The protective effect of humic-rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.).

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic-rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic-rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite-derived humic-rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic-rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic-rich water from the recirculating system, the synthetic humic acid or the Leonardite humic-rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic-rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.

A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus.

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2-8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.

Influence of visible light and ultraviolet irradiation on motility and fertility of mammalian and fish sperm.

OBJECTIVE: The effects of visible light irradiation on sperm motility, fertility, and reactive oxygen species (ROS) formation were investigated and compared in ram and fish (tilapia). BACKGROUND DATA: Low-energy visible light has previously been found to modulate various processes in different biological systems. In the literature, it is accepted that the first step following visible light irradiation is the formation of ROS by endogenous cellular photosensitizers. METHODS: Sperm of ram and tilapia were irradiated with various light sources (400-800 nm white light, 660 nm red light, 360 nm blue light, 294 nm UV), and their motility and fertility rates were measured. The amount of ROS generated by irradiation was estimated using electron paramagnetic resonance (EPR) technique. RESULTS: Sperm taken from tilapia showed higher motility and fertility following red and white light irradiation. In contrast, the motility and fertility of ram sperm were slightly increased only by red light. A negative effect on motility and fertility of sperm of both species was obtained following irradiation with UV and blue light. The amount of ROS produced in irradiated tilapia sperm was much higher than that of ram sperm. CONCLUSIONS: The results show that different wavelengths differentially affect tilapia and ram sperm motility and fertilization. The difference in response to the various light sources might be explained by the different amounts of ROS formation by ram and tilapia, which are in agreement with the physiology of fertilization appropriate to each of these species. Based on these results, it is suggested that in vitro fertilization in mammals should be performed in darkness or at least under red light.

Immunisation of fish by bath immersion using ultrasound.

Ultrasonic irradiations (USI) as a means to open routes in the skin, thus facilitating the transdermal delivery of vaccines that will improve the effectiveness of vaccination by immersion, are reviewed in this paper. Based on our recent results in goldfish and carp it could be summarized that: (i) USI significantly improved the antigen uptake and enhanced antibody response; (ii) the requirements for high antigen concentrations, which are needed for simple bath immersion, could be considerably reduced in presonicated fish; (iii) after bath immersion, the antigen was slowly released from the skin to the blood in which its presence could still be detected 24 hours later. This retardation of the antigen in the skin was suggested to be due to a possible interaction with cells of the local immune system, in which it is processed and recognized. It is concluded that the recent advances in biotechnology of immunization with recombinant DNA and the use of DNA vaccines, together with the improvement of their administration using USI, provide interesting prospects for the further application of vaccines against viral and even parasitic diseases of fish.

Association between loci with deleterious alleles and distorted sex ratios in an inbred line of tilapia (Oreochromis aureus).

Three microsatellite markers (UNH159, UNH231, and UNH216) were examined for association with both deleterious genes and sex-ratio distortions in a full-sib family of 222 progeny from the fourth generation of a meiogynogenetic tilapia line (Oreochromis aureus). The three markers were mapped previously to different linkage groups and were shown to be associated with genes with deleterious alleles in this line. A restricted maximum likelihood model was used for analysis of major effects and their interactions on sex ratio and viability. This model was based on selective mortality of genders, ignoring effects of possible sex-determining genes. The results showed that deleterious genes linked to UNH216 and UNH231 exert higher lethality in females than in males (P < .0005 and P < .05, respectively). UNH159 was not associated directly with sex ratio distortion, but acts strongly as a modifier of sex ratio in combination with UNH216 and UNH231. Each of the three loci was found to have a significant effect on viability (P < .05) in the maximum likelihood analysis. The deleterious single-locus effects act strongly against females, while most of the epistatic interactions exert higher lethality in males. This contradiction results in a close to 1:1 sex ratio at maturity. The genetic mechanism and significance of such a balance between genders are still unknown. A detailed analysis of sex-specific lethality may be applied by screening in appropriate series of matings and fine mapping with additional markers. Our data suggest that UNH216 and UNH231 are linked to sex ratio distortion genes and that UNH159 may be linked to a modifier of these genes.

Spectral imaging of red blood cells in experimental anemia of Cyprinus carpio.

In the present work we have studied the effect of experimental anemia induced at both low and optimal temperatures on erythropoiesis in Cyprinus carpio. The results showed that hemoglobin concentration per cell was similar in both temperature conditions, however, red blood cell (RBC) concentration was higher at the optimal temperature. Induced anemia caused an abrupt decrease in RBC concentration, while the hemoglobin concentration per cell remained unchanged. Recovery, as shown by electron microscopy, was characterized by the release of differentiating young and intermediate cells to the peripheral blood. It was revealed that with the progression of differentiation the nucleus/cytoplasm ratio decreases, the chromatin condenses and the shape of the nucleus changes from round to elliptical. Spectral imaging revealed an increase in the optical density of chromatin with the maturation of the cells. The chromatin that was dispersed over the nuclear volume in the young cells becomes highly ordered in the mature cells. Spectral similarity mapping revealed the formation of a novel structure of high symmetry, representing chromatin rearrangement during the process of cellular differentiation.

Preparation of a diagnostic antigen of human melanoma based on lymphocyte activation as measured by intracellular fluorescein fluorescence polarization.

The intracellular fluorescein fluorescence polarization (IFFP) test indicates that peripheral blood lymphocytes (PBL) of cancer patients display stimulatory sensitivity to a short incubation with specific tumor protein extracts. In this work, a human lymphocyte activation melanoma antigen (LAMA) was purified from supernatant of a human melanoma cell line (L1M1), which could specifically stimulate lymphocytes of melanoma patients. The results showed a significant stimulation of lymphocytes from healthy donors after incubation with phytohaemagglutinin (PHA), while no stimulation was observed after incubation with LAMA. On the other hand, lymphocytes from melanoma patients showed a significant stimulation with LAMA, while generally showing minor or no stimulation with PHA. Melanoma specificity of LAMA was demonstrated by no response in lymphocytes from patients of lung, colon, or breast cancer. The purified fraction is therefore considered to be a shared tissue-specific antigen which may be useful in immunodiagnosis and immunotherapy of melanoma.

Acute stress causes skin ulceration in striped bass and hybrid bass (Morone).

Exposure of striped bass (Morone saxatilis) and hybrid bass (M. saxatilis female x Morone chrysops male) to an acute (2-hour) confinement stress caused skin ulceration on the fins but not on the body of all confined fish. Striped bass displayed more severe lesions than did hybrid bass. Histologically, lesions had varying degrees of epithelial erosion and ulceration, which was most severe at the distal portion of the fins. Ulceration was associated with dermal and hypodermal edema and necrosis of the remaining stromal tissue and tips of bone in the fin rays. No hemorrhage or thrombosis was present to suggest any obvious vascular derangement. No evidence was found for either trauma or an infectious agent initiating the lesions. Injecting fish with epinephrine caused a similar response, although the degree of ulceration was less severe. These findings may explain why many opportunistic skin pathogens can rapidly develop into serious infections in fish.

Infection of K562 cells with influenza A virus increases their susceptibility to natural killer lysis.

Natural killer (NK) cells play a role in the natural immunity against tumor cells. In the present study, we demonstrate that infection of the NK-sensitive tumor cell line K562 with influenza A virus caused a substantial increase in lysis of up to sevenfold when compared to noninfected cells. Similar to NK cells, IL-2-activated killer cells exhibited higher lytic activity against virus-infected K562 cells. This effect of the virus correlated with the increase in the expression of intracellular adhesion molecule-1 (ICAM-1) on K562 cells. Changes in the susceptibility to NK lysis were accompanied by alterations, within minutes, in the cytoskeleton as detected by intracellular fluorescein fluorescence polarization measured on the Cellscan, a static cytometer. The possible role of iCAM-1 and the cytoskeleton in the cytotoxic response of NK cells is discussed.

Whole blood assay for examining lymphocyte blastogenesis of percichthyid bass (Morone) [corrected].

A simple and reproducible method was developed for the measurement of blastogenesis of peripheral blood lymphocytes using whole blood of hybrid bass (striped bass [Morone saxatilis] female x white bass [M. chrysops] male) stimulated with Concanavalin A, phytohemagglutinin-P, lipopolysaccharide or pokeweed mitogen. Compared to traditional methods which use leucocyte separation procedures, whole blood culture is faster and less expensive. Only small aliquots of blood (10 microliters per culture well) were needed, which would be beneficial for sampling small fish as well as for taking multiple samples from single animals. Optimal culture conditions for hybrid bass, including mitogen concentration, incubation temperature and incubation period, were determined. This is the first report to demonstrate a blastogenic response of whole blood cells in fish.

Production of F1 and F 2 diploid gynogenetic tilapias and analysis of the "Hertwig curve" obtained using ultraviolet irradiated sperm.

In this study, a Hertwig effect with a non-typical biphasic curve was obtained using sperm irradiated with increasing intensities of UV. The first phase of the UV curve appeared to be quite different from that normally demonstrated using γ or x-ray irradiation. This difference is characterised throughout the length of the first phase by (1) low and stable embryo hatching rates of about 3.5%, and (2) exclusive formation of haploid embryos at any irradiation intensity. Additionally, at both phases, the ability of the sperm to induce morula formation was not affected at all, and no aneuploidy nor chromosomal fragments could be seen. Therefore, it was suggested that in this fish the lethal effect of UV irradition on sperm is mainly expressed on early differentiative events during embryogenesis, which lead to a degeneration of the embryos during early stages of their development. The possible mechanism by which haploidy is achieved during the first phase is discussed. Two generations of diploid gynogenetic tilapias were induced by activating Oreochromis aureus eggs with UV-irradiated O. niloticus sperm and by using the heat-shock technique, at optimized conditions, for the prevention of the second polar body extrusion. Species specific dominant genetic markers (serum esterases and tail striation) were used to confirm the exclusive content of the maternal genome in gynogenetic offspring. Very low survival rates (0.36%) were shown in F1 gynogenetic fish as well as a high incidence of malformations among survivors. In the second gynogenetic generation, both significantly higher survival rates (3.6%) and a considerably reduced incidence of malformations were obtained. We suggest that low frequencies of recombination occur in this species and cause a rapid increase in the inbreeding level. This is followed by the expression of lethal and defective genes that are considerably reduced after second generation selection.

The induction of triploidy in Oreochromis aureus by heat shock.

Triploid fish were obtained using heat-shock treatment. The optimal conditions for the heat shock (39.5±0.2°C for 3.5-4 min) as well as the exact zygote age (3 min) at which this heat shock was applied were studied. Results showed that this treatment gives rise to 100% of triploid fish with a satisfactory survival rate of 61% beyond the yolk sac resorption. The genital papillae of this triploid fish were underdeveloped in comparison to normal diploid fish. However, no morphological or growth-rate differences between diploid and triploid fish could be observed up to the age of 6 months. Triploidy was assessed by the karyotyping of embryo cells or adult PHA-stimulated lymphocytes, or by erythrocyte measurements. The occurrence of a heat-shock sensitive event at the zygotic age of 6 min is discussed.

The mixed leukocyte reaction (MLR) in carp: bidirectional and undirectional MLR responses.

Two-way and one-way mixed leukocyte reaction (MLR) was demonstrated in peripheral blood leukocytes (PBL) of carp. Primary two-way MLR in randomly selected donor pairs were highly variable. Weak primary responses could be strongly augmented by mutual in vivo priming of the reacting donors. One-way MLR was performed using irradiated (16,000 R) allogeneic PBL as stimulators. Reciprocal responses of randomly paired donors were usually unequal, suggesting the usefulness of this method for genetic analysis of MLR-recognized histocompatibility antigens in carp. Kinetics of the primary and secondary two-way MLR were studied, as well as the kinetics of primary one-way MLR against pooled allogenic stimulator cells.

Evidence for the existence of an IL-2-like lymphocyte growth promoting factor in a bony fish, Cyprinus carpio.

Activity promoting the growth of carp T-like cells has been found in supernatants of mitogen (PHA)- and alloantigen (MLR)-stimulated carp leukocyte cultures. Activity level in culture supernatants was elevated by phorbol myristate acetate. Proliferation of carp T-like lymphoblasts was also promoted in the presence of Il-2-containing supernatants of mammalian origin. The significance of these findings is discussed.

Specific activity of carp antisera against beta 2-microglobulin (beta 2m) and evidence for a beta 2m homologue in carp (Cyprinus carpio).

Antisera against rabbit and human beta 2-microglobulin were produced in carp (Cyprinus carpio). These antisera were found to be specific for beta 2m, and detected beta 2m epitopes in various vertebrates including guinea-pig, cow, mouse, rat, dog, horse, cat, sheep, goat, parrot, chicken and frog. In addition, these antisera were also inhibited by an extract from oyster. The differences between the reactivity of these two antisera and the ability of the anti-rabbit beta 2m to distinguish between mouse and rat beta 2m's suggested that the carp itself carries beta 2m epitopes. This hypothesis was corroborated by the ability of a variety of mammalian anti-beta 2m antisera to induce a mitogenic response in carp leukocytes. Finally, a rabbit antiserum against dog beta 2m was shown to precipitate a low molecular weight molecule from carp leukocyte extract. This molecule is likely to represent the carp homologue of mammalian beta 2m.

Heterogeneity of mitogen-responsive lymphocytes in carp (Cyprinus carpio).

Peripheral blood leukocytes (PBL) of carp respond in vitro to a variety of phytomitogens, shown to be T-cell specific or B-cell specific in mammalian systems. Some basic differences have been observed in the proliferative response of carp PBL to PHA (phytohemagglutinin), ConA ( concanvalin A) and LPS (lipopolysaccharide): (1) The response to PHA and ConA was found to be highly dependent upon the continuous presence of mitogen in the medium, in contrast to LPS, where after the initial stimulation, cells could continue to proliferate for several days without mitogen. (2) Lymphoblasts grown in long term culture with either PHA or Con A could be transferred into medium containing the other mitogen without impairing cell proliferation, but cell growth was reduced to background level following transfer into LPS-containing medium. LPS grown cells continue to proliferate independently of the mitogen content of the medium. (3) Co-stimulation with LPS+PHA or LPS+ConA results in a synergistic response, while co-stimulation with PHA+ConA results in inhibition of DNA synthesis. (4) Several morphological differences have been observed between cells proliferating in the presence of PHA and those proliferating in the presence of LPS. It is suggested that while the PHA and ConA responsive cells may belong to the same lymphocyte subpopulation, they are distinct from the LPS-responsive subpopulation.

The cells involved in the immune response of fish. III. Culture requirements of PHA-stimulated carp (Cyprinus carpio) lymphocytes.

The optimalization of the culture conditions for carp peripheral blood lymphocytes was studied using the thymidine uptake technique. The quantitative and/or qualitative requirements of this culture were determined with respect to the source of serum, the concentrations of 2-ME, glutamine and PHA, and with respect to the initial cell density. High individual variations, shown along the experiments, pointed out the existence of low and high responders to the mitogenic stimulus. These individual variations were considerably reduced and the mitogenic response extremely enhanced by the addition to the culture medium of optimal concentrations of charcoal adsorbed pooled homologous serum (4%), 2-ME (125-250 microM), glutamine (8 mM), PHA (10-40 microliter per ml culture, for the batch used) and by seeding the cells at the optimal density of 2 x 10(5) per microplate well. Finally, the kinetics of cell growth were studied at optimal concentrations of all these factors. The peak response was obtained between days 6 and 7 at 28 degrees C, and the mean doubling time at the exponential phase of the culture, at this temperature, was about 14 hours.