Effect of dietary β-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton)

The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B₁(AFB₁)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB₁, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB₁at 1·25 mg kg⁻¹body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB₁-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB₁exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

The effect of dietary immunomodulation upon Edwardsiella tarda vaccination in healthy and immunocompromised Indian major carp (Labeo rohita)

In order to determine the impact on disease resistance of four dietary immunomodulators viz., beta-1,3 glucan, levamisole, vitamins C and E, in an important farmed Indian major carp species, rohu (Labeo rohita Ham.), fish were fed diets containing various levels of these substances during a 60 day trial. Aflatoxin B1 (AFB,) at 125 mg kg(-1) body weight was injected intraperitoneally (i.p.) into fish to induce an immunosuppressive state on the first day of the experiment in some individuals. The fish were vaccinated against formalin-killed Edwardsiella tarda vaccine on day 30 of the experiment. Specific immunity, as measured by bacterial agglutination titre and disease resistance against E. tarda, was determined at the end of the trial. The results demonstrate that all the four immunomodulators were capable of significantly (P<0.05) increasing specific immunity and reducing mortality in immunocompromised fish but failed to enhance specific immunity and protection in healthy fish. The increased bacterial agglutination titre by beta-1,3 glucan, and reduced mortality losses by both beta-1,3 glucan and levamisole were marked in healthy vaccinated fish compared with their controls. Similarly, all four substances significantly reduced the mortality rates in immunocompromised and healthy unvaccinated fish. Out of these four substances, glucan was recorded to be the most effective immunomodulator in rohu. The present results suggest that the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions could increase their resistance to infection by reducing mortality rates and offer economic benefits.     

Dietary immunostimulants influence specific immune response and resistance of healthy and immunocompromised Asian catfish Clarias batrachus to Aeromonas hydrophila infection

In order to determine the efficacy and immunoreversal effect of the 4 dietary immunomodulators, viz. lactoferrin, beta-1,3 glucan, levamisole and vitamin C, on disease resistance of a commercially important catfish, Clarias batrachus, fish were fed diets supplemented with various levels of these substances in 2 subgroups, healthy and immunocompromised, during a 30 d trial. An artificial immunosuppressive state was induced by giving 3 intraperitoneal (i.p.) injections of cyclophosphamide (CYP) at a dose level of 200 mg kg(-1) body weight at 1 wk intervals in the immunocompromised vaccinated subgroup and 3 consecutive injections 3 d before challenge in the immunocompromised non-vaccinated subgroup. On the first day of the experiment, the fish were vaccinated against a formalin-killed Aeromonas hydrophila bacterin. After 30 d, antibody titre (as measured through bacterial agglutination titre) and disease resistance against A. hydrophila were determined. The results demonstrate that all 4 immunomodulators were capable of significantly (p < 0.05) enhancing the specific immune response; this was evident through raised antibody titre and protection against A. hydrophila in both healthy and immunocompromised vaccinated subgroups compared to their respective controls. Similarly, all 4 substances significantly raised the survival rates in immunocompromised and healthy non-vaccinated fish. Thus, these substances were capable of reducing the immunosuppression induced by CYP injections in both vaccinated and non-vaccinated fish compared to their respective controls. Among the 4 substances studied, beta-1,3 glucan was found to be the most effective immunomodulator, followed by levamisole, lactoferrin and vitamin C in Asian catfish. Therefore, the results support the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions in order to enhance protection against infection and offer economic benefits.     

Effects of cyclophosphamide on the immune system and disease resistance of Asian catfish Clarias batrachus

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mgkg(-1) body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant (P<0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.     

Effects of polyherbal formulation 'ImmuPlus' on immunity and disease resistance of Indian major carp, Labeo rohita at different stages of growth

A series of experiments were performed to determine the impact of polyherbal immunomodulatory formulation 'ImmuPlus' (AquaImmu) on growth, immunity and disease resistance of rohu (Labeo rohita), one of the Indian major carp at different stages of growth. Rohu larvae were fed on plankton, ImmuPlus-mixed compound feed, and plankton plus ImmuPlus-mixed compound feed (ImmuPlus added at three dose levels of 0.25, 0.50, and 0.75 g/kg feed) from 4th day of hatching to 14th day. ImmuPlus-mixed diets enhanced growth of larvae, survival and disease resistance against Aeromonas hydrophila challenge, compared to only plankton-fed group. In two other experiments, advanced rohu larvae and fingerlings were fed with ImmuPlus-mixed compound feed (at 0, 0.5, 1.0 and 2.0 g/kg) for 15, 30 and 45 days. At the end of 45 days for advanced larvae and 30 days for fingerlings, the fish fed with ImmuPlus at 1.0 g/kg level showed significantly higher growth and disease resistance against A. hydrophila challenge. In a separate experiment, juveniles of rohu were fed with 1 g/kg of ImmuPlus incorporated feed for 15 and 30 days. At the end of the trial, the ImmuPlus fed fish showed enhanced non-specific immunity (as measured through nitroblue tetrazolium reduction assay, serum lysozyme activity, serum haemolysin titre and resistance against A. hydrophila challenge in non-vaccinated fish as well as specific immunity levels (as measured through bacterial agglutination titre against A. hydrophila in vaccinated fish). Incorporation of ImmuPlus at 1 g/kg level in the diet of rohu may be beneficial for enhancing disease resistance.     

