
(a)  ‘continental compar tment’ means one or more farm(s) situated on the continental part of one or more Member 
State(s), being under a common biosecur ity system and containing an aquatic animal population with a distinct 
health status with respect to a specifi c disease; 
(b)  ‘continental farm’ means a farm keeping aquaculture animals situated on the continental part of the territory of 
one Member State; 
(c)  ‘continental zone’ means a precise geographic area situated on the continental part of one or more Member 
State(s) with a homog enous hydrological system compr ising parts of a water catchment area from the source(s) 
to a natural or artificial barrier that prevents the upward migration of aquatic animals from lower stretches of 
the water catch ment area, an entire water catchm ent area from its source(s) to its estuar y, or more than one 
water catchment area, including their estuar ies, due to the epidemiological link between the catchme nt areas 
through the estuar y; 23.9.2015 L 247/5 Official Jour nal of the European Union EN     (d)  ‘offi cially declared infected farm’ means a farm keepin g aquatic animals where one or more of the listed diseases 
has been conf irmed by the compet ent author ity in accordance Articles 28(a), Article 29 and Article 57(b) of 
Directive 2006/88/EC. 
(e)  ‘contact farm’ means a farm keeping aquatic animals which in any way is demonstrated or strong ly suspected to 
have been contaminate d by infectious mater ial from an officiall y declared infected farm. 
PART 1 
SUR VEILL ANCE AND CONTR OL METHODS FOR VIRAL HAEMORRHA GIC SEPTIC AEMIA (VHS) AND INFECTIOUS 
HEMA TOPOIETIC NECR OSIS (IHN) 
I.  Requirements for surveillance and eradication prog rammes to obta in and maint ain disease free-health statuses with 
regard to VHS and IHN and containment measures for those listed diseases 
I.1.  General requirements for health inspections and sam pling for VHS and IHN: 
(a)  the health inspections and, where appropr iate, the sampling shall be carried out during the period of the 
year when the water temperature is belo w 14 °C or whenever the water temperature is likely to reach its 
lowest annual points; 
(b)  when targe ted surveillance in wild populations is required in accordance with the second paragraph of 
point 2 of Part I of Annex V to Directive 2006/88/EC, the number and geographical distr ibution of 
sampli ng points shall be determi ned to obtain a reasonable coverag e of the Member State, zone or 
compar tment. The sampli ng points shall be representative of the different ecosystems where wild 
populations of suscep tible species are locat ed; 
(c)  when farms or wild populations are to be subject to health inspections or sampled more than once per year , 
the inter vals between the health inspections and between the collection of samples shall be at least 
4 months and as long as possible, taking into account the temperature requirements provided for in 
point (a); 
(d)  all production units, such as ponds, tank s and net cage s, shall be subject to health inspections for the 
presence of dead, weak or abnor mally behaving fish. Particular attention shall be paid to the water outlet 
area where weak fish tend to accumulate because of the water current ; 
(e)  fish of suscept ible species to be collected as samples shall be selected as follows: 
(i)  if rainbo w trout are present, only fish of that species shall be selected for sampling, excep t where other 
suscep tible species are present which show typical signs of VHS or IHN; if rainbo w trout are not 
present, the sample must be representative of all other susceptible species which are present ; 
(ii)  if weak, abnor mally beha ving or freshly dead but not decomposed fish are present, such fish shall be 
select ed; if more than one water source is utilised for fish production, fish representing all water sources 
shall be included in the sample; 
(iii)  the fish selected shall include fish collected in such a way that all parts of the farm, as well as all year 
classes, are propor tionally represented in the sample. 
I.2.  Specific requirements to obtain disease-free health status (categor y I) with regard to VHS and IHN 
I.2.1.  Surveillance programmes: 
(a)  a Member State , zone or compar tment that has Categor y III health status as refer red to in Part B of 
Annex III to Directive 2006/88/EC with regard to VHS or IHN or both, may achieve Categor y I health status 
with regard to those listed diseases provided that all farms keeping susceptible species listed in Part II of 
Annex IV to that Directive within that Member State, zone or compar tment comply with the requirements 
laid down in Annex V to that Directive and all those farms and, when required by the second paragraph of 
point 2 of Part I of Annex V thereto, sampli ng points in wild populations select ed in accordance with that 
Part, have been subject to one of the following surveillance programmes: 23.9.2015 L 247/6 Official Jour nal of the European Union EN     (i)  model A — 2-year surveillance programme: 
The farms or sampling points must have been subject to health inspections and sam pled for a minimum 
period of two consecutive years as laid down in Table 1.A set out in Section II. 
Dur ing that 2-yea r period, the testing of all samples using the diagnostic methods set out in point II.2 
must have produced negative results for either VHS or IHN or both, and any suspicion of either VHS or 
IHN or both must have been ruled out in accordance with the sampling and diagnostic methods set out 
in point II.3; 
(ii)  model B — 4-ye ar surveillance programme with reduced sample size: 
The farms or sampling points must have been subject to health inspections and sam pled for a minimum 
period of four consecutive years as laid down in Table 1.B set out in Section II. 
Dur ing that 4-yea r period, the testing of all samples using the diagnostic methods set out in point II.2 
must have produced negative results for either VHS or IHN or both and any suspicion of either VHS or 
IHN or both must have been ruled out in accordance with the sampling and diagnostic methods set out 
in point II.3; 
(b)  if during the imp lementation of the surveillance programme refer red to in point (a), infection with either 
VHS or IHN or both is conf irmed in a farm included in that surveillance programme, and theref ore the 
farm's Categor y II health status has been withdrawn, that farm may immediately regain its Categor y II health 
status and continue the implementation of the surveillance programme to obtain disease-free status without 
implementing an eradication programme as set out in point I.2.2 provided that the farm compl ies with the 
following conditions: 
(i)  it is a continental farm whose health status rega rding either VHS or IHN or both is independent of the 
health status of aquatic animal populations in the surrounding natural water s as regards those liste d 
diseases in accordance with point 3 of Part II of Annex V to Directive 2006/88/EC; 
(ii)  it is emptied, cleansed, disinf ected and fallowed; the duration of the fallo wing period shall be at least 
6 weeks; 
(iii)  it has been restocked with fish sourced from Member States, zones or compar tments with a Categor y I 
health status with rega rd to either VHS or IHN or both. 
I.2.2.  Eradication programmes 
I.2.2.1.  General requirements 
A Member State , zone or a compar tment with Categor y V health status with rega rd to either VHS or IHN or 
both may achieve Categor y I health status with regard to those listed diseases, provided that all farms keep ing 
suscep tible species listed in Part II of Annex IV to Directive 2006/88/EC within that Member State, zone or 
compar tment have been subject to an eradication programme that compl ies with points (a) to (e): 
(a)  the minimum control measures laid down in Section 4 of Chapt er V of Directive 2006/88/EC must have 
been effectively applied and a containment area, as refer red to in Article 32(b) of that Directive comprising a 
prote ction zone and surveillance zone, must have been established in the vicinity of the farm(s) officially 
declared infec ted with either VHS or IHN or both those listed diseases. 
The containment area must have been defined on a case-by- case basis taking into account factors 
influencing the risks for the spread of the listed disease to farmed and wild fish, such as: the number , rate 
and distr ibution of the mor talities of fish on the farm infec ted with either VHS or IHN or both; the distance 
and density of neighbour ing farms; the proximity to slaughterh ouses; contact farms; species present at the 
farms; the farming practices applied in the affected farms and in the neighbour ing farms to the affected 
farms; the hydrodynamic conditions and other factors of epidemiological significance identified. 23.9.2015 L 247/7 Official Jour nal of the European Union EN     For the establishment of the protection and surveillance zones, the following minimum requirements shall 
apply as regards the geographical demarcation of those zones: 
(i)  a prot ection zone shall be established in the immediate vicinity of a farm officially declared infec ted with 
either VHS or IHN or both those listed disease and shall correspond to:  
(1) in coastal areas: an area included in a circle with a radius of at least one tidal excursion or at least 
5 km, whic hever is larger , centred on the farm officially declared infect ed with either VHS or IHN or 
both, or an equivalent area deter mined according to appropr iate hydrodynamic or epidemiological 
data;  
(2) in inland areas: the entire water catch ment area of the farm officially declared infect ed with VHS or 
IHN or both; the compet ent author ity may limit the exte nsion of the zone to parts of the wate r 
catchm ent area, or the area of the farm, provided that the prevention of the spread of either VHS or 
IHN or both is not compr omised; 
(ii)  a surveillance zone shall be established by the compet ent author ity outside the prote ction zone and shall 
correspond to:  
(1) in coastal areas: an area, surrounding the prote ction zone, of overlapping tidal excur sion zones; or 
an area, surrounding the protect ion zone, and included in a circle of radius 10 km from the centre 
of the prot ection zone; or an equivalent area determi ned according to appropr iate hydrodynamic or 
epidemiological data;  
(2) in inland areas: as an extended area outside the established protection zone; 
(b)  all farms keepin g susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
prote ction zone not officially declared infected with either VHS or IHN or both shall be subject to an offici al 
investigation compr ising at least the following elements: 
(i)  the collection of samples for testing of 10 fish, when clinical signs or post-mor tem signs consistent with 
infec tion with either VHS or IHN or both are obser ved or minimum 30 fish, when clinical or post- 
mortem signs are not obser ved; 
(ii)  one health inspection: in those farms where the tests refer red to in (i) have produced negativ e results; 
health inspections shall continue once per month during the period when the water temperature is 
belo w 14 °C, excep t when fish ponds or net cages are covered with ice, until the prote ction zone is 
withdrawn in accordance with point I.2.2.1(c); 
(c)  all farms officiall y declared infec ted with either VHS or IHN or both shall be emp tied, cleansed, disinf ected 
and fallowed. The duration of the fallo wing period shall be at least 6 weeks. When all farms officiall y 
declared infect ed within the same protection zone are emptied, at least 3 weeks of synchr onised fallo wing 
shall be carried out. This paragraph also applies to new farms officiall y declared infec ted during the 
implementation of the eradication programme. 
When fallowing of the officially declared infect ed farms is carried out, the protection zones shall be 
conver ted into surveillance zones. 
The compet ent author ity may decide to require the emp tying, cleansing, disinfection and fallo wing of other 
farms within the established prot ection and surveillance zones. The length of the fallowing period for those 
farms shall be determined by the compet ent author ity following a case-by- case risk evaluation; 
(d)  all farms officiall y declared infected with either VHS or IHN or both those listed diseases and all other farms 
fallowed within the established prote ction and surveillance zones as refer red to in point (c), shall be 
restocked with fish sourced from Member States, zones or compar tments with a disease-free health status 
(Cat egor y I) with regard to either VHS or IHN or both. 23.9.2015 L 247/8 Official Jour nal of the European Union EN     Restoc king shall only take place when all farms officially declared infected have been emp tied, cleansed, 
disinf ected and fallowed in accordance with point I.2.2.1(c); 
(e)  all farms keepin g susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
Member State , zone or compar tment covered by the eradication programme and when surveillance in wild 
populations is required, sampling points select ed in accordance with point I.1, shall subsequently be subject 
to the surveillance scheme laid down in point I.2.1. 
I.2.2.2.  Requirements for regaining disease-free status for continental compar tments compris ing one sing le farm 
previously being declared free of either IHN or VHS or both 
A continental compar tment compr ising one sing le farm previously declared free of either VHS or IHN or both 
of those listed diseases, whose health status with regard to those listed diseases is independent of the 
surrounding natural water s in accordance with point 3 of Part II of Annex V to Directive 2006/88/EC, and 
whose Categor y I health status has been withdra wn in accordance with Article 53(3) of that Directive, may 
regai n Categor y I health status immediately after the compet ent author ity has conf irmed that the following 
conditions have been complied with: 
(a)  the officiall y conf irmed infect ed farm with either VHS or IHN or both must have been emptied, cleansed, 
disinf ected and fallowed; the duration of the fallowing period must be at least 6 weeks; 
(b)  the officially confir med infec ted farm with either VHS or IHN or both has been restocked with fish sourced 
from Member State s, zones or compar tments with a cate gory I health status with regar d to either VHS or 
IHN or both. 
I.3.  Specific requirements for the mainte nance of disease-free health status (Category I) with regar d to either VHS or 
IHN or both 
When target ed surveillance is required in order to maintain Categor y I health status, as provided in Article 52 of 
Directive 2006/88/EC, all farms keeping susceptible species liste d in Part II of Annex IV to that Directive within 
the Member State, zone or compar tment concer ned shall be subject to health inspection and fish shall be 
sam pled in accordance with Table 1.C set out in Section II of this Part, taking into account the risk level of the 
farm for the contraction of either VHS or IHN or both of those listed diseases. 
When deter mining the health inspection frequency for Categor y I health status compar tments with regard to 
either VHS or IHN or both, whic h are placed in continental areas and where the health status regarding VHS or 
IHN is dependent on the health status of the aquatic animal populations in surrounding natural water s in 
accordance with point 2 of Part II of Annex V to Directive 2006/88/EC, the risk for the contraction of either 
VHS or IHN or both shall be considered as high. 
Disease-free status shall only be maintained as long as all sam ples tested, using the diagnostic methods set out in 
point II.2., produce negative results for either VHS or IHN or both of those listed diseases and any suspicion of 
either VHS or IHN or both are ruled out in accordance with the diagnostic methods set out in point II.3. 
I.4.  Requirements for the lifting of containment measures provided for in Article 39 of Directive 2006/88/EC, 
namely the change from Categor y V to Categor y III health status 
A Member State , zone or a compar tment whic h has Categor y V health status with regard to either VHS or IHN 
or both may achi eve Categor y III health status with regar d to those listed diseases provided that: 
(a)  the requirements set out in points I.2.2.1(a), (b) and (c) have been met. In case fallowing is not technically 
possible, the farms concer ned shall be subject to an alternative measure which provides almost similar 
guarant ee for exte rmination of either IHNV or VHSV or both from the environment of the farm; 
(b)  all farms officially declared infected and all other farms fallowed/been subject to altern ative measures in 
accordance with (a) within the established protection and surveillance zones have been restocked with fish 
sourced from Member State s, zones or compar tments with a Categor y I, II or III health status with regar d to 
either VHS or IHN or both; 23.9.2015 L 247/9 Official Jour nal of the European Union EN     (c)  the restocking has only taken place after all farms officially declared infec ted, have been emp tied, cleansed, 
disinf ected and fallowed/been subject to alternative measures in accordance with (a). 
II.  Diagnostic and sampling methods 
II.1.  Org ans to be sampled: 
The tissue mat erial to be examined is spleen, anter ior kidne y, and either hear t or encephalon. When sampling 
broodsto ck ovarian or seminal fluid may also be examined. 
In case of small fry, whole fish less than 4 cm long can be minced with sterile scissors or a scalpel after remo val 
of the body behind the gut opening. If a samp le consists of whole fish with a body length between 4 cm 
and 6 cm, the viscera including kidne y shall be collected. 
Org an pieces from a maximum of 10 fish may be pooled. 
II.2.  Diagnostic methods to obtain and maintain disease-free status for either VHS or IHN or both 
The diagnostic method, in accordance with the appro ved diagnostic methods and procedures set out in point I. 
of Part 1 of Annex II, to achi eve or to maintain disease-free status for VHS or IHN or both shall be either: 
(a)  virus isolation in cell cultures followed by identif ication using enzyme-linked immunosorbent assa y (ELISA ), 
indirect fluorescent antibody test (IFAT), virus neutralisation test or real-time reverse transcr iptase 
polymerase chain reaction (RT-qPCR); or 
(b)  RT-qPCR. 
II.3.  Sam pling and diagnostic methods to rule out or to confir m the presence of VHS or IHN 
When a suspicion of either VHS or IHN or both is required to be conf irmed or ruled out in accordance with 
Article 28 of Directive 2006/88/EC, the following inspection, sampling and testing procedures shall be compl ied 
with: 
(a)  the farm under suspicion shall be subject to at least one health inspection and one sampli ng of 10 fish, 
when clinical signs or post-mor tem signs consistent with infection with either VHS or IHN or both are 
obser ved or minimum 30 fish, when clinical or post-mor tem signs are not obser ved. Samples shall be tested 
using one or more of the diagnostic methods set out in points (i) and (ii) in accordance with the detailed 
diagnostic methods and procedures as set out in Section II of Part 1 of Annex II: 
(i)  conventional virus isolation in cell culture with subsequent immunoche mical or molecular virus 
identification; 
(ii)  virus detection by RT-qPCR; 
(iii)  Other diagnostic techniques of proven similar efficacy such as Indirect fluorescent antibody test (IFAT), 
Enzyme-link ed immuno-sorbent assa y (ELISA), RT-PCR and Immunohist ochemistry (IHC). 
(b)  the presence of VHS shall be considered as confir med, if one or more of those diagnostic methods are 
positive for VHSV . The presence of IHN shall be considered as conf irmed, if one or more of those diagnostic 
methods are positive for IHNV . The confir mation of the first case of VHS or IHN in Member States, zones 
or compar tments previously not infect ed shall be based on conven tional virus isolation in cell culture or 
RT-qPCR; 
(c)  Suspicion of either VHSV or IHNV or both may be ruled out, if cell cultivation or RT-qPCR tests reveal no 
further evidence of the presence of either VHSV or IHNV or both. 23.9.2015 L 247/10 Official Jour nal of the European Union EN     Table 1.A 
Sur veillance scheme for zones and compar tments for the 2-year control period referred to in point I.2.1(a)(i) 
which precedes the achiev ement of disease-free status for VHS or IHN 
Type of farm Number of health 
inspections per 
year (2 years) Number of 
samplings per 
year (2 years) Number of fish in the sample (1) 
Number of growing fish Number of broodstoc k 
fish (2) 
(a)  Farms with broodsto ck 2 2 50 (first inspection) 
75 (second inspection) 30 (first or second  
inspection) 
0 (first or second  
inspection) 
(b)  Farms with broodsto ck only 2 1 0 75 (first or second  
inspection) 
(c)  Farms without broodsto ck 2 2 75(3) (first and second 
inspection) 0 
Maximum number of fish per pool: 10 
(1)  The samples must be collected no sooner than 3 weeks after the transfer of the fish from fresh to saltwater . 
(2)  Ovar ian or seminal fluid of broodstoc k shall be collected at the time of maturation, in connection with stripping. 
(3)  Samples must be taken from the number of fish that will ensure the detect ion of VHSV or IHNV with a 95 % confidence if the 
design prevalence is 5 %.  
Table 1.B 
Sur veillance scheme with a reduced sample size for the 4-year control period refer red to in point I.2.1(a)(ii) 
which precedes the achiev ement of disease-free status for VHS or IHN 
Type of farm Number of health 
inspections per 
year Number of 
samplings per 
year Number of fish in the sample (1) 
Number of growing fish Number of broodstoc k 
fish (2) 
First 2 years of the surveillance period 
(a)  Farms with broodsto ck 2 1 0 (first inspection) 
30 (second inspection) 0 (first inspection) 
0 (second inspection) 
(b)  Farms with broodsto ck only 2 1 0 30 (first or second  
inspection) 
(c)  Farms without broodsto ck 2 1 30(3) (first or second 
inspection) 0 
Last 2 years of the surveillance period 
(a)  Farms with broodsto ck 2 2 30 (first inspection) 
0(second inspection) 0 (first inspection) 
30 (second inspection) 23.9.2015 L 247/11 Official Jour nal of the European Union EN     Type of farm Number of health 
inspections per 
year Number of 
samplings per 
year Number of fish in the sample (1) 
Number of growing fish Number of broodstoc k 
fish (2) 
(b)  Farms with broodsto ck only 2 2  30 (first and second 
inspection) 
(c)  Farms without broodsto ck 2 2 30(3) (first and second 
inspection)  
Maximum number of fish per pool: 10 
(1)  The samples must be collected no sooner than 3 weeks after the transfer of the fish from fresh to saltwater . 
(2)  Ovar ian or seminal fluid of broodstoc k shall be collected at the time of maturation, in connection with stripping. 
(3)  Samples must be taken from the number of fish that will ensure detection of VHSV or IHNV with a 95 % confidence if the design 
prevalence is 10 %.  
Table 1.C 
Sur veillance schemes for zones or compar tments to maintain disease-free status for VHS or IHN as refe rred to 
in point I.3 
Risk level Number of health inspections Number of fish in the sample (3) 
High 2 ever y year 30(1)(2) 
Medium 1 ever y year 30(1) 
Low 1 ever y 2 years 30(1) 
Maximum number of fish per pool: 10 
(1)  The samples must be collected no sooner than 3 weeks after the transfer of the fish from fresh to saltwater . 
(2)  Samples must be taken from the number of fish that will ensure the detect ion of VHSV or IHNV with a 95 % confidence if the 
design prevalence is 10 %. 
(3)  There shall be minimum one sample for ever y health inspection.  
PART 2 
SUR VEILL ANCE AND CONTR OL METHODS FOR KOI HERPESVIR US DISEASE (KHVD) 
I.  Requirements for surveillance and eradication prog rammes to obtain and maintain disease-free health status with 
regard to KHVD and to contain infection with koi herpesvir us (KHV) 
I.1.  General requirements 
When targe ted surveillance in wild populations is required in accordance with the second paragraph of point 2 
of Part I of Annex V to Directive 2006/88/EC, the number and geographical distr ibution of sampli ng points 
shall be determi ned to obtain a reasonable coverag e of the Member State , zone or compar tment. The sampli ng 
points shall also be representative of the differ ent ecosyste ms where the wild suscep tible populations are locat ed, 
namely , river system s and lakes . 23.9.2015 L 247/12 Official Jour nal of the European Union EN     Target ed surveillance shall rely on the regular monitori ng of sites holding suscep tible species. Sites shall be 
monitor ed when water temperatures have reached levels that are permissive for the development of the disease 
(> 15 °C) and no sooner than 2 weeks from the date such temperatures have been reac hed. Any diseased fish or 
fish showing abnor mal behavior that are found on the site shall be sam pled and tested. 
Whenever possible, fish that have been kept for a prolonged time period at the virus permissive temperature 
rang e, namely 2 to 3 weeks at 15 °C to 26 °C, shall be sampled. The following approac h may, however , be 
accept ed: 
(a)  to collect a sub-population at transfe r from winter to summer ponds and hold the fish in the same water 
body as the summer pond until minimum temperature requirements have been obtained, or 
(b)  to collect samples at harvesting or during other fish handling as part of normal manag ement practices. If 
possible, samples shall be collect ed between 24 and 72 hours after such manag ement practices to enhance 
the chance of KHV detection. 
When farms or wild populations have to be subject to health inspections or sample d more than once per year, 
the inter vals between the health inspections or collections of sam ples shall be as long as possible within the 
season when the water temperature is likely to reach its highest annual points without exceed ing the limit of 
28 °C. 
All production units, such as ponds and tank s, must be subject to health inspections for the presence of dead, 
weak or abnor mally behaving fish. 
Cypr inus carpio and its hybr ids, such as Cypr inus carpio × Carassius auratus, shall be collect ed when present in the 
farm. 
