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.

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.

[Leukocyte nitric oxide status in patients with chronic recurrent furunculosis].

The basal and in vitro pyrogenal-stimulated nitric oxide-producing capabilities of leukocytes versus their oxygen-producing and phagocytic activity were studied in patients with chronic recurrent furunculosis during remission (n=62) and exacerbation (n=12). The basal nitric oxide-producing activity of leukocytes was found to be similar. The stimulated leukocyte cultures from the patients showed mainly a perverted reaction--suppression of nitric oxide production instead of its increase detectable in donors. A direct correlation was found between the nitric oxide-producing activity of leukocytes and the results of HCT test in healthy individuals and patients during exacerbation rather than remission.

[Assessment of the level of immunoglobulin E in patients with pyo-inflammatory diseases and its influence on the course of the infectious process].

The level of immunoglobulin E (IgE) in patients with different pyo-inflammatory diseases was assessed and it was found to elevate in patients with acute appendicitis in 49% of cases, in patients with chronic relapsing furunculosis (CRF) in 41%, with chronic osteomyelitis--in 66.6%. In 8 out of 16 examined patients with the elevated level of IgE there were antibodies to toxocars in titers 1:800, 1:400. No correlation with the IgE level was found in assessing the level of IL-4 and gamma-IFN in blood serum of patients with CRF. In patients with acute appendicitis, CRF and chronic osteomyelitis the elevated level of IgE can be considered as a marker of unfavorable course of pyo-inflammatory diseases. The elevated level of IgE does not influence the oxygen-dependent mechanisms of bactericidal action of neutrophils.

Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma.

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.

Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish.

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 degrees C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Phagocytosis and oxidative burst by neutrophils in patients with recurrent furunculosis.

Neutrophil phagocytosis of fluorescently labelled Staphylococcus aureus and oxidative burst by the neutrophils were assessed by flow cytometry in 22 patients with recurrent furunculosis and in 17 controls. Phagocytosis and oxidative burst were not found to be significantly different between the patients and controls. Low serum iron concentrations were demonstrated in six patients (27%). In these patients with hypoferraemia, oxidative burst was significantly lower than in the patients without hypoferraemia and in the controls. These data suggest that hypoferraemia may be an important predisposing factor in a subgroup of patients with recurrent furunculosis in impairing oxidative killing by neutrophils.

Development of an enzyme-linked immunosorbent assay for the detection of IgG subclasses in the serum of normal and diseased dogs.

The present study describes the development of an enzyme-linked immunosorbent assay capable of quantifying serum antibody of all four canine IgG subclasses. A panel of subclass-restricted and subclass-specific monoclonal antibodies was used to measure IgG subclasses in the serum of healthy dogs, as well as in dogs with a range of clinical diseases. The subclasses have been redefined as IgG1, IgG2, IgG3 and IgG4 based on a comparison with the relative concentration and electrophoretic mobilities of human IgG subclasses. In serum samples from healthy dogs, the concentration of IgG1 (mean, 8.17 +/- 0.95 mg ml-1) and IgG2 (mean, 8.15 +/- 3.16 mg ml-1) were very similar and considerably higher than the levels of IgG3 (mean, 0.36 +/- 0.43 mg ml-1) and IgG4 (mean, 0.95 +/- 0.45 mg ml-1). There was no apparent difference in the level of subclasses between the different breeds comprising this normal population. Sera from dogs with a range of immune-mediated or inflammatory diseases all had markedly elevated levels of IgG2 (more than 13 mg ml-1), but IgG1 decreased (less than 4 mg ml-1) to levels below the normal range.

Amoxicillin concentrations in the serum of Atlantic salmon (Salmo salar L) during furunculosis therapy.

The effectiveness of the delivery of amoxicillin to Atlantic salmon, undergoing chemotherapy in natural outbreaks of furunculosis in sea-cages, was investigated by measuring the concentration of the drug in serum samples. Five groups of 50 sera from three outbreaks were collected two hours after oral treatment with doses of 80 or 120 mg/kg bodyweight. Amoxicillin was detected in 82, 82, 92, 100 and 90 per cent of the sera in the five groups (limit of detection 0.16 microgram/ml). Many sera contained less than the minimum inhibitory concentration of amoxicillin for the causative agent Aeromonas salmonicida (0.3 microgram/ml), but a concentration more than double the minimum inhibitory concentration was achieved in 2, 2, 56, 32 and 44 per cent of the samples. There was wide variation in the serum concentrations between individuals in the same population and between populations receiving the same treatment; this variation was associated with population factors, the severity of infection and the accuracy of medicating the feed.

The role of alpha 2-macroglobulin in furunculosis: a comparison of rainbow trout and brook trout.

1. The molecular basis for the high survival rate of rainbow trout, Oncorhynchus mykiss, infected with furunculosis was investigated. 2. Alpha 2-macroglobulin (alpha 2M), a major serum protease inhibitor, was partially purified from rainbow trout and brook trout, Salvelinus fontinalis, sera; the latter species shows marked disease susceptibility. 3. It is shown that a 10-fold species-based difference in alpha 2M inhibitory activity exists against a furunculosis associated bacterial protease. 4. A possible basis for the observed disparity is discussed. 5. Results suggest that the high mol. wt form of teleost (trout) albumin is a dimer composed of two 85,000 subunits.

Preliminary report: furunculosis and hypoferraemia.

16 patients with recurrent staphylococcal furunculosis but without anaemia had significantly lower serum iron concentrations than normal laboratory reference values, 8 controls with single furuncles, or 10 controls with acne conglobata. There were no significant differences in serum glucose or iron binding capacity between the groups studied. Furunculosis resolved after 3-4 weeks' treatment with iron supplements in all but 1 patient. The relation between iron and susceptibility to infection is unclear, but merits further investigation.

[Ultraviolet irradiation of the blood in the complex treatment of suppurative-inflammatory diseases]. / Ultrafioletovoe obluchenie krovi bolnogo v kompleksnom lechenii gnoino-vospalitelnykh zabolevanii.

The experience with the use of ultraviolet irradiation (UVI) of the blood in 98 patients with purulent-inflammatory disease is presented. UVI of the blood has considerably improved the results of treatment of the patients. The highest effectiveness of UVI of the blood is noted in treatment of chromosepsis. The treatment of psoriasis by the mentioned method appeared ineffective.

Bestatin treatment for the correction of granulocyte dysfunction in patients with recurrent furunculosis.

Bestatin, a new immunomodulator which is chemically well-defined, was examined for its capacity to enhance the phagocytic activity of neutrophilic granulocytes from patients with furunculosis. The ability of the granulocytes to ingest fluorescein-labelled yeast particles was significantly decreased in 19 patients with recurrent furunculosis (p less than 0.01). Oral administration of 40 mg bestatin to ten patients increased the phagocytic function of their granulocytes significantly (p less than 0.01).

Measurement of immunoreactive alpha-melanocyte-stimulating hormone in the blood of rainbow trout kept under various conditions.

Initial attempts to measure alpha-MSH in trout blood by radioimmunoassay proved unsuccessful due to apparent losses of immunoreactive hormone, especially in the plasma of trout adapted to a black background. An extraction protocol for alpha-MSH from plasma is described. Subsequent radioimmunoassay revealed progressive increases in immunoreactive alpha-MSH as trout became adapted to a black background. Plasma alpha-MSH titres were raised during fungal infection, and also showed significant differences in fish obtained from different fish farms.

Neutrophil chemotaxis in patients with Staphylococcus aureus furunculosis.

Neutrophil chemotaxis was evaluated in patients with staphylococcal furunculosis using a modified Boyden chamber assay. Neutrophil chemotactic response to Staphylococcus aureus-derived chemotactic factor was compared with response to Escherichia coli-derived chemotactic factor and zymosan-activated serum. Twenty-one patients with active furunculosis were compared with 29 patients with a history of furunculosis but no recent infection and with 29 healthy control subjects. Chemotactic response to the staphylococcal chemotactic factor was significantly higher in patients with active furunculosis (mean 61.6) than in patients with a history of furunculosis (mean 36.4) or controls (mean 31.4), P less than 0.001. Neutrophils from patients with active staphylococcal infections also had higher chemotactic activity toward E. coli chemotactic factor, but not significantly so (P = 0.09). Chemotactic response to zymosan-activated serum and background neutrophil motility was comparable among the three groups. The increased neutrophil chemotactic response of patients with active infection to bacterial factors, but not zymosan-activated serum, may represent a specific neutrophil response to products of infecting organisms. The differential response of the patients' neutrophils to these attractants supports evidence for the presence of separate categories of chemotaxin receptor on the surface of neutrophils.