Administration of yeast glucan enhances survival and some non-specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila

Effects of beta-glucan administration on survival and immune modulations were studied in Cyprinus carpio against the bacterial pathogen, Aeromonas hydrophila. Beta-glucan was extracted from Saccharomyces cervisiae and purified. A virulent strain of the pathogen A. hydrophila was collected from infected fish. Different concentrations of beta-glucan were administered to test animals on day 1, 3 and 5 through different routes (intraperitoneal injection (ip), bathing and oral administration). Control and test animals were challenged by ip injection of LD50 concentration of A. hydrophila on day 7 and mortality was observed and Relative Percent Survival (RPS) was calculated. Intraperitoneal injection of 500 microg of glucan significantly enhanced the RPS; bathing and oral administration of glucan did not influence the RPS. On day 7, test animals injected with 100, 500 and 1000 microg of glucan had a significant increase in total blood leucocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by kidney macrophages was also elevated. RT-PCR and northern blot analysis of interleukin-1 mRNA showed elevated expression in kidney on day 7 in fish injected with glucan. Glucan had an adjuvant effect on antibody production as pretreatment by injection of 100-1000 microg glucan/fish resulted in the highest antibody titer against A. hydrophila following vaccination. Classical and alternative complement pathways were not affected by glucan administration by any of the three routes.     

Effect of multiple injections of beta-glucan on non-specific immune response and disease resistance in Labeo rohita fingerlings

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.     

The auxotrophic aroA mutant of Aeromonas hydrophila as a live attenuated vaccine against A. salmonicida infections in rainbow trout (Oncorhynchus mykiss)

An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A. hydrophila infection in rainbow trout (Oncorhynchus mykiss). The protection conferred by the live attenuated vaccine against A. salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised. Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A. salmonicida (relative percent survival RPS >60%). This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout.     

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3

The acute hepatotoxicity induced by aflatoxin B1 (AFB1) and the potential protective effect of (+)-cyanidanol-3 (Catergen) were evaluated in both human and rat hepatocytes in primary cultures. AFB1-induced acute toxicity was visualized by light microscope observation and quantified by measurement of lactic dehydrogenase activity in the medium. Human hepatocytes were susceptible to AFB1-induced cytotoxicity but no evident relationship between the concentration of mycotoxin and the extent of cellular damage was established. (+)-Cyanidanol-3 was not toxic at concentrations up to 2 x 10(-3)M, but no obvious protective effect from AFB1-induced injury was evidenced in human cells. By contrast, rat hepatocytes responded in a dose-related manner to AFB1. (+)-Cyanidanol was toxic at 10(-3)M, but even at this concentration exerted a strong protective effect against AFB1-induced cytotoxicity. Such species differences suggest the existence of metabolic differences in both AFB1 and (+)-cyanidanol-3 activating and deactivating mechanisms.     

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10? fold increase of LD?? dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10? CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10? or 10? CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10?-10? CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10?-10? CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.     

黄连须和小檗碱对草鱼非特异性免疫系统的影响及对嗜水气单胞菌感染的抵抗作用

为了评估黄连须(FRC)和小檗碱(BBR)对草鱼(Ctenopharyngodon idella)的非特异性免疫及对抵抗嗜水气单胞菌感染的影响, 鱼喂食含1.00%、0.50%、0.25%, 0.10%, 0.00%的FRC和0.05%BBR的饲料28d。喂养28d后, 鱼注射嗜水气单胞菌, 记录感染14d后的存活率。检测NBT活性、血清溶菌酶活性、吞噬活性和补体C3水平来评价药物对草鱼的免疫反应。结果表明, 与模型组相比, 喂养饲料含有FRC和BBR的各组提高NBT活性、血清溶菌酶活性、吞噬活性和草鱼补体C3水平。喂食0.50%FRC组感染细菌后的存活率最高, 与模型组比较增加44%。FRC和BBR的体外抗菌活性采用最小抑菌浓度的方法进行了研究, 并显示出较强的抑制作用。结果表明, FRC和BBR可以用作免疫刺激剂以提高草鱼非特异性免疫和对嗜水气单胞菌引起疾病的抵抗。       

The immunomodulatory effects of tuftsin on the non-specific immune system of Indian Major carp, Labeo rohita

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p相似文献   

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.     

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater inter-moult sub-adult prawn Macrobrachium rosenbergii (15-20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200mg kg(-1) feed for 7 or 14 days. M. rosenbergii fed a diet containing 100mg Lf kg(-1) diet for 7 days showed significant (P<0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200mg kg(-1) for 14 days compared to control. Feeding of Lf at 50mg kg(-1) diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50mg kg(-1) for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100mg kg(-1) diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200mg kg(-1) diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100mg kg(-1) for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.     