The fish to be collect ed as sam ples shall be select ed as follows: 
(i)  if weak, abnor mally beha ving or freshly dead but not decomposed fish are present, such fish must be 
selected; 
(ii)  if more than one water source is utilised for fish production, fish representing all water sources must be 
included for sam pling; 
(iii)  the fish selected must include fish collect ed in such a way that all parts of the farm, as well as all year 
classes, are propor tionally represented in the samp le. 
I.2.  Specific requirements to achieve disease-free health status (Cat egor y I) with regard to KHVD 
I.2.1.  Surveillance programmes 
(a)  a Member State, zone or a compar tment which has Categor y III health status with regar d to KHVD may 
achi eve Categor y I health status when all farms keeping suscep tible species listed in Part II of Annex IV to 
Directive 2006/88/EC within that Member State , zone or compar tment compl y with the requirements for 
disease-free status set out in Annex V to that Directive and all those farms, and, when required by the 
second paragraph of point 2 of Part I of that Annex, sampling points in wild populations selected in 
accordance with that Part, have been subject to one of the following surveillance programmes: 
(i)  model A — 2-year surveillance programme: 
The farms or sampling points must have been subject to health inspections and sam pled for a minimum 
period of two consecutive years as laid down in Table 2.A set out in Section III. 
Dur ing that 2-yea r period, the testing of all samples using the diagnostic methods set out in point II.2 
must have produced negative results for KHV and any suspicion of KHVD must have been ruled out in 
accordance with the diagnostic methods set out in point III.2; 23.9.2015 L 247/13 Official Jour nal of the European Union EN     (ii)  model B — 4-ye ar surveillance programme with reduced sample size: 
The farms or sampling points must have been subject to health inspections and sam pled for a minimum 
period of four consecutive years as laid down in Table 2.B set out in Section III. 
Dur ing that 4-yea r period, the testing of all samples using the diagnostic methods set out in point II.2 
must have produced negative results for KHV and any suspicion of KHVD must have been ruled out in 
accordance with the diagnostic methods set out in point III.2; 
(b)  if during the implementation of the 4-ye ar surveillance programme set out in point (a), infection with KHV 
is conf irmed in a farm included in that surveillance programme, and theref ore its Categor y II health status 
has been withdrawn, that farm may immediately regain the Categor y II health status and continue the 
implementation of the surveillance programme to obtain disease- free status without implementing an 
eradication programme as descr ibed in point I.2.2 provided that the farm complies with the following 
conditions: 
(i)  it is a continental farm whose health status regar ding KHVD is independent of the health status of 
aquatic animal populations in the surrounding natural water as regar ds that listed disease in accordance 
with point 3 of Part II of Annex V to Directive 2006/88/EC; 
(ii)  it has been emptied, cleansed, disinfect ed and fallo wed; the duration of the fallowing period shall be at 
least 6 weeks; 
(iii)  it has been restocked with fish sourced from Member States, zones or compar tments with a Categor y I 
health status with rega rd to KHVD. 
I.2.2.  Eradication programmes 
I.2.2.1.  General requirements 
A Member State, zone or a compar tment that has Categor y V health status with regard KHVD, may achieve 
Categor y I health status with rega rd to that listed disease when all farms keeping susceptible species listed in 
Part II of Annex IV to Directive 2006/88/EC within that Member State, zone or compar tment have been subject 
at least to the following eradication programme: 
(a)  the minimum control measures laid down in Section 4 of Chapt er V of Directive 2006/88/EC have 
effectively been applied, and a containment area as referred to in Article 32(b) of that Directive, comprising 
a prot ection zone and surveillance zone, has been established in the vicinity of the farm(s) officially declared 
infected with KHV . 
The containment area must have been defined on a case-by- case basis taking into account factors 
influencing the risks for the spread of KHVD to farmed and wild fish, such as: the number , rate and 
distr ibution the mor talities of fish on the farm infected with KHV ; the distance and density of neighbour ing 
farms; the proximity to slaughterhouses; contact farms; the species present at the farms; the farming 
practices applied in the affect ed and neighbour ing farms; the hydrodynamic conditions and other factors of 
epidemiological signif icance identif ied. 
For the establishment of the protection and surveillance zones, the following minimum requirements shall 
apply as regards the geographical demarcation of those zones: 
(i)  a prot ection zone shall be established in the immediate vicinity of a farm officially declared infec ted with 
KHV and shall correspond to the entire water catchment area of the farm officiall y declared infec ted 
with KHV ; the compet ent author ity may limit the exte nsion of the zone to parts of the wate r catchme nt 
area provided that the prevention of the spread of KHVD is not compromised; 
(ii)  a surveillance zone shall be established outside the protection zone and shall correspond to an extende d 
area surrounding the established prot ection zone; 23.9.2015 L 247/14 Official Jour nal of the European Union EN     (b)  all farms keepin g susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
prote ction zone not officially declared infected with KHV shall be subject to an official investigation 
comprising at least the following elements: 
(i)  the collection of samples for testing of 10 fish, when clinical signs or post-mor tem signs consistent with 
KHVD are obser ved or 30 fish, when clinical or post-mor tem signs are not obser ved; 
(ii)  one health inspection; in those farms, where the tests refer red to in point III.2 have produced negative 
results; health inspections shall continue once per month during the season when the wate r temperature 
is likely to reac h > 15 °C until the protect ion zone is withdrawn in accordance with point I.2.2.1(c); 
(c)  all farms officially declared infected with KHV shall be emptied, cleansed, disinfect ed and fallowed. The 
duration of the fallowing period shall be at least 6 weeks. When all farms within the same protection zone 
officiall y declared infec ted have been emp tied, at least 3 weeks of synch ronised fallo wing shall be carried 
out. This paragraph also applies to new farms officially declared infected during the implementation of the 
eradication programme. 
When fallowing of the officially declared infect ed farms is carried out, the protection zones shall be 
conver ted into surveillance zones. 
The compet ent author ity may decide to require the emp tying, cleansing, disinfection and fallo wing of other 
farms within the established protection and surveillance zones. The length of the fallowing period shall be 
determined by the compet ent author ity following a case-by-case risk evaluation; 
(d)  All farms officially declared infect ed with KHV and all other farms fallowed within the established prot ection 
and surveillance zones shall be restocked: 
(i)  with fish sourced from Member States, zones or compar tments with a Categor y I health status with 
regard to KHVD; or 
(ii)  for a transitional period until 31 December 2020, with fish from Member States, zones or 
compar tments with an appro ved KHVD surveillance programme. 
Restoc king shall only take place when all farms officiall y declared infected with KHV have been emp tied, 
cleansed, disinf ected and fallowed in accordance with point I.2.2.1(c); 
(e)  All farms keeping susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
Member State, zone or compar tment covered by the eradication programme and, when surveillance in wild 
populations is required, sam pling points selecte d in accordance with Point I.1., shall have subsequently been 
subject at least to the surveillance programme laid down in point I.2.1. 
I.2.2.2.  Requirements for regai ning disease free status for continental compar tments comprising one sing le farm 
previously declared free of KHVD 
A continental compar tment comprising one sing le farm that has Categor y I health status with regar d to KHVD, 
whose health status with regard to KHVD is independent of the surrounding natural water s in accordance with 
point 3 of Part II of Annex V to Directive 2006/88/EC, and whose Categor y I status has been withdrawn in 
accordance with Article 53(3) of that Directive, may regain Categor y I health status with rega rd to KHVD 
immediate ly after the compet ent author ity has conf irmed that it has compl ied with the following conditions: 
(a)  it has been emptied, cleansed, disinfect ed and fallo wed; the duration of the fallowing period must have been 
be at least 6 weeks; 
(b)  it has been restocked with fish sourced from Member States, zones or compar tments with a Categor y I 
health status or compar tments with an appro ved KHVD surveillance programme (Category II health status). 23.9.2015 L 247/15 Official Jour nal of the European Union EN     I.3.  Specific requirements for the mainte nance of Categor y I status with regar d to KHVD 
When target ed surveillance is required in order to maintain Categor y I health status, as provided for in 
Article 52 of Directive 2006/88/EC, all farms keeping susceptible species listed in Part II of Annex IV to that 
Directive within the Member State, zone or compar tment concer ned shall be subject to health inspection and 
sam pled in accordance with Table 2.B set out in Section III of this Part, taking into account the risk level of the 
farm for the contraction of KHV . 
The inspection frequency for health inspections of Categor y I compar tments with regar d to KHVD placed in 
continental areas and comprising one or more farms whose health status regarding KHVD is dependent on the 
health status for that listed disease of surrounding natural wate rs in accordance with point 2 of Part II of 
Annex V to Directive 2006/88/EC, shall be in accordance with the number set out for high risk level in that 
Table 2.C. 
In Member States, zones or compar tments in whic h the number of farms is limited and target ed surveillance on 
these farms does not provide suffi cient epidemiological data, the surveillance schem es to maintain disease-free 
status shall include sam pling points selecte d in accordance with the requirements set out in point I.1. 
Those sampli ng points shall be inspected and sampled by rotation of 50 % of the sampling points each year. 
The sampli ng shall be carried out in accordance with Table 2.C set out in Section III. The samples shall be 
select ed, prepared and examined as descr ibed in Section II and the laborat ory examinations must be negat ive 
with regar d to the presence of the agent of KHVD. 
Disease-free status shall only be maintained as long as all samples tested using the diagnostic methods set out in 
point II.2 produce negative results for KHVD and any suspicion of KHVD must be ruled out in accordance with 
the diagnostic methods set out in point III.2. 
I.4.  Specific requirements for lifting the containment measures provided for in Article 39 of Directive 2006/88/EC 
to obtain Categor y III health status with rega rd to KHVD in Member States, compar tments or zones that have 
Categor y V health status 
A Member State, zone or a compar tment that has Categor y V health status with regard to KHVD may achi eve 
Categor y III health status with rega rd to that listed disease provided that: 
(a)  the requirements set out in points I.2.2.1(a), (b) and(c) have been met. In case fallo wing is not technically 
possible, the farms concer ned shall be subject to an alternative measure which provides almost similar 
guarant ee for exte rmination of KHV from the environment of the farm; 
(b)  all farms officially declared infected and all other farms fallowed/been subject to altern ative measures in 
accordance with (a) within the established protection and surveillance zones have been restocked with fish 
sourced from Member State s, zones or compar tments with a Categor y I, II or III health status with regar d to 
KHVD; 
(c)  the restoc king has only take n place when all farms officially declared infect ed have been emp tied, cleansed, 
disinf ected and fallowed/been subject to alternative measures in accordance with (a). 
II.  Diagnostic and sampling methods for surveillance to obtain and maint ain disease-free status with regard to KHVD 
II.1.  Sam ples 
The tissue mate rial to be examined shall be parts of gill and kidne y. Org an pieces from a maximum of two fish 
may be pooled. 
II.2.  Diagnostic methods for the surveillance to obtain and maintain disease-free status with regar d to KHVD 
The diagnostic method to achi eve or to maintain disease-free status for KHVD shall be Real-time PCR (qPCR) in 
accordance with the detailed diagnostic methods and procedures as set out in point II of Part 2 of Annex II. 23.9.2015 L 247/16 Official Jour nal of the European Union EN     III.  Diagnostic and sampling methods for official investigations for conf irmation or to rule out a suspicion of KHVD 
III.1.  Sam ples 
The tissue mate rial to be examined shall be parts of gill and kidne y. Org an pieces from a maximum of two fish 
may be pooled. 
III.2.  Official investigation and diagnostic methods to rule out and to conf irm the presence of infection with KHV 
When a suspicion of KHVD is required to be conf irmed or ruled out in accordance with Article 28 of Directive 
2006/88/EC, the following inspection, sampling and testing procedure shall be compl ied with: 
(a)  the official inve stigation shall include at least one health inspection and one sam pling of 10 fish, when 
clinical signs or post-mor tem signs consistent with infection with KHV are obser ved or 30 fish when clinical 
or post-mor tem signs are not obser ved. Samples shall be tested using the diagnostic method set out in 
point (b) in accordance with the detailed diagnostic methods and procedures set out in point II of Part 2 of 
Annex II; 
(b)  the presence of the infection with KHV shall be considered as conf irmed if KHV is detected by PCR; 
suspicion of KHVD may be ruled out, if this test reveals no further evidence of the presence of KHV . 
Table 2.A 
Sur veillance scheme for zones and compar tments for the 2-year control period which precedes the 
achiev ement of disease-free status for KHVD as refer red to in point I.2.1   
Number of clinical 
inspections per year 
(2 years) Number of labora ­
tory examinations 
per year (2 years) Number of fish in 
the sample 
Farms/sampling sites First 2 years of the surveillance 
period 2 2 75(1)  
Maximum number of fish per pool: 2 
(1)Samples must be taken from the number of fish that will ensure the detecti on of KHV with a 95 % confidence if the design preva ­
lence is 5 %.  
Table 2.B 
Sur veillance scheme for zones and compar tments for the 4-year control period which precedes the 
achiev ement of disease-free status for KHVD as refer red to in point I.2.1   
Number of clinical 
inspections per year Number of labora ­
tory examinations 
per year Number of fish in 
the sample 
Farms/sampling sites First 2 years of the surveillance 
period 1 1 30 
Farms/sampling sites Last 2 years of the surveillance 
period 2 2 30  
Maximum number of fish per pool: 2  23.9.2015 L 247/17 Official Jour nal of the European Union EN     Table 2.C 
Sur veillance schemes for zones or compar tments to maint ain disease-free status for KHVD as referred to in 
point I.3 
Risk level Number of health inspections Number of fish in the sample 
High 2 ever y year 30 
Medium 1 ever y year 30 
Low 1 ever y 2 years 30 
Maximum number of fish per pool: 2  
Table 2.D 
Sur veillance scheme to maint ain KHVD disease-free status in Member States, zones or compar tments where 
the number of farms is limited and targete d surveillance on these farms does not provide suff icient epidemio ­
logical data as refer red to in point I.3  
Number of clinical inspections 
per year Number of laborato ry exami ­
nations per year Number of fish in the sample 
Samp ling points 1 ever y 2 years 1 ever y 2 years 30 
Maximum number of fish per pool: 2  
PART 3 
SUR VEILL ANCE AND CONTR OL METHODS FOR INFECTIOUS SALMON ANEMIA (ISA) 
I.  Requirements for surveillance and eradication prog rammes to obtain and to maint ain disease-free health statuses 
with regard to ISA and to contain infection with HPR deleted ISAV 
I.1.  General requirements 
When health inspections and sampli ng of farms in accordance with the second paragraph of point 2 of part I of 
Annex V to Directive 2006/88/EC is required to be carried out more than once per year, the inter vals between 
the health inspections or collection of samples shall be as long as possible. 
When targe ted surveillance in wild populations is required in accordance with the second paragraph of point 2 
of Part I of Annex V to Directive 2006/88/EC, the number and geographical distr ibution of sampli ng points 
shall be determi ned to obtain a reasonable coverag e of the Member State , zone or compar tment. The sampli ng 
points shall also be representative of the differ ent ecosystems where the wild susceptible populations are locat ed. 
The health inspections shall be carried out in all production units, such as ponds, tanks and net cages, for the 
presence of dead, weak or abnor mally beha ving fish. Particular attention shall be paid to the water outlet area 
where weak fish tend to accumulate because of the water current. 23.9.2015 L 247/18 Official Jour nal of the European Union EN     The fish to be collect ed as sam ples shall be select ed as follows: 
(a)  only mor ibund or freshly dead fish, but not decom posed fish shall be selected; in particular fish 
demonstrating anaemia, bleedings or other clinical signs suggest ing circulator y disturbances shall be 
prioritised for collection; 
(b)  if Atlantic salmon is among the susceptible species on the site, sam ples from Atlanti c salmon shall be 
prioritised. If there is no Atlanti c salmon in the fish farm, other susceptible species must be sampled; 
(c)  if more than one water source is utilised for fish production, fish representing all water sources shall be 
included in the sam ple; 
(d)  the fish selected shall include fish collected in such a way that all production units, such as net cages, tank s 
and ponds, of the farm as well as all year classes are propor tionally represented in the sample. 
I.2.  Specific requirements to achieve Categor y I health status with regard to ISA 
I.2.1.  Surveillance programmes 
A Member State, zone or compar tment which has Categor y III health status in accordance with part B of 
Annex III to Directive 2006/88/EC with regard to ISA may achieve Categor y I health status with regar d to that 
listed disease when all farms keeping susceptible species listed in Part II of Annex IV to Directive 2006/88/EC 
within the Member State , zone or compar tment meet the relevant requirements set out in Annex V to that 
Directive and all those farms and, when required by the second paragraph of point 2 of Part I of Annex V 
theret o, sam pling points in wild populations select ed in accordance with that point, have been subject to the 
following surveillance programme: 
(a)  the farms or sampli ng points have been subject to health inspections and samp led for a minimum period of 
two consecutive years as laid down in Table 3.A set out in Section II; 
(b)  during that 2-year period, the testing of all samples using the diagnostic methods set out in point II.2 must 
have produced nega tive results for HPR -delet ed ISAV and any suspicion of ISA must have been ruled out in 
accordance with the diagnostic methods set out in point II.3; 
(c)  if during the implementation of the surveillance programme, ISA is confir med in a farm included in that 
surveillance programme, and theref ore its Categor y II health status has been withdrawn, an eradication 
programme in accordance with point I.2.2 must have been carried out. 
I.2.2.  Eradication programmes 
I.2.2.1.  General requirements 
A Member State , zone or a compar tment that has Categor y V health status with regar d to ISA may achi eve 
Categor y I health status with rega rd to that listed disease when all farms keeping susceptible species listed in 
Part II of Annex IV to Directive 2006/88/EC within the Member State , zone or compar tment have been subject 
to an eradication programme that compl ies with the following points (a) to (e). 
(a)  the minimum control measures laid down in Section 3 of Chapt er V of Directive 2006/88/EC have 
effectively been applied, and in particular a containment area as referred to in Article 32(b) of that Directive, 
comprising a prote ction zone and surveillance zone, has been established in the vicinity of the farm(s) 
officiall y declared infected with HPR -deleted ISAV or conf irmed ISA. 
The containment area must have been defined on a case-by- case basis taking into account factors 
influencing the risks for the spread of ISA to farmed or wild fish, such as: the number , rate and distr ibution 
of the mor tality of fish on the farm infec ted with HPR -delete d ISAV or conf irmed ISA; the distance and 
density of neighbour ing farms; the proximity to slaughterh ouses; contact farms; the species present at the 
farms; the farming practices applied in the affect ed and neighbour ing farms; the hydrodynamic conditions 
and other factors of epidemiological signif icance identified. 23.9.2015 L 247/19 Official Jour nal of the European Union EN     For the establishment of the protection and surveillance zones, the following minimum requirements shall 
apply as regards the geographical demarcation of those zones: 
(i)  a prot ection zone shall be established in the immediate vicinity of a farm officially declared infec ted with 
ISA and shall correspond to:  
(1) in coastal areas: an area included in a circle with a radius of at least one tidal excursion or at least 
5 km, whic hever is larger , centred on the farm officially declared infect ed with ISA, or an equivalent 
area determined according to appropr iate hydrodynamic or epidemiological data;  
(2) in inland areas: the entire water catchm ent area of the farm officially declared infect ed with ISA; the 
compet ent author ity may limit the extensi on of the zone to parts of the water catchme nt area 
provided that the prevention of the spread ISA is not compromised; 
(ii)  a surveillance zone shall be established outside the protection zone and shall correspond to:  
(1) in coastal areas: an area, surrounding the prote ction zone, of overlapping tidal excur sion zones; or 
an area, surrounding the protect ion zone, and included in a circle of radius 10 km from the centre 
of the prot ection zone; or an equivalent area determi ned according to appropr iate hydrodynamic or 
epidemiological data; or  
(2) in inland areas: an exte nded area outside the established prot ection zone; 
(b)  all farms keepin g susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
prote ction zone not officially declared infected with ISA shall be subject to an official inve stigation 
comprising at least the following elements: 
(i)  the collection of samples for testing of minimum 10 mor ibund fish, when clinical signs or post-mor tem 
signs consistent with ISA are obser ved, or minimum 30 fish when no clinical or post mor tem signs are 
obser ved; 
(ii)  one health inspection; in those farms where the tests refer red to in (i) have produced negativ e results, 
the health inspections shall continue once per month until the prot ection zone is withdrawn in 
accordance with point I.2.2.1(c); 
(c)  all farms officially declared infected with HPR -delete d ISAV or confir med ISA shall be emp tied, cleansed, 
disinf ected and fallo wed for a period of at least 3 months. The prote ction and surveillance zones may be 
lifted when all farms within the prot ection zone are emptied, cleaned, disinf ected and followed by a 
synchr onized fallo wing period of at least 6 weeks. 
When fallowing of the officially declared infect ed farms is carried out, the protection zones shall be 
conver ted into surveillance zones. 
The compet ent author ity may decide to require the emp tying, cleansing, disinfection and fallo wing of other 
farms within the established prot ection and surveillance zones. The length of the fallowing period for those 
farms shall be determined by the compet ent author ity following a case-by- case risk evaluation; 
(d)  all farms officially declared infec ted with HPR -delete d ISAV or conf irmed ISA and all other farms fallowed 
within the established protection and surveillance zones shall be restocked with fish sourced from Member 
States, zones or compar tments with a Categor y I with regard to ISA. 
Restoc king shall only take place when all farms officially declared infected have been emp tied, cleansed, 
disinf ected and fallowed in accordance with point I.2.2.1(c); 
(e)  all farms keepin g susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
member State, zone or compar tment covered by the eradication programme and when surveillance in wild 
populations is required, sampling points select ed in accordance with point I.1, shall subsequently be subject 
to the surveillance scheme set out in point I.2.1. 23.9.2015 L 247/20 Official Jour nal of the European Union EN     I.2.2.2.  Requirements concer ning regai ning disease-free status for continental compar tments compris ing one sing le farm 
that was previously declared as having Categor y I health status 
A continental compar tment compris ing one sing le farm that has Categor y I health status with regar d to ISA, 
whose health status is independent of the surrounding natural water s in accordance with point 3 of Part II of 
Annex V to Directive 2006/88/EC, and whose Categor y I health status has been withdrawn in accordance with 
Article 53(3) of that Directive, may regain it again immediately after the compet ent author ity has conf irmed 
that it compl ies with the following conditions: 
(a)  it has been emp tied, cleansed, disinf ected and fallowed; the duration of the fallo wing period shall be at least 
6 weeks; 
(b)  it has been restocked with fish sourced from Member States, zones or compar tments with a Categor y I 
health status as regards ISA. 
I.3.  Minimum control measures for the maintenance of Categor y I status with regard to ISA 
When target ed surveillance is required to maintain Categor y I health status, as provided for in Article 52 of 
Directive 2006/88/EC, all farms keeping susceptible species liste d in Part II of Annex IV to that Directive within 
the Member State, zone or compar tment concer ned shall be subject to health inspections and sampled in 
accordance with Table 3.B (1) set out in Section II of this Part, taking into account the risk level of the farm for 
the contraction of ISA. 
