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The surveillance and control programme for bonamiosis and marteiliosis in European flat oysters, Ostrea edulis, and blue mussels, Mytilus sp. in Norway in 2020

Sammendrag

The surveillance programme is carried out by the Institute of Marine Research according to a contract with the Norwegian Food Safety Authority. Samples were collected from one oyster farm, four wild oyster populations, one cultivated mussel population and two wild mussel beds. Samples were collected in April/May and in October, in order to be able to detect Bonamia sp. and Marteilia sp. during the periods when the potential prevalence is at the highest. No abnormal mortalities were observed during the surveillance. Bonamia ostreae / B. exitiosa were not detected. There have been several reports on mortality or “disappearance” of mussels along the Norwegian coast. The reason(s) for the mortalities have not been determined. However, The parasite Marteilia sp. was detected for the first time in mussela, Mytilus edulis, collected at Bømlo, western Norway in 2016 and Tysnes in 2019. In 2020, Marteilia was detected in wild populations in Sunnfjord and in an open water mussel farm nearby. All detections were M. pararefringens . The work will be continued in 2021, linked to research on the distribution of M. pararefringens in wild mussels. We propose a revision of the surveillance programme combined with the establishment of a new model for health control in mollusk farms, application for disease free status for Norwegian flat oysters and a categorization of zones.

1 Introduction

The surveillance programme for bonamiosis and marteiliosis in European flat oysters, Ostrea edulis, and blue mussels, Mytilus sp.

The surveillance programme for bonamiosis and marteiliosis in European flat oysters, Ostrea edulis, and blue mussels, Mytilus sp. is carried out by the Institute of Marine Research according to a contract with the Norwegian Food Safety Authority. The over-all aim is to gain knowledge on the health situation of farmed Norwegian oysters and mussels. There is however a connection between farmed and wild populations, due to the geographically widespread and extensive nature of the bivalve industry. The work therefore requires a tight link between the Surveillance programme and the on-going research projects, as well as the building of a time-series of data on wild stocks. After the detection of Marteilia sp. in 2016, we have increased the effort to study this case – including distribution and parasite life cycle. This report gives a brief overview of the present situation, results from 2020 and the prospects for the work in 2021.

 

Norwegian flat oyster, Ostrea edulis, are used in re-stocking projects

There is a growing interest in the re-establishment of wild oyster beds in Northern Europe, and initiatives united in the NORA project (https://noraeurope.eu/) now work to develop a protocol on how to select, treat and seed oysters on some of the historical oyster beds. We are collaborating with this project, with contributions to their biosecurity manual and linking the selection of potential oyster source population to the on-growing health monitoring and research. 

Both the oyster farming industry and re-stocking projects focus on where to find naïve flat oyster populations that are free from Bonamia spp, as well as other pathogens that may affect the populations. In the present situation – and after the re-occurrence of Bonamia ostreae in Limfjorden, Denmark, in 2014 – safe sources of oysters can probably only be found in Sweden and Norway. Norwegian populations of European flat oysters have been monitored since 1989 and are considered free from notifiable diseases (Mortensen et al. 2016; 2020). Oysters from Hafrsfjord in Rogaland are being used in re-stocking trials in the North Sea. Oysters from this area are included in the surveillance program.

 

Marteilia pararefringens sp. nov. in blue mussels, Mytilus edulis in Norway

The blue mussel, Mytilus sp, populations are changing, and there are numerous reports on an un-explained “disappearance” or mortalities from many Nordic areas. It is not known to which extent diseases play a role in these changes. The wild mussel beds are not monitored on a regular basis, and there is a limited control of mussel farms, using traditional longline cultivation based on the collection of wild seed.

Marteilia pararefringens has been found in blue mussels, Mytilus edulis, in two traditional heliothermic marine oyster lagoons in western Norway. These lagoons have much higher water temperatures than the ambient fjord water during the summer season, and have been used for breeding oysters since the 1880’s. The semi-closed system of the oyster ponds represents a unique opportunity to study the life cycle and parasite progression of M. pararefringens in M. edulis. We use the pond at Aga, on the island of Bømlo, Western Norway, as a study site to elucidate the infection and life cycle of M. pararefringens in mussels.

M. pararefringens have never been detected during our health surveillance of mussels from open water farms or wild mussel beds along the coast. There is however one report of Marteilia sp. was observed in blue mussels, Mytilus edulis used for environmental monitoring around 2010 (Aarab et al. 2011), but this finding was not notified. In 2020, the Norwegian Veterinary Institute reported Marteilia-infected mussels from a health control in an open-water farm. Our sampling in 2020 was therefore extended to include mussels from wild mussel populations in the vicinity of this farm.

2 Material and methods

The surveillance was performed according to EU directive 2006/88 and Decision 2015/1554.

Sampling periods were defined according to the periods when the prevalence of Bonamia ostreae and Marteilia sp. (sporulating stage) are highest in the northernmost areas where they have been detected (Engelsma et al. 2010; A. Alfjorden pers. comm). The selected sampling sites are shown in Figure 1 and listed in Table 1.

Usually, the surveillance includes an on-site survey, as the state of the population (density, reproduction, signs of mortality) are considered important meta-data. At Hafrsfjord, Haugevågen, Innerøyen, Flekke and Ostrevig oysters and mussels were collected by skin-diving or wading and transported to the Institute of Marine Research (IMR) in Bergen. At Strømmen the mussels were collected by personnel from the Food Safety Authority. At Vågstranda, mussels and oysters were collected by the local landowner and sent to IMR Bergen by over-night mail (Table 1). At Langesand, the site was inspected by skin diving. Samples were not collected, as this site is no longer included in the surveillance programme.

All oysters and mussels were processed at the IMR laboratory in Bergen, according to standard methodology, and under ISO 17025 QA. The samples with 100 specimens were split in two. Half were analyzed using histology, the other half with Polymerase Chain Reaction analysis (PCR). Samples from Flekke, Haugland (Table 1) were analyzed in parallel. Briefly; Histology was performed using dorso-ventral cross sections, fixed in Davidson’s fixative, embedded in paraffin, sectioned at 3µm, stained with Hematoxylin Eosin Saffron (HES), mounted with a cover slip and observed at 100 to 1000 x magnification. Samples for PCR were fixed in ethanol. DNA was extracted from ethanol fixed digestive gland tissue. Marteilia detection and typing was done with by PCR as described by Le Roux et al (2001). Positive samples were sequenced.

 

Figure 1. Sampling sites of flat oysters, Ostrea edulis (yellow) and blue mussels, Mytilus sp. (blue) in the surveillance programme for Bonamia sp. and Marteilia sp. See also Table 1. The flat oyster population at Langesand (orange) was inspected but not sampled in 2020.

Table 1. Sampling and surveillance of flat oysters (Ostrea edulis) and mussels (Mytilus sp.) in 2020.

Sampling site

Oysters

Mussels

 

 

 

  Spring-summer

Autumn

Spring

Autumn

 

Hafrsfjord, Rogaland

100

 

 

 

52 obs by microscopy, 48 by PCR

Haugevågen, Rog.

99

 

 

 

51 by microscopy, 48 by PCR

Innerøy, Vestland

30

 

 

 

PCR only

Ostrevig, Rogal.

 

30

 

 

Microscopy only

Vågstranda

 

18

 

22

Microscopy only

 

 

 

 

 

 

Flekke, river

 

 

 

50

30 obs by histology

Flekke, Haugland

 

 

 

50

30 obs by histology, all by PCR

Strømmen, Rissa, Trøndelag

 

 

 

100

52 obs by microscopy, 48 by PCR

 

 

 

 

 

 

 

3 Results

Bonamia spp. was not observed in any sample in 2020. Marteilia sp. was not observed in oysters but M. pararefringens were present in mussels from two sites in Flekke. Results from the sites listed in Table 1 are briefly described below.

Examination of flat oysters

Langesand (58.539225, 8.937646)

Langesand has a large population of flat oysters. The site was previously included in the surveillance programme and subjected to a targeted Bonamia survey (see previous reports).

The site has an apparent stable sub-population of flat oysters growing from 3-6 m depth mostly protected from harvesting and climatic events. From 3m to surface the population has variable recruitment success and survival due to predation, harvesting and ice conditions. Both sub-populations contain specimens of several year-classes. There was no sign of elevated mortality. Oysters were not sampled for further examination in 2020.

Hafrsfjord (58.926864, 5.6436861)

Hafrsfjord is located west of the city of Stavanger in Rogaland county, Western Norway. The fjord has a narrow inlet and appears as a semi-closed marine fjord-system. Hafrsfjord has a large population of flat oysters. These oysters have been harvested and used in re-stocking projects in the North Sea. Six sites in Hafrsfjord were visited in June 2020. At five sites, low to moderate densities and several year-classes of flat oysters were observed. Oysters observed by microscopy appeared normal, had a good condition and variable gonad status. Some shells were infested by Polydora sp. Haemic neoplasia was observed in two oysters. 

Haugevågen, Rogaland (59.3665555, 5.2489903)

Haugevågen is a sheltered lagoon on the north-western side of the island of Karmøy, in Rogaland. The lagoon and inlet have a dense population of flat oysters, down to 4 – 5 m depth. Oyster farmers have applied for a permission to use this site to collect flat oyster spat, and a visit was performed to gain information and data on the oyster population there. The population is dominated by a few strong year-classes. There are patches of mussels in some areas along the shoreline. A few Pacific oysters were found and removed. The site was visited in June. Oysters had a good condition, and most were sexually mature. Some were in a state of spawning. Rickettsia-like organisms (RLOs) were observed in the digestive gland of six oysters. Haemic neoplasia was observed in three oysters.

Innerøy (60.1508985, 5.4122928)

Innerøyen is presently the only site in Norway with a traditional production of flat oyster spat.

Oysters were collected from suspended ropes in the mid part of the lagoon in July. The condition index of the oysters was low but variable. All samples were Bonamia negative by PCR analysis.

Ostrevig (58.3271717, 6.2350926)

Ostrevig is a traditional oyster lagoon that was abandoned in the 1990’s. The lagoon has no direct contact with the sea. Seawater splashes over the rocks on high tide and windy weather and fills the lagoon. There is an isolated population of flat oysters in the lagoon. Oysters were sampled for genetical studies and histology. The oysters had a low condition index but normal digestive epithelia, and passive / empty gonadal tissues. Early-stage haemic neoplasia was observed in two oysters. In one oyster, we observed heavy secretion of eosinophilic granules in the digestive tissues, indicating a lysosomal destabilization, probably as an effect of an external stressor.  

Vågstranda (62.6040173, 7.3325886)

Vågstrandapollen (Vågen) is an approximately 1,8-kilometer-long estuary connected to the Romsdal fjord by a shallow sill with a tidal current. The estuary is sheltered and has been used as an oyster lagoon from the 1930’s until the 1990’s. The site is abandoned, but there is a remaining population of flat oysters in the estuary. Oysters were sampled during winter. Sections from 22 oysters were observed by microscopy. All oysters appeared healthy. Gonads were empty. Some damage was observed on the gills, presumably due to low temperatures before shipment. RLOs were observed in one oyster.

Examination of Mussels

Flekke (61.3134761, 5.3467415)

After the detection of Marteilia sp. in mussels in an open water longline farm, we surveyed two local mussel populations in the Flekke fjord:

1. There is a mussel population in the Flekke river estuary, in the river mouth and along hard bottom areas. In the river mouth we found only large mussels in a low density on the rocks, and a mussel bed on shallow water along the west side, with a high number of empty/dead shells. No oysters or oyster shells were observed. Thirty mussels were sampled. Twenty-two were infected by Marteilia sp.

2. Mussels were sampled at Haugland, in a shallow, sheltered area between a cluster of small islands. There were patches of mussels on ropes, rocks and hard bottom areas. Here were numerous empty shells, and several year classes of mussels. No oysters were observed. Thirty mussels were sampled for histology. Mussels had empty gonads and low condition. Four were infected by Marteilia sp. Digestive gland samples from 50 mussels were analyzed by PCR. Eleven were Marteilia positive. There was correspondence between microscopy and PCR.

A Restriction Fragment Length Polymorphism (RFLP) analyse (Le Roux et al. 2001) was performed on positive samples and showed the presence of M. refringens Type M / M. pararefringens. Sequencing of samples from the RFLP analyse verified the Type M, based on the ITS-sequence. Blast results from sequences from this region give the highest score for M. refringens / M. pararefringens and sequences have the same signature sequence as M. pararefringens (DQ426550) (see Kerr et al. 2018).

Strømmen, Rissa, Trøndelag (63.5776946, 9.940414)

Mussels from Rissastrømmen come from a wide tidal current used as intermediate grow-out and harvest of farmed mussels. Fifty-two mussels were examined by microscopy. Mussels appeared in good but variable condition. Haemic neoplasia was observed in two mussels. RLOs were observed in two mussels. No other abnormal finding was recorded.

Vågstranda (62.6040173, 7.3325886)

Twenty-two mussels were collected at the same site as the oysters (see above). Mussels had a normal condition. Eleven exhibited microcolonies, presumably Coccomyca parasitica in connective tissues and gonads. Trematode cysts were observed in connective tissues of 3 mussels. Marteilia sp. was not observed.

4 Discussion and conclusions

The health status of flat oysters

Norwegian flat oysters, O. edulis, appear free from Bonamia spp. Healthy flat oysters – which in the present situation particularly means free from Bonamia spp. - is a valuable resource, both for European oyster growers, as well as programs aiming at re-stocking the disappearing, historical wild oyster beds. It is an important task to monitor Norwegian stocks and disseminate the information on their health status in order to obtain a consensus on how to protect and care for this resource. The institute of Marine Research has started a monitoring of Norwegian flat oyster populations and aims at using the data gained in a Nordic and European context. The monitoring of stocks is linked to the national health surveillance, and through the contact with European scientists to both genetic studies (Margen project) and re-stocking programmes (https://noraeurope.eu/). Flat oysters from Hafrsfjord in Rogaland have already been translocated and seeded on hard bottom sites off the coast of The Netherlands.

The wild flat oyster populations examined in 2020 appear healthy, with a normal reproductive cycle pattern. Haemic neoplasia and the presence of intracellular Rickettsia-like colonies were occasionally observed, but at low prevalence and intensity. This is a common observation, and not considered a problem, although the neoplasia may develop into disease problems and potentially induce winter mortalities of affected flat oysters as their nutrient uptake and immune defense are knocked out (Mortensen et al. 2013).

Examination of mussels

Marteilia pararefringens has been found in blue mussels, Mytilus edulis, in two traditional heliothermic marine oyster lagoons (Aga and Espevik) in western Norway. These lagoons have much higher water temperatures than the ambient fjord water during the summer season, and have been used for breeding oysters since the 1880’s. The semi-closed system of the oyster ponds represents a unique opportunity to study the life cycle and parasite progression of M. pararefringens in M. edulis.

M. pararefringens have never been detected during our health surveillance of mussels from open water farms or wild mussel beds along the coast. There is however one report of Marteilia sp. was observed in blue mussels, Mytilus edulis used for environmental monitoring around 2010 (Aarab et al. 2011), but this finding was not notified. In 2020, the Norwegian Veterinary Institute reported Marteilia-infected mussels from an open-water farm. Sampling of mussels from two near-by wild beds revealed a high prevalence of M. pararefringens and heavily infected mussels. These findings indicate that M. pararefringens is not restricted to the heliothermic lagoons. This finding represents a new situation, where the parasite occurs outside a closed lagoon environment. In the continuation of this study, we will look more closely at the traditional oyster lagoons, mussels in closed, sheltered areas and try to understand the distribution and possible movements of M. pararefringens in the mussel populations. In order to optimize the sampling regime in the surveillance programme, we will collaborate with all active mussel producers in order to map the farms, spat collection areas and translocations and initiate sampling in the vicinity of a number of lagoons.

The Norwegian mussel production is concentrated in Trøndelag and Nordland, where two dispatch centers receive mussels from several producers in a large geographic area. Although two selected sites are included in the surveillance programme, we do not have a proper overview of the production in this area. The dispatch centers will be followed up and their sources of mussels mapped. The situation illustrates the need for combining the surveillance with the health control of shellfish farms.

Based on our findings, M. pararefringens does not seem to infect flat oysters. It is however important to be sure that oysters do act as vectors. We will thus continue to combine surveillance and research activity in order to obtain as much data as possible, also from oysters at the infected site, and from more sites that have been in contact with the former network of oyster producers.

Conclusions and recommendations

Revision of the programme. Selection of sites and increased sample size

The surveillance will be continued according to the plan discussed with the Norwegian Food Safety Authority, and with the aim of obtaining a time series of data on the health status of flat oysters and mussels along the Norwegian coast:

The surveillance programme will be focused on mussel and oyster farms and/or populations that are commercially harvested for on-growth, transport to the markets or used in re-stocking programmes.

The detection of Marteilia pararefringens in mussels in new areas indicate that this parasite is more widely distributed than previously known. We consider the sample size of 30 specimens (or 20 as applied in the Veterinary health control of commercial farms) as too low. This sample size should only be used if the health status is known, after a previous two-year surveillance based on 150 specimens every 6 months, based on the principles laid down in EU Decision 2015/1554. To improve the screening and ensure a rapid detection, we started using a method based on 100 specimens per sampling, whereas 50 was analyzed by PCR and 50 by histology. We will increase the sample size to 150 in 2021 to ensure full compliance with the new EU regulations which are implemented in 2021.

The health surveillance is linked to monitoring of stocks, oyster genetic studies and European oyster projects, strengthening the scientific basis for a strong and adequate management of the few remaining, healthy flat oyster populations in Europe. 

Knowledge on Marteilia pararefringens – progress of the work

Flat oysters do not appear susceptible to M. pararefringens. Future scientific studies will focus on understanding the infection and life cycle of M. pararefringens in mussels, as a background for a risk assessment and an epidemiological study of the spreading of this parasite in North European mussel populations.

Categorization of mussel and flat oyster production areas in Norway

The samples from Trøndelag collected between 2018 and 2020, and the present mussel sample from Vågstranda represent the first study of the health status of mussels north of Bergen. Due to the lack of data on the health status of mussels from most of the Norwegian coast, the categorization should be discussed and potentially revised.

Absence of Bonamia spp. in Norway

We consider Norwegian populations of flat oysters as free from Bonamia spp. This makes the Norwegian flat oysters a valuable resource, for the oyster producers as well as initiatives of re-stocking the vanishing populations of native European oyster beds.

 

Acknowledgements

Thanks to Ingrid U. Fiksdal for technical assistance, to Øivind Strand, Tore Strohmeier and Antonio Aguera Garcia for help with sampling in the field, to David Bass for the collaboration on the M. pararefringens life cycle.

 

5 References

Aarab, N., Godal, B.F., Bechmann, R.K. (2011). Seasonal variation of histopathological and histochemical markers of PAH exposure in blue mussel (Mytilus edulis L.). Marine Environmental Research 71: 213-217.

Engelsma MY, Kerhoff S, Roozenburg I, Haenen OLM, van Gool A, Sistermans W, Wijnhoven S, Hummel H (2010). Epidemiology of Bonamia ostreae infecting European flat oysters Ostrea edulis from Lake Grevelingen, The Netherlands. Marine Ecology Progress Series 409: 131 – 142.

EU. 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. Official Journal of the European Union L 328/14.

EU. Decision 2015/1554 of 11 September 2015, laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods. Official Journal of the European Union L 247/1.

Kerr R, Ward GM, Stentiford GD, Alfjorden A, Mortensen S, Bignell JP, Feist SW, Villalba A, Carballal MJ, Cao A, Arzul I, Ryder D, Bass D (2018). Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions. Parasitology 1–10. https://doi.org/10.1017/ S003118201800063X

Le Roux F, Lorenzo G, Peyret P, Audemard C, Figueras A, Vivares C, Gouy M, Berthe F (2001). Molecular evidence for the existence of two species of Marteilia in Europe. Journal of Eukaryote Microbiology 48: 449-454.

Mortensen S, Skår CK, Harkestad LS (2013). Hemisk neoplasi hos norsk flatøsters, Ostrea edulis. Norsk Veterinærtidsskrift 125 (7): 438 -442.

Mortensen S, Sælemyr L, Skår CK, Bodvin T, Jelmert A (2016). Health surveillance of the flat oyster populations in Aust-Agder County, southern Norway in the period 2009 – 2015. Rapport fra havforskningen nr 11, 2016, 11s.

Mortensen S, Skår C, Sælemyr L (2020). Summarizing the screening for Bonamia ostreae in Norwegian populations of flat oysters, Ostrea edulis. Rapport fra Havforskningen no 24, 2020. ISSN:1893-4536,  https://www.hi.no/hi/nettrapporter/rapport-fra-havforskningen-en-2020-24