嗜水气单胞菌菌蜕的制备及其对银鲫的口服免疫

菌蜕系统是一个自身具有佐剂性质的新型疫苗体系,不含细胞质内容物但具有细菌的完整表面抗原结构,可诱导机体的体液、细胞免疫应答及增强黏膜免疫反应.本研究通过将带有裂解基因E的质粒pElysis转化至嗜水气单胞菌J-1株中,对Ah J-1(pElysis)进行温度诱导,温度从28℃升至42℃,每隔15min检测菌液的OD600值,测定其溶菌动力学,并做无菌检验,用扫描电镜观察裂解后的细菌形态,研究其作为口服疫苗对银鲫的效果.结果显示,通过温度诱导,嗜水气单胞菌J-1(pElysis)OD值在诱导30min后开始持续下降,75min时开始趋于平稳,到120min溶菌效率达99.99%,诱导16h后进行无菌检验,证实其无活菌.扫描电镜观察绝大部分菌体经诱导后形成菌蜕,细胞两端有溶菌通道.动物试验表明,用菌蜕口服免疫的银鲫,在第5周产生较高的凝集抗体,达到27,并能维持2周;而甲醛灭活苗组为26,维持时间仅一周;生理盐水对照组效价仅2.攻击试验表明,菌蜕疫苗组和甲醛灭活疫苗组对嗜水气单胞菌强毒株J.1的攻击均有保护作用,其相对保护率分别为16/20(78.95%)和12/20(57.9%),显示菌蜕疫苗比普通灭活疫苗能更有效地激活机体的免疫保护.     

Environmental stress and bacterial infection in channel catfish, Ictalurus punctatus Rafinesque

Channel catfish, Ictalurus punctatus , were injected intraperitoneally with a sublethal dose of Aerornonas hydrophila and then stressed for 144 h by being maintained either in a dissolved oxygen concentration of 1·5 mg/1, 1·2 mg/1 total ammonia, and/or 6·5 mg/1 free CO₂ with a continuous inflow of water. A significant difference in percentage of mortality was noted between treatments ( P < 0·05). The trunk kidneys of surviving stressed fish had significantly higher total bacterial counts than non-stressed controls. A. hydrophila was isolated from 67% of the stressed fish and 9% of the control fish. Edwardsiella tarda , apparently endemic in the population, was isolated from 43% of the stressed fish and 7% of the control fish. Histopathological lesions were in the gills, liver, spleen, trunk kidney, and head kidney of stressed fish, but not control fish.     

Immune response and disease resistance of Oreochromis mossambicus to Aeromonas hydrophila after exposure to hexavalent chromium

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.     

An attenuated plasmid-cured strain of Aeromonas hydrophila elicits protective immunity in Clarias batrachus L

Wild type Aeromonas hydrophila (Strain AO1) isolated from the lesions of ulcerative disease syndrome (UDS) affected fish bears a 21 kb virulence plasmid. With plasmid curing the isolates became attenuated and failed to induce fatal haemorrhagic ulcers in fish. The objective of the present work was to check the immunogenicity of these plasmid-cured derivatives and determine whether such strains could be used as candidate antigens for eliciting protective immunity to A. hydrophila infections in the Indian catfish Clarias batrachus L. It was observed that the plasmid-cured strains were immunogenic since infection with live plasmid-cured AO1 isolates generated effective T cell responses and led to increase in serum antibacterial agglutinin titres in C. batrachus. Plasmid-cured AO1 strains injected into C. batrachus could disseminate into head kidney (HK) and spleen but never attained the same bacterial loads obtained with wild type AO1 and were cleared rapidly from the host. Immunisation with plasmid-cured bacteria prevented systemic spread and conferred protection against lethal challenge (10 x LD(50)) with wild type A. hydrophila as well as other pathogenic strains of Aeromonas sp. These results demonstrate the potentials of plasmid-cured A. hydrophila derivatives as candidate antigens for eliciting protective immunity in fish and the possibility of using such isolates as shuttle vectors in aquaculture.     

凡纳滨对虾血蓝蛋白与病原菌凝集作用靶标的鉴定

血蓝蛋白是一种具有多种非特异性免疫学活性的多功能蛋白,以前的研究发现,血蓝蛋白具有凝集活性.本研究采用凝集抑制实验和亲和蛋白质组学等方法探索凡纳滨对虾血蓝蛋白与病原菌的凝集作用靶标.结果显示,大肠杆菌K12和副溶血弧菌外膜蛋白可以抑制血蓝蛋白对7种细菌的凝集活性,其中大肠杆菌K12中2种分子质量分别为16 kD、18 kD （命名为 p16、p18）的外膜蛋白可以与血蓝蛋白发生特异性的结合,经MALDI-TOF/MS鉴定,p16、p18 分别与大肠杆菌外膜蛋白OmpC、OmpX具有高度同源性.尤其是与大肠杆菌K12野生菌株相比,血蓝蛋白对 ΔOmpX 的凝集特异性明显降低,后者仅为前者的25％.由此推测,OmpX 应为血蓝蛋白与病原菌的凝集作用靶标.