Investigation into Potential Allergenicity and Digestion-Resistant Linear Epitopes of Fish Skin Gelatin in Cell-Cultured Meat Scaffolds.

As a key component of cell-cultured fish, fish skin gelatin (FSG)-based cell scaffold provides support structures for cell growth, proliferation, and differentiation. However, there are potential allergenicity risks contained in FSG-based scaffolds. In this study, 3D edible scaffolds were prepared by phase separation method and showed a contact angle of less than 90°, which indicated that the scaffolds were favorable for cell adhesion. Besides, the swelling ratio was greater than 200%, implying a great potential to support cell growth. The sequence homology analysis indicated that FSG was prone to cross-reaction with collagen analogues. Additionally, a food allergic model was constructed and represented that mice gavaged with cod FSG exhibited higher levels of specific antibodies, mast cell degranulation, vascular permeability, and intestinal barrier impairment than those gavaged with pangasius and tilapias FSG. Its higher allergenicity might be attributed to a higher number of digestion-resistant linear epitopes. Moreover, the higher hydrolysis degree linked to the exposure of linear epitopes to promote the combination with IgE, which was also responsible for maintaining the higher allergenicity of cod FSG. This study clarifies allergenic risks in cell-cultured fish and further study will focus on the allergenicity reduction of FSG-based cell scaffolds.

IgE reactivity to fish allergens from Pacific cod (Gadus macrocephalus) in atopic dogs.

BACKGROUND: IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS: The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS: The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

Oil from hake (Merluccius merluccius): Characterization, antioxidant activity, wound healing and anti-inflammatory effects.

The aim of this work was to evaluate some biological properties of hake head oil (HHO) as well its lipid composition. The fatty acid profiles showed a dominance of unsaturated fatty acids overtaking 55% of the total fatty acids. Omega-3 polyunsaturated fatty acid profiles exhibited a dominance of EPA (eicosapentaenoic acid) (3.96%) and DHA (docosahexaenoic acid) (25.39%). The antioxidant activity was determined through two different assays: DPPH scavenging activity and ß-carotene bleaching by linoleic acid assay. Eighteen mice were excised on their back and divided into 3 groups, treated with sterile saline, commercial healing cream and HHO, respectively. The wound closure rate, the hydroxyproline contents and the histopathology evolution in skin tissue were elaborated. Also, the anti-inflammatory activity was studied by carrageenan-induced mouse paw edema. Mice were divided into 3 groups treated respectively with sterile saline, anti inflammatory drug reference and HHO. The anti-inflammatory evaluation of HHO in mice exhibited an important inhibition of carrageenan-induced hind paws edema, as confirmed by the histological analysis, the superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) level. HHO displayed a significant wound healing effect probably due to the anti-inflammatory and antioxidant activities of its EPA and DHA contents. The overall results proved that HHO might be favorable drugs who exert a great therapeutic potential wound healing and anti-inflammatory effects in animal model.

A method to detect allergenic fish, specifically cod and pollock, using quantitative real-time PCR and COI DNA barcoding sequences.

BACKGROUND: Fish are one of eight major allergens defined in the US Food Allergen Labeling and Consumer Protection Act, and cod and pollock are two of the major fish allergens. This paper describes development and validation of a method to detect cod and pollock in complex food matrices using real-time polymerase chain reaction (PCR). Mitochondrial cytochrome oxidase I (COI) sequences obtained through DNA barcoding were used to design a single set of primers and probe which detected three species in the genus Gadus: Atlantic cod, Pacific cod, and walleye pollock. RESULTS: Cod spiked into three different food matrices (cooking oil, clam chowder, and hushpuppy mix) yielded high linearity, dynamic range spanning six orders of magnitude, and lower limits of detection at 1-10 ppm (ppm; mg kg-1 ). Frying had an adverse effect on the lower limit of detection, but not on linearity. CONCLUSIONS: This work shows that COI DNA barcoding sequences can be used to effectively design real-time PCR assays for detection of food allergens in complex matrices. While full-length DNA barcodes distinguish individual species, the PCR assay designed here detected three different species. This is likely because real-time PCR assays are tolerant to basepair mismatches and do not utilize the full length of the DNA barcode. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Molecular characterization and expression analysis of the interleukin 1b gene in Pacific cod (Gadus macrocephalus).

Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24 h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.

Molecular characterization of caspase members and expression response to Nervous Necrosis Virus outbreak in Pacific cod.

Multiple functions of caspases include normal cell turnover, proper development and function of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell injury. During artificial propagation of Pacific cod, Gadus macrocephalus, high mortality occurred during early development stages. Here, we performed various analyses on the cDNA and protein sequences of six different G. macrocephalus caspases namely GmCasp3, 6, 7, 8, 9 and 10, and tried to investigate the contributions of caspase family to the development and Nervous Necrosis Virus (NNV) resistance. Sequence analysis of GmCaspase proteins showed that each caspase shared conserved domains like "HG", "QACXG (X for R, G or Q)" and "GSWF" except GmCasp10. Sequence alignment and phylogenetic tree showed that GmCasp8 and GmCasp10 were quite different from those of other fishes. 3-D models indicated that structure of GmCasp3 is very conservative, but GmCasp6, 7, 8, 9 and 10 are less conservative. Tissue distribution analysis showed that six Gmcaspases mRNA transcripts were detected in tissues of intestine, gill, thymus, head-kidney and spleen with different abundance, but Gmcasp7 were not detected in the brain. GmCasp3 transcript was kept at very low level in the early development stages, while the expression levels of GmCasp6, 7, 8, 10 were different at various development stages. GmCasp8 level seemed to be much higher than other caspases in the heads of 65dph and 75dph juveniles. To understand the role of caspases during NNV outbreak, modulation in expression of each Gmcaspases were investigated. The results showed that GmCasp3 transcript level increased significantly when NNV broke out, while GmCasp7, 8, 9 and 10 in cod heads decreased obviously at 69dph and 77dph. The results suggest that caspases in Pacific cod should be diverse in their structure and function, and their unique features and response to NNV outbreak add more evidences for the specificity of immune system in Pacific cod.

Increased ratio of pollock roe-specific IgE to salmon roe-specific IgE levels is associated with a positive reaction to cooked pollock roe oral food challenge.

BACKGROUND: Anaphylaxis and immediate-type fish roe allergies have been reported worldwide, and, in Japan, fish roe is the sixth most common food allergen. No oral food challenges (OFCs) have used pollock roe (PR), which is reported to have high cross-reactivity with salmon roe (SR). Therefore, we administered an OFC using cooked PR to evaluate PR- and SR-specific immunoglobulin E (IgE) levels and allergic reactions in patients with PR sensitivity. METHODS: This retrospective study evaluating patient characteristics and responses to OFCs was conducted with 10-20 g of cooked PR, between April 2006 and November 2016. RESULTS: We assessed 51 patients (median age: 6.8 years). All had PR sensitization, 6 (12%) with a history of immediate reactions to PR, and 18 (35%) of immediate reactions to SR. Median PR-specific and SR-specific IgE values were 3.4 kUA/L and 9.9 kUA/L, respectively. Seven patients (14%) had a positive OFC. There was no anaphylaxis. Induced symptoms were mild and included localized urticaria, throat pruritus, intermittent cough, and mild abdominal pain. We treated one patient with mild abdominal pain with oral antihistamines. There were no significant differences in history of immediate reaction to PR and PR-specific IgE titers between OFC-positive and OFC-negative patients, although significant differences were found for PR-specific IgE titers adjusted for SR-specific IgE (p = 0.025) and PR-specific IgE/SR-specific IgE ratio (p = 0.009). CONCLUSIONS: Increased PR-specific IgE/SR-specific IgE ratio or PR-specific IgE levels adjusted for SR-specific IgE levels were risk factors for OFC positivity.

Atrial Fibrillation in Anaphylaxis.

BACKGROUND: The relationship between anaphylaxis and cardiovascular events has been reported in the past. While skin and respiratory symptoms are usually the most common and the first to appear, cardiovascular complications play a key role and represent the leading cause of death in anaphylaxis. METHODS: We report 3 episodes of atrial fibrillation triggered by anaphylaxis. Allergy and cardiology studies were performed. In both patients, the etiological agent was identified: Anisakis simplex hypersensitivity and food allergy. RESULTS: The heart is the source and target of chemical mediators released during an allergic reaction. In the heart, there are plenty of mast cells, and they are predominantly located around the coronary adventitia and in close contact with small vessels in the muscle wall. The release of mediators can influence ventricular function, heart rate, and coronary artery tone. Anaphylaxis can trigger any kind of arrhythmia. In these cases, the very interesting point of discussion was: which should be first, treating anaphylaxis or cardiac events? The other controversial point was the use of epinephrine, the first line of treatment for anaphylaxis. Recommendations about epinephrine in cardiac patients during an anaphylactic event are still a major dilemma. CONCLUSIONS: We emphasize the importance of the priority of establishing protocols between cardiologist and allergist in treatment of cardiac complications during anaphylaxis, and we warn about the correct diagnosis of arrhythmias in anaphylaxis in order to treat them as soon as possible, to prevent other consequences and complications.

Successive Losses of Central Immune Genes Characterize the Gadiformes' Alternate Immunity.

Great genetic variability among teleost immunomes, with gene losses and expansions of central adaptive and innate components, has been discovered through genome sequencing over the last few years. Here, we demonstrate that the innate Myxovirus resistance gene (Mx) is lost from the ancestor of Gadiformes and the closely related Stylephorus chordatus, thus predating the loss of Major Histocompatibility Complex class II (MHCII) in Gadiformes. Although the functional implication of Mx loss is still unknown, we demonstrate that this loss is one of several ancient events appearing in successive order throughout the evolution of teleost immunity. In particular, we find that the loss of Toll-like receptor 5 predates the loss of Mx involving the entire Paracanthopterygii lineage. Using a time-calibrated phylogeny, we show that loss of MHCII and Mx overlap with major paleoclimatic and geological events indicating that these genetic changes were adaptive responses to the changing environment at the time.

Cloning and expression analysis of interferon regulatory factor 7 in the Pacific cod, Gadus macrocephalus.

Interferon regulatory factor 7 (IRF7) plays an important role in regulating the response of type I interferon (IFN) to viral infection. To understand the mechanisms underlying immune reactions in the Pacific cod, Gadus macrocephalus, the gene encoding G. macrocephalus IRF7 was cloned and characterized. The cDNA of G. macrocephalus IRF7 was also cloned and sequenced. A cDNA sequence of 2032 bp was assembled using polymerase chain reaction (PCR) products. It contains an open reading frame of 1323 bp in length, which encoded a 440-amino acid polypeptide that comprised a DNA-binding domain (DBD), an IRF association domain (IAD), and a serine-rich domain (SRD). In the DBD, the tryptophan cluster consisted of only four tryptophans, which is a unique characteristic in fish IRF7. The mRNA of IRF7 was detected in various tissues, including in the spleen, thymus, kidney, intestine, and gills, using relative quantification PCR (R-qPCR). Dynamic expression of IRF7 was observed in larvae throughout post-hatching (ph) development, with the highest level detected at day of ph (dph) 25. Response to immune stimulation was examined by challenging larvae with polyriboinosinic polyribocytidylic acid (pIC) to mimic viral infection and elicit an immune reaction. R-qPCR revealed that the expression of IRF7 significantly increased in pIC-treated groups relative to that in the control groups, in a time-dependent manner, with peak responses at 48 and 72 h after pIC-treatment. These results show that IRF7 is expressed in various tissues of adult fish and larvae and is sensitive to viral infection, suggesting that it plays a role in antiviral immune defense in G. macrocephalus.

Transcriptomic analysis and biomarkers (Rag1 and Igµ) for probing the immune system development in Pacific cod, Gadus macrocephalus.

Mortality (>90%) is a big concern in larval rearing facilities of Pacific cod, Gadus macrocephalus, limiting its culture presently still in the experimental stages. Understanding the immune system development of G. macrocephalus is crucial to optimize the aquaculture of this species, to improve the use of economic resources and to avoid abuse of antibiotics. For the transcriptome analysis, using an Illumina sequencing platform, 61,775,698 raw reads were acquired. After a de novo assembly, 77,561 unigenes were obtained. We have classified functionally these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 27 genes mainly related to hematopoietic or lymphoid organ development and somatic diversification of immune receptors have been reported for the first time in Pacific cod, and 14 Ig heavy chain (µ chain) locuses were assembled using Trinity. Based on our previous achievement, we have chosen Rag1 and Igµ as immune system development biomarkers. Full length cDNA of Rag1 and Igµ as biomarkers were obtained respectively using RACE PCR. Concerning Rag1, the deduced amino acid of Rag1 and protein immunodetection revealed a Rag1 isoform of 69 kDa, significantly different from other fish orthologs, such as Oncorhynchus mykiss (121 kDa). Phylogenetic analysis reveals a unique immune system for the Gadus genre, not exclusive for Atlantic cod, among vertebrates. Meanwhile, full length cDNA of Igµ included an ORF of 1710 bp and the deduced amino acid was composed of a leader peptide, a variable domain, CH1, CH2, Hinge, CH3, CH4 and C-terminus, which was in accordance with most teleost. Absolute quantification PCR revealed that significant expression of Rag1 appeared earlier than Igµ, 61 and 95 dph compared to 95 dph, respectively. Here we report the first transcriptomic analysis of G. macrocephalus as the starting point for genetic research on immune system development towards improving the Pacific cod aquaculture.

Unraveling the evolution of the Atlantic cod's (Gadus morhua L.) alternative immune strategy.

Genes encoding the major histocompatibility complex (MHC) have been thought to play a vital role in the adaptive immune system in all vertebrates. The discovery that Atlantic cod (Gadus morhua) has lost important components of the MHC II pathway, accompanied by an unusually high number of MHC I genes, shed new light on the evolution and plasticity of the immune system of teleosts as well as in higher vertebrates. The overall aim of this study was to further investigate the highly expanded repertoire of MHC I genes using a cDNA approach to obtain sequence information of both the binding domains and the sorting signaling potential in the cytoplasmic tail. Here we report a novel combination of two endosomal sorting motifs, one tyrosine-based associated with exogenous peptide presentation by cross-presenting MHCI molecules, and one dileucine-based associated with normal MHC II functionality. The two signal motifs were identified in the cytoplasmic tail in a subset of the genes. This indicates that these genes have evolved MHC II-like functionality, allowing a more versatile use of MHC I through cross-presentation. Such an alternative immune strategy may have arisen through adaptive radiation and acquisition of new gene function as a response to changes in the habitat of its ancestral lineage.

A multibiomarker approach on the Atlantic tomcod (Microgadus tomcod) in the St. Lawrence Estuary.

A multibiomarker approach was developed on juvenile Atlantic tomcod (Microgadus tomcod) to evaluate the pertinence of this approach for low-cost screening assessment of the environmental quality of various coastal sites within estuaries. Several biometric indices and biomarkers (ethoxyresorufin-O-deethylase (EROD) activity, metallothionein concentration, and immune responses) were investigated on immature and maturing tomcods (≤ 31 months) collected in four environmentally contrasted sites in the St. Lawrence Estuary (SLE). Simultaneous examination of various age classes provides the opportunity to detect short-term responses in sensitive young-of-the-year fish (e.g., EROD induction) and longer-time effects associated with chronic exposure and bioaccumulation (e.g., metallothionein induction). Principal component analysis was helpful to discriminate between responses possibly related to contaminant exposure (EROD, metallothionein) and responses that could be affected by upstream-downstream gradient (immune response, biometric indices). Measurement of a battery of biomarkers in young tomcods at several sites along the shore of the SLE is a low-cost screening investigation useful to identify hot spots requiring further investigation with chemical analysis and additional reference sites.

[Features of lysozyme content in cods (order Gadiformes) and flatfish (order Pleuronectiformes)].

The content of lysozyme as an indicator of nonspecific immunity in kidney, liver, spleen, and blood serum in 8 fish species of the order Gadiformes and the order Pleuronectiformes was analyzed. The Gadiformes are characterized by a very low level of enzyme or the absence of its activity in individual organs and serum, regardless of the fish habitat location. The Pleuronectiformes demonstrated diversity in content and distribution of lysozyme in the body of various fish species. Two plaice species with the unique distribution inhabiting the Sea of Okhotsk were found. This feature is due, first, to the highest lysozyme content in serum of the previously studied fish; second, to the 2-3-fold excess of the enzyme content in serum as compared with the studied immunocompetent organs. The obtained results confirm the opinion about the existence of various related groups among fishes which essentially differ in the lysozyme role in nonspecific immunity.

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function.

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout-Salmo trutta, perch-Perca fluviatilis, burbot-Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml(-1)) and Ig concentrations (T-Ig: 1.11-1.61 microg ml(-1), IgG [measured only in brown trout]: 1.49 microg ml(-1)) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 microg/g protein, IgG: 0.198 microg ml(-1)) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.

Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.