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1.
In practice, protection of fish against disease by immunization is of limited effectiveness. Therefore, research is concentrated on how to improve the potency and efficacy of vaccines and how to optimally activate the cell-mediated immunity and the specific antibody response. In the present study, the influence of HMB (beta-Hydroxy-beta-methylbutyrate) on the antibody secreting cells (ASC) after both in vitro and in vivo immunization of rainbow trout (Oncorhynchus mykiss) with the anti-yersiniosis vaccine was studied. For in vitro immunization, the spleens from 160 fish were sampled and placed each in 35 mm sterile wells with medium containing HMB at concentrations of 0, 0.1, 1, 5, 10, 25, 50 or 100 microg/mL of medium. The spleens from 80 fish were injected with the vaccine and incubated at 14 degrees C for 10 days. For the in vivo study, fish were fed pellets containing HMB at doses of 0, 10, 25 and 50 mg/kg bw per day. After 2 weeks of HMB supplementation, the fish were immunized by intraperitoneal injection of the vaccine. At 7, 14, 18, 21, 28 and 35 days after immunization, pronephros were taken from 10 fish in each group for testing. When analyzed by the ELISPOT assay, HMB increased the number of splenic ASC after in vitro immunization at concentrations between 10 and 100 microg/mL (P < 0.05). Dietary HMB also increased the number of total and specific ASC when the fish were vaccinated in vivo. In conclusion, the results of the present study showed that HMB increases the levels of specific ASC after both in vitro and in vivo immunization of rainbow trout with the anti-Yersinia ruckeri vaccine.  相似文献   

2.
Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.  相似文献   

3.
Melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) are widespread vertebrate neuropeptides. In teleost fish the peptides are involved in the hormonal control of skin pigmentation, but they have also been shown to modulate corticosteroid secretion in both fish and mammals. alpha-MSH has additional potent anti-inflammatory actions in mammals and both peptides stimulate leucocyte phagocytosis in rainbow trout in vitro. The effects of these peptides on phagocytosis and the release of immunomodulatory factors by rainbow trout head kidney leucocytes were investigated in vitro. Neither MCH nor alpha-MSH had any effect on the adherence of phagocytes to glass slides or the activity of isolated phagocytes. When added to mixed leucocyte suspensions, however, MCH (50 and 100nM) and alpha-MSH (1 and 10nM) significantly increased the percentage of cells undergoing phagocytosis and MCH (50nM), but not alpha-MSH, stimulated the phagocytic index. In subsequent experiments, isolated phagocytes were exposed to supernatants derived from mixed leucocyte suspensions exposed to MCH (50 and 100nM) and alpha-MSH (1 and 10nM). Supernatants from leucocytes exposed to all doses of the peptides significantly increased the percentage phagocytosis and those from cells stimulated with MCH (100nM) and alpha-MSH (1 and 10nM) increased the phagocytic index of the phagocytes. The results suggest that cells other than phagocytes are required for MCH and alpha-MSH to exert their stimulatory effects on trout phagocytic cells through the release of one or more macrophage-activating factors.  相似文献   

4.
Immunomodulation is a commonly used method of prophylaxis in humans and animals. Lysozyme dimer (KLP-602) was used at a dose of 50 ug/kg b.w. in order to correct the immunosuppression caused by the action of herbicide glyphosate (Roundup- Monsanto), which was used in a single bath for 10 minutes in a concentration of 100 mg/l of water. The investigations were carried out on 2 species of fish: the carp (Cyprinus carpio L.) and european catfish (Silurus glanis L.). Herbicide glyphosate caused a decrease in metabolic and phagocytic activity (RBA and PKA) and in proliferative response stimulated by Con A and LPS in carp and european catfish. The immunosuppression sustained for about 2 weeks. The results obtained indicate the possibility of correction of immunosuppression applying lysozyme dimmer (KLP-602) after use of which, the level of the studied indexes increased.  相似文献   

5.
Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.  相似文献   

6.
OBJECTIVES: To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes. SAMPLE POPULATION: Blood samples from 10 clinically normal domestic shorthair cats. PROCEDURE: Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry. RESULTS: Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.  相似文献   

7.
Three hundred and seventy-seven monoclonal antibodies (mabs) directed against human CD antigens and non-classified human leukocyte surface antigens were assayed for their reactivity with common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBL) and thymocytes within the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8). Four of the mabs clearly reacted with rainbow trout PBL and two with carp PBL. Positive mabs were investigated further by two-colour flow cytometry with established mabs directed against carp and rainbow trout leukocyte subpopulations. None of these mabs were suitable for Western blotting and immunoprecipitation. Three mabs were found to stain cells in fixed cryostate sections of the lymphatic organs thymus, pronephros and spleen. In this study, for the first time an anti-CD14 mab was found to cross-react with fish cells. This mab could be a valuable tool complementing the limited toolbox of population-specific mabs in fish. The low number of cross-reactive mabs analyzed in this workshop is another indication for the great phylogenetic difference between mammals and osteichthyes.  相似文献   

8.
Rhodiola quadrifida (Rq) roots and rhizomes are traditionally used in Asia as a tonic, adaptogen, antidepressant and anti-inflammatory drug. The aim of this work was to study the in vivo effect of aqueous and 50% hydro-alcoholic extracts of Rq rhizomes on some parameters of cellular immunity in mice and rats. The metabolic activity of blood phagocyting cells was determined based on the measurement of intracellular respiratory burst after stimulation by PMA in RBA test. Potential bactericidal activity of phagocyting cells was determined in isolated blood leukocytes stimulated with killed microorganisms, according to the PKA test. Proliferative response of lymphocytes stimulated by mitogen concanavaline A (ConA) or lipopolysaccharide (LPS) were determined by MTT assay. Both extracts stimulated granulocytes activity in vitro and increased lymphocyte response to mitogens. The ability of parental strain mice lymphocytes to induce local cutaneous graft-versus-host reaction (GVH) in F1 hybrids was stimulated by 50% hydro-alcoholic extract only.  相似文献   

9.
This study examined the effects of the dimerized lysozyme (KLP-602) on the immunocompetence cell activity in sheatfish (Silurus glanis) and its influence in vivo on the non-specific defence mechanisms and protection against motile aeromonad septicaemia (MAS). The in vitro study showed that the lysozyme dimer (KLP-602), at concentrations between 5 and 50 micrograms/mL of medium significantly (P < 0.05) increased the respiratory burst activity and potential killing activity of pronephric macrophages, as well as the proliferative ability of pronephric lymphocytes stimulated by ConA and LPS. The in vivo study showed that injecting lysozyme dimer (Lydium-KLP) intraperitoneally at doses of 50 micrograms/kg bw stimulated cell-mediated and humoral-mediated imunity. On day 5, after application of Lydium-KLP in vivo, a statistically higher (P < 0.05) respiratory burst activity and potential killing activity of blood and pronephros phagocytes were observed. A higher proliferative ability of blood and pronephros lymphocytes stimulated by Concanavaline A (ConA) or lipopolysaccharide (LPS) was also observed. At the same time, the myeloperoxidase activity in the PMN cells and the lysozyme activity and total Ig levels in serum were significantly higher (P < 0.05), compared to the control group. A challenge test with Aeromonas hydrophila showed that dimerized lysozyme increased the protection against MAS. Dimerized lysozyme stimulates non-specific cellular and humoral mechanisms and protection against MAS in sheatfish.  相似文献   

