Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

Fate of MHCII in salmonids following 4WGD.

Major histocompatibility complex (MHC) genes are key players in the adaptive immunity providing a defense against invading pathogens. Although the basic structures are similar when comparing mammalian and teleost MHC class II (MHCII) molecules, there are also clear-cut differences. Based on structural requirements, the teleosts non-classical MHCII molecules do not comply with a function similar to the human HLA-DM and HLA-DO, i.e., assisting in peptide loading and editing of classical MHCII molecules. We have previously studied the evolution of teleost class II genes identifying various lineages and tracing their phylogenetic occurrence back to ancient ray-finned fishes. We found no syntenic MHCII regions shared between cyprinids, salmonids, and neoteleosts, suggesting regional instabilities. Salmonids have experienced a unique whole genome duplication 94 million years ago, providing them with the opportunity to experiment with gene duplicates. Many salmonid genomes have recently become available, and here we set out to investigate how MHCII has evolved in salmonids using Northern pike as a diploid sister phyla, that split from the salmonid lineage prior to the fourth whole genome duplication (4WGD) event. We identified 120 MHCII genes in pike and salmonids, ranging from 11 to 20 genes per species analyzed where DB-group genes had the most expansions. Comparing the MHC of Northern pike with that of Atlantic salmon and other salmonids species provides a tale of gene loss, translocations, and genome rearrangements.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB.

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

Are cortisol and melatonin involved in the immune modulation by the light environment in pike perch Sander lucioperca?

The pineal gland is the main organ involved in the transduction process converting environmental light information into a melatonin response. Since light environment was described as an important factor that could affect physiology of teleosts, and because melatonin is a crucial hormone regulating numerous physiological processes, we hypothesized that environmental light may act on both stress and circadian axes which in turn could influence the immune status of pike perch. Therefore, we investigated the effects of two light spectra (red and white) and two light intensities (10 and 100 lx) with a constant photoperiod 12L(8:00-20:00) /12D on pike perch physiological and immune responses. Samples were collected at 04:00 and 16:00 at days 1 and 30 of the experiment. Stress markers, plasma melatonin levels, humoral innate immune markers, and expression of key immune genes in the head kidney were assessed. Light intensity clearly affected pike perch physiology. This included negative growth performances, increase in stress status, decrease in plasma melatonin levels, and immune depression. Light spectrum had only little influences. These results demonstrate that high stress status may have impacted melatonin production and secretion by the pineal organ. The drop in circulating melatonin and the increase in stress status may both be involved in the immune suppression.

Development of neutralizing antibody responses in muskellunge, Esox masquinongy (Mitchill), experimentally exposed to viral haemorrhagic septicaemia virus (genotype IVb).

A complement-dependent 50% plaque neutralization test was used to assess the neutralizing antibody response in sera of muskellunge, Esox masquinongy, experimentally infected with viral haemorrhagic septicaemia virus (VHSV, genotype IVb) by immersion. Groups of muskellunge were challenged with varying concentrations of VHSV: Group 1 with 10(2) plaque-forming units (pfu) mL(-1) , Group 2 with 4 × 10(3) pfu mL(-1) , Group 3 with 10(5) pfu mL(-1) and Group 4 with 0 pfu mL(-1) . The fish were held at a temperature of 11 ± 1 °C and were sampled over a 20-week period. Neutralizing antibodies were not detected in sera of any of the negative control fish throughout the study. Low neutralizing titres were detected in Groups 1-3 by 6 days post-infection (p.i.). Neutralizing titres of ≥80 [corrected]. were not detected again until 3, 4 and 7 weeks p.i. for Groups 2, 3 and 1, respectively, with peak titres for those groups occurring 16, 11 and 17 weeks p.i., respectively. VHSV was detected in serum for up to 11 weeks p.i. Results of this study show that survivors can be detected by a serological technique, despite being virus negative. This may benefit the investigation of VHSV IVb distribution in the Great Lakes and the study of host immune responses to this emerging sublineage.

Isolation and characterization of the immunoglobin of pike (Esox lucius L.).

The purification of the immunoglobul in from pike serum and its physicochemical characterization is presented. The immunoglobul in was prepared by means of gel filtration and ion exchange chromatography. Measurements in the analytical ultracentrifuge showed a sedimentation constant of 15.0 S. A molecular weight of 650.000 was calculated. The immunoglobulin was composed of heavy and light chains of molecular weights 60.000 and 22.500, respectively. It is likely that the immunoglobulin of pike is composed of 8 heavy and 8 light chains and possesses a tetrameric structure. The heavy chains contain 9.2% sugars and amino sugars. The amino acid composition of the chains is similar to that of other fish immunoglobulins.