Influence of chronic stress on the mechanism of the cytotoxic system in common carp (Cyprinus carpio).

Aquaculture conditions expose fish to internal and environmental stressors that increase their susceptibility to morbidity and mortality. The brain accumulates stress signals and processes them according to the intensity, frequency duration and type of stress, recruiting several brain functions to activate the autonomic or limbic system. Triggering the autonomic system causes the rapid release of catecholamines, such as adrenaline and noradrenaline, into circulation from chromaffin cells in the head kidney. Catecholamines trigger blood cells to release proinflammatory and regulatory cytokines to cope with acute stress. Activation of the limbic axis stimulates the dorsolateral and dorsomedial pallium to process emotions, memory, behaviour and the activation of preoptic nucleus-pituitary gland-interrenal cells in the head kidney, releasing glucocorticoids, such as cortisol to the bloodstream. Glucocorticoids cause downregulation of various immune system functions depending on the duration, intensity and type of chronic stress. As stress persists, most immune functions, with the exception of cytotoxic functions, overcome these effects and return to homeostasis. The deterioration of cytotoxic functions during chronic stress appears to be responsible for increased morbidity and mortality.

Acute stress promotes post-injury brain regeneration in fish.

The central nervous system and the immune system, the two major players in homeostasis, operate in the ongoing bidirectional interaction. Stress is the third player that exerts strong effect on these two 'supersystems'; yet, its impact is studied much less. In this work employing carp model, we studied the influence of preliminary stress on neural and immune networks involved in post-injury brain regeneration. The relevant in vivo models of air-exposure stress and precisely directed cerebellum injury have been developed. Neuronal regeneration was evaluated by using specific tracers of cell proliferation and differentiation. Involvement of immune networks was accessed by monitoring the expression of selected T cells markers. Contrast difference between acute and chronic stress manifested in the fact that chronically stressed fish did not survive the brain injury. Neuronal regeneration appeared as a biphasic process whereas involvement of immune system proceeded as a monophasic route. In stressed fish, immune response was fast and accompanied or even preceded neuronal regeneration. In unstressed subjects, immune response took place on the second phase of neuronal regeneration. These findings imply an intrinsic regulatory impact of acute stress on neuronal and immune factors involved in post-injury brain regeneration. Stress activates both neuronal and immune defense mechanisms and thus contributes to faster regeneration. In this context, paradoxically, acute preliminary stress might be considered a distinct asset in speeding up the following post-injury brain regeneration.

Application of ultrasound in vaccination against goldfish ulcer disease: a pilot study.

Antigenicity and immunogenicity of Aeromonas salmonicida soluble A-layer protein (AP) and formalin-killed bacterin were assessed in goldfish (Carassius auratus L.) by applying ultrasound-immersion (USI) and injection (Inj) routes. No adjuvant was used in USI while IFA was used in Inj. The USI treated fish did not produce detectable acquired antibodies; however, this group stimulated by AP proved to be best protected against challenge infection with a homologous virulent strain of A. salmonicida. The results reveal the asset of USI technique in fish vaccination and indicate the option of designing a pure AP-based ballast-free vaccine.

Vertical and horizontal transmission of tilapia larvae encephalitis virus: the bad and the ugly.

Impairment of innate immunity in tilapia larvae after vertical and horizontal infection with the newly characterized tilapia larvae encephalitis virus (TLEV) was accessed by evaluation of cell-mediated reactive oxygen species (ROS) production in affected fish with the use of horseradish peroxidase-amplified luminol-dependent chemiluminescence assay. The priming in-vivo infection with TLEV resulted in downregulation of ROS response in both vertically- and horizontally-infected fish; this suppression was further exacerbated by specific in-vitro booster infection with the same virus. Application of Ca ionophore and phorbol myristate acetate as alternative nonspecific boosters enabled restoration of ROS release in vertically-infected but not in horizontally-infected larvae. The results indicate severe TLEV-imposed phagocyte dysfunction in affected larvae. The difference in restoration potential of ROS production after vertical and horizontal virus transmission is interpreted in the frame of principal distinctions between the two modes.

Viral encephalitis of tilapia larvae: primary characterization of a novel herpes-like virus.

We report here an outbreak of an acute disease that caused high mortality rate in laboratory-reared tilapia larvae. The disease was initially observed in inbred gynogenetic line of blue tilapia larvae (Oreochromis aureus) and could be transmitted to larvae of other tilapia species. Based on the clinical manifestation (a whirling syndrome), we refer to the disease as viral encephalitis of tilapia larvae. The disease-associated DNA virus is described and accordingly designated tilapia larvae encephalitis virus (TLEV). A primary morphological, biophysical and molecular characterization of TLEV is presented. By virtue of these properties, the newly discovered virus is a herpes-like virus. Phylogenetic analysis, albeit limited, confirms this assumption and places TLEV within the family of Herpesviridae and distantly from the families Alloherpesviridae and Iridoviridae. By using PCR with virus-specific primers, diseased larvae and adult TLEV carriers were also identified in tilapia delivered from external hatcheries.

Determinants of pathology in light-irradiated cells.

This work is devoted to further evaluation of pathological states in light-irradiated cells in terms of relationship between the level of reactive oxygen species (ROS) and polymorphonuclear neutrophil (PMN) count. Human leukocytes were isolated from 34 donors exhibiting flu-like symptoms, irradiated with a nonlaser blue light (2 and 5 mW cm(-2)) and examined for ROS production by a chemiluminescence (CL) assay. The level of ROS was measured as a basal chemiluminescence before (BCL0) and after (BCL) irradiation. In our preceding study (Sinyakov, M. et al. [2007] Photomed. Laser Surg.25, 257-263), PMN-oriented group analysis was applied to a data set divided into light-resistant and light-sensitive points, and cell responsiveness to light was suggested for prognostic evaluation of pathology. In the present work, BCL-oriented analysis was performed on the whole data set to evaluate the individual contribution of every point. BCL level and PMN count were confirmed to be independent variables. Sign-specific delta (DEL) values (DEL = BCL - BCL0) were assessed in relation to respective levels of BCL, BCL0 and PMN counts. DEL and BCL0 were found to be reliable photochemical indices of pathology. Taken together, the results obtained from group and individual analyses provide a novel outlook on the dynamics of pathological development and indicate a promising potential of the assay as a fast discriminator between the nonpathological state and different levels of pathology.

Immunogenicity of bioactive magnetic nanoparticles: natural and acquired antibodies.

Uniform magnetic nanoparticles (MNP) were prepared by nucleation followed by controlled growth of maghemite thin films onto porcine gelatin nuclei. The formed gelatin containing MNP (Gel-MNP) were then coated with dextran (Gel-MNP-Dex) followed by human serum albumin (Gel-MNP-Dex-HSA). Since these MNP are designated for clinical applications, studies concerning the immunogenicity of their antigenic components (porcine gelatin, dextran, and HSA) have been performed in BALB-C mice. These studies demonstrated that plasma of nonimmunized mice already contains basal levels of natural antibodies against all of these antigenic components. This work also demonstrated that the conjugated gelatin is a weak immunogen: Intraperitoneal injection of the various MNP (Gel-MNP, Gel-MNP-Dex dispersed in PBS emulsified with Incomplete Freund's Adjuvant (IFA) mineral oil and Gel-MNP-Dex-HSA dispersed in PBS) did not increase significantly the acquired anti-gelatin antibody titers. Exceptional behavior was observed following immunization with Gel-MNP-Dex-HSA dispersed in PBS emulsified with IFA, which exhibited an adjuvant effect and turned gelatin into a stronger immunogen. In contrast to gelatin, the conjugated HSA and dextran were found to be strong immunogens. The possibility that the various MNP will not induce an autoimmune response as a result of their clinical use is discussed in the contest of the protective role of the natural antibodies.

Chemiluminescent analysis of light-irradiated leukocytes as a diagnostic tool for fast identification of pathological states.

OBJECTIVE: Reactive oxygen species (ROS), mainly produced by polymorphonuclear neutrophils (PMN), are a significant part of host defense in pathologic states. We attempted to relate numbers of PMN and ROS generated within PMN to develop an alternative photochemical approach for evaluation of the potential of these cells to resist the development of inflammatory pathology. BACKGROUND DATA: Lack of sensitivity to light has been reported in healthy cells, while sensitivity to light characterizes cell pathology. METHODS: Human leukocytes from 34 donors were isolated and irradiated with a non-laser blue light (2 and 5 mW/cm(2) for 2 minutes), and a luminol-dependent chemiluminescence assay that reflects intracellular production of ROS was applied thereafter. The levels of basal chemiluminescence (BCL) were related to respective numbers of PMN. RESULTS: A light-insensitive cluster was discovered within the total sample and was considered to be a discrete nonpathological group. Following elimination of this group, the rest of the sample was divided into three well-defined light-sensitive groups, which were attributed to various pathological states, and differed in PMN numbers and BCL counts. Within these groups the two traits were interrelated, and each PMN range was associated with a respective level of intracellular ROS. CONCLUSIONS: Leukocyte responsiveness to light can be used for discrimination between pathological and nonpathological states and prognostic evaluation of pathological development. Patients exhibiting similar clinical symptoms could be divided into separate groups with potentially different outcomes. A novel definition of nonpathological states as well as the mechanism underlying the bell-shaped curve that delineates the relationship between PMN number and intracellular ROS is suggested in pathological states.

Nano- and microparticles as adjuvants in vaccine design: success and failure is related to host natural antibodies.

Bovine serum albumin (BSA) and the surface A-layer protein (AP) of an atypical strain of fish bacterial pathogen Aeromonas salmonicida were covalently linked with polymeric nano- and microparticles, and antigenicity of the resulted conjugates was compared in mice and goldfish. Distinct albeit different levels of natural BSA and AP antibodies were present in both animal species. Significant stimulation of the anti-AP antibody response in mice strikingly contrasted to unresponsiveness or even suppression in fish. The results negatively correlate with the levels of respective natural antibodies in the host and are discussed in context of problems related to fish vaccination. The work reinforces the instructive role of natural antibodies in adaptive immune response.

Altered self-erythrocyte recognition and destruction in an inbred line of tilapia (Oreochromis aureus).

Carboxyfluorescein diacetate (cFDA)-stained autologous and syngeneic tilapia (Oreochromis aureus) erythrocytes are recognized by effector peripheral blood leukocytes and lysed after a short culture period of 4 h. The hemolysis level was evaluated by measuring the fluorescence of the released cFDA. The degree of lysis of stained target erythrocytes of 60 individuals revealed a trimodal distribution statistically stratified into three groups of low (LR), intermediate (IR), and high (HR) responders. Depletion of the majority of phagocytes from leukocytes lowered the lysis level of HR to that of LR. A highly significant increase of LR cytotoxicity was obtained after the addition of conditioned medium from HR but only in the presence of phagocytes. Genetic analysis of offspring from four crosses (IR x HR, IR x LR, HR x LR, and LR x LR) revealed a quantitative trait locus (QTL) segregating for the level of response linked to markers UNH207 and UNH231 on linkage group 6 of tilapia. Based on segregation analysis of 58 gynogenetic BIU-1 offspring, the distances from the centromere were estimated as 21.5, 11.5, and 9.0 cM for UNH207, UNH231, and the QTL, respectively. It is suggested that 1) self-target recognition and destruction requires both cFDA-altered self-erythrocyte membrane and membrane structures normally present in autologous, syngeneic, and xenogeneic targets; 2) natural cytotoxic cells and/or macrophages are involved in erythrocyte lysis; and 3) the lysis level is codominantly inherited by a QTL segregating on tilapia linkage group 6.

Upregulation of carp GDNF mRNA by the immunomodulator AS101.

Although the glia derived neurotrophic factor (GDNF) is defined as a molecule that maintains neuronal cells, it possesses a range of functions outside the nervous system. For example, it is essential for uretric branching in kidney morphogenesis and for regulating the differentiation of stem cells during spermatogenesis, cardiac, hair follicle and vascular differentiation and the maintenance of immune cells. In the present work, the presence of GDNF in carp peripheral blood leukocytes (PBL) and head kidney cells (HK) was evidenced and its evolutionary importance in both neural and immune systems development was suggested. Using the northern-blot technique, we could observe the expression of two different transcripts of this gene. GDNF upregulation was detected using semi-quantitative PCR, following ex vivo treatment of PBL and HK cells with the immunomodulator AS101 which was previously shown to inhibit IL-10 and to up-regulate GDNF protein levels in human SVG astrocyte cell line, in 6-OHDA hemi-parkinsonian mice in vivo and in rat glomerular mesengial cells in vitro.

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease.

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.

Enhancement of antigen uptake and antibody production in goldfish (Carassius auratus) following bath immunization and ultrasound treatment.

Ultrasound irradiation and hyperosmotic treatments were compared as facilitators of antigen (BSA) penetration through the skin by bath vaccination and as enhancers of the antibody response in goldfish. The kinetics of BSA penetration and accumulation into the skin, and via it to the blood, and the consequent specific stimulation of the humoral immune response, were studied. The main findings are: (1). ultrasonic treatment is more effective than hyperosmotic treatment in enhancing both antigen transport through the skin and antibody production; (2). the requirements for high antigen concentrations, which are needed for simple bath immersion, could be reduced five times in presonicated fish; and (3). anesthesia, which significantly reduced gill uptake following hyperosmotic treatment, had no effect on skin uptake. The importance of these finding for mass vaccination of adult fish and larvae is discussed.

betaFSH, betaLH and growth hormone gene expression in blue gourami (Trichogaster trichopterus, Pallas 1770) during spermatogenesis and male sexual behavior.

The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (betaFSH and betaLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of betaFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of betaLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.

Natural antibodies and their significance in active immunization and protection against a defined pathogen in fish.

Natural antibody activity against Aeromonas salmonicida extracellular A-layer protein (A-protein) showed large individual variations in a farmed group of 101 goldfish (Carassius auratus L.). Statistical analyses of these variations led us to divide this group into homogeneous high and low naturally active (HNA and LNA) subgroups. The HNA fish were largely protected against experimental infection with a virulent atypical A. salmonicida strain, while 100% morbidity was recorded in the LNA group. In the course of active immunization with a particulate form of A-protein, a significant antibody response was exhibited by the LNA group only. Significance and implication of these results in vaccination practice are discussed.