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Despite the importance and success of vaccination against bacterial diseases in fish, little is known about the mechanisms of vaccine-induced disease resistance. In this study a known efficacious bacterial vaccine, to Enteric Redmouth Disease (ERM), was used to vaccinate rainbow trout, and sixty days later the fish were challenged with the causative agent of the disease, Yersinia ruckeri. The bacterial burden in the spleen, the spleen index, and the expression profiles of pro- and anti-inflammatory cytokines and marker genes for T helper (Th) cells in the spleen and gills were analyzed, in comparison to the profiles in naïve/challenged fish. As expected, the bacterial burden in the spleen of naïve fish increased over time and was correlated with the spleen index after Y. ruckeri challenge. The gene expression data showed that pro-inflammatory cytokines were upregulated post-infection in the spleen of both naïve and vaccinated fish after Y. ruckeri challenge although the pro-inflammatory cytokine expression was much lower in vaccinated fish compared to the naïve fish. A correlated expression between pro-inflammatory cytokines and anti-inflammatory cytokines was only seen in spleen of ERM vaccinated fish, where a Th1-like response was indicated by the correlated gene expression of IFN-γ, T-bet and IL-2. In contrast, in the gills, the inflammatory gene response was enhanced in vaccinated fish compared to naïve fish, but perhaps more importantly there was a strong upregulation of IL-22 which was negatively correlated with IFN-γ gene expression at this site. Thus, it is possible that different types of adaptive responses are on-going within the vaccinated fish during infection with Y. ruckeri, potentially affected by the site and stage of infection.

Arsenic toxicity in an aquatic environment is a major concern, and its elimination has become a global challenge. In the current study, histopathology, serum biomarkers and cytokine gene expression were comparatively examined in fish fed with a control diet or diets containing Chlorella vulgaris (Ch) after exposure to sodium arsenite (NaAsO2) in Nile tilapia (Oreochromis niloticus) with the aim of evaluating the protective role of Ch against arsenite-induced toxicity. Severe histopathological alterations were evident in fish exposed to 7 ppm (parts per million) arsenite for 21 days, compared to unexposed fish. Levels of serum biomarkers ALT, AST, ALP, urea and creatinine were increased, but the levels of Na+, total proteins, albumins and globulins were decreased. Moreover, the expression of all the cytokine genes examined, including IL-1β (7-fold), TNF-α (14-fold) and TGF-β1 (13-fold), were significantly upregulated after arsenite exposure. However, in fish fed with diets containing 5% or 10% Ch, the histopathological alterations in the gills, liver and head kidney were reduced, the biomarkers were stabilized, and the upregulation of cytokine gene expression was lowered, with the high Ch diet (10%) showing more prominent effects. These results suggest the protective and therapeutic roles of Ch as a feed supplement in Nile tilapia against arsenic induced toxicity.

Th2 immunity is a primary host defence against metazoan pathogens and two of the important cytokines involved in this immune response in mammals are IL-4 and IL-13. Recently the origin and evolution of Th2 immune responses have been investigated in fish where a molecule with relatedness to both IL-4 and IL-13 is present, termed IL-4/13. Different IL-4/13 paralogues (IL-4/13 A and IL-4/13B) exist in teleost fish. In this paper, we have focused on the IL-4/13 isoforms found in the European sea bass ( Dicentrarchus labrax L.). Two tandem duplicated but divergent IL-4/13 A isoforms and one IL-4/13B are present, a unique situation compared to other teleosts. These genes were studied in terms of their in vitro and in vivo transcript levels after different treatments and their biological activities after production of the recombinant isoforms. The results show that the presence of these three paralogues is associated with different activities, both in terms of their expression profiles and the ability of the proteins to modulate the expression of immune genes in head kidney leukocytes. It is clear that the initiation and control of type-2 responses in seabass is complex due to the presence of multiple IL-4/13 isoforms with overlapping but distinct activities.

The following research was conducted to elucidate the evolution and expression of salmonid selenoprotein P (SelP), a selenoprotein that is unique in having multiple selenocysteine (Sec) residues, following supranutritional selenium supplementation and infection in rainbow trout. We show that in salmonids SelP is present as four paralogues and that the diversification of SelP genes during vertebrate evolution relates to whole genome duplication events. With 17 and 16 selenocysteine residues for rainbow trout (Oncorhynchus mykiss)/Atlantic salmon (Salmo salar) SelPa1 and SelPa2 proteins respectively and 1 or 2 (trout or salmon) and 4 or 3 (trout or salmon) selenocysteine residues for salmonid SelPb1 and SelPb2 proteins respectively, this is the highest number of (predicted) multiple selenocysteine containing SelP proteins reported for any vertebrate species to date. To investigate the effects of selenium form on SelP expression we added different concentrations (1 nM- 10 μM) of organic or inorganic selenium to a trout cell line (RTG-2 cells) and analysed changes in mRNA abundance. We next studied the impact of supplementation on the potential modulation of these transcripts by PAMPs and proinflammatory cytokines in RTG-2 and RTS-11 cells. These experiments revealed that selenium type influenced the responses, and that SelP gene subfunctionalisation was apparent. To get an insight into the expression patterns in vivo we conducted a feeding trial with 2 diets differing in selenium content and 5 weeks later challenged the trout with a bacterial pathogen (Aeromonas salmonicida). Four tissues were analysed for SelP paralogue expression. The results show a significant induction of SelPa1 in gills and intestine following infection in selenium supplemented fish and for SelPa2 in gills. SelPb1 was significantly reduced in head kidney of both diet groups following infection, whilst SelPb2 was significantly upregulated in skin of both diet groups post infection. Overall these findings reveal differential expression profiles for the SelPa/SelPb paralogues in trout, influenced by selenium supply, cell type/tissue and stimulant. The increase of multiple Sec containing SelP proteins in salmonids could indicate an enhanced requirement for selenium in this lineage.

The thymus in vertebrates plays a critical role in producing functionally competent T-lymphocytes. Phylogenetically, the thymus emerges early during evolution in jawed cartilaginous fish, and it is usually a bilateral organ placed subcutaneously at the dorsal commissure of the operculum. In this review, we summarize the current understanding of the thymus localization, histology studies, cell composition, and function in teleost fishes. Furthermore, we consider environmental factors that affect thymus development, such as seasonal changes, photoperiod, water temperature fluctuations and hormones. Further analysis of the thymus cell distribution and function will help us understand how key stages for developing functional T cells occur in fish, and how thymus dynamics can be modulated by external factors like photoperiod. Overall, the information presented here helps identify the knowledge gaps and future steps needed for a better understanding of the immunobiology of fish thymus.

Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5'-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense.

•Intramuscular VHSV DNA vaccination induces the infiltration of B cells in trout.•These B cells are both IgM+ and IgT+ cells.•CXCL11_L1, CK5B, CK6 and CXCR3B transcription is up-regulated in vaccinated fish.•CK5B and CK6 have chemotactic capacities. In fish, intramuscular (i.m) injection of plasmid DNA encoding viral proteins has proved a highly effective vaccination strategy against some viral pathogens. The efficacy of DNA vaccination in teleost fish is based on the high level of viral antigen expression in muscle cells inducing a strong and long-lasting protection. However, the mechanisms through which this protection is established and effectuated in fish are still not fully understood. Moreover, similarities to mammalian models cannot be established since DNA vaccination in mammals usually induces much weaker responses. In this work, we have focused on the characterization of the immune cells that infiltrate the muscle at the site of DNA injection in vaccinated fish and the chemokines and chemokine receptors that may be involved in their infiltration. We have demonstrated through diverse techniques that B lymphocytes, both IgM+ and IgT+ cells, represented a major infiltrating cell type in fish vaccinated with a viral haemorrhagic septicaemia virus (VHSV) glycoprotein-encoding DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocyte/monocyte-type cells were attracted. Among twelve chemokine genes studied, only CXCL11_L1, CK5B and CK6 mRNA levels were up-regulated in DNA vaccinated fish compared to fish injected with the corresponding vector backbone. Furthermore, the transcription of CXCR3B, a possible receptor for CXCL11_L1 was also significantly up-regulated in vaccinated fish. Finally, experiments performed with recombinant trout CK5B and CK6 and chemokine expression plasmids revealed that these chemokines have chemotactic capacities which might explain the recruitment of B cells to the site of DNA injection. Altogether, our results reveal that there is an early chemokine-related B cell recruitment triggered by i.m. DNA vaccination against VHSV which might play an important role in the initial phase of the immune response.

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins’ abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish.

We collected, processed, identified, and analyzed the spores and pollen samples from the Zhuanchengzi Bed of the Yixian Formation in the Yingwoshan area of western Liaoning. As a result, we confirm a palynomorph assemblage of Cicatri- cosisporites-Protoconiferus. The pollen was primarily from gymnosperms, dominated especially by conifer pollen. Pterido- phyte spores were less common and some questionable angiosperm pollen occurred occasionally. The age of the palynomorph assemblage is dated as the late Valanginian or Hauterivian-Barrernian stage, the Early Cretaceous. The study applies the con- cept of Palynological Vegetation based on palynological spectra and the paleoecological characteristics of palynological taxa for the first time. Palynological vegetation type, climatic zone type, and humidity type are divided quantitatively for the Zhuanchengzi Bed in the Yixian Formation of western Liaoning. We then obtained the evolutionary trends. The results showed that the overall climate was warm and humid during the deposition period of the Zhuanchengzi Bed in the Yixian Formation. Palynological vegetation types are various and include coniferous forest, deciduous broadleaf forest, evergreen broad-leaved forest, grass, and shrubs. The local temperature changed from warm to much warmer and from a semi-humid to humid climate. Palynological vegetation types are always dominated by coniferous forest. The coexistence of deciduous broad-leaved forest, evergreen broad-leaved forest, shrubs, grass, and some xerophytic plants indicates vertical zonation and seasonal climate change The vertical vegetation types and the warm humid climate may imply a large geomorphological contrast in the Yixian Formation of western Liaoning.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer‐related death. Although clinicopathological parameters provide invaluable prognostic information, the accuracy of prognosis can be improved by using molecular biomarker signatures. Using a large dataset of immunohistochemistry‐based biomarkers (n = 66), this study has developed an effective methodology for identifying optimal biomarker combinations as a prognostic tool. Biomarkers were screened and assigned to related subsets before being analysed using an iterative algorithm customised for evaluating combinatorial interactions between biomarkers based on their combined statistical power. A signature consisting of six biomarkers was identified as the best combination in terms of prognostic power. The combination of biomarkers (STAT1, UCP1, p‐cofilin, LIMK2, FOXP3, and ICOS) was significantly associated with overall survival when computed as a linear variable (χ2 = 53.183, p < 0.001) and as a cluster variable (χ2 = 67.625, p < 0.001). This signature was also significantly independent of age, extramural vascular invasion, tumour stage, and lymph node metastasis (Wald = 32.898, p < 0.001). Assessment of the results in an external cohort showed that the signature was significantly associated with prognosis (χ2 = 14.217, p = 0.007). This study developed and optimised an innovative discovery approach which could be adapted for the discovery of biomarkers and molecular interactions in a range of biological and clinical studies. Furthermore, this study identified a protein signature that can be utilised as an independent prognostic method and for potential therapeutic interventions.