The use of salmonid epithelial cells to characterize the toxicity of Tenacibaculum maritimum soluble extracellular products.

AIMS: This study assessed how the etiological agent of mouth rot in farmed Atlantic salmon, Tenacibaculum maritimum, induces toxicity in host salmonid barrier cells, and determined whether environmental changes are relevant for these effects. METHODS AND RESULTS: Tenacibaculum maritimum soluble extracellular products (ECPs) were collected and used to treat Atlantic salmon and rainbow trout intestinal barrier cell lines as a comparative model of bacterial-salmonid cell interactions. Cellular assays that examine cell membrane integrity, marker expression, and metabolic activity revealed that T. maritimum ECPs induced salmonid epithelial cell death through an apoptosis mechanism. Changes in salinity (25, 29, and 33 ppt) and temperature (12°C, 18°C, and 24°C) within the natural ranges observed in Pacific Northwest aquaculture facilities affected bacterial growth and cytotoxicity of T. maritimum ECPs. CONCLUSIONS: Our results suggest epithelial barriers as targets of T. maritimum-mediated toxicity in farmed mouth rot-infected Atlantic salmon. The induction of apoptosis by T. maritimum soluble ECPs may also help to explain the absence of overt inflammation typically reported for these fish.

Expression analysis of Carassius auratus-leukocyte-immune-type receptors (CaLITRs) during goldfish kidney macrophage development and in activated kidney leukocyte cultures.

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements.

Mechanical replication of natural fever enhances protection against Aeromonas veronii infection in a teleost fish.

There is a long-standing debate on the attributes of temperature for fish health. We recently showed that thermoregulatory programs exerted through natural behavioural fever drive molecular and cellular responses that contribute to pathogen clearance, inflammation control, and tissue repair. These offered a mechanistic basis for the survival advantage conferred through fever. Herein, we show the attributes of mechanical replication of this fever response. Central to our approach was consideration of both, the maximal temperatures naturally selected by fish after infection, as well as the dynamics of thermal changes induced through this response. Coarse replication of the febrile thermal program as well as shorter truncated thermal schedules offered immune-regulatory capacity. Most notably, these promoted induction of acute inflammation and significant enhancements to pathogen clearance. However, the coarse protocols tested only partially recapitulated enhancements to induction and control of tissue repair. Our findings highlight a promising new alternative to combat infections in fish using a natural, drug-free, sustainable approach.

Acute Inflammation in Tissue Healing.

There are well-established links between acute inflammation and successful tissue repair across evolution. Innate immune reactions contribute significantly to pathogen clearance and activation of subsequent reparative events. A network of molecular and cellular regulators supports antimicrobial and tissue repair functions throughout the healing process. A delicate balance must be achieved between protection and the potential for collateral tissue damage associated with overt inflammation. In this review, we summarize the contributions of key cellular and molecular components to the acute inflammatory process and the effective and timely transition toward activation of tissue repair mechanisms. We further discuss how the disruption of inflammatory responses ultimately results in chronic non-healing injuries.

Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.

Application of imaging flow cytometry for characterization of acute inflammation in non-classical animal model systems.

Phagocytes display marked heterogeneity in their capacity to induce and control acute inflammation. This has a significant impact on the effectiveness of antimicrobial immune responses at different tissue sites as well as their predisposition for inflammation-associated pathology. Imaging flow cytometry provides novel opportunities for characterization of these phagocyte populations through high spatial resolution, statistical robustness, and a broad range of quantitative morphometric cell analysis tools. This study highlights an integrative approach that brings together new tools in imaging flow cytometry with conventional methodologies for characterization of phagocyte responses during acute inflammation. We focus on a comparative avian in vivo challenge model to showcase the added depth gained through these novel quantitative multiparametric approaches even in the absence of antibody-based cellular markers. Our characterization of acute inflammation in this model shows significant conservation of phagocytic capacity among avian phagocytes compared to other animal models. However, it also highlights evolutionary divergence with regards to phagocyte inflammation control mechanisms based on the internalization of apoptotic cells.

Neutrophils exert protection in early Aeromonas veronii infections through the clearance of both bacteria and dying macrophages.

Aeromonas veronii is a gram-negative opportunistic pathogen capable of infecting both fish and mammals. Left untreated, natural infection in fish can prove fatal and result in irreparable damage to the aquaculture industry. Neutrophils are essential innate effector cells that play critical roles in pathogen defense. Our aim was to investigate the immunological roles of teleost neutrophils during infection with A. veronii. We began by examining the functional defenses of neutrophils in vitro, where neutrophils efficiently killed the pathogen. In addition, we developed an in vivo infection model to assess the roles of neutrophils during an infection in goldfish. This allowed us to explore the complex dynamics between immune cells and Aeromonas veronii. Interestingly, our studies found that neutrophils are capable of sensing a diverse range of dead and dying cells, resulting in varying downstream responses. Herein, we report that neutrophils internalized dead or dying macrophages previously infected with A. veronii. Moreover, once internalized, neutrophils went on to display classical pro-inflammatory ROS responses, in contrast to the more typical anti-inflammatory responses seen in cells following the uptake of a dead host cell. This led us to hypothesize that during infection, neutrophils are capable of simultaneously clearing dead and dying cells as well as A. veronii. This study provides additional insights into the complex mechanisms by which neutrophils operate within an inflammatory site and contribute to the induction and regulation of acute inflammatory responses.

Early activation of teleost B cells in response to rhabdovirus infection.

UNLABELLED: To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. IMPORTANCE: Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

An approach to classification and capacitance expressions in electrochemical capacitors technology.

The proliferation of novel types and designs of electrochemical capacitors makes it necessary to obtain a better understanding of the behavior of these systems together with a more systematic classification of them. In this study a rational classification of supercapacitors based on the charge storage mechanism and the active material of each electrode is proposed. The internationally accepted terminology - the terms symmetric, asymmetric and hybrid - is also clarified in an attempt to standardize the current definitions and facilitate the systematic classification of each device. Additionally, the selection of suitable mathematical expressions to calculate the capacitance of each kind of system is rationalized throughout the discussion taking into account the behavioral characteristics of each electrode. An examination of the potential evolution profile of each electrode during the galvanostatic cycling of the supercapacitor is presented as a key tool for understanding the fundamental behavior of these devices.

Control of CSF-1 induced inflammation in teleost fish by a soluble form of the CSF-1 receptor.

The colony-stimulating factor-1 (CSF-1) is the principal regulator of the survival, proliferation, differentiation, and function of macrophages and their precursors, and has been shown to play a role in the etiology of inflammation. We recently identified a novel mechanism for the control of CSF-1 activity in teleost fish, through the production of an inhibitory soluble form of the CSF-1 receptor (sCSF-1R). Primary goldfish kidney macrophages selectively expressed sCSF-1R during the senescence phase, which corresponds to a defined stage of in vitro culture development where inhibition of macrophage proliferation and apoptotic cell death are prominent. In contrast, primary macrophage cultures undergoing active proliferation displayed low levels of sCSF-1R expression. Addition of purified recombinant sCSF-1R to developing primary macrophage cultures leads to a dose-dependent decrease in macrophage proliferation and inhibits macrophage antimicrobial functions including chemotaxis, phagocytosis, and production of reactive oxygen intermediates. Using a goldfish in vivo model of self-resolving peritonitis, we found that sCSF-1R plays a role in the inhibition of inflammation, following an initial acute phase of antimicrobial responses within an inflammatory site. Soluble CSF-1R inhibits pro-inflammatory cytokine production, inhibits leukocyte recruitment to the inflammatory site and decreases ROS production in a dose-dependent manner. This sCSF-1R-dependent regulation of inflammation appears to be an elegant mechanism for the control of macrophage numbers at inflammatory sites of lower vertebrates. Overall, our results provide new insights into the evolutionary origins of the CSF-1 immune regulatory axis.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells.

Nitric oxide (NO) and Ca(2+) are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca(2+) signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca(2+)-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca(2+) availability and voltage-sensitive Ca(2+) channels, as well as intracellular Ca(2+) store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca(2+)-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca(2+)-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca(2+)-dependent transduction pathways.

Unveiling the potential of cellulose, chitosan and polylactic acid as precursors for the production of green carbon nanofibers with controlled morphology and diameter.

Carbon nanofibers (CNFs) are very promising materials with application in many fields, such as sensors, filtration systems, and energy storage devices. This study aims to explore the use of eco-friendly biopolymers for CNF production, finding novel, suitable and sustainable precursors and thus prioritising environmentally conscious processes and ecological compatibility. Polymeric nanofibers (PNFs) using cellulose acetate, polylactic acid, and chitosan as precursors were successfully prepared via electrospinning. Rheological testing was performed to determine suitable solution concentrations for the production of PNFs with controlled diameter and appropriate morphology. Their dimensions and structure were found to be significantly influenced by the solution concentration and electrospinning flow rate. Subsequently, the electrospun green nanofibers were subject to stabilisation and carbonisation to convert them into CNFs. Thermal behaviour and chemical/structural changes of the nanofibers during stabilisation were investigated by means of thermogravimetric analysis and Fourier-transform infrared spectroscopy, while the final morphology of the fibers after stabilisation and carbonisation was examined through scanning electron microscopy to determine the optimal stabilisation parameters. The optimal fabrication parameters for cellulose and chitosan-based CNFs with excellent morphology and thermal stability were successfully established, providing valuable insight and methods for the sustainable and environmentally friendly synthesis of these promising materials.

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity.

BACKGROUND: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. METHODS: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. RESULTS: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). CONCLUSIONS: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.

Mycobacterial phenolic glycolipids with a simplified lipid aglycone modulate cytokine levels through Toll-like receptor 2.

Phenolic glycolipids (PGLs) are virulence factors present in the cell walls of many pathogenic mycobacteria. PGLs have been implicated in various aspects of mycobacterial disease, but there are limited structure-activity data available for these molecules. We report here the preparation of seven synthetic PGL analogues, differing from the native compounds in the replacement of the complex phenolic lipid moiety with a p-methoxyphenyl group. The ability of these compounds to stimulate or inhibit the production of cytokines (TNF-α, IL-1ß, IL-6, MCP-1) and nitric oxide (NO) was then evaluated by ELISA-based assays. None of the compounds stimulated the production of these biological signalling molecules. In contrast, they each displayed concentration-dependent inhibitory activity, related to the methylation pattern of the molecule and mediated by Toll-like receptor 2. Additional studies revealed that native PGL-I from Mycobacterium leprae and a synthetic PGL-I analogue containing a simplified lipid domain had enhanced inhibitory activities relative to the corresponding analogues containing the p-methoxyphenyl aglycone; however, the natural lipid phenolthiocerol was only weakly active. These studies reveal that synthetic molecules of this type can be used as probes for PGL function. Moreover, their ease of synthesis relative to the natural glycolipids, as well as their more favourable aqueous solubility, should allow for more thorough structure-activity relationship studies.

The acute inflammatory response of teleost fish.

Acute inflammation is crucial to the immune responses of fish. The process protects the host from infection and is central to induction of subsequent tissue repair programs. Activation of proinflammatory signals reshapes the microenvironment within an injury/infection site, initiates leukocyte recruitment, promotes antimicrobial mechanisms and contributes to the resolution of inflammation. Inflammatory cytokines and lipid mediators are primary contributors to these processes. Uncontrolled or persistent induction results in delayed tissue healing. The kinetics by which inducers and regulators of acute inflammation exert their actions is essential for understanding the pathogenesis of fish diseases and identifying potential treatments. Although, a number of these are well-conserved across, others are not, reflecting the unique physiologies and life histories of members of this unique animal group.

Early-life ß-glucan exposure enhances disease resilience of broiler chickens to a natural Clostridium perfringens infection.

Necrotic enteritis (NE) is an economically important disease in poultry. Colonization by the opportunistic pathogen C. perfringens occurs early after hatch and induces host immune tolerance, which allows it to persist as part of the bird's commensal microflora. ß-glucan, a yeast cell wall component, is well characterized for its immunomodulatory capacity, and is a strong driver of innate immune memory. In this study, we assessed the effectiveness of ß-glucan to reduce severity of NE, when co-administered with heat-killed C. perfringens via intra-abdominal route at day 1 of age. We found that this early-life exposure in the presence of ß-glucan did not reduce intestinal C. perfringens loads or lesion severity during a subsequent NE outbreak. However, it improved ileal morphology, prevented liver and spleen weight decline, and preserved feed efficiency in challenged birds. Molecular analyses revealed metabolic changes consistent with innate immune memory. Together, our results suggest that ß-glucan can reduce the negative impacts of NE by influencing the context in which C. perfringens is first encountered.

The importance of the timing of microbial signals for perinatal immune system development.

Background: Development and maturation of the immune system begin in utero and continue throughout the neonatal period. Both the maternal and neonatal gut microbiome influence immune development, but the relative importance of the prenatal and postnatal periods is unclear. Methods: In the present study, we characterized immune cell populations in mice in which the timing of microbiome colonization was strictly controlled using gnotobiotic methodology. Results: Compared to conventional (CONV) mice, germ-free (GF) mice conventionalized at birth (EC mice) showed few differences in immune cell populations in adulthood, explaining only 2.36% of the variation in immune phenotypes. In contrast, delaying conventionalization to the fourth week of life (DC mice) affected seven splenic immune cell populations in adulthood, including dendritic cells and regulatory T cells (Tregs), explaining 29.01% of the variation in immune phenotypes. Early life treatment of DC mice with Limosilactobacillus reuteri restored splenic dendritic cells and Tregs to levels observed in EC mice, and there were strain-specific effects on splenic CD4+ T cells, CD8+ T cells, and CD11c+ F4/80+ mononuclear phagocytes. Conclusion: This work demonstrates that the early postnatal period, compared to the prenatal period, is relatively more important for microbial signals to influence immune development in mice. Our findings further show that targeted microbial treatments in early life can redress adverse effects on immune development caused by the delayed acquisition of the neonatal gut microbiome.

Fever integrates antimicrobial defences, inflammation control, and tissue repair in a cold-blooded vertebrate.

Multiple lines of evidence support the value of moderate fever to host survival, but the mechanisms involved remain unclear. This is difficult to establish in warm-blooded animal models, given the strict programmes controlling core body temperature and the physiological stress that results from their disruption. Thus, we took advantage of a cold-blooded teleost fish that offered natural kinetics for the induction and regulation of fever and a broad range of tolerated temperatures. A custom swim chamber, coupled to high-fidelity quantitative positional tracking, showed remarkable consistency in fish behaviours and defined the febrile window. Animals exerting fever engaged pyrogenic cytokine gene programmes in the central nervous system, increased efficiency of leukocyte recruitment into the immune challenge site, and markedly improved pathogen clearance in vivo, even when an infecting bacterium grew better at higher temperatures. Contrary to earlier speculations for global upregulation of immunity, we identified selectivity in the protective immune mechanisms activated through fever. Fever then inhibited inflammation and markedly improved wound repair. Artificial mechanical hyperthermia, often used as a model of fever, recapitulated some but not all benefits achieved through natural host-driven dynamic thermoregulation. Together, our results define fever as an integrative host response that regulates induction and resolution of acute inflammation, and demonstrate that this integrative strategy emerged prior to endothermy during evolution.

A Poultry Subclinical Necrotic Enteritis Disease Model Based on Natural Clostridium perfringens Uptake.

Necrotic enteritis (NE) in poultry is an opportunistic infection caused by Clostridium perfringens. Well-known as a multifactorial disease, NE development is under the influence of a wide range of environmental risk factors that promote the proliferation of pathogenic C. perfringens at the expense of nonpathogenic strains. Current in vivo NE challenge models typically incorporate pre-exposure to disease risk factors, in combination with exogenous C. perfringens inoculation. Our goal was to enhance current models using a natural uptake of C. perfringens from the barn environment to produce a subclinical infection. We incorporated access to litter, coccidial exposure (either 10× or 15× of the manufacturer-recommended Coccivac B52 Eimeria vaccine challenge; provided unspecified doses of E. acervulina, E. mivati, E. tenella, and two strains of E. maxima), feed composition, and feed withdrawal stress, and achieved the commonly observed NE infection peak at 3 weeks post-hatch. NE severity was evaluated based on gut lesion pathology, clinical signs, and mortality rate. Under cage-reared conditions, 15× coccidial vaccine-challenged birds showed overall NE lesion prevalence that was 8-fold higher than 10× coccidial vaccine-challenged birds. NE-associated mortality was observed only in a floor-reared flock after a 15× coccidial vaccine challenge.

Isolation of Skin Leukocytes Uncovers Phagocyte Inflammatory Responses During Induction and Resolution of Cutaneous Inflammation in Fish.

Leukocytes offer a critical layer of protection to the host following skin infections. Delineating the kinetics of cutaneous leukocyte recruitment as well as their anti-microbial and regulatory profiles is challenging since it requires the isolation of adequate cell numbers and maintenance of their functional properties. Herein, we took advantage of a modified procedure to gain insights into the contributions of fish phagocytes through induction and resolution phases of acute cutaneous inflammation in goldfish (Carassius auratus). Our data shows early upregulation of pro-inflammatory cytokines and chemokines, which was paired with neutrophil-dominant leukocyte migration of neutrophils from circulation to the injury site. Recruited neutrophils were associated with high levels of reactive oxygen species (ROS). Following pathogen elimination, a reduction in ROS levels and pro-inflammatory cytokines expression preceded the resolution of inflammation. These results provide a better understanding of the cutaneous immune responses in fish. Moreover, the increased viability and functionality of isolated skin leukocytes opens the door to better understand a range of additional skin diseases.