Carbohydrate and lectin interactions with Edwardsiella ictaluri and channel catfish, Ictalurus punctatus (Rafinesque), anterior kidney leucocytes and hepatocytes

Abstract. Seven isolates of Edwardsiella ictaluri were evaluated for channel catfish red blood cell and neutrophil agglutination properties and reactivity with lectins and carbohydrates. All isolates were agglutinated by lectin derived from Ricinus communis and the intensity of the agglutination reaction could be lessened or abrogated by premixing the lectin derived from Ricinus communis with β-D-galactose or α-L-fructose. Edwardsiella ictaluri isolates also sedimented much more rapidly when mixed with lectin derived from Ricinus communis than when mixed with other lectins. Premixing Ricinus communis with β-D-galactose had the greatest effect on slowing the rate of sedimentation. Two out of the seven E. ictaluri isolates were capable of agglutinating channel catfish red blood cells and anterior kidney neutrophils, and this agglutination could be prevented by the addition of β-D-mannose. Likewise, the per cent phagocytosis of E. ictaluri isolate 1389 (the most intense neutrophil agglutinator) could be dramatically reduced by the inclusion of mannose in the assay mixture. Anterior kidney leucocytes and hepatocytes primarily reacted with lectins derived from Canavalia ensiformis, Ricinus communis and Triticum vulgaris. The presence on channel catfish cells of carbohydrate-lectin receptors similar in function to those found on mammalian cells and the reaction of E. ictaluri with specific lectins suggest that bacterial clearance and lectinophagocyotosis may occur in a similar manner in catfish to that in mammals.     

Cross-protection elicited in channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri strains

Cross-protection of channel catfish ( Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri and challenged with six E. ictaluri strains was examined in four trials. The relative per cent survival among low-dose immunized and then challenged fish ranged from 27.7% to 100%. Significant protection ( P <0.05), with the exception of strain ATCC-33202, was conferred by immunization with a given E. ictaluri strain challenged either with a homologous or a heterologous strain. Antibody titres of pooled serum collected on day 22 from surviving fish examined by enzyme-linked immunosorbent assay (ELISA) ranged from 1:40 to 1:320, but no differences were apparent among different vaccinated groups. The protein profiles of six E. ictaluri strains examined by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a relatively homogeneous pattern. Immuno-blots probed with pooled serum from immunized and challenged fish showed a pattern similar to LPS-reaction patterns observed with E. ictaluri in other studies. Since the present studies further corroborate that E. ictaluri is a clonal bacterial species with no apparent antigenic differences, it is possible that immunization with a single E. ictaluri field strain should confer protection against any other strain.     

Protective immunity against enteric septicaemia in channel catfish, Ictalurus punctatus (Rafinesque), following controlled exposure to Edwardsiella ictaluri

Protective immunity against enteric septicaemia of catfish (ESC) following immunization with Edwardsiella ictaluri bacterins and exposure to live E . ictaluri was investigated. Mean cumulative percentage survival was significantly higher ( P 0.05) in controlled live vaccinates (100%) than in immersion and oral bacterin vaccinates (68.3% and 50.0%, respectively). Bactericidal activity against E . ictaluri by peritoneal macrophages from controlled live vaccinates (85.9%) was significantly greater ( P 0.05) than bactericidal activity of macrophages from immersion bacterin vaccinates (71.4%) or non-vaccinates (68.1%). No significant ( P > 0.05) difference was found in the bactericidal activity of macrophages from oral bacterin vaccinates and macrophages from controlled live vaccinates. The E . ictaluri -specific antibody response of controlled live (0.08 OD) and immersion bacterin vaccinates (0.11 OD) was significantly higher ( P 0.05) than that of oral bacterin vaccinates and non-vaccinates (0.01 OD) 15 days post-vaccination. A significantly higher antibody response was seen in controlled live vaccinates (0.17 OD), when compared to other vaccinates or non-vaccinates 33 days after vaccination. Neither immersion nor oral bacterins protected the vaccinates against ESC. Controlled live E . ictaluri immunization of channel catfish resulted in production of specific antibodies, increased macrophage bactericidal activity and protection against ESC.     

Genetic Effects for Response to Live Edwardsiella ictaluri, Killed E. ictaluri, and Stress in Juveniles from All Crosses Among USDA 103, USDA 102, and Norris Channel Catfish Ictalurus punctatus Strains

Juveniles from all possible crosses among USDA 102. USDA 103, and Norris channel catfish Ictalurus punctatus strains were compared for: 1) survival and mix-Edwardsiella ictaluri antibody after exposure to live E. ictaluri bacterium (isolate 597-458); 2) antibody level after injection with formalin-killed E. ictaluri (597-458); and 3) pre-stress. post-stress, and stress-recovery serum Cortisol levels. Purebred and crossbred USDA 102 strain fish had higher survival (mean of five genetic groups = 87%) and lower anti- E.ictaluri antibody (mean optical density (OD) of five genetic groups = 0.167) 30 d after live E. ictaluri challenge than purebred Norris and USDA 103 strains and their crosses (means of four genetic groups = 60% survival and 0.210 OD antibody level). Significant general combining ability, line effects, and heterosis indicated that the USDA 102 strain contributed additive and dominance genetic effects for increased survival and lower antibody level after live E. ictaluri challenge. Antibody response to formalin-killed, intra-peritoneally injected E. ictaluri was not different among genetic groups (overall mean = 0.198 OD). Serum Cortisol was measured prior to (pre-stress), immediately after (post-stress), and 2 h after (stress-recovery) a standard stressor. Serum Cortisol level was highest in post-stress fish (35.8 ng/mL), intermediate in stress-recovery fish (10.9 ng/mL), and lowest in pre-stress fish (6.5 ng/mL), but was not different among genetic groups within a stress time period. Results indicate diat differences exist among genetic groups of channel catfish for survival and antibody production after live E. ictaluri challenge, but these differences were not related to antibody response to killed E. ictaluri or serum Cortisol levels.     

A comparison of the antigenicity of the extracellular products and whole-cell sonicates from Mycobacterium spp. in rabbits, mice and fish by immunoblotting and enzyme-linked immunosorbent assay

The antigenicity of extracellular products (ECPs) derived from Mycobacterium spp. isolated from snakehead, Channa striata (Bloch), and Siamese fighting fish, Betta splendens (Regan), were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera collected from immunized rabbits, mice and fish (rainbow trout). All three species responded to a 65-kDa protein present in both the ECPs and whole cell sonicates (WCSs) from a variety of Mycobacterium spp. Cross-reactivity of anti- M. tuberculosis and anti-human heat-shock protein monoclonal antibodies (MAbs), and the presence of fibronectin binding proteins secreted into ECPs of mycobacteria were also examined. The MAbs against human 60-kDa heat-shock protein cross- reacted with the band at 65 kDa in the ECPs of TB1 (isolated from snakehead fish) and the type strain M. marinum, while the anti- M. tuberculosis MAb F29–47 elicited a strong reaction with a band at 21 kDa with most of the ECPs from mycobacterial strains examined. The major fibronectin-binding proteins were located between 21 and 25 kDa. The 65-kDa protein from ECPs of Mycobacterium spp. proved strongly immunogenic to rabbits, mice and fish. Rabbit antiserum against the 65-kDa protein from strain TB267 reacted with many non- Mycobacterium WCSs, and therefore, the 65-kDa protein from Mycobacterium spp. is believed to be a common protein found in many fish bacterial pathogens.     

Characteristics of monoclonal antibodies against Piscirickettsia salmonis

A panel of 28 monoclonal antibodies against Piscirickettsia salmonis was produced using a purified fraction of the bacterium. To determine their specificity to the pathogen, the antibodies were assayed by ELISA and indirect immunofluorescence microscopy. Six monoclonal antibodies were selected based on their strong reaction against P. salmonis and absence of cross-reactivity with other common fish pathogens. Western blot analysis showed that the antibodies reacted to several antigens of P. salmonis . Immunofluorescence assays revealed that these antibodies reacted with the same specificity to different isolates of P. salmonis obtained from the south of Chile. This panel of monoclonal antibodies represents an important tool to develop simple, rapid, sensitive and highly specific methods for the detection of the pathogen and diagnosis of the disease.     

Comparison of rhabdoviruses associated with epizootic ulcerative syndrome (EUS) with respect to their structural proteins, cytopathology and serology

Abstract. Seven rhabdoviruses isolated from fish suffering from epizootic ulcerative syndrome (EUS) were compared in terms of their morphology, cytopathogenicity, antigenic relatedness and structural polypeptide composition. All strains exhibited a bullet-shaped morphology, but the T9204 isolate was found to be longer and more variable in length than the other strains. Sixteen fish cell lines investigated showed some variation in susceptibility to each isolate, but the cytopathic changes induced by T9204 in SSN-2, RSN, GCP, ONP, FHM, AS and MUL lines were significantly different from the other isolates. Polyclonal antisera raised against the BPV, 20E and SL11 strains neutralized six isolates (BPV, 02, 19, 20E, A4 and SL11), but not T9204. Conversely, anti-T9204 serum only neutralized homologous virus. Polyacrylamide gel electrophoresis demonstrated that BPV, 02, 19, 20E, A4 and SL11 had virtually identical protein profiles, whereas T9204 differed both in the number of protein bands and in their migration pattern. Western blots of these gels identified the proteins specific to T9204 that reacted with anti-T9204 serum. Therefore, the isolates represent two distinct species of fish rhabdoviruses, but as yet, no causal relationship with EUS, or any other disease condition, has been established.     

A comparative study of Edwardsiella ictaluri parent (EILO) and E. ictaluri rifampicin-mutant (RE-33) isolates using lipopolysaccharides,outer membrane proteins,fatty acids,Biolog, API 20E and genomic analyses

The biological properties of Edwardsiella ictaluri RE-33 rifampicin-mutant and its parent strain EILO were analysed. RE-33 is an avirulent isolate used as a modified live vaccine against enteric septicaemia of catfish. Electrophoretic analysis of lipopolysaccharide (LPS) patterns showed high homology between both isolates. Further characterization of LPS by immunoblotting revealed the main differences in LPS composition. The RE-33 isolate lacks the high molecular weight bands of LPS (HMW-LPS). Outer membrane protein analysis also showed some immunological differences between RE-33 and the EILO parent strain. Only two fingerprinting techniques, fatty acid composition analysis and Biolog phenotypic profiles, were able to discriminate between both isolates.     

Antigenic analysis of reference strains and Norwegian field strains of aquatic birnaviruses by the use of six monoclonal antibodies produced against the infectious pancreatic necrosis virus N1 strain

Abstract. Six monoclonal antibodies (MAbs) produced against the infectious pancreatic necrosis virus (IPNV) N1 strain were used in a dot-blot assay to examine reference strains of the nine proposed serotypes and a representative selection of 81 Norwegian aquatic birnavirus isolates. These isolates had earlier been serotyped by use of a panel of 11 MAbs produced against other strains of IPNV. Correlations between the reaction patterns of the two panels of MAbs were found. All reference strains and field isolates shared two epitopes, one on VP2 and one on VP3. Seventy-seven of the field isolates reacted identically with the N1 strain (positive with all six MAbs). The Sp type strain was positive with five of the MAbs and was different from all the field strains. The other reference strains (WB, VR299, Ab, Ja, Te, He, C1, C2 and C3) were positive with two to four of the MAbs. Together with previously published data, these findings indicate that most Norwegian isolates are closely related to, or identical with, the N1 strain and belong to the Sp serotype. No correlation between the health status of Atlantic salmon and antigenicity of the isolates was found. Testing of the reference strains in ELISA revealed some discrepancies with the dot-blot results.     

鲑鱼甲病毒E1基因高保守区融合表达及免疫特性的分析

根据Gen Bank中公布的鲑鱼甲病毒(salmonid alphavirus,SAV)SAV 1、SAV 2和SAV3三个基因型中E1基因,选择高保守序列702 bp(436-1137)合成基因,命名为SAV E1,将其克隆到原核表达载体p Cold TF中构建重组质粒。然后将重组质粒转化到大肠杆菌感受态细胞BL21中,经终浓度为1.0 mmol/L的IPTG诱导表达,SDSPAGE和Western blot鉴定,重组蛋白均获得了表达,表达E1重组蛋白约95 k D。用镍离子亲和层析柱纯化重组蛋白,制备抗血清。间接ELISA结果显示,鼠抗重组蛋白E1血清效价为1∶25 600;间接免疫荧光结果显示,鼠抗重组E1蛋白血清可与SAV发生特异反应,由此表明表达的E1重组蛋白具有良好的免疫原性和免疫反应性,为SAV检测方法的建立提供理论依据。     

Uptake and Clearance of Edwardsiella ictaluri in the Peripheral Blood of Channel Catfish Ictalurus punctatus Fingerlings during Immersion Challenge

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish Ictalurus punctatus fingerlings were monitored for 216 h after exposure to E. ictaluri for 4 h and 8 h under static conditions. Most fish exposed to E. ictaluri developed bacteriemia 24 h post-exposure, and by 72 h post-exposure E. ictaluri was recovered from all the blood of all exposed fish. The number of E. ictaluri colony forming units (CFU) in the blood of moribund fish ranged between 1.7 × 10³ to 1.6 × 10⁵ CFU/50 μL whole blood. Clearance of bacteria from the blood was observed by 216 h post-exposure and all fish surviving bacterial exposure developed agglutinating antibody against E. ictaluri . The pathogenesis of the infection was accompanied by the shedding of viable E. ictaluri into the water which may serve as a mechanism by which fish to fish transmission occurs.     

Serological detection of Edwardsiella ictaluri Hawke lipopolysaccharide antibody in serum of channel catfish, Ictalurus punctatus Rafinesque

Abstract. Bacterial agglutination, passive haemagglutination, complement-dependent passive haemolysis, indirect immunofluorescence, agar gel immunodiffusion and agglutination with fractions of immunized fish serum were compared for detecting humoral antibody to the lipopolysacchande (LPS) of Edwardsiella icialuri Hawke in channel catfish. Bacterial agglutination titres averaged 1: 672; passive haemagglutination titres averaged 1: 1152; and complement-dependent haemolysis titres averaged 1: 2360. Serum from non-vaccinated fish ranged from 0 to 1:32. Indirect fluorescence and immunodiffusion demonstrated positive reactions to the LPS antibody. Fractionation of immune sera produced three fractions, one of which strongly haemagglutinated E. ictaluri but the other two did not. All six serological techniques were sensitive to E. ictaluri LPS antibody.     

Recovery and Viability of Edwardsiella ictaluri from Great Blue Herons Ardea herodias Fed E. ictaluri-Infected Channel Catfish Ictalurus punctatus Fingerlings

Feeding activities of great blue herons Ardea herodias in catfish ponds during outbreaks of enteric septicemia of catfish have been implicated as a mechanism for the transmission of the disease from infected to uninfected ponds. Although Edwardsiella ictaluri , the causative agent, has been identified in gastrointestinal tracts of great blue herons, the role of these birds as a vector of E. ictaluri is not well documented. The potential of these birds to contaminate catfish ponds with E. ictaluri was investigated by feeding captive herons over a 4-d period with catfish fingerlings injected intraperitoneally with live E. ictaluri . Daily fecal samples, throat and rectal swabs, and feather samples were collected, cultured and examined for E. ictaluri using both a selective media and a monoclonal indirect fluorescent antibody test specific for E. ictaluri . Gastrointestinal tracts sampled at the conclusion of the feeding trial were similarly examined. While E. ictaluri was detected using the indirect fluorescent antibody test, no viable E. ictaluri was cultured from either feces, gastrointestinal tracts or feathers. Growth of E. ictaluri was not observed at 40 C, the rectal temperature observed in captive great blue herons. Prior incubation at 40 C suppressed the growth of E. ictaluri at 24 C, an optimal temperature for growth of this bacterium. These results indicate that great blue herons appear to play little or no role in the transmission of E. ictaluri among catfish ponds.     

Epitope analysis of infectious pancreatic necrosis virus (IPNV) isolated from lake trout, Salvelinus namaycush (Walbaum), by monoclonal antibodies

Abstract. A panel of 15 monoclonal antibodies (MAbs) were raised against infeetious panercretic mecrosis virus (IPNV) associated with lake trout. salvelinus namaycush (Walwaum). (LT-IPNV) in Cornwall Lake Alberta, for LT-IPNV epietope analysis and comparison with other Canadian IPNV isolates. All the MAbs reacted with IPNV VP2 polypeptide in western blot and 10 MAbs were neutralizing. Both conformation and sequence dependent epitopes were found to be present on the IPNV VP, protein. The antibodies reeognized different epitopes on VP, protein in reeiproeal bloeking ELISA. Twelve MAbs reeognized common epitopes present on LT-IPNV and IPNV from Aretic char. Salvelinus alpinus (L.), (AC-IPNV) in binding and neutralization assays. Three MAbs reacted only with LT-IPNV indicating that it has distinct epitopes, and thus clerly differentiaing it from AC-IPNV isolated from the adjacent Northwest Trritories.Only two MAbs bound to Ja and BCI-IPNV isolate and none of the MAbs neutralized these two IPNV isolates. LT-IPNV was found to be distinct isolate, more colosly related to AC-IPNV and Canda -2 than to Ja-IPNV from alberta or other isolates in Canda. Additionally, the panel of MAbs could differnciate all the propsed Canadian IPNV scrotypes, namely C1. C2. C3 and Ja.     

大鲵虹彩病毒贵州分离株MCP基因的克隆与原核表达

根据Gen Bank中大鲵虹彩病毒主衣壳蛋白MCP(major capsid protein,MCP)基因序列(序列号:KF512820),设计一对特异性引物,以大鲵虹彩病毒贵州分离株基因组DNA为模板,PCR扩增大鲵虹彩病毒MCP基因并测序,与Gen Bank中大鲵虹彩病毒MCP基因进行比对,然后将其亚克隆到原核表达载体p ET-32a(+)中,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后进行Western blot分析。结果显示:PCR扩增出长度为1 392 bp的片段,与Gen Bank中大鲵虹彩病毒MCP基因核苷酸序列相似性为99.7%~99.9%,SDSPAGE电泳显示该重组蛋白的相对分子质量约为67×103。免疫原性检测结果表明,该重组蛋白可与兔抗大鲵虹彩病毒阳性血清特异性反应,具有免疫原性。     

中华鳖虹彩病毒单克隆抗体的制备及其抗原表位的初步分析

以纯化的中华鳖虹彩病毒为抗原免疫Balb/C小鼠,取免疫鼠脾细胞经杂交融合获得了8个能稳定分泌抗中华鳖虹彩病毒特异性单克隆抗体的杂交瘤细胞株。单克隆抗体亚级份分析结果表明,Mab2A4属IgA,Mab8E1是IgG2a,其他的6株单抗Mab1D3、Mab2H1、Mab3A1、Mab4B5、Mab5E1和Mab6F2均为IgG1。酶联免疫吸附剂测定（ELISA）分析表明,8株单抗均能特异性地识别中华鳖虹彩病毒,与EPC、CO、FHM等宿主细胞不产生交叉反应,腹水抗体的ELISA效价在10⁵～10⁶。IFA分析表明,8株单抗中仅Mab5E1没有免疫荧光反应特性,其余7株单抗均能对染毒病灶产生特异性的荧光染色。中和试验结果证实8株单抗均没有中和病毒的特性。应用Western-blotting进行中华鳖虹彩病毒的抗原表位初步分析,结果显示：Mab1D3和Mab2A4分别识别分子量为84 ku和35 ku中华鳖虹彩病毒结构蛋白,Mab3A1能够同时识别分子量分别为14 ku和16 ku的两条多肽,说明这3株单抗结合位点是非构象依赖性抗原决定簇,其余单抗不具备Western-blotting反应特性。这些结果提示上述单抗对中华鳖虹彩病毒抗原特异、灵敏,可用于中华鳖虹彩病毒的检测和结构蛋白分析。     

Organization and sequence of four flagellin-encoding genes of Edwardsiella ictaluri

Edwardsiella ictaