The teleost polymeric Ig receptor counterpart in ballan wrasse (Labrus bergylta) differs from pIgR in higher vertebrates

As mucosal barriers in fish are the main sites where pathogens are encountered, mucosal immunity is crucial to avoid infection in the aquatic environment. In teleost fish, immunoglobulins are present in gut, gill and skin mucus, although not in the same amounts as in higher vertebrates. In mammals, the poly-Ig receptor (pIgR) is synthesized in epithelial cells and mediates the active transport of poly-immunoglobulins (pIgs) across the epithelium. During transport, a component of the pIgR, the secretory component (SC), is covalently bound to pIgs secreted into the mucus providing protection against proteases and avoiding degradation. The teleost pIgR gene does not show synteny to higher vertebrates, the overall structure of the protein is different (comprising two Ig domains) and its functional mechanisms remain unclear. The J-chain which is essential for pIgR-mediated transport of IgA and IgM in higher vertebrates is absent in teleost fish. The aim of the present study was to characterize the ballan wrasse (Labrus bergylta) pIgR and use it as a marker for further studies of mucosal immunity in this species. The pIgR gene was unambiguously identified. Unexpectedly, reverse transcription real time PCR (RT-qPCR) revealed highest abundance of pIgR mRNA in liver and significantly lower expression in mucosal organs such as foregut, hindgut, and skin. In situ hybridization showed pIgR-positive cells dispersed in the lamina propria while it was undetectable in epithelial cells of foregut and hindgut of ballan wrasse. A similar pattern was observed in Atlantic salmon. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of IgM enriched mucus samples from gut, gill, skin, and bile gave relatively few matches to wrasse pIgR. Notably, the matching peptides were from the transmembrane (TM) and cytoplasmatic (Cy) region as well as the putative SC, indicating leakage from lysed cells rather than covalent bonds between IgM and SC. Altogether, the results indicate that pIgR has another (or at least an additional) function in wrasse. Another pIgR-like molecule (pIgRL) in ballan wrasse (comprising three Ig domains) was analyzed to see if this could be an alternative functional pIgR homolog. However, the presence of pIgRL mRNA in blood leukocytes and a relatively high expression in immune organs like spleen and head kidney pointed to a receptor function on a circulating leukocyte population. As significant amounts of IgM were found in bile of ballan wrasse further studies should consider the hepato-biliary route regarding IgM delivery to the gut lumen.publishedVersio

Alternatives to Mineral Oil Adjuvants in Vaccines Against <i>Aeromonas salmonicida</i> subs. <i>salmonicida </i>in Rainbow Trout Offer Reductions in Adverse Effects

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Injection vaccines formulated with nucleotide, liposomal or mineral oil adjuvants induce distinct differences in immunogenicity in rainbow trout

Protection facilitated by the widespread use of mineral oil adjuvanted injection vaccines in salmonid fish comes with adverse effects of varying severity. In this study, we characterized the immunological profiles of two alternative vaccine formulations, both with proven efficacy and an improved safety profile in rainbow trout. Experimental injection vaccines were prepared on an identical whole-cell Aeromonas salmonicida bacterin platform and were formulated with CpG oligodeoxynucleotides, a liposomal (CAF01) or a benchmark mineral oil adjuvant, respectively. A naïve group, as well as bacterin and saline-injected groups were also included. Following administration, antigen-specific serum antibody titers, the tissue distribution of immune cell markers, and the expression of immune-relevant genes following the in vitro antigenic restimulation of anterior kidney leukocytes was investigated. Immunohistochemical staining suggested prolonged antigen presentation for the particulate formulations and increased mucosal presence of antigen-presenting cells in all immunized fish. Unlike the other immunized groups, the CAF01 group only displayed a transient elevation in specific antibody titers and immunohistochemical observations, and the transcription data suggest an increased role of cell-mediated immunity for this group. Finally, the transcription profile of the CpG formulation approached that of a TH1 profile. When compared to the benchmark formulation, CAF01 and CpG adjuvants induce slight, but distinct differences in the resulting protective immune responses. This is important, as it allows a broader immunological approach for the future development of safer vaccines.publishedVersio

Immunopathological characterization of red focal changes in Atlantic salmon (Salmo salar) white muscle

Farmed Atlantic salmon (Salmo salar) are prone to various conditions affecting the quality of the fillet. A well-known but so far poorly understood condition is the focal red changes in muscle, often referred to as haemorrhages. Such changes are characterized by muscle necrosis, haemorrhages and acute inflammation. They can progress into focal melanised changes, a chronic inflammatory condition with melanin-producing leukocytes. The initial cause of intramuscular haemorrhages is unknown. In this study, we aimed to reveal some of their key immunological features. Samples of red focal changes were investigated by immunohistochemistry (IHC), in situ hybridization (ISH) and RT-qPCR for various immune markers. The results were compared with samples of melanised changes and control muscle, subjected to the same analyses. In all red changes, infiltrates with mononuclear cells were detected, consisting mostly of MHC class I/II+ cells, but also of CD3+ and CD8+ cells. ISH studies on IgM showed few to moderate amounts of B-cells in red focal changes. Trends in the RT-qPCR showed upregulation of genes related to innate immunity in the red changes, whereas genes related to adaptive immunity were upregulated in the melanised changes. An important result was the significant downregulation of the anti-inflammatory cytokine IL10 in all red changes. Our findings indicate that we can rule out an auto invasive nature of the changes. The downregulation of IL10 at an early phase is a trait for the condition.publishedVersio

Comparative analysis of IgM sub-variants in salmonid fish and identification of a residue in mu 3 which is essential for MAb4C10 reactivity

In rainbow trout (Onchorhynchus mykiss) it has been shown that high affinity IgM antibodies havea higher degree of disulfide polymerization and a longer half life time. In the present study, distinct IgMsub-variants related to ancestral tetraploidy in salmonidfish were analyzed to reveal possible charac-teristic differences between these. A monoclonal antibody (MAb4C10) which distinguishes between IgM-A and IgM-B in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) was further characterized. Itwas shown that substitution of a proline located in the loop between the B and C beta strands of the thirdconstant domain (m3) of salmonmA eliminated MAb4C10 reactivity. Accordingly, the reverse substitutionin salmonmB restored MAb4C10 reactivity. Molecular cloning ofmcDNA from arctic char (Salvelinusalpinus) revealed two sub-variants (mA-1 andmA-2), i.e. a similar situation as in Atlantic salmon andbrown trout. However, arctic char IgM eluted in one peak by anion exchange chromatography, in contrastto salmon and brown trout IgM that are eluted in two peaks. The only characteristic residue of salmonand brown troutmB is an additional cysteine in the C-terminal part ofm4. Most likely, this cysteine isinvolved in inter-chain disulfide bonding and influences the elution profiles of IgM-A and IgM-B on anionexchange chromatography. Neither of themsub-variants in arctic char have the additional cysteine, andchar IgM, as well as salmon and brown trout IgM-A, showed a lower degree of inter-chain disulfidebonding than IgM-B when subjected to denaturation and gel electrophoresis under non-reducingconditions. Hybrids of char/salmon expressedmA-1,mA-2,mA andmB, indicating that there are twoparalogous Ig heavy chain gene complexes in the haploid genome of char, like in Atlantic salmon. Acomparison of salmonidmsequences is presented, including representatives ofSalmoninae(trout, salmonand char),Thymallinae(grayling) andCoregoninae(whitefish)submittedVersio

Comparative cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and pancreas disease (PD)

Author's accepted version (post-print)

Cellular heterogeneity in red and melanized focal muscle changes in farmed Atlantic salmon (Salmo salar) visualized by spatial transcriptomics

Spatial transcriptomics is a technique that provides insight into gene expression profiles in tissue sections while retaining structural information. We have employed this method to study the pathological conditions related to red and melanized focal changes in farmed Atlantic salmon (Salmo salar). Our findings support a model where similar molecular mechanisms are involved in both red and melanized filet discolorations and genes associated with several relevant pathways show distinct expression patterns in both sample types. Interestingly, there appears to be significant cellular heterogeneity in the foci investigated when looking at gene expression patterns. Some of the genes that show differential spatial expression are involved in cellular processes such as hypoxia and immune responses, providing new insight into the nature of muscle melanization in Atlantic salmon.publishedVersio

Anatomical and pathological characteristics of ribs in the Atlantic salmon (Salmo salar L.) and its relevance to soft tissue changes

Studies on the anatomical and pathological characteristics of ribs in farmed Atlantic salmon (Salmo salar L.) are warranted due to their possible association with red and melanized focal changes (RFC and MFC) in the fillet, a major quality and animal welfare concern. In this work, we provide an anatomical description of ribs based on radiographical and histological analyses. We also address various pathological rib changes and their association to RFC and MFC. In total, 129 fish were investigated; captured wild (n = 10) and hatchery reared (n = 119) Atlantic salmon (3.5–6.1 kg). The fish were selected based on the macroscopic presence of RFC, MFC or no changes (controls). Radiographic results revealed costal abnormalities in all fish groups. By histological investigations of the variations herein, our results provide new insight into the anatomical characteristics including vascularization within the ribs; a potential site for haemorrhage following costal fractures. Costal fractures were detected by radiology in 40 of 129 samples (RFC: 38.4%, MFC: 47.2%, controls: 9.5 %). A statistically significant association was found between costal fractures and red (p = 0.007) and melanized changes (p = 0.000). However, red and melanized changes were also observed in samples with no costal fractures (n = 45), indicating that also other factors influence the development of RFC/MFC.publishedVersio

Red and melanized focal changes in white skeletal muscle in Atlantic salmon (Salmo salar): Comparative analysis of farmed, wild and hybrid fish reared under identical conditions

Selective breeding plays a vital role in the production of farmed Atlantic salmon and has shown success in many aspects. Still, challenges related to fish health and welfare continue to result in significant economic losses. One such challenge is red and melanized focal changes (RFC/MFC), which result from acute and chronic inflammation, respectively, in the skeletal muscle. Importantly, RFC/MFC has not been observed in wild Atlantic salmon, suggesting that both external and genetic factors may contribute to the development of inflammation. To investigate the underlying cause of RFC/MFC, we conducted a study involving 1854 Atlantic salmon of farmed, wild and hybrid origin. All fish were reared under identical conditions to minimize the influence of external factors. Throughout the production cycle, the fish was monitored for growth parameters and examined for RFC/MFC using macroscopic and histological analysis. We found no association between the experimental groups and the presence of RFC/MFC. Histological investigations revealed melano-macrophages in the soft tissue in freshwater smolt, although no macroscopic discoloration was observed. MFC showed granulomas in various stages, suggesting a complex progression of the condition. In summary, we conclude that RFC/MFC is primarily caused by external factors found in the rearing facilities of farmed Atlantic salmon.publishedVersio