Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

Comportamiento de la IgE y las subclases de IgG en las infecciones por Staphylococcus aureus / Manifestation of IgE and IgG subclasses in Staphylococcus aureus infections

RESUMEN Introducción: Las infecciones cutáneas por Staphylococcus aureus, se presentan con alta frecuencia en la comunidad. La forma superficial de esta es la foliculitis. Una extensión hacia el tejido subcutáneo da como resultado la formación de una lesión supurativa local llamada forúnculo. Aproximadamente el 20 % de los pacientes con forúnculo presentan una o más recidivas durante el año siguiente, y cierto número presenta forunculosis recidivante crónica durante meses o años. Objetivo: Conocer el comportamiento de los niveles de IgE y las subclases de IgG expresadas en pacientes y controles, para inferir el patrón de la respuesta inmune a la infección. Métodos: Se realizó un estudio prospectivo donde se determinaron las subclases de inmunoglobulina G (IgG) y los niveles de inmunoglobulina E (IgE) en suero de 25 enfermos con lesiones cutáneas por Staphylococcus aureus, y 25 controles sanos, provenientes del banco de sangre. Se elaboró un antígeno (Bacterina de Staphylococcus aureus) y en su enfrentamiento se procedió con la metodología de normalización y validación de ensayos inmunoenzimáticos para cuantificar IgG humana. Los resultados se expresaron en densidades ópticas y gráficamente como la relación matemática (IgG1/3) para Th1 e (IgE/IgG4) para Th2, en el suero de los pacientes. Resultados: Se obtuvo respuesta IgG1 e IgG4 en pacientes y controles y respuesta IgE anti Staphylococcus aureus en pacientes. Conclusiones: Como Staphylococcus aureus es flora normal de la piel, los controles ya han tenido contacto con él, lo que provoca el desarrollo de respuesta Th1 (IgG1) y anticuerpos bloqueadores (IgG4). Contrariamente, los enfermos desarrollan respuesta Th2 (IgE) y la infección.

ABSTRACT Introduction: Staphylococcus aureus skin infections occur with high frequency in the community. Folliculitis is its superficial form. An extension into the subcutaneous tissue results in the formation of a local suppurative lesion known as a furunculosis. Approximately 20% of patients with a furuncle have one or more recurrences over the next year, and a certain number have chronic relapsing furunculosis for months or years. Objective: to know the manifestation of IgE levels and IgG subclasses expressed in patients and controls, to infer the pattern of the immune response to infection. Methods: we conducted a prospective study where immunoglobulin G (IgG) subclasses and immunoglobulin E (IgE) levels were determined in serum of 25 ill patients with Staphylococcus aureus skin lesions, and 25 healthy controls, from the blood bank. An antigen (Staphylococcus aureus bacterin) was elaborated and in its confrontation, the normalization and validation methodology of immunoenzymatic assays to quantify human IgG was carried out. The results were expressed in optical densities and graphically as the mathematical ratio (IgG1/3) for Th1 and (IgE/IgG4) for Th2, in the serum of the patients. Results: IgG1 and IgG4 responses were obtained in patients and controls, and anti-Staphylococcus aureus IgE responses in patients. Conclusions: the controls have already had contact with Staphylococcus aureus, as it is normal flora of the skin, which causes the development of Th1 response (IgG1) and antibodies blockers (IgG4). On the contrary, ill patients develop a Th2 response (IgE) and the infection.

Transcriptional Signatures of Immune, Neural, and Endocrine Functions in the Brain and Kidney of Rainbow Trout (Oncorhynchus mykiss) in Response to Aeromonas salmonicida Infection.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with "sickness behaviors" and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the "sickness behaviors" of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

GAS1: A New ß-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

ß-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two ß-glucan types extracted from wild-type baker's yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-ß-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that ß-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each ß-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard ß-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT ß-glucan, at D36, WT-ß-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-ß-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of ß-glucans (p<0.05). In conclusion, our results suggest that Gas1-ß-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other ß-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

CXC chemokines and their receptors in black rockfish (Sebastes schlegelii): Characterization, evolution analyses, and expression pattern after Aeromonas salmonicida infection.

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, seventeen CXC chemokine ligands (SsCXCLs) and nine CXC chemokine receptors (SsCXCRs) were systematically identified from Sebastes schlegelii genome. Phylogeny, synteny, and evolutionary analyses were performed to annotate these genes, indicating that the tandem duplications (CXCL8, CXCL11, CXCL32, CXCR2, and CXCR3), the whole genome duplications (CXCL8, CXCL12, CXCL18, and CXCR4), and the teleost-specific members (CXCL18, CXCL19, and CXCL32) led to the expansion of SsCXCLs and SsCXCRs. In addition, SsCXCLs and SsCXCRs were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in head kidney, liver, gill and spleen. Moreover, most SsCXCLs and SsCXCRs were significantly differentially expressed in head kidney, liver, and gill after Aeromonas salmonicida infection, and exhibited tissue-specific and time-dependent manner. Finally, protein-protein interaction network (PPI) analysis indicated that SsCXCLs and SsCXCRs interacted with a few immune-related genes such as interleukins, cathepsins, CD genes, and TLRs, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokines and receptors in teleost.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria).

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis.

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103  CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A-layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin-killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post-vaccination, respectively, in challenge test.

A transcriptome analysis focusing on splenic immune-related mciroRNAs of rainbow trout upon Aeromonas salmonicida subsp. salmonicida infection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that can regulate the immune responses during pathogen infection. Aeromonas salmonicida (A. salmonicida) subsp. salmonicida is the causative agent of furunculosis in salmon and trout. To identify the miRNAs and investigate the specific miRNAs in rainbow trout upon A. salmonicida subsp. salmonicida infection, we performed high throughput sequencing using the spleens of rainbow trout infected with and without an A. salmonicida subsp. salmonicida clinical isolate. A total of 381 known miRNAs and 926 novel miRNAs were identified. Eleven known and 16 novel miRNAs were found to be differentially expressed upon infection. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the target genes of the differentially expressed miRNAs were closely associated with immune responses and biological regulations. Additionally, over- and suppressed expression of miR-155-5p significantly enhanced and reduced the IL-2 and IL-1ß expressions in RTG-2 cells induced by A. salmonicida, respectively. To our knowledge, this is the first experimental study on the miRNAs of rainbow trout upon A. salmonicida infection. The results here might lay a foundation for the further understanding of the roles of miRNAs in the immune responses during A. salmonicida infection in rainbow trout.

Staphylococcus aureus and Host Immunity in Recurrent Furunculosis.

Staphylococcus aureus is one of the severest and most persistent bacterial pathogens. The most frequent S. aureus infections include impetigo, folliculitis, furuncles, furunculosis, abscesses, hidradenitis suppurativa, and mastitis. S. aureus produces a great variety of cellular and extracellular factors responsible for its invasiveness and ability to cause pathological lesions. Their expression depends on the growth phase, environmental factors, and location of the infection. Susceptibility to staphylococcal infections is rooted in multiple mechanisms of host immune responses and reactions to bacterial colonization. Immunological and inflammatory processes of chronic furunculosis are based on the pathogenicity of S. aureus as well as innate and acquired immunity. In-depth knowledge about them may help to discover the whole pathomechanism of the disease and to develop effective therapeutic options. In this review, we focus on the S. aureus-host immune interactions in the pathogenesis of recurrent furunculosis according to the most recent experimental and clinical findings.

A pentavalent vaccine for rainbow trout in Danish aquaculture.

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens - Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA, C3, IL-1ß, IL-6, IL-8, IgD and MHCII.

Frequencies of PD-1- and PD-L1- positive T CD3+CD4+, T CD3+CD8+ and B CD19+ lymphocytes and its correlations with other immune cells in patients with recurrent furunculosis.

Programmed cell death receptor 1 (PD-1) is one of the major immune checkpoints. Due to the lack of reports on PD-1- and PD-L1-positive lymphocyte proportions in patients with recurrent furunculosis, we aimed to evaluate percentages of those cells in the peripheral blood and to assess their correlations with other lymphocyte subsets, and the level of cell activation measured by the expression of CD25 and CD69 molecules on T lymphocytes. We recruited 30 patients with recurrent furunculosis and 15 controls. The amount of 5 mL of peripheral blood was collected for laboratory tests. Patients with chronic furunculosis presented with the similar number of lymphocytes, CD4+ T lymphocytes, CD8+CD3+ T suppressor lymphocytes, and CD19 + B lymphocytes to controls, but significant differences were found between subpopulation of those cells. Furunculosis patients had the significantly elevated percentage of lymphocytes with PD-1 and PD-L1 on their surface. Early onset of furunculosis was correlated with a higher percentage of CD19 + PD1 B lymphocytes. Greater number of skin lesions correlated with a decrease in the CD4PDL1+ cells, which subsequently was associated with an increase in the percentage of Treg cells, NKT cells, CD8+CD3+ lymphocytes and B lymphocytes. Changes in the proportion of immune cells may lead to reduced inflammatory reactions in patients with recurrent furunculosis. In the light of mechanisms of S. aureus invasion, the degree of immune impairments in the scope of adaptive immunity seems to play a significant role in the course of furunculosis. PD-1 and PD-L1 molecules change the host response and affect the ongoing inflammatory process.

Four selenoprotein P genes exist in salmonids: Analysis of their origin and expression following Se supplementation and bacterial infection.

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.

TNF overproduction impairs epithelial staphylococcal response in hyper IgE syndrome.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.

Inducers of salmon innate immunity: An in vitro and in vivo approach.

Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE-214. We found that the butyrate derivatives 4-phenyl butyrate (PBA) and ß-hydroxy-ß-methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24-72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG-1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL-families had lower basal levels of cathelicidin in gills, than non QTL-families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future.

NK and NKT-Like Cells in Patients with Recurrent Furunculosis.

To analyze changes in the number and percentage of NK and NKT-like cells in relation to other immune cells as well as to examine associations between increased susceptibility to infections and NK and NKT-like status in patients with recurrent furunculosis (RF) and healthy controls. Thirty patients with RF and 20 healthy age- and sex-matched volunteers were recruited. Blood samples were examined. Lymphocyte count and cytometric analyses were conducted. For statistical analysis, the Student's t test, F test, and Brown-Forsythe test were used for comparison between groups of variables. Associations were assessed with Pearson coefficient. Patients with RF had lower lymphocyte count than controls. Additionally, they presented with the following changes in the blood picture: a significant increase in the number of NK cells with a CD3+CD16+CD56+ phenotype; a proportional increase in the number and percentage of NKT-like cells with a CD3+CD16+CD56+ phenotype; a significant decrease in the number and percentage of T CD3+ cells. The number of NK cells was strongly positively correlated with the number of CD3 cells (r = 0.6162). The number of NKT cells was strongly positively correlated with CD3 cells (r = 0.6885) and CD3CD8 cells (r = 0.5465). Periodic exacerbations in RF are associated with the development of furuncles, which are a result of many already discovered as well as just being examined mechanisms. One of them is a significant increase in the number and most likely activation of NK and NKT-like cells during the formation of the inflammatory process and furuncles.

Possible Immunomodulating Effect of Retinol on Cytokines Secretion in Patients with Recurrent Furunculosis.

Recurrent furunculosis is an infection of hair follicles which results in formation of abscesses. Previous studies showed that the pathogenesis of the disease may include an immune-mediated component as the proliferative response of peripheral blood lymphocytes to staphylococcal antigen is depressed. The aim of our study was to evaluate cytokines concentration in the plasma of patients with recurrent furunculosis and to determine whether retinol affects the secretion of those cytokines in patients with recurrent furunculosis and healthy subjects. Blood samples were taken from 15 patients with recurrent furunculosis and 15 age-matched healthy subjects. A quantitative determination of selected cytokines (IL-17, 13, 2, 10, 4, IFN-γ, TNF-α) was performed in the plasma at baseline and after 72-h culture of peripheral blood mononuclear cells with and without retinol in both groups. In the plasma of patients with recurrent furunculosis, concentration of IL-10, 2, and TNF-α was significantly higher, whereas IL-13 significantly lower when compared with healthy subjects. After retinol stimulation, the concentration of IL-17 and IFN-γ increased significantly in both groups. Secretion of anti-inflammatory cytokines, especially IL-10 (p < 0.002) and 13 (p < 0.01), achieved lower levels in recurrent furunculosis samples than in those of healthy controls. Network of cytokines differs in patients with recurrent furunculosis from healthy subjects. Retinol stimulation affects secretion of both pro-inflammatory and anti-inflammatory cytokines. Further studies are recommended for better understanding the pathomechanism of recurrent furunculosis and potential clinical use of retinol in patients affected by recurrent furunculosis.

