Relationship between Southern Bluefin Tuna, Thunnus maccoyii, melanomacrophage centres and Cardicola spp. (Trematoda: Aporocotylidae) infection.

Southern Bluefin Tuna (SBT), Thunnus maccoyii, is ranched off Port Lincoln, South Australia and is Australia's second largest economic finfish aquaculture industry. The biggest threats to SBT health identified by the industry are the blood flukes Cardicola forsteri and C. orientalis (Trematoda: Aporocotylidae). Melanomacrophage centres (MMCs) are aggregations of pigmented macrophage like cells present in spleen, kidney and liver of teleost fish. The aim of this study was to quantify MMCs in SBT anterior kidney, liver and spleen to investigate changes in relation to Cardicola spp. Infection. Samples were collected at the end of ranching from pontoons where SBT were treated with PZQ and pontoons with untreated SBT. SBT MMC percentage of surface area cover was highest in SBT spleen and lowest in the liver. Significant positive correlations were identified between SBT MMC area and SBT size in all three organs (p < 0.05). MMC area and parasite infection showed significant positive correlations in the kidney and spleen for Cardicola spp. gill egg counts, and in the kidney for C. forsteri DNA from SBT hearts and gills (p < 0.05). MMCs area increased with increased intensity of Cardicola spp. Infection and MMCs have the potential to be used as an indicator to assess health effects that Cardicola spp. have on SBT.

In vivo and in vitro assessment of host-parasite interaction between the parasitic copepod Brachiella thynni (Copepoda; Lernaeopodidae) and the farmed Atlantic bluefin tuna (Thunnus thynnus).

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1ß (il1ß), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

Canned tuna tolerance in children with IgE-mediated fish allergy: an allergological and nutritional view.

Scientific research, diagnostic tools and clinical experience have shown that children suffering from IgE-mediated fish allergy do not need to follow a strict exclusion diet. In fact, they could tolerate some species of fish, which could be reintroduced in the diet by verifying their tolerance with an oral food challenge in a clinical setting. Consequently, it is possible to look a new insight on diagnosis and management of IgE-mediated fish allergy in children, considering the use of canned tuna in clinical settings. Authors performed a literature search through the Cochrane Library and Medline/PubMed databases. All quantitative and qualitative pediatric studies involving diagnosis and management of IgE-mediated fish allergy and the use of canned tuna in clinical settings were considered. Articles related to allergological and nutritional features of fish, and especially canned tuna, were selected. This research was conducted on May 2020. Canned tuna shows peculiar allergological and nutritional characteristics. Relating to allergy, canning process, characterized by cooking the fish under pressure for a time equal to about 7 hours, can lead a conformational change in parvalbumin, making it less allergenic. In terms of nutrition, canned tuna contains B, D and A vitamins and, above all, omega-3 fatty acids and shows a favourable and significantly sustainable nutritional profile. Lower allergenicity, adequate nutritional value and its rich availability in markets at reasonable costs, could make the use of canned tuna as a solution with an excellent risk/benefit ratio in the field of IgE-mediated fish allergy.

Non-ST-Elevation Myocardial Infarction-Like Syndrome in Scombroid Tuna Fish Poisoning.

Mistreated fish products ingestion can lead to a histaminergic illness known as Scombroid Syndrome (SS). The disease usually causes cutaneous rash, stomach cramps, nausea, vomiting, breathing disorder and further histamine-related symptoms. To date, however, SS has been disregarded among the potential triggers of acute coronary syndrome (ACS), in spite of prior published occasional case reports. In the present study we describe 3 consecutive patients presenting with signs and symptoms of ACS associated to SS. Two men and a woman with no history of coronary artery disease and food allergy were studied. Clinical characteristics, electrocardiographic presentation and outcomes are described. Non-ST-elevation myocardial infarction-like pattern was observed in all patients. The 2 men underwent unremarkable coronary angiography, whereas the woman was just monitored at emergency department. All individuals had uneventful follow-up. The present study confirms non-ST-elevation myocardial infarction-like ACS as a possible histaminergic toxic, not allergic, epiphenomenon of mistreated raw tuna fish ingestion, likely due to transient epicardial and/or microvascular coronary vasospasm.

Kounis syndrome following canned tuna fish ingestion.

Kounis syndrome (KS) is a complex of cardiovascular symptoms and signs following either allergy or hypersensitivity and anaphylactic or anaphylactoid insults. We report the case of 57-year-old man, with hypertension and history of allergy, referred for facial rash and palpitations appeared after consumption of canned tuna fish. Suddenly, the patient collapsed: electrocardiogram showed ST-elevation in inferior leads. The patient was transferred from the spoke emergency room for coronary angio, which did not show any sign of coronary atherosclerosis. A transient coronary spasm was therefore hypothesized and the final diagnosis was KS. To the best of our knowledge, this is one of the first cases of KS following the ingestion of tuna fish. KS secondary to food allergy has also been reported, and shellfish ingestion has been considered as one of the most active KS inducer foods. Canned tuna fish too is well known as an allergy inducer. Tuna fish allergy should be considered, however, within the context of scombroid food poisoning, also called histamine fish poisoning. Fish with high levels of free histidine, the enzyme substrate converted to histamine by bacterial histidine decarboxylase, are those most often implicated in scombroid poisoning. Inflammatory mediators such as histamine constitute the pathophysiologic basis of Kounis hypersensitivity-associated acute coronary syndrome. Patients with coronary risk factors, allergic reaction after food ingestion, and suspected scombroid poisoning should be therefore carefully monitored for a prompt diagnosis of possible coronary complications.

Effect of ranching time on melanomacrophage centres in anterior kidney and spleen of Southern bluefin tuna, Thunnus maccoyii.

Melanomacrophage centres (MMCs) are common in the haemopoietic tissue of fish and are an important component of the humoral immune response. Increasingly they are being studied as a biomarker of environmental stress in both wild and aquaculture fish stocks. Computer image analysis of histological sections of the head kidney and spleen of ranched Southern bluefin tuna (SBT) was used to investigate changes in MMCs over various stages of the ranching cycle. Histochemical pigment differentiation indicated haemosiderin, lipofuscin and melanin were present in MMCs of both the kidney and spleen, with kidney MMCs being highly positive for lipofuscin and melanin and spleen having high levels of haemosiderin and a lower level of melanin than kidney. The MMC area in the kidney of wild sampled SBT ranged from 0.41 ± 0.13% to 2.95 ± 0.26% and that in the spleen ranged from 0.35 ± 0.09% to 2.75 ± 0.86%. The MMC area in the spleen of SBT increased over the latter stages of ranching and at harvest compared to SBT sampled in the wild and at the end of tow. There was no statistically significant difference in the MMC area in the kidney of ranched SBT sampled at various stages. SBT appear to have a higher MMC area in haemopoietic organs than numerous other fish species, perhaps providing a greater capacity to deal with 'wear and tear' and increased immune-competence.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Antimicrobial function of SHßAP, a novel hemoglobin ß chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis.

A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin ß-chain; thus, this peptide was designated as the Skipjack Hemoglobin ß chain-related Antimicrobial Peptide (SHßAP). SHßAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0 µg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0-19.0 µg/mL), and against Candida albicans (MEC; 12.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHßAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin ß-chain from marine fish.

Characterization of three pro-inflammatory cytokines, TNFα1, TNFα2 and IL-1ß, in cage-reared Atlantic bluefin tuna Thunnus thynnus.

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1ß, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna, Katsuwonus pelamis.

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2-17.0 µg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1-12.0 µg/mL), and against Candida albicans (MEC, 16.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel ß-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.

Expressed IgH µ and τ transcripts share diversity segment in ranched Thunnus orientalis.

It is now appreciated that in addition to the immunoglobulin (Ig)M and D isotypes fish also make the mucosal IgT. In this study we sequenced the full length of Ig τ as well as µ in the commercially important Thunnus orientalis (Pacific bluefin tuna), the first molecular analysis of these two Ig isotypes in a member of the order Perciformes. Tuna IgM and IgT are each composed of four constant (CH) domains. We cloned and sequenced 48 different variable (VH) domain gene rearrangements of tuna immunoglobulins and grouped the VH gene sequences to four VH gene segment families based on 70% nucleotide identity. Three VH gene families were used by both IgM and IgT but one group was only found to be used by IgM. Most interestingly, both µ and τ clones appear to use the same diversity (DH) segment, unlike what has been described in other species, although they have dedicated IgT and IgM joining (JH) gene segments. We complemented this repertoire study with phylogenetic and tissue expression analysis. In addition to supporting the development of humoral vaccines in this important aquaculture species, these data suggest that the DH-JH recombination rather than the VH-DH recombination may be instructive for IgT versus IgM/D bearing lymphocyte lineages in some fish.

Identification of parvalbumin and two new thermolabile major allergens of Thunnus tonggol using a proteomics approach.

BACKGROUND: The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach. METHODS: Raw and boiled extracts of the fish were prepared. Fish proteins were separated by means of SDS-PAGE and two-dimensional (2-DE) electrophoresis. 1-DE immunoblotting of raw extract was performed with sera from fish-allergic patients. Ten sera were further analysed by 2-DE immunoblotting. Selected major allergenic protein spots were excised, trypsin digested and analysed by means of mass spectrometry. RESULTS: SDS-PAGE of raw extract revealed 26 protein fractions, while boiled extract demonstrated fewer bands. The 2-DE gel profile of the raw extract further fractionated the protein bands to more than 100 distinct protein spots. 1-DE immunoblotting of raw extract exhibited two thermolabile protein fractions of 42 and 51 kDa as the major allergens, while the boiled extract only revealed a single IgE-binding band at 151 kDa. 2-DE immunoblotting of raw extract further detected numerous major IgE-reactive spots of 11-13, 42 and 51 kDa. Mass spectrometry analysis of the peptides generated from the 12, 42 and 51 kDa digested spots indicated that these spots were parvalbumin, creatine kinase and enolase, respectively. CONCLUSIONS: In addition to parvalbumin, two new thermolabile allergens were identified as major allergenic proteins of T. tonggol. This study proved that both thermostable and thermolabile proteins are important in local tuna allergy and should be included in diagnostic strategies.

Temperature-induced transcription of inflammatory mediators and the influence of Hsp70 following LPS stimulation of southern bluefin tuna peripheral blood leukocytes and kidney homogenates.

Temperature is known to influence inflammatory signalling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signalling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators - TNFα (1 and 2), IL-1ß, IL-8, and Cox2 - and demonstrate induction of four - TNFα (2), IL-1ß, IL-8, and Cox2 - following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 °C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFα, IL-1ß and IL-8 following LPS stimulation at 25 °C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 °C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24 h incubation at 25 °C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 °C incubation caused Hsp70 up-regulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 °C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish.

Correlation of humoral immune response in southern bluefin tuna, T. maccoyii, with infection stage of the blood fluke, Cardicola forsteri.

The blood fluke, Cardicola forsteri, is a prevalent infection in ranched southern bluefin tuna. This project aimed to define the timing and intensity of the various developmental stages of C. forsteri within southern bluefin tuna as well as to relate infection to host pathology and immune response. Archival samples from several cohorts of T. maccoyii sampled from 2008 to 2010 were used in this study. The prevalence and intensity of C. forsteri infection was described using heart flushes and histological examination. Humoral immune response, i.e. C. forsteri specific antibody, lysozyme activity, and alternative complement activity, was also described. Based on the validated and detailed C. forsteri infection timeline, relationships between infection events, physiological response, and diagnosis were proposed. Immune response developed concurrently with C. forsteri infection, with the majority of physiological response coinciding with commencing egg production. Further research is needed to confirm the origin of C. forsteri antigen which is responsible for immune response development and how T. maccoyii immune response works against infection. To aide this research, further diagnostic methods for confirmation of infection need to be developed.

Exercise-induced anaphylaxis after ingestion of sandwich with soy containing canned tuna: case report.

We report a case of a 17-year-old female adolescent who experienced an episode of exercise-induced anaphylactic reaction following ingestion of tuna sandwich (from soy containing canned tuna). Her medical history revealed that she had previously had one episode of urticaria after ingestion of sulfamethoxazole with trimethoprim and anaphylactic reaction after ingestion of sunflower nuts. Skin prick tests and specific immunoglobulin E antibody to tuna were negative, and to soy were positive. Treadmill exercise induced test in fasting state and 1 hour after a fresh tuna meal and meal not containing soy were negative. However, an exercise challenge test one hour after soy ingestion resulted in pruritus of hands, shoulders and back, urticarial lesions of the face and neck with angioedema of the lips and eyelids, hoarseness, tachycardia and anxiety.

Effects of immunostimulants on ranched southern bluefin tuna Thunnus maccoyii: immune response, health and performance.

Ranched southern bluefin tuna Thunnus maccoyii were fed baitfishes supplemented with vitamins (predominantly E and C) or vitamins and immunostimulants, nucleotides and ß-glucans, over 12 weeks after transfer and monitored for enhancement in immune response, health and performance through their 19 week grow-out period. Fish from two different tows were sampled separately at three different sampling points: at transfer to grow-out pontoons, at 8 weeks post-transfer and at harvest, 19 weeks post-transfer. Lysozyme activity was enhanced during vitamin supplementation compared to control fish. Performance (i.e. survival, condition index and crude fat), health (i.e. blood plasma variables including pH, osmolality, cortisol, lactate and glucose) and alternative complement activity were not commonly improved through diet supplementation. There were some tow-specific improvements in performance through vitamin supplementation including survival, selected parasite prevalence and intensity, and alternative complement activity. Immunostimulant supplementation also showed a tow-specific improvement in plasma cortisol level. Tow-specific responses may suggest that life history, previous health condition and husbandry can affect the success of vitamin and immunostimulant enhancement of immune response, health and performance of ranched T. maccoyii.

Expression of cytokines IL-1beta and TNF-alpha in tissues and cysts surrounding Didymocystis wedli (Digenea, Didymozoidae) in the Pacific bluefin tuna (Thunnus orientalis).

Tuna long distance migrations and exposure to wide range of ambient water temperatures facilitate infections with several parasitic groups. This is reflected in the remarkable diversity of tuna parasite communities, especially members of Didymozoidae superfamily (Poche, 1907) (Trematoda, Digenea). Didymocystis wedli is the most frequent species encountered in bluefin tuna parasitizing gill filaments, therefore suggested as a biological marker to differentiate between discrete tuna Atlantic stocks. Because of its high occurrence in gill tissue and inflammatory reaction as the consequence, the aim of our study was to asses if inflammatory madiation through expression of IL-1beta and TNF-alpha is present locally at the site of D. wedli encystment, as well as if the systematic expression of cytokines can be detected in different tissues of infected versus uninfected fish. Quantification of localized cytokine expression was done on paraffine embedded gill sections by in situ hybridization, while quantitative PCR was used to mesured cytokine transcripts in skin mucus, kidney, spleen, gills and liver. Our results suggest that tuna constitutive expression of IL-1beta and TNF-alpha in gills and skin implies a well-adapted innate immunity present at the barrier between the organism and environment. Upregulation of both cytokines in Didymocystis-infected gills not followed by a systematic response evidences the ongoing of an inflammatory process specific for the parasitation site. However, the lack of intensive cytokines response to D. wedli observed by molecular and histological data that fails to eliminate the parasite, could be related to the "old" age of the parasitic process.

Comparison of allergenic properties of salmon (Oncorhynchus nerka) between landlocked and anadromous species.

BACKGROUND: Salmon is one of the most widely consumed seafoods in Japan and many other countries around the world. Due to the confirmed cases of salmon-induced allergy, the food sanitation law in Japan stipulates salmon as one of the specific food items for which labeling is recommended when used as an ingredient of processed foods. However, trout, the landlocked form of anadromous salmon, is not subject to the allergen-labeling requirements, even though both populations belong to a single species. Since no supporting data have been demonstrated to make a clear distinction between these two populations in terms of allergenicity, we comparatively examined their allergenic properties using sera from patients allergic to fish. METHODS: Extracts of Oncorhynchus nerka from different habitats were obtained: kokanee (landlocked) and red salmon (anadromous). Control extracts were derived from four other species. This study focused on the (1) IgE-binding capacity of the fish extracts in patients' sera (n = 50), (2) ELISA inhibition test (n = 6), and (3) inhibition immunoblot test (n = 8) between the kokanee and red salmon. RESULTS: The extracts from kokanee and red salmon showed the highest correlation with each other in terms of the IgE-binding capacity, and showed complete (100%) reciprocal cross-inhibition in the ELISA inhibition test. On immunoblotting, there was no marked difference in the staining pattern between the two extracts, and each IgE-binding band gradually disappeared when the patients' sera were preincubated with the counterpart antigen in a dose-dependent manner. CONCLUSIONS: These results suggest that kokanee has similar allergenic properties to red salmon.

Two types of tumor necrosis factor-alpha in bluefin tuna (Thunnus orientalis) genes: Molecular cloning and expression profile in response to several immunological stimulants.

Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator and has also the potential as a prominent biomarker of innate immunity. In this study, we identified and characterized TNF-alpha from bluefin tuna, which is an important cultured species. Two types of TNF-alpha were also cloned incidentally (TNF1 and TNF2). The open reading frame of TNF1 and TNF2 cDNA encoded 247 and 245 amino acids, respectively. The amino acid sequence identity among sea perch, red sea bream, and tiger puffer was 73, 70, 59% for TNF1 and 49, 51, 45% for TNF2, respectively. The identity between TNF1 and TNF2 amino acid sequences of the bluefin tuna was only 43%. The positions of cysteine residues, transmembrane sequence, and protease cleavage site in bluefin tuna TNFs were similar with other reported fish and mammalian TNF-alpha. In a phylogenetic analysis, TNF1 is grouped with other reported Perciformes TNF-alpha. On the other hand, TNF2 is grouped with ayu TNF and is quite distant from the fish TNF-alpha group and lymphotoxin-beta group. While TNF1 mRNA showed no significant difference in all tissues, TNF2 mRNA was expressed significantly higher in the blood than in the gill, intestine, head kidney, spleen, heart, and ovary. In peripheral blood leucocytes (PBL), expressions of TNF2 mRNA were significantly increased by stimulation with lipopolysaccharide, phytohemagglutinin, concanavalin A, pokeweed mitogen, phorbol myristate acetate in vitro, but those of TNF1 were not. Recombinant mature TNF1 and TNF2 proteins significantly enhanced phagocytic activity of PBL. Our results suggest that bluefin tuna possess two types of TNF-alpha homologue, and TNF2 is a potential biomarker for innate immunity.

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri.

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort(2005)) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE=1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U microl(-1) and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U microl(-1) (SE=6.26 U microl(-1)) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort(2005) were compared to a 2006 cohort (Cohort(2006)) in March 2006 and August 2006 to determine if prior infection in Cohort(2005) elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort(2005) had significantly lower abundances and prevalences of blood fluke infection than Cohort(2006). Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort(2006) was significantly greater than that of Cohort(2005) in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.