10.
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11.
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12.
The objective of this study was to determine the stimulating effect of the Inter Yeast S dietary supplement on selected parameters of specific and non-specific humoral and cellular immunity in lambs. The study involved 32 lambs aged 30 +/- 3 days, divided into two equal groups: II--control, and II--experimental. Experimental group animals were fed a C-J concentrate mixed with a prebiotic, the Inter Yeast S, commercially available, containing dried brewer's yeast Saccharomyces cerevisiae in the amount of 3 g/kg of the concentrate. At the beginning of the experiment (day 0) and on the 15th, 30th and 60th day of the study, blood was sampled from the jugular vein to determine selected parameters of biochemical, specific and non-specific humoral and cellular immunity in lambs (total protein levels, gamma globulin levels, lysozyme activity, ceruloplasmin activity, proliferative response of blood lymphocytes (MTT) after stimulation with LPS or ConA, the metabolic activity (RBA) and potential killing activity (PKA) of phagocytes). As regards humoral immunity parameters, significantly higher gamma globulin levels and higher lysozyme and ceruloplasmin activity were found in blood serum of experimental lambs administered the Inter Yeast S, compared with those determined in control lambs not fed the supplement. No statistically significant differences in serum total protein were found between the control and experimental groups. An analysis of cellular immunity indicators revealed significantly higher levels of RBA and PKA, and higher proliferative response of blood lymphocytes (MTT) after stimulation with LPS and ConA in the experimental group, compared with those observed in the control group.  相似文献   

13.
The present work provides information concerning the immunostimulatory activity of Ergosan, an algal based product, injected intraperitoneally in the rainbow trout (Oncorhynchus mykiss). Ergosan is composed of 0.002% unspecified plant extract, 1% alginic acid from Laminaria digitata, and 98.998% algal based carrier. Migration of leucocytes into the peritoneal cavity was stimulated at doses > or =1 mg ml(-1). A single dose of 1mg significantly augmented the proportion of neutrophils, degree of phagocytosis, respiratory burst activity and expression of interleukin-1beta (IL-1beta), interleukin-8 (IL-8) and one of the two known isoforms of trout tumour necrosis factor-alpha (TNF2) in peritoneal leucocytes at 1 day post-injection. Humoral immune parameters were less responsive to intraperitoneal Ergosan administration, with complement stimulation only evident in the 1mg treated group at 2 days post-injection. Antiprotease and lysozyme activity were unaffected by Ergosan over a 7-day time period at the doses examined.  相似文献   

14.
The pharmacokinetic-pharmacodynamic predictor of antimicrobial activity for tetracyclines is reported to be the area under the concentration-time curve at steady state (AUC(ss)) divided by the minimal inhibitory concentration of the targeted pathogen. Here, we estimate AUC(ss) values for oxytetracycline (OTC) in serum of rainbow trout Oncorhynchus mykiss by using a destructive sampling study design. Seventy-two rainbow trout were fed OTC-medicated feed at 74.7 +/- 1.5 mg/kg (mean +/- SD) body weight (BW) by oral gavage for 10 consecutive days. Serum was collected from nine fish at 1, 3, 6, 8, 10, 12, 15, and 22 d after dosing began. Serum OTC concentrations were measured by high-performance liquid chromatography with a 0.01-microg/mL limit of detection. The average OTC AUC(ss) was 29.2 microg x h/mL and was estimated using nonlinear mixed-effects modeling and bootstrap resampling techniques. The elimination half-life was estimated as 85.0 h, and the fraction of steady state achieved was estimated as 0.85. The calculated AUC(ss) (24.8 microg x h/mL) following 10 d of oral dosing with 75 mg OTC/kg BW was less than the estimated AUC(ss). Results suggest that the pharmacokinetics of OTC exposure, including the AUC(ss), is better evaluated by using multiday dosimetry than by using a standard single-dose protocol.  相似文献   

15.
The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 microL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 microL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 +/- 1 degrees C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 +/- 1 degrees C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 +/- 1 degrees C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.  相似文献   

16.
We studied the safety and efficacy of an inactivated vaccine (Ichtiovac-Lg) against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss). In an initial dose-response experiment to test safety, we injected 50 rainbow trout weighing 30-40 g with a double dose of vaccine (0.2 ml) intraperitoneally. We observed these fish three times a day until day 50 post-vaccination when they were killed to evaluate visceral reactions, adhesions and intraperitoneal absorption. Survival was 100% in both the treatment and control groups and no significant differences were found in percentage of severe adhesions and pigmentation of peritonea and viscera. In a second trial, we injected 50 rainbow trout weighing 30-40 g with 0.1 ml of vaccine and a control group was injected with 0.1 ml of PBS intraperitoneally. On day 29 post-vaccination, both groups were challenged by intraperitoneal injection with 0.1 ml of a virulent heterologous strain of L. garvieae at 3 x 10(6) cfu ml(-1) and fish were observed for a further 21 days. At the end of the experiment, the survivals of the vaccinated fish and control group were 94 and 4%, respectively.  相似文献   

17.
Proliferation of rainbow trout peripheral blood leucocytes in vitro is usually assessed by measuring incorporated tritiated thymidine. In this report we monitored the in vitro proliferative response to the mitogen Concanavalin A (Con A) by means of flow cytometry (FCM) and 3H-thymidine incorporation. When analysed by FCM, blood leucocytes displayed two main cell populations with distinct forward and side scatter (FSC/SSC) characteristics: lymphocytes with low FSC/SSC values and non-lymphoid leucocytes (NLL) with increased FSC/SSC values. The nature of these cell types were confirmed by microscopy. Interestingly, the FSC/SSC pattern of lymphocytes remained unchanged after in vitro stimulation with Con A, whereas cells from the NLL population showed a marked shift towards increased FSC values. In stimulated cultures, the increase of FSC values of the NLL population significantly correlated with contemporarily measured 3H-thymidine incorporation (r = 0.7, P < 0.001). The mitogenic response of blood leucocytes originating from different individual fish varied over wide ranges. It was found to be related to the numbers of NLL present in the leucocyte sample. The present results show that qualitative and quantitative FCM analysis of morphological parameters (FSC/SSC) of blood leucocytes makes it possible to discriminate between leucocyte populations of the rainbow trout and to monitor cell proliferation experiments.  相似文献   

18.
Abstract

Triploid (heat-shocked) and diploid groups of rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, coho salmon Oncorhynchus kisutch, and reciprocal hybrids were produced, monitored for early life stage survival, and evaluated for susceptibility to infectious hematopoietic necrosis virus (IHNV). The female rainbow trout × male brook trout triploid hybrids had significantly greater (P < 0.01) survival than the diploid hybrids of this cross. The heat-shocked hybrid group of the female rainbow trout × male coho salmon also exhibited significantly greater survival to the eyed egg stage of development than the untreated group of this hybrid. Studies of the susceptibility of treatment groups to a 1990 IHNV isolate from the Hagerman Valley were conducted by using a standardized immersion exposure procedure at one or two different mean body weights. The diploid brook trout and coho salmon and two triploid hybrids (female rainbow trout × male brook trout or male coho salmon) were significantly less (P < 0.05) susceptible to IHNV than the pure-species diploid and triploid rainbow trout groups.  相似文献   

19.
Abstract

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 μL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 μL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 ± 1°C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 ± 1°C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 ± 1°C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.  相似文献   

20.
A procedure established for the selective isolation of the species of Streptococcus responsible for rainbow trout streptococcosis in South Africa, consisted of the inoculation of samples into nutrient broth which had been supplemented with 100 micrograms/ml of nalidixic acid, 160 micrograms/ml of oxolinic acid or 200 micrograms/ml of sodium azide. After incubation, the sample was plated onto tetrazolium agar on which the rainbow trout pathogenic Streptococcus species grew as a red colony. The colonies were isolated from the tetrazolium agar and identified as rainbow trout pathogenic isolates by biochemical and serological tests. In the laboratory the selective procedure is capable of detecting about 2 bacteria per ml. This procedure was used in the field and biochemically identical Streptococcus species were found in the mud and a freshwater crab from the water source of a site with a history of streptococcosis.  相似文献   

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