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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Impact of Reduced Dietary Crude Protein in the Starter Phase on Immune Development and Response of Broilers Throughout the Growth Period.

Crude protein (CP) levels in commercial broiler (Gallus gallus) diets, optimized for maximum yield production vs. feed cost, have only begun to be assessed for impact on immune function. In order to study immune effects of dietary CP levels, different starter phase (day 1-14) diets were fed to 230 Ross 708 male broiler chicks randomly assigned at 1 day of age into two treatment groups. Group 1: Standard diet (STD) contained 3,000 kcal AMEn/kg energy and 23.78% CP; and Group 2: Reduced crude protein diet (RCP) contained 3,000 kcal AMEn/kg energy and 21.23% CP. From day 15-35 a common standard grower/finisher diet (3,150 kcal AMEn/kg energy and 22.18% CP) was allocated to both groups. Zymosan, a glycan derived from yeast cell walls that binds to TLR 2 and Dectin-1, was used for intra-abdominal challenge. Results demonstrated that a reduced crude protein starter diet (21.23 vs. 23.78% CP) between age 1-14, while maintaining the same levels of metabolizable energy and essential amino acids, did not affect broilers growth performance or lymphoid organ weights (P > 0.05). Interestingly, basal leukocyte levels in the RCP group significantly (P < 0.01) increased in the blood compartment at d35 in the unchallenged birds. Significant enhancements to leukocyte infiltration into the abdominal cavity were also detected post-immune challenge with zymosan (day 14 and day 35; P < 0.01). Post-challenge levels of TNF-α, IL-1ß, and CXCL8 gene expression cells collected from the abdominal cavity were not affected by the diets (P > 0.05). Moreover, dietary treatments did not influence percentage of ROS producing cells in the abdominal cavity (P > 0.05). To our best knowledge, this is the first study that reports the impacts of reduced crude protein diet on the innate immune response of poultry to an acute inflammation model in the abdominal cavity. Overall, our results highlight that reduced crude protein diets can be used without negatively impacting broiler performance and may enhance the capacity of broilers to recruit leukocytes upon infection.

Differential Effects of Drinking Water Quality on Phagocyte Responses of Broiler Chickens Against Fungal and Bacterial Challenges.

Combinatorial effects of xenobiotics in water on health may occur even at levels within current acceptable guidelines for individual chemicals. Herein, we took advantage of the sensitivity of the immune system and an avian animal model to examine the impact of xenobiotic mixtures on animal health. Water was derived from an underground well in Alberta, Canada and met guidelines for consumption, but contained a number of contaminants. Changes to chicken immunity were evaluated following acute (7d) exposure to contaminated water under basal and immune challenged conditions. An increase in resident macrophages and a decrease in CD8+ lymphocytes were identified in the abdominal cavity, which served as a relevant site where immune leukocytes could be examined. Subsequent intra-abdominal immune stimulation detected differential in vivo acute inflammatory responses to fungal and bacterial challenges. Leukocyte recruitment into the challenge site and activation of phagocyte antimicrobial responses were affected. These functional responses paralleled molecular changes in the expression for pro-inflammatory and regulatory genes. In all, this study primarily highlights dysregulation of phagocyte responses following acute (7d) exposure of poultry to contaminated water. Given that production food animals hold a unique position at the interface of animal, environmental and human health, this emphasizes the need to consider the impact of xenobiotic mixtures in our assessments of water quality.

Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals.

Imaging flow cytometry (IFC) is a powerful tool which combines flow cytometry with digital microscopy to generate quantitative high-throughput imaging data. Despite various advantages of IFC over standard flow cytometry, widespread adoption of this technology for studies in aquatic sciences is limited, probably due to the relatively high equipment cost, complexity of image analysis-based data interpretation and lack of core facilities with trained personnel. Here, we describe the application of IFC to examine phagocytosis of particles including microplastics by cells from aquatic animals. For this purpose, we studied (1) live/dead cell assays and identification of cell types, (2) phagocytosis of degradable and non-degradable particles by Atlantic salmon head kidney cells and (3) the effect of incubation temperature on phagocytosis of degradable particles in three aquatic animals-Atlantic salmon, Nile tilapia, and blue mussel. The usefulness of the developed method was assessed by evaluating the effect of incubation temperature on phagocytosis. Our studies demonstrate that IFC provides significant benefits over standard flow cytometry in phagocytosis measurement by allowing integration of morphometric parameters, especially while identifying cell populations and distinguishing between different types of fluorescent particles and detecting their localization.

Neonatal Exposure to Amoxicillin Alters Long-Term Immune Response Despite Transient Effects on Gut-Microbiota in Piglets.

Antibiotic exposure during neonatal development may result in transient or persistent alterations of key microbes that are vital for normal development of local and systemic immunity, potentially impairing immune competence later in life. To further elucidate the relationship between antibiotic exposure and immune development, newborn pigs were exposed to a therapeutic pediatric dose (30 mg/kg/day) of amoxicillin (AB) or placebo (PL) from post-natal day (PND) 0-14. Subsequently, immune cell phenotype, microbial composition, and immune response to an intraperitoneal (IP) challenge with Salmonella enterica serovar Typhimurium were evaluated. AB exposure caused significant changes in fecal microbial composition on PND 3 (P = 0.025). This stemmed from a 2-fold increase in Enterobacteriaceae with live cecal coliforms on PND 7 indicating at 10-fold increase (P = 0.036). Alterations in microbial composition were transient, and successional patterns were normalizing by PND 14 (P = 0.693). Differences in PBMC (peripheral blood mononuclear cell) immune cell subtypes were detected, with the percentage of CD3+CD4+ T cells among the broader T cell population (CD3+CD4+/CD3+) being significantly higher (P = 0.031) in AB pigs and the numbers of CD4+CD45RA+ (naïve) T cells per liter of blood were lower on PND 21 in AB pigs (P = 0.036). Meanwhile, PBMCs from AB pigs produced significantly more IFNγ upon stimulation with a T-cell mitogen on PND 21 and 49 (P = 0.021). When AB pigs were challenged with heat-killed Salmonella (IP) on PND 49, IFNγ gene expression in peripheral blood was upregulated compared to those treated with PL (P = 0.043). Additionally, AB pigs showed stronger activation among neutrophils infiltrating the peritoneal cavity after in vivo immune challenge, based on higher levels of NF-κB nuclear translocation (P = 0.001). Overall, our results indicate that early life treatment with a therapeutically relevant dose of a commonly prescribed antibiotic has a programming effect on the immune system. Despite antibiotics only causing a transient disruption in gut-associated microbial communities, implications were long-term, with antibiotic treated pigs mounting an upregulated response to an immune challenge. This research adds to the growing body of evidence indicating adverse immune outcomes of early life antibiotic exposures.

Teleost contributions to the understanding of mycobacterial diseases.

Few pathogens have shaped human medicine as the mycobacteria. From understanding biological phenomena driving disease spread, to mechanisms of host-pathogen interactions and antibiotic resistance, the Mycobacterium genus continues to challenge and offer insights into the basis of health and disease. Teleost fish models of mycobacterial infections have progressed significantly over the past three decades, now supplying a range of unique tools and new opportunities to define the strategies employed by these Gram-positive bacteria to overcome host defenses, as well as those host antimicrobial pathways that can be used to limit its growth and spread. Herein, we take a comparative perspective and provide an update on the contributions of teleost models to our understanding of mycobacterial diseases.

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.

Perspective on the Development and Validation of Ab Reagents to Fish Immune Proteins for the Correct Assessment of Immune Function.

Understanding of immune function in humans and model organisms, such as mice, has advanced in the last few decades because of technological breakthroughs and availability of reagents. While novel genomic technologies have helped to increase knowledge of many aspects of immunology, most developments in immunology have occurred because of the availability of antibodies to identify and sort different cell types, as well as to identify and quantify the protein products of cells. Unfortunately, many studies performed in fish make use of poorly characterized antibody reagents that may affect the conclusions of those studies. In light of this, we would like to offer some insight and discussion points based on our research experience on the strategies and techniques that are required for proper validation of antibody reagents to fish immune molecules. Our main goal is to encourage a much needed discussion in our field to foster the use of correctly validated reagents that enable the study of fish immune function.

Innate Immunity Provides Biomarkers of Health for Teleosts Exposed to Nanoparticles.

In recent years, the unique properties of nanoparticles have fostered novel applications in various fields such as biology, pharmaceuticals, agriculture, and others. Unfortunately, their rapid integration into daily life has also led to environmental concerns due to uncontrolled release of nanoparticles into the aquatic environment. Despite increasing awareness of nanoparticle bioaccumulation in the aquatic environment, much remains to be learned about their impact on aquatic organisms and how to best monitor these effects. Herein, we provide the first review of innate immunity as an emerging tool to assess the health of fish following nanoparticle exposure. Fish are widely used as sentinels for aquatic ecosystem pollution and innate immune parameters offer sensitive and reliable tools that can be harnessed for evaluation of contamination events. The most frequent biomarkers highlighted in literature to date include, but are not limited to, parameters associated with leukocyte dynamics, oxidative stress, and cytokine production. Taken together, innate immunity offers finite and sensitive biomarkers for assessment of the impact of nanoparticles on fish health.

Contribution of leukocytes to the induction and resolution of the acute inflammatory response in chickens.

A successful immune response against invading pathogens relies on the efficient activation of host defense mechanisms and a timely return to immune homeostasis. Despite their importance, these mechanisms remain ill-defined in most animal groups. This study focuses on the acute inflammatory response of chickens, important both as an avian model with a unique position in evolution as well as an increasingly notable target of infectious zoonotic diseases. We took advantage of an in vivo self-resolving intra-abdominal challenge model to provide an integrative view of leukocyte responses during the induction and resolution phases of acute inflammation. Our results showed rapid leukocyte infiltration into the abdominal cavity post zymosan challenge (significant increase as early as 4 h), which was dominated by heterophils. Peak leukocyte infiltration and ROS production reached maximum levels at 12 h post challenge, which was significantly earlier than comparative studies in teleost fish and mice. Both heterophils and monocyte/macrophages contributed to ROS production. Local leukocyte infiltration was preceded by an increase in peripheral leukocytes and a drop in the number of bone marrow leukocytes. The proportion of apoptotic leukocytes increased following peak of acute inflammation, rising to significant levels within the abdominal cavity by 48 h, consistent with other indicators for the resolution of inflammation. Importantly, comparison of chicken phagocytic responses with those previously shown in agnathan, teleost and murine models suggested a progressive evolutionary shift towards an increased sensitivity to pro-inflammatory pathogen-derived particles and decreased sensitivity towards homeostatic stimuli. Thus, while significant conservation can be noted across the immune systems of endotherms, this study highlights additional unique features that govern the induction and resolution of acute inflammation in the avian system, which may be relevant to disease susceptibility and performance.