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1.
A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C. salmositica (SDS-PAGE and Western immuno-blotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immunoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody cross- reacted with the 47-kDa polypeptides of C. bullocki Srrout and C. catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1–9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C, salmositica antigen in whole cell lysate preparations at concentrations as low as 0.5 μg ml-1. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections. 相似文献
2.
A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti‐S. iniae antibody was produced in HSB by immunization with formalin‐killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non‐immunized, immunized and non‐challenged fish respectively. Live S. iniae were recovered only from moribund non‐immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti‐S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti‐S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae. 相似文献
3.
《Journal Of Applied Aquaculture》2013,25(1-2):43-52
ABSTRACT The humoral antibody response of channel catfish, Ictalurus punctatus, to experimental Flavobacterium columnare infection was measured in control (non-infected) and infected (intraperitoneal- and intramuscular-injected) fish by an indirect enzyme-linked immunoassay (ELISA). The antibody response of the experimentally infected fish was significantly higher (P<0.0001) than that of the non-infected channel catfish by both indirect ELISA and agglutination. The indirect ELISA utilized goat anti-channel catfish IgM and a commercially available rabbit anti-goat IgG enzyme conjugate. The antibody response was directed against a cell-free sonicated antigen preparation derived from live whole F. columnare cells. Indirect ELISA and agglutination results correlated (r2 = 0.60) for anti-F. columnare antibody response in experimentally and non-infected fish. We were also able to correlate ELISA and agglutination results of 60 fish naturally infected with F. columnare(r2 = 0.76). Indirect ELISA will allow for rapid monitoring of humoral antibody, following natural exposure or vaccination against F. columnare, with a small sample of serum. 相似文献
4.
Abstract. The specificity and kinetics of the immune response of rainbow trout ( Salmo gairdneri ) to single injections of an O-antigen extracted from the bacterial pathogen Yersinia ruckeri , which causes enteric redmouth in fish, were investigated by the passive haemolytic plaque assay and serum antibody quantitation. Doses ranging from 5 ng to 500 mg in 10-fold increments were injected intraperitoneally into groups of trout held at 17 × 1°5°C. The occurrence of plaque forming cells (PFC) and humoral antibody was followed for 35 days after injection. Trout gave an immune response to doses of 500 ng and above. Seven days after injection no humoral antibody was detected, but PFC were found in the spleen. The maximum PFC numbers occurred 11 days after injection. On day 21, few PFC were found, whereas serum antibody titres were highest. The antibody from immunized trout showed little or no cross-reactions with sheep red blood cells passively labelled With antigens from other fish pathogens. 相似文献
5.
鱼类免疫应答机制研究进展 总被引：1，自引：0，他引：1
鱼类免疫应答可以分为固有免疫和适应性免疫,但固有免疫发挥主要作用。固有免疫对病原体的识别是通过模式识别受体PRR与病原相关分子模式PAMP的相互结合实现,这与哺乳类相似。但为适应水生生活,鱼类固有免疫对PAMP的识别范围更广,免疫应答的启动条件更低。固有免疫的效应细胞主要是单核/巨噬细胞、嗜中性粒细胞、自然杀伤细胞等,具有吞噬和杀伤功能,还可分泌多种免疫相关的细胞因子,介导发生炎症反应。适应性免疫中,T淋巴细胞通过抗原提呈细胞分解吸收抗原,由主要组织相容性复合物(MHC)类分子递送到细胞表面才能识别。B淋巴细胞分泌产生以免疫球蛋白Ig M为主的抗体分子,而发挥抗体中和作用及免疫调理作用的Ig G在鱼类中比较少见,说明鱼类抗体的免疫功能还处于较低水平。本文综述了近二十年内鱼类免疫应答机制的相关研究进展,为进一步了解鱼类免疫应答机制提供参考。 相似文献
6.
Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells. 相似文献
7.
Heidi Mikkelsen Thomas Lindenstrøm Michael Engelbrecht Nielsen 《Journal of the World Aquaculture Society》2006,37(4):518-522
Abstract.— The effect of temperature on production and affinity of antibodies against antigens from the parasitic ciliate Ichthyophthirius multifiliis were studied in rainbow trout ( Oncorhynchus mykiss ). Fish were immunized with I. multifiliis antigens and reared at three different temperatures, 5, 12, and 20 C for 56 d. The production of specific antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that temperature has a pronounced effect on the assay result of the ELISA technique. Plasma samples analyzed at 5 C showed that the specific antibody response of fish reared at 5 C was similar to fish reared at 12 and 20 C. However, when samples were assayed at 12 and 20 C, the measured antibody response tended to be higher for the samples from trout reared at 12 and 20 C. Additionally, it was found that rainbow trout reared at 5 C showed a delayed but not hampered antibody response compared to fish reared at 12 and 20 C, indicating a correlation between low temperature and delayed antibody production. 相似文献
8.
应用间接ELISA方法,研究经嗜水气单胞菌(Aeromonas hydrophila)疫苗浸泡免疫后鳜(Siniperca chuatsi)皮肤黏液中抗体消长规律,以揭示鳜局部黏膜免疫在免疫保护中的作用及浸泡免疫的保护效果。结果表明,鳜皮肤黏液中抗体滴度在免疫后第7天达到峰值211,抗体从开始形成到消失持续21 d;添加佐剂(IMS1312、葡聚糖、莨菪碱、食盐)可以提高鳜皮肤黏液及血清中抗体滴度和相对免疫保护率,其中添加IMS1312组免疫保护率最高,达77.8%,免疫保护与抗体滴度成正相关(R2=0.79,P<0.05);以溶菌酶为代表的非特异性免疫在免疫后1周内即抗体未形成时,对鱼体起主要保护作用。此外,通过比较鳜血清与皮肤黏液中的抗体消长规律,发现血清中抗体滴度峰值出现时间较迟(第14天),抗体持续时间较长(42 d),初步推断鳜皮肤黏膜中可能存在相对独立于全身免疫系统的局部黏膜免疫系统。 相似文献
9.
Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries. 相似文献
10.
为了从对虾体内分离出的菌株中快速筛选出蜡样芽孢杆菌和溶藻胶弧菌,将蜡样芽孢杆菌与溶藻胶弧菌作为抗原,免疫SPF新西兰大白兔,获得免疫血清,效价均高于1:2000。将免疫兔血清作为一抗,HRP-羊抗兔血清作为二抗,建立了蜡样芽孢杆菌和溶藻胶弧菌快速检测的间接ELISA方法。此方法中,兔抗血清最佳稀释度为1:10000,菌液最佳包被浓度为106 CFU/ml;HRP-羊抗兔血清最佳稀释浓度为1:1000,可检测细菌最低浓度为104 CFU/ml。抗蜡样芽孢杆菌血清与苏云金芽孢杆菌有交叉反应,抗溶藻胶弧菌血清与其亲缘相近菌株无交叉反应,具有较强的特异性。2012年和2013年,分别从斑节对虾、中国对虾、凡纳滨对虾等9批样本中分离纯化到109株海洋细菌,利用建立的多抗间接ELISA方法对其中部分菌株进行快速检测,共检测出6株溶藻胶弧菌,未检测出蜡样芽孢杆菌。 相似文献
11.
Humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from Tetramicra brevifilum Matthews & Matthews, 1980 (Microspora) 总被引：1，自引：0，他引：1
J. LEIRO J. ESTÉVEZ M. T. SANTAMARINA M. L. SANMARTÍN F. M. UBEIRA 《Journal of fish diseases》1993,16(6):577-584
Abstract. The humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from the microsporean parasite Tetramicra brevifilum Matthews & Matthews, 1980, was studied. Thirty days after intraperitoneal immunization with whole T. brevifilum spores in Freund's complete adjuvant, double indirect ELISA indicated that initial production of antibodies to parasite surface antigens was considerably higher than production of antibodies to the antigens contained in a crude extract (CE) of spores. Following re-immunization without adjuvant on day 30, levels of antibodies to surface antigens gradually declined, whilst levels of antibodies to CE antigens increased. The antibody response of intraperitoneally immunized fish was characterized by Western blotting of total soluble antigens obtained by heating and reduction of T. brevifilum spores at 95–100°C in Tris-HCl buffer containing SDS and dithiothreitol: a series of bands with molecular weights between 20 and 53 kDa was recognized by immunized turbot sera. Four additional bands (with molecular weights between 15 and 18kdA) were recognized by serum from re-immunized fish. ELISA studies of sera from naturally infected fish revealed a surprisingly low incidence of strong T. brevifilum seropositivity (61% individuals); antibodies to surface antigens predominated in seropositive individuals. The low background response levels and high sensitivity of the ELISA used in this study indicate that the assay is of value for the monitoring of serum antibody levels in turbot. However, given the relatively low seropositivities observed in naturally infected turbot, particularly to CE antigens, the use of anti- T. brevifilum serum antibody levels for the diagnosis of infection by this parasite may lead to false negative results. 相似文献
12.
It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme. 相似文献
13.
Abstract. There are two basic antigenic components in Cryptobia that are responsible for the anaemia in infected rainbow trout. A 'lytic component', which is dosage-dependent, causes lysis of red blood cells independent of antibody or complement. The second, an 'immune-complex-forming component', attaches to red blood cells, forms immune complexes with specific antibody and activates complement resulting in haemolysis. These two antigenic components, from both live and lysed Cryptobia , were present in the serum of infected fish. When sonicated antigen or heat-inactivated antiserum (from infected fish) was incubated with red cells from uninfected fish, a portion of the red cells was lysed and a positive Coombs' reaction was observed with the remaining intact red cells. The positive Coombs' reaction was due to immune complexes adsorbed onto the red cells and these lysed when incubated with complement. Antibody by itself did not adsorb onto the red cells. From the fourth week post-infection, a positive Coombs' reaction was observed in all infected fish and haemolysis occurred with complement. The authors suggest that, in infected fish, one or more components of the complement cascade is depleted continually during infection and that the anaemia is due to the lytic action of the antigen and immune complex formation on red cells. These lead to intra-vascular haemolysis as well as erythrophagocytosis. In general, the mechanism of anaemia in cryptobiosis appears similar to that in African trypanosomiasis. 相似文献
14.
温度对牙鲆皮肤黏液抗体产生的影响 总被引：1，自引：0，他引：1
在9℃、15℃、21℃和26℃4种不同水温下,用淋巴囊肿病毒(LCDV)灭活疫苗腹腔注射牙鲆,应用间接酶联免疫吸附试验(ELISA)研究了其皮肤黏液中特异性抗体水平的变化,以分析温度对牙鲆皮肤黏液抗体产生的影响.ELISA结果表明,9℃和15℃水温下牙鲆黏液OD值分别在注射LCDV后第9周和第7周达到峰值(9℃:OD=0.179; 15℃:OD=0.233); 21℃水温下OD值上升最快,5周达到峰值(0.316); 26℃水温下OD值较21℃无显著差异,也于5周达到峰值(0.295).采用硫酸铵分步盐析等技术粗提不同温度下OD值最高时的牙鲆皮肤黏液中的免疫球蛋白(Ig),SDS-PAGE检测发现,各温度组黏液蛋白中均含有72 kD和26 kD蛋白条带.Western blotting结果显示,抗牙鲆血清Ig重链的单克隆抗体只与黏液蛋白中72 kD条带发生反应,确定为牙鲆皮肤黏液Ig重链.综上结果表明,牙鲆在最适生活温度(21℃)下,抗体应答强度最大.牙鲆粗提黏液蛋白中Ig的初步确定,为探索牙鲆黏液免疫机制提供了材料. 相似文献
15.
Lancefield group C Streptococcus dysgalactiae (GCSD) causes severe necrotic lesions in the caudal peduncle in the genus Seriola farmed in Japan. To develop a sero‐diagnostic method for GCSD infection in farmed fish, we attempted to identify a surface immunogenic protein that induces an antibody after infection with GCSD by immunoblot analysis using sera collected from infected fish. A protein obtained from sodium dodecyl sulfate (SDS) extracts of GCSD was identified as S. dysgalactiae surface immunogenic protein (Sd‐Sip). Sd‐Sip exhibited more than 94% homology with a surface antigen or a hypothetical protein from S. dysgalactiae mammalian isolates at the nucleotide sequence level. Expression of the recombinant Sd‐Sip (rSd‐Sip) was confirmed by immunoblot analysis, that is, its reactivity to GCSD‐infected sera. Antibody detection ELISA using rSd‐Sip and their usefulness for diagnosis of GCSD infection were examined. GCSD‐infected sera collected from farmed amberjack, Seriola dumerili (Risso), showed strong reaction with immobilized rSd‐Sip. Meanwhile, sera immunized by other pathogenic bacteria of fish were showed ELISA values similar to those of non‐infected sera. These results of this study suggest that the antibody detection ELISA using rSd‐Sip is an effective diagnostic method for GCSD infection in fish. 相似文献
16.
Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment. 相似文献
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18.
The deceptive marketing of imported basa, Pangasius bocourti (Sauvage), fillets as catfish has resulted in serious economic losses to the channel catfish, Ictalurus punctatus (Rafinesque), industry in the US. The similar appearance of channel catfish and basa fillets created a need for a rapid method to differentiate uncooked, cooked and/or marinated channel catfish fillets from basa fillets and other fish products. A monoclonal antibody (MAb) specific for a 36.8 kDa channel catfish fillet protein was produced and characterized by an indirect enzyme‐linked immunoabsorbent assay (ELISA) and Western blotting. This MAb was used to develop an indirect ELISA specific for a fillet protein unique to fish of the genus Ictalurus. Using this ELISA, 100% of raw and cooked channel catfish fillets were correctly identified and differentiated from other fish in a single‐blind study. These results show that the indirect ELISA using MAbs specific for unique Ictalurus sp. fillet proteins is a rapid and sensitive method for the identification of raw and cooked catfish fillets. 相似文献
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20.
鱼类粘膜免疫研究进展 总被引：7，自引：1，他引：7
Fish immunology has achieved great progress in recent years. While before 1990s, most researches focused on the fish systematic immunity, and the mucosal immunity of fish had not been given enough attention. Indeed, it has been shown that fish mucosal immunity plays an important role in disease defense. Fish mucosal immunity research has made some exciting progress in this decade. This review will focus on such progress: Constitution of mucosal-associated tissues and distribution of different immune cells, including T/B lymphocytes, granules, monocytes, macrophages, goblet cells, etc, in these sites have been well described with the development of some monoclonal antibody to these cells and associated techniques. Non-specific immune response mechanism of mucosal tissues reported these years, such as secretion of non-specific anti-bacteria and anti-fungi substances in mucus, the respiratory burst, enzyme activity of immune cells and so on, is believed important for fish disease defense. The specific immunity of mucosal tissues also attracts much interest and makes great achievement in antigen presenting, MHC genes, antibody producing and antibody secreting cells, comparison of serum and mucus immunoglobulin, relationships of immune response between different mucosal immune tissues. Whether mucosal immune system is independent of systematic immune system is another interesting question and causes great concern. In recent years, some evidences from phyletic evolution and ontogenesis show that mucosal immunity is prior to systematic immunity in evolution. Dynamics of antibody producing of mucosal tissues and serum in immersion or oral vaccines immunized fish also shows immune response can be elicited in mucosal tissues independent of systematic immune system. Some researchers also begin to pay attention to factors involved in mucosal immune regulations, for instance, neuromodulators and cytokines. The level of these factors changes in fish immune response process but the mechanisms of regulation still remain unknown. Prospect of the promising future of fish mucosal immunity has also been discussed in this review. 相似文献