When determining the health inspection frequency for categor y I health status with regar d to ISA for 
compar tments which are placed in continental areas and where the health status with regard to ISA is 
dependent of the health status of surrounding natural water s that are housing Atlanti c salmon (Salmo salar), the 
risk for the contraction of ISA shall be regar ded as high. 
Disease-free status with regard to ISA may only be maintained as long as all samples tested using the diagnostic 
methods set out in point II.2 have produced negative results for HPR -delet ed ISAV and any suspicion of ISA has 
been ruled out in accordance with the diagnostic methods set out in point II.3. 
I.4.  Specific requirements to achieve Categor y III health status with regard to HPR -delete d ISAV in Member State s, 
zones or compar tments that previously held Categor y V health status 
A Member State, zone or compar tment that has Categor y V health status with regar d to ISA may achi eve 
Categor y III status provided that : 
(a)  the requirements set out in point I.2.2.1 (a), (b) and (c) have been met. In case fallowing is not technically 
possible, the farms shall be subject to an altern ative measure whic h provides almost similar guarant ee for 
exterminati on of ISAV from the envi ronment of the farm; 
(b)  all farms officially declared infected and all other farms fallo wed or been subject to altern ative measures in 
accordance with (a) within the prote ction and surveillance zones established, have been restocked with fish 
sourced from Member State s, zones or compar tments with a Categor y I, II or III health status with regar d to 
ISA; 
(c)  that such restocking has only take n place after all farms officially declared infect ed have been emptied, 
cleansed, disinf ected and fallowed/been subject to alternative measures in accordance with (a). 
(d)  no confir mation of HPR -deleted ISAV has occur red during the period of 2 years that follows the compl etion 
of the measures referred to in (a), (b) and (c), and suspicions during this period have been ruled out in 
accordance with the procedures established in point II.3. 23.9.2015 L 247/21 Official Jour nal of the European Union EN     
(1)Shall not apply to farm s only rear ing rainbo w trout (Oncorhynchus mykiss) or brown trout (Salmo trutta) or both and where the water 
supply is exclusively based on fresh water sources not housing Atlantic salmon (Salmo salar). II.  Diagnostic methods and official investigations 
II.1.  Sam ples 
The tissue mate rial to be examined shall be: 
(a)  Histology: head-kidney , liver , hear t, pancreas, intest ine, spleen and gill; 
(b)  Immunohistoc hemistr y: mid-kidney and hear t including valves and bulbus arteriosus; 
(c)  RT-qPCR analysis: mid-kidney and hear t; 
(d)  Virus culture: mid-kidney , hear t, liver and spleen. 
Org an pieces from a maximum of five fish may be pooled. 
II.2.  Diagnostic methods to obtain or maintain disease-free status with regar d to ISA 
The diagnostic method to be used to obtain or to maintain disease-free status with regard to ISA in accordance 
with points I.2 and I.3 shall be RT-qPCR, followed by sequencing of positive samples in accordance with the 
detailed methods and procedures set out in Part 3 of Annex II. 
In the case of a positive result to RT-qPCR, further sam ples shall be tested before the implementation of the 
initial control measures provided for in Article 28 of Directive 2006/88/EC. 
Those samples shall be tested as follows in accordance with the detailed methods and procedures set out in 
Part 3 of Annex II: 
(a)  screening of the samples by RT-qPCR, including sequencing of the HE-g ene to verify HPR -deletion; 
and 
(b)  examination in tissue preparations by means of specific antibodies against ISAV (namely IHC on fixed 
sections or IFAT on tissue imprints); or 
(c)  isolation and identification of ISAV in cell culture from at least one samp le from any fish sampled from the 
farm. 
II.3.  Official investigation and diagnostic methods to rule out or to conf irm the presence of ISA 
When a suspicion of ISA shall be conf irmed or ruled out in accordance with Article 28 of Directive 
2006/88/EC, the following inspection, sampling and testing procedure shall be compl ied with: 
(a)  the official investigation whic h shall include at least one health inspection and one samp ling of 
10 mor ibund fish, when clinical signs or post-mor tem signs consist ent with ISA are obser ved. If no clinical 
signs or post-mor tem signs consiste nt with ISA are obser ved, the health inspection shall be followed by 
target ed sampling of minimum 30 mor ibund fish or fresh post-mor tems with normal constitution in 
accordance with point I.1. Samples shall be tested in accordance with the diagnostic methods set out in 
point (b); 
(b)  in the case of a positive result of RT-qPCR for HPR -delet ed ISAV, further sam ples shall be tested before the 
implementation of the initial control measures provided in Article 28 of Directive 2006/88/EC. A suspecte d 
case of infection with ISA shall be conf irmed in accordance with the following criteria using the detailed 
methods and procedures set out in Part 3 of Annex II: 
(i)  Detection of ISAV by RT-qPCR, including sequencing of the HE-g ene to verify HPR -deletion, and 
detec tion of ISAV in tissue preparations by means of specifi c antibodies against ISAV (namely IHC on 
fixed sections or IFAT on tissue imp rints) 
or 23.9.2015 L 247/22 Official Jour nal of the European Union EN     (ii)  detec tion of ISAV by RT-qPCR, including sequencing of the HE-g ene to verify HPR -deletion, and 
isolation and identif ication of ISAV in cell culture from at least one sample from any fish from the farm; 
(c)  where the presence of clinical, gross pathological change s or histopathological findings consist ent with ISA 
are obser ved, the findings must be corroborat ed by virus detection by two diagnostic methods with 
independent principles of detection, such as RT-qPCR and IHC, in accordance with Part 3 of Annex II. 
The suspicion of ISA may be ruled out, if tests and inspections over a period of 12 months from the date of 
the suspicion are found to reveal no further evidence of the presence of ISA. 
Table 3.A 
Sur veillance scheme for zones and compar tments for the 2-year control period which precedes the 
achiev ement of disease-free status for ISA as refer red to in point I.2.1 
Year of surveillance Number of health inspections 
per year (2 years) Number of laborato ry exami ­
nations per year (2 years) Number of fish to be sampled 
per year 
Year 1 6 2(1) 2 * 75(2) 
Year 2 6 2(1) 2 * 75(2) 
(1)  Samples must be collected and stored and examined during two 1 month test periods per year (namely spring and autumn) or 
when appropr iate in accordance with practical considerations. 
(2)  Maximum number of fish per pool: 5.  
Table 3.B 
Sur veillance schemes for zones or compar tments to maint ain disease- free status for ISA as refer red to in 
point I.3(2) 
Risk level Number of health inspections 
per year Number of laborato ry exami ­
nations per year Number of fish to be sampled 
per year 
High 2 2(1) 2 * 30 
Medium 1 1(1) 30 
Low 1 ever y 2 years 1 ever y 2 years 30 ever y 2 years 
(1)Samples must be collected and examined during two 1 month test periods per year (namely spring and autumn) or when appropr i­
ate in accordance with practical considerations. 
(2)  Shall not apply to farms only rear ing rainbo w trout (Oncorhynchus mykiss) or brown trout (Salmo trutta) or both and where the water 
supply is exclusively based on fresh water sources not housing Atlantic salmon (Salmo salar).  
PART 4 
SUR VEILL ANCE AND CONTR OL METHODS FOR INFECTION WITH MARTEILIA REFRINGENS 
I.  Requirements for surveillance and eradication prog rammes to obtain and to maint ain disease-free health statuses with 
regard to infect ion with Mar teilia refringens 
I.1.  General requirements 
Health inspections and, where appropr iate, the sam pling for laborat ory examination shall be carried out in the 
period of the year when prevalence of the parasite in the Member State , zone or compar tment is known to be 
maximal. When such data is not available, sampli ng shall be carried out just after the water temperature has 
excee ded 17 °C. 23.9.2015 L 247/23 Official Jour nal of the European Union EN     When molluscs are to be sam pled in accordance with the requirements set out in Part 4, the following criteria 
shall apply: 
(a)  if Ostrea spp. and Mytilus spp. are present in the production units or production area, both genera shall be 
sampled equally in sample size. If only one of those genera is present, that genus shall be sampled. If neither 
Ostrea nor Mytilus genera are present, the samp le must be representative of all other susceptible species 
present ; 
(b)  if weak , gaping or freshly dead but not decomp osed molluscs are present in the production units, such 
molluscs shall primar ily be selected. If such molluscs are not present, the molluscs select ed shall include the 
oldest health y molluscs; 
(c)  when sampling in mollusc farms whic h utilise more than one wate r source for mollusc production, mollusc 
representing all water sources shall be included for samp ling in such a way that all parts of the farm are 
propor tionally represent ed in the sample; 
(d)  when sampling in mollusc farming areas, mollusc from a sufficient number of sam pling points, shall be 
included in the sample in such a way that all parts of the mollusc farming area are propor tionally represente d 
in the sample. The main factors to be considered for the selection of these sampling points are previous 
sampli ng points where Mar teilia refringens was detected , stoc king density , water flows, presence of susceptible 
species, presence of vect or species, bathymet ry and manage ment practices. Natural beds shall be included in 
the sampli ng. 
I.2.  Specific requirements to achieve Categor y I health status with regard to Mar teilia refringens 
I.2.1.  Surveillance programmes 
A Member State , zone or compar tment whic h has Categor y III health status with regar d to infection with Mar teilia 
refrin gens may achi eve Categor y I health status with regard to that liste d disease when all farms or mollusc farming 
areas keeping suscept ible species listed in Part II of Annex IV to Directive 2006/88/EC within the Member State , 
zone or compar tment have been subject at least to the following surveillance programme comprising health 
inspections and the collection of sam ples for testing. 
2-yea r surveillance programme: 
(a)  the farms or mollusc farming areas have been subject to health inspections and sampled for a minimum 
period of two consecutive years as laid down in Table 4.A set out in Section II; 
(b)  during that 2-year period, the testing of all samples using the diagnostic methods set out in point II.2 have 
produced negative results for Mar teilia refringens and any suspicion of Mar teilia refringens has been ruled out in 
accordance with the diagnostic methods set out in point II.3; 
(c)  when Ostre a edulis, Mytilus edulis or Mytilus galloprovincialis sourced from a Member State, zone or 
compar tment of Categor y I health status are to be included in the sample , they must have been introduced in 
the farm or mollusc farming area at least in the spring just preceding the period when surveillance 
programme is carried out. 
I.2.2.  Eradication programmes 
The eradication of Mar teilia refringens is considered to be impossible in most cases, but where the Member State 
judg es it to be feasible, the following model for an eradication programme shall apply . 
A Member State , zone or compar tment that has Categor y V health status as regards infection with Mar teilia 
refrin gens may achi eve Categor y I health status with regard to that liste d disease when all farms or mollusc farming 
areas keeping suscep tible species listed in Part II of Annex IV to Directive 2006/88/EC within that Member State, 
zone or compar tment have been subject at least to the following eradication programme: 
(a)  the measures laid down in Section 3 of Chapt er V of Directive 2006/88/EC have effectively been applied, and 
in particular a containment area as referred to in Article 32(b) of Directive 2006/88/EC, comprising a 
prote ction zone and surveillance zone, has been established in the vicinity of the farm(s) or mollusc farming 
area(s) officially declared infec ted with Mar teilia refrin gens. 23.9.2015 L 247/24 Official Jour nal of the European Union EN     The containment area shall be defined on a case-by- case basis taking into account factors influencing the risks 
for the spread of Mar teilia refrin gens, such as: the number , age, rate and distr ibution of the mor talities of 
molluscs on the farm or mollusc farming area infected with Mar teilia refrin gens including wild molluscs; the 
distance and density of neighbour ing farms or mollusc farming areas including wild molluscs; the proximity 
to processing establishments, contact farms or mollusc farming areas; the species, especially susceptible 
species and vector species, present at the farms or mollusc farming areas; the farming practices applied in the 
affected and neighbour ing farms and mollusc farming areas; the hydrodynami c conditions and other factors of 
epidemiological signif icance identif ied. 
For the establishment of the protection and surveillance zones, the following minimum requirements shall 
apply: 
(i)  a protect ion zone shall be established in the immediate vicinity of a farm or mollusc farming area 
officially declared infect ed with Mar teilia refringens and shall correspond to an area deter mined according 
to appropr iate hydrodynamic or epidemiological data; 
(ii)  a surveillance zone shall be established outside the protection zone and shall correspond to an area, 
surrounding the prote ction zone, deter mined according to appropr iate hydrodynamic or epidemiological 
data; 
(b)  all farms and mollusc farming areas keeping susceptible species listed in Part II of Annex IV to Directive 
2006/88/EC within the protection zone not officiall y declared infec ted with Mar teilia refringens shall be subject 
to an official investigat ion compris ing at least the collection of samples for the testing of 150 molluscs after 
the beginning of the transmission period of Mar teilia refrin gens. When the transmission period is not kno wn, 
the sampli ng shall begin in the period after the temperature of the water exceeds 17 °C; 
(c)  all farms and mollusc farming areas offici ally declared infected with Mar teilia refringens shall be emp tied, 
fallowed and if possible cleansed, and disinf ected. 
The duration of the fallo wing period shall be at least: 
(i)  2 months in case of the farms and mollusc farming areas with limited connections with the surrounding 
wate rs such as hatch eries and nurser ies; 
(ii)  2 months in case of the farms and mollusc farming areas with unlimit ed connections with the 
surrounding water s provided that the infected molluscs of the suscep tible species and those molluscs of 
the suscep tible species with epidemiological link s with the infected farm or mollusc farming area have 
been harvested or remo ved before the period of the year when the prevalence of Mar teilia refrin gens is 
kno wn to be maximal, or when that period is not kno wn, before the period when water temperature 
excee ds 17 °C; 
(iii)  14 months in case of the farms and mollusc farming areas with unlimit ed connections with the 
surrounding water s provided that the infected molluscs of the suscep tible species and those molluscs of 
the suscept ible species with epidemiological link s with the infected farm or mollusc farming area have not 
been harvested or remo ved before the period of the year when the prevalence of Mar teilia refrin gens is 
kno wn to be maximal or when such data is not kno wn, when molluscs of the suscept ible species have 
not been harvested or remo ved before the period when water temperature exceeds 17 °C. 
When all farms and mollusc farming areas officially declared infec ted are emptied, at least 4 weeks of 
synchr onised fallo wing shall be carried out. 
The compet ent author ity may decide to require the emp tying, cleansing, disinfection and fallo wing of other 
farms or mollusc farming areas, as appropr iate within the established protect ion and surveillance zones. The 
length of the fallo wing period shall be determined by the compe tent author ity following a case by case risk 
evaluation; 
(d)  all farms or mollusc farming areas officially declared infected and all other farms or mollusc farming areas 
fallowed within the established protection and surveillance zones shall be restocked with molluscs sourced 
from Member States, zones or compar tments with a Categor y I health status as regar ds infection with 
Mar teilia refringens. 23.9.2015 L 247/25 Official Jour nal of the European Union EN     Restoc king shall only take place when all farms official ly declared infected have been emp tied, cleansed, 
disinf ected and fallo wed in accordance with point I.2.2(c); 
(e)  all farms and mollusc farming areas keeping susceptible species listed in Part II of Annex IV to Directive 
2006/88/EC within the Member State, zone or compar tment covered by the eradication programme shall 
subsequently be subject to the surveillance sche me set out in point I.2.1 of this Part. 
I.3.  Specific requirements for the maintenance of disease-free health status (Cate gory I) with regar d to infection with 
Mar teilia refringens 
When target ed surveillance is required to maintain Categor y I health status, as provided for in Article 52 of 
Directive 2006/88/EC, all farms or mollusc farming areas keeping susceptible species liste d in Part II of Annex IV 
to Directive 2006/88/EC within the Member State, zone or compar tment concer ned shall be subject to health 
inspections and sampled in accordance with Table 4.B set out in Section II, taking into account the risk level of 
the farm or mollusc farming area for the contraction of Mar teilia refringens. 
The disease-free status may only be maintained as long as all samples using the diagnostic methods set out in 
point II.2 produced negativ e results for Mar teilia refringens and any suspicion of Mar teilia refrin gens is ruled out in 
accordance with the diagnostic methods set out in point II.3. 
I.4.  Requirements for the lifting of the containment measures provided for in Article 39 of Directive 2006/88/EC 
(change from Categor y V to Categor y III health status) with regar d to infection with Mar teilia refringens 
A Member State, zone or compar tment that has Categor y V health status with regard to infection with Mar teilia 
refrin gens may achi eve Categor y III health status with regar d to that listed disease provided that: 
(a)  the requirements laid down in points I.2.2(a), (b) and (c) have been met. In case fallo wing is not technically 
possible, the farms shall be subject to an altern ative measure whic h provides almost similar guarant ee for 
exterm ination of Mar teilia refringens from the environment of the farm; 
(b)  all farms or mollusc farming areas officially declared infected and all other farms or mollusc farming areas 
fallowed/been subject to alternative measures in accordance with (a) within the established prote ction and 
surveillance zones have been restocked with molluscs sourced from Member States, zone or compar tments 
with a Categor y I, II or III health status with regard to infection with Mar teilia refringens; 
(c)  restocking has only taken place when all farms or mollusc farming areas official ly declared infected have been 
emp tied, cleansed, disinf ected and fallo wed or been subject to altern ative measures in accordance with (a); 
(d)  no conf irmation of infection with Mar teilia refrin gens has occur red during the period of 2 years that follows 
the compl etion of the measures refer red to in (a), (b) and (c), and suspicions during this period have been 
ruled out according to the procedures established in point II.3. 
II.  Diagnostic methods and official investigations 
II.1.  Sam ples 
The whole animal shall be submitt ed to the laborat ory for the perf ormance of the diagnostic tests provided for in 
points II.2 and II.3. 
II.2.  Diagnostic methods to obtain or maintain disease-free status for infection with Mar teilia refrin gens 
The diagnostics methods to be used to obtain or maintain disease-free status for infection with Mar teilia refringens 
following the detailed diagnostic methods and procedures set out in of Part 4 of Annex II, shall be histo pathology , 
tissue impr ints or PCR. 23.9.2015 L 247/26 Official Jour nal of the European Union EN     II.3.  Offi cial investigation and diagnostic methods to confir m the presence of or to rule out the suspicion of infection 
with Mar teilia refrin gens 
When a suspicion of infection with Mar teilia refrin gens is required to be conf irmed or ruled out in accordance with 
Article 28 of Directive 2006/88/EC, the following inspection, sampling and testing procedure shall be complied 
with: 
(a)  the official inve stigation shall include at least one sam pling of 30 molluscs of suscep tible species if the 
suspicion is based on a mor tality repor t or if not, 150 molluscs of susceptible species after the beginning of 
the transmission period of Mar teilia refringens. When the transmission period is not kno wn, the sampli ng shall 
begin in the period after the temperature of the water exce eds 17 °C; 
(b)  Sample s shall be tested using the diagnostic methods set out in point (i) following the detailed diagnostic 
methods and procedures set out in Section I of Part 4 of Annex II: 
(i)  the presence of Mar teilia refringens shall be considered as conf irmed when a positive result by 
histo pathology , tissue imp rints or in situ hybridisation is combined with a positive result PCR complet ed 
by sequencing; 
(ii)  the suspicion of infection with Mar teilia refringens may be ruled out, if the tests refer red to in (i) reveal no 
further evidence of the presence of Mar teilia refringens. 
Table 4.A 
Sur veillance scheme for Member States, zones or compar tments for the control period which precedes the 
achiev ement of disease-free status for Mar teilia refringens as refer red to in point I.2.1  
Number of health inspections 
per year Number of laborato ry exami ­
nations per year Number of molluscs in the 
sample 
Farms/mol lusc farming areas 1 1 150  
Table 4.B 
Sur veillance schemes for Member States, zones or compar tments to maintain disease-free status for Mar teilia 
refringens as referred to in point I.3 
Risk level Number of health inspections Number of laborato ry exami ­
nations Number of molluscs in the 
sample 
High 1 ever y year 1 ever y 2 years 150 
Medium 1 ever y 2 years 1 ever y 2 years 150 
Low 1 ever y 2 years 1 ever y 4 years 150  
PART 5 
SUR VEILL ANCE AND CONTR OL METHODS FOR INFECTION WITH BONAMIA OSTREAE 
I.  Requirements for surveillance or eradication prog rammes to obtain and maintain disease-free health statuses with 
regard to infect ion with Bonamia ostr eae 
I.1.  General requirements 
Health inspections and, where appropr iate, the sampli ng of production units shall be carried out in the period of 
the year when prevalence of Bonamia ostreae in the Member State, zone or compar tment is kno wn to be maximal. 
When such data is not available, sampli ng shall be carried out in winte r or at the beginning of spring. 23.9.2015 L 247/27 Official Jour nal of the European Union EN     When molluscs are to be sam pled in accordance with the requirements set out in Part 5, the following criteria 
shall apply: 
(a)  if Ostrea edulis are present, only oysters of that species shall be selected for sampling. If Ostrea edulis are not 
present, the sam ple shall be representative of all other suscep tible species present ; 
(b)  if weak, gaping or freshly dead but not decomposed molluscs are present, such molluscs shall primar ily be 
selecte d. If such molluscs are not present, the molluscs selecte d shall include the oldest health y molluscs; 
(c)  when sampli ng in farms which utilise more than one water source for mollusc production, mollusc 
representing all water sources shall be included for samp ling in such a way that all parts of the farm are 
propor tionally represent ed in the sample; 
(d)  when sam pling in mollusc farming areas, molluscs from a suffi cient number of sampling points shall be 
included in the sample. The main factors to be considered for the selection of those sampli ng points are 
previous points where Bonamia ostreae was detected , stoc king density , water flows, the presence of suscept ible 
species, the presence of vect or species, bath ymetr y and manage ment practices. Natural beds within or adjacent 
to the farming areas shall be included in the sampling. 
I.2.  Specific requirements to achieve Categor y I health status as regar ds Bonamia ostreae 
I.2.1.  Surveillance programmes 
A Member State, zone or compar tment that has Categor y III health status with regar d to Bonamia ostreae may 
achi eve Categor y I health status again with regard to that listed disease when all farms keeping susceptible species 
listed in Part II of Annex IV to Directive 2006/88/EC within the Member State, zone or compar tment have been 
subject at least to the following surveillance programme compris ing health inspections and the collection of 
samp les for testing. 
2-yea r surveillance programme: 
(a)  the farms and mollusc farming areas keeping suscep tible species listed in Part II of Annex IV to Directive 
2006/88/EC have been subject to health inspections and sampled for a minimum period of two consecutive 
years as laid down in Table 5.A. set out in this Part; 
(b)  during that 2-yea r period, the testing of all sam ples using the diagnostic methods set out in point II.2 
produced nega tive results for Bonamia ostreae and any suspicion of Bonamia ostreae has been ruled out in 
accordance with the diagnostic methods set out in point II.3; 
(c)  when Ostrea edulis sourced from a Member State, zone or compar tment of Categor y I health status are to be 
included in the sample, they must have been introduced into the farm or mollusc farming area at least in the 
autumn just preceding the period when the surveillance programme is carried out. 
I.2.2.  Eradication programmes 
Eradication of Bonamia ostreae is considered to be impossible in most cases, but where the Member State judge s it 
to be feasible, the following model for an eradication programme shall apply . 
A Member State, zone or compar tment that has Categor y V health status as regards Bonamia ostreae may achi eve 
Categor y I health status again with regar d to that listed disease when all farms or mollusc farming areas keeping 
suscep tible species listed in Part II of Annex IV to Directive 2006/88/EC within the Member State , zone or 
compar tment have been subject at least to the following eradication programme: 
(a)  the minimum control measures laid down in Section 3 of Chapt er V of Directive 2006/88/EC have effectively 
been applied, and in particular a containment area as refer red in point (b) of Article 32 of that Directive, 
compris ing a prot ection zone and surveillance zone, has been established in the vicinity of the farm(s) or 
mollusc farming area(s) officially declared infected with Bonamia ostreae. 23.9.2015 L 247/28 Official Jour nal of the European Union EN     The containment area shall be defined on a case-by- case basis taking into account factors influencing the risks 
for the spread of that liste d disease, such as: the number , rate, age and distr ibution of the mor talities of 
molluscs on the farm or mollusc farming area infect ed with Bonamia ostreae including wild molluscs; the 
distance and density of neighbour ing farms or mollusc farming areas, including wild molluscs; the proximity 
to processing establishments, contact farms or mollusc farming areas; the species present at the farms or 
mollusc farming areas, especially susceptible species and vector species; farming practices applied in the 
affected and neighbour ing farms or mollusc farming areas; hydrodynamic conditions and other factors of 
epidemiological signif icance identif ied. 
For the establishment of the protection and surveillance zones, the following minimum requirements shall 
apply: 
(i)  a protect ion zone shall be established in the immediate vicinity of a farm or mollusc farming area 
officially declared infected with Bonamia ostreae and shall correspond to an area determined according to 
appropr iate hydrodynamic or epidemiological data; 
(ii)  a surveillance zone shall be established outside the protect ion zone and shall correspond to an area 
surrounding the prote ction zone, deter mined according to appropr iate hydrodynamic or epidemiological 
data; 
(b)  all farms and mollusc farming areas keeping susceptible species listed in Part II of Annex IV to Directive 
2006/88/EC within the protection zone not officially declared infected with Bonamia ostreae shall be subject to 
an official investigation comprising at least the collection of sam ples for testing of 150 molluscs of suscept ible 
species after the beginning of the transmission period of Bonamia ostreae. When the transmission period is not 
kno wn, the sampli ng shall begin in winter or at the beginning of spring; 
(c)  all farms and mollusc farming areas officially declared infec ted with Bonamia ostreae shall be emp tied, fallo wed 
and if possible cleansed and disinf ected. The duration of the fallo wing period shall be at least 6 months. 
When all farms or mollusc farming areas officially declared infect ed are emptied, at least 4 weeks of 
synchr onised fallo wing shall be carried out. 
The compet ent author ity may decide to require the emp tying, cleansing, disinfection and fallo wing of other 
farms or mollusc farming areas, as appropr iate within the established protect ion and surveillance zones. The 
length of the fallo wing period shall be determined by the compe tent author ity following a case by case risk 
evaluation; 
(d)  all farms or mollusc farming areas officially declared infected and all other farms or mollusc farming areas 
fallowed within the established protection and surveillance zones shall be restocked with molluscs sourced 
from Member States, zones or compar tments with a Categor y I health status with regard to infection with 
Bonamia ostreae. Rest ocking shall only take place when all farms officiall y declared infec ted have been emptied, 
cleansed, disinf ected and fallowed in accordance with point I.2.2(c); 
(e)  all farms and mollusc farming areas keeping susceptible species listed in Part II of Annex IV to Directive 
2006/88/EC within the Member State, zone or compar tment covered by the eradication programme must 
subsequently be subject to the surveillance programme laid down in point I.2. 
I.3.  Specific requirements for the maintenance of disease-free health status (Cate gory I) with regar d to infection with 
Bonamia ostreae 
When target ed surveillance is required to maintain Categor y I health status, as laid down in Article 52 of Directive 
2006/88/EC, all farms or mollusc farming areas keeping susceptible species listed in Part II of Annex IV to that 
Directive within the Member State, zone or compar tment concer ned shall be subject to health inspections and 
samp led in accordance with Table 5.B set out in Section II of this Part, taking into account the risk level of the 
farm or mollusc farming area for the contraction of infection with Bonamia ostreae. 
Disease-free status with regard to infection with Bonamia ostreae may only be maintained as long as all sam ples 
using the diagnostic methods set out in point II.2 have produced negat ive results for Bonamia ostreae and any 
suspicion of Bonamia ostreae has been ruled out in accordance with the diagnostic methods set out in point II.3. 23.9.2015 L 247/29 Official Jour nal of the European Union EN     I.4.  Requirements for the lifting of the containment measures provided for in Article 39 of Directive 2006/88/EC (the 
change from Categor y V to Categor y III health status) with regar d to infection with Bonamia ostreae. 
A Member State, zone or compar tment that has Categor y V health status with regard to infection with Bonamia 
ostreae may achi eve Categor y III health status with regar d to that disease provided that : 
(a)  the requirements set out in points I.2.2(a), (b) and (c) have been met. In case fallo wing is not technically 
possible, the farms shall be subject to an altern ative measure whic h provides almost similar guarant ee for 
exterm ination of Bonamia ostreae from the environment of the farm; 
(b)  all farms or mollusc farming areas officially declared infected and all other farms or mollusc farming areas 
fallowed/been subject to alternative measures in accordance with (a) within the established prote ction and 
surveillance zones have been restocked with molluscs sourced from Member States, zones or compar tments 
with a Categor y I, II or III health status with regard to infection with Bonamia ostreae. 
(c)  the restocking has only taken place when all farms or mollusc farming areas officially declared infected have 
been emptied, cleansed, disinf ected and fallowed/been subject to altern ative measures in accordance with (a); 
(d)  no conf irmation of infec tion with Bonamia ostreae has occur red during the period of 2 years that follows the 
compl etion of the measures referred to in (a),(b) and (c), and suspicions during this period have been ruled out 
according to the procedures established in point II.3. 
II.  Diagnostic methods and diagnostic criter ia 
II.1.  Sam ples 
The whole animal shall be submitt ed to the laborat ory for the perf ormance of the diagnostic tests provided for in 
points II.2 and II.3. 
II.2.  Diagnostic methods to obtain or maintain disease-free status for infection with Bonamia ostreae 
The diagnostics methods to be used to obtain or maintain disease-free status for infection with Bonamia ostreae, 
shall be histopathology , tissue imp rints or PCR. When applying these diagnostic methods, the corresponding 
detailed methods and procedures set out in of Part 5 of Annex II shall be followed. 
II.3.  Diagnostic criteria to conf irm of the presence of or to rule out the suspicion of infection with Bonamia ostreae 
When a suspicion of infection with Bonamia ostreae is required to be conf irmed or ruled out in accordance with 
Article 28 of Directive 2006/88/EC, the following inspection, sampling and testing procedure shall be complied 
with: 
The official investig ation shall include at least one sampling of 30 molluscs of suscep tible species if the suspicion 
is based on a mor tality repor t, or if not 150 molluscs of suscep tible species after the beginning of the 
transmission period of Bonamia ostreae. When the transmission period is not kno wn, the sampling shall begin in 
the winter or at the beginning of spring. The samples shall be tested using the diagnostic methods set out in 
point (i) following the detailed diagnostic methods and procedures set out in Section I of Part 5 of Annex II. 
(i)  the presence of Bonamia ostreae shall be considered as conf irmed when a positive result by histopathology , 
tissue impr ints or in situ hybridisation is combined with a positive result by PCR complet ed by sequencing in 
accordance with the appro ved methods and procedures set out in Part 5 of Annex II; 
(ii)  the suspicion of the presence of infection with Bonamia ostreae shall be ruled out, if those tests reveal no 
further evidence of the presence of Bonamia ostreae. 23.9.2015 L 247/30 Official Jour nal of the European Union EN     Table 5.A 
Sur veillance scheme for Member States, zones or compar tments for the control period which precedes the 
achiev ement of disease-free status for Bonamia ostr eae as refer red to in point I.2.1  
Number of health inspections 
per year Number of laborato ry exami ­
nations per year Number of molluscs in the 
sample 
Farms/Mo llusc farming areas 1 1 150  
Table 5.B 
Sur veillance schemes for Member States, zones or compar tments to maint ain disease-free status for Bonamia 
ostr eae as refer red to in point I.3 
Risk level Number of health inspections Number of laborato ry exami ­
nations Number of molluscs in the 
sample 
High 1 ever y year 1 ever y 2 years 150 
Medium 1 ever y 2 years 1 ever y 2 years 150 
Low 1 ever y 2 years 1 ever y 4 years 150  
PART 6 
SUR VEILL ANCE AND CONTR OL METHODS FOR WHITE SPOT DISEASE (WSD) 
I.  Requirements for surveillance and eradication prog rammes to obta in and maint ain disease-free health statuses with 
regard to WSD and to contain infection with WSSV 
I.1.  General requirements for inspections and sam pling 
The sampling of crustaceans for laboratory examination shall be carried out whenever the water temperature is 
likely to reach its highest annual point. That requirement concer ning water temperature shall also apply to 
health inspections where these are feasible and appropr iate. 
When farmed crustaceans are to be sample d in accordance with the requirements set out in this Part, the 
following criteria shall apply: 
(a)  if weak or mor ibund crustaceans are present in the production units, such crustaceans shall primar ily be 
selected. If such crustaceans are not present, those selected shall include crustaceans of differ ent size cohor ts 
namely juveniles and adults, of the select ed susceptible species, propor tionally represente d in the sam ple; 
(b)  if more than one water source is utilised for crustacean production, suscep tible crustacean representing all 
water sources must be included for sampling. 
When target ed surveillance in wild populations is required in accordance with the second paragraph of point 2 
of Part I of Annex V to Directive 2006/88/EC, the number and geographical distr ibution of the sampling points 
shall be determined to obtain a reasonable coverage of the Member State , zone or compar tment. The sam pling 
points shall also be representative of the differ ent ecosystems where the wild populations of suscep tible species 
are locat ed namely mar ine, estuar y, river and lake syste ms. 23.9.2015 L 247/31 Official Jour nal of the European Union EN     When target ed surveillance in wild populations is required in accordance with the second paragraph of point 2 
of Part I of Annex V to Directive 2006/88/EC, the crustaceans to be sam pled shall be selected as follows: 
(i)  in mar ine and estuar y systems areas, one or more of the following species shall be selecte d: Carcinus maenas, 
Cancer pagur us, Eriocheir sinensis, Liocarcinus depur ator, Liocarcinus puber , Crangon crangon, Homar us gammar us, 
Palaemon adspersus or penaeid shrimp species namely Penaeus japonicus, Penaeus kerathur us, Penaeus 
semisulcatus. If those species are not present, the samp le must be representative of other susceptible decapod 
species present. Given the broad susceptible host range , hosts may be selected from genera or families of 
the Decapoda where susceptibility has been exper imentally or naturally demonstrated ; 
(ii)  in river and lake systems, one or more of the following species shall be selecte d: Pacifastacus leniusculus, 
Astacus leptodactylus, Austro potamobius pallipes or Orconectes limosus. If those species are not present, the 
samp le must be representative of other susceptible decapod species present. Given the broad susceptible 
host rang e, hosts may be selected from genera or families of the Decapoda where suscep tibility has been 
exper imentally or naturally demonstrate d; 
(iii)  if weak or mor ibund crustaceans are present, such crustaceans shall primar ily be select ed. If such 
crustaceans are not present, those selected shall include crustaceans of diffe rent size cohor ts namely 
juveniles and adults of the selected susceptible species, propor tionally represent ed in the sample. 
I.2.  Specifi c requirements to obtain Categor y I health status with regar d to WSD 
I.2.1.  Surveillance programmes 
(a)  a Member State, zone or compar tment that has Categor y III health status with rega rd to WSD in accordance 
with part B of Annex III to Directive 2006/88/EC may achi eve Categor y I health status with regar d to that 
listed disease when all farms keeping susceptible species liste d in Part II of Annex IV to that Directive within 
the Member State, zone or compar tment, meet the relevant requirements set out in Annex V to that 
Directive and all those farms and, when required by the second paragraph of point 2 of Part I of Annex V 
to Directive 2006/88/EC, the sampling points in wild populations selected in accordance with that point, 
have been subject to the following 2-ye ar surveillance programme comprising health inspections and 
collection of samples for testin g. 
The farms or sampling points have been subject to health inspections and sampled for a minimum period of 
two consecutive years as laid down in Table 6.A set out in Section II. 
Dur ing that 2-year period, the testing of all samples using the diagnostic methods set out in point II.2 have 
produced negative results for infection with WSD and any suspicion of WSD has been ruled out in 
accordance with the diagnostic methods set out in point II.3. 
(b)  when during the implementation of the surveillance programme refer red to in (a), infection with WSSV is 
conf irmed in a farm included in that surveillance programme, and theref ore its Categor y II health status has 
been withdra wn, that farm may immediate ly rega in the Categor y II health status and continue the 
implementation of the surveillance programme to obtain disease-free status without implementing an 
eradication programme as set out in point I.2.2. provided that: 
(i)  it is a continental farm whose health status regar ding WSD is independent of the health status 
regarding that liste d disease of the surrounding natural wate rs in accordance with point 3 of Part II of 
Annex V to Directive 2006/88/EC; 
(ii)  it has been emp tied, cleansed, disinf ected and fallowed; the duration of the fallo wing period must be at 
least 6 weeks; 
(iii)  it has been restocked with crustaceans sourced from Member State s, zones or compar tments with a 
Categor y I health status with regar d to WSD. 23.9.2015 L 247/32 Official Jour nal of the European Union EN     I.2.2.  Eradication programmes 
I.2.2.1.  General requirements 
A Member State, zone or a compar tment that has Categor y V health status with regard WSD may achieve 
Categor y I health status with regard to that listed disease when all farms keeping suscep tible species liste d in 
Part II of Annex IV to Directive 2006/88/EC within the Member State , zone or the compar tment have been 
subject at least to the following eradication programme: 
(a)  the minimum control measures laid down in Section 4 of Chapt er V of Directive 2006/88/EC have 
effectively been applied, and a containment area referred to in Article 32(b) of that Directive, comprising a 
protection zone and surveillance zone, has been established in the vicinity of the farm(s) officially declared 
infec ted with WSD. 
The containment area must have been defined on a case-by- case basis taking into account factors 
influencing the risks for the spread of WSD to farmed and wild crustaceans, such as: the number , rate and 
distr ibution of the mor talities of crustaceans on the farm infected with WSD; the distance and density of 
neighbour ing farms; contact farms; the species present at the farms; the farming practices applied in the 
affect ed and neighbour ing farms; hydrodynamic conditions and other factors of epidemiological signif icance 
identif ied. 
For the establishment of the prot ection and surveillance zones, the following minimum requirements shall 
apply: 
(i)  a protection zone shall be established in the immediate vicinity of a farm official ly declared infected with 
WSD and shall correspond to:  
(1) in mar ine and estuar ine areas: the area included in a circle with a radius of at least one tidal 
excursion or at least 5 km, whic hever is larger , centred on the farm officially declared infected with 
WSD, or an equivalent area determined according to appropr iate hydrodynamic or epidemiological 
data; or  
(2) in freshwater s: the entire water catchment area of the farm officially declared infected with WSD; the 
compet ent author ity may limit the extension of the protection zone to parts of the water catchment 
area provided that the prevention of the spread of WSD is not compr omised; 
(ii)  a surveillance zone shall be established outside the prote ction zone and shall correspond:  
(1) in mar ine areas: an area, surrounding the prote ction zone, of overlapping tidal excur sion zones; or 
an area, surrounding the prot ection zone, and included in a circle of a radius of 10 km from the 
centre of the protection zone; or an equivalent area deter mined according to appropr iate 
hydrodynamic or epidemiological data; or  
(2) in freshwater s: as an extended area outside the established protection zone; 
(b)  all farms keeping susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
protection zone not officially declared infec ted with WSD shall be subject to an official inve stigation 
compr ising at least the following: 
(i)  the collection of sam ples for testing of 10 crustaceans, when clinical signs or post-mor tem signs 
consist ent with infection WSD are obser ved, or 150 crustaceans, when clinical or post-mor tem signs are 
not obser ved; and 
(ii)  one health inspection; in those farms where the tests refer red to in (i) have produced negat ive results, 
health inspections shall continue once per month during the season when the wate r temperature is 
likely to reach its highest annual points, until the prot ection zone has been withdra wn in accordance 
with point I.2.2.1(c). 23.9.2015 L 247/33 Official Jour nal of the European Union EN     (c)  all farms officially declared infect ed with WSD shall be emp tied, cleansed disinf ected and fallowed. The 
duration of the fallowing period shall be at least 6 weeks. When all farms officially declared infected are 
emp tied, at least 3 weeks of synchronised fallo wing shall be carried out. This paragraph shall also apply to 
new farms officially declared infec ted during the implementation of the eradication programme. 
When fallo wing of the officially declared infec ted farms is carried out, the prot ection zones shall be 
conver ted into surveillance zones. 
The compet ent author ity may decide to require the emptying, cleansing, disinf ection and fallowing of other 
farms within the established protection and surveillance zones. The length of that fallowing period shall be 
deter mined by the compet ent author ity following a case by case risk evaluation. 
(d)  all farms officiall y declared infected and all other farms fallowed within the established protection and 
surveillance zones shall be restocked: 
(i)  with crustaceans sourced from Member States, zones or compar tments with a Categor y I health status 
as regards WSD; or 
(ii)  for a transitional period until 31 December 2020, with crustaceans from Member State s, zones or 
compar tments with an appro ved WSD surveillance programme. 
Restoc king shall only take place when all farms officially declared infected with WSD have been emptied, 
cleansed, disinfect ed and fallo wed in accordance with point I.2.2.1(c). 
(e)  all farms keeping susceptible species listed in Part II of Annex IV to Directive 2006/88/EC within the 
Member State, zone or compar tment covered by the eradication programme and, when surveillance in wild 
populations is required, sam pling points select ed in accordance with the second paragraph of point 2 of 
Part I of Annex V to that Directive, must subsequently be subject at least to the programme laid down in 
point I.2.1. 
I.2.2.2.  Requirements for regai ning disease-free status with regar d to WSD for continental compar tments comprising 
one sing le farm that previously was declared free of WSD 
A continental compar tment compris ing one sing le farm that has Categor y I health status with regard to WSD, 
whose health status with regar d to that listed disease is independent of the surrounding natural wate rs in 
accordance with point 3 of Part II of Annex V to Directive 2006/88/EC, and whose Categor y I status has been 
withdrawn in accordance with Article 53(3) of that Directive, may regai n Categor y I health status immediately 
after the compet ent author ity has confir med that the following conditions have been complied with: 
(a)  the farm with WSD has been emp tied, cleansed, disinf ected and fallowed; the duration of the fallo wing 
period must have been at least 6 week s; 
(b)  the farm with WSD has been restocked with crustaceans sourced from Member States, zones or 
compar tments with a Categor y I health status with regar d to WSD. 
I.3.  Specifi c requirements for the maintenanc e of disease-free health status (Cat egor y I) with regar d to WSD 
When target ed surveillance is required to maintain cate gory I health status, as laid down in Article 52 of 
Directive 2006/88/EC, all farms keeping suscep tible species listed in Part II of Annex IV to that Directive within 
the Member State, zone or compar tment concer ned shall undergo a health inspection and sampling in 
accordance with Table 6 B set out in Section II, taking into account the risk level of the farm for the contraction 
of WSD. 
In Member States, zones or compar tments where the number of farms is limite d and target ed surveillance on 
those farms does not provide suffi cient epidemiological data, the surveillance programmes to maintain disease- 
free status shall include sampli ng points selected in accordance with the requirements laid down in point I.1. 23.9.2015 L 247/34 Official Jour nal of the European Union EN     Those sam pling points shall be inspecte d and sam pled by rotation of 50 % of the sampling points each year . 
The sam pling shall be carried out in accordance with Table 6 B set out in Section II. The samples shall be 
selecte d, prepared and examined in accordance with the diagnostic and sampling methods set out in Section II 
and the laboratory examinations must have produced negative results as regar ds to the agent of WSD. 
Disease-free status shall only be maintained as long as all sam ples tested using the diagnostic and sampli ng 
methods set out in point II.2 produce negative results for WSD and any suspicion of WSD has been ruled out 
in accordance with the offici al investig ation and diagnostic methods set out in point II.3. 
I.4.  Requirements for the lifting of the containment measures provided for in Article 39 of Directive 2006/88/EC 
(the chang e from Categor y V to Categor y III health status) with regard to WSD 
A Member State, zone or a compar tment which has Categor y V health status with regard to WSD may achi eve 
Categor y III health status with regar d to that listed disease provided that: 
(a)  the requirements set out in points I.2.2.1(a), (b) and (c) have been met. In case fallowing is not technically 
possible, the farms shall be subject to an alternative measure which provides almost similar guarantee for 
exterm ination of WSSV from the envi ronment of the farm; 
(b)  all farms officiall y declared infected with WSD and all other farms fallowed/been subject to alternati ve 
measures in accordance with (a) within the established prote ction and surveillance zones have been 
restocked with crustacean sourced from Member States, zones or compar tments with a Categor y I, II or III 
health status with regard to WSD. 
(c)  the restocking has only taken place when all farms officially declared infect ed with WSD have been emp tied, 
cleansed, disinfect ed and fallo wed/been subject to alternati ve measures in accordance with (a); 
(d)  no detection of WSD has occur red during the period of 2 years that follows the compl etion of the measures 
set out in paragraphs (a) and (b) and suspicions during this period have been ruled out according to the 
procedures established in point II.3. 
II.  Diagnostic and sampling methods 
II.1.  Samples 
Samples of integumental epider mis, either dissected or contained within walking legs, pleopods, mouthpar ts or 
gills of the test animal shall be fixed in 95 % ethanol prior to the preparation of sample s for two- step PCR. 
Other sam ples, fixed for histo logy and transmission electron microscop y may be collect ed to suppor t diagnostic 
data arising from PCR. 
II.2.  Diagnostic methods to obtain or maintain disease-free status with regard to WSD 
The diagnostic method to be used to obtain or to maintain disease-free status with regar d to WSD following the 
detailed methods and procedures set out in Part 6 of Annex II, shall be two-st ep PCR. 
In the case of a positive result of the two-st ep PCR, the result shall be corroborat ed by the sequencing of the 
amplicon before the initial control measures provided for in Article 28 of Directive 2006/88/EC are 
imp lemented, if possible under practical conditions by the demonstration of pathognomonic signs of WSD in 
those susceptible hosts select ed, via histo logy and transmission electron microscop y. 
II.3.  Offi cial investig ation and diagnostic methods to rule out the suspicion of or to conf irm the presence of 
infection with WSD 
When the presence of infection with WSD is required to be conf irmed or the suspicion of such infection is 
required be ruled out in accordance with Article 28 of Directive 2006/88/EC, the following inspection, 
sampli ng and testing procedure shall be compl ied with: 
(a)  the official investigation shall include at least one health inspection and one sam pling of 10 crustaceans 
when clinical or post-mor tem signs consistent with infection with WSD are obser ved or 150 crustaceans 
when clinical or post-mor tem signs are not obser ved. The samples shall be tested using the diagnostic 
method set out in point II.2 (two-step PCR); 23.9.2015 L 247/35 Official Jour nal of the European Union EN     (b)  the presence of WSD shall be considered as confir med when two- step PCR followed by sequencing, 
following the detailed methods and procedures set out in this Part 6 of Annex II, is positive for WSSV and, 
when pathognomonic signs of WSD are present in the select ed hosts. 
The suspicion of WSD may be ruled out, if those tests reveal no further evidence of the presence of WSD. 
Table 6 A 
Sur veillance scheme for Member States, zones and compar tments for the 2-year control period which precedes 
the achiev ement of WSD disease-free status as refer red to in point I.2.1  
Number of clinical inspec ­
tions per year Number of laborato ry exami ­
nations per year Number of crustaceans in the 
sample 
Farms/sampling sites 1 1 150  
Table 6 B 
Sur veillance schemes for Member States, zones or compar tments to maint ain WSD disease-free status as 
refer red to in point I.3 
Risk level Number of health inspections Number of laborato ry exami ­
nations Number of crustaceans in the 
sample 
High 1 ever y year 1 ever y 2 years 150 
Medium 1 ever y 2 years 1 ever y 2 years 150 
Low 1 ever y 2 years 1 ever y 4 years 150   23.9.2015 L 247/36 Official Jour nal of the European Union EN     ANNEX II 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES 
I.  Introduction 
This Annex sets out the detailed procedures for the diagnostic methods to be used for the laborat ory 
examination in the eradication and surveillance programmes set out in Annex I to this Decision, and in order to 
conf irm or rule out the suspected presence of the following non-ex otic diseases listed in Part II to Annex IV to 
Directive 2006/88/EC (‘the listed diseases’) in accordance with Article 57 (b) to that Directive: 
1. Viral haemor rhagic septicaemia (VHS) Part 1 
2. Infectious hemat opoietic necrosis (IHN) Part 1 
3. Koi herpesvir us (KHV) disease Part 2 
4. Infectious salmon anaemia (ISA) Part 3 
5. Infection with Mar teilia refringens Part 4 
6. Infection with Bonamia ostreae Part 5 
7. White spot disease (WSD) Part 6  
II.  Def initions 
For the purpose of this Annex, ‘transpor t medium’ means a cell culture medium with 10 % calf serum and with 
200 iu penicillin, 200 μg strept omycin, and 200 μg kanam ycin per millilitre or with other antibiotics of proven 
efficacy . 
PART 1 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR THE SUR VEILL ANCE AND CONFIRMA TION OF IHN 
AND VHS 
I.  Diagnostic methods and procedures for the surveillance of VHS and IHN 
When sampling and laborat ory examination for the purpose of obtaining or maintaining disease-free health 
status with regard to IHN or VHS as set out in Section I of Part 1 of Annex I are carried out, using the 
diagnostic methods set out in points II.1 and II.2 of Part 1 of that Annex, the detailed diagnostic methods and 
procedures set out in following points I.1 to I.6 shall apply . 
I.1.  Preparation and shipment of samples from fish 
I.1.1.  Tissues for virological examination on cell culture 
Before shipment or transfer to the laboratory , pieces of the organs to be examined shall be remo ved from the 
fish with sterile dissection tools and transferred to steri le plastic tubes containing transpor t medium. 
The quantity of fish mate rial suitable for virological examination on cell culture and by RT-qPCR is dependent 
on fish size. Thus, whole alevin (body length < 4 cm), viscera including kidney (4 cm < body length < 6 cm) or, 
for larger size fish, kidney , spleen, hear t and/or encephalon, and ovarian fluid from brood fish at the time of 
spawn ing shall be the tissues to be sampled. 
Ovar ian or seminal fluid or organ pieces from a maximum of 10 fish may be collect ed in one steri le tube 
containing at least 4 ml transpor t medium and represent one pooled sample. The tissue in each sam ple shall 
weigh a minimum of 0,5 gram (g). 
The virological examination on cell culture shall be started as soon as possible and no later than 48 hours after 
the collection of the samples. In excep tional cases, the virological examination may be started at the latest 
within 72 hours after the collection of the mater ial, provided that the mater ial to be examined is protect ed by a 
transpor t medium and that the temperature requirements during transpor tation can be fulfilled . 23.9.2015 L 247/37 Official Jour nal of the European Union EN     I.1.2.  Samples for reversed transcr iptase polymerase chain reaction (RT-PCR or RT-qPCR) analysis 
Samples shall be take n from the fish in accordance with the procedure descr ibed in point I.1.1 using a sterile 
instr ument and transferr ed to a sterile plastic tube containing transpor t medium. Tissue from 10 fish may be 
collect ed in one tube and shall represent one pooled sample. However , in case the amount of inoculum is small, 
tissue from up to five fish may be used. Alternatively , sample s may be pooled in RNA stabilization reagents, 
such as 0,2 g tissue/ml reag ent according to the recommendation from the manufacturers , although each fish 
shall be processed individually and shall not be pooled in the sam ples because of the small amount of mater ial 
to be used for extraction. 
Whole fish may also be sent to the laboratory . 
I.2.  Shipment of samples for fish 
Tubes containing fish tissues in transpor t medium for cell cultivation or RT-PCR/RT -qPCR analysis shall be 
placed in insulat ed containers, such as thick-walled polystyrene boxes, together with suffic ient ice or an 
alternati ve cooling medium with the similar cooling effect to ensure chilling of the samples during transpor ­
tation to the laborat ory. However , freezing of the samples shall be avoided. The temperature of a sam ple during 
transit must never exce ed 10 °C and ice must still be present in the transpor t box at receipt or one or more 
freeze bloc ks must still be partly or compl etely frozen. 
Whole fish may be sent to the laborat ory if the temperature requirements refer red to in the first paragraph 
during transpor tation can be fulfilled. Whole fish shall be wrapped up in paper with absor ptive capacity and 
shall finally be shipped in a plastic bag. Live fish may also be shipped. 
I.3.  Collection of supplementar y diagnostic mater ial 
When appro ved by the diagnostic laborat ory, other fish tissues may be collected as well and prepared for supple ­
mentar y examinations. 
I.4.  Preparation of sam ples for cell culture examination and RT-qPCR 
I.4.1.  Freezing in excep tional cases 
Where practical difficulties arise, which make it impossible to process the sam ples within 48 hours after the 
collection of the fish tissues, it may be acceptable to freeze the tissue specimens in transpor t medium at – 20 °C 
or belo w and to carry out virological examination within 14 days. However , the fish tissue shall only be frozen 
and thawed once before examination. Records shall be kept with details on the reason for each freezing of fish 
tissue sam ples. 
I.4.2.  Homogen isation of organs 
In the laborat ory, the fish tissue in the tubes shall be compl etely homogenised , either by stoma cher , blender or 
mor tar and pestle with sterile sand, and subsequently suspended in the original transpor t medium. 
If a sam ple consists of a whole fish less than 4 cm long, it shall be minced with steri le scissors or scalpel after 
remo val of the body behind the gut opening. If a sample consists of a whole fish with body length between 
4 cm and 6 cm, the viscera including kidney shall be collect ed. If a sample consists of a whole fish more than 
6 cm long, the tissue specimens shall be collect ed as descr ibed in point I.1. The tissue specimens shall be 
minced with sterile scissors or scalpel and homog enised as descr ibed in the first paragraph of this point and 
suspended in transpor t medium. 
The final ratio between tissue mate rial and transpor t medium shall be adjuste d in the laborat ory to 1:10. 
I.4.3.  Centr ifugation of homog enate 
The homog enate shall be centr ifuged in a refrigerat ed centr ifuge at 2 °C to 5 °C at 2 000 to 4 000 × g for 
15 minute s and the super natant collect ed and may be treated for either four hours at 15 °C or overnight at 4 to 
8 °C with antibiotics. If the sample has been shipped in a transpor t medium, the treatment of the super natant 
with antibiotics may be omitt ed. 
Where practical difficulties arise, such as incubator breakdo wn or problems with cell cultures, whic h make it 
imp ossible to inoculat e cells within 48 hours after the collection of the fish tissue samples , the super natant may 
be frozen at – 80 °C and virological examination may be carried out within 14 days. 23.9.2015 L 247/38 Official Jour nal of the European Union EN     If the collect ed super natant is store d at – 80 °C within 48 hours after the sampli ng, it may be reused only once 
for virological examination. 
Prior to the inoculation of the cells, the super natant shall be mixed with equal parts of a suitably diluted pool 
of antisera to the indige nous serotypes of Infectious pancreatic necrosis (IPN) virus and incubated with this for a 
minimum of one hour at 15 °C or a maximum of 18 hours at 4 °C. The titre of the antiser um shall be at least 
1/2 000 in a 50 % plaque neutralisation test. 
Treatment of all inocula with antiser um to IPN virus aims at preventing cytopathic effect (CPE) due to IPN virus 
from developing in inoculat ed cell cultures. This will reduce the duration of the virological examinations as well 
as the number of cases in whic h occur rence of CPE would have to be considered potentially indicative of VHSV 
or IHNV . 
When samples come from production units, which are considered free from IPN, the treatment of inocula with 
antiser um to IPN virus may be omitt ed. 
I.4.4.  Sample preparation for RT-PCR and RT-qPCR based surveillance programmes 
If samples were collect ed in transpor t medium, the procedure set out in points I.4.2 and I.4.3 shall be carried 
out. After centr ifugation, super natant shall be collected and RNA extract ed. If further examination is not to be 
under take n directly after centr ifugation, the samples shall be immediately frozen at – 20 °C or belo w. 
For the analysis of fish tissues preser ved in RNA stabilization reagent, subsequent work shall be carried out 
within the following time scales for samples stored at different temperatures: 
samples stored at 37 °C: 1 day; 
samples stored at 25 °C: 1 week; 
samples stored at 4 °C: 1 month; 
samples stored at – 20 °C: indefinitely 
Pooled samples in RNA stabilization reagent shall be treated like sing le samples in RNA stabilization reagent. 
For samples pooled in RNA stabilization reage nt, the sample amount shall not exceed that recommended by the 
manufacturer for extraction with RNA kits, such as RNeasy Mini kits (Qiag en) or similar . If larger samples are 
pooled, the extraction kits or methods must reflect this pooling. 
Samples collected in RNA stabilization reage nts shall not be used for cell cultivation. 
I.4.5.  Pooling of samples for RT-qPCR 
As the RT-qPCR protocols given are of similar or higher sensitivity than the cell cultivation methods, it may be 
acceptable to use super natant from homogenised fish tissue mat erial of pooled organs from up to 10 fish in cell 
culture medium for PCR. However , due to the much smaller inoculum used for PCR compared to cell 
cultivation, all fish tissues shall be carefully homogenised before collating mat erial for extraction. 
The same principle shall also be applied if samples are collect ed in RNA stabilization reagents. However , in that 
case it is often difficult to collect representative mate rial from up to 10 fish in one tube, and the number of fish 
per pool shall theref ore be reduced to 2 to 5. 
I.5.  Virological examination on cell culture 
I.5.1.  Cell cultures and media 
Bluegill fry cell line -2 (BF-2) or Rainbow trout gonad cell line - 2 (RTG-2) and either Epithelioma papulosum 
cyprini (EPC) or Fathead minno w (FHM) cells shall be grown at 20 to 30 °C in suitable medium, namely Eagle's 
Minimum essential medium (MEM) or modifications thereof, with a supplement of 10 % foetal bovine serum 
and antibiotics in standard concentrations. 23.9.2015 L 247/39 Official Jour nal of the European Union EN     When the cells are cultivated in closed vials, the medium shall be buffe red with bicarbonat e. The medium used 
for cultivation of cells in open units may be buff ered with tris(hydro xymethyl)aminomethane-HCl (Tris-HCl) 
(23 mM) and sodium bicarbonat e (6 mM). The pH must be 7,6 ± 0,2. 
The cell cultures to be used for inoculation with fish tissue mater ial shall be young, normally 1 day old cell 
culture monola yers where possible; however , a range between 4 to 48 hours old may be accept ed. The cells 
must be actively growing at inoculation. 
I.5.2.  Inoculation of cell cultures 
Antibiotic-treat ed organ suspension shall be inoculat ed into cell cultures in two dilutions, namely the primar y 
dilution and, in addition, a 1:10 dilution thereof, resulting in final dilutions of tissue mate rial in cell culture 
medium of 1:100 and 1:1 000, respectively , in order to prevent homologous interference. At least two cell lines 
shall be inoculat ed as referred to in point I.5.1. The ratio between inoculum size and volume of cell culture 
medium shall be about 1:10. 
For each dilution and each cell line, a minimum of about 2 cm2 cell area, corresponding to one well in a 
24-well cell culture tray, shall be utilised. Cell culture trays shall be used where possible. 
I.5.3.  Incubation of cell cultures 
The inoculat ed cell cultures shall be incubated at 15 °C for seven to 10 days. If the colour of the cell culture 
medium chang es from red to yellow indicating medium acidification, pH adjustment with sterile bicarbonat e 
solution or equivalent substances shall be performe d to ensure cell susceptibility to virus infection. 
At least ever y 6 months or if decreased cell susceptibility is suspect ed, titration of frozen stoc ks of VHSV and 
IHNV shall be performe d to verify the suscept ibility of the cell cultures to infection. The procedure set out in 
Section III shall be used, if possible. 
I.5.4.  Microscop y 
Inoculated cell cultures shall be inspected regularly , at least three times a week , for the occur rence of CPE at 40 
to 150 × magnific ation. If obvious CPE is obser ved, virus identif ication procedures in accordance with point I.6 
shall be initiate d immediately . 
I.5.5.  Subcultivation 
If no CPE has developed after the primar y incubation for 7 to 10 days, subcultivation shall be performe d to 
fresh cell cultures utilising a cell area similar to that of the primar y culture. 
Aliquots of medium (super natant) from all cultures or wells constituting the primar y culture shall be pooled 
according to cell line 7 to 10 days after inoculation. The pools shall then be inoculat ed into homologous cell 
cultures undiluted and dilut ed 1:10 (resulting in final dilutions of 1:10 and 1:100, respectively , of the 
super natant) as descr ibed in point I.5.2. Alternatively , aliquots of 10 % of the medium constituting the primar y 
culture shall be inoculat ed directly into a well with fresh cell culture (namely , well to well subcultivation). The 
inoculation may be preceded by pre-incubation of the dilutions with the antiser um to IPN virus at appropr iate 
dilution as descr ibed in point I.4.3. 
The inoculat ed cultures shall then be incubated for 7 to 10 days at 15 °C and inspected in accordance with 
point I.5.4. 
If toxic CPE occurs within the first 3 days of incubation, subcultivation shall be perf ormed at that stage, but the 
cells shall then be incubate d for 7 days and sub cultivated again with further 7 days incubation. When toxic CPE 
develops after 3 days, the cells shall be passed once and incubated to achi eve the total of 14 days from the 
primar y inoculation. There must be no evidence of toxicity in the final 7 days of incubation. 
If bacterial contamination occurs despite treatment with antibiotics, subcultivation shall be preceded by centr ifu­
gation at 2 000 to 4 000 × g for 15 to 30 minutes at 2 to 5 °C, or filtration of the super natant through a 
0,45 µm filter or both (low protein-binding membrane). In addition to this, subcultivation shall follow the same 
procedures as descr ibed for toxic CPE in the fourth paragraph of this point. 
If no CPE occurs, the test may be declared negative. 23.9.2015 L 247/40 Official Jour nal of the European Union EN     I.6.  Virus identification 
If evidence of CPE has been obser ved in a cell culture, medium (super natant) shall be collect ed and examined by 
one or more of the following techniques: Enzyme-linked immunosorbent essa y (ELISA ), Immunof luorescence 
(IF), neutralisation, RT-PCR or RT-qPCR. If these tests have not allowed definitive identification of the virus 
within 1 week, the super natant shall be forwarded to the national reference laborat ory or to the EU reference 
laboratory for fish diseases referred to in Annex VI to Directive 2006/88/EC for immediate identif ication. 
I.6.1.  ELISA 
A double antibody sandwich ELISA shall be performe d in order to identify the virus isolat e. Micro well plates 
shall be coated with 50 µl/well (0,9 pg) of proven quality prot ein-A purified immunog lobulins(Ig) from rabbit 
antisera against IHNV or VSHV dilut ed in carbonate buffer (pH 9,6) containing 15 mM sodium azide and 
incubated from 18 hours to 2 weeks at 4 °C. 
On a dilution plate, each sample containing 1 % Triton X-100 and the positive controls shall be diluted with 
buffe r solution (namely , phosphate buffered saline (PBS)- T-BSA, 1 % BSA) in a 4-fold dilution: undiluted, 1:4, 
1:16, 1:64. The ELIS A plates shall be washed in PBS containing 0,05 % Tween-20 (PBS- T) and 50 μl of each 
dilution shall be transferr ed from the dilution plate to the washed and coated ELISA- plate. 
ELISA plates shall then be incubated for 30 minutes at 37 °C. Subsequently plates shall be washed and 
incubated for 30 minutes at 37 °C with specific monoclonal antibodies (namely for VHSV identif ication MAb 
IP5B11 and for IHNV Hyb 136-3, respectively). 50 μl of horseradish-pero xidase (HRP) conjugat ed rabbit anti 
mouse antibodies diluted 1:1 000 in PBS- T-BSA shall be transferred to the ELISA plate. 
Finally , after renewed washing the reactions shall be developed adding 50 µl/well of ortho-pheny lenediamine 
(OPD). The ELISA plates shall be incubated for 20 minutes at room temperature in the dark and the reaction 
shall be stopped by adding 100 µl/well 0,5 M H2SO4. 
The absorbance shall be monitore d at a wavelength of 492 and 620 nm in an ELISA reader . Samp les shall be 
designate d positive or negative after comparing the test results to the absorbance values for the positive and 
negativ e controls. In general, samples with combined absorbance (A) < 0,5 for undiluted mate rial shall be 
considered negative, samples with A values between 0,5 and 1,0 shall be considered suspicious and samples 
with A values > 1,0 shall be considered positive. 
Other ELISA versions with a proven similar efficacy may be used instead of those refer red to in this point. 
I.6.2.  Immunof luorescence- IF 
The identif ication of listed pathogen s VHSV and IHNV shall be performe d by infecting cells in ‘Black’ 96-well 
plates, conventional 24-well plates or cover slips into 24-well plate s. When IHNV or VHSV or both are 
identif ied by infecting cells on cover slips, the following prot ocol shall apply: 
(a)  cover slips shall be seeded with cells at a density leading to between 60 % and 90 % conf luence after 
24 hours of cultivation. EPC cells shall be used where possible for this purpose because of their strong 
adherence to glass surfaces, but other cell lines such as BF-2, RTG- 2 or FHM may be used as well. 150 µl 
cell culture super natant in two differ ent dilutions (1:10 and 1:1 000) shall be inoculat ed in duplicate onto 
1-da y-old monola yers and incubated at 15 °C for 24 hours; 
(b)  subsequently , cell culture medium shall be remo ved, and the infected cell monola yers fixed with 0,5 ml ice- 
cold, aqueous acetone solution (80 % vol:vol). Fixation shall take place in fume hood for 15 minut es at 
room temperature, then the aceton e solution shall be remo ved and the cover glasses shall be air dried for at 
least 30 minutes. At this stage, the plates shall either be processed immediate ly or stored at – 20 °C for 
further use; 
(c)  specifi c monoclonal antibodies (namely for VHSV identif ication, MAb IP5B11 and for IHNV , Hyb 136-3 
respectively) shall be dilut ed in 0,01 M PBST , pH 7,2 in the dilution recommended by the provider of the 
MAbs; 50 to 100 µl/well shall be added to the fixed monola yer and plate s shall be incubate d for one hour at 
37 °C in a humid chamber; 23.9.2015 L 247/41 Official Jour nal of the European Union EN     (d)  cover glasses shall be washed gently three times with PBS containing 0,05 % Tween-20 (PBS- T), and 
the buffer shall be remo ved compl etely after the last rinse. The cells shall subsequently be incubated for 
one hour at 37 °C with fluorescein isothiocyanat e (FITC) — or tetrameth ylrhodamine-5-(and-6-) isothio ­
cyanate (TRITC)-conjug ated antibodies against mouse immunog lobulin used as the primar y antibody , diluted 
according to the supplier instr uctions, washed again in PBS- T, and dried. Stained cultures shall be mount ed 
onto glass slides using glycerol saline and examined under incident ultraviolet (UV) light. Use 10 × or 
12 × eyepieces and a × 25 or × 40 objective lens with numer ical aper tures > 0,7 and > 1,3 respectively . 
Other IF techniques, with regard to cell cultures, fixation and antibodies of reference quality , of proven similar 
efficacy may be used instead. 
I.6.3.  Neutralisation 
Cells from the collected super natant shall be remo ved by centr ifugation (2 000 to 4 000 × g) or membrane 
filtration (0,45 μm) with a low prot ein binding membrane and the super natant shall be dilut ed 1:100 
and 1:10 000 in cell culture medium. 
Aliquots of a minimum of two super natant dilutions shall be mixe d and incubated for 60 minutes at 15 °C with 
equal parts of the following reagents separat ely: 
(a)  serum containing group specific antibody against VHSV at a 1:50 (vol: vol) dilution; 
(b)  serum containing group specific antibody against IHNV at a 1:50 (vol:v ol) dilution; 
(c)  pool of antisera against the indigenous serotypes of IPNV at a 1:50 (vol:v ol) dilution; 
(d)  medium alone (positive control). 
From each virus super natant-ser um mixture, at least two cell cultures shall be inoculated with 50 μl each and 
then incubat ed at 15 °C. The development of CPE shall be chec ked as descr ibed in point I.5.4. 
VHSV strains and isolat es that do not react in neutralisation tests shall be identified by IF or ELISA. 
Other neutralisation tests of proven similar efficacy may be used inste ad. 
I.6.4.  RT-PCR/RT -qPCR 
I.6.4.1.  Preparation of viral RNA 
All work with RNA shall be performe d on ice, using gloves. 
RNA shall be extract ed using the phenol-c hlorof orm method or by RNA affinity spin columns, according to the 
manufacturer's instr uctions. Commercially available RNA extraction kits that will produce high quality RNA 
suitable for use with the RT-PCR protocols detailed in the points belo w may be used. 
RNA shall be re-suspended in distilled RNAse-free water , (namely water treated with 0,1 % dieth yl pyrocar ­
bonat e) or an appropr iate elution buffe r. 
I.6.4.2.  RT-PCR 
The following primers shall be used for detec tion of IHNV : 
Forward Primer 5′-A GA-GA T-CCC- TAC-A CC-A GA-GA C-3′; 
Reverse Primer 5′-GGT -GGT -GTT -GTT -TCC-GTG-CAA -3′. 
The following cycles shall be used (one-ste p RT-PCR): 1 cycle: 50 °C for 30 minutes; 1 cycle: 95 °C for 
2 minutes ; 30 cycles: 95 °C for 30 seconds, 50 °C for 30 seconds, 72 °C for 60 seconds; 1 cycle: 72 °C for 
7 minutes and soak at 4 °C. 
The following primers shall be used for detec tion of VHSV : 
VN For 5′-A TG-GAA-GGA-GGA-A TT-CGT -GAA-GCG-3′; 
VN Rev 5′-GCG-GTG-AA G-TGC- TGC-A GT-TCC-C-3′. 23.9.2015 L 247/42 Official Jour nal of the European Union EN     The following cycles shall be used (one-st ep RT-PCR): 50 °C for 30 minut es, 95 °C for 15 minut es, 35 cycles at 
94 °C for 30 seconds, 55 °C for 30 seconds, and 68 °C for 60 seconds. Subsequently , the reaction shall be held 
at 68 °C for 7 minutes . 
Quantity and specific ity of the RT-PCR reactions shall be evaluated by gel electrophoresis in 1,5 % agarose gel 
with ethidium bromide and obser ved using UV transillumination. A 693 bp PCR amplicon may be obser ved for 
IHNV . For VHSV , the size shall be 505 bp. 
The results of the PCR may vary depending on the conditions under which it is performe d, namely the ther mal 
protocols might need optimisation, depending on the ther mal cycler in use. Further more, false-positive results 
may occur because of false primer annealing or laboratory contamination. Adequate positive and negative 
controls and sequence amplicons shall theref ore be included to avoid any doubts. For the VHSV primers, special 
care shall be taken when using BF-2 cells, as the primers may react with the cell line DNA/RNA producing false- 
positive results of similar size. When testing super natant from BF-2 cells, any ampli fied PCR fragments shall be 
sequenced. 
I.6.4.3.  RT-qPCR for VHSV 
For VHSV , amplification shall be performed using the following primers and probe: 
Forward primer: 5′-AAA-CTC-GCA-GGA- TGT -GTG-CGT -CC-3′; 
Reverse primer: 5′-TCT -GCG-A TC-TCA-GTC-A GG-A TG-AA-3′; 
and probe: 5′-F AM- TAG-A GG-GCC- TTG-GTG-A TC-TTC- TG-BHQ1. 
One-step RT-qPCR: 
Nega tive template controls and positive controls shall be included on each plate run. Cycling conditions: 50 °C 
for 30 minutes, 95 °C for 15 minute s, 40 cycles of 94 °C for 15 seconds, 60 °C for 40 seconds, 72 °C for 
20 seconds; adjust if necessar y. Other RT-PCR or RT-qPCR versions of proven similar efficacy may be used 
instead. 
I.6.4.4.  RT-qPCR for IHNV 
For IHNV , ampli fication shall be performe d using the following primers and probe: 
Forward primer: 5′- AGA-GCC-AA G-GCA-CT G-TGC-G-3′; 
Reverse primer: 5′- TTCTTTGCGGCTTGGTTGA - 3′; 
and probe: 5′ 6FAM- TGA GACTGA GCGGGA CA-NFQ/MGB. 
Two-step RT-qPCR: 
As the following assa y depends on a two- step amplification, extra care shall be taken when handling the tubes 
from one reaction to the other in order to prevent contamination. 
Cycling conditions (after RT-step): 50 °C for 2 minutes , 95 °C for 10 minute s, followed by 40 cycles of 95 °C 
for 15 seconds and 60 °C for 1 minutes; adjustments shall be made if necessar y. 
Other RT-PCR or RT-qPCR versions of proven similar efficacy may be used instead. 
II.  Detail ed diagnostic methods and procedures for the conf irmation of or to rule out the suspicion of VHS or IHN or 
both in suspected outbreaks 
When a laborat ory examination is required to confir m or rule out the presence of IHN or VHS or both in 
accordance with Article 57(b) of Directive 2006/88/EC using the diagnostic methods set out in point II.3 of 
Part 1 of Annex I, the following detailed diagnostic methods and procedures shall apply: 
(a)  convention al virus isolation with subsequent seroneutralisation, immune-che mical or molecular virus 
identif ication; 
(b)  virus detection by RT-PCR or RT-qPCR; 
(c)  other diagnostic techniques such as IFAT, ELIS A, RT-PCR, IHC. 23.9.2015 L 247/43 Official Jour nal of the European Union EN     II.1.  Conventiona l virus isolation with subsequent virus identif ication 
II.1.1.  Selection of samples 
At least 10 fish showing typical signs of IHN or VHS shall be select ed for examination. 
II.1.2.  Preparation and shipment of samples from fish 
The preparation and shipment for the purpose of conven tional virus isolation shall follow the methods and 
procedures laid down in point I.2. 
II.1.3.  Collection of supplementar y diagnostic mater ial 
The collection of supplementar y mater ial for the purpose of conventional virus isolation shall follow the 
methods and procedures laid down in point I.3. 
II.1.4.  Preparation of sam ples for cell culture examination 
The preparation of samples for cell culture examination for the purpose of conventional virus isolation shall 
follow the methods and procedures laid down in point I.4. 
II.1.5.  Virological examination on cell culture 
The virological examination for the purpose of conventional virus isolation shall follow the methods and 
procedures laid down in point I.5. 
II.1.6.  Virus identification 
The virus identification for the purpose of conven tional virus isolation shall follow the methods and procedures 
laid down in point I.6. 
II.2.  Virus detection by RT-qPCR 
II.2.1.  Selection of samples 
The selection of sam ples for the purpose of virus detection by RT-qPCR shall follow the methods and 
procedures laid down in point I.1.2. 
II.2.2.  Preparation and shipment of samples from fish 
The preparation and shipment for the purpose of virus detection by RT-qPCR shall follow the methods and 
procedures laid down in point I.2. 
II.2.3.  Collection of supplementar y diagnostic mater ial 
The collection of supplementar y diagnostic mater ial for the purpose of virus detection by RT-qPCR shall follow 
the methods and procedures laid down in point I.3. 
II.2.4.  Sample preparation for RT-qPCR 
The samp le preparation for the purpose of virus detection by RT-qPCR shall follow the methods and procedures 
laid down in point I.6.4.1. 
II.2.5.  RT-qPCR 
The virus detection by RT-qPCR shall follow the methods and procedures laid down in points I.6.4.1, I.6.4.3 
and I.6.4.4. 
II.3.  Other diagnostic techniques 
Super natant prepared as descr ibed in point I.4.3 may be submitte d to ELISA, Indirect fluorescent antibody test 
(IFAT), or RT-PCR in accordance with point I.6.1, point I.6.2 or point I.6.4 respectively . Tissue mat erial may be 
subjected to other diagnostic techniques, such as IFAT on frozen sections, immunohist ochemistry on formalin 
fixed tissue mate rial. Those rapid techniques shall be supplement ed with a virological examination in 
accordance with either point II(a) or point II(b) within 48 hours after sampli ng, if: 
(a)  a negat ive result is obtained; or 
(b)  a positive result is obtained with mater ial representing the first case of IHN or VHS. 23.9.2015 L 247/44 Official Jour nal of the European Union EN     III.  Procedure for titration to verify the susceptibility of the cell cultures to infection 
When titration to verify the susceptibi lity of the cell cultures to infection as referred to in point I.5.3 is carried 
out, the procedures set out in the following paragraphs of this point shall be followed. 
At least two VHSV isolates and one isolate of IHNV shall be used. The isolat es shall represent the major group 
of viruses within the European Union, namely for VHSV one pathogenic isolat e from rainbo w trout in 
freshwater and one mar ine isolate pathogenic for turbot, and for IHNV one rainbo w trout pathogen ic strain 
from the European Union. Well-def ined isolat es from the Member States shall be used. Batch es of virus in low 
cell culture passage numbers shall be propagat ed in cell culture flasks on BF-2 or RTG- 2 cells for VHSV and on 
EPC or FHM cells for IHNV . Cell culture medium with at least 10 % serum shall be used. Low MOI for 
inoculation (< 1) shall be used. 
At total CPE, the virus shall be harvested by centr ifugation of cell culture super natant at 2 000 × g for 
15 minut es, filter sterilised through 0,45 µm membrane filter and distr ibuted in labelled cryotubes. The virus 
shall be kept at – 80 °C. 
1 week after freezing, three replicate vials with each virus shall be thawed under cold water and titrat ed on their 
respective cell lines. At least ever y 6 months, or if it is suspected that the suscep tibility of a cell line has 
decreased, each virus isolat e shall be thawed and titrated. 
Titration procedures must be descr ibed in detail and the same procedure followed each time. 
Titration by end point dilution shall include at least six replicates at each dilution step . The titres shall be 
compare d with previously obtained titres. If the titre of any of the three virus isolat es drops by a factor of 
2 logs or more, compare d with the initial titre, the cell line shall no long er be used for surveillance purposes. 
If different cell lines are kept in the laboratory each line shall be examined separately . 
Records shall be kept for a period of at least 10 years. 
PART 2 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR THE SUR VEILL ANCE AND CONFIRMA TION OF KHV 
DISEASE (KHVD) 
I.  Detail ed diagnos tic methods and procedures for the conf irmation of the presence of or to rule out the suspicion of 
KHVD 
When a laborat ory examination is required for the purpose of conf irmation of the presence of or to rule 
out the suspicion of KHVD in accordance with Article 57(b) of Directive 2006/88/EC, using the diagnostic 
methods set out in Section III of Part 2 of Annex I, the detailed diagnostic methods and procedures set out in 
points I.1 – I.2 of this Part shall apply . 
I.1.  Preparation of sam ples from fish 
For diagnostic purposes the fish (sent alive or killed and pack ed separately in sealed asep tic containers) or 
alternati vely frozen organs or organ pieces preser ved in 80 % to absolute ethanol or viral transpor t medium (to 
be processed within 48 hours after collection) may be used for testing with conventional PCR or qPCR based 
methods. 
For the detec tion of KHV , gill and kidne y shall be collect ed; in addition spleen, encephalon and intest ine may be 
included in an additional separated sample. In acute cases, tissue mater ial of up to five fish may be pooled. 
Further more, non-lethal samples such as blood, gill swabs, gill biopsy , mucus scrape may be used in certain 
cases (namely very valuable fish may be used in the case of the suspicion of the presence of KHV). 
I.1.1.  DNA extraction 
DNA shall be extract ed in accordance with standard procedures. 
The commercially available DNA extraction kits that produce high quality DNA suitable for use with the PCR 
protocols referred to in point I.2 may be used. 23.9.2015 L 247/45 Official Jour nal of the European Union EN     I.2.  Agen t detection and identification by Polymerase chain reaction (PCR) based methods 
I.2.1.  qPCR for KHV detection 
For qPCR detec tion of KHV , the following qPCR assa y shall be used: 
Forward primer (KHV -86f): 5′- GACGCCGGA GACCTTGTG -3′; 
Reverse primer (KHV -163r): 5′- CGGGTTCTT ATTTTTGTCCTTGTT -3′; 
and probe (KHV -109p): 5′-F AM- CTTCCTCTGCTCGGCGA GCA CG -3′. 
Cycling conditions: one cycle of 95 °C at 15 minutes, followed by 40 cycles of 94 °C at 15 seconds and 60 °C 
for 60 seconds. Nega tive template controls and positive controls shall be included on each plate run. However , 
other qPCR versions of proven similar efficacy may be used inste ad. 
I.2.2.  Conventiona l PCR for KHV detection 
The assa y descr ibed in this point targe ting the Thymine kinase (TK) gene of KHV shall be used. However , other 
PCR assa ys with demonstrate d similar sensitivities and specificities to the descr ibed assa y may be used instead. 
Forward primer (KHV -TKf): 5′-GGGTT ACCTGT AC GAG-3′; 
Reverse primer (KHV -TKr): 5′-CA CCCA GTAGATTA TGC-3′. 
Cycling conditions: one cycle of 95 °C for 5 minute s followed by 35 cycles of 95 °C for 30 seconds, 52 °C for 
30 seconds, 72 °C for one minute and one cycle of 72 °C for 10 minut es. Product size should be 409 bp. 
The results of the PCR may vary depending on the conditions under which it is performe d, namely the ther mal 
protocols might need optimisation, depending on the ther mal cycler in use. Further more, false-positive results 
may occur because of false primer annealing or contamination. Neg ative templat e controls and positive controls 
shall be included on each plate run. However , other PCR versions of proven similar efficacy may be used 
instead. 
The first detec tion in an area shall be confir med by sequencing or sent to a to a national reference laboratory or 
to the EU reference laboratory for fish diseases refer red to in Annex VI to Directive 2006/88/EC, for immediate 
identif ication. 
II.  Detail ed diagnos tic methods and procedures for the surveillance of KHVD 
When sampli ng and laborat ory examination for the purpose of obtaining or maintaining certain health statuses 
with regard to KHVD as set out in Section I of Part 2 of Annex I are carried out using the diagnostic methods 
set out in Sections II or III of Part 2 of that Annex, the detailed diagnostic methods and procedures set out in 
the following points II.1 and II.2 of this Part shall apply . 
II.1.  Preparation of sam ples from fish 
If possible, fish that have been kept for a prolonged time period at the virus permissive temperature range 
(namely , 2 to 3 weeks at 15 °C to 26 °C) shall be sampled. If possible, samples shall be collect ed 24 hours but 
not later than 72 hours after manag ement practices that may reactivate the virus in fish with a carrier status, 
such as netting or transpor t, in order to enhance the chance of KHV detec tion. 
For the purpose of the surveillance of KHVD, the fish may be sent alive or killed and pack ed separate ly in 
sealed aseptic containers or altern atively frozen organs or organ pieces preser ved in 80 % to 100 % alcohol or 
viral transpor t medium (to be processed within 48 hours after collection) may be used for testing PCR based 
methods. For the surveillance of KHVD, gill and kidney tissue shall be collected. 
For the purpose of surveillance of KHVD, pooling shall be avoided where possible. If pooling is necessar y, tissue 
mater ial from a maximum of two fish may be pooled. Larger samples shall be homogenised in mor tar and 
pestle or stomac her, and subsamples retrieved for DNA extraction before clarification. Alte rnatively , subsam ples 
may be collect ed from each tissue included in the sam ple and placed in ‘lysis-tubes’. 23.9.2015 L 247/46 Official Jour nal of the European Union EN     II.1.1.  DNA extraction 
DNA shall be extract ed in accordance with standard procedures. Commercially available DNA extraction kits 
that produce high quality DNA suitable for use with the PCR protocols set out in point II.2 may be used. 
The accept able tissue medium ratio shall be 1:9 w/v. 20 to 25 mg tissue mate rial shall be included in the tests. 
II.2.  Surveillance of KHVD by PCR based methods 
For the surveillance of KHV , a qPCR shall be used. If positive samples appear in an area not previously 
conf irmed positive, the test results shall be confir med either: 
(a)  by sequencing of a PCR or neste d PCR product from the samp les. 
The obtained clean consensus sequence shall match (by at least 98 %) with these referen ce sequences. 
(b)  or alternatively , sam ples may be sent to a national reference laborat ory for conf irmation. 
II.2.1.  qPCR for KHV detection 
The qPCR descr ibed as follows shall be used: 
Forward primer (KHV -86f): 5′- GACGCCGGA GACCTTGTG -3′; 
Reverse primer (KHV -163r): 5′- CGGGTTCTT ATTTTTGTCCTTGTT -3′; 
and probe (KHV -109p): 5′-F AM- CTTCCTCTGCTCGGCGA GCA CG -3′. 
Cycling conditions: one cycle of 95 °C at 15 minutes, followed by 50 cycles of 94 °C at 15 seconds and 60 °C 
for 60 seconds. 
The results of the qPCR may vary depending on the conditions under which it is performe d, namely the 
ther mal protocols might need optimisation, depending on the ther mal cycler in use. Further more, false-positive 
results may occur because of false primer annealing or laboratory contamination. Negative template controls 
and positive controls shall be included on each plate run. However , other qPCR versions of proven similar 
efficacy may be used instead. 
II.2.2.  Conventiona l PCR for confir mation of KHV detection 
For conf irmation of the presence of infection with KHV , the gener ic nested PCR descr ibed in the following 
Table 2.1 shall be used followed by sequencing of the amp lified product. 
Table 2.1 
Primers and conditions for the nested PCR assay targeting all cypr inid herpesvir uses (CyHV -1, CyHV -2 
and CyHV -3) 
Primer name Sequence Cycling conditions Product size 
CyHVpol-f orward 5′-CC AGCAACATGTGCGA CGG-3′ First round PCR 
1 cycle: 
95 °C 2 minutes 
40 cycles: 
95 °C for 30 seconds 
55 °C for 30 seconds 
72 °C for 45 seconds 
1 cycle: 
72 °C for 10 minutes 362 bp 
CyHVpol-reverse 5′-CCGT ARTGA GAGTTGGCGCA -3′ 23.9.2015 L 247/47 Official Jour nal of the European Union EN     Primer name Sequence Cycling conditions Product size 
CyHVpol-inter nal 
forward 5′-CGA CGGV GGY ATCAGCCC-3′ Second round PCR 
1 cycle: 
95 °C 2 minutes, 
40 cycles: 
95 °C for 30 seconds 
55 °C for 30 seconds 
72 °C for 45 seconds 
1 cycle: 
72 °C for 10 minutes 339 bp 
CyHVpol-inter nal 
reverse 5′-GA GTTGGCGCA YACYTTC ATC-3′  
The results of the PCR may vary depending on the conditions under which it is performe d, namely the ther mal 
protocols might need optimisation, depending on the ther mal cycler in use. Further more, false-positive results 
may occur because of false primer annealing or laboratory contamination. Negative template controls and 
positive controls shall be included on each plate run. PCR versions of proven similar efficacy may be used 
instead. 
Sequencing may be performe d by the laboratory or at external specialised sequencing companies. Sequencing 
results shall be analysed by aligning the sequences to the kno wn reference sequences of KHV (Gen Bank 
accession numbers AP008984, DQ657948 and DQ177346). The obtained clean consensus sequence must 
match at least to 98 % with those reference sequences. 
PART 3 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR THE SUR VEILL ANCE AND CONFIRMA TION OF 
INFECTIOUS SALMON ANEMIA (ISA) 
I.  Sampling procedures for the surveillance and control of ISA 
When samp ling and laboratory examination are carried out, for the purpose of the surveillance or eradication 
programmes set out in Part 3 of Annex I or to conf irm or rule out the presence of ISA in accordance with 
Article 57(b) of Directive 2006/88/EC, the detailed methods and procedures set out in points I.1, I.2 and I.3 of 
this Section shall apply . 
I.1.  Preparation of sam ples from fish 
For the purpose of laboratory examination for the presence of ISA, fish samp les shall not be pooled where 
possible. However , for the purpose of surveillance for ISA, the pooling of 2 to be 5 fish is accept ed. 
Samples for Reverse Transcr iptase polymerase chain reaction (RT-PCR) analysis shall be taken from all of the 
fish sam pled. A piece of mid-kidne y shall be remo ved from the fish using a steri le instr ument and transferr ed to 
a microfuge tube containing one ml RNA preser vative solution of proven efficacy . Tissue from up to five fish 
may be collect ed in one tube of transpor t solution and shall represent one pooled sam ple. The weight of tissue 
in one sam ple shall be 0,5 g. When the fish are too small to obtain a sample of the required weight, pieces of 
kidne y, hear t, spleen, liver or pylor ic caeca may be taken, in that order of preference, to make up 0,5 g. 
Tissue for histo logical examination shall only be taken from freshly killed fish with a normal constitution, 
exhibiting clinical signs or post-mor tem findings consistent with the presence of ISA. Any exte rnal or intern al 
lesions shall be sampled and in any case sam ples of mid-kidne y, hear t, liver , pancreas, gut, gills and spleen shall 
be remo ved from individual fish using a scalpel and transferr ed to 8 % to 10 % (vol:v ol) buff ered formol saline. 
The ratio of fixative to tissue shall be at least 20:1 to ensure satisf actor y preser vation of the tissues. For 
immunohistoc hemistr y (IHC), samples from mid-kidne y and hear t shall be taken. 23.9.2015 L 247/48 Official Jour nal of the European Union EN     Tissues for virological examination on cell culture shall be take n from all of the fish sampled. Pieces of the liver , 
anterior or mid-kidney , hear t and spleen shall be remo ved from the fish using a steri le instr ument and 
transferr ed to plastic tubes containing 9 ml transpor t medium. Tissues from up to five fish may be collected in 
one tube containing transpor t solution and represent one pooled sam ple. The weight of tissue in one sample 
shall be 1,0 ± 0,5 g. 
I.2.  Shipment of samples from fish 
Whole fish may be transpor ted to the laboratory if the temperature requirements during transpor tation, as 
descr ibed in paragraph 3 of this point can be fulfilled. Whole fish shall be wrapped in absorbent paper and 
shipped in a plastic bag, chilled as descr ibed in that paragraph. 
Live fish may also be shipped, but only under the super vision of the National referen ce laboratory for fish 
diseases and taking into account the additional disinf ection and biosecur ity issues when transpor ting live fish. 
Blood samples and tubes containing fish tissues for virological examination or RT-PCR analysis shall be placed 
in insulat ed containers, such as thick-walled polystyrene boxes, together with suffic ient ice or freeze bloc ks to 
ensure chilling of the samples during transpor tation to the laborat ory. Freezing shall be avoided and ice must 
still be present in the transpor t box at receipt of the shipment or one or more of the ‘freeze blocks ’ must still be 
partly or compl etely frozen. In excep tional circumstances, RT-PCR sam ples and sample s for virological 
examination may be snap-frozen and transpor ted to the laboratory at – 20 °C or belo w. 
For RT-PCR analysis of tissues preser ved in Ribonucleic acid(RNA)later , RNA extraction shall be carried out 
within the following time frames depending on the temperature the samples are stored at: 
samples stored at 37 °C: 1 day; 
samples stored at 25 °C: 1 week; 
samples stored at 4 °C: 1 month; 
samples stored at – 20 °C: indefinitely . 
If fish tissues are transpor ted in fixative for histo logical examination, they shall be shipped in leak proof tubes 
in impact-resistant containers. Freezing of those samples shall be avoided. 
The virological examination on cell culture shall be started as soon as possible and no later than 48 hours after 
the collection of the samples. In excep tional cases, the virological examination may be started at the latest 
within 72 hours after the collection of the mater ial, provided that the mater ial to be examined is protect ed by a 
transpor t medium and that the temperature requirements during transpor tation can be fulfilled . 
I.3.  Collection of supplementar y diagnostic mater ial 
Subject to the appro val of the diagnostic laboratory , fish tissues other than those referred to in point I.1 may be 
collect ed and prepared for supplementar y examination. 
II.  Detail ed diagnostic methods and procedures for the surveillance and for the conf irmation of the presence of or to 
rule out the suspicion of ISA 
When laborat ory examination for the purpose of obtaining or maintaining a certain health status with regar d to 
ISA as set out in Section I of Part 3 of Annex I, or for the purpose of the conf irmation of the presence of or to 
rule out a suspicion of ISA in accordance with Article 57(b) of Directive 2006/88/EC, are carried out, using the 
diagnostic methods set out in Section II of Part 3 of Annex I, the detailed methods and procedures set out in 
the following points II.1 to II.5 shall apply . 
II.1.  Examination of samples by RT-PCR 
The diagnostic method to be used for the screening for ISAV shall be RT-qPCR. As the results of the RT-qPCR 
can vary depending on the conditions under which it is performed, adequate positive and negative controls and 
amplicons shall be included to avoid any doubts. 
II.1.1.  Total RNA extraction 
All work with RNA shall be performe d on ice, using gloves. 23.9.2015 L 247/49 Official Jour nal of the European Union EN     Total RNA shall be extract ed using the phenol-chlorof orm method or RNA affinity spin columns, according to 
the manufacturer's instr uctions. 
Purified RNA shall be re-suspended in distilled RNase-free wate r (namely water treated with 0,1 % dieth yl 
pyrocarbonate). 
The concentration and purity of the extract ed RNA shall be estimate d by measur ing the optical density at 
260 nm and at 280 nm. An altern ative approach may be to include intern al controls target ed against the virus 
genome as referred to in point II.1.3. 
II.1.2.  RT-PCR for ISAV detection 
Several RT-PCR methods may be used for ISAV genome amplification. A two- step RT-PCR may be performe d 
whereby the RT and the PCR reactions steps are run in two separate tubes. However , a one-st ep reaction, where 
the two reactions are run in one tube, may also be performe d. The one-st ep method shall be used where 
possible, as the one tube assa y minimises the risk of cross-contamination as no transfer of content have to be 
made and it is regar ded to be as sensitive as the two- step method. 
The primers and assa y descr ibed in this point, namely the ILA1 or ILA2 primer pair that target segment 8 and 
which have been found suitable for detection of ISAV in outbreaks and in carrier fish, shall be used. The ILA2 
reverse primer does not match isolate s from Nor th Amer ica and an alternative primer set shall be used in those 
cases. 
Forward primer (ILA1): 5′-GGCT ATCT ACCA TGAA CGAA TC-3′; 
Reverse primer (ILA2): 5′-GCCAA GTGT AAGTAGCACTCC-3′. 
Cycling conditions: one cycle of 50 °C for 30 minutes , 1 cycle of 94 °C for 15 minut es, 40 cycles of 94 °C for 
30 seconds, 55 °C for 30 seconds, 72 °C for 60 seconds; one cycle of 72 °C for 5 min. Product size 155 bp. 
The results of the PCR may vary depending on the conditions under which it is performe d, namely the ther mal 
protocols might need optimisation, depending on the ther mal cycler in use. Further more, false-positive results 
may occur because of false primer annealing or laboratory contamination. Negative template controls and 
positive controls shall be included on each plate run. However , other RT-PCR versions of proven similar efficacy 
may be used inste ad. 
II.1.3.  RT-qPCR for ISAV detection 
The use of RT-qPCR may increase specificity and probably also sensitivity . The method can be performed more 
rapidly as no gel electrophoresis step is required and it reduces the risk of cross contamination as it is possible 
to estimate the amount of viral genomic RNA within the sample tube. A drawback of the RT-qPCR assa y is that 
it is often not possible to sequence amplified products. However , if there is doubt on the specifi city of the 
amplified product, another ISAV specific assa y must be run to verify the result. 
The assa y descr ibed in this point, whic h is an assa y that target segment 8, shall be used. This assa y shall cover 
isolate s from the European Union, the European Free Trade Association and Nor th Amer ica. The one-st ep 
method shall be used where possible, because the one tube assa y minimises the risk of cross-contamination. 
Forward primer: 5′- CTACACAGCAGGA TGCA GATGT -3′; 
Reverse primer: 5′- CAGGA TGCCGGAA GTCGA T -3′; 
and probe: 5′-F AM- CATCGTCGCTGCA GTTC – MGBNFQ-3′. 
Nega tive templat e controls and positive controls shall be included on each plate run. Cycling conditions: one 
cycle of 50 °C for 30 minutes, one cycle of 95 °C for 15 minut es, 40 cycles of 94 °C for 15 seconds, 60 °C for 
60 seconds; it shall be adjusted if necessar y. Other RT-PCR or RT-qPCR versions of proven similar efficacy may 
be used instead. 23.9.2015 L 247/50 Official Jour nal of the European Union EN     II.1.4.  Sequencing of ampli fied PCR products 
Forward primer (ILAs6-3F): 5′-A TGA GGGA GGT AGCATTGC A -3′; 
Reverse primer (ILAs6-2R): 5′-CA TGCTTTCCAA CCTGCT AGGA -3′. 
Nega tive template controls and positive controls shall be included on each plate run. Cycling conditions (One- 
step RT-PCR): one cycle of 50 °C for 30 minute s, one cycle of 94 °C for 15 minute s, 40 cycles of 94 °C for 
30 seconds, 55 °C for 30 seconds, 72 °C for 60 seconds, one cycle of 72 °C for 5 minute s; it shall be adjuste d if 
necessar y. Other RT-PCR or RT-qPCR versions of proven similar efficacy may be used instead. 
Alte rnatively , the following method for sequencing HPR in segment 6 may be used: 
Forward primer: 5′-GA C-CA G-A CA-A GC- TTA-GGT -AA C-A CA-GA-3′; 
Reverse primer: 5′-GA T-GGT -GGA-A TT-CTA-CCT -CTA-GA C-TTG- TA-3′; 
Product size: 304 nt if HPR0. 
RT-PCR assa ys with similar sensitivities and specifi cities to the assa ys descr ibed in this point may also be used. 
The purity of the amplified RT-PCR product shall be check ed by gel electrophoresis before sequencing. If only 
one pure fragment appears, it shall be purified directly from the PCR reaction. If multiple amplified fragments 
are present, the fragment of interest shall be purified by gel electrophoresis. The purification of PCR fragments 
from solutions or agarose gels shall be made using PCR fragment affinity spin columns, according to the 
manufacturer's instr uctions. 
Sequencing shall be performe d using amplification primers at exte rnal specialised sequencing compan ies. The 
results shall be analysed with the search tool BLAST and the sequences shall be compar ed with other kno wn 
sequences in the US National Centre for Biot echn ical Information (NCBI) nucleotide database. 
Sequencing must eliminate any doubt on the specifi city of an amplified RT-PCR product. 
II.2.  ISAV isolation on cell cultures 
II.2.1.  Preparation of sam ples 
The tissue may be kept at – 80 °C. The tissue shall only be frozen and thawe d once before examination. For 
surveillance and control purposes, the examination shall be under taken as fast as possible. 
Each sam ple (tissue pool in transpor t solution) shall be complet ely homogenised using a validat ed homog eniser , 
centr ifuged at 2 000 to 4 000 × g for 15 minutes at 0 to 6 °C, and the super natant shall be filtered (0,45 μm) 
and incubate d with an equal volume of a suitably dilut ed pool of antisera to the indigenous serotypes of IPNV . 
The titre of the antiser um must be at least 1:2 000 in a 50 % plaque neutralisation test. The mixture shall be 
incubated for one hour at 15 °C. This shall represent the inoculum. 
Treatment of all inocula with antiser um to Infectious pancreatic necrosis virus (a virus which in some parts of 
the Europe occurs in 50 % of fish samples) aims at preventing cytopatic effect (CPE) due to IPN virus from 
developing in inoculat ed cell cultures. To reduce the duration of the virological examinations as well as the 
number of cases in which occur rence of CPE would have to be considered poten tially indicative of ISAV, such 
treatment may be carried out. When sample s come from production units which are considered free from IPN, 
the treatment of inocula with antiser um to IPN virus may be omitted. 
II.2.2.  Inoculation on cell cultures 
Atlanti c salmon kidne y (ASK) cells shall be used for primar y ISAV isolation. Other cell lines of proven efficacy 
and sensitivity in isolating ISAV may be used, taking into consideration strain variability and the ability of 
differ ent strains to replicate in differ ent cell lines. The ASK cells seem to suppor t isolation and growth of the 
hither to known virus isolat es, as long as a low passage level is used. A more distinct cytopathic effect (CPE) may 
appear in ASK cells than in other susceptible cell lines like SHK -1 (Salmon head kidne y-1). 23.9.2015 L 247/51 Official Jour nal of the European Union EN     ASK (pass 65 or lower) cells shall be grown in L-15 medium containing 10 % foetal bovine serum, 2 % (vol:v ol) 
200 mM L-glutamine, and 0,08 % (vol:v ol) 50 mM 2-mercap toethanol in multi-well plates. Antiser um-treated 
organ suspension shall be inoculat ed into young actively growing cell cultures to give a final dilution of tissue 
mater ial to culture medium of 1:1 000. For each organ, suspension 40 μl of inoculum shall be added to one 
well containing 2 ml of culture medium. To minimise the risk of cross-contamination, separate 12- or 24-well 
plates shall be used for sam ples from different fish farm sites. 
One plate shall be left uninoculated to serve as a negative control. A separate plate shall be inoculat ed with a 
referen ce isolate of ISAV as a positive control, as follows. One hundred μl of a stoc k preparation of ISAV 
(minimum titre 107 Tissue culture infective dose at the 50 % end point (TCID50 ml– 1)) shall be inoculat ed into 
the first well and mixed. A volume of this mater ial shall be transferr ed from the first well to the second well to 
make a 1:10 dilution and mixe d. This shall be repeate d across the plate to make six 10-f old dilutions. Stoc k 
ISAV may be stored at – 80 °C for at least 2 years but once thawed must be used within 3 days. Care shall be 
taken to prevent cross-contamination of test plate s with positive control mater ial. To avoid that risk, positive 
controls shall be set up and handled separat ely from test plates. A sensitivity test ever y 6 months of ASK cells 
towards ISAV isolat es may replace the use of including a positive control at each inoculation. 
Samples shall be incubated at 15 ± 2 °C for up to 15 days. Using a microscope, cell cultures shall be examined 
for CPE twice, between 5 to 7 and 12 to 14 days following inoculation. If any pool shows CPE, virus identifi ­
cation procedures shall be initiated immediately in accordance with point II.2.4. If no CPE is obser ved by 
day 14, an Indirect fluorescent antibody test (IFAT), haemadsor ption or RT-PCR shall be performed. 
II.2.3.  Subcultivation 
Subcultivation shall be carried out between days 13 to 15. Culture super natant shall be added to wells 
containing fresh actively growing cells in appropr iate dilution (1/10) in multi-well plates and incubate d at 
14 ± 2 °C for up to 18 days. Using a microscope, cell cultures shall be examined for CPE twice, between days 
five to seven and days 14 to 18 following inoculation. If any pool shows CPE, virus identif ication procedures 
shall be initiat ed immediately in accordance with point II.2.4. If no CPE is obser ved by days 14 to 18, a haemad ­
sorption or an RT-PCR test shall be performe d. 
If cytot oxicity occurs within the first 7 days of incubation, subcultivation shall be performed at that stage, and 
the cells shall be incubat ed for 14 to 18 days and sub cultivated again with a further period of 14 to 18 days 
incubation. If cytotoxicity occurs after 7 days, subcultivation shall be performe d once and the cells shall be 
incubated to achieve the total period of 28 to 36 days incubation from the primar y inoculation. 
If bacteri al contamination occurs in the primar y culture, the test shall be set up again using the tissue 
homog enate stored at – 80 °C. Prior to inoculation, the tissue homog enate shall be centr ifuged at 4 000 × g for 
15 to 30 minut es at 0 to 6 °C and the super natant shall be filtered at 0,22 μm. If bacteri al contamination 
occurs during the subcultivation step the super natant shall be filtered at 0,22 μm, inoculat ed onto fresh cells 
and incubate d for a further 14 to 18 days. 
II.2.4.  Virus identification tests 
If evidence of CPE is obser ved at any stage, or if a haemadsor ption test is positive, virus identification shall be 
carried out. The methods of choice for the identif ication of ISAV shall be RT-PCR in accordance with point II.1 
and Immunof luorescence (IF) in accordance with point II.2.6. If it is considered that other viruses may be 
present, supplementar y virus identification tests shall be carried out. If those tests have not result ed in a 
definit ive identification of the virus within 1 week, the super natant shall be forwarded for immediate identifi ­
cation to: 
(a)  the World Organisation for Animal Health (OIE) reference laboratory for ISA, or; 
(b)  a national reference laboratory or the EU reference laborat ory for fish diseases as refer red to in Annex VI to 
Directive 2006/88/EC. 
II.2.5.  Haemadsor ption 
As replication of ISAV in cell cultures does not alwa ys result in a CPE, ever y well shall be subject to an RT-PCR 
test or a haemadsor ption test in accordance with this point, or an IF test in accordance with point II.2.6. 23.9.2015 L 247/52 Official Jour nal of the European Union EN     Cell culture medium shall be remo ved from each well, including those of positive and negative controls, and 
placed in labelled steril e tubes. 500 μl of a 0,2 % (vol:vol) suspension of washed rabbit or horse red blood cells, 
or a 0,05 % (vol:v ol) suspension of washed rainbo w trout or Atlantic salmon red blood cells, shall be added to 
each well and incubate d at room temperature for 45 minut es. The red blood cells shall be remo ved and each 
well shall be washed twice with L-15 medium. Each well shall be examined using a microscope. 
The presence of cluster s of red blood cells attaching to the surface of ASK cells shall be indicative of 
presump tive infection with an orthom yxovirus. If a haemadsor ption test is positive, a virus identification test 
shall be performe d immediately in accordance with point II.2.4. 
II.2.6.  Immunof luorescence (IF) 
ASK (pass 65 or lower) shall be grown in a L-15 medium containing 10 % foetal bovine serum, 2 % (vol:v ol) 
200 mM L-glutamine, and 0,08 % (vol:v ol) 50 mM 2-mercap toethanol in multi-well plates and used at greate r 
than 50 % conf luence. Other cell lines or growth medium of proven efficacy may also be used. 225 μl of 
putative virus-inf ected culture super natant shall be added to each of two wells, mixed and 225 μl transferr ed to 
two further wells, namely a 1:5 dilution. Two additional wells shall be left uninoculated to act as controls. 
Samples from each fish farm site shall be handled on separate plates , as shall the virus control. A virus control 
shall be established using a reference isolate of ISAV. 
Plates shall be incubated at 14 ± 2 °C and examined microscopically for up to 7 days. When early CPE is 
obser ved, or if no CPE is obser ved within 7 days, the next step shall be fixation. Wells shall be washed with 
Phosphate buffe red saline (PBS) and fixed by incubation with 80 % acet one for 20 minutes at room temperature. 
Plates shall be air-dried and stained immediately or stored at 0 to 6 °C for no more than 24 hours prior to 
staining. 
Replicat e wells shall be stained with a mix of monoclonal antibodies (MAb) 3H6F8 and 1OC9F5 against ISAV, 
or other MAb of proven efficacy and specifi city, dilut ed in PBS and incubated at 37 ± 4 °C for 30 minutes. MAb 
shall be remo ved and plates washed three times with 0,05 % Tween 20 in PBS. Anti-mouse IgG Fluorescein 
isothiocyanat e (FITC) conjug ate diluted in PBS shall be added to each well and incubat ed at 37 ± 4 °C for 
30 minut es. The dilutions of different batche s of MAb and FITC conjugat e shall be optimised in each laborat ory. 
Antibody shall be remo ved and plates shall be washed three times with 0,05 % Tween 20 in PBS. 
Wells shall be examined immediately using an inver ted microscope set up for fluorescence microscop y with a 
suitable filter for excitation of FITC. A test shall be considered positive if fluorescent cells are obser ved. For a 
test to be valid, the positive controls shall score positive and the negative controls shall score negativ e. 
II.3.  Examination of other tissues 
The technique refer red to in point II.2.6 may be applied to other fish tissues such as liver , spleen and hear t 
providing a reasonable quantity of endothelial cells, leucocyt es or lymphocyt es can be deposited on the slide. 
The staining procedure shall remain the same for each tissue, although for some tissues it may be preferable to 
omit the propidium iodide staining and rely on the phase illumination to identify the cell types present in the 
imp rint. 
II.4.  Histology 
Paraff in-embedded sections shall be cut at 5 μm and stained using haemato xylin and eosin. 
Histological changes in clinically diseased Atlantic salmon are variable, but may include the following: 
(a)  numerous erythrocytes in the central venous sinus and lamellar capillar ies of the gills, where erythrocyte 
thrombi also may be formed; 
(b)  multif ocal to conf luent pete chiae or hepat ocyte necrosis or both at some distance from larger vessels in the 
liver; multif ocal accumulation of erythrocytes in dilat ed hepatic sinusoids; 23.9.2015 L 247/53 Official Jour nal of the European Union EN     (c)  accumulation of erythrocytes in blood vessels of the intes tinal lamina propr ia and eventually haemor rhage 
into the lamina propr ia; 
(d)  spleen stroma diste nded by erythrocyte accumulation; 
(e)  slight multif ocal to extensive diffuse interstitial haemor rhage with tubular necrosis in the haemor rhagic 
areas, erythrocyte accumulation in the glomer uli in the kidne y; 
(f)  erythrophagocyt osis in the spleen and secondar y haemor rhages in liver and kidne y. 
II.5.  Immunohistoc hemistr y (IHC) 
Polyclonal antibody against ISAV nucleoprotein shall be used on paraff in sections from formalin-fi xed tissue. 
The orga ns to be examined shall be mid-kidne y and hear t (transitional area including all three chambers and 
valves). Suspect ed cases due to pathological signs shall be verified with a positive IHC. Histolog ical sections shall 
be prepared in accordance with standard methods.  
(1) Preparation of tissue sections 
The tissues shall be fixed in neutral phosphat e-buffered 10 % formalin for at least 1 day, dehydrated in a 
graded ethanol series, cleared in xylene and embedded in paraff in, according to standard prot ocols. Appro xi­
mat ely 5 µm thick sections (for IHC placed on poly-L-ly sine-coat ed slides) shall be heat ed at 56 °C to 58 °C 
(maximum 60 °C) for 20 minut es, dewaxed in xylene, rehydrat ed through a graded ethanol series, and 
stained with haemato xylin and eosin for pathomor phology and IHC in accordance with point (2).  
(2) Staining procedure for IHC 
All incubations shall be carried out at room temperature on a rocking platf orm, unless other wise provided 
for in this decision: 
(a)  antig en retrieval shall be done by boiling sections in 0,1 M citrate buffer pH 6,0 for 2 × 6 minutes 
followed by block ing with 5 % non-fa t dry milk and 2 % goat serum in 50 mM TBS (TBS; Tris/HCl 
50 mM, NaCl 150 mM, pH 7,6) for 20 minutes ; 
(b)  sections shall then be incubated overnight with primar y antibody (monospecif ic rabbit antibody against 
ISAV nucleoprot ein) dilut ed in TBS with 1 % non-fat dry milk, followed by three washes in TBS with 
0,1 % Tween 20; 
(c)  for detection of bound antibodies, sections shall be incubate d with Alkaline phosphatase-conjug ated 
antibodies to rabbit IgG for 60 minutes . Follo wing a final wash, Fast Red (1 mg ml– 1) and Naphthol 
AS-MX phosphate (0,2 mg ml– 1) with 1 mM Levamisole in 0,1 M TBS (pH 8,2) shall be added to 
develop for 20 minutes. Sections shall then be washed in tap water before counterstaining with Harris 
haemato xylin and mounted in aqueous mounting medium. ISAV positive and ISAV negative tissue 
sections shall be included as controls in ever y setup.  
(3) Interpretation of the result of IHC 
The interpr etation of the result of the IHC test shall be as set out in points (a) and (b): 
(a)  control sections shall be considered as positive, when it is obser ved that the control sections have 
clearly visible red-coloured (reddish) cytoplas mic and intranuclear staining of endothelia cells in blood 
vessels of endocardium. A test sample section shall only be regarded as positive if such clear , 
intranuclear red staining of endothelial cells is found; 
(b)  control sections shall be considered as negative if they don't have any signif icant colour reaction. 
Since the intranuclear localisation is particular to the orthom yxovirus nucleoprot ein during a stage of virus 
replication but concur rent cytoplasmic staining is often dominant, cytoplasmic and other staining patter ns 
without intranuclear localisation shall be considered as nonspecifi c or inconclusive. 23.9.2015 L 247/54 Official Jour nal of the European Union EN     The strong est positive staining reactions are usually obtained in endothelial cells of hear t and kidney . 
Endothelial staining reactions within very extensive haemor rhagic lesions may be slight or absent, possibly 
because of lysis of infected endothelial cells. 
PART 4 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR THE SUR VEILL ANCE AND CONFIRMA TION OF 
INFECTION WITH MARTEILIA REFRINGENS 
I.  Detail ed diagnos tic methods and procedures for the diagnosis of infection with Mar teilia refringens 
When sampling and laborat ory examination are carried out for the purpose of obtaining or maintaining a 
health status with regar d to infection with Mar teilia refrin gens as set out in Section I of Part 4 of Annex I or to 
conf irm or rule out the presence of that listed disease in accordance with Article 57(b) of Directive 
2006/88/EC, using the diagnostic methods set out in Section II of Part 4 of Annex I, the detailed diagnostic 
methods and procedures set out in points I.1, I.2 and I.3 of this Part shall apply . 
I.1.  Sampli ng procedure 
Gaping or freshly dead individual molluscs shall be sampled by priority, to increase the chances of finding 
infected animals. 
Once samp led, oysters or mussels shall be maintained at 4 °C or on refrigerated ice for no more than 24 hours 
if samples include gaping molluscs and no more than 72 hours if not, in a plastic bag including a label with 
details relat ed to the nature and the origin of the oysters or mussels. Gaping or freshly dead molluscs shall be 
maintained separately from other molluscs. 
A 3 to 5 mm thick section of tissues including gills and hear t tissue shall be used for the diagnostic of Mar teilia 
refrin gens by histology . A piece of diges tive gland shall be used for some tests, including impr ints and 
polymerase chain reaction (PCR). 
I.2.  Microscopic techniques 
I.2.1.  Cytology (imprint cytolog y) 
After drying digestive gland tissues on absorbent paper , several imp rints shall be made on a glass slide. Slides 
shall be air-dried, fixed in methanol or in absolute ethanol and stained using a commercially available blood- 
staining kit such as Diff-Quik®/Hemacolor®, in accordance with the manuf acturer's instr uctions. After rinsing 
in tap water and drying, the slides shall be mounted with a cover-slip using an appropr iate synthetic resin. The 
slides shall be obser ved first at × 200 magnifi cation and then under oil immersion at × 1 000 magnifi cation. 
A positive result shall be the obser vation of cells ranging in size up to between 30 to 40 µm. The cytoplasm 
stains basophilic, whereas the nucleus stains eosinophilic. Pale halos around large, strong ly stained (refr inge nt) 
granules and, in larger cells, cell within cell arrang ements are obser ved. 
The technique is not parasit e species specif ic. 