ALDH7A1 is a protein that protects Atlantic salmon against Aeromonas salmonicida at the early stages of infection.

Aldehyde dehydrogenases (ALDHs) belong to a super-family of detoxifying proteins and perform a significant role in developing epithelial homeostasis, protecting cells from toxic aldehydes and drug resistance. However, the activity and function of these detoxifying proteins remain unknown, especially in fish. In our research, we aimed to study functions of aldehyde dehydrogenase 7A1 (ALDH7A1) in Atlantic salmon infected by Aeromonas salmonicida. Recombinant ALDH7A1 (rALDH7A1) was verified by SDS-PAGE and western blot. The molecular mass of the deduced amino acid sequence of rALDH7A1 is 58.9 kDa with an estimated pI of 7.09. Only a low complexity region (141yvegvgevqeyvdv153) without a signal peptide existed in rALDH7A1. Results of ELISA indicated that rALDH7A1 exhibited apparent binding activities with A. salmonicida and its expression was highest in fish kidney. A Real-Time PCR (qRT-PCR) assay in kidneys confirmed that fish in this experiment were authentically infected and bacterial loads in rALDH7A1-adminsitered fish were significantly reduced at an early stage of infection. Meanwhile, we found the mRNA expression of NF-kß, P-38 MAPK, caspase-3 and TNF-α were mainly up-regulated at 72 h in the kidneys and livers of highly infected fish injected with rALDH7A1, and the same variation trend existed in fish spleens at 12 h. Consistent with these observations, neutralization experiments in vivo indicated that rALDH7A1 could obviously reduce the death rate compared to the BSA and control group. Taken together, we concluded that rALDH7A1 could act in host immune defense against bacterial infection and decrease the mortality rate of Atlantic salmon at early stages of infection with A. salmonicida.

Analysis of adipose tissue immune gene expression after vaccination of rainbow trout with adjuvanted bacterins reveals an association with side effects.

Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. Whilst very effective they are frequently associated with inflammatory responses that can result in clinically significant side-effects often involving the adipose tissue that is in direct contact with the vaccine. To explore the potential of immune gene expression changes in the adipose tissue of fish to be markers of vaccination efficacy or development of side-effects we have studied the response to a bacterial (Aeromonas salmonicida) vaccine administered with two different adjuvants. The first adjuvant was Montanide™ ISA 763A VG, thought to induce a mostly humoral response, and the second was Montanide™ ISA 761 VG that gives a more balanced humoral and cell mediated response. Following vaccination tissue samples were collected at days 3, 14 and 28 for RTqPCR analysis. Fifty immune genes were studied with a focus on a) pro-inflammatory associated molecules and b) adaptive immune response related molecules linked with possible Th1, Th2, Th17 and T-regulatory pathways, with the expression data analysed for associations with Speilberg post-vaccination side effect scores. The results showed that the adipose tissue is a particularly sensitive and discriminatory tissue for studying adjuvant effects. A clear upregulation of many immune genes occurred in response to both vaccine groups, which persisted over time and overlapped with the appearance of visible adhesions. Our analysis revealed a relationship between adipose tissue immune function and the development of vaccine-induced adhesions giving the potential to use immune gene expression profiling in this tissue to predict the side-effects seen.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L).

Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co-operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer-2 (AI-2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold-water teleost, little information has been known about the function of AI-2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI-2 activity and luxS defective mutant strain fail to produce AI-2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with ΔluxS strain. Overall, we hypothesis that AI-2 quorum sensing could regulate the expression of A-layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida.