Parasitic nematodes
Physicochemical properties of nematodes present in Atlantic cod (Gadus morhua)
Snorri Karl Birgisson 2016
Supervisors: Sigurjón Arason & Magnea G. Karlsdóttir
Thesis for the degree of Master of Science in food science
Hringormar í sjávarútvegi Efna- og eðlisfræðilegir eiginleikar hringorma í Atlantshafsþorski
Snorri Karl Birgisson
Leiðbeinendur: Sigurjón Arason og Magnea G. Karlsdóttir
Einingar 90
Febrúar 2017
Ritgerð til meistaragráðu í matvælafræði
Ritgerð þessi er til meistaragráðu í matvælafræði og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa.
© Snorri Karl Birgisson 2017
Prentun: Prentsmiðja xxx
Reykjavík, Ísland 2017
Ágrip
Megin markmið verkefnisins var að rannsaka eðlis- og efnaeiginleika hringorma sem finnast í Atlantshafs þorski. Hringormar voru flokkaðir eftir lengd og staðsetningu í þorskflaki og rannsakaðir með myndbandsupptökum í fiskvinnslu. Efnaeiginleikar hringorma voru rannsakaðir með því að mæla efnasamsetningu þeirra. Ásamt efnasamsetningu voru ýmis stein- og snefilefni mæld. Að auki var amínósýrusamsetning hringorma mæld. Til að skoða heildarmyndina voru efnisþættir bornir saman við efnainnihald þorskflakahluta. Eðliseiginleikar hringorma voru rannsakaðir með því að skoða áhrif þeirra á vinnslu þar sem áhersla var lögð á vinnsluafköst og hringormafjölda í flakahlutum. Hringormar voru einnig tegundagreindir eftir flakahlutum. Þol hringorma gagnvart frystingu var rannsakað, með þvi að koma lifandi hringormum milli tveggja þorsksmarningslaga og fryst við mismunandi hitastig og tíma. Að lokum var þykkt hringormahams rannsakað með stærðargreiningu á þverskurðarsniði hringorms.
Greining á hringormum leiddi í ljós að hringormar höfðu ekki mikil áhrif á vinnsluafköst en höfðu í stað áhrif á nýtingu. Hringormarhópar mældust með líka efnasamsetningu, en þegar kom að samanburði við þorskflakahluta þá höfðu hringormar hærra magn af kolvetnum og fitu. Hringormar voru einnig með hærri steinefnagildi í kopar(Cu), kalki(Ca) og járni(Pb) ef miðað var við þroskflökin. Hringormar mældust með minna af snefilefnum miðað við þorskflök. Hringormar og þorskflök voru með svipuð hlutföll í níu amínósýrum, og innhéldu báðir hópar töluvert magn af lífsnauðsynlegum amínósýrum. Frysting á lifandi hringormum sýndi fram á að þol hringorma lækki með lækkuðu hitastigi og auknum tíma við það hitastig.
Það var ljóst í upphafi að þetta verkefni myndi ekki svara öllum spurningum er snúa að fiskvinnslunni í dag varðandi hringorma og kostnað samfara þeim. Heldur er vonast um að niðurstöður verkefnisins séu eitt nytsamlegt skref í þeirri vinnu sem liggur fyrir að vinna áfram með.
3 Abstract
The main objective of this research was to study the physical and chemical properties of nematodes found in Atlantic cod. Nematodes were categorized by length and location in the cod fillet, by analyzing video footage from the industry. Chemical properties of nematodes were studied by measuring their chemical composition. Along with the chemical composition, several minerals and trace elements were also analyzed. In addition, the amino acid composition of nematodes was measured. To view the full picture, the components were compared to the chemical content of cod fillet portions.
The physical characteristics of nematodes were studied by examining their effects on processing where the emphasis was on processing rate and the number of nematodes in muscle portions. Nematodes were also analyzed by their position in the fillet. Nematodes resistance to freezing was examined by freezing live nematodes in a fish mince medium using specific sub-zero temperatures and time intervals. Finally, the thickness of nematode´s protective wall was studied by analyzing cross- sectional cuts in a microscope.
Analysis of nematodes revealed that nematodes had no major impact on the processing performance but instead affected the utilization of fillets. Nematode groups measured with similar chemical composition, but when compared to cod fillets, the nematodes had higher amounts of carbohydrates and fats. Nematodes included higher values of copper (Cu), calcium (Ca) and iron (Fe) if compared to cod fillets. Nematodes had fewer contents of all measured micronutrients when compared to cod fillets. Nematodes and cod fillets had a similar proportion of nine individual amino acids. In addition, both groups included considerable amounts of essential amino acids. Freezing the live nematodes showed that the survival of nematodes is decreased by lowering temperatures and increasing time at that specific sub-zero temperature.
It was clear at the beginning that this project would not answer all questions related to the fish processing and costs associated with them. But hopefully, the findings can be a useful step forward in the work related to nematodes.
4 Acknowledgment (and funding)
This study was conducted at Matís ohf in Reykjavík.
I would like to thank my supervisors, Sigurjón Arason and Magnea G. Karlsdóttir, and special adviser Erlingur Hauksson for their knowledge and guidance through this study. I will also like to send sincere thanks to Ína Björg Össurardóttir at Icelandic Nýfiskur, Kristján Guðmundur Jóakimsson at Hraðfristihúsið Gunnvör; Kristján Þórarinsson at SFS; Óðinn Gestsson at Íslandssaga and Lovísa Ásgeirsdóttir at Toppfiskur, for all help and assistances during my studies.
I want to thank the sponsors of the project, Matís, AVS (Added Value of Seafood) fund of Ministry of Fisheries and Agriculture in Iceland (project no. R030-15), HB Grandi, Icelandic Nýfiskur, Toppfiskur, Hraðfrystihúsið Gunnvör, Íslandssaga and other project´s processing companies. I want to send sincere thanks to the staff of the chemical lab of Matís laboratories for their help and understanding.
I want to thank my girlfriend Ingibjörg Kristjánsdóttir and my family for all their support throughout my studies.
5
Table of contents
Ágrip ...... 3 Abstract ...... 4 Table of contents ...... 7 List of tables ...... 10 List of figures ...... 11 Abbreviations ...... 14 1 Introduction ...... 15 1.1 Nematode species ...... 15 1.1.1 Other nematode species ...... 15 1.2 Challenges associated with nematodes ...... 15 1.3 Physical and chemical properties of nematodes ...... 16 1.4 Aim of the study ...... 16 1.4.1 Research questions ...... 16
2 Literature review ...... 17 2.1 Parasites of Atlantic cod ...... 17 2.2 Anatomy and physiology of nematodes ...... 17 2.3 Distinction between nematode species ...... 18 2.4 Chemical composition of nematodes ...... 20 2.5 The seal-worm ...... 20 2.5.1 The life cycle of seal-worms ...... 21 2.5.2 Seal hosts ...... 21 2.5.3 Macroinvertebrate host ...... 22 2.5.4 Fish hosts ...... 22 2.6 The whale-worm ...... 22 2.6.1 The life cycle of whale worms ...... 23 2.6.2 Epidemiology of whale-worms ...... 23 2.7 Human infection nematode diseases...... 24 2.7.1 Anisakiasis ...... 24 2.7.2 Pseudoterranovosis ...... 25 2.8 Allergies induced by whale-worms, diagnostics, and symptoms ...... 25 2.8.1 Anaphylaxis ...... 26 2.8.2 Acute urticarial reaction ...... 26 2.8.3 Other allergic symptoms ...... 26 2.8.4 Is it possible for a dead Anisakis larvae to induce allergic reactions? ...... 26 2.9 Geographical distribution patterns of fish hosts ...... 27 2.9.1 Distribution patterns of nematodes in the fish host ...... 28 2.10 Physical attributes of parasitic nematodes ...... 28
7 2.10.1 High temperature treatment ...... 28 2.10.2 Sub-zero temperatures ...... 29 2.10.3 Pressure ...... 29 2.10.4 Modified atmosphere (MAP) ...... 29 2.10.5 Electromagnetic radiation and electric current ...... 29 2.11 Nematodes and the fishing industry ...... 30 2.12 Efficiency of candling ...... 30 2.12.1 Other nematode detection methods ...... 31 2.12.2 The cost of removing nematodes in fish fillets ...... 31 2.13 Current laws and regulations concerning parasites in fish ...... 33 2.13.1 Europe ...... 33 2.13.2 North America (USA & Canada)...... 33
3 Methods & Material ...... 34 3.1 Experimental design ...... 34 3.2 Nematode sampling procedures ...... 35 3.3 Fish sampling ...... 36 3.4 Video analysis from the industry ...... 36 3.5 Chemical composition of nematodes and cod fillets ...... 37 3.5.1 Water content ...... 37 3.5.2 Sodium content...... 37 3.5.3 Protein content ...... 37 3.5.4 Carbohydrate content ...... 37 3.5.5 Lipid content ...... 37 3.5.6 Ash content ...... 37 3.6 Mineral & trace element analysis ...... 38 3.7 Amino acid composition ...... 38 3.7.1 Determination of free amino acids...... 38 3.8 The freezing survival of nematodes in fish muscle ...... 38 3.9 PCR analysis of nematode species ...... 41 3.9.1 DNA extraction ...... 41 3.9.2 PCR sequencing...... 41 3.10 Estimation of thickness of nematode protective wall...... 42 3.11 Data handling ...... 43
4 Results ...... 45 4.1 Video analysis from the industry ...... 45 4.2 Chemical composition ...... 46 4.2.1 Chemical composition of nematodes ...... 46 4.2.2 Comparisons between nematodes and cod muscles...... 47 4.3 Amino acid composition of nematodes and cod proteins ...... 49
8 4.4 Nematode survival in sub-zero temperatures ...... 50 4.5 Estimation of thickness of the nematodes skin layer ...... 56 4.6 Nematode species identification using PCR ...... 57
5 Discussions ...... 59 5.1 Video analysis from the industry ...... 59 5.2 Chemical composition of nematodes and cod muscle ...... 60 5.3 Nematode survival in sub-zero temperatures ...... 61 5.4 Estimation of thickness of the nematodes skin layer ...... 62 5.5 PCR analysis of nematode species in cod muscle ...... 62
6 Conclusion...... 63 7 Future perspectives ...... 64 References ...... 65 Appendix I - Chemical composition ...... 74 Appendix II – Amino acid composition ...... 76 Appendix III – Nematode survival against sub-zero temperatures ...... 77 Appendix IV – PCR analysis ...... 80
9 List of tables
Table 2.1. List of parasitic fauna reported from the cod muscle (Gadus morhua) (Hemmingsen et al., 2000)...... 17 Table 2.2. The chemical composition of nematodes vs. the chemical composition of cod muscle surrounding the nematodes (Dagbjartsson, 1973)...... 20 Table 3.3. The experimental design for the scientific study ...... 34 Table 3.4. Results registration for the survival analysis of nematodes in sub-zero temperatures. The nematodes were frozen at four different temperatures, with the freezing time from 1 to 6 hours...... 40 Table 3.5. The ingredients and runs information for each PCR sample. For each nematode sample, only 1 uL of the DNA extract was needed...... 41 Table 3.6. The order of all groups during PCR sequencing. Each group was marked with a letter and a number for the sample number in each cluster. 10 nematode samples were collected for each group. The group P represents test samples for calibration...... 42 Table 4.7. The chemical composition comparison of large and small nematodes...... 47 Table 4.8. The contents of minerals and trace elements in nematodes (mean) and cod muscle groups...... 49 Table 4.9. Species identification of nematodes found in Atlantic cod (Gadus morhua). *Nematodes collected from the loin and the belly flap of the cod fillets were analyzed as well as large and small nematodes ...... 57 Table 12. The comparison in composition between nematodes and the cod muscle parts ...... 75 Table 13. The mineral composition of nematodes (mean) and cod muscle groups...... 75 Table 14. The amino acid composition of nematodes and cod proteins ...... 76 Table 13. The survival rate of small nematodes (< 4 cm) ...... 77 Table 14. The survival rate of large nematodes (> 4 cm) ...... 77 Table 17. The first experimental reactions in PCR analysis of nematodes DNA. For the second reaction Magnesium Chloride was added. For the third reaction Betaine was added.
However, the MgCl2 gave the best results...... 80 Table 18. The second experimental PCR reactions ...... 81
10 List of figures
Figure 2.1. The nematode anatomy; A) Full scale anatomical view of the nematode. B) Drawings of cross-sectional anatomical view.l ...... 18 Figure 2.2. Morphology of nematodes; ...... 19 Figure 2.3. The life cycle of the seal-worm larvae showing all infection paths between hosts except for the human host (McClelland, 2002)...... 21 Figure 2.4. The life cycle of whale-worm (Leversen, 2010)...... 23 Figure 2.5. Variety of culinary dishes associated with Anisakiasis outbreaks (Audicana & Kennedy, 2008) ...... 24 Figure 2.6. A number of nematodes per 1 kg fish, according to fishing areas in Icelandic waters. December, January, February from 2001 to 2005 to the right. To the left: Mars, April, May, from 2001 to 2005 (Guðmundsson et al., 2006; Margeirsson, et al. 2010; Margeirsson et al., 2006)...... 27 Figure 2.7. Processing setup of a trimming station which includes a candling table. Far to the right is a worker trimming cod...... 31 Figure 2.8. A) Trimming of cod fillet B) Nematodes that have been removed from cod fillets...... 32 Figure 3.9. Length measurement of nematodes. The nematode to the left is around 6 cm, the nematode to the right is around 4 cm ...... 35 Figure 3.10. A) Collected nematodes from fish processors, B) The Nematode separation, weighing and cleaning, C) Prepared samples for measurement. All nematodes were first collected into plastic boxes that included water. The nematodes were taken out one by one, measured in length, and put in an empty dry container until further analysis...... 36 Figure 3.11. The positioning of each muscle part of the fish as targeted in the study. The lines in the picture mark the cuttings of the fillet. A = Loin, B = belly flap, C = Tail of the loin side, D = Tail of the belly flap side...... 36 Figure 3.12. The angle of the recordings in the processing step. The angle of the camera was straight on top of fillet, which would give visual accessibility on nematode infestation...... 37 Figure 3.13. The making of the fish mince used in the experiment. The fish parts were all put in the HALLDE grinder and ground into a small grained mince. The same mince was utilized for the whole experiment...... 39 Figure 3.14. Fish mince used in this trial, to the top left, sampling boxes with the bottom layer of fish mince to the top right. The fish mince was stored in a closed polystyrene box which included a cooling medium between experiments...... 39 Figure 3.15. A) Inside the ILSA-Convochill-blast freezer, B) Outside the blast freezer, C) The setup of for the analysis of nematode´s survival ratio...... 40
11 Figure 8.16. The equipment used for the cross-sectional cuts. The cutting blade setup is seen on the left; it includes a nematode sample. The figure in the middle is the full look of the machine...... 43 Figure 4.17. Distribution of nematodes in cod fillets (n = 120) as visually detected with video recordings from a trimming station...... 45 Figure 4.18. Effects of nematode infection intensity on the trimming speed of fillets caught with trawl nets far from the shore...... 46 Figure 4.19. Water, protein and carbohydrate content of fresh cod muscles (loin and belly flap) and nematodes present in the cod fillet...... 48 Figure 4.20. Ash, lipid and sodium contents of fresh cod muscles (loins and belly flaps) and nematodes present in the cod fillet...... 48 Figure 4.21. The amino acid composition of nematode and cod proteins ...... 50 Figure 4.22. The survival ratio of nematodes frozen in fish mince at -5 °C. Each sample box included 15 nematodes...... 51 Figure 9.23. The temperature within the mince sample frozen at -5 °C...... 52 Figure 4.24. The survival ratio of nematodes frozen at -10 °C. Each sample box included 15 nematodes...... 52 Figure 4.25. The temperature within the fish mince sample at -10 °C...... 53 Figure 4.26. The survival rate of nematode groups frozen at -15°C. Each sample box included 15 nematodes...... 53 Figure 4.27. The temperature in the sample at -15°C...... 54 Figure 4.28. The survival rate of nematodes frozen at -20°C. Each sample box included 15 nematodes...... 54 Figure 4.29. The temperature in the sample at -20 °C...... 55 Figure 9.30. The overall comparison of tolerance against sub-zero temperatures for both nematode groups. It´s seen that the decreasing temperature is very effective. At -10 °C nematodes die after 4 h, and at -15 °C they all die after 3 h...... 55 Figure 4.31. The nematode thickness was measured at 1.0 mm. There are three different layers marked, the inner and outer protective layers and the core in the middle...... 56 Figure 5.32. To the left, the lack of light permeability in large fillets. The black spot is heavily nematode infected area in the right sided figure...... 59 Figure 33. Chemical composition of small and large nematodes...... 74 Figure 34. Mineral contents of small and large nematodes...... 74 Figure 35. The temperature recordings from inside the freezer at -5°C ...... 78 Figure 36. The temperature recordings from inside the freezer at -10°C ...... 78 Figure 37. The temperature recordings inside the freezer at -15°C ...... 79 Figure 38. The temperature recordings from inside the freezer at -20°C ...... 79
12 Figure 39. The first PCR experimental reaction ...... 80
13 Abbreviations
Whale-worm = Anisakis simplex
Seal-worm = Pseudoterranova decipiens
Seal-worm A = Pseudoterranova krabbei
Seal-worm B = Pseudoterranova senso stricto
Seal-worm C = Pseudoterranova bulbosa
Seal-worm D = Pseudoterranova azarasi
H. aduncum = Hysteriolacum aduncum
PCR = Polymerase chain reaction
DNA = Deoxyribonucleic acid
HACCP = Hazard analysis and critical control points
EtOH = Ethanol
h = hours
Min = minutes
g = grams
mg = milligrams
mL = millilitre
L = litre
°C = Degrees Celsius
cm = centimetre
ssp. = Sub species
14 1 Introduction
Nematodes are round and thread-like parasites which are naturally found in wild animals all around the world. Overall, there are mainly two types of nematodes which found in the muscle of Atlantic cod (Gadus morhua) which is caught around Iceland. Those species are known as “whale-worm” (Anisakis simplex) and “seal-worm” (Pseudoterranova decipiens), but both species can drill through the stomach wall and infect the host’s intestines and muscle parts.
1.1 Nematode species The seal-worm, also known as cod-worm is a complexed species. Seal-worms go through five different developing stages which include for molts and five to six hosts during its life cycle. The nematodes eggs are spread with seals faeces, in which are transmitted to copepod hosts, then through macroinvertebrate hosts, which then get eaten by pelagic species which then get eaten by demersal fish species. The life cycle is completed when the infected fish is eaten by seals (McClelland, 2002).
The whale-worm larvae are spread with marine mammals (dolphins, porpoises, and baleen whales), where they are embedded in the mucosa. The eggs of the larva hatch in the sea and become free- swimming in the second developing stage. The second stage larvae are ingested by krill crustaceans, where they mature into stage three, which becomes infective. The larvae are then transferred between hosts through predator-prey (Smith & Wootten, 1975).
1.1.1 Other nematode species H. aduncum is a nematode species which gets sexually matured in fish. The larvae are usually found in the digestive tract of the fish and are only found in cold-blooded marine animals. In right circumstances, the nematode can mature into fully grown larvae inside fish. The nematode does not penetrate the fish muscle and is often confused with seal worm by fishermen because it can be seen, when the larvae exit its former host, either outside the mouth or the anal cavity (González, 1998).
1.2 Challenges associated with nematodes Nematodes can cause several challenges within the seafood industry. They are both difficult to detect and to remove from the fish muscle. The methods used today are not efficient enough to get 100% detection rates. It is highly costly for producers to detect and remove nematodes because it takes a long time to process each fillet. The time also increases the temperature of the product. The removal of nematodes which are deep in the muscle can also damage the muscle which is visually unattractive for consumers. In addition, visual scars and gaping on fillets can result in lowered values of products. Nematodes which get detected by consumers can have detrimental effects on product sales. Today, posting videos and photos online is very simple, and a bad review can be reflected in lowered sales of the product, similar to what happened in the 70s (van Thiel & van Houten, 1966).
Some nematodes have been found to be harmful to consumers. Nematodes can cause infection diseases such as Anisakiasis and Pseudoterranovosis, which occurs when a live larva is eaten. Also, some nematodes have been found to cause allergic reactions (Fernández et al., 1995).
15 1.3 Physical and chemical properties of nematodes Nematodes have been found to have special properties against harsh conditions which make them hard to detect and destroy. They have been shown to tolerate to some extent sub-zero temperatures and extreme temperatures from 60 °C up to 70 °C (Ronald, 1960). This tolerance can be explained by their chemical structure. The nematodes are composed of a thick protective wall/skin which contains oligosaccharides with cryoprotectant functions (Wharton & Aalders, 2002).
1.4 Aim of the study The main objectives of the study were to investigate the physical- and chemical properties of parasitic nematodes. By knowing the physical properties of nematodes, such as what effects nematodes have on the industry. In addition, size factors or tolerance against extreme condition might assist in developing new detection techniques in the future. Same can be said for the chemical composition of nematodes, as the results of those studies might give hints on chemical indicators that can be used in developing new detection techniques.
The chemical composition of nematodes and fish muscle was analyzed and compared including amino acid composition, water, carbohydrates, protein, ash, lipid, and sodium content, as well as the mineral and micronutrient content. The physical factors that were investigated include the position of nematodes inside the cod muscle by using video recordings of the processing line. A PCR analysis of nematodes DNA found in different places in the cod muscle was also investigated. The nematode´s ability to survive sub-zero temperatures was also analyzed.
1.4.1 Research questions What are the similarities and differences between nematodes and fish muscle with regard to chemical composition?
Is freezing an effective way in killing nematodes occurring in the cod loins?
What areas in cod fillets are mostly infected and how do nematode affect the processing time of fillets?
Is it possible to categorize whale-worms from seal-worms by length or position in the fillet?
Is there a compatibility between the amino acid compositions of nematodes compared to the fish muscle?
16 2 Literature review
This literature review represents current knowledge of the most important biological and physical properties of nematodes in commercially important fish species and the challenges associated with them in the fishing industry. It is well known in Iceland, and other fishing countries in the North Atlantic, that the presence of nematodes such as the seal-worms (P. decipiens), and whale-worms (A. simplex) in commercial fish have been causing problems for decades.
2.1 Parasites of Atlantic cod Parasites occur naturally in virtually all wild animals. Unfortunately, if the wild animals are then caught and used for human consumption, the presence of such parasites can be problematic as some parasites can be pathogenic to humans. Currently, there are 107 species of protozoan and metazoan parasites reported in the Atlantic cod. Most parasites are present in the viscera and the intestines of the fish. However, there are some species that have been found in the muscle, mainly seal-worms and whale- worms, with few other species as well (Table 2.1) (Hemmingsen, Halvorsen, & MacKenzie, 2000).
Table 2.1. List of parasitic fauna reported from the cod muscle (Gadus morhua) (Hemmingsen et al., 2000). Major taxa Species
Protozoa, Microsporidia Plisptophora gadi (Polyansky, 1955)
Pleistophora sp. (Drew, 1909 and Young, 1969)
Microsporidia gen. sp. (Karasev, 1984)
Myxosporea Kudoa thyrsites (Gilchrist, 1924)
Nematoda (larvae) Anisakis simplex (Rudolphi, 1809)
Pseudoterranova decipiens (Krabbe, 1878)
Hysterothylacium aduncum (Rudolph, 1802)
Of all parasitic species found in the seafood industry, the most problematic are the nematodes. Around the world, there have been discovered at least over 23 marine species known to be the final hosts for the whale-worm. Therefore, the whale-worm is known for not being particularly host specific (Ugland, Stromnes, Berland, & Aspholm, 2004). Also, seal-worms have been found in at least 79 species of paratenic fish species (McClelland, 2002).
2.2 Anatomy and physiology of nematodes Nematodes (roundworms) are thin, elongated, circular worms without segmentation. They are easily distinguishable from other worm groups because of their uniform shape, however, it is more complicated to determine the species of nematodes. Nematodes have a complete digestive tract with mouth, esophagus, intestines and anus (fig. 2.1). The main criteria for determination of species are size, fine structure of the head and tail, position of the excretory pore, and structure of the transitional area between esophagus and intestines. Adult male worms carry a spicule near the end of the tail (fig 2.1.) and in some species a characteristic sucker (Hauksson, 1991).
17
A) B)
Figure 2.1. The nematode anatomy; A) Full scale anatomical view of the nematode. B) Drawings of cross-sectional anatomical view. Retrieved from: http://www.ucmp.berkeley.edu/phyla/ecdysozoa/nematoda.html
2.3 Distinction between nematode species The nematode species can be quite similar in shape, size, and color and therefore it can be difficult and nearly impossible to distinguish between seal-worm and whale-worm. However, it is known that whale- worms can be smaller and have a different color when found in cod (Ugland et al., 2004).
The colors of whale-worms are usually translucent white, like the fish muscle which also makes it harder to detect them. Also, whale-worms are known to be a little smaller than seal-worm species (3 cm vs. 6 cm). However, the size or length is not considered to be the best parameter for determining nematode species, because the developing stages of nematodes can vary, and thus species of an early 3rd stage seal-worm could be confused with whale-worm species (Ugland et al., 2004).
By dividing nematodes into two size groups could exclude most of the seal-worms from the whale-worm types of nematodes. There is, however, a method that has been used for decades, comprised of the nematode anatomy (Hauksson, 2015). This method includes the use of microscopes where the nematodes are usually put in contact with glycerol for some time, usually 24 hours. Glycerol has a special ability to make the skin of the nematodes transparent. With transparent skin, it is possible to see the intestines of this parasite but it is known that the intestines vary between species, namely seal-worm have an appendix but whale-worm has none (fig. 2.2). Moreover, the shape and arrangement of the tail can be different between species (fig. 2.2). This method can be effective but can be time consuming and can include deviations and errors (Hauksson, 2015).
18
However, the most effective method to distinctive between nematodes is by far the scanning of DNA extracts with polymorphism chain reaction and restriction fragment length polymorphism (PCR-RFLP). PCR samples are digested with restriction enzymes to identify the characteristic of RFLPs. The larvae are distinguished based on combinations of different RFLP patterns. Kawakani and others distinguished whale-worm sub-species based on Hinfl and Hhal restriction profiles, and C. Osculatum, H. aduncum and seal-worms were distinguished based on Rsal and Haelll restriction profiles (Umehara, Kawakami, Araki, & Uchida, 2008).
Figure 2.2. Morphology of nematodes; A) Whale worm, B) Seal worm, the colors represent the intestines. The orange represents the ventriculus, blue represents the intestinal caecum and yellow the ventricular appendix (Milligan, 2008).
19 2.4 Chemical composition of nematodes Dagbjartsson (1973) studied the chemical composition of nematodes found in Atlantic cod in comparison with the chemical composition of the cod muscle (Table 2.2). The results showed that the larvae were denser (less water) than the cod muscle and contained a higher amount of carbohydrates which may result in a difference in absorption because of the higher numbers of OH components in nematodes (Dagbjartsson, 1973).
Table 2.2. The chemical composition of nematodes vs. the chemical composition of cod muscle surrounding the nematodes (Dagbjartsson, 1973). Chemical components Nematodes Cod muscle
Water (%) 71.0 81.0 Protein (%) 13.5 18.0 Fat (ether-extract) (%) 0.8 0.2 Ash (%) 0.7 1.1 Salt (NaCl) (%) 0.05 0.1 Carbohydrates (%) 14.0 0.0 Sodium (mg/g) 0.7 1.5 Copper (mg / g) 0.02 0.005 Proline (% of total proteins) 6.4 3.2 Glycine (% of total proteins) 7.2 4.3 Steric acid (% of Chloroform extract) 6.0 1.5 Oleic acid (% of Chloroform extract) 10.5 4.5 Linoleic acid (% of Chloroform extract) 9.6 1.5
2.5 The seal-worm The larvae of Pseudoterranova decipiens, also known as “seal-worm “or “cod-worm,“ is a complexed species (Nematoda, Superfamily Ascaridoidea, subfamily Anisakinae). Other related species include P.decipiens B ( Krabbe, 1878). P. decipiens A (Paggi et al., 2000). P.decipiens C (Cobb, 1889). P.decipiens D, (Yamaguti & Arima, 1942) and P.decipiens E (Mattiucci et al., 1997). Additional species of Pseudoterranova include P.cattani, P. kogiae (Johnston and Mawson, 1939) infecting the pygmy sperm whale (Kogia breviceps) and P. ceticola (Deardorff & Overstreet, 1981) infecting dwarf sperm whale (Kogia simus). Nematodes of the genus Pseudoterranova have proven to be a costly problem for the seafood industry. Also, seal-worms can affect human health. Due to the severe pathology, when consumed alive in raw or undercooked fish, they can provoke in humans a fish-borne zoonotic disease named Pseudoterranovosis (McClelland, 2002; Mattiucci & Nascetti, 2008).
20 2.5.1 The life cycle of seal-worms The seal-worm larvae are involved in five different developing stages which include four molts and five to six hosts during their life cycle. The definitive host is usually a pinniped, in which the larvae will grow from stage three to stage four and then mature into an adult. The eggs of these nematodes are shed in the seal´s feces where the larvae grow to stage three and hatch (Koie, Berland, & Burt, 1995).
rd The 3 stage larva can infect crustaceans (Jackson, Marcogliese, & Burt, 1997) before it is passed on to fish or squid host (fig. 2.3) (McClelland, 2002). The fish hosts are usually bentophagous or piscivorous demersal species (e.g. gadoids) but can rarely be pelagic crustaceans or fish (Marcogliese, Boily, & Hammill, 1996). The life cycle of the larvae is completed when seals eat infected fish or squid. It is possible for unsuitable hosts to become infected with this parasite. Such hosts include organisms that will never be consumed by the definitive host, and thereby preventing the parasite
Figure 2.3. The life cycle of the seal-worm larvae from completing its life cycle. Such hosts showing all infection paths between hosts except for the become „dead ends“ and may include human host (McClelland, 2002). secondary fish hosts (e.g. large Atlantic cod), seabirds (Riley, 1972) and humans for example (Sakanari & Mckerrow, 1989).
2.5.2 Seal hosts Grey seal (Halichoreus grypus) (fig. 2.4), and common harbor seal (Phoca vitulina) (fig. 2.5) are the principle, definitive hosts of seal worms in the North Atlantic, although small numbers of these nematodes have been found in harp seals (Phoca groenlandicus). Seal-worms reside in the stomach, occasionally in large numbers (>10.000) in gray seals. In Icelandic waters, the gray seal is recognized as the main final host for seal worms to the infestation of cod and other commercially valuable fish (E., Hauksson, 2005; Ólafsdóttir, 2014). Studies have shown that gray seals are far more infected with seal-worm larvae than harbor seals (Olafsdottir & Hauksson, 1998). Even though the Icelandic harbor seal population is two times greater in numbers than the gray seal population (Hauksson, 2006; Hauksson & Einarsson, 2010), the average nematode burden was several times higher in the gray seals than the harbor seals. This discovery indicates the relative importance of the gray seal as the final host (Aznar, Balbuena, Fernández, & Raga, 2014).
21 2.5.3 Macroinvertebrate host Seal-worms in the 1-9 mm length range have been observed from thirteen crustacean and one Polychaeta species in the North Atlantic and adjacent waters. Natural macroinvertebrate hosts include sea mouse (Polychaeta), a gammaridean amphipod and caprellid from the White Sea, Russia, shrimps (Decapod) from the Barents Sea, an isopod from Norway, a mysid from Elbe estuary, Germany, and three mysids and five gammaridean species from Canada (Marcogliese, 2001).
Laboratory studies have shown that juvenile gammarideans are susceptible to infection by exposure to freshly hatched unsheathed larvae, but the transmission is enhanced with the participation of copepod carrier hosts. Macroinvertebrates (Polychaeta, nudibranchs, mysids, mature amphipods, isopods, cetaceans, and decapods), are not susceptible to infection by ensheathed larvae. However, they are readily infected by larger exsheated larvae from copepods. After penetration into the hemocoel of an amphipod, larval Sealworms grow at an exponential rate until they reach 2-3 mm in length. There is no evidence that nematodes molt in the macroinvertebrate host (McClelland, 1990).
2.5.4 Fish hosts Fishes become infected by ingesting larvae infected macroinvertebrates. The larvae of seal-worm penetrate the stomach wall of the fish and gain access to the body cavity, and from there on gain access to the muscle of the host. Numerous fish species have been found to host larvae of seal-worm. The prevalence and abundance of the parasite tend to be higher in large benthic piscivorous fishes such as sculpins (Myoxocephalus spp), sea ravens (Hemitripterus spp), anglerfish (Lophius spp), cod (Gadus morhua), and cusk (Brosme brosme). However, the highest densities of infection (number of units per host weight) are usually found in smaller specimens such as smelt, ocean pout and young flatfish (McClelland et al., 1983a; 1983b; 1985; 1987). The primary fish hosts of seal-worms are benthic consumers who acquire the parasite directly from invertebrate hosts (McClelland & Martell, 2014). Experimental evidence of fish to fish transmissions has been presented (Burt et al, 1990; McClelland, 1995; Jensen, 1997). The heavy infections found in the major demersal piscivores (> 50 cm in total length) has indicated that seal worms may pass through one or more host through predator-prey (McClelland, 1990). Secondary fish host in the North Atlantic Ocean includes Atlantic cod, monkfish, cusk and sea raven. Those species may be considered primary hosts as juveniles but become increasingly piscivorous as they mature (McClelland & Martell, 2014).
2.6 The whale-worm Whale worms (Anisakis simplex) belongs to the subfamily Anisakinae, family Anisakidae, superfamily Ascaridoidea, suborder Ascaridina, and order Ascaridia (Smith & Wootten, 1975). These nematodes are distributed worldwide, occurring in fish species from all oceans. Several whale-worm genera are found as adults in marine mammals (dolphins & whales), and as larvae in diverse tissues of fish, and in some invertebrates (Mattiucci & Nascetti, 2008). At least 23 different species of baleen and toothed whales are known to serve as the final hosts (Dailey & Walker, 1978; Davey, 1971; Smith, 1983; van Thiel & van Houten, 1966). In the eastern North Atlantic, Anisakis simplex type B is most common. The
22 third stage nematode, found in captured fish, is about 25 mm long. In small hosts, such as the porpoise (Phocoena phocaena) it grows to reach a mean body length of about 6-7 cm, in the larger long-finned pilot whale (Globicephala melas) it is slightly larger, and in the much greater minke whale (Balaenoptera acutorostrata) the mean female body length is 12.6 cm (Ugland et al., 2004). Whale-worms are the potential risk to humans as they may cause a disease known as anisakiasis, whose main etiologic agents belong to the genera Anisakis Dujardin, and Pseudoterranova Mozgovoi (Mattiucci et al., 2013).
2.6.1 The life cycle of whale worms
Whale-worms have a complex life cycle that passes through many different hosts (figure 2.4). Whale- worm has a complete life cycle comprising of five developing stages separated by four molts. Aquatic invertebrates serve as the first intermediate hosts, and various fishes and squid are used as other transport or paratenic hosts. Adult stages of whale- worms reside in the stomach of marine mammals (dolphins, porpoises, and baleen whales), where they are embedded in the mucosa. Unembryonated eggs produced by adult females are expelled through the feces (figure 2.4) of marine mammals and become embryonated in the seawater, where 1st stage larvae are formed in the eggs (Berland, Figure 2.4. The life cycle of whale-worm (Levsen, 2010). 1961; Koie, 1993). The larvae molt to become free- swimming in the 2nd stage and are ingested by krill crustaceans, usually euphausiids, in which they mature into the 3rd stage, which is infective to fish and squid. Through predator-prey, the larvae transfer between hosts. Upon the hosts ‘death, Anisakis larvae migrate from the intestine to the tissues in the coelomic cavity and the muscle tissues, growing up to 3 cm in length. When marine mammals ingest fish or squid containing the 3rd stage larvae, the larvae molt twice and develop into adult worms, completing the nematode lifecycle (Koie et al., 1995).
2.6.2 Epidemiology of whale-worms Anisakiasis is a fish born parasitic disease caused by the consumption of raw, marinated, undercooked fish or cephalopods contaminated by 3rd stage larvae of the Anisakidae family. Anisakiasis was first described as “worm-herring disease “in the 1960s in the Netherlands by Van Thiel, who associated different cases of patients suffering from acute abdominal pain with the consumption of lightly salted herring (Van Thiel et al., 1960). The majority of anisakiasis cases occurs in Japan, Spain, Italy, South America, USA (Hawaii), Netherlands and Germany. The sources are from traditional raw, marinated or brined fish dishes like sushi, sashimi, pickled anchovies, ceviche, lomilomi and salted herring (figure 2.5) (Audicana & Kennedy, 2008; Consortiumcr, 2011). Scandinavian countries are known for proportionally high per-capita fish consumption. However, the number of anisakiasis incidents are low, possibly because the fish is well cooked when eaten (Lin
23 et al., 2012; Levsen et al., 2013). However, this might change due to a more globalized cuisine, especially concerning Asian-inspired seafood. Also, chefs are now more fond of undercooking fish for gaining desirable texture and taste (Audicana & Kennedy, 2008). In recent years, several new cases have been reported. Of the approximately 20.000 anisakiasis cases reported worldwide, over 90% of all cases are reported from Japan, which accounts for approximately 2000 cases yearly. Most other cases occur in Spain, Netherlands, and Germany (Panel on Biological Hazards, 2010). The presence of live whale-worms in previously discussed foods can elicit two types of nematode diseases: the parasitic digestive infection (Anisakiasis), and allergic reactions with or without digestive symptoms (Fernández et al., 1995).
A. Lightly salted herring B. Ceviche C. Pickled anchovies fillets
D. Sashimi E. Salmon Lomi-lomi
Figure 2.5. Variety of culinary dishes associated with Anisakiasis outbreaks (Audicana & Kennedy, 2008)
2.7 Human infection nematode diseases
2.7.1 Anisakiasis Anisakiasis occurs only when live larvae of whale-worm is ingested. Symptoms appear within few hours after ingestion. Symptoms that are associated to anisakiasis have been described as gastric, intestinal, and ectopic (extra-gastrointestinal), and allergic. Gastric anisakiasis starts within few hours after ingestion, generally 1-2 hours, when a live Anisakis larvae reach the human stomach. Within the stomach, the larvae adhere to the gastric mucosa by a projection surrounding its mouth and produce
24 proteolytic enzymes, which are mainly secreted by a dorsal esophageal gland and other excretory glands around the mouth. These proteases can cause erosive and hemorrhagic lesions which include the formulation of tunnels through the gastric mucosa to the submucosa (Cespedes et al., 2000). The acute phase of the infection elicits severe gastric pain, vomiting, diarrhea and mild fever. Acute symptoms are resolved within few days, but the untreated gastric disease can lead to chronic, ulcer-like symptoms which can last for weeks. Intestinal anisakiasis is characterized by either intermittent or constant abdominal pain starting five to six days after ingestion. Infected individuals may develop ascites or peritoneal signs. Intestinal infection and inflammatory responses occur mainly in the terminal ileum and less commonly in the colon. Rare complications have been reported and include small bowel obstructions, ileal stenosis, intussusception, intestinal perforation and pneumoperitoneum (Cespedes et al., 2000). Granulomas, ulcerative lesions, and hypersensitivity response occurs the first or second week after ingestion. After two weeks, the larvae die, but a persistent inflammation or granulomas remain. After the death of the larva two different situations can occur:
i) The loss of parasite with ulcerative lesions or the endowing of a dead larva into a granuloma. ii) The dead larvae induce an adaptive response characterized by T-lymphocyte proliferation with polyclonal and monoclonal, which is responsible for Anisakis allergic symptoms.
The clinical manifestation of anisakiasis varies depending on the organ where the person was infected, and which Anisakis simplex caused the infection. Gastric infection occurs primarily in Japan, whereas intestinal disease is more common in Europe (Hochberg & Hamer, 2010).
2.7.2 Pseudoterranovosis Occurs when live seal-worm larvae are ingested. Pseudoterranovosis has been reported in Japan but is less common than Anisakiasis. In the 90s there were at least 335 human cases of pseudoterranovosis in Japan and 19 cases in other countries, including the United States, Canada, Greenland, Chile, the United Kingdom, and Korea (Ishikura et al., 1993). Pseudoterranovosis has frequently been reported in northern Japan which resulted from dietary differences compared to the south of Japan. Also, due to increased populations of pinnipeds, such as sea lions and seals. Recently, pseudoterranovosis has been reported from Mexico and South America (Torres et al., 2007).
2.8 Allergies induced by whale-worms, diagnostics, and symptoms Whale-worm is the only known fish parasite so far that can provoke clinical allergic responses by the ingestion of processed seafood containing anisakid proteins (Daschner & Pascual, 2005; Fernández et al., 1995). The whale-worm proteome structure is complexed and contains considerable numbers of proteins with allergic potential (Fæste et al., 2014). Several allergens have been shown to be relatively resistant to digestion or heat treatments. The diagnosis of IgE-mediated A. simplex allergy is based on the following criteria; an individual with a compatible history of typical allergy symptoms wich is followed by consumption of fishery products. Individual who is positive for skin prick test (SPT) and positive serum-specific IgE levels of Anisakis simplex. If an individual has a negative history of allergy symptoms and positive in vivo and in-vitro test to fish and other possible cross-reactive allergens (crustaceans, dust mites, and insects) (Caballero & Moneo, 2004; Moneo et al., 2005).
25 2.8.1 Anaphylaxis Anaphylaxis is a potentially life-threatening allergic response that is marked by swelling, hives, lowered blood pressure and dilated blood vessels. Anaphylactic allergic reactions to Anisakis simplex occurs mainly in Mediterranean and Asian countries, because of high consumption of raw, raw-marinated and undercooked seafood (Anibarro et al., 2007; Choi et al., 2009; Hoshino & Narita, 2011). In a total of 12 studies describing allergic and anaphylactic reactions with 448 patients with Anisakis allergy, 130 (29%) experienced anaphylaxis. In a study that analyzed the causes of anaphylaxis in two different hospitals in Spain concluded that if whale worms were considered as a causative agent, idiopathic anaphylaxis dropped from 14 to 4%. Therefore, as food allergy, whale worms accounted for 10% of the total recorded anaphylactic reactions (Audicana & Kennedy, 2008).
2.8.2 Acute urticarial reaction Acute urticaria is described as an itchy uplifted rash that suddenly develops. The rash may be triggered by an allergy and it is a very common allergic reaction to A. simplex which was demonstrated in 10 Anisakis simplex allergic patients. All patients had acute urticaria (100%) followed by abdominal pain (30%) and anaphylaxis (30%) (Choi et al., 2009). In the Basque region in Spain, Anisakis simplex was considered the leading cause of urticaria and angioedema in adults from fish consumption. Also, whale- worm was seen as responsible for 8% of all acute urticaria (Rioja et al., 1998).
2.8.3 Other allergic symptoms Along with occupational exposure, other routes of transmission symptoms, such as inhalation or skin contact, can be involved, and allergic conjunctivitis, dermatitis, and asthma have been described (Armentia et al., 2006; Purello-D'Ambrosio et al., 2000; Scala et al., 2001). However, among fishery and aquaculture workers, fishmongers, and seafood handlers, whale worm allergy is relatively rare, with the consideration that over 38 million people work in this field (Nieuwenhuizen et al., 2006). Anisakis simplex is a stronger sensitizer than fish since the prevalence of Anisakis induced allergy which exceeded fish allergy 8% against 6% (Nieuwenhuizen et al., 2006). The incidence of whale-worm sensation in fish workers is higher, up to 64%. However, in the general population, whale-worm allergy remains rare (Anadon et al., 2009).
2.8.4 Is it possible for a dead Anisakis larvae to induce allergic reactions? Some authors have demonstrated an in vitro IgE reactivity of Anisakis simplex allergic patients through thermally treated Anisakis extracts (Rodriguez-Mahillo et al., 2010). Allergens from whale-worms may also be present in the fish flesh surrounding the larvae, therefore if a whale-worm has been removed, some allergens could be present in the fish muscle and might cause allergic symptoms (Audicana & Kennedy, 2008). Another study on 5 Anisakis-allergic patients with one or more nematodes detected by gastroscopy in the stomach was submitted to two different single-blinded challenges versus placebo. The first challenge was performed with 11 Anisakis larvae frozen at -20°C for 48 hours, while the second was carried out with the offending seafood after freezing at -20°C for 48 hours. All patients tolerated both challenges without any allergic or gastric symptoms (Alonso et al, 1999).
26 One study challenged 22 Anisakis-allergic patients with up to 20 frozen larvae without reporting any allergic reactions. The subjects were followed up for more than two years, in which patients consumed deep-frozen fish without problems. However, there is a general agreement that in most cases, an active infection is required to initiate allergic sensitivity to Anisakis, even if a prior sensitization via exposure to thermally resistant Anisakis allergens, including dead larvae, could not be excluded (Alonso-Gomez et al., 2004).
2.9 Geographical distribution patterns of fish hosts Studies from Iceland, Norway, and Sweden have shown that seal-worm is more abundant in cod from coastal regions, particularly around seal colonies and haul-outs since seals are the definitive host for this parasite (Aspholm, 1995; Desclers & Andersen, 1995; Olafsdottir & Hauksson, 1998). However, seals and cod can both be highly mobile species. Therefore the distribution patterns can vary seasonally (Guðmundsson et al., 2006). Around Iceland, the highest numbers of seals are often found in the coastal areas in the west and northwest (Hauksson, 2006), and cod from these regions have a correspondingly high prevalence of seal-worms (Olafsdottir & Hauksson, 1998). The Atlantic cod around Iceland has been described as “sedentary“ or “accurate homers,“ meaning that they will either remain in relatively small geographical areas or perform seasonal movements before returning to a relatively small area, for example, to spawn (Robichaud & Rose, 2004). This factor can affect the overall prevalence of parasites in cod from different regions. For example, the southwest of Iceland is known to contain major spawning ground of Atlantic cod, and cod in this region have lower numbers of parasitic nematodes, potentially due to the influx of “clean“ cod from Greenland (Platt, 1975). As seen in fig. 2.6, the numbers of nematodes per kg of fish caught vary between seasons. In some cases, there are possibilities of some areas further from the shore containing more quantities of nematodes than areas near the coast (Margeirsson et al., 2006).
Figure 2.6. A number of nematodes per 1 kg fish, according to fishing areas in Icelandic waters. December, January, February from 2001 to 2005 to the right. To the left: Mars, April, May, from 2001 to 2005 (Guðmundsson et al., 2006; Margeirsson, et al. 2010; Margeirsson et al., 2006).
By contrast, there are relatively few data on the distribution of whale-worms. It is noted that the highest density found in Norwegian waters was typically in the upper waters of the open sea which
27 potentially relates to the habitat of the euphausiid hosts (Stromnes & Andersen, 2000). However, it has been studied that the prevalence of whale-worms in the Barents Sea was higher in coastal areas compared to offshore areas (Aspholm, 1995).
2.9.1 Distribution patterns of nematodes in the fish host In general, fish will naturally have different immunities and tolerance towards different parasites, but the geographical location of the fish and its life history can also be an influential factor. The age and length of cod have shown to correlate with the prevalence and mean intensity of both whale-worms and seal- worms in studies from Icelandic and Scottish waters (Hemmingsen et al., 2000; Platt, 1975; Stromnes & Andersen, 2003; R. Wootten & Waddell, 1977). Within the Atlantic cod, whale-worms have shown to be most abundant in the viscera and abdominal flaps (Brattey & Bishop, 1992; Platt, 1975; Stromnes & Andersen, 2000). Studies have shown that both whale-worm and seal-worm species are reported in higher numbers on the left side of the fish (Brattey & Bishop, 1992). These factors could be substantial for fish processors, as they could suggest ways of targeting cod with naturally lower numbers of parasites (Smith & Hemmingsen, 2003). Post-capture movements of whale-worms in various fish species have been studied in Scottish waters. These authors reported significant post-capture migration of whale-worms from the viscera to the muscle of mackerel and herring. However, such movements have not been noticed for lean fish, but could again be critical for commercial fish processors if those movements occur in cod (Smith, 1984; Smith & Wootten, 1975).
2.10 Physical attributes of parasitic nematodes The physical behavior of parasitic nematodes has not been widely studied, and most investigations on whale-worms and seal-worms have focused on their pathology and life cycles. Physical and behavioral information is often extremely valuable when attempting to manipulate or kill these parasitic organisms.
2.10.1 High temperature treatment The effects of various physical stimuli on the behavior and mortality of seal-worm have been studied by Ronald (1960). The survivability of nematodes increased from 7.5 °C to 25 °C. Viability increased sharply again at 35 °C before decreasing at 70 °C. Above 40 °C, survival times fell below 6 hours within the muscle tissue, dropping to only a few minutes at 70 °C (Ronald, 1960, 1962, 1963). The survival peaks are likely related to the life cycle of nematodes, as they correspond to the temperature range that it would encounter in both invertebrate and fish hosts (-2.5 °C to 7.5 °C). Also, in the mammalian or seal host (33 °C to 37 °C) (Gustafson, 1953; Deardorff et al., 1984). It has also been reported a thermostatic response in the “free nematodes“ which moved towards temperature up to 35 °C. Increasing the temperature of the cod fillets for a specific period may, therefore, enhance the mobility of the nematodes and encourage them to migrate out of the muscle (Ronald, 1960).
28 2.10.2 Sub-zero temperatures The stimuli of induced temperature changes showed to be important for influencing mortality rates within the cod muscle, where 100% mortality occurred within 20 hours at temperatures between -70 °C and - 25 °C. Longer times were required to achieve 100% mortality the temperature was increased towards 0 °C. Nematodes are known to have tolerance towards sub-zero temperatures (Wharton & Aalders, 2002). An experimental study was performed on 3rd stage whale-worms where individual nematode was exposed to minimum temperatures in the range of -2 °C to -15 °C with the total of five to eight larvae being exposed to each test temperature. The temperature at which 50% of the nematodes froze and were killed was calculated using the analysing methods of Finney (1952). Nematodes did not survive an exposure to -15 °C for 10 min. Frozen nematodes did, however, survive exposure to -5 °C and -10 °C for five hours. The survival rate declined with time spent at both temperatures (Wharton & Aalders, 2002).
2.10.3 Pressure By subjecting infected cod muscle to a negative hydrostatic pressure (near vacuum) at 25 °C can cause mortality of nematodes within 78 hours of treatment (Ronald, 1962). However, high pressure up to 10 bars (1 MPa), has no effect on the mortality (Ronald, 1962). Recent studies on whale-worms have shown that it can be killed in fish at pressures between 140 MPa and 300 MPa over different timescales. Treatment times could be decreased with increased pressure (Molina-Garcia & Sanz, 2002).
2.10.4 Modified atmosphere (MAP) Studies have shown that hydrogen, oxygen, and methane gasses have no effects on mortality of nematodes if compared to untreated, whereas nitrogen and carbon dioxide slightly increased survival ratio (Ronald, 1962). Chlorine gas and solutions could achieve 100% mortality but current EC regulations state that chlorine can no longer be used as food preservatives (Regulation EC No 2032/2003). It has been suggested recently that exposure to different gasses may affect the coiling behavior and maybe the mortality of whale-worms. It has been reported that carbon dioxide (CO2) may encourage the nematodes to uncoil, while oxygen encouraged coiling within the body cavity of silver scabbardfish (Lepidopus caudatus) (Panebianco et al., 2000). It was however noted, that these results might be reflected by the different timescales employed in each treatment. In addition, such manipulation could encourage the nematodes to migrate and make it easier to detect them during candling, which would be beneficial for the seafood industry (Panebianco et al., 2000).
2.10.5 Electromagnetic radiation and electric current Studies have shown that 100% mortality was achieved in “free” nematodes that were exposed to ultrasound or certain wavelengths of light (Ronald, 1963). However, electromagnetic radiation treatments were all ineffective when the nematodes were seeded into the cod muscle. Also, there was no evidence of any photobiotic behavior (Ronald, 1963). A wide range of electric currents was also
29 tested but had no effect on parasites within the fish muscle (Ronald, 1963). However, other species of nematodes have shown to be susceptible to electricity (Caveness & Caveness, 1970).
2.11 Nematodes and the fishing industry Fish processing companies in Iceland are aware of the risk in buying fish which is caught in shallow coastal areas, where it might contain higher infestation numbers than fish caught far from the shore. The reason for fishing near shore is usually due to inclement weather conditions. In the last 50 years, extensive evidence has been supplied to recognize the presence of whale-worms in viscera and fillet of many economically important cephalopod and fish species. The presence of nematodes in fish was recognized as early as the thirteenth century (Stern et al., 1976), but due to recent media coverage, nematodes are nowadays identified as an aesthetic problem by the public which has led to adverse effects on marketing. Removing parasites add appreciably to the cost of packaging, reducing the value of the product and representing a financial loss in marketing value (McClelland et al., 1985; J. Smith & Wootten, 1979).
2.12 Efficiency of candling Candling is a detection technique that is commonly used by most fish processors today. The simplest candling table is a box (50 cm square) with a flat 6 mm thick plexiglas on top. The box is lit inside by two fluorescent tubes giving a white uncolored light (figure 2.7). The usage of candling tables is as follows; the fillets are laid down on the plexiglas. The worms show up inside the fillet as dark shadows in the flesh. After visual confirmation, the nematodes are removed from the muscle either with forceps or a knife (figure 2.7). Detection of nematodes in skinless cod fillets is far from being 100% effective, and even with best available light conditions, efficiency higher than 75% can hardly be expected (Valdimarsson, Einarsson, & King, 1985). The manual detection efficiency for nematodes in Atlantic cod is reported to be 65% under ideal conditions, and as low as 50% under industrial working conditions (Hafsteinsson & Rizvi, 1987) and only 25% for cod fillets with skin on (Hauksson, 1991). Other studies have reported nematode detection varying from 33 to 93% without the specification of the species of the fish or the nematode (Bublitz & Choudhury, 1993; Hafsteinsson & Rizvi, 1987). The relatively low detection efficiency reported for manual inspection is due to multiple scattering of visible light in the cod muscle (Peturson, 1991). That is the biggest issue because nematodes which are embedded deeper than 6 mm in the muscle get undetected by manual inspection (Bublitz & Choudhury, 1993; Hafsteinsson et al., 1989).
30
Figure 2.7. Processing setup of a trimming station which includes a candling table. Far to the right is a worker trimming cod. The efficiency of candling can depend on the fish species, nematode species, fish conditions, and other factors. The fishing ground can also play a part in the efficiency of candling, probably through the difference in the texture of the fish from different grounds. Proper bleeding of fishes is also crucial. In cod made to bleed alive, nematodes are more easily detected, than in cod which was already dead when gutted and made to bleed. This difference in efficiency can amount up to 15%. There are also human factors involved. Proper working conditions, without noise and with periodic breaks in working at a candling table, proper training and good eyesight all increase the candling efficiency (Hauksson, 1988; 1991).
2.12.1 Other nematode detection methods Due to the high cost of manual inspection, it is of keen interest for the fish processing industry to have this operation automated. Various techniques have been tested. Techniques such as fluorescence (Pippy, 1970), ultrasound (Hafsteinsson et al., 1989), magnetometer measurements (Choudhury, Jenks, Wikswo, & Bublitz, 2002). Computer tomography and magnetic resonance imaging (Heia et al., 1997), multispectral imaging (Wold et al., 2001) and imaging spectroscopy (Heia et al., 2007) are methods that have been proposed for detection of nematodes in fish muscle. One of the most recent techniques proposed imaging spectroscopy or hyperspectral imaging combines both spatial and spectral information in a 3D data cube called a hyperspectral image (Heia et al., 2007 Sigernes et al., 2000). Many methods have achieved promising result under lab conditions and small segments of cod fillets, but none has so far been set up and tested in industrial conditions on full- size cod fillets (Sigernes et al, 2000).
2.12.2 The cost of removing nematodes in fish fillets Today, the most expensive parts of the cod are the loins. Loins are mainly processed fresh and sent out to buyers the same day, either by airplanes or vessels. The fillets are skinned, deboned and the visible nematodes removed. The removal of nematodes from fillets is usually the part of the processing which is the most time consuming, and therefore can be expensive for fish processing companies. According to Bergsveinsson & Pálsson (1986), as cited by Margeirsson et al. (2010), it will take approximately 4-5 seconds to remove one nematode manually from a cod fillet during candling. The labor cost of removing nematode from an average cod fillet can be around 10 to 13 IKR. Other expenses
31 related to nematodes include the cost of the quality decrease due to increased processing time for the product (Margeirsson et al., 2010). It is hard to plan the processing of fillets when the processors do not know how much infected their raw material is, for example; at one point of the processing they get highly infected fish, and at another point, they get no infections. This uncertainty results in less efficiency, less amount of raw material processed for a period of time, so nematodes may actually be the reason for the increased temperature in the fish during processing, especially when the fillets are separated into smaller parts (figure 2.8). The third costly factor of nematodes is the change in the appearance of fillets. It is likely that the removal of nematodes itself can leave negative effects or scars on the fillets, which in return can degrade the value of the product (Margeirsson et al., 2010). The fourth factor, and may be the most important one, is that nematodes must be removed from the fillet and do not appear for the consumers. Only one live nematode larva showing in processed or cooked fish can end up in a negative media coverage and therefore reduce sales. Like what happened in Germany in 1987, where nematodes in Danish herring triggered a strong reaction of the German people towards nematodes. After a television program had been subjected in Germany, it reduced the sales of the product down to one-third of its normal sales volume (Hauksson, 1991).
A) B)
Figure 2.8. A) Trimming of cod fillet B) Nematodes that have been removed from cod fillets.
If approximately 215.000 tons of cod are caught annually in Iceland, with the average weight of 3 kg per fish, indicates that approximately 72 million individuals are caught every year (equation 1). If its assumed that each individual cod contains eight nematodes on average, and the cost of removing one nematode is assumed to be around 2 IKR (equation 2), the cost of nematodes could be estimated around 1.147 million-IKR (equation 3). According to Statistical Iceland (2015), the value of all cod caught in the Iceland jurisdiction was around 61.000 million IKR in 2015. The cost of nematodes can have real effects on the profitability of the Icelandic seafood industry.
ퟐퟏퟓ.ퟎퟎퟎ.ퟎퟎퟎ 풌품/퐲퐞퐚퐫 퐂퐨퐝퐬 퐂퐨퐝퐬 퐜퐚퐮퐠퐡퐭 퐞퐚퐜퐡 퐲퐞퐚퐫 = = ퟕퟏ. ퟔퟕퟎ. ퟎퟎퟎ Equation 1 ퟑ 풌품 (풂풗풆풓풂품풆) 퐲퐞퐚퐫
ퟏퟖퟎퟎ 푰푺푲/풉풐풖풓 풔풆풌 퐓퐡퐞 퐜퐨퐬퐭 퐨퐟 퐧퐞퐦퐚퐭퐨퐝퐞퐬 퐝퐮퐫퐢퐧퐠 퐭퐫퐢퐦퐦퐢퐧퐠 = ( ) ∗ ퟒ = ퟐ 푰푺퐊/퐰퐨퐫퐦 Equation 2 ퟑퟔퟎퟎ 풔풆풌/풉풐풖풓 풘풐풓풎
32
푾풐풓풎풔 푰푺푲 퐂퐨퐝퐬 푰푺푲 푬풔풕풊풎풂풕풊풐풏 풐풇 풕풐풕풂풍 풄풐풔풕 = (ퟖ ∗ ퟐ ) ∗ ퟕퟏ. ퟔퟕퟎ. ퟎퟎퟎ = ퟏ. ퟏퟒퟕ 풎풊풍퐥퐢퐨풏 Equation 1 퐂퐨퐝 풘풐풓풎 퐘퐞퐚퐫 퐘퐞퐚퐫
2.13 Current laws and regulations concerning parasites in fish
2.13.1 Europe The European Food Safety Authority (EFSA) has concluded a scientific opinion that routine testing of seafood products for the presence of whale-worm is needed (BIOHAZ, 2010). As a safeguard measure for prevention of anisakiasis, regulations for raw, cold smoked, marinated or salted fishery products have been implemented in the European Union requiring freezing or heat treatments (European Commission 2004; Adams et al. 2005). EFSA has evaluated the risk for whale-worm’s contamination in Atlantic salmon farmed in floating cages and fed on an artificial diet to be negligible (BIOHAZ, 2010), and thus, this product has been exempted from mandatory freezing since November 2006 (European commission, 2011). The European commission recommends freezing ambient temperature of -20 °C for at least 24 hours or -35 °C for at least 15 hours. A heat treatment of at least 60 °C core temperature or more for 10 minutes before consumption was also included (Panel of biological hazards, 2010).
2.13.2 North America (USA & Canada) Similar regulations exist in the USA and Canada (FDA, 2012; Weir, 2005). These measure requirements have been adopted by the fish industry as parts of their Hazard Analysis, and Critical Control Points systems (HACCP) (FDA, 2011). The US Food and Drugs Administration agency (FDA) recommends freezing and storing -20 °C or below for at least 7 days, or freezing at -35 °C or below until solid and storing at -35 °C for 15 hours. This is explained by the higher resistance of Pseudoterranova spp., which is found in Canadian and Northern USA waters (Deardorff, Kayes, & Fukumura, 1991). The FDA also noted that the freezing may not be suitable for large fish (thicker than 6 inches). The time and temperature of cooking are similar as with the European Commission. These factors should reduce infection rates and, if whale-worm infection predisposes an individual to a related allergic response, there should also be a reduction in the prevalence of allergic reactions by ingesting nematode material in fish (Audicana et al, 2002).
33 3 Methods & Material
3.1 Experimental design Table 3.3. The experimental design for the scientific study
Experiment Title / descirption Factors Groups
1 Video analysis from the Trimming rate of fillets - None industry - Nematodes numbers and positions in fillets
2 Cheimical compositon of - Water, protein, - Large nematodes carbohydrates, lipids, nematodes ash and sodium - Small - Minerals and trace nematodes elements
3 The comparison in chemical - Water, protein, - Nematodes composition between carbohydrates, lipids, nematodes and cod muscle ash and sodium - Loins groups - Minerals and trace - Belly flaps elements
4 The amino acid composition of - Composition - Nematodes nematodes and cod proteins comparison of individual amino acids - Cod fillets
5 The freezing tolerance of - Sub-zero temperatures - Large nematodes nematodes - Time intervals - Small - Survival ratios nematodes
6 The cross-sectional cuts of - Thickness - All visible nematodes layers
7 The analysis of nematode - Species analysis - Loins species within cod fillets - Belly flaps - Large nematodes - Small nematodes
34 3.2 Nematode sampling procedures Prior to sampling of nematodes from Atlantic cod (Gadus morhua), it was decided to divide nematodes into two groups in an attempt to distinguish between whale-worm and seal-worm larvae. The speculation for the separation was the length of the nematodes, where the whale-worm larvae were assumed to be < 4 cm in length when found in cod, while seal-worm larvae can be from 3 to 6 cm in length (fig. 8.13). The nematode groups were therefore defined as follows:
Small nematodes (< 4 cm in length) Figure 3.9. Length measurement of nematodes. The nematode to the left is around 6 cm, the Large nematodes (> 4 cm in length) nematode to the right is around 4 cm
For the first sampling of nematodes, sampling packages were prepared and sent to selected fish processing companies. The sampling packages included ten 250 mL plastic containers with lids, 2 L of saline solution (9 g / L of NaCl) and instructions sampling procedures (e.g. sampling, handling and storage of the samples, as well as posting information). The collected nematodes samples were stored in saline solution as a precaution of drying and hence the structure and chemical composition of the nematodes was preserved. At each candling table, the nematodes were collected in the 250-mL plastic container with 100 mL of the saline solution (40 g of nematodes / 100-mL saline) (fig. 8.14). For the second sampling, a similar procedure was conducted as in the first sampling beside the storing solution because the sodium in the saline solution influenced the results of the analysis, therefore the solution was substituted with clean tap water. Based on the results of obtained in the first two sampling trials, the third and final sampling of nematodes was designed to explore the effects of storing solution on the proximate composition of nematodes, and hence no storing solution was applied in the final sampling. The nematodes were collected from the fish muscle and put in a plastic container at a trimming station. The process also included washing contaminants of the nematodes, categorization (length) and weighing.
A) B) C)
35 Figure 3.10. A) Collected nematodes from fish processors, B) The Nematode separation, weighing and cleaning, C) Prepared samples for measurement. All nematodes were first collected into plastic boxes that included water. The nematodes were taken out one by one, measured in length, and put in an empty dry container until further analysis.
3.3 Fish sampling During the third and last sampling of nematodes, samples of Atlantic cod (Gadus morhua) muscle were also collected for comparison purpose. The fish was caught near shore southwest of Iceland. The cod was bled, gutted and stored on ice until it was processed the next day. The day after the fish was beheaded and filleted. The fillets were washed and trimmed where bones, visual nematodes, and blood spots were removed. The loin part and the belly flaps of the fillets were separate (figure. 3.11) in a similar manner as for the collected nematode samples.
Figure 3.11. The positioning of each muscle part of the fish as targeted in the study. The lines in the picture mark the cuttings of the fillet. A = Loin, B = belly flap, C = Tail of the loin side, D = Tail
of the belly flap side.
3.4 Video analysis from the industry The candling procedure during fillet processing of cod, caught with trawls, was recorded by using the specific camera (GoPro Inc. America. Type; HERO3). The cameras were placed above the trimming table (figure 3.12) in order to revies the handling procedures, as well as to evaluate the distribution and position of nematodes in the fish muscle. The camera recorded video footage for 15 min at a time. All in all, there were 3 hours of recorded material. In addition, the trimming speed of cod fillets was compared to the infection numbers seen in the videos. The objective in that part was to analyze if the nematodes had a direct effect on the trimming rate of an individual.
36
Figure 3.12. The angle of the recordings in the processing step. The angle of the camera was straight on top of fillet, which would give visual accessibility on nematode infestation.
3.5 Chemical composition of nematodes and cod fillets
3.5.1 Water content The water content was measured by weighing 5 grams of mashed/minced sample with ceramic bowl and sand. The sample was then mixed with the sand and dried in an oven at the temperature of 103 °C for 4 hours. The samples were next allowed to cool down to ambient temperature in a desiccator. The water content was determined by the change in total weight of the sample before and after the drying process (ISO, 1999).
3.5.2 Sodium content The total content of sodium (NaCl) was determined by the Volhard Titrino method (AOAC, 2000).
3.5.3 Protein content Nitrogen was measured by the Kjeldahl-method (NMK, 2007). The protein content was achieved by multiplying the produced nitrogen quantity by 6.25.(ISO, 2009).
3.5.4 Carbohydrate content The carbohydrate content was calculated as the part or proportion of the total weight by deducting the rate of other nutrients from the total weight of the sample as is seen in the following equation: 𝑔 푇표푡푎푙 퐶푎푟푏표ℎ푦푑푟푎푡푒푠 ( ) = 100𝑔 − (푤푎푡푒푟(𝑔) + 푝푟표푡푒𝑖푛(𝑔) + 푙𝑖푝𝑖푑푠(𝑔) + 푎푠ℎ(𝑔) + 푠표푑𝑖푢푚(𝑔)) 100𝑔
3.5.5 Lipid content The lipids were extracted using Soxhlet method, according to the official methods and recommended practices of American Oil Chemists’ Society (AOCS, 1997).
3.5.6 Ash content The sample is weighted into an incineration dish. The incineration dish is placed on a hot plate over a gas burner and heated progressively until the test portion has carbonized. The dish is then transferred
37 to the muffle furnace, where it is heated to 550°C, and left for 3 hours. The sample is inspected visually on whether the ash is free from carbonaceous particles. The dish is then allowed to cool in the desiccator down to ambient temperature and weighed exactly (ISO, 2002).
3.6 Mineral & trace element analysis The method used for degradation of the samples prior to analysis was based on Sloth et alt. (2005) and the protocol of NMKL (2007), with modification. A microwave (Mars5, CEM, North Carolina, USA) was used for degradation of the samples. The nematode samples were freeze-dried followed by grinding. Each sample was measured in triplicates. Each triplicate weighted at 200 mg (accuracy = 0.1 mg) in degradation bombs, followed by addition of 3 mL HNO3 and 1.5 mL H2O2. The bombs were closed and put in the microwave oven where the samples were degraded. After degradation, samples were moved into 50 mL polypropylene tubes and diluted. The minerals in the sample were next measured with an inductively coupled plasma mass spectrometer (ICP-MS).
Reference samples were analysed parallel to each measurement. The reference samples are prepared with known mineral and trace elements content, to monitor the quality of the results. In addition, blanks samples were also measured, to observe if there were any contamination within the laboratory or during the handling of the samples. The limit of quantification was defined as four times the concentration of the blanks (NMKL, 2007; Gunnlaugsdóttir et al., 2010).
3.7 Amino acid composition The amino acid analysis was conducted at the Eurofins laboratory in Hamburg, Germany. Tryptophan content was analyzed using the method EU 152/2009, IC-UV (No). Other oxidative amino acids, including calculation of Tyrosine, was analyzed using ISO 13903:2005, IC-UV method (ISO 13903., 2005).
3.7.1 Determination of free amino acids Samples were weighted at 0.2 mg. The weighted sample is put in a conical flask, and 100 mL of extraction mixture is added. The mixture is shaken for 60 min using a mechanical shaker or a magnetic stirrer. The sediment is allowed to settle, and 10 mL of the supernatant solution is pipetted into a 100- mL beaker. Around 5 mL of sulfosalicylic acid solution is added with stirring with the aid of magnetic stirrer for 5 minutes. The supernatant is then filtrated, to remove precipitates. After, 10 mL of solution is placed into a 100-mL beaker; pH is adjusted to 2.20 using sodium hydroxide solution. The supernatant transferred to a volumetric flask of appropriate volume using a citrate buffer and is filled up with the buffer solution (ISO 13903, 2005).
3.8 The freezing survival of nematodes in fish muscle The main objective of the trial explored the effects of freezing and extended frozen storage on the survival of both Anisakiasis and Pseudoterranovosis, where different temperatures and time were compared. Boneless and skinless cod fillets were minced using a grinder (HALLDE.VCB-62. Grinder) (fig. 3.13). The sample preparation was executed by putting the right amount of cod mince in a plastic
38 sample container, compressed into a layer of around 2 cm thickness (figure 3.14), followed by a layer of selected nematodes, and finally topped with another layer of compressed cod mince. Prior to selection of nematodes, the nematodes could reach room temperature to increase their movements. It also made it easier in detecting live nematodes because they had to show mobility to be selected.
Figure 3.13. The making of the fish mince used in the experiment. The fish parts were all put in the HALLDE grinder and ground into a small grained mince. The
same mince was utilized for the whole experiment.
Figure 3.14. Fish mince used in this trial, to the top left, sampling boxes with the bottom layer of fish mince to the top right. The fish mince was stored in a closed polystyrene box which included a cooling medium between experiments. The prepared samples were frozen by using a blast freezer (Ilsa-CONVOCHILL-blast-freezer, Germany) which had been calibrated for the needed temperature. The samples were arranged on crates, to make a larger contact area for the wind flow (fig. 3.15). For each temperature step, temperature loggers (iButton temperature data logger type; DS1922L) were used to confirm and record the temperature. Four temperature loggers were employed in this part of the study. Two temperature loggers were used
39 to record the surrounding temperature inside the freezer during the experiment, and the other two loggers were used to record the temperature in the middle of the samples. The prepared samples were frozen at different temperatures without caps. The temperature steps employed in the trial were -5, -10, -15 and -20 °C. The time of freezing at each temperature was set at 6 hours, with the temperature interval set at one hour (table 3.3). At each time interval, two sample containers were removed from the freezer. The samples were thawed at a room temperature. After thawing, the nematodes were carefully separated from the mince and put in a clean container which included a 100 mL of warm (20 °C) deionized water (fig. 3.15). The survival rate was then visually analyzed. If nematodes showed movements, they were removed from the solution and counted. The nematodes which showed no movements were left in the solution for 1 hour more, or until they started moving, if they did not, then nematodes were estimated as dead. In the end, the survival number was calculated.
A) B) C)
Figure 3.15. A) Inside the ILSA-Convochill-blast freezer, B) Outside the blast freezer, C) The setup of for the analysis of nematode´s survival ratio.
Table 3.4. Results registration for the survival analysis of nematodes in sub-zero temperatures. The nematodes were frozen at four different temperatures, with the freezing time from 1 to 6 hours. Freezing time Frozen at (-5°C) Frozen at (-10°C) Frozen at (-15°C) Frozen at (-20°C)
Dead live Dead Live Dead live Dead live
1 h
2 h
3 h
4 h
5 h
6 h
40 3.9 PCR analysis of nematode species Prior to analysis, nematodes were categorized into two groups depending on their position in the fillet (loins vs. belly flaps). Moreover, nematodes were also analysed dependent on their length. The nematodes were stored for 3 weeks in a refrigerator in small glass flasks which included 1.5 mL of 96% EtOH. All flasks were marked specifically with a letter for the group and a number within each cluster (table 3.5).
The groups for PCR analysis:
1. Nematodes from the belly flaps of the cod fillet. 2. Nematodes from the loins of the cod fillet. 3. Large nematodes (>4cm) 4. Small nematodes (< 4cm)
3.9.1 DNA extraction A small part of the tissue of each sample was taken and placed in a tube. Next, 75 µL of Alkaline Lysis Reagent was added to each sample, followed by heating to 95 °C for 10 minutes. After heating, the samples were cooled down to 4 °C, and 75 µL of a Neutralization Buffer added (Truett et al., 2000).
3.9.2 PCR sequencing The test samples for the PRC reaction was composed with 10x Standard (1.50 µL) (table 3.4), 22 mM of Magnesium chloride (MgCl2) (1.50 µl) and 10 mM dNTP (1.50 µL)(table 8.5). The primers that were used for the sequencing were 211.F-100 µM- TTTTCTAGTTATATAGATTGRTTYAT (0.05 µL) and 210.R-100 µM-CACCAACTCTTAAAATTATC (0.05 µL). The reaction also included 1.0 µL of the DNA extraction, Taq Polymerase (0.12 µL) and water (9.28 µL). All sample at the total volume of 15 µL and at the annealing temperature of 50 °C. All samples were sequenced with 98% probability (p < 0.02).
Table 3.5. The ingredients and runs information for each PCR sample. For each nematode sample, only 1 uL of the DNA extract was needed. 15 uL reaction 1 reaction Runs Total cycles
10x Standard 1,50 5 min at 95°C
25 mM MgCl2 1,50
10 mM dNTP 1,50 1 min at 95°C 211.F 100 µM 0,05 210.R 100 µM 0,05 1 min at 50°C 35 DNA 1,00 Taq Polymerase 0,12 1 min at 50°C Water 9,28
Total 15 5 min at 72°C
Annealing 50°C
41 Table 3.6. The order of all groups during PCR sequencing. Each group was marked with a letter and a number for the sample number in each cluster. 10 nematode samples were collected for each group. The group P represents test samples for calibration. Sequencing 1 Sequencing 2 Nematodes 1 2 3 4 5 6 7 8 9 10 A H-1 H-2 H-3 H-4 H-5 H-6 H-7 H-8 H-9 H-10 B L-1 L-2 L-3 L-4 L-5 L-6 L-7 L-8 L-9 L-10 C S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 D P-1 P-2 P-3 P-4 P-5 P-6 P-7 P-8 E Þ-1 Þ-2 Þ-3 Þ-4 Þ-5 Þ-6 Þ-7 Þ-8 Þ-9 Þ-10 * H = Loins (row A) * Þ = Belly Flaps (row E) * S = Large nematodes (row B) * L = Small nematodes (row C) * P = Test sample (row D)
3.10 Estimation of thickness of nematode protective wall The analysis of the thickness of nematode´s protective skin was performed in to order to gain a necessary knowledge regarding their physicochemical properties. Nematodes of different size were selected for this trial. Individual nematodes were collected and measured in length and thickness. The nematodes were placed in a small plastic tube (2 cm thick), where it was important for nematodes to have a vertical arrangement. After arranging the nematodes correctly, the medium (Leica Biosystems- tissue freezing medium) was sprayed around the nematodes. This was conducted inside a Cryostat machine (Leica Biosystems; Cryostat type; CM1800, Germany) where the surrounding temperature was calibrated at -20 °C (fig. 8.21). After exposing the medium, it waited until the sample was frozen solid. The sample was removed from the tube and placed at the right cutting angle in the Cryostat machine for cross-sectional cuts using a microtome blade (Thermo Fisher Scientific, premium disposable microtome blade type; MX35-ULTRA, United States). The thickness of the cuttings varied from 20 µm up to 40 µm. The cross-sectional slices were taken and put on a microscope glass slides. Each sample was washed with deionized water for protection against drying, followed by washing with ethanol (70%). At last, the samples were analysed using a binocular microscope (OLYMPUS-stereo microscope type; SZX9, Olympus Corporation, Japan) and a magnification lens (OLYMPUS-stereo microscope lens type;
DF-PLAPO – 1X-2. Olympus Corporation, Japan). The cross-sectional cuttings were then visually analysed.
42
Figure 8.16. The equipment used for the cross-sectional cuts. The cutting blade setup is seen on the left; it includes a nematode sample. The figure in the middle is the full look of the machine.
3.11 Data handling All data handling such as tables and graphs were saved and performed using spreadsheet application (Microsoft Office Excel. Year: 2013; USA). Most of the statistical analysis were descriptive, because of lack of measuring point which in most of the experiments. The nematodes were difficult to sample, and in most cases only allowed for one analysis each time.
43
44 4 Results
4.1 Video analysis from the industry The distribution of nematodes within fillets were analyzed visually with video recordings from a trimming station. From 120 cod fillets, around 131 nematodes were observed. From the 131 nematodes, 86 and 19 nematodes were found in the belly flaps and in the tail section of the belly flaps, respectively (figure 9.22). Moreover, 19 nematodes were detected in the loin section and 3 in the tail part of the loin section. The trimming capacity was also evaluated to explore how the nematodes affected the processing time of the fillets. The task was to evaluate if there was a direct relationship between the trimming time and the number of nematodes per cod fillets. The average trimming time was calculated 1.88 fillets/min and the average nematodes per fillets were 0.7 nematodes/fillet (figure 4.18). As figure 4.18 shows, there was no correlation between trimming speed and the infection intensity of cod fillets caught with trawls far from the shore. The correlation coefficient of the trimming rate was 0.1182 compared to 0.8722 of the infection intensity (figure 4.18). In the video, there were other factors that affected the trimming time.
100
90
80
70
60
50
40 Nematodes(N) 30
20
10
0 Belly flaps Loins Tail of the belly flaps Tail of the loins
Figure 4.17. Distribution of nematodes in cod fillets (n = 120) as visually detected with video recordings from a trimming station.
45 1,4 Nematodes per fillet Trimming speed (Fillets / min) 2,5
1,2 2 1
1,5 0,8
0,6 1
0,4 0,5
0,2 (fillets/min)speedTrimming Infection intensity (nematodes / / fillet)(nematodesintensityInfection 0 0 1 2 3 4 5 6
Figure 4.18. Effects of nematode infection intensity on the trimming speed of fillets caught with trawl nets far from the shore.
4.2 Chemical composition
4.2.1 Chemical composition of nematodes The chemical composition of both the large and small nematode groups was evaluated (Table 4.6). The results indicated that the carbohydrate content was higher in larger nematodes, however, the difference was not significant. In addition, the water content of both groups was not significantly different. The protein, lipid, and sodium contents were a little higher in smaller nematodes, but there was no significant difference. The ash contents were measured at 0.8 % for both large and small nematodes.
The iron content was measured at the same levels in both nematode groups at 1.1 mg/100g. The copper content was 0.31 mg/100g for large nematodes and 0.26 mg/100g for small nematodes. The large nematode group had a higher content of copper, sodium, potassium, phosphorus, calcium and magnesium while the small nematode group had a higher amount of arsenic. Both nematode groups had equal amounts of other trace elements (Hg, Pb, and Cd). For the lack of measuring points, there was no statistical analysis performed on these results.
46 Table 4.7. The chemical composition comparison of large and small nematodes. Chemical composition Large nematodes Small nematodes
(g / 100g) (g / 100g)
Water 74,02 ± 2,9 75,9 ± 3,0
Protein 10,3 ± 0,3 10,7 ± 0,32
Carbohydrates 14,25 11,8
Ash 0,8 ± 0,016 0,8 ± 0,016
Lipids 0,5 ± 0,04 0,6 ± 0,048
Sodium chloride 0,15 ± 0,004 0,2 ± 0,006
Potassium (K)* 180 mg 160 mg
Phosphorus(P)* 162 mg 140 mg
Sodium (Na)* 45 mg 42 mg
Calcium (Ca)* 26 mg 21 mg
Magnesium (Mg)* 16 mg 15 mg
Iron (Fe) 1,1 mg ± 0,22 1,1 mg ± 0,22
Copper (Cu) 0,308 mg ± 0,06 0,258 mg ± 0,05
Arsenic (As) 37,6 µg ± 7 µg 41,2 ± 0,008 µg
Mercury (Hg) < 2 µg ± 0,4 < 2 µg ± 0,4
Lead (Pb) < 1 µg ± 0,2 < 1µg ± 0,2
Cadmium (Cd) 2 µg ± 0,4 2 µg ± 0,4
4.2.2 Comparisons between nematodes and cod muscles The water content of the cod muscle groups was higher compared to the nematodes, or 83 % ± 0.6 s. d. and 75.0 % ± 1.3 s.d., respectively (figure 4.20). The protein content was lower in nematodes (10.5 % ± 0.3 s.d) compared to cod muscle groups (16.1 % ± 0.42 s.d). The difference between groups was recorded in the carbohydrate contents. No carbohydrates were present in the cod muscle while the nematodes measured with 13% ± 1.7% s.d of carbohydrates (figure 4.20).
Despite the differences, there were similarities between all groups. The sodium chloride (NaCl) content was around 0.2 g/100g. In addition, the lipid contents were similar for all groups where the nematodes were measured with the mean of 0.55 g/100g compared to 0.4 g/100g for the cod muscle parts, see in fig. 4.21 and table 13 in appendix I.
Comparison of minerals in nematodes and cod muscle revealed that the copper (Cu) content of nematodes measured 0.28 mg/100g compared to 0.01 mg/100g for both muscle groups (table. 4.7). The iron (Fe) content of nematodes measured 1.1 mg/100g compared to 0.05 mg/100g for both muscle groups. In addition, the calcium (Ca) contents of nematodes measured 23.5 mg/100g compared to 8
47 mg/100g for both muscle groups. Both cod muscle groups had higher contents of potassium, sodium, magnesium when compared to nematodes (table. 4.7).
The arsenic content of nematodes was much lower, or at 39.5 µg/100g compared with 123 µg/100g for the belly flap muscle and 243 µg/100g for the loins. The mean mercury levels of nematodes were also lower than in both muscle groups, or < 2 µg/100g compared with 5 µg/100g, for both loins and belly flaps. The lead contents of nematodes were also lower with < 1 µg/100g compared with < 7 µg/100g for cod muscle. In addition, the cadmium contents of cod were higher, or < 5 µg/100g compared to 2 µg/100g in nematodes (table 4.7).
90 83,3 82,4 80 75
70
60
50 Nematodes Belly Flap 40 g / / g 100g Loins 30
20 15,8 16,4 13 10,5 10 0 0 0 Water Protein Carbohydrate
Figure 4.19. Water, protein and carbohydrate content of fresh cod muscles (loin and belly flap) and nematodes present in the cod fillet.
1,2 1,1
1,0
0,8 0,8
Nematodes 0,6 0,55 Belly Flaps g / / g 100g 0,4 Loins 0,4
0,175 0,2 0,2
0,0 Ash Lipids Sodium chloride
Figure 4.20. Ash, lipid and sodium contents of fresh cod muscles (loins and belly flaps) and nematodes present in the cod fillet.
48 Table 4.8. The contents of minerals and trace elements in nematodes (mean) and cod muscle groups. Minerals and trace Nematodes mean Belly flap Loins
elements (mg / 100g) (mg / 100g) (mg / 100g)
Iron (Fe) 1,1 ± 0,22 0,06 ± 0,012 0,04 ± 0,008
Copper (Cu) 0,283 ± 0,056 0,011 ± 0,002 0,009 ± 0,0018
Sodium (Na)* 43,5 65 49
Potassium (K)* 170 415 449
Phosphorus (P)* 151 156 164
Calcium (Ca)* 23,5 8 7
Magnesium (Mg)* 15,5 24 23
Arsenic (As) 39,4 ± 7,8 µg 123 ± 24,6 µg 243 ± 48,6 µg
Mercury (Hg) < 2,0 ± 0,4 µg 5,1 ± 1,0 µg 5,4 ± 1,0 µg
Lead (Pb) < 1,0 ± 0,2 µg < 7,0 ± 1,4 µg < 7,0 ± 1,4 µg
Cadmium (Cd) 2,0 ± 0,4 µg < 5,0 ± 1 µg < 5,0 ± 1 µg
* = Measuring method is not accredited
4.3 Amino acid composition of nematodes and cod proteins Th amino acid composition of nematodes and cod proteins were analyzed and compared (figure 4.22). The highest difference in amino acid contents was nematode exceeded the contents of the cod fillet was seen in cystein+cystine, or 2.5 % ±. 0.25 against 1.2 % ± 0,12 for the cod proteins. Also, nematodes had higher ratios of glycine (7 % vs. 4.5 %) and tyrosine (3.5% vs.2%). There was also a high difference measured for proline, where nematodes were measured with 6.3% of total proteins against only 3.3% for the cod muscle proteins (figure,4.22) and table 15 in appendix I. The amino acids which did not show significant difference in both groups were glutamic acid, histidine, hydroxyproline, isoleucine, phenylalanine, ornithine, serine, threonine, valine, and tryptophan respectively (figure 4.22). It was not possible to perform statistical analysis in this section because the samples were only measured once, and the lack of measurements resulted in no significant difference between the tested groups.
49 18,0% 16,0% 14,0% 12,0% 10,0% 8,0% 6,0% 4,0% 2,0% 0,0%
Nematodes (% of total proteins) Cod muscle (% of total proteins)
Figure 4.21. The amino acid composition of nematode and cod proteins
4.4 Nematode survival in sub-zero temperatures In the analysis of nematodes tolerance against sub-zero temperatures, both small and large nematode groups tolerated -5 °C (figure. 4.23). All nematodes survived the first two hours of freezing at -5° C). However, after 3 hours, the survival number decreased slightly down to 93% for the small nematodes and 87% for the large nematodes. After 4 hours, both groups had the same survival ratio of 84%. However, after 5 hours at -5 °C the survival rate decreased down to 78% for the large nematodes and 68% for the small nematodes. After 6 hours, the survival ratio for both groups was measured at 67% (figure. 4.23). The temperature data log from the core of the sample did not reach the acceptable mark (figure 4.24). The core temperature was ranging from -0.5 °C down to -1.5 °C. This might be explained by the unstable temperature inside the freezer, but the inside temperature was fluctuating from -2 °C down to -9.8°C, (figure 35) in appendix III.
The treatment of -10 °C freezing resulted in 100% survival ratio for both nematode groups after 1 h (figure. 4.25). After 2 h, the survival ratio had decreased slightly down to 93% for the small nematodes and 89% for the large nematodes. After 3 h of storage, the survival ratio dropped drastically, which resulted in 50% survival ratio. However, no nematode survived after 4 h of freezing at -10 °C. The temperature data loggers of the core of the sample were -1 °C after 1 h, -1.3 °C after 2 h, -4 °C after 3 h, and -9.4 °C after 4 h (figure 4.26). The temperature inside the freezer resulted in temperature fluctuations from -6 °C down to -14 °C, (figure 36) in appendix III. When the nematodes were frozen at -15 °C, 77% of the large nematodes and 81% of the small nematodes survived after 1 h (figure 4.27). After 2 h, only 46% of the small nematodes and 23% of the large nematodes survived. However, after 3 h, the nematodes showed no signs of survival.
50 The temperature inside the mince samples after 1 h was -0.6 °C, and -3.5 °C after 2 h (figure. 4.28). However, after 3 h of freezing, the sample temperature was recorded at -15.5°C. The temperature loggers from inside the freezer recorded temperature fluctuations from -11° C down to -18 °C, (figure 37) in appendix III. At -20 °C freezing, the nematodes showed a steeper decline in survival compared with survival at -15 °C (figure. 4.29). The survival ratio after 1 h was measured at 43% of the small nematodes and 33% for the large nematodes. However, after only 2 h of freezing, there were no signs of survival in both groups. The temperature was -1.0 °C after 1 h freezing time (fig. 4.30). After 2 h of freezing, the temperature was measured at -15.6°C (time of no survival). The overall comparison between the survival ratios of nematodes at different freezing temperature is summarized in the figure. 4.31.
120,0%
100,0%
80,0%
60,0%
40,0% Survival Survival ratio
20,0%
0,0% 0 1 2 3 4 5 6 Freezing time (h)
Small nematodes (-5 °C) Large nematodes (-5 °C)
Figure 4.22. The survival ratio of nematodes frozen in fish mince at -5 °C. Each sample box included 15 nematodes.
51 8
6 C) ° 4
2
0 Temperature( -2
-4 0 60 120 180 240 300 360 Time (min)
Logger 3 Logger 4
Figure 9.23. The temperature within the mince sample frozen at -5 °C.
120%
100%
80%
60%
40% Survival Survival ratio
20%
0% 0 1 2 3 4 5 6 Freezing time (h)
Small nematodes (-10 °C) Large nematodes (-10 °C)
Figure 4.24. The survival ratio of nematodes frozen at -10 °C. Each sample box included 15 nematodes.
52 6 4
2 C) ° 0 -2 -4 -6
Temperature( -8 -10 -12 0 60 120 180 240 300 360 Time (min)
Logger 3 (-10°C) Logger 4 (-10°C)
Figure 4.25. The temperature within the fish mince sample at -10 °C.
100%
80%
60%
40% Survival Survival ratio 20%
0% 0 1 2 3 4 5 6 Freezing time (h)
Small nematodes (-15°C) Large nematodes (-15°C)
Figure 4.26. The survival rate of nematode groups frozen at -15°C. Each sample box included 15 nematodes.
53 10
5 C) ° 0
-5
-10
Temperature( -15
-20 0 60 120 180 240 Time (min)
Logger 3 Logger 4
Figure 4.27. The temperature in the sample at -15°C.
100%
80%
60%
40% Survival Survival ratio 20%
0% 0 1 2 3 4 5 6 Freezing time (h)
Small nematodes ( -20°C) Large nematodes (-20°C)
Figure 4.28. The survival rate of nematodes frozen at -20°C. Each sample box included 15 nematodes.
54
10
5
C)
° 0
-5 -10
-15 Temperature( -20
-25 0 60 120 180 240 300 360
Time (min)
Logger 3 (-20°C) Logger 4 (-20°C)
Figure 4.29. The temperature in the sample at -20 °C.
100,0%
80,0%
60,0%
40,0% Survival Survival ratio
20,0%
0,0% 0 1 2 3 4 5 6
Freezing time (h)
Small nematides (-5 °C) Large nematodes (-5 °C) Small nematodes (-10 °C) Large nematodes (-10 °C) Small nematodes (-15 °C) Large nematodes (-15 °C) Small nematodes (-20 °C) Large nematodes (-20 °C)
Figure 9.30. The overall comparison of tolerance against sub-zero temperatures for both nematode groups. It´s seen that the decreasing temperature is very effective. At -10 °C nematodes die after 4 h, and at -15 °C they all die after 3 h.
55 4.5 Estimation of thickness of the nematodes skin layer The cross-sectional shape of the nematode body was more oval than circular (figure 4.32) with at least three layers. The outer layer was rather thick or approximately 36% of the nematode´s total width or 180 µm all around the core for a nematode with the average with of 1000 µm in diameter. However, the thinnest part of the outer layer was measured at 120 µm around the core (figure. 4.32).
The thickness of the inner layer was measured at 60 µm all around the core. In addition, the inner layer looked like having equal width all around the core. The inner part of the nematode, or the core, was more circular shaped than the rest of the body. The width of the core of a nematode with 1 mm in diameter was estimated to be approximately 470 µm of the width of a nematode with 1000 µm in diameter (figure 4.32).
Figure 4.31. The nematode thickness was measured at 1.0 mm. There are three different layers marked, the inner and outer protective layers and the core in the middle.
56 4.6 Nematode species identification using PCR The results for nematode species identification using polymerase chain reaction (PCR) analysis (table 4.8). Eight nematodes removed from the loins were identified as seal-worms, and two nematodes were identified for other species (table 4.8). In the belly flaps, nine out of ten nematodes were identified as seal-worms and one from another nematode species.
For large (> 4 cm) randomly found nematodes, all were identified for the seal-worm genus, while nine out of ten of the small (< 4 cm) nematodes were determined for the seal-worm genus and one from another nematode species.
In the species analysis, no nematodes were identified for the whale-worm genus (table 4.8). The other nematode species were identified as Pseudoterranova krabbei, a nematode species that is very related to seal-wroms.
Table 4.9. Species identification of nematodes found in Atlantic cod (Gadus morhua). *Nematodes collected from the loin and the belly flap of the cod fillets were analyzed as well as large and small nematodes Nematode groups* N Seal-worms Whale-worms Others
From the cod loin 10 8 0 2
From the cod belly flap 10 9 0 1
Large nematodes (>4 cm) 10 10 0
Small nematodes (<4 cm) 10 9 0 1
57
5 Discussions
5.1 Video analysis from the industry The candling technique while trimming is the usual way of detecting and removing parasites. During the visual inspection of trimming cod fillets, it was noticed that the most detected nematodes were moved by slicing or cutting. For example, if there were large numbers of nematodes in the belly flaps the trimmer would rather cut a large piece off rather than pick out individual nematodes (5.32). The use of the cutting technique may increase the processing performance of a trimmer because the time in cutting large parts from the rest of the fillet is much shorter than the removing nematodes using the picking technique. The video recordings showed that it took a second to remove nematodes by cutting compared to 4 seconds using picking. For those reasons, it was not possible to get a clear view on the effects nematodes can have on the trimming rate of cod fillets. In addition, there are other factors that have similar influences on the trimming rate, for example, filleting defects like bones, dirt, and leftover fish skin. The size of fillets can also affect the rate of trimming (fig. 5.32). Where the lack of light permeability in thicker fillets makes it harder for the trimmer to detect and remove nematodes. The results of the video analysis supported similar older studies, where the highest numbers of nematodes were also found in the belly flaps of cod fillets (Sigurðsson, 2016). The reason for higher numbers of nematode in belly flaps is because of the infection sites. Fish will eat nematode infected marine animal; through digestion, the nematodes penetrate the stomach wall of its new host and becomes free in the visceral cavity. From there it is thought that nematodes penetrate the nearest muscles which are the belly flap (Stromnes & Andersen, 2000). The lower number of nematodes found in the loins do not specify the true infection number of nematodes as the detection technique only covers 0.6 mm on each side of the loin (Bublitz and Choudhury, 1992).
Figure 5.32. To the left, the lack of light permeability in large fillets. The black spot is heavily nematode infected area in the right sided figure.
59 5.2 Chemical composition of nematodes and cod muscle The results showed that nematodes had more carbohydrates than the cod muscle parts, as it is known that the nematode outer body structure is a complexed compound of oligo and polysaccharides (Wharton & Aalders, 2002). The carbohydrate content in the present study was slightly lower than in previous studies (Dagbjartsson, 1973). The cod muscle contained, however, no carbohydrates. This difference in chemical composition might give opportunities to identify nematodes deep in the fish muscle. There might be a way for a detection technology which could detect either the density of these sugars or the detection of (-OH) components which are commonly found in the oligosaccharide’s chemical structure.
The protein content of the nematodes in the present study was rather lower compared to other studies, or 10.5% and 13.5%, respectively (Dagbjartsson, 1973). The difference might be due to different sampling procedures. Where the earlier study might have had dry nematode samples, or that nematodes in this study had higher water contents than previously measured. However, when the ratio of water is increased, the ratio of dry matter (proteins and minerals) decreases. This is described with the principle of conservation of mass (Reddy, 1993).
The lipid content of nematodes varied between measuring points. In the first analysis, nematodes were measured with 0.3 % lipids compared to 0.55 % of lipids in the latest analysis. This might give hints on a seasonal variation in the fat contents of nematodes or inconsistency in analysing method. When the lipid content was compared with the older study (Dagbjartsson, 1973), it was noticed that the lipid content of nematodes was 0.8%, this difference could also be because of the loss of water.
The mineral contents in the two nematode groups showed similar quantities. However, differences were detected between nematodes and the cod muscle. The nematodes had a higher content of calcium, iron, and copper compared to cod muscle parts. The calcium level was 68% higher in the nematodes compared to the cod muscle. The iron and copper content were also of 95% and 96% higher content than in the cod muscle, respectively (table 14 in appendix I). When these results were compared to an older study (Dagbjartsson, 1973) there were similarities in copper contents (0.2 mg/100g vs. 0.27 mg/100g). The sodium contents between studies were, however, different (70 mg/100g vs. 40 mg/100g). In respect to the old study, it did not measure all minerals that were analysed in this study.
The minerals calcium and copper might both be promising indicators for the future. Possibly with technology that can either detect calcium or copper densities deep in the muscle. The iron, however, might not be so successful, because the processing of the fish might include blood stains in which would damage the outcome of the scanning technique.
The trace elements results did not indicate any significant danger or indicators for nematodes. However, for all trace elements, the cod measured with higher quantities. The trace element contents in the Atlantic cod might vary with age, size and feeding areas of the individual fish. Older and matured fish is e.g. more likely to have higher contents of, for example, mercury (Gunnlaugsdóttir et al., 2010).
Looking at the amino acid composition the main difference between nematodes and cod muscle was in the amount of glycine, cysteine, lysine, proline, and tyrosine, where the nematodes had higher amounts of glycine, cysteine, proline and tyrosine.
60 Proline was the amino acid which was found in higher quantities in the nematodes compared to cod. Proline can act as a structural disruptor in the middle of secondary structure elements such as alpha helixes and beta sheets. It is also found in turns and aids the formation of beta turns (Morris et al., 1992). The higher proline contents of nematode might suggest nematode having proteins which contain higher numbers of beta turns.
Cysteine is often in the same category as hydrophobic amino acids, though it is sometimes also classified as slightly polar. However, cysteine is generally covalently bonded to other cysteine residues to form disulfide bonds. This might suggest that nematode protein are less likely to fold compared to cod muscle proteins, as disulphide bonds play an important role in folding stability of some proteins (Sevier & Kaiser, 2002).
The amino acid composition of proteins is not a good indicator in determining if there are specific amino acids that have more allergenic potential. The allergens are more complexed in size and function, and cannot be investigated using only the composition of the proteins. The comparison between both groups shows some similarities in amino acid composition.
5.3 Nematode survival in sub-zero temperatures The effects of temperature and time on freezing tolerance of nematodes was measured in the present study. There were factors that could have influenced the physical and survival state of the nematodes. For example, nematodes that were removed from the fish muscle in the processing might have been damaged during sampling. Such damage might have affected the survival state of the sampled nematdoes and increase the uncertanity in the analysis.
The study showed that the survival ratio declined with lowered temperature and increasing time. The effects of lowered temperature were substantial, where nematodes did not survive -15 °C and -20 °C even after 2 hours of storage. Nematodes did, however, show some tolerance against freezing at -5 °C and -10 °C. At -5 °C, around 70% of the nematodes survived after 6 hours of freezing.
The comparison between large and small nematodes did not give a significant difference. However, the smaller nematodes tended to have little more tolerance towards sub-zero temperatures than the larger nematodes. In the present study, the small nematodes had higher lipid content than the large nematodes. Moreover, the nematodes have oligosaccharides in their structure which have cryoprotectant function, which protects biological tissues from freezing damages (i.e. due to ice formation) (Wharton and Alders, 2002).
The results showed that time and temperature could both be powerful attributes in lowering the survival ratio of nematodes in fish muscle and therefore lowering the odds of the consumer being exposed to nematodes.
The temperature loggers showed that the core temperature of the sample did not reach an acceptable temperature when the samples were frozen at -5 °C. The reason behind that might be dependent on the low freezing rate and the thickness of the sample. The insulation properties of the fish mince made the freezing process harder in killing nematodes. Also, the results showed that there was
61 a slight decline in survival with increasing time, meaning that the time might have affected the survivability at -5 °C.
Nematodes only survived 3 hours at -10 °C. After 3 hours, the core temperature of the sample had reached around -3 °C. The time and temperature of which no nematode survived were 4 hours, where the core temperature was measured at -8 °C. The temperature loggers also showed that by decreasing the freezing temperature increased the freezing rate. When the results were compared to an older study of Wharton and Alders (2002), where whale-worms did not survive exposure to -15°C, even after 10 min at that temperature, confirms these findings. In addition, the results indicated nematode´s partial tolerance of exposure to -5°C and -10 °C (Wharton and Aalders, 2002), which also relates to the findings of this study.
5.4 Estimation of thickness of the nematodes skin layer The cross-sectional cuts of nematodes gave useful information regarding their physical structure. The oval shape was mainly formed by the protective skin layer. Also, the skin layer appeared to be thicker on the top and the bottom of their structure. This factor could be the reason for the nematodes round and spiral shape when they are found in the cod muscle. The rounded structure might give them more overall protection and could serve as a defensive mechanism (Wharton and Alders, 2002). It could also be the reason why they are found in spiral shapes in cod fillets. The estimation of the thickness of the nematode layers indicated that the outer layer is a large part of the nematode structure. However, the results also indicated that the thickness of the outer layer is not of equal width all around the nematode.
The applied cross-sectional cuts were from the thickest parts of the nematodes. Some factors might have influenced the results. The handling of nematodes during that process might have had an effect on the structure, as the nematodes were frozen in a freezing medium. Meaning, when the nematode froze their structure expanded because of the effects of phase change of water molecules (from liquid to solid) (Mishima & Stanley, 1998).
5.5 PCR analysis of nematode species in cod muscle The PCR analysis of nematode present in cod muscle indicated that there were no whale-worm larvae present in either the belly flaps or loins. There were only two nematode species found, seal-worm and P. krabbei. The results indicated that the cod was caught near shore and that it had been exposed to seal colonies or other infection related lifeforms (Hauksson, 1991).
However, this study cannot clearly confirm that there are no whale-worms in the Atlantic cod because it has yet to be researched on a larger scale. In these studies, it had been suggested to analyze dark coloured nematodes, to get the confirmation if they were whale-worm larvae or not. However, the dark coloured nematodes came out negative. This could mean that older studies (Sigurdsson, 2016) which indicate dark nematodes as whale-worms might not be so accurate.
62 6 Conclusion
Differences were noticed in the chemical composition comparison between nematodes and cod muscle groups. These differences in chemical contents suggest that nematodes may include specific indicators that can be used for new detection technologies.
The experiment on freezing survival did show that freezing is very effective in killing nematodes in various sizes and shapes which are isolated in a thick fish medium. The preservation method of freezing fish is therefore very effective in preventing nematode diseases such as Anisakiasis and/or Pseudoterranovois. Also, it can be included that the experiment gave hints on nematodes not having as much tolerance against freezing like older studies have shown (Wharton and Aalders, 2002). However, it should be included, that the parameters used in this part of the study may not be as suitable for larger muscles, or larger than 4 cm.
From the videos in the industry, it was noticed that fillets caught with trawl-nets far from the shore, had lower infection intensity compared to fish caught near shore with line-fishing gear (Sigurdsson, 2016). The nematode prevalence in certain parts of the fillets was noted and showed similarities compared with older studies (Sigurdsson, 2016). For the trawl caught cod, there were no signs of nematodes having any effects on the trimming time of fillets. Looking at all areas, there are few other factors that do also effected the trimming time of the fillets. Factors such as handling and filleting defects, like bones, skin, dirt, blood spots/bruises and gaping. The nematode effect on the industry was clearly seen in the video footage. The loss in the utilization of individual fillets due to nematodes was visible.
The nematodes extracted from cod were all categorized into two groups depending on their length. The length being a factor in the speculation of separating whale-worms from seal-worms. However, all nematode samples were collected from the same processing plant which was specialized in processing of line-caught fish. The species analysis using PCR suggested that there were no whale- worms in either length dependent groups. The separation by length is therefore not as reliable as was previously thought. In the species analysis, there were only 10 larvae included in each group, which does not give much reliability. The PCR analysis of nematode DNA is needed to be researched on a larger scale with the more diversified raw material to see if whale-worms are present.
The amino acid composition comparison between nematodes and cod muscle indicates some amino acids being ratios of total proteins of nematodes and cod muscle. Some amino acids had similar ratios in nematodes and cod muscle and some amino acids had more variable contents. It was noted that both nematode and cod fillets inlcuded high contents of all essential aminoa acids that were measured.
63 7 Future perspectives
The analysis of nematode species and comparison between nematodes positioned in the loins vs. belly flaps is needed to analyzed on a larger scale. Such research would give clearer hints in finding out if whale-worms are found in the loins and/or belly flaps of the Atlantic cod in Icelandic waters, because of the dangers that follow whale-worm larvae. Future researches are needed on nematodes, especially on their distribution with regards to fishing areas and their presence in muscles of all commercial marine species that have been known to host nematodes. The reason behind that; to increase knowledge on the distribution of whale-worms, as they can be harmful to consumers.
Future research would be to develop a new detection technology for the industry. A technology that is more efficient and effective in detecting nematodes than current methods. The lack of efficiency ins current methods affects both the profitability and liquidity of the raw material. In addition, the increased efficiency would make the product safer for consumers. In addition, further research is needed in developing new technology specialised in removing nematodes from fillets. Furthermore, a technology that is more efficient and effective than an employee at a trimming station. It would possibly increase the utilization of cod fillets and leave fewer visible scars on fillets.
Further knowledge is needed on the processing methods that can possibly be applied to reduce survivability of nematodes. In other words, the effects of salting, drying and superchilling on the survivability of nematodes. Such studies could assist in knowing which processing method is more effective in preventing nematode diseases. Other areas require further research on e.g. increase the knowledge on the frequency and the causes of a nematode allergies. Such research would assist in identifying the severity of the hypersensitivity caused by the specific nematode allergens.
64 References
AOAC. (2000). Official methods of analysis 937.18. Salt (Chlorine as Sodium Chloride) in seafood. 17th
Ed. Association of Official Analytical Chemists.
American Oil Chemists’ Society. (1997). Official methods and recommended practices of the AOCS (5th
ed.). Champaign: AOCS Press.
Alonso-Gomez, A., Moreno-Ancillo, A., Lopez-Serrano, M. C., Suarez-de-Parga, J. M., Daschner, A., Caballero, M. T., Cabanas, R. (2004). Anisakis simplex only provokes allergic symptoms when the worm parasitises the gastrointestinal tract. Parasitology Research, 93(5), 378-384. doi:10.1007/s00436-004-1085-9 Alonso, A., Moreno-Ancillo, A., Daschner, A., & Lopez-Serrano, M. C. (1999). Dietary assessment in five cases of allergic reactions due to gastroallergic anisakiasis. Allergy, 54(5), 517-520. Anadon, A. M., Romaris, F., Escalante, M., Rodriguez, E., Garate, T., Cuellar, C., & Ubeira, F. M. (2009). The Anisakis simplex Ani s 7 major allergens as an indicator of true Anisakis infections. Clin Exp Immunol, 156(3), 471-478. doi:10.1111/j.1365-2249.2009.03919.x Anibarro, B., Seoane, F. J., & Mugica, M. V. (2007). Involvement of hidden allergens in food allergic reactions. J Investig Allergol Clin Immunol, 17(3), 168-172. Armentia, A., Martin-Gil, F. J., Pascual, C., Martin-Esteban, M., Callejo, A., & Martinez, C. (2006). Anisakis simplex allergy after eating chicken meat. J Investig Allergol Clin Immunol, 16(4), 258- 263. Aspholm, P. E. (1995). Anisakis Simplex Rudolphi, 1809, Infection in Fillets of Barents Sea Cod Gadus- Morhua L. Fisheries Research, 23(3-4), 375-379. doi: Doi 10.1016/0165-7836(94)00348-Z Audicana, M. a. T., Ansotegui, I. J., de Corres, L. F., & Kennedy, M. W. (2002). Anisakissimplex: dangerous—dead and alive? Trends in parasitology, 18(1), 20-25. Audicana, M. T., & Kennedy, M. W. (2008). Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity. Clin Microbiol Rev, 21(2), 360-379, table of contents. doi:10.1128/CMR.00012-07 Aznar, F. J., Balbuena, J. A., Fernández, M., & Raga, J. A. (2014). Establishing the relative importance of sympatric definitive hosts in the transmission of the sealworm, Pseudoterranova decipiens a host-community approach. NAMMCO Scientific Publications, 3, 161-171. Berland, B. (1961). Use of glacial acetic acid for killing parasitic nematodes for collection purposes. Nature, 191, 1320-1321. Brattey, J., & Bishop, C. A. (1992). Larval Anisakis-Simplex (Nematoda, Ascaridoidea) Infection in the Musculature of Atlantic Cod, Gadus-Morhua, from Newfoundland and Labrador. Canadian Journal of Fisheries and Aquatic Sciences, 49(12), 2635-2647. doi:Doi 10.1139/F92-292 Bublitz, C. G., & Choudhury, G. S. (1993). Effect of light intensity and color on worker productivity and parasite detection efficiency during candling of cod fillets. Journal of Aquatic Food Product Technology, 1(2), 75-89.
65 Burt, M. D. B., Campbell, J. D., Likely, C. G., & Smith, J. W. (1990). Serial Passage of Larval Pseudoterranova-Decipiens (Nematoda, Ascaridoidea) in Fish. Canadian Journal of Fisheries and Aquatic Sciences, 47(4), 693-695. doi:Doi 10.1139/F90-077 Caballero, M. L., & Moneo, I. (2004). Several allergens from Anisakis simplex are highly resistant to heat and pepsin treatments. Parasitology Research, 93(3), 248-251. Caveness, F. E., & Caveness, C. E. (1970). Nematode Electrocution. Journal of Nematology, 2(4), 298- &. Cespedes, M., Saez, A., Rodriguez, I., Pinto, J. M., & Rodriguez, R. (2000). Chronic anisakiasis presenting as a mesenteric mass. Abdominal Imaging, 25(5), 548-550. doi:DOI 10.1007/s002610000089 Choi, S. J., Lee, J. C., Kim, M. J., Hur, G. Y., Shin, S. Y., & Park, H. S. (2009). The clinical characteristics of Anisakis allergy in Korea. Korean J Intern Med, 24(2), 160-163. doi:10.3904/kjim.2009.24.2.160 Choudhury, G., Jenks, W., Wikswo, J., & Bublitz, C. (2002). Effects of parasite attribute and injected current parameters on electromagnetic detection of parasites in fish muscle. Journal of food science, 67(9), 3381-3387. Cobb, N. (1889). Neue parasitische Nematoden. W [illy] Kükenthal, Beiträge zur Fauna Spitzbergens. Archiv für Naturgeschichte, 55(1), 149-151. Consortiumcr, A. I. A. (2011). Anisakis hypersensitivity in Italy: prevalence and clinical features: a multicenter study. Allergy, 66(12), 1563-1569. Dagbjartsson, B. (1973). Rannsóknir varðandi hringormavandamálið (in Icelandic). Tækni tíðindi, Rannsóknir fiskiðnaðarins(35). Dailey, M. D., & Walker, W. A. (1978). Parasitism as a factor (?) in single strandings of southern California cetaceans. The Journal of parasitology, 593-596. Daschner, A., & Pascual, C.-Y. (2005). Anisakis simplex: sensitization and clinical allergy. Current opinion in allergy and clinical immunology, 5(3), 281-285. Davey, J. T. (1971). The case of Anisakis Sp Infection in a Brown Bear from Alaska. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65(4), 433-&. doi: Doi 10.1016/0035- 9203(71)90141-6 Deardorff, T., Kayes, S., & Fukumura, T. (1991). Human anisakiasis transmitted by marine food products. Hawaii medical journal, 50(1), 9-16. Deardorff, T. L., & Overstreet, R. M. (1981). Larval Hysterothylacium (= Thynnascaris)(Nematoda: Anisakidae) from fishes and invertebrates in the Gulf of Mexico. Paper presented at the Proc Helminthol Soc Wash. Deardorff, T. L., Raybourne, R. B., & Desowitz, R. S. (1984). Description of a third-stage larva, Terranova type Hawaii A (Nematoda: Anisakinae), from Hawaiian fishes. J Parasitol, 70(5), 829- 831. Desclers, S., & Andersen, K. (1995). Sealworm (Pseudotervanova-Decipiens) Transmission to Fish Trawled from Hvaler, Oslofjord, Norway. Journal of Fish Biology, 46(1), 8-17.
66 Fernández, d. C. L., Audicana, M., Del Pozo, M., Muñoz, D., Fernández, E., Navarro, J., Diez, J. (1995). Anisakis simplex induces not only anisakiasis: report on 28 cases of allergy caused by this nematode. Journal of investigational allergology & clinical immunology, 6(5), 315-319. Fæste, C. K., Jonscher, K. R., Dooper, M. M., Egge-Jacobsen, W., Moen, A., Daschner, A., Christians, U. (2014). Characterisation of potential novel allergens in the fish parasite Anisakis simplex. EuPA Open Proteomics, 4, 140-155. González, L. (1998). The life cycle of Hysterothylacium aduncum (Nematoda: Anisakidae) in Chilean marine farms. Aquaculture, 162(3), 173-186. Guðmundsson, R., Margeirsson, S., & Arason, S. (2006). Ákvarðanataka og bestun í sjávarútvegi. Rannsóknastofnun fiskiðnaðarins, 27-06. Gunnlaugsdóttir, H., Viðarsson, J. R., Ásmundsdóttir, Á. M., Garate, C., Jörundsdóttir, H. Ó., Jónsdóttir, I. G., . . . Margeirsson, S. (2010). Grandskoðum þann gula frá miðum í maga‐rannsókn á þáttum sem hafa áhrif á verðmæti þorskafla. Gustafson, P. V. (1953). The effect of freezing on encysted Anisakis larvae. J Parasitol, 39(6), 585-588. Hafsteinsson, H., Parker, K., Chivers, R., & Rizvi, S. S. H. (1989). Application of Ultrasonic-Waves to Detect Sealworms in Fish Tissue. Journal of Food Science, 54(2), 244-&. doi:DOI 10.1111/j.1365-2621.1989.tb03053.x Hafsteinsson, H., & Rizvi, S. S. H. (1987). A Review of the Sealworm Problem - Biology, Implications and Solutions. Journal of Food Protection, 50(1), 70-84. Hauksson, E. (1988). Er munur á lifandiblóðguðum and dauðblóðguðum fiski. Fiskvinnslan, 1:15. Hauksson, E. (1991). Parasitic nematodes in commercially important fish (pp. 77): Marcel Dekker, New York. Hauksson, E. (2005). Nematode infestation and diet of fish, cormorants (Phalacrocorax carbo) and common seals (Phoca vitulina) at Melrakkanes, Hamarsfjördur, Eastern Iceland. Marine Research Institute Report, 115(1), 21-30. Hauksson, E. (2006). Growth and reproduction in the Icelandic common seal (Phoca vitulina L., 1758). Marine Biology Research, 2(1), 59-73. doi:10.1080/17451000600650038 Hauksson, E., & Einarsson, S. T. (2010). Review on utilization and research on harbour seal (Phoca vitulina) in Iceland. NAMMCO Scientific Publications, 8, 341-353. Hauksson, E. (2015). Personal communication, November 13, 2015 Heia, K., Lauritzen, K., Nilsen, H., Wold, J., & Wedberg, T. (1997). Studies of methods for detection and removal of nematodes in white-fish: x-ray, CT, NMR, time-gating and optical measurements. Norwegian Institute of Fisheries and Aquaculture research Ltd, Report no, 11. Heia, K., Sivertsen, A. H., Stormo, S. K., Elvevoll, E., Wold, J. P., & Nilsen, H. (2007). Detection of nematodes in cod (Gadus morhua) fillets by imaging spectroscopy. Journal of Food Science, 72(1), E11-E15. doi:10.1111/j.1750-3841.2006.00212.x Hemmingsen, W., Halvorsen, O., & MacKenzie, K. (2000). The occurrence of some metazoan parasites of Atlantic cod, Gadus morhua L., in relation to age and sex of the host in Balsfjord (70 degrees N), North Norway. Polar Biology, 23(5), 368-372. doi:DOI 10.1007/s003000050457
67 Hochberg, N. S., & Hamer, D. H. (2010). Anisakidosis: Perils of the Deep. Clinical Infectious Diseases, 51(7), 806-812. doi:10.1086/656238 Hoshino, C., & Narita, M. (2011). Anisakis simplex-induced anaphylaxis. Journal of Infection and Chemotherapy, 17(4), 544-546. Ishikura, H., Kikuchi, K., Nagasawa, K., Ooiwa, T., Takamiya, H., Sato, N., & Sugane, K. (1993). Anisakidae and anisakidosis Progress in clinical parasitology (pp. 43-102): Springer. ISO (2002). ISO 5984Animal feeding stuffs– Determination of crude ash. International Organization for Standardization, Geneva, Switzerland. ISO (1999). ISO 6496. Animal feeding stuffs - Determination of moisture and other volatile matter content. International Organization for Standardization, Geneva, Switzerland. ISO (2009). ISO 5983. Animal feeding stuffs – Determination of nitrogen content and calculation of crude protein content–Part 2: Block digestion/steam distillation method. International Organization for Standardization, Geneva, Switzerland. Jackson, C., Marcogliese, D., & Burt, M. D. (1997). The role of hyperbenthic crustaceans in the transmission of marine helminth parasites. Canadian Journal of Fisheries and Aquatic Sciences, 54(4), 815-820. Jensen, T. (1997). Experimental infection/transmission of sculpins (Myoxocephalus scorpius) and cod (Gadus morhua) by sealworm (Pseudoterranova decipiens) larvae. Parasitology Research, 83(4), 380-382. Koie, M. (1993). Nematode Parasites in Teleosts from 0-M to 1540-M Depth Off the Faroe Islands (the North-Atlantic). Ophelia, 38(3), 217-243. Koie, M., Berland, B., & Burt, M. D. B. (1995). Development to 3rd-Stage Larvae Occurs in the Eggs of Anisakis-Simplex and Pseudoterranova-Decipiens (Nematoda, Ascaridoidea, Anisakidae). Canadian Journal of Fisheries and Aquatic Sciences, 52, 134-139. Køie, M., & Fagerholm, H.-P. (1995). The life cycle ofContracaecum osculatum (Rudolphi, 1802) sensu stricto (Nematoda, Ascaridoidea, Anisakidae) in view of experimental infections. Parasitology Research, 81(6), 481-489. Levsen, A., Fæste, C., Lin, A., Van Do, T., Florvag, E., & Egaas, E. (2013). Consumer health risk posed by Anisakis simplex in northern Europe-an update. Paper presented at the TROPICAL MEDICINE & INTERNATIONAL HEALTH. Lin, A. H., Florvaag, E., Van Do, T., Johansson, S., Levsen, A., & Vaali, K. (2012). IgE sensitization to the fish parasite Anisakis simplex in a Norwegian population: a pilot study. Scandinavian journal of immunology, 75(4), 431-435. Marcogliese, D. J. (2001). Pursuing parasites up the food chain: Implications for food web structure and function on parasite communities in aquatic systems. Acta Parasitologica, 46(2), 82-93. Marcogliese, D. J., Boily, F., & Hammill, M. O. (1996). Distribution and abundance of stomach nematodes (Anisakidae) among grey seals (Halichoerus grypus) and harp seals (Phoca groenlandica) in the Gulf of St. Lawrence. Canadian Journal of Fisheries and Aquatic Sciences, 53(12), 2829-2836. doi:DOI 10.1139/cjfas-53-12-2829
68 Margeirsson, S., Hrafnkelsson, B., Jónsson, G. R., Jensson, P., & Arason, S. (2010). Decision making in the cod industry based on recording and analysis of value chain data. Journal of food engineering, 99(2), 151-158. Margeirsson, S., Jensson, P., Jónsson, G. R., & Arason, S. (2006). Hringormar í Þorski. Árbók VFÍ / TFÍ. Mattiucci, S., Fazii, P., De Rosa, A., Paoletti, M., Megna, A. S., Glielmo, A., . . . Nascetti, G. (2013). Anisakiasis and Gastroallergic Reactions Associated with Anisakis pegreffii Infection, Italy. Emerging Infectious Diseases, 19(3), 496-499. Mattiucci, S., & Nascetti, G. (2008). Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. Advances in Parasitology, Vol 66, 66, 47-148. doi:10.1016/S0065-308x(08)00202- 9 Mattiucci, S., Nascetti, G., Cianchi, R., Paggi, L., Arduino, P., Margolis, L., . . . Bullini, L. (1997). Genetic and ecological data on the Anisakis simplex complex, with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). Journal of Parasitology, 83(3), 401-416. doi: Doi 10.2307/3284402 McClelland, G. (1990). Larval sealworm (Pseudoterranova decipiens) infections in benthic macrofauna. Population biology of sealworm, 47-65. McClelland, G. (1995). Experimental infection of fish with larval sealworm, Pseudotertanova decipiens (Nematoda, Anisakinae), transmitted by amphipods. Canadian Journal of Fisheries and Aquatic Sciences, 52(S1), 140-155. McClelland, G. (2002). The trouble with sealworms (Pseudoterranova decipiens species complex, Nematoda): a review. Parasitology, 124, S183-S203. doi:10.1017/S0031182002001658 McClelland, G., & Martell, D. (2014). Surveys of larval sealworm (Pseudoterranova decipiens) infection in various fish species sampled from Nova Scotian waters between 1988 and 1996, with an assessment of examination procedures. NAMMCO Scientific Publications, 3, 57-76. McClelland, G., Misra, R., & Marcogliese, D. J. (1983a). Variations in abundance of larval anisakines, sealworm (Phocanema decipiens) and related species in cod and flatfish from the southern Gulf of St. Lawrence (4T) and the Breton Shelf (4Vn): Department of Fisheries and Oceans, Fisheries Research Branch, Halifax Fisheries Research Laboratory. McClelland, G., Misra, R., & Marcogliese, D. J. (1983b). Variations in abundance of larval anisakines, sealworm (Phocanema decipiens) and related species in Scotian Shelf (4Vs and 4W) cod and flatfish: Department of Fisheries and Oceans, Fisheries Research Branch, Halifax Fisheries Research Laboratory. McClelland, G., Misra, R., & Martell, D. (1985). Variations in abundance of larval anisakines, sealworm (Pseudoterranova decipiens) and related species, in eastern Canadian cod and flatfish: Department of Fisheries and Oceans, Fisheries Research Branch, Scotia-Fundy Region, Halifax Fisheries Research Laboratory. McClelland, G., Misra, R., & Martell, D. (1987). Temporal and Geographical Variations in Abundance of Larval Sealworm, Pseudoterranova (Phocanema) Decipiens in the Fillets of American Plaice
69 Hippoglossoides Platessoides in Eastern Canada: 1985-86 Surveys: Department of Fisheries and Oceans, Biological Sciences Branch, Scotia-Fundy Region, Halifax Fisheries Research Laboratory. Milligan, R. J. (2008). The occurrence and behaviour of Pseudoterranova decipiens and Anisakis
simplex (Nematoda) in Gadus morhua and their impacts on commercial processing. University of Glasgow
Mishima, O., & Stanley, H. E. (1998). The relationship between liquid, supercooled and glassy
water. Nature, 396(6709), 329-335.
Molina-Garcia, A. D., & Sanz, P. D. (2002). Anisakis simplex larva killed by high-hydrostatic-pressure processing. Journal of Food Protection, 65(2), 383-388. Moneo, I., Caballero, M. L., González-Muñoz, M., Rodríguez-Mahillo, A. I., Rodríguez-Perez, R., & Silva, A. (2005). Isolation of a heat-resistant allergen from the fish parasite Anisakis simplex. Parasitology Research, 96(5), 285-289. Morris, A. L., MacArthur, M. W., Hutchinson, E. G., & Thornton, J. M. (1992). Stereochemical quality of protein structure coordinates. Proteins: Structure, Function, and Bioinformatics, 12(4), 345-364. Nieuwenhuizen, N., Lopata, A. L., Jeebhay, M. L. F., Herbert, D. R., Robins, T. G., & Brombacher, F. (2006). Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. Journal of Allergy and Clinical Immunology, 117(5), 1098-1105. doi:10.1016/j.jaci.2005.12.1357 No, E. 152/2009. Commission regulation laying down the methods of sampling and analysis for the official control of feed. Official Journal of the European Union L, 54, 1-130. Olafsdottir, D., & Hauksson, E. (1998). Anisakid nematodes in the common seal (Phoca vitulina L.) in Icelandic waters. Sarsia, 83(4), 309-316. Ólafsdóttir, D. (2014). Review of the ecology of sealworm, Pseudoterranova sp (p)(Nematoda: Ascaridoidea) in Icelandic waters. NAMMCO Scientific Publications, 3, 95-111. Paggi, L., Mattiucci, S., Gibson, D. I., Berland, B., Nascetti, G., Cianchi, R., & Bullini, L. (2000). Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters, and genetic markers. Syst Parasitol, 45(3), 185-197. Panebianco, A., Giuffrida, A., Minniti, A., Ziino, G., Calabro, P., & Triglia, F. (2000). Effect of some gases on Anisakis larvae uncoiling in Lepidopus caudatus. Industrie Alimentari, 39(391), 467-+. The panel, E. B. (2010). Scientific Opinion on risk assessment of parasites in fishery products. EFSA Journal, 8(4), 1543. Peturson, J. (1991). Optical spectra of fish flesh and quality defects in fish. Fish quality control by computer vision, 45. Platt, N. E. (1975). Infestation of Cod (Gadus-Morhua L) with Larvae of Codworm (Terranova-Decipiens Krabbe) and Herringworm, Anisakis Sp (Nematoda Ascaridata), in North-Atlantic and Arctic Waters. Journal of Applied Ecology, 12(2), 437-450. doi: Doi 10.2307/2402166
70 Purello-D'Ambrosio, F., Pastorello, E., Gangemi, S., Lombardo, G., Ricciardi, L., Fogliani, O., & Merendino, R. A. (2000). The incidence of sensitivity to Anisakis simplex in a risk population of fishermen/fishmongers. Annals of Allergy Asthma & Immunology, 84(4), 439-444. Reddy, J. N. (1993). An introduction to the finite element method (Vol. 2): McGraw-Hill New York. Riley, J. (1972). THE PATHOLOGY OF ANZSAKZS NEMATODE INFECTIONS OF THE FULMAR FULMARUS GLACZALZS. Ibis, 114(1), 102-104. Rioja, L. R., Gil, M. D. D., Labairu, T. L., Cucalon, V. I., Sarramian, A. B., & Revuelta, J. A. O. (1998). Allergy to Anisakis simplex. Report of three cases and review of the literature. Revista Clinica Espanola, 198(9), 598-600. Robichaud, D., & Rose, G. A. (2004). Migratory behaviour and range in Atlantic cod: inference from a century of tagging. Fish and Fisheries, 5(3), 185-214. doi:DOI 10.1111/j.1467- 2679.2004.00141.x Rodriguez-Mahillo, A. I., Gonzalez-Munoz, M., de las Heras, C., Tejada, M., & Moneo, I. (2010). Quantification of Anisakis simplex Allergens in Fresh, Long-Term Frozen, and Cooked Fish Muscle. Foodborne Pathogens and Disease, 7(8), 967-973. Ronald, K. (1960). The Effects of Physical Stimuli on the Larval Stage of Terranova Decipiens (Krabbe, 1878)(Nematoda: Anisakidae): I. Temperature. Canadian Journal of Zoology, 38(3), 623-642. Ronald, K. (1962). THE EFFECTS OF PHYSICAL STIMULI ON THE LARVAE OF TERRANOVA DECIPIENS: II. RELATIVE HUMIDITY, PRESSURE, AND GASES. Canadian Journal of Zoology, 40(7), 1223-1227. Ronald, K. (1963). THE EFFECTS OF PHYSICAL STIMULI ON THE LARVAL STAGE OF TERRANOVA DECIPIENS: III. ELECTROMAGNETIC SPECTRUM AND GALVANOTAXIS. Canadian Journal of Zoology, 41(2), 197-217. Sakanari, J. A., & Mckerrow, J. H. (1989). Anisakiasis. Clinical Microbiology Reviews, 2(3), 278-284. Scala, E., Giani, M., Pirrotta, L., Guerra, E. C., Cadoni, S., Girardelli, C. R., . . . Puddu, P. (2001). Occupational generalised urticaria and allergic airborne asthma due to anisakis simplex. European Journal of Dermatology, 11(3), 249-250. Sevier, C. S., & Kaiser, C. A. (2002). Formation and transfer of disulphide bonds in living cells. Nature reviews Molecular cell biology, 3(11), 836-847. Sigernes, F., Lorentzen, D. A., Heia, K., & Svenoe, T. (2000). Multipurpose spectral imager. Applied Optics, 39(18), 3143-3153. doi:Doi 10.1364/Ao.39.003143 Sigurðsson, E. (2016). Áhrif hringorma við þorskvinnslu. Slominski, A., Semak, I., Pisarchik, A., Sweatman, T., Szczesniewski, A., & Wortsman, J. (2002). Conversion of L-tryptophan to serotonin and melatonin in human melanoma cells. FEBS letters, 511(1), 102-106. Smith, J., & Wootten, R. (1979). Recent surveys of larval anisakine nematodes in gadoids from Scottish waters. Int Counc Explor Sea CM/G, 46. Smith, J. W. (1983). Anisakis simplex (Rudolphi, 1809, det. Krabbe, 1878)(Nematoda: Ascaridoidea): morphology and morphometry of larvae from euphausiids and fish, and a review of the life- history and ecology. Journal of helminthology, 57(03), 205-224.
71 Smith, J. W. (1984). The Abundance of Anisakis-Simplex-L3 in the Body-Cavity and Flesh of Marine Teleosts. International Journal for Parasitology, 14(5), 491-495. doi: Doi 10.1016/0020- 7519(84)90030-4 Smith, J. W., & Hemmingsen, W. (2003). Atlantic cod Gadus morhua L.: Visceral organ topography and the asymetrical distribution of larval ascaridoid nematodes in the musculature. Ophelia, 57(3), 137-144. Smith, J. W., & Wootten, R. (1975). Experimental Studies on Migration of Anisakis Sp Larvae (Nematoda-Ascaridida) into Flesh of Herring, Clupea-Harengus L. International Journal for Parasitology, 5(2), 133-136. doi: Doi 10.1016/0020-7519(75)90019-3 Stern, L., Myers, B. J., Amish, R. A., Knollenberg, W., & Chew, K. (1976). Anisakine Nematodes in Commercial Marine Fish from Washington State. Transactions of the American Microscopical Society, 95(2), 264-264. Stromnes, E., & Andersen, K. (2000). "Spring rise" of whaleworm (Anisakis simplex; Nematoda, Ascaridoidea) third-stage larvae in some fish species from Norwegian waters. Parasitology Research, 86(8), 619-624. doi:Doi 10.1007/Pl00008541 Stromnes, E., & Andersen, K. (2003). The growth of whaleworm (Anisakis simplex, Nematodes, Ascaridoidea, Anisakidae) third-stage larvae in paratenic fish hosts. Parasitology Research, 89(5), 335-341. doi:10.1007/s00436-002-0756-7 Torres, P., Jercic, M. I., Weitz, J., Dobrew, E., & Mercado, R. (2007). Human pseudoterranovosis, an emerging infection in Chile. Journal of Parasitology, 93(2), 440-443. Truett, G., Heeger, P., Mynatt, R., Truett, A., Walker, J., & Warman, M. (2000). Preparation of PCR- quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques, 29(1), 52, 54. Ugland, K. I., Stromnes, E., Berland, B., & Aspholm, P. E. (2004). Growth, fecundity and sex ratio of adult whaleworm (Anisakis simplex; Nematoda, Ascaridoidea, Anisakidae) in three whale species from the North-East Atlantic. Parasitol Res, 92(6), 484-489. doi:10.1007/s00436-003- 1065-5 Umehara, A., Kawakami, Y., Araki, J., & Uchida, A. (2008). Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii, and the other anisakid nematodes. Parasitology International, 57(1), 49-53. Valdimarsson, G., Einarsson, H., & King, F. J. (1985). Detection of Parasites in Fish Muscle by Candling Technique. Journal of the Association of Official Analytical Chemists, 68(3), 549-551. van Thiel, P., Kuipers, F., & Roskam, R. T. (1960). A nematode parasitic to herring, causing acute abdominal syndromes in man. Tropical and geographical medicine, 12(2), 97-113. van Thiel, P., & van Houten, H. (1966). The herring worm Anisakis marina as a human parasite outside the wall of the gastrointestinal tract. Nederlands tijdschrift voor geneeskunde, 110(35), 1524- 1528. Wharton, D., & Aalders, O. (2002). The response of Anisakis larvae to freezing. Journal of helminthology, 76(04), 363-368. Wilson, K. (2014). Microsoft Excel 2013 Using Microsoft Office 2013 (pp. 59-79): Springer.
72 Wold, J. P., Westad, F., & Heia, K. (2001). Detection of parasites in cod fillets by using SIMCA classification in multispectral images in the visible and NIR region. Applied Spectroscopy, 55(8), 1025-1034. doi: Doi 10.1366/0003702011952929 Wootten, R., & Waddell, I. F. (1977). Studies on Biology of Larval Nematodes from Musculature of Cod and Whiting in Scottish Waters. Journal Du Conseil, 37(3), 266-273. Yamaguti, S., & Arima, S. (1942). Porrocaecum azarasi n. sp.(Nematoda) from the Japanese seal. Transactions of the Sapporo Natural History Society, 17(2), 113-116.
73 Appendix I - Chemical composition
80 74 76 70 60 50 40 30 20 14,25 Content Content (g/ 100g) 10,3 10,7 11,8 10 0,15 0,8 0,8 0,5 0,6 0,2 0
Small nematodes Large nematodes
Figure 33. Chemical composition of small and large nematodes.
200
180
160
140
120
100 Small nematodes 80 Large nematodes 60
Contents Contents /100g) (mg 40 1,1 20 1,1 0,258 0,308 0 Potassium Phosphorus Sodium Calcium Magnesium Iron Copper
Figure 34. Mineral contents of small and large nematodes.
74 Table 10. The comparison in composition between nematodes and the cod muscle parts Chemical composition Nematodes mean Belly Flap Loins
(g / 100g) (g / 100g) (g/100g)
Water 75,0 ± 3 83,3 ± 3,3 82,4 ± 3,3
Protein 10,5 ± 0,315 15,8 ± 0,474 16,4 ± 0,492
Carbohydrates 13,0 0,0 0,0
Ash 0,8 ± 0,016 1,1± 0,022 1,1 ± 0,022
Lipids 0,55 ± 0,044 0,4 ± 0,032 0,4 ± 0,032
Sodium chloride 0,175 ± 0,005 0,2 ± 0,006 0,2 ± 0,006
TOTAL 100 100,8 100
Table 11. The mineral composition of nematodes (mean) and cod muscle groups. Nematodes mean Bellyflap Loins
Minerals (mg / 100g) (mg / 100g) (mg / 100g)
Iron (Fe) 1,1 ± 0,22 0,06 ± 0,012 0,04 ± 0,008
Copper (Cu) 0,283 ± 0,056 0,011 ± 0,002 0,009 ± 0,0018
Sodium (Na)* 43,5 65 49
Potassium (K)* 170 415 449
Phosphorus (P)* 151 156 164
Calcium (Ca)* 23,5 8 7
Magnesium (Mg)* 15,5 24 23
* = Measuring method is not accredited
75 Appendix II – Amino acid composition
Table 12. The amino acid composition of nematodes and cod proteins Amino acids Nematodes (mean) cod muscle (% of total proteins) (% of total proteins) Alanine 4,9 ± 0,294 6,2 ± 0,372 Arginine 7,0 ± 0,42 6,3 ± 0,378 Aspartic acid 10,2 ± 0,612 11,9 ± 0,714 Cystein + Cystine 2,5 ± 0,25 1,2 ± 0,12 Glutamic acid 15,6 ± 1,1 16,1 ± 1,12 Glycine 6,9 ± 0,483 4,5 ± 0,315 Histidine* 2,3 ± 0,23 2,2 ± 0,22 Hydroxyproline < 0,13 ± 0,026 < 0,13 ± 0,026 Isoleucine* 4,3 ± 0,344 4,7 ± 0,376 Leucine* 7,0 ± 0,49 8,3 ± 0,581 Lysine* 7,6 ± 0,608 10,2 ± 0,816 Methionine* 2,8 ± 0,28 3,6 ± 0,36 Ornithine < 0,02 ± 0,001 < 0,02 ± 0,001 Phenylalanine* 3,8 ± 0,228 4,2 ± 0,252 Proline 6,3 ± 0,378 3,3 ± 0,198 Serine 4,8 ± 0,288 4,4 ± 0,264 Threonine* 4,6 ± 0,276 4,6 ± 0,276 Tyrosine 3,5 ± 0,385 1,9 ± 0,209 Valine* 4,6 ± 0,368 5,1 ± 0,408 Tryptophan* 1,3 ± 0,091 1,2 ± 0,084 *= Essential amino acids
-
76 Appendix III – Nematode survival against sub-zero temperatures
Table 13. The survival rate of small nematodes (< 4 cm) Freezing time (h) Survival at -5°C Survival at -10°C Survival at -15°C Survival at -20°C 0 100,0% 100% 100% 100% 1 100,0% 100% 81,3% 43% 2 100,0% 93,3% 46,2% 0% 3 93,3% 55,6% 0,0% 0% 4 84,2% 0,0% 0% 0% 5 68,4% 0,0% 0% 0% 6 66,7% 0,0% 0% 0%
Table 14. The survival rate of large nematodes (> 4 cm) Freezing time (h) Survival at -5°C Survival at -10°C Survival at -15°C Survival at -20°C 0 100,0% 100% 100% 100% 1 100,0% 100% 76,9% 33% 2 100,0% 88,9% 23,1% 0% 3 87,5% 50,0% 0,0% 0% 4 84,6% 0,0% 0% 0% 5 78,6% 0,0% 0% 0% 6 66,7% 0,0% 0% 0%
77 4
2 C) ° 0 -2 -4 -6 -8
Temperature( -10
-12
0
15 30 45 60 75 90
225 105 120 135 150 165 180 195 210 240 255 270 285 300 315 330 345 360
Time (min) Logger 1 (-5°C) Logger 2 (-5°C)
Figure 35. The temperature recordings from inside the freezer at -5°C
0
-2 C)
° -4 -6 -8 -10
-12 Temperature( -14 -16 0 30 60 90 120 150 180 210 240 270 300 330 360 Time (min)
Logger 1 Logger 2
Figure 36. The temperature recordings from inside the freezer at -10°C
78 0
C) -4 °
-8
-12
-16 Temperature( -20 0 30 60 90 120 150 180 210 240 Time (min)
Logger 1 Logger 2
Figure 37. The temperature recordings inside the freezer at -15°C
0
C) -5 °
-10
-15
-20 Temperature Temperature ( -25 0 30 60 90 120 150 180 210 240 270 Time (min)
Logger 1 (-20°C) Logger 2 (-20°C)
Figure 38. The temperature recordings from inside the freezer at -20°C
79 Appendix IV – PCR analysis
Table 15. The first experimental reactions in PCR analysis of nematodes DNA. For the second reaction Magnesium Chloride was added. For the third reaction Betaine was added. However, the MgCl2 gave the best results. 1 2 3 15 ul reaction reaction 15 ul reaction reaction 15 ul reaction reaction 10x Standard 1,50 10x Standard 1,50 10x Standard 1,50 10 mM dNTP 1,50 25 mM MgCl2 1,50 Betaine 1,50 211.F 100 µM 0,05 10 mM dNTP 1,50 10 mM dNTP 1,50 210.R 100 µM 0,05 211.F 100 µM 0,05 211.F 100 µM 0,05 DNA 2,00 210.R 100 µM 0,05 210.R 100 µM 0,05 Taq Polymerase 0,12 DNA 2,00 DNA 2,00 Taq Taq 9,78 0,12 0,12 Water Polymerase Polymerase Total 15 Water 8,28 Water 8,28 Annealing 50°C Total 15 Total 15 Annealing 50°C Annealing 50°C
Figure 39. The first PCR experimental reaction
80 Table 16. The second experimental PCR reactions
15 ul reaction 1 reaction 15 ul reaction 1 reaction 10x Standard 1,50 10x Standard 1,50 10 mM dNTP 1,50 25 mM MgCl2 1,50 211.F 100 µM 0,05 10 mM dNTP 1,50
210.R 100 µM 0,05 211.F 100 µM 0,05 DNA 2,00 210.R 100 µM 0,05 Taq DNA 2,00 0,12 Polymerase Taq Polymerase 0,12 Watn 9,78 Watn 8,28 Total 15 Total 15 Annealing 50°C Annealing 50°C
81
82