I.2.2.  Histology 
Sections of tissue that include gills, diges tive gland, mantle, and gonad shall be fixed for at least 24 hours in 
Davi dson's fixative followed by normal processing for paraff in histo logy and staining, for example with 
haemato xylin and eosin. Obser vations shall be made at increasing magnifications up to × 1 000. 
A positive result shall be the obser vation of cells ranging in size from 4 up to 40 µm. Early stages consist of 
multinucleate, spher ic to elongat ed cells. These are mainly found in the epithelium of oesophagus and stomac h 
and sometimes of labial palps. Spor ulation involves divisions of cells within cells and takes place in the dige stive 
gland tubules and ducts. Refr ingent granules appear in the course of spor ulation, but are not obser ved in early 
stages. In late phases of infection, sporangia are obser ved free in the lumen of the digestive tract. The cytoplasm 
stains basophilic, whereas the nucleus stains eosinophilic. The granules can rang e from deep orange to deep red. 
The technique is not parasit e species specif ic. 23.9.2015 L 247/55 Official Jour nal of the European Union EN     I.3.  Molecular techniques 
I.3.1.  DNA extraction 
DNA shall be extract ed in accordance with standard procedures. 
DNA extraction kits, which are available commercially and usually produce high quality DNA suitable for use 
with the PCR protocols as descr ibed in point I.3.2, may be used. 
I.3.2.  Polymerase chain reaction (PCR) 
Several PCR protocols have been developed and published. 
PCR primers that target the internal transcr ibed spacer (ITS1) region shall be used as they are able to amplify 
only M. refringens. 
PCR shall be carried out in a 50 µl volume. PCR mixtures shall contain buffer (500 mM KCl, 100 mM Tris/HCl 
[pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 µM forward and reverse 
primers, 0,02 units µl– 1 Taq DNA polymerase, and 10 to100 ng of extracted DNA . After denaturation of DNA 
at 94 °C for five minutes, 30 cycles shall be run as follows: denaturation at 94 °C for one minute, annealing at 
55 °C for one minute, and elongati on at 72 °C for one minut e per kilo-base pair . A final elongati on step of 
10 minutes at 72 °C shall be performe d. For the detection of M. refringens, PCR shall be performe d with primers 
that target the ITS1 region (5′-CCG-CA C-A CG- TTC- TTC-A CT-CC-3′ and 5′-CTC-GCG-A GT-TTC-GA C-A GA- 
CG-3′). 
Positive controls shall consist of genomic DNA from a highly infec ted host or plasmidic DNA including the 
target region. 
Nega tive controls shall consist of genomic DNA from non-infe cted hosts and PCR reactives without target DNA . 
A positive result shall be positive PCR amplification at the expected size (412 bp), with all negative controls 
being negat ive and all positive controls being positive. 
I.3.3.  In-situ hybridisation (ISH) 
Several ISH prot ocols have been developed and published. 
Probe that targets the SSU of the rRNA gene compl ex shall be used because it has been validat ed against 
histo logy . 
Sections of tissue that include gills, digestive gland, shall be fixed for at least 24 hours in Davi dson's fixative 
followed by normal processing for paraffin histo logy . Sections of 5 µm shall be cut and placed on aminoalkyl ­
silane-coated slides, which shall then be bake d overnight in an oven at 40 °C. The sections shall be dewax ed by 
immersing in xylene for 10 minutes . This step shall be repeated once and then the solvent shall be eliminated 
by immersion in two successive absolute ethanol baths for 10 minutes each. The sections shall then be 
dehydrated by immersion in a graded ethanol series. The sections shall be treated with prot einase K (100 µg 
ml– 1) in TE buff er (Tris [50 mM], EDT A [10 mM]), at 37 °C for 30 minut es. Slides shall be dehydrat ed by 
immersion in a graded ethanol series and then air dried. Sections shall be incubat ed with 100 µl of hybridisation 
buffe r (4 × SSC [standard saline citrate], 50 % formamide, 1 × Denhardt's solution, 250 µg ml– 1 yeast tRNA, 
10 % dextran sulphate) containing 10 ng (1 µl of the PCR reactants prepared as descr ibed in point I.3.2 using 
primers CCG-GTG-CCA-GGT -ATA-TCT -CG and TTC-GGG- TGG- TCT -TGA-AA G-GC) of the digo xigenin-labelled 
probe. Sections shall be covered with in-situ plastic cover-slips and placed on a heating block at 95 °C for five 
minutes . Slides shall then be cooled on ice for one minute before overnight hybridisation at 42 °C in a humid 
chamber . Sections shall be washed twice for five minutes in 2 × SSC at room temperature, and once for 
10 minute s in 0,4 × SSC at 42 °C. The detection steps shall be performed according to the manufacturer's 
instr uctions. The slides shall then be rinsed in steri le distilled water (dH2O). The sections shall be counte r- 
stained with Bismarck Brown Yellow, rinsed in dH2O, and cover-slips shall be applied using an aqueous 
mounting medium. 
Positive and negativ e controls shall be sections from kno wn infected and non-inf ected hosts respectively . 23.9.2015 L 247/56 Official Jour nal of the European Union EN     A positive result shall be demonstrated by the purple-blac k labelling of M. refringens cells within known target 
tissues, with all negativ e controls being negative and all positive controls being positive. 
I.3.4.  Sequencing 
Sequencing shall be carried out as one of the final steps for conf irmatory diagnostic. Target ed regions are the 
SSU rDNA and ITS1. 
II.  Detail ed diagnos tic methods and procedures for surveillance and conf irmation of infection with Mar teilia refringens 
For the purpose of surveillance programmes and to confir m the presence of an infection with Mar teilia refrin gens 
or to rule out a suspicion of that listed disease, in accordance with the requirements laid down in Section II of 
Part 4 to Annex I, the diagnostic methods and corresponding procedures to be used shall be in accordance with 
the guidelines laid down in Table 4.1 as follows: 
Table 4.1 
Guidelines for the use of diagnost ic methods for the surveillance prog rammes and to conf irm or rule 
out infection with Mar teilia refringens 
Method Target ed surveillance Presump tive diagnosis Confir mat ory  
diagnosis 
Dige stive gland impr ints X X X, or 
Histopath ology X  X, or 
In situ hybr idisation   X, and 
PCR X X X, and 
Sequencing   X  
PART 5 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR THE SUR VEILL ANCE AND CONFIRMA TION OF 
INFECTION WITH BONAMIA OSTREAE 
I.  Procedures for diagnosis of Infection with Bonamia ostr eae 
When sampling and laborat ory examination are carried out for the purpose of obtaining or maintaining a 
certain health status with regar d to Bonamia ostreae as set out in Section I of Part 5 of Annex I or to conf irm or 
rule out the presence of that listed disease in accordance with Article 57(b) of Directive 2006/88/EC, using the 
diagnostic methods set out in Section II of Part 5 of Annex I, the detailed diagnostic methods and procedures 
set out in following points I.1, I.2 and I.3 shall apply . 
I.1.  Sample process 
Gaping or freshly dead individual molluscs shall be sampled by priority, to increase the chances of finding 
infected animals. 
Once sampled, oysters shall be maintained at 4 °C or on refrigera ted ice for no more than 24 hours if samples 
include gaping molluscs and 72 hours if not, in a plastic bag including a label with details relat ed to the nature 
and the origin of the oysters. Gaping or freshly dead molluscs shall be maintained separately from other 
molluscs. 
A 3 to 5 mm thick section of tissues including gills and hear t tissue shall be used for the diagnostic of Bonamia 
ostreae by histology . A piece of diges tive gland shall be used for some tests, including imprints and polymerase 
chain reaction (PCR). 23.9.2015 L 247/57 Official Jour nal of the European Union EN     I.2.  Microscopic techniques 
I.2.1.  Cytology (imprint cytolog y) 
After drying gills or hear t tissues on absorbent paper , several impr ints shall be made on a glass slide. Slides shall 
be air-dried, fixed in methanol or in absolute ethanol and stained using a commercially available blood-staining 
kit (namely such as Diff- Quik®/Hemacolor®.), in accordance with the manuf acturer's instr uctions. After 
rinsing in tap water and drying, the slides shall be mount ed with a cover-slip using an appropr iate synthetic 
resin. Slides shall be obser ved first at × 200 magnifi cation and then under oil immersion at × 1 000 magnifi ­
cation. 
A positive result shall be the presence of small spher ical or ovoid organisms (2 to 5 µm wide) within 
haemocytes . However , the parasite might also occur extracellularly . These organisms show a basophilic 
cytoplas m and an eosinophilic nucleus (colours may vary with the stain used) and, because they spread on the 
slide, they can appear wider on impr ints than on histological examination. Multinucleated cells may be 
obser ved. The technique is not parasite species specifi c. 
I.2.2.  Histology 
Sections of tissue that include gills and dige stive gland, shall be fixed for a period of at least 24 hours in 
Davi dson's fixative followed by normal processing for paraff in histo logy and staining, for example with 
haemato xylin and eosin. Obser vations shall be made at increasing magnifications up to × 1 000. 
A positive result shall be the presence of parasites as very small cells of 2 to 5 µm wide within the haemocytes 
or free in the connective tissue or sinuses of gill, gut and mantle epithelium, often associated with an intense 
inflammat ory reaction. To avoid any doubt, the parasit e shall be obser ved inside the haemocyte for a positive 
diagnosis. The technique is not parasite species specifi c. 
I.3.  Molecular techniques 
I.3.1.  DNA extraction 
DNA shall be extract ed in accordance with standard procedures. 
DNA extraction kits, which are available commercially and usually produce high quality DNA suitable for use 
with the PCR protocols as detailed belo w, may be used. 
I.3.2.  Polymerase chain reaction (PCR) 
Several PCR protocols have been developed and published. 
Two PCR protocols target ing the small subunit (SSU) rDNA may be used: 
(a)  the first one is a conven tional PCR that amplify several members of the Haplospor idia including Bonamia spp. 
Primers designat ed Bo and Boas are 5′-CA T-TTA-A TT-GGT -CGG-GCC-GC-3′ and 5′-CTG-A TC-GTC- TTC- 
GAT-CCC-CC-3′ respectively , and amplify a 300 bp product. PCR mixtures contain buffer (500 mM KCl, 
100 mM Tris/HCl [pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 µM 
forward and reverse primers, 0,02 units µl– 1 Taq DNA polymerase, and 0,2 ng µl– 1 of the DNA templat e in 
a total volume of 50 µl. Sample s shall be denatured in a ther mocy cler for five minut es at 94 °C before being 
submitted to 30 cycles (94 °C for one minute, 55 °C for one minut e, 72 °C for one minute) followed by a 
final extensi on of 10 minutes at 72 °C. 
Positive controls shall consist of genomic DNA from a highly infected host or plasmidic DNA including the 
target region. 
Negative controls shall consist of genomic DNA from non-infe cted hosts and PCR reactives without target 
DNA . 
A positive result shall be positive PCR ampli fication at the expected size (namely , 300 bp), with all negative 
controls being negat ive and all positive controls being positive; 23.9.2015 L 247/58 Official Jour nal of the European Union EN     (b)  the second PCR protocol is a SYBR® Green Real time PCR assa y. It allows the specifi c detection of B. ostreae 
(descr ibed belo w) and may be combined with a SYBR® Green Real time PCR assa y allowing the specifi c 
detection of B. exitiosa (Ramilo et al. 2013). 
Primers BOSTRE-F (5′- TTACGTCCCTGCCCTTTGT A-3′) and BOSTRE-R (5′- TCGCGGTTGAA TTTT ATCGT ­
-3′) amp lify a 208 bp product. PCR mixtures contain SYBR® Green Master Mix (1X), 0,3 µM forward and 
reverse primers, and 200 ng of extract ed DNA. Sam ples shall be denatured in a Real Time Detection System 
for 10 minutes at 95 °C before being submitted to 35 cycles (95 °C for 30 seconds, 55 °C for 45 seconds 
and 72 °C for one minute). Melting temperature curve analysis shall be carried out with temperature 
increments of 0,5 °C/s starting at 55 °C and ending at 95 °C and recording fluorescence at each change of 
temperature. 
Positive controls shall consist in genomic DNA from a highly infec ted host or plasmidic DNA including the 
target region. 
Negative controls shall consist in genomic DNA from non-infect ed hosts and PCR reactives without target 
DNA . 
A positive result shall be positive PCR ampli fication with a unique melting temperature peak 
(78,25 ± 0,25 °C in the conditions published by Ramilo et al. 2013), with all negative controls being 
negative and all positive controls being positive. 
I.3.3.  In-situ hybridisation (ISH) 
Several ISH prot ocols have been developed and published. 
A probe that targe ts the SSU of the rDNA gene compl ex shall be used although it was shown to cross react 
with some other members of the family Haplospor idia. 
Sections of tissue that include gills and digestive gland shall be fixed for a period of at least 24 hours in 
Davi dson's fixative followed by normal processing for paraff in histo logy . Sections of 5 µm shall be cut and 
placed on aminoalkylsilane-coat ed slides, whic h shall then be baked overnight in an oven at 40 °C. The sections 
shall be dewaxe d by immersing in xylene for 10 minute s. This step shall be repeated once and then the solvent 
shall be eliminate d by immersion in two successive absolute ethanol baths for 10 minutes each. The sections 
shall then be dehydrated by immersion in a graded ethanol series. The sections shall be treated with prote inase 
K (100 µg ml– 1) in TE buffe r (Tris [50 mM], EDT A [10 mM]), at 37 °C for 30 minutes. Slides shall be 
dehydrated by immersion in a graded ethanol series and then air dried. Sections shall be incubated with 100 µl 
of hybridisation buffer (4 × SSC [standard saline citrate], 50 % formamide, 1 × Denhardt's solution, 250 µg 
ml– 1 yeast tRNA, 10 % dextran sulphate) containing 20 ng (2 µl of the PCR reaction prepared as descr ibed in 
point I.3.2 using primers Bo and Boas) of the digo xigenin-labelled probe. Sections shall be covered with in-situ 
plastic cover-slips and placed on a heating bloc k at 95 °C for five minutes. Slides shall then be cooled on ice for 
one minute before overnight hybridisation at 42 °C in a humid chamber . Sections shall be washed twice for five 
minutes in 2 × SSC at room temperature, and once for 10 minutes in 0,4 × SSC at 42 °C. The detection steps 
shall be performe d according to the manuf acturer's instr uctions. The slides shall then be rinsed in sterile 
distilled water (dH2O). The sections shall be counter -stained with Bismarck Brown Yellow, rinsed in dH2O, and 
cover-slips shall be applied using an aqueous mounting medium. 
Positive and negativ e controls shall be sections from kno wn infected and non-inf ected hosts respectively . 
A positive result shall correspond to labelled parasites inside the haemocyte s, with all negative controls negative 
and all positive controls positive. 
I.3.4.  Sequencing 
Sequencing shall be carried out as one of the final steps for conf irmat ory diagnostic. Target ed regions shall be 
the SSU rDNA and ITS1. 23.9.2015 L 247/59 Official Jour nal of the European Union EN     II.  Procedures for surveillance and conf irmation of infection with Bonamia ostr eae 
For the purpose of surveillance and conf irmation of the presence of or to rule out a suspicion of infection with 
Bonamia ostreae in accordance with the requirements laid down in Section II of Part 5 to Annex I, the diagnostic 
methods and corresponding procedures to be used shall be in accordance with the guidelines laid down in the 
following Table 5.1. 
Table 5.1 
Guidelines for the use of diagnosti c methods for the surveillance prog rammes and to rule out or 
conf irm infection with Bonamia ostr eae 
Method Target ed surveillance Presump tive diagnosis Confir mat ory  
diagnosis 
Hear t or gill impr ints X X X, or 
Histopath ology X  X, or 
In situ hybr idisation   X, and 
PCR X X X, and 
Sequencing   X  
PART 6 
DET AILED DIA GNOSTIC METHODS AND PROCEDURES FOR SUR VEILL ANCE AND CONFIRMA TION OF WHITE SPOT 
DISEASE (WSD) 
1.  Diagnostic procedures for detection of WSSV 
When sampli ng and laborat ory examination for the purpose of the surveillance and eradication programmes set 
out in Section I of Part 6 of Annex I and for the confir mation of the presence of or to rule out a suspicion of 
infection with WSSV are carried out in accordance with Article 57(b) of Directive 2006/88/EC, using the 
diagnostic methods set out in Section II of Part 6 of Annex I, the detailed diagnostic methods and procedures 
set out in points 2 to 7 of this Part shall apply . 
The methods and procedures descr ibed in this Part of Annex II are adap ted from the ISO 17025 accredited test 
applied at the European Union Reference Laboratory for Crustacean Diseases. Alter native approac hes, using 
equivalent conditions or kits produced by differ ent manufacturers , but whic h offer equivalent sensitivity and 
specifi city to those descr ibed in this Part may be applied. In all instances, PCR amplified product shall be 
sequenced to confir m identity as White spot syndrome virus (WSS V). 
2.  Sample process 
Tissue (pleopods and gills) containing WSSV from crustaceans may be stored in ethanol, RNAlater or snap 
frozen at – 80 °C. The stages required for the identif ication of WSSV from tissue samples shall be as follows: 
Homogen isation of the tissue, extraction of the DNA, specif ic amplification of WSSV DNA using PCR, visuali ­
sation of the ampli fied product on a gel, purification of the DNA and sequencing to conf irm the identity of the 
pathogen . 
3.  Tissue homogenisation 
The disrupti on of the tissues and the preparation of a homog enate in an appropr iate buffe r shall be carried out 
using the Fast Prep tissue disruptor and Lysing matr ix A tubes (MP Biomedicals). The tissue shall be weighed, 
placed in Lysing Matr ix A tubes, dilut ed 1 in 10 w/v or according to manufa cturer's instr uctions, in an 
appropr iate buffer (G2 and 10 µl Proteinase K for use with DNA Tissue kit (Qiag en)) and homog enised using 
the Fast Prep 24 homog eniser for two minute s. Homogen ised samples shall be incubated at 56 °C for a 
minimum period of four hours or overnight. Samples shall be vortexed, centr ifuged at 9 000 rpm for two 
minutes and 50 µl of super natant or a volume equivalent to 5 mg of tissue (weight of tissue optimal for 
extraction kit) shall be added to a sam ple tube for DNA extraction and volume made up to 200 µl using G2 
buffe r. 23.9.2015 L 247/60 Official Jour nal of the European Union EN     4.  DNA extraction 
Total DNA shall be extracted using a DNA tissue extraction kit and the EZ1 Advanced XL Biorobot (Qiag en) 
following the manufact urers' instr uctions. An extraction control (Calf Thymus DNA) and a nega tive control 
(G2 buffer) shall run with each batch of samples . DNA shall be eluted into 50 µl volume. To ensure that the 
extraction has compl eted successfully , the DNA concentration for all sample s and controls shall be deter mined 
using a Nano Drop machi ne. Extracte d DNA shall be frozen at – 20 °C if not required immediately . 
5.  WSSV Polymerase Chain Reaction (PCR) 
The method to be used for the detection of WSSV shall be the protocol for the detection of WSSV by neste d 
PCR set out in the following paragraphs whic h ampli fies a 1 447 bp and an 848 bp ampli con of the 18s rRNA 
gene in the first and second round PCRs respectively . 
The 1st round PCR reaction shall be set up in a 50 µl volume which contains final concentrations of 1 × GoTaq 
Buffe r (Promega) , 5 mM MgCl2, 1 pmol/µl WSSV 146 F1 primer , 1 pmol/µl WSSV 146 R1 primer (Table 1), 
0,25 mM dNTPs, 1,25U Taq polymerase and 2,5 µl of DNA . Each sample shall be run in duplicate alongside a 
negativ e extraction control, a negativ e PCR control, (2,5 µl H2O added instead of DNA) and a positive control. 
The positive control shall be diluted WSSV plasmid made and validat ed for use in-house (available from the 
EURL). 
The 2nd round WSSV PCR reaction shall be set up in the same way as the first round but using the WSSV 146 
F2/R2 primer set and using a second positive control to check that this stage of the PCR has worked. 
Primer Sequence 
WSSV 146 F1 ACTACTAACTTCA GCCT ATCT AG 
WSSV 146 R1 TAATGCGGGTGT AATGTTCTT ACGA 
WSSV 146 F2 GTAACTGCCCCTTCC ATCTCC A 
WSSV 146 R2 TACGGC AGCTGCTGCA CCTTGT  
Both 1st and 2nd round PCRs shall be run using the following cycling conditions on a DNA Engine Tetrad 2 
Peltier Ther mal Cycler (or equivalent). An initial denaturation step of 94 °C for two minutes followed by 94 °C 
for 30 seconds, 62 °C for 30 seconds, 72 °C for 30 seconds repeated for 30 cycles, a 72 °C extension step for 
two minutes and held at 4 °C. 
6.  Gel electrophoresis 
Amplified PCR products from both the 1st and 2nd round PCRs shall be visualised on 2 % agarose gels made 
using TAE buff er. 15 µl of each sam ple shall be run at 120 Volts for appro ximat ely 20 minutes and viewed 
under UV light. Positive samples will produce a band of 1 447 bp in the 1st round PCR and 848bp in the 
second round PCR. Sam ples of that size shall be cut out and placed into a 1,5 ml microfuge tube. The DNA 
contained within the gel slices shall be purified using Promega Wizard® SV Gel and PCR clean-Up System 
according to manuf acturers' instr uctions. The concentration of the DNA shall be estimate d using a Nano Drop 
machi ne. The purified DNA shall be frozen at – 20 °C if not being used immediate ly. 
7.  Sequencing of PCR products 
DNA shall be sequenced using the Big Dye Terminator Kit v3,1 (Applied Biosystems). The total volume in each 
reaction is 20 µl, the final concentrations being 1 × Big Dye term inator , 1 × sequencing buffer , 10 pmol/µl 
forward or reverse primer and 10 µl of purified DNA (diluted to appro ximat ely 10 ng/µl) run using the 
following cycling conditions on a DNA Engine Tetrad 2 Peltier Ther mal Cycler (or equivalent): 94 °C for 
30 seconds followed by 96 °C for 10 seconds, 50 °C for 10 seconds and 60 °C for four minute s, cycling the last 
three steps 30 times. 23.9.2015 L 247/61 Official Jour nal of the European Union EN     The PCR products shall be precipitate d using a sodium acetate method where 20 µl DNA is added to 10 µl 
NaA c, 70 µl H2O and 250 µl ethanol, vortexed and centr ifuged at 13 000 rpm for 20 minut es, the super natant 
shall be remo ved and the pellet shall be washed with 200 µl absolute ethanol, centr ifuging at 13 000 rpm for 
five minutes. The pellet shall be dried for five minutes at 37 °C. 25 µl of Hi-Di formamide shall be added to the 
pellets, heat ed to 95 °C for two minute s and vortexed thoroughly . Samp les shall be sequenced using the 
ABI3130xl Avant Genetic analyser according to manuf acturers' instr uctions. Sequencing results shall be 
analysed using Sequenc her software and sequences matc hed to sequences on the NCBI database using the 
BLAST function.  23.9.2015 L 247/62 Official Jour nal of the European Union EN