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.

Longitudinal evaluation of the cutaneous and rectal microbiota of German shepherd dogs with perianal fistulas undergoing therapy with ciclosporin and ketoconazole.

BACKGROUND: Perianal fistulas are painful ulcers or sinus tracts that disproportionately affect German shepherd dogs and are proposed as a spontaneous animal model of fistulising Crohn's disease. OBJECTIVES: To characterise the rectal and cutaneous microbiota in German shepherd dogs with perianal fistulas and to investigate longitudinal shifts with lesion resolution during immunomodulatory therapy. ANIMALS: Eleven German shepherd dogs with perianal fistulas and 15 healthy German shepherd dogs. MATERIALS AND METHODS: Affected dogs were evaluated and swabbed at three visits, 30 days apart, while undergoing treatment with ciclosporin and ketoconazole. Healthy German shepherd dogs were contemporaneously sampled. Sites included the rectum, perianal skin and axilla. The microbiome was evaluated following sequencing of the V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene. RESULTS: Alpha diversity was not significantly different between healthy and affected dogs at each of the three body sites (p > 0.5), yet rectal and perianal beta diversities from affected dogs differed significantly from those of healthy dogs at Day 0 (p = 0.004). Rectal and perianal relative abundance of Prevotella spp. increased and perianal Staphylococcus spp. relative abundance decreased in affected dogs over time, coincident with lesion resolution. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in lesional cutaneous and rectal microbiota occur in German shepherd dogs with perianal fistulas and shift over time with lesion resolution during immunomodulatory therapy. Further investigations of the role of cutaneous and enteric microbiota in the pathogenesis of perianal fistulas, and whether manipulation of microbial populations may ameliorate disease, are needed.

Common superficial and deep cutaneous bacterial infections in domestic animals: A review.

The skin covers the external surface of animals, and it is constantly exposed to and inhabited by different microorganisms, including bacteria. Alterations in the skin barrier allow commensal and/or pathogenic bacteria to proliferate and penetrate deep into the lower layers of the skin. Being the first barrier to the external environment, the skin is prone to injuries, allowing the penetration of microorganisms that may lead to severe deep infections. Companion animals, especially dogs, are prone to bacterial infections, often secondary to allergic dermatitis. When environmental conditions are unfavorable, horses, cattle, sheep, and goats can develop superficial infections, such as those caused by Dermatophilus congolensis. Deep inflammation is commonly caused by Mycobacterium spp., which results in granulomatous to pyogranulomatous dermatitis and panniculitis. Likewise, bacteria such as Nocardia spp. and Actinomyces spp. can cause deep pyogranulomatous inflammation. Bacteria that lead to deep necrotizing lesions (eg, necrotizing fasciitis/flesh-eating bacteria) can be severe and even result in death. This review includes an overview of the most common cutaneous bacterial infections of domestic animals, highlighting the main features and histologic morphology of the bacteria, cutaneous structures involved, and the type of inflammatory infiltrates.

Early Molecular Immune Responses of Turbot (Scophthalmus maximus L.) Following Infection with Aeromonas salmonicida subsp. salmonicida.

Turbot aquaculture production is an important economic activity in several countries around the world; nonetheless, the incidence of diseases, such furunculosis, caused by the etiological agent A. salmonicida subsp. salmonicida, is responsible for important losses to this industry worldwide. Given this perspective, this study aimed to evaluate early immune responses in turbot (S. maximus L.) following infection with A. salmonicida subsp. salmonicida. For this, 72 fish were individually weighed and randomly distributed into 6 tanks in a circulating seawater system. For the bacterial challenge, half of the individuals (3 tanks with 36 individuals) were infected using a peritoneal injection with the bacterial suspension, while the other half of individuals were injected with PBS and kept as a control group. Several factors linked to the innate immune response were studied, including not only haematological (white blood cells, red blood cells, haematocrit, haemoglobin, mean corpuscular volume, mean cell haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, monocytes, lymphocytes, thrombocytes) and oxidative stress parameters, but also the analyses of the expression of 13 key immune-related genes (tnf-α, il-1ß, il-8, pparα-1, acox1, tgf-ß1, nf-kB p65, srebp-1, il-10, c3, cpt1a, pcna, il-22). No significant differences were recorded in blood or innate humoral parameters (lysozyme, anti-protease, peroxidase) at the selected sampling points. There was neither any evidence of significant changes in the activity levels of the oxidative stress indicators (catalase, glutathione S-transferase, lipid peroxidation, superoxide dismutase). In contrast, pro-inflammatory (tnf-α, il-1ß), anti-inflammatory (il-10), and innate immune-related genes (c3) were up-regulated, while another gene linked with the lipid metabolism (acox1) was down-regulated. The results showed new insights about early responses of turbot following infection with A. salmonicida subsp. salmonicida.

Ultrasonographic findings may be useful for differentiating interdigital abscesses secondary to migrating grass awns and interdigital furunculosis in dogs.

Grass awn migration and furunculosis are common diseases in dogs that can lead to interdigital subcutaneous lesions with a similar clinical presentation, and occasionally similar ultrasonographic images, but different treatments are required. This retrospective, multicentric, analytical study aimed to determine whether epidemiological, clinical, and ultrasonographic features could be used to differentiate interdigital furunculosis and abscesses secondary to migrating grass awns. Fifty-nine dogs that underwent interdigital ultrasonography were included (interdigital furunculosis [IDF], n = 27; interdigital abscess secondary to a migrating grass awn [IAGA], n = 32). Ultrasonographic images were reviewed by two observers blinded to the diagnosis, who graded nine qualitative and four quantitative parameters for each patient. In both groups, pruritus/licking (IDF 74%, IAGA 70%), a discharging interdigital wound (63% for each group), and thoracic limb involvement (IDF 88%, IAGA 75%) were common features. On ultrasound, a subcutaneous multilinear hyperechoic main element was identified in most dogs (IDF 85%, IAGA 100%). The ability to display this element in a single plane (P < 0.01), the absence of additional hyperechoic linear elements in different planes (P < 0.01), and a surrounding hypoechoic halo (P < 0.05) were significantly more common in dogs with interdigital abscesses secondary to migrating grass awns. A cut-off value of 0.83 cm for the length of the main multilinear element provided a sensitivity of 91% and a specificity of 87% in the diagnosis of a migrating grass awn. Findings supported prioritizing a differential diagnosis of interdigital abscesses secondary to grass awns in dogs with these ultrasonographic characteristics.

Antibacterial treatment of lumpfish (Cyclopterus lumpus) experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica.

Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg-1  day-1 of FFC, 25 mg kg-1  day-1 of OA and 25 mg kg-1  day-1 FLU, whereas only FFC (20 mg kg-1  day-1 ) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg-1  day-1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish.

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.

The Intersection of Human and Veterinary Medicine-A Possible Direction towards the Improvement of Cell Therapy Protocols in the Treatment of Perianal Fistulas.

The effective treatment of perianal fistulizing Crohn's disease is still a challenge. Local administration of mesenchymal stromal cells (MSCs) is becoming a part of accepted treatment options. However, as a fledgling technique, it still can be optimized. A new trend in translational research, which is in line with "One Health" approach, bases on exploiting parallels between naturally occurring diseases affecting humans and companion animals. Canine anal furunculosis (AF) has been indicated as condition analogous to human perianal Crohn's disease (pCD). This narrative review provides the first comprehensive comparative analysis of these two diseases based on the published data. The paper also outlines the molecular mechanisms of action of MSCs which are likely to have a role in modulating the perianal fistula niche in humans, and refers them to the current knowledge on the immunomodulatory properties of canine MSCs. Generally, the pathogenesis of both diseases shares main determinants such as the presence of genetic predispositions, dysregulation of immune response and the relation to intestine microbiota. However, we also identified many aspects which should be further specified, such as determining the frequency of true fistulas formation in AF patients, elucidating the role of TNF and Th17 pathway in the pathogenesis of AF, or clarifying the role of epithelial-to-mesenchymal transition phenomenon in the formation of canine fistulae. Nevertheless, the available data support the hypothesis that the results from testing cell therapies in dogs with anal furunculosis have a significant translational value in optimizing MSC transplants procedures in pCD patients.

Furunculosis and its complications: A cause of morbidity in renal transplant recipients: A retrospective observational study from Sindh Institute of Urology and Transplantation Karachi.

Furunculosis in renal transplant recipients may be associated with increased morbidity. With the aim to study the presentation, morbidity, and risk factors for furunculosis, this observational study was conducted at the Sindh Institute of Urology and Transplantation, between January to December 2014. All patients with furuncles or abscesses were included. The clinical presentation and risk factors were recorded. A morbidity scale of 0 and 1 was made on the basis of hospital stay for ≥7 days, bacteraemia, large abscesses and repeated furunculosis. Out of 38 patients, 29 (76%) had large abscesses and 9 (24%) had furuncles, with gluteal region being the most common site. Twelve (32%) had severe disease; 29 (76%) had morbidity scale of ≥1. High dose immunosuppression was significantly associated with severe disease while repeated furunculosis had significantly more risk factors. Furunculosis is a severe disease with high morbidity in renal transplant recipients and more studies are needed on skin colonisation and preventive strategies.

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.

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.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes.

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post-infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.

Aeromonas salmonicida Growth in Response to Atlantic Salmon Mucins Differs between Epithelial Sites, Is Governed by Sialylated and N-Acetylhexosamine-Containing O-Glycans, and Is Affected by Ca2.

Aeromonas salmonicida causes furunculosis in salmonids and is a threat to Atlantic salmon aquaculture. The epithelial surfaces that the pathogen colonizes are covered by a mucus layer predominantly comprised of secreted mucins. By using mass spectrometry to identify mucin glycan structures with and without enzymatic removal of glycan residues, coupled to measurements of bacterial growth, we show here that the complex Atlantic salmon intestinal mucin glycans enhance A. salmonicida growth, whereas the more simple skin mucin glycans do not. Of the glycan residues present terminally on the salmon mucins, only N-acetylglucosamine (GlcNAc) enhances growth. Sialic acids, which have an abundance of 75% among terminal glycans from skin and of <50% among intestinal glycans, cannot be removed or used by A. salmonicida for growth-enhancing purposes, and they shield internal GlcNAc from utilization. A Ca2+ concentration above 0.1 mM is needed for A. salmonicida to be able to utilize mucins for growth-promoting purposes, and 10 mM further enhances both A. salmonicida growth in response to mucins and binding of the bacterium to mucins. In conclusion, GlcNAc and sialic acids are important determinants of the A. salmonicida interaction with its host at the mucosal surface. Furthermore, since the mucin glycan repertoire affects pathogen growth, the glycan repertoire may be a factor to take into account during breeding and selection of strains for aquaculture.

Concise Review: Stem Cell Trials Using Companion Animal Disease Models.

Studies to evaluate the therapeutic potential of stem cells in humans would benefit from more realistic animal models. In veterinary medicine, companion animals naturally develop many diseases that resemble human conditions, therefore, representing a novel source of preclinical models. To understand how companion animal disease models are being studied for this purpose, we reviewed the literature between 2008 and 2015 for reports on stem cell therapies in dogs and cats, excluding laboratory animals, induced disease models, cancer, and case reports. Disease models included osteoarthritis, intervertebral disc degeneration, dilated cardiomyopathy, inflammatory bowel diseases, Crohn's fistulas, meningoencephalomyelitis (multiple sclerosis-like), keratoconjunctivitis sicca (Sjogren's syndrome-like), atopic dermatitis, and chronic (end-stage) kidney disease. Stem cells evaluated in these studies included mesenchymal stem-stromal cells (MSC, 17/19 trials), olfactory ensheathing cells (OEC, 1 trial), or neural lineage cells derived from bone marrow MSC (1 trial), and 16/19 studies were performed in dogs. The MSC studies (13/17) used adipose tissue-derived MSC from either allogeneic (8/13) or autologous (5/13) sources. The majority of studies were open label, uncontrolled studies. Endpoints and protocols were feasible, and the stem cell therapies were reportedly safe and elicited beneficial patient responses in all but two of the trials. In conclusion, companion animals with naturally occurring diseases analogous to human conditions can be recruited into clinical trials and provide realistic insight into feasibility, safety, and biologic activity of novel stem cell therapies. However, improvements in the rigor of manufacturing, study design, and regulatory compliance will be needed to better utilize these models. Stem Cells 2016;34:1709-1729.

Aeromonas salmonicida type III secretion system-effectors-mediated immune suppression in rainbow trout (Oncorhynchus mykiss).

Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model.

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss).

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic-3',5'-adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A. salmonicida virulent and non-virulent isolates. We found that A. salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A. salmonicida ∆crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A. salmonicida ∆crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A. salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging.

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual-labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild-type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24-h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.

Detection and quantification of Aeromonas salmonicida in fish tissue by real-time PCR.

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real-time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real-time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real-time PCR compared to 30% by a culture approach). Also, no real-time PCR-negative samples were found positive by culturing. A. salmonicida was detected by real-time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real-time PCR showed the presence of the bacterium in all examined organs (1-482 genomic units mg-1 ). With a limit of detection of 40 target copies (1-2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real-time PCR assay provides a sensitive tool for the detection of A. salmonicida.

A comparison of the response of diploid and triploid Atlantic salmon (Salmo salar) siblings to a commercial furunculosis vaccine and subsequent experimental infection with Aeromonas salmonicida.

Sterile triploid fish represent a solution to the problems associated with sexual maturation and escapees in aquaculture. However, as disease outbreaks continue to cause significant economic losses to the industry, it is essential that the response of triploids to disease and disease treatments be characterised. The aim of this study was to compare the response of triploid Atlantic salmon to a commercial furunculosis vaccine with that of diploid fish, and to assess the vaccine efficacy in the two ploidies through an experimental infection with Aeromonas salmonicida. Diploid and triploid Atlantic salmon were injected intraperitoneally with either phosphate buffered saline, liquid paraffin adjuvant or a commercial furunculosis vaccine. Following vaccination, growth, adhesion scores and a variety of assays to assess immune function, such as respiratory burst and antibody response, were measured. Vaccination did not have a significant effect on the weight of either ploidy prior to challenge at 750° days. Adhesion scores were significantly higher in vaccinated fish compared to unvaccinated fish, although no effect of ploidy was observed. Ploidy significantly affected respiratory burst activity following vaccination, however, with triploids exhibiting higher activity than diploids. Combined with lower white blood cell numbers observed in the triploids, it may be that this low cell number is compensated for by increased cellular activity. Ploidy however, did not have a significant effect on complement activity or antibody response, with significantly higher antibody levels detected in all vaccinated fish compared to unvaccinated controls. In addition, both ploidy groups were well protected following challenge with no difference in the relative percentage survival. Based on these results, it appears that ploidy does not affect the severity of adhesions that result post-vaccinate or in the fish's immune response following vaccination, and the furunculosis vaccine performs equally well in both diploid and triploid Atlantic salmon.