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Cefas contract report

TECHNICAL REPORT FOR THE UK NATIONAL REFERENCE LABORATORY FOR ANISAKIS (APRIL 2015 – March 2016)

(C5936 - Anisakis NRL)

Author: Tom Hill

Issue date: 30/3/2016

Cefas Document Control

TECHNICAL REPORT FOR THE UK NATIONAL REFERENCE LABORATORY FOR ANISAKIS (APRIL 2015 – MARCH 2016)

Submitted to: Chelvi Leonard Date submitted: 30/3/2016 Project Manager: Stephen Feist Report compiled by: Tom Hill Quality control by: Stephen Feist

Approved by & Stephen Feist date: 30/3/2016

Version: 1.4 (FINAL)

Version Control History Author Date Comment Version Tom Hill 21/03/2016 1st draft 1.0 Tom Hill 22/03/2016 1.1 Tom Hill 23/03/2016 Sent to SWF for comment 1.2 Stephen Feist 23/03/2016 Amendments made to v1.2 1.3 Tom Hill 30/3/2016 PT results added. Minor changes 1.4 to document Tom Hill 30/3/2016 Sent to SWF for approval FINAL Tom Hill 25/4/2016 Report amended with acronym FINAL definitions

TECHNICAL REPORT FOR THE UK NATIONAL REFERENCE LABORATORY FOR ANISAKIS Page i

TECHNICAL REPORT FOR THE UK NATIONAL REFERENCE LABORATORY FOR ANISAKIS (APRIL 2015 – MARCH 2016)

(C5936 – Anisakis NRL)

Author: Tom Hill

Issue date: 30/3/2016

Head office

Centre for Environment, Fisheries & Aquaculture Science Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK

Tel +44 (0) 1502 56 2244 Fax +44 (0) 1502 51 3865 www.cefas.defra.gov.uk

Cefas is an executive agency of Defra

Page ii

Page iii Table of contents

1 Introduction ...... 1 2 Updates to website ...... 1 2.1 Overview ...... 1

2.1.1 Website structure ...... 2

2.1.2 Email enquiries ...... 2

3 Participation in European Union Reference Laboratory for Parasites (EURLP) proficiency test scheme (2015 and 2016)...... 3 3.1 Completion of 2015 EURLP proficiency test ...... 3

3.2 Completion of 2016 EURLP proficiency test ...... 3

4 Reference material ...... 4 4.1 Maintenance of tissue bank ...... 4

5 Report of the molecular analysis of Anisakis from six species of fish ...... 5 5.1 Methods ...... 5

5.2 Results: ...... 8

6 Attendance at the EURLP annual meeting ...... 9 6.1 Participation at EURLP annual meeting ...... 9

6.2 Implementation of EURLP recommendations ...... 9

7 International Council for the Exploration of the Seas (ICES) Working Group on Pathology and Diseases in Marine Organisms ...... 9 8 Ongoing surveillance ...... 11 9 ISO9001:2008 Audit ...... 12 9.1 Overview ...... 12

9.1.1 Outcome of audit ...... 12

10 2015/2016 Work plan progress ...... 14 10.1 Overview ...... 14

10.1.1 Anisakis NRL Work plan - progress 2015/16 ...... 14

11 Annex ...... 17 11.1 EURLP Proficiency Testing Report 2016 ...... 17

TECHNICAL REPORT FOR THE UK NATIONAL REFERENCE LABORATORY FOR ANISAKIS Page iv

11.2 ICES WGPDMO meeting. ICES Working Group on Pathology and Diseases in Marine Organisms (WGPDMO). Virginia Institute of Marine Science, 16–20 February 2016...... 18

Page v

1 Introduction

The Centre for Environment, Fisheries and Aquaculture Science (Cefas) Weymouth is designated as the UK National Reference Laboratory (NRL) for Anisakis. This annual technical report summarises the activities carried out by the NRL between April 2015 and March 2016 according to the requirements of Regulation (EC) No. 882/2004 and as defined in the Service Level Agreement between the Food Standards Agency and Cefas.

2 Updates to website

2.1 Overview The website continues to deliver information on the responsibilities and function of the NRL. During the period covered by this report, the website was migrated to a new server, and has been updated with quality controlled standard operation procedure documents. Further scientific references and other information resources have also been added.

A website analytical report was run 21/03/2016. This determined that the NRL website has had 720 visits to date Figure 1 - Website traffic (page views) 1/1/2014 to 21/3/2016Figure 1) from approx. 362 unique users. The sections of the website which experienced the most traffic were the methodology, proficiency testing, references, and contact sections. Although there is only a small level of traffic moving through the website at this time, the report suggests that interested parties are actively searching for advice and techniques related to Anisakis.

Figure 1 - Website traffic (page views) 1/1/2014 to 21/3/2016

2.1.1 Website structure Although content and formatting updates have occurred, the website structure has not changed since the previous reporting period (2015).  NRL Home o Background o Regulations . Links to regulations relating to Anisakis o Methods . List of methods from EURL o Proficiency testing o References . List of references o Contact NRL

2.1.2 Email enquiries We have received one email enquiry through the Anisakis mailbox during 2015/2016. This related to an enquiry from the Food Safety and Veterinary Institute in Albania regarding proficiency testing for Anisakis. Advice was provided in a timely manner and contact details were provided for the EURLP who organises such proficiency testing.

3 Participation in European Union Reference Laboratory for Parasites (EURLP) proficiency test scheme (2015 and 2016)

3.1 Completion of 2015 EURLP proficiency test Due to a breakdown in communication with the Union Reference Laboratory for Parasites (EURLP), we were not informed of the 2015 Proficiency Test until after the samples had been sent to participating laboratories. However, following contact with the EURLP director the error was acknowledged and we were included on their ‘Corrective Action’ proficiency test normally offered to those laboratories which had failed the first round.

Fillet samples were received on the 15th April 2015 and screened using compression and the ultraviolet transillumination system following internal SOP NRL001. Our results were reported to the EURLP shortly afterwards. We are pleased to report successful completion of the exercise with a score of 100%.

3.2 Completion of 2016 EURLP proficiency test Samples for the 2016 Proficiency Test were received 14th March 2016 and screened using the ultraviolet transillumination system following internal SOP NRL001. Our results were returned to the EURLP 17th March 2016. We received our PT report from the EURLP 29th March and are delighted to confirm successful completion of the exercise with a score of 100% (see annex).

4 Reference material

4.1 Maintenance of tissue bank We are maintaining the Anisakis ‘tissue bank’ on-site. This contains a substantial number of frozen parasites removed from Plaice, Haddock and Mackerel sampled at two recognised sites (St. Bees and Celtic Deep) within the geographical region of the Irish Sea. Although not identified to their species at this time, these samples are available for use in gross (fillet) screening proficiency testing or training programmes.

In addition to this material, we have screened a small number of samples of a deep-sea fish species – Aphanopus carbo – caught during a research cruise. Although the material has been held in long term frozen storage a very large number of well-preserved Anisakidae worms were found to be present within the viscera. A number of these parasites (Figure 2) were isolated and transferred into suitable reagents. These samples will be formally identified using traditional morphological techniques alongside contemporary molecular tests.

Figure 2 - Anisakidae worms isolated from the viscera of A. carbo. Images from left to right: 1) mass of parasites removed from visceral bundle; 2) parasites in classic coiled position; and, 3) an individual parasite.

We will continue to add to the content of the tissue bank as and when sampling opportunities present themselves. We are able to obtain Specific Pathogen Free (SPF) fillets for use in training programs from within our aquarium facility. The production of any PT material required for PT or training will follow SOP NRL007 (in draft) – see section 7.

We are also holding a small quantity of identified parasites, and extracted DNA provided by the EURLP.

5 Report of the molecular analysis of Anisakis from six species of fish

A number of nematode worms from seven fish (six species, Table 1) were placed in ETOH during the annual Cefas cruise in July 2015.

Table 1 - Sample providence

Number Fish species sampled for Anisakid worms 1 Dab Limanda limanda 2 Whiting Merlangius merlangus 3 Plaice Pleuronectes platessa 4 Herring Clupea harengus 5 Gurnard 6 Mackerel I Scomber scombrus 7 Mackerel II Scomber scombrus

The worms were difficult to separate so more than one worm was placed into the incubation buffer for extraction. If the DNA extraction consisted of more than one species this could be resolved as each PCR was to be run separately.

5.1 Methods Buffers and solutions: Proteinase K: 5.5ml of Incubation Buffer (IB) was added to 100mg Proteinase K powder. DTT: 32.4ml of molecular grade water was added to the 5g of DTT powder. IB / Proteinase K / DTT mix: 8 parts incubation buffer (800µl), 1 part Proteinase K (100µl) and 1 part DTT (100µl).

15ml of Isopropyl alcohol and 15ml of ETOH was added to the 2 x Wash Buffer concentrate. Lysis buffer: 1µl of Proteinase K per 100µl of Lysis Buffer. Detailed DNA IQ Tissue and Hair extraction method:

One worm was removed from the ETOH and 100µl of Incubation Buffer was added and the sample was incubated at 55oC for 2 hours vortexing every 30 minutes. The sample was centrifuged briefly

and 200µl of Lysis Buffer and DTT was added to the sample followed by 10µl of Resin and incubated for 10 minutes at 25oC, vortexing regularly. The sample was placed in the magnetic stand and the Lysis Buffer removed before 100µl fresh Lysis Buffer was added. The sample was vortexed briefly and the buffer removed as before. One hundred microlitres of Wash Buffer was added, the sample vortexed to resuspend the Resin and the Wash Buffer was removed as previously described using the magnetic stand. This step was repeated three times. After the last wash the tubes were left to air

o dry for 15 minutes and 25µl of molecular grade H20 added and the sample was incubated at 65 C for 5 minutes with vortexing. The eluate was removed from the Resin using the magnetic separation stand and the concentration estimated using a fluorometer.

Separate single round PCR reactions were performed for each sample and each of the eight PCRs in

50l reaction mix consisting of 1 X Green Go Taq buffer, 2.5mM MgCl2, 0.25mM dNTPs, 50pmol each of the forward and reverse primer, 0.25 units Go Taq Flexi (Promega), and 2.5l extracted nucleic acid. These PCRs were then run using the Anisakis 40 thermal cycler program on a Peltier PTC-225: denaturation of 95oC x 2 minutes followed by 40 cycles of 95 oC x 30 seconds, 55oC x 30 seconds and 72oC x 45 seconds followed by 72oC x 7 minutes. Performed using a Peltier PTC-225 thermal cycler.

Resulting PCR amplifications were resolved on a 2% agarose gel stained with ethidium bromide and visualised using a UV illuminator. Correct size products (Table 2) were excised from the gels, purified using the Bio 101 Geneclean kit (MP Biomedicals) and sequenced using the ABI PRISIM Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems) following manufacturers’ instructions. DNA was sequenced using the ABI 3130xl Avant Genetic analyser (Applied Biosystems). Analysis of the sequences was completed using Sequencher software (Gene Codes Corporation).

Table 2 - Product sizes and species amplified.

PCR code Primer set Product size Species amplified A ASPf/RevBT 588bp A. pegreffi, A. simplex s.i, and A. simplex/pegreffi hybrid. B APE1/ RevBT 672bp A. pegreffi C APyf/RevBT 143bp A. physeteris, A. brevispiculata, A. paggiae D ATf/RevBT 427bp A. typica E COf/RevBT 799bp Contracaecum osculatum

F CRf/RevBT 307bp Contracaecum rudolphii (A,B,C) G PDf/RevBT 370bp Pseudoterranova spp. H HAf/RevBT 991bp Hysterotilacium aduncum

Anisakis PCRs A Aspf/Revbt and B APEI/Revbt 100bp A1 A2 A3 A4 A5 A6 A7 –ve B1 B2 B3 B4 B5 B6 B7 –ve 100bp

Anisakis PCRs C APyf/ Rev bt and D ATf / Rev bt 100bp C1 C2 C3 C4 C5 C6 C7 -ve D1 D2 D3 D4 D5 D6 D7 –ve 100bp

Anisakis PCRs E COf/Revbt and F CRf/ Rev bt 100bp E1 E2 E3 E4 E5 E6 E7 –ve F1 F2 F3 F4 F5 F6 F7 –ve 100bp

Anisakis PCRs G PDf /Rev bt and H HAf /Rev bt 100bp G1 G2 G3 G4 G5 G6 G7 –ve H1 H2 H3 H4 H5 H6 H7 –ve 100bp

The correct size of product for each of the samples highlighted in red was cut out and sequenced as described above.

5.2 Results: Number Fish species % Match Accession Species sampled for number Anisakid worms 1 Dab Limanda 100 KP857649 Anisakis limanda simplex 2 Whiting 100 KM273050 Contracaecum Merlangius osculatum merlangus 3 Plaice 100 KP979763 Hysterotilacium Pleuronectes aduncum platessa 4 Herring Clupea 100 KP857649 Anisakis harengus simplex 5 Gurnard 100 KP857649 Anisakis simplex 6 Mackerel I 100 KP857649 Anisakis Scomber simplex scombrus 7 Mackerel II 100 KP857649 Anisakis Scomber simplex scombrus

When the consensus sequences (amplified using the primer set ASPf/RevBT) from samples 1, 4, 5, 6 and 7 were compared against sequences on the NCBI database all samples matched with both A. simplex and A. pegreffi. However none of the Samples 1, 4, 5, 6 and 7 produced amplification products of the correct size when amplified using the primer set APE1/ RevBT which indicates that these are all A. simplex and not A. pegreffi.

6 Attendance at the EURLP annual meeting

6.1 Participation at EURLP annual meeting Two members of the NRL team will attend this year’s meeting at the EURLP, in May 2016. We will be giving a short oral presentation on the activities of the laboratory with emphasis on parasitic infections with zoonotic potential.

6.2 Implementation of EURLP recommendations None received.

7 International Council for the Exploration of the Seas (ICES) Working Group on Pathology and Diseases in Marine Organisms

Anisakis prevalence in dab from waters of England and Wales remains high (13.0-55.4 %) at the majority of fishing stations in the North Sea. The Tyne Tees region typically exhibits the highest prevalence of 37.2 %, 55.4 % and 20.0 % at Amble, Flamborough and Tees Bay respectively. In ICES area 1, prevalence in polar cod decreased from 64.0% to 33.2% and in capelin from area 2b, from 52% to 28%. Prevalence in cod from area 2b increased from 93.3% to 100% and in herring from area 1, there was an increase from 46.6% to 63.8%. In Polish waters, prevalence in cod decreased from 30.6% (n=278) to 14.5% (n=303). The prevalence of infection was higher in the Gulf of Gdansk (29.4%) than in the Western Baltic (8.9%) and middle coast (5.0%). Previously, higher values were recorded in western and middle Baltic. For herring in Polish waters, analysis of prevalence data using a GLM revealed that estimated year effects for 2013 2014 and 2015 strongly decreased in comparison to effects of 2011 and 2012. A similar pattern was revealed for intensity.

Along with the reports of Anisakis in UK wild fish, data from Denmark, Poland, Russia, and Sweden suggests that the increasing trend of Contracaecum osculatum frequency in Baltic sea cod continued into 2015. Another species Pseudoterranova decipiens from in fish from the Barents Sea was also reported as an increasing trend.

A summary of the full meeting is annexed to this report and contains information on other marine pathogens reported from wild and cultures marine fish and shellfish.

8 Ongoing surveillance

As part of our ongoing surveillance of papers concerning anisakiasis (in fish and humans) we identified a significant increase in the published during 2015/2016 related to human infections to date compared to previous years (see listing below). However, we have not determined if this is an increasing trend reflecting the increased occurrence of the condition in humans or increased research activity in Europe (only one paper originated from Asia, see no. 5). We will maintain vigilance on the published literature as part of our deliverables under the contract.

1. E. Heffler, M. E. Sberna, S. Sichili, R. Intravaia, G. Nicolosi, M. Porto, M. T. Liuzzo, G. Picardi, S. Fichera, N. Crimi (2016) High prevalence of Anisakis simplex hypersensitivity and allergy in Sicily, Italy. Annals of Allergy, Asthma & Immunology, 116(2), 146-150. 2. E. Madrid, F. Gil, M. García, Á. L. Debenedetti, M. Trelis, M. V. Fuentes (2016) Potential risk analysis of human anisakiasis through the consumption of mackerel, Scomber scombrus, sold at Spanish supermarkets. Food Control, Volume 66, August 2016, 300-305. 3. B. Brieau, G. Rahmi, H. Benosman, C. Cellier (2015). Acute dysphagia and odynophagia revealing an unusual case of oesophageal anisakiasis. Digestive and Liver Disease, 47(12), e21. 4. J. Ivanovic, M. Z. Baltic, M. Boskovic, N. Kilibarda, M. Dokmanovic, R. Markovic, J. Janjic, B. Baltic (2015) Anisakis Infection and Allergy in Humans. Procedia Food Science, 5, 101-104. 5. S.-W. Li, S.-H. Shiao, S.-C. Weng, T.-H. Liu, K.-E. Su, C.-C. Chen (2015) A case of human infection with Anisakis simplex in Taiwan. Gastrointestinal Endoscopy, 82(4), 757-758. 6. D. Ljubojevic, N. Novakov, V. Djordjevic, V. Radosavljevic, M. Pelic, M. Cirkovic (2015) Potential Parasitic Hazards for Humans in Fish Meat. Procedia Food Science, 5, 172-175. 7. E. Heffler, M. E Sberna, S. Sichili, R. Intravaia, G. Nicolosi, M. Porto, M. T. Liuzzo, G. Picardi, S. Fichera, N. Crimi (2016) High prevalence of Anisakis simplex hypersensitivity and allergy in Sicily, Italy. Annals of Allergy, Asthma & Immunology, 116(2), 146-150. 8. A. J. Brooker, R. Wootten, A. P. Shinn, J. E. Bron (2016) An assessment of the potential for zoonotic parasitic nematode infections arising from the consumption of maricultured Atlantic halibut, Hippoglossus hippoglossus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum), in Scotland. Food Control, Volume 66, 198-204. 9. D. Ljubojevic, N. Novakov, V. Djordjevic, V. Radosavljevic, M. Pelic, M. Cirkovic (2015) Potential Parasitic Hazards for Humans in Fish Meat. Procedia Food Science, 5, 172-175. 10. G. Gironé, C. Mateo, V. Gaya, J. Usó, C. Mínguez, B. Roca, J. M. Ramos (2015) Admissions for imported and non-imported parasitic diseases at a General Hospital in Spain: A retrospective analysis. Travel Medicine and Infectious Disease, 13(4), 322-328.

9 ISO9001:2008 Audit

9.1 Overview On the 24 and 25th June 2015, a number of projects at Cefas were externally audited to verify that compliance with ISO 9001:2008 was being maintained. One of the projects selected for audit was the Anisakis NRL.

The auditor carried out an in depth review of the project’s management procedures through documentation assessment and interviews with the NRL director and a member of the project team. The auditor was particularly interested in document version control and reporting processes. Other activities reviewed also included:

 Contract defining deliverables and target dates  Production of deliverables, for example Code of Practice and SOPs  Project handover / transfer of projects  Customer satisfaction  Project correspondence  Project review  Completion of Proficiency Tests  Project planning

9.1.1 Outcome of audit The auditor was satisfied with the management processes in place, and concluded that the NRL project was adhering to the accredited system. No major non-compliances were identified and the auditor only made two minor suggestions for improvement:

a) The issue of the Code of Practice and SOPs had not used the formal document issue process. The available copies of the SOPs did not bear any document control information (version number / revision date), although they were reported to be now published on the Cefas web site. This was easily corrected. All documentation released by the NRL (both internally and externally) will follow the correct format and process. b) It was noted that the contract did not specifically define the required content of the Code of Practice. In practice, the content was defined by the project team’s experience and was subsequently agreed by the customer.

10 2015/2016 Work plan progress

10.1 Overview A work plan for 2015/2016 was accepted by the NRL director 9/9/15 and implemented during the following months. The plan predominantly set objectives to continue website maintenance and updates, and improve stakeholder engagement.

There was a general lack of interest from the Official Control Laboratories (OCLs) when we previously contacted them. This continued during this year’s attempts to re-engage them. We continue to offer advice and/or training should they require it.

A small number of new SOPs are currently under draft or planned i.e. production of PT material. SOPs have been published on the NRL website once approved by the NRL director. The current SOPs and the NRL Code of Practice were recently transferred into a new document management system. As part of this process, all documents were reviewed and updated as required.

We will continue to monitor the EURLP website for relevant updates, and contact the EURLP director as required.

10.1.1 Anisakis NRL Work plan - progress 2015/16 1. Continued maintenance of NRL Website (Ongoing): a. Update the website with relevant information, to include: literature (including food safety policy), reference, SOPs, and Code of Practice updates (Complete but ongoing review). b. The background section will be updated to include more information and accessibility for consumers/laypersons (by end of FY 2015/16). c. News section to be added to website, continual updates as required (ongoing). d. Ensure open access to up to date versions of Cefas SOPs and Code of Practice (Complete but ongoing, dependent on updated versions).

2. Code of Practice and SOP review a. Review food safety policy, and scientific literature. Update the CoP as required (complete). b. Review and update SOPs (complete).

i. Provide to OCLs if requested (all are available on website). ii. SOP for the production of SPF PT material to be produced and uploaded to the website (in draft).

3. Continuing OCL engagement a. Re-approach OCLs with offer of training, quality control assurance, and PT programs. Arrange and carryout training programs as required (complete this FY, but ongoing).

4. Attend EURLP meeting if requested/relevant. a. If attendance is not required, maintain contact with current UK representative (attending May 2016).

5. Participate in year 3 proficiency testing, as organised by the EURLP (awaiting results of 2016 PT).

6. Implement year 3 EURLP recommendations if provided (none provided). a. Monthly checks/search of EURLP website for relevant updates and information (ongoing).

7. Continue to offer advice to FSA as required (ongoing).

8. Molecular method validation. Including identification of multiple species. Additional sample material collected/sourced if required (2015/2016 planned molecular work complete; sample material acquisition and parasite identification ongoing).

9. Produce six monthly (complete) and annual reports (complete).

Tom Hill

Research Scientist.

Environment and Animal Health,

Centre for Environment Fisheries and Aquaculture Science (Cefas),

Barrack Road, The Nothe, Weymouth, DT4 8UB.

+44 (0)1305 206758 www.cefas.defra.gov.uk

11 Annex

11.1 EURLP Proficiency Testing Report 2016

11.2 ICES WGPDMO meeting. ICES Working Group on Pathology and Diseases in Marine Organisms (WGPDMO). Virginia Institute of Marine Science, 16–20 February 2016.

This meeting was attended by S. W. Feist who provided a national report for England and Wales on disease occurrence during 2015 as well as an update on the Leaflet series on diseases of marine fish and shellfish. A key Term-of-Reference for the group is the assessment of emerging disease trends in fish and shellfish. The following information is a summary of the national reports from 13 ICES member countries. Please note the section on Nematoda which includes reference to Anisakis in UK wild fish and Contracaecum osculatum in cod from the Baltic sea where the increasing trend reported previously continued in 2015 with data from Denmark, Poland, Russia and Sweden. Another species Pseudoterranova decipiens from in fish from the Barents Sea was also reported as an increasing trend. Additional information is available if required.

Emerging disease trends. WILD FISH Viruses Salmon Gill Poxvirus (SGPV) – Reported for the first time in Canada from a healthy adult Atlantic salmon in the Magaguadavic River, New Brunswick. The finding was based on cytopathology and high-throughput sequencing.

Piscine Reovirus (PRV) – Reported from Denmark in 2014 for the first time, 6.3% of 176 salmon brood-stock tested positive by qPCR. The virus was later detected in progeny (fry) from the affected brood fish despite disinfection of eggs. Eight wild brown trout were found to be negative for the virus.

Infectious Pancreatic Necrosis Virus (IPNV) – In mid-Norway, the virus was detected in 0 to 5.6% of returning salmon.

Infectious Salmon Anaemia Virus (ISAV) – In mid-Norway, the virus was detected in 0.7 to 12.5 % of returning salmon.

Viral Haemorrhagic Septicaemia Virus (VHSV) – Genotype 1b detected in Baltic cod from Hanö Bay, Sweden (ICES district SD 25). The fish also showed signs of fin rot, purulent exudate, splenic granulomas, endo- and pericarditis, anaemia and peritoneal haemorrhage.

Bacteria Vibrio anguillarum – In Sweden, mass mortality among herring from the island of Orust (ICES district 21) associated with serotype O3 (“Pacific herring serotype”), not previously reported from Sweden. Morbid fish were lethargic with balance and buoyancy problems and had haemorrhages, ascites and superficial skin ulcers.

Acute/healing skin ulcerations – In Baltic cod, prevalences in Polish, Russian and Swedish waters were 2.5% (n=17,748), 3.3% (n=945) and 2.8% (n=3,940), the lowest in Polish waters since 2010. In Swedish waters, prevalence in flounder was 0.6% (n=4,895).

Fungi Paranucleospora theridion - In mid-Norway, prevalence ranged from 7.4% to 70% during testing of nearly 900 returning salmon between 2013 and 2014. Oomycetes Saprolegnia - In July-October 2015, in the Kola River (Barents Sea basin of Russia), infection in adult Atlantic salmon (Salmo salar), whitefish (Coregonus lavaretus) and minnow (Phoxinus phoxinus) was observed for the first time with associated mortality. Similar observations, reported previously from Finland and Sweden, persisted in 2015 in Sweden. Affected salmon were lethargic and a diagnosis of UDN was made from a sample of 5 not showing oomycete growth.

Parasites Protists

Sphaerothecum-like parasite (Mesomycetozoa) – prevalence in dab from the North Sea stations West Dogger Bank and North Dogger Bank was 2.5% (n=80) and at Indefatigable Bank 1.3% (n=80). Infections were observed in the liver and kidney at low intensity, usually associated with a granulomatous host response.

Ichthyobodo salmonis – In mid-Norway, prevalence ranged from 12% to 35% during testing of nearly 900 returning salmon between 2013 and 2014.

Myxozoa

Parvicapsula pseudobranchicola - In mid-Norway, prevalence ranged from 18% to 22% during testing of nearly 900 returning salmon between 2013 and 2014.

Nematoda

Contracaecum osculatum – The increasing trend reported earlier from Baltic cod continued in 2015 with data from Denmark, Poland, Russia and Sweden. Small cod (35-40 cm) harboured intense infections of up to 300 parasites per cod (n= 66 liver samples).

Pseudoterranova decipiens – In the Barents Sea (ICES areas 1 and 2b), infections in long-rough dab increased between 2014 and 2015 from 21% to 30% and 12% to 35%, respectively. In cod from area 2b, prevalence increased from 17% to 24%. In Baltic cod caught east of Bornholm, prevalences up to 55 % and intensities up to 56 worms per fish were reported. In Swedish waters, over 60% of cod and 100% of bullheads (Myoxocephalus scorpius) were infected, indicating that the upward trend reported earlier continues. Further up into the Baltic, around Gotland, prevalence in cod was 7 % and 9% in bullheads. In the archipelago of Stockholm and the sea of Åland, infection rates decreased to nearly zero, with only a single nematode found in over 200 examined fish. It is not known why the prevalence was so low in northern areas, which are also home to the largest concentrations of grey seal in the Baltic Sea. Another factor such as salinity or the absence of suitable intermediate hosts may be responsible.

Anisakis simplex – Prevalence in dab from waters of England and Wales remains high (13.0-55.4 %) at the majority of fishing stations in the North Sea. The Tyne Tees region typically exhibits the highest prevalence of 37.2 %, 55.4 % and 20.0 % at Amble, Flamborough and Tees Bay respectively. In ICES area 1, prevalence in polar cod decreased from 64.0% to 33.2% and in capelin from area 2b, from 52% to 28%. Prevalence in cod from area 2b increased from 93.3% to 100% and in herring from area 1, there was an increase from 46.6% to 63.8%. In Polish waters, prevalence in cod decreased from 30.6% (n=278) to 14.5% (n=303). The prevalence of infection was higher in the Gulf of Gdansk (29.4%) than in the Western Baltic (8.9%) and middle coast (5.0%). Previously, higher values were recorded in western and middle Baltic. For herring in Polish waters, analysis of prevalence data using a GLM revealed that estimated year effects for 2013 2014 and 2015 strongly decreased in comparison to effects of 2011 and 2012. A similar pattern was revealed for intensity.

Monogenea

Gyrodactylus salaris - The parasite was discovered for the first time in the River Rolfsån on the Swedish west coast. Together with the nearby River Kungsbackaån, this was one of two water systems previously recognised as free of G. salaris.

Other diseases Eye Pathology – Persisting in the Barents Sea as acute exophthalmos, cataract, red eyes, or ocular degeneration in 2.83 % of capelin, in 1.54 % of polar cod and in 0.41 % of cod.

Hyperpigmentation - continues to be observed in dab at relatively high prevalence compared to the Irish Sea, Severn and English Channel regions: Amble (29.5 %), Tees Bay (15.0 %), Flamborough (43.2 %), North Dogger (35.4 %), Central Dogger (48.7 %), West Dogger (49.3 %), Indefatigable Bank (39.0 %), and Off Humber (39.0 %). Moreover, North Sea regions continue to show an increasing trend concerning prevalence of this condition.

Effects of munition dumpsites - Based on German data, no major differences in the health status of cod have been observed between munition dumpsites and reference areas in the western and eastern Baltic Sea. However, condition factors of cod in the main dumpsite east of Bornholm were significantly lower compared to all other study areas.

Conclusion A salmon gill poxvirus (SGPV) sequence was obtained from a healthy Atlantic salmon in New Brunswick, eastern Canada Piscine reovirus in Atlantic salmon was reported for the first time from Denmark in 2014 In Sweden, mass mortality among herring from the island of Orust associated with Vibrio anguillarum serotype O3 (“Pacific herring serotype”). High mortality and morbidity among migrating salmon in Russia (Kola River) and Sweden (Baltic, west coast) were associated with Oomycete infection. UDN was diagnosed from samples not showing oomycete growth. Prevalence of Pseudoterranova decipiens in cod and bullheads declined along a salinity gradient in the Baltic Sea despite the presence of large seal populations. No measurable differences in health status of Baltic cod were attributed to proximity to munition dumpsites. A reduction in condition factor among cod collected near dumpsites was measured.

FARMED FISH

Viral diseases

Viral Haemorrhagic Septicemia Virus (VHSV) – In Western Canada, seven cases have been reported, North American strain. Denmark is still declared free from VHS; no outbreaks after the eradication program ran from 2009 till 2011. Infectious Haematopoietic Necrosis Virus (IHNV) – No new trends.

Infectious pancreatic necrosis virus (IPNV): In Sweden, two cases of IPN serotype ab were diagnosed in rainbow trout in a national screening program. One of the farms was in the Baltic Sea, the other in an inland lake. In Norway, the number of cases has continued to decline, from 48 in 2014 to 30 in 2015. The steady decrease in numbers of clinical outbreaks since 2010 is mainly caused by the breeding of IPN-resistant fish. IPN is not considered to be a serious problem in Norway anymore. IPNV was found in fry-size halibut on two occasions in Norway. In western Canada, none of 759 Atlantic salmon or 61 Pacific salmon tested positive for IPN-virus by qRT-PCR.

Infectious salmon anemia virus (ISAV): The disease was diagnosed in 15 farms; for both the two previous years, the number was 10 farms. Only three cases were considered primary outbreaks, one in brood fish, one at a sea site, and the third in a smolt. Four secondary cases received fish from the smolt farm. The remaining cases are considered secondary probably caused by horizontal spread from neighboring farms. Two epidemics in Northern Norway started in 2013 and 2014 and are still not declared eradicated. At two sites, rainbow trout were infected, after the disease had attacked Atlantic salmon at the same site. These cases are the first registered in this fish species under ordinary farming conditions. In eastern Canada, sporadic outbreaks with the North American genotype of ISAV persist, however surveillance revealed a high prevalence of European type HPR0 strains. In western Canada, none of 3703 Atlantic salmon or 61 Pacific salmon tested positive for the virus by qRT-PCR.

Pancreas disease virus – Salmonid alphavirus (SAV) – In Norway, there are two endemic regions with two different subtypes of the virus, SAV2 and SAV3, and the northernmost part of the country is surveilled to keep a free-status. One case was seen in this region, and this population was eliminated immediately. During 2014 and 2015, there have been cases of PD caused by SAV2 in the

SAV3-zone. The number of PD cases in 2015 was 135, close to the historically high number of 142 in 2014. Ireland experienced seven outbreaks of PD, and only three in 2014.

Piscine orthoreovirus (PRV) – Heart and skeletal muscle inflammation (HSMI) was diagnosed for the first time in Ireland on one marine Atlantic salmon site, and detection of the piscine orthoreovirus was confirmed by qPCR. Mortality was reported to be low. In Norway, the number of HSMI outbreaks in Atlantic salmon was 135. Of these, 129 cases were sea-sites with ongrowers, three were brood fish, and three were in smolt farms. This is a reduction from the highest number ever in 2014 (181). The disease was listed till 2014, delisting has probably contributed to the reduction in cases reported. Using qRT-PCR, the virus was detected for the first time in eastern Canada in all Atlantic salmon from one lot held in quarantine. In addition, 6 of 11 salmon originating from another hatchery and held at a government research facility tested positive. No fish were examined histologically for evidence of HSMI.

Onchorhynchus mykiss reovirus – A new viral disease in rainbow trout was first reported in 2013 in Norway, disease was seen in four different hatcheries. Mortalities also occurred in fish originating from these hatcheries after they were transferred to seawater. Sequencing of the new viral agent showed that it is related to Piscine orthoreovirus in Atlantic salmon, and the pathological picture is similar to HSMI. No disease outbreaks have been registered in 2015, however, a screening program in 50 farms has resulted in virus detection in nine sea-farms.

Piscine myocarditis virus (PMCV) - The number of cases of cardiomyopathy syndrome (CMS) outbreaks in Atlantic salmon in Norway was 105, two less than the year before.

Salmon gill poxvirus (SGPV) – Salmon gill poxvirus disease has been known in Norway since 1995. The first genome sequence of this DNA-virus was described in 2015. Salmon pox was diagnosed in a total of 18 fish farms last year, 15 sea sites and three smolt farms.

Nodavirus - Nervous necrosis virus (NNV) – Nodavirus was found in fry-size halibut on three occasions in Norway.

Bacterial diseases

Renibacterium salmoninarum – Bacterial kidney disease (BKD) in rainbow trout was

reported in three Swedish inland fish farms during 2015. Sweden has additional guarantees from the EU to keep the country (inland) free from BKD. Aeromonas salmonicida – In Sweden (Baltic Sea), one case of furunculosis was diagnosed in rainbow trout, and one case was diagnosed in Atlantic salmon, marine site, in Ireland. A. salmonicida subsp. salmonicida was isolated in one case with increased mortalities in lumpfish transferred to an Atlantic salmon sea-site in Norway. The salmon (vaccinated) were not affected. In Scotland, atypical A. salmonicida is detected in moribund ballan wrasse, being used as cleaner fish for farmed Atlantic salmon. In Norway, atypical A. salmonicida has been diagnosed in lumpfish used as cleaner fish in 51 cases, and in wrasses in 32 cases. In farmed halibut, atypical A. salmonicida was found in three cases in small, fry-size fish. In Russia, Far East region, seasonal occurrence of A. hydrophila and A. eucrenophila were registered in pink and chum salmon larvae. Yersinia ruckeri – In Sweden, one case of yersiniosis (serotype II) was found in farmed rainbow trout in mid-Sweden. Norway had 34 cases of yersiniosis in 2015, eight in smolt farms, 25 in sea farms, and one in brood fish, all Atlantic salmon. The number has increased gradually from year to year the last four to five years. Several smolt farms use vaccines against yersiniosis.

Moritella viscosa / winter ulcers - Winter ulcer syndrome was diagnosed in three sites in Scotland, and three sites in Ireland, all in Atlantic salmon. Most salmonids are vaccinated against M. viscosa. In Norway, 57 cases of winter ulcers in Atlantic salmon and four cases in rainbow trout were diagnosed. In 2014, there were 44 cases in salmonids.

Vibrio-infections - In Norway, Vibrio anguillarum has been isolated from cleaner fish used to control salmon lice, 12 cases in lumpfish and two cases in wrasses. Three cases of V. ordalii have been reported in lumpfish.

Flavobacterium / Flexibacter – Three cases of infection with Flavobacterium psychrophilum in rainbow trout were reported in Norway, two in seawater sites, the third in an inland farm. In 2014, two cases were registered. From 2014, septicemic flavobacteriosis in rainbow trout is a list 3 disease in Norway. In the Netherlands, Flexibacter maritimus-like bacteria have been isolated from sole and dab.

Pasteurella / Pseudomonas - In lumpfish (cleaner fish), Pasteurella sp. was isolated in 14 cases and Pseudomonas anguilliseptica in four. In Russia, Far East region, seasonal occurrence of Pseudomonas anguilliseptica, P. alcaligenes, and P. putida was seen in pink and chum salmon larvae.

Parasites

Crustacea

Lepeophtheirus salmonis – Salmon lice are reported to be the most important cause of disease in Faroese salmon farming. Norway found a slight decrease in the overall number of reported lice accompanied by a reduction in the amount of drugs used for lice treatment. However, problems with lice that developed resistance against the pharmaceuticals applied continue to exist.

Paramoebida

Paramoeba perurans – In Canada, infection has been reported in two new management zones and an increased mortality in zones that had earlier been identified as affected. Norway reports an ADG level due to Paramoeba perurans that seems to have stabilized after an increase in previous years. Scotland has found a similar behaviour over time, but it is pointed out that AGD is also a significant contributor to further ‘compound’ gill diseases.

Other parasites Norway reports Ichthyobodo in salmonids (freshwater: I. necator, seawater: I. salmonis), as well as Eubotrium sp.

Conclusions Salmon lice continues to pose great problems to salmon aquaculture, and infection pressure in wild salmonids is significant in regions. Amoebic gill disease (AGD) caused by Paramoeba perurans continues to exist with an increase in Canada and a constant tendency elsewhere. Cleaner fish to control salmon lice are raised in aquaculture systems, and specific disease problems have developed.

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