Occurrence of zoonotic nematodes Pseudoterranova decipiens, Contracaecum osculatum and Anisakis simplex in cod (Gadus morhua) from the Baltic Sea

Veterinary Parasitology 205 (2014) 581–587

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Occurrence of zoonotic nematodes Pseudoterranova decipiens, Contracaecum osculatum and Anisakis simplex in cod (Gadus morhua) from the Baltic Sea Foojan Mehrdana, Qusay Z.M. Bahlool, Jakob Skov, Moonika H. Marana, Diana Sindberg, Mai Mundeling, Bettina C. Overgaard, Rozalia Korbut, Sverri B. Strøm, Per W. Kania, Kurt Buchmann ∗ Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark

a r t i c l e

i n f o

Article history: Received 30 April 2014 Received in revised form 20 August 2014 Accepted 23 August 2014 Keywords: Cod Pseudoterranova Contracaecum Anisakis

a b s t r a c t Baltic cod Gadus morhua (a total of total 224 specimens) captured east of the island of Bornholm in the southern Baltic Sea were subjected to a parasitological investigation between March 2013 and April 2014. Full artificial digestion of fillets from 188 cod and additional investigation of livers from 36 cod were performed. Cod or seal worm Pseudoterranova decipiens was recorded in musculature (prevalences up to 55% and intensities up to 56 worms per fish) associated with a negative correlation between worm intensity and condition factor. Liver worm Contracaecum osculatum (100% prevalence with intensities up to 320 worms per fish) in liver tissue were recorded but only a slight negative correlation between intensity and condition factor was noted. Seals act as final host for both worm species and the increased occurrence during recent years is associated with the increasing grey seal population in the area. Infection with Anisakis simplex (the herring or whale worm) in Baltic cod was found at a low level corresponding to previous studies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The Baltic Sea is a semi-enclosed brackish sea surrounded by Sweden, Finland, Russia, Estonia, Latvia, Lithuania, Poland, Germany and Denmark and it is connected to the North Sea through the Danish straits. The Baltic is populated by a stationary stock of the Atlantic cod (Gadus morhua) which is divided into an eastern and western sub-stock with a mixing zone around the island of

∗ Corresponding author at: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark. Tel.: +45 35332700; fax: +45 35332755. E-mail address: [email protected] (K. Buchmann). http://dx.doi.org/10.1016/j.vetpar.2014.08.027 0304-4017/© 2014 Elsevier B.V. All rights reserved.

Bornholm in the southern Baltic (Bagge et al., 1994) where the main spawning grounds are located as well. Several parasitological investigations during the 20th and 21st centuries have documented occurrence of various parasites in Baltic cod by Kahl (1939), Petrushevski and Shulman (1955), Fagerholm (1982), Myjak et al. (1994), PerdigueroAlonso et al. (2008), Haarder et al. (2014) and Nadolna and Podolska (2014). Zoonotic nematodes of the genus Pseudoterranova were recorded in some cod in the 1930s (Kahl, 1939) but later studies indicated a decreased occurrence as Myjak et al. (1994) detected only one Pseudoterranova decipiens infected Baltic cod out of 3036 investigated. The low infection level of this parasite, which uses seals as final hosts (McClelland, 2002), may be explained by their rare occurrence in the last part of the 20th century. The

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grey seal (Halichoerus grypus) population, however, has experienced a remarkable and successful expansion in the Baltic since the year 2000 (Harding et al., 2007; Buchmann and Kania, 2012; Haarder et al., 2014; Galatius and Olsen, 2014) and Perdiguero-Alonso et al. (2008) discovered a low but increasing seal worm Pseudoterranova infection level in Baltic cod along the Swedish east coast in 2002–2003. This trend was further confirmed by Buchmann and Kania (2012) who reported a prevalence of 2% (compared to 0% prevalence in 1982/83) and a mean intensity of 4 worms per fish caught in the spawning area east of the island Bornholm. The liver worm Contracaecum osculatum, which also uses grey seal as the final host (Skrzypczak et al., 2014), was reported to occur in significantly higher abundances in 2012 in the same area compared to the 1980s (Haarder et al., 2014; Nadolna and Podolska, 2014). Due to these reports and the steadily increasing seal population, which now has reached 40,000 individuals in the Baltic (Galatius and Olsen, 2014), we have conducted a one year parasitological survey of cod caught in the marine area immediately east of the island of Bornholm in order to document the parasitic infections. We have focused on the occurrence of larval nematodes (in musculature and liver) belonging to the family Anisakidae. These larvae include Anisakis simplex (herring or whale worm) and P. decipiens (cod or seal worm) which are considered the two main species that may cause anisakidosis (Torres et al., 2007; Audicana and Kennedy, 2008; Levsen and Berland, 2012; Baird et al., 2014). Also the liver worm C. osculatum may be associated with this disease but merely few reports recording human infections have been published (Schaum and Müller, 1967; Nagasawa, 2012). 2. Materials and methods 2.1. Sampling area and fish The fishery took place in the southwestern Baltic Sea, more specifically located east of the island of Bornholm near the islet Christiansø. Baltic cod were caught by a local fishing vessel (trawler) whereby captured cod represent fish from various areas in the area. A total of 224 cod (188 for fillet inspection and 36 for liver investigation) were immediately purchased when reaching the harbour (Svaneke, Bornholm) and then air-transported to the Laboratory of Aquatic Pathobiology at the University of Copenhagen for investigation. Gutted fish for fillet investigation: A total of 188 cod (collected March 2013–March 2014) was examined for nematodes in the fish musculature. Ungutted fish for liver investigation: A total of 36 Baltic cod (collected February, March and April 2014) was used to investigate the occurrence of liver nematodes. 2.2. Cod investigation 2.2.1. Measurements of cod Gutted weight (g) and total length (cm) were measured for each fish. Liver weight (g) was also recorded for fish examined for liver nematodes.

2.2.2. Fillet recovery Each fish was decapitated and the cod musculature (fillets) was removed and skinned. Both right and left side fillets were divided into four parts in order to detect any preference by worms for microhabitats in the fish: frontal epaxial musculature, frontal hypaxial musculature, caudal epaxial and caudal hypaxial musculature. 2.2.3. Artificial digestion Each fillet part was weighed and incubated in a pepsin solution with magnetic stirring (300 rpm) at 37 ◦ C (Skov et al., 2009). A volume of 10 ml pepsin solution was used per gram fish tissue. Complete digestion of the musculature or liver was completed between 2 and 4 h, whereafter the digest was filtered through a 200 ␮m sieve and the isolated material was examined for the presence of nematodes in a glass Petri dish on dark background. 2.3. Nematode identification Larvae recovered were first examined live in PBS using a Leica light microscope (Leica Microsystems© ) and identified to genus level, according to Möller and Anders (1986), based on morphology and presence/absence of ventriculus, intestinal caecum, ventricular appendage, nerve ring location, excretory pore location, and caudal end morphology. All nematodes were subsequently preserved in 96% ethanol (Kemetyl, Køge, Denmark) for further molecular identification. The middle part of the nematode larva was incubated in 100 ␮l lysis buffer [Tween 20 (0.45%), Proteinase K (60 ␮l ml−1 ), 10 mM Tris and 1 mM EDTA] at 55 ◦ C, 800 rpm in the Eppendorf Thermomixer comfort (Eppendorf AG, Hamburg, Germany). Incubation time varied but continued until complete digestion of nematode parts was achieved (confirmed by microscopy). Proteinase was then inactivated at 95 ◦ C for 10 min whereafter the lysate was used for PCR amplification. 2.4. Molecular identification 2.4.1. PCR amplification PCR was performed in a Biometra T3 thermocycler (Fisher Scientific) using 60 ␮l reaction volumes. The reaction mixtures consisted of 5 ␮l lysate as template, 1 unit of BioTaq DNA polymerase (DNA-Technology), 1 mM dNTP, 1.5 mM MgCl2 and 1 ␮M of the two primers. In order to amplify the ITS region, the primers NC5 (5 -GTA GGT GAA CCT GCG GAA GGA TCA TT-3 ) and NC2 (5 -TTA GTT TCT TTT CCT CCG CT-3 ) were used as forward and reverse primer, respectively (Zhu et al., 2007). The PCR procedure, DNA purification and evaluation were performed as described by Skov et al. (2009). 2.4.2. Gene sequencing Species identification was based on the sequences (GenBank accession numbers in Table 4) encoding the internal transcribed spacer region (18S gene (3 end), ITS-1 gene, 5.8S rRNA gene, ITS-2 gene, and 28S rRNA gene (5 end)). PCR products were purified using the illustraTM GFXTM PCR

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Table 1 Presence of cod worms P. decipiens and herring worm A. simplex in musculature of Baltic cod sampled in 2013/14. Cod size (body length and gutted weight) is shown with prevalence and mean intensity of parasite infections. The seasonal variation was not significant (p > 0.05). Period

Number of cod

Mean total length (cm) ± SD

Range (Min–max)

Mean gutted weight (g) ± SD

Range (Min–max)

Pseudoterranova decipiens Prevalence % Mean intensity (SD)

Anisakis simplex Prevalence % Mean intensity(SD)

14.8% 3.0 (1.2) 23.3% 6.4 (3.4) 45.8% 6.9 (2.7) 29.0% 2.2 (0.6) 28.7% 5.2 (1.4)

4.9% 2.8 (1.2) 10.0% 4.3 (2.4) 15.6% 1.0 (0.0) 5.3% 1.0 (0.0) 8.1% 2.0 (0.9)

Spring

61

46.4 ± 7.9

34–70

741 ± 452

314–2359

Summer

30

55.2 ± 10.2

42–79

1288 ± 791

525–3634

Autumn

59

53.8 ± 8.7

40–81

1207 ± 611

451–3793

Winter

38

49.3 ± 6.6

38–68

939 ± 411

398–2288

50.7 ± 9

34–81

1014 ± 600

314–3793

Total

188

Table 2 Cod examined for presence of liver worms in February to April 2014. Period

Number of cod examined

Mean total length cm (SD)

Mean gutted weight g (SD)

Mean liver weight g (SD)

Mean liver index (SD)

Contracaecum osculatum Prevalence % Mean intensity (SD)

February–April 2014

36

48.2 (6.9)

821.5 (424.3)

46.3 (23.3)

4.11 (1.6)

100% 84.6 (77.2)

DNA and Gel Band Purification Kit (GE Healthcare) and sequenced at Macrogen Inc. (Korea). 2.5. Statistics and calculations Condition factor (expressing the weight in relation to body length) – Fish weight (g) × 102 /fish length (cm)3 (Buchmann, 1986) – was calculated for all fish and liver index (expressing liver weight in relation to body length) – Liver weight (g) × 104 /cod length (cm)3 (Buchmann, 1986) – was calculated for fish examined for liver worms. Prevalence (percentage of the cod population infected), mean intensity (mean number of worms per infected fish) were calculated according to Bush et al. (1997). Differences between mean intensities in different size groups were evaluated by the Mann–Whitney U-test. Spearman rank correlation coefficients (r) were calculated for associations between condition factor and worm burden (intensity) and for associations between liver index and worm burden. Preference by worms for fillet parts were analyzed by a Chisquare test. Microsoft Excel 2007 and Graph Pad Prism 5.04 were used for statistical calculations. A probability level of 5% was used for all analyses. 3. Results The number of fish examined and the infection data with regard to P. decipiens (the number of isolated worms: 283) and A. simplex (total number of worms isolated: 33) in the four seasons are presented in Table 1. Number of cod examined and infection data for C. osculatum (total number of worms isolated: 3046) recovered from in cod liver February to April 2014 are found in Table 2. Nematode larvae occurred in cod muscle tissue throughout the

year (Fig. 1). ITS sequence data for a total of 7 A. simplex, 5 C. osculatum and 37 P. decipiens are presented in Table 3. P. decipiens larvae were referred to genus level based on morphological criteria and ITS sequences obtained from 37 of these worms were 905 bp long. Molecular comparison with available GenBank sequences showed that 35 of these confirmed 100% similarity to P. decipiens sensu stricto (Zhu et al., 2002) (Table 3). Two sequences were heterozygous at the position A221 where both A and T occurred. Overall infection prevalence of cod was 28.7% with a mean intensity of 5.2 cod worms per fish (Table 1). Prevalence of infection increased from spring 2013 (18% infected, mean intensity 3.0) and summer (23.3% infected, mean intensity 6.4) to autumn (45.8% infected, mean intensity 6.9). It decreased slightly in winter 2013/14 (28.7% infected, mean intensity 2.0) (Fig. 1). Infection prevalence and intensity was significantly higher in larger cod (>50 cm) compared to smaller cod. Thus, larger cod in the autumn sample had a prevalence of 56.4% and intensity could reach 56 worms per fish. Nematode larvae isolated from livers of Baltic cod corresponded by morphometric evaluation to the genus Contracaecum. ITS sequences (from a subsample of 5 worms) were 964 bp long (Table 3) and showed 100% similarity to C. osculatum sensu stricto from Baltic cod reported by Zhu et al. (2000) and Haarder et al. (2014). Infection prevalence was 100% and overall mean intensity 84.6 worms per fish. The infection intensity ranged from 2 to 320 nematode larvae per fish (Table 2). C. osculatum larvae were found in cod livers but in two cases (April 2013) liver remnants with worm larvae were found in the gutted fish associated with the fillet. Mean condition factors of cod (based on gutted weight to reflect musculature status) were 0.68 in spring, 0.70 in summer, 0.74 in autumn and 0.69 in winter samples. Correlation analyses showed a significant negative correlation

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Fig. 1. Seasonal occurrence of cod worm P. decipiens in Baltic cod sampled in 2013/14. Prevalence (percentage of fish population infected) for smaller cod (<50 cm total body length), larger cod (>50 cm total body length), and all cod combined is shown for spring, summer, autumn and winter samples. Table 3 Number of worms processed and sequenced. Comparison of sequences revealed in this study with sequences from GenBank. Species A. simplex s.s C. osculatum s.s. P. decipiens s.s.

No. of fisha

No. of worms

5 5

7b 5

17

37c

GenBank acc. no. This study

Identity % GenBank acc. no.

Reference

KM273043 to KM273049 KM273050 to M273051 and KM491171 to KM491173 KM273052 to KM273086

100% JN968760 to JN968769d 100% AJ250412 and AJ25042e

Kuhn et al. (2011) Zhu et al. (2000)

100%A413968 and A413969ee

Zhu et al. (2002)

a

Two fish were infected with both A. simplex and P. decipiens. b One A simplex had two heterozygous nucleotides (C174 →Y and T306 →Y). c In two P. decipiens originating from the same fish one heterozygous nucleotide was present (A221 →W), however 7 nematodes from the same fish were 100% identical to the one described by Zhu et al. (2002). d All sequences contain both ITS1 and ITS2. e The first sequence represents the ITS1, and the second represents ITS2.

(r: −0.67) (p < 0.05) between P. decipiens infection intensity and condition factor for fish between 500 and 1000 g and for cod with body lengths between 41 and 49 cm (r: −0.63) (p < 0.05). Correlations were not significant in larger fish. With regard to C. osculatum and fish parameter analyses only cod with body lengths from 41 to 49 cm represented a homogenous sample and a weak negative correlation (r: −0.13) between liver worm count and condition factor was found. No correlation (r: 0.032) between liver index and C. osculatum infection was seen. The small livers with high worm burdens were often haemorrhagic and numerous worms were macroscopically visible. P. decipiens larvae showed a clear and significant preference for the anterior parts of the fillets and the hypaxial parts contained most of the larvae (2 = 51.4, df = 1, p < 0.05) (Table 4). Thus, the belly flaps contained 58.4% of all worms (with a majority in the left belly flap) whereas the dorsal frontal musculature contained 21.3%. However, a considerable part (20.3%) of the worm population was still found in the caudal fillet parts (Table 4). The preference for the frontal hypaxial muscle tissue increased with rising worm burden (Fig. 2). No clear trend for a seasonal variation was found with regard to site preference. Anisakis larvae were identified to genus level by morphometric evaluation and a subsample of these was subjected to molecular characterization. The seven ITS

sequences obtained were 953 bp long (Table 3). They showed 100% similarity to A. simplex sensu stricto corresponding to GenBank accession no. GQ472924 reported from Danish wild marine fishes (Skov et al., 2009) and JN968760 to JN968769 (Kuhn et al., 2011). One sequence was heterozygous at positions C174 and T306 where both C and T occurred. A. simplex infections occurred at low prevalences of 4–15% and mean intensities between 1 and 4 parasites per fish. 4. Discussion The present survey has confirmed that the infection level with regard to P. decipiens and C. osculatum in Baltic cod captured east of the island of Bornholm has increased markedly during the latest decade. Several previous investigations on Baltic cod by Möller (1975), Fagerholm (1982), Thulin et al. (1989) and Myjak et al. (1994) have shown that P. decipiens has been absent or extremely rare during the last part of the 20th century. P. decipiens was not detectable in Baltic cod during the 1980s and in 2011 a prevalence of 2% and a maximum intensity of 4 larvae per fish were found (Buchmann and Kania, 2012). In 2013/14 we recorded a prevalence of infection of more than 50% in larger cod (>50 cm) and up to 20% in smaller cod (<50 cm). Infection intensity increased correspondingly with the highest

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Table 4 Location of P. decipiens larvae (number and percentage of all worms) in the different parts of the cod musculature from 188 Baltic cod. Data shown for the different seasons 2013/14.

Spring Summer Autumn Winter Total

L1

L2

L3

L4

R1

R2

R3

R4

3 (11.1%) 7 (15.5%) 19 (10.3%) 5 (20.8%) 34 (12.1%)

9 (33.4%) 19 (42.2%) 78 (42.2%) 3 (12.5%) 109 (38.8%)*

0 (0%) 2 (4.5%) 5 (2.7%) 5 (20.8%) 12 (4.3%)

3 (11.1%) 1 (2.2%) 12 (6.5%) 3 (12.5%) 19 (6.8%)

2 (7.4%) 4 (8.9%) 17 (9.2%) 3 (12.5%) 26 (9.2%)

6 (22.2%) 10 (22.2%) 36 (19.4%) 3 (12.5%) 55 (19.6%)*

3 (11.1%) 0 (0%) 6 (3.2%) 0 (0%) 9 (3.2%)

1 (3.7%) 2 (4.5%) 12 (6.5%) 2 (8.4%) 17 (6.0%)

Musculature designation: L = left side of the fish, R = right side of the fish, 1 = Frontal epaxial, 2 = Frontal hypaxial, 3 = Caudal epaxial, 4 = Caudal hypaxial. * p < 0.05.

Fig. 2. Location of P. decipiens larvae in different parts of the cod musculature. The columns show the percentage of all worms recovered from fish with different infection intensities.

occurrence of 56 P. decipiens nematode larvae in fillets from one fish. The nematode larvae were detected in all parts of the musculature but a preference for the frontal hypaxial part (belly flaps) was seen. This trend was especially evident in fish carrying more than five worms corresponding to investigations on cod from Canadian waters (McClelland, 2002) and the North Atlantic (Hafsteinsson and Rizvi, 1987). The pyloric caeca and the ventricle are oriented towards the left side of the cod and the dominance of worms in the left belly flap may suggest that worm larvae, in their search for the microhabitat in the fish host, mostly penetrate these body parts when migrating towards the muscle tissues. C. osculatum infection has also risen dramatically during the same period. Recently Nadolna and Podolska (2014) reported up to 22% prevalence in Baltic cod livers and Haarder et al. (2014) documented an increase of C. osculatum infection level from a low level in the 1980s (prevalence 22%, mean intensity 4.2) to the year 2012 where the prevalence was 55.1% and the mean intensity was 20.2 parasites per fish. Previous studies on Baltic cod by Fagerholm (1982) and Valtonen et al. (1988) showed maximum prevalences of 33.0% and 15.4%, respectively. However, the present study on liver worms performed during the first months of 2014 showed 100% prevalence with a mean intensity of 84 (ranging from 2 to 320) worms per fish. The impact of these worms on the health of the fish hosts may be associated with several physiological reactions. Field studies on parasitic impact on fish and liver conditions may only partly reflect a physiological association because knowledge of the previous life

of the fish (and especially their food intake) is unknown (Buchmann and Børresen, 1988). However, the lowered condition factor of infected cod in this study may suggest a pathogenic effect of these worms. The infection with nematode larvae in experimental fish elicits a systemic inflammatory reaction (Haarder et al., 2013) which is likely to affect food uptake and growth. A low number of P. decipiens larvae in the cod muscles may probably affect fish welfare to a minor extent but it must be expected that the impact on the host increases with a raising worm burden. Some of the highly parasitized cod were clearly cachectic with shrunken haemorrhagic livers. Previous studies have shown that liver indices of Baltic cod are very sensitive to even non-migrating and non-pathogenic intestinal worms (Echinorhynchus gadi acanthocephalans) (Buchmann, 1986) but with no influence on muscle tissues. Russian studies from the eastern Baltic conducted during the 1940s and 1950s (where seals were relatively abundant in the eastern part of the Baltic) showed similar negative associations between Contracaecum worm burdens and cod condition (Petrushevski and Shulman, 1955) and it should be investigated experimentally to which extent C. osculatum infections may affect both the individual cod and the population. Nematode larvae in the fish tissue produce a series of pharmacologically active compounds. Immune depressing components excreted by A. simplex third stage larvae were studied by Bahlool et al. (2013) and volatile ketones released by P. decipiens larvae may inactivate the fish musculature (Ackman and Gjelstad, 1975) which can explain the decreased swimming ability of infected fish (Sprengel and Lüchtenberg, 1991; Rowling

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et al., 1998). From an evolutionary viewpoint this would optimize the life cycle because parasitized cod lose weight and become more vulnerable to predation by the final hosts. It cannot be excluded that C. osculatum larvae produce similar substances and it is therefore noteworthy that we found 100% prevalence and extremely high infection intensities in cod with significantly lower condition factors. Thus, we found mean condition factors of 0.68 in spring, 0.70 in summer, 0.74 in autumn and 0.69 in winter which are markedly lower compared to similar parameters of sized cod obtained from the same geographic region in the 1980s (mean condition factors ranging from 0.82 to 0.95) (Buchmann, 1986). As described by Mattiucci and Nascetti (2008) P. decipiens sensu lato is a species complex comprising P. decipiens sensu stricto (type B), P. krabbei (type A), P. bulbosa (type C), P. azarasi (type D), P. decipiens (type E) and P. cattani. In the present work ITS sequences were similar to P. decipiens sensu stricto (type B). Likewise, the C. osculatum complex comprises five members (types A–E) where C. osculatum sensu stricto corresponds to type C. The present study on C. osculatum ITS sequences showed 100% similarity to type C. The genus Anisakis comprises six species of which A. simplex sensu stricto is the only species recovered from fish in the Baltic and ITS sequences of Anisakis isolates from Baltic cod in the present investigation showed 100% similarity with this. A. simplex, P. decipiens and C. osculatum have indirect and complex life cycles, which involve various definitive and transport hosts. Definitive hosts for adult Anisakis spp. are usually cetaceans (whales and dolphins) (Audicana and Kennedy, 2008), while adult P. decipiens and C. osculatum reach maturity in pinnipeds (e.g., grey seals and common seals) (Valtonen et al., 1988; Lunneryd, 1991; Skrzypczak et al., 2014). Although the salinity in the Baltic Sea is low around the island of Bornholm, it is still adequate for supporting the life cycle of P. decipiens. Thus, it has been demonstrated that even these low salinities allow hatching of eggs (Measures, 1996). The recovered anisakid worms detected in this study are all able to cause anisakidosis in humans. Most published cases are associated with Anisakis spp. infection (Audicana and Kennedy, 2008; Baird et al., 2014). This parasite is widely spread in fish populations (Kuhn et al., 2013) but in this investigation it was recovered in only few cod and at a low level and no evidence for an increase during the latest decades is indicated. This corresponds to data from the 1970s presented by Grabda (1976) showing low A. simplex infections of Baltic cod which was explained by lack of life cycle completion in the Baltic. These fish acquire the worm only by consumption of infected and migrating spring spawning herring migrating in from the North Sea (Grabda, 1974). In contrast, our survey detected a remarkable increase of both P. decipiens and C. osculatum infections of cod in the Baltic which corresponds perfectly to the increasing grey seal population in the area. Pseudoterranova spp. larvae may infect humans following ingestion of raw infected fish meat (Skirnisson, 2006; Torres et al., 2007). Even light infections may cause diarrhoea, stomach pain, tickling feelings, vomiting and coughing from the gastro-intestinal

system (Pinel et al., 1996). Likewise, C. osculatum may – although reported more rarely – cause a similar disease in man (Schaum and Müller, 1967; Nagasawa, 2012). The present investigation suggests that the infections represent a problem for the Baltic cod stock which at present is at a critically low level (Köster et al., 2009). Canadian studies have pointed to elevated cod mortality due to grey seal abundance (Chouinard et al., 2005) and Swain and Chouinard (2008) predicted an accelerated reduction of the cod population through seal predation. Based on theoretical considerations and modelling on the predation of seals on local cod populations Mackenzie et al. (2011) argued that increasing grey seal populations – under certain favourable conditions – would not counteract recovery of the Baltic cod stock. However, even though seal predation alone may affect fish stocks we suggest that the pathogenic effects of nematode larvae on the Baltic cod must be incorporated into future modelling but it should be framed that this issue should be experimentally investigated under controlled laboratory conditions. Thereby precise impact parameters would be available for future modelling. Acknowledgements This study was supported by EFF (European Fisheries Fund) and the Danish Ministry of Food, Agriculture and Fisheries grant 33010-13-k-0262. References Ackman, R.G., Gjelstad, R.T., 1975. Gas chromatographic resolution of isomeric pentanols and pentanones in the identification of volatile alcohols and ketones in the codworm Terranova decipiens. Anal. Biochem. 67, 684–687. Audicana, M.T., Kennedy, M.W., 2008. Anisakis simplex: from obscure infectious worms to inducer of immune hypersensitivity. Clin. Microbiol. Rev. 21, 20–25. Bagge, O., Thurow, F., Steffensen, E., Bay, J., 1994. The Baltic cod. Dana 10, 1–28. Bahlool, Q.Z.M., Skovgaard, A., Kania, P.W., Buchmann, K., 2013. Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 35, 734–739. Baird, F.J., Gasser, R.B., Jabar, A., Lopata, A.L., 2014. Food borne anisakiasis and allergy. Mol. Cell. Probes 28, 167–174. Buchmann, K., 1986. On the infection of Baltic cod (Gadus morhua L.) by the acanthocephalan Echinorhynchus gadi (Zoega) Müller. Nord. Vet. Med. 38, 308–314. Buchmann, K., Børresen, T., 1988. The effect of different food types and rations on the liver and muscle of cod (Gadus morhua L.). Acta Vet. Scand. 29, 57–59. Buchmann, K., Kania, P.W., 2012. Emerging Pseudoterranova decipiens (Krabbe, 1878) problems in Baltic cod, Gadus morhua L., associated with grey seal colonization of spawning grounds. J. Fish Dis. 35, 861–866. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, J.W., 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83, 575–583. Chouinard, G.A., Swain, D.P., Hammill, M.O., Poirier, G.A., 2005. Covariation between grey seal (Halichoerus grypus) abundance and natural mortality of cod (Gadus morhua) in the southern Gulf of St. Lawrence. Can. J. Fish. Aquat. Sci. 62, 1991–2000. Fagerholm, H.P., 1982. Parasites of fish in Finland. VI. Nematodes. Acta Acad. Abo. B. 40, 5–128. Galatius, A., Olsen, M.T., 2014. Seals in the Baltic. Nat. Bornholm (Natur på Bornholm). 12, 68–74 (in Danish). Grabda, J., 1974. The dynamics of the nematode larvae Anisakis simplex (Rud.) invasion in the south-western Baltic herring (Clupea harengus L.). Acta Ichthyol. Piscat. 4, 3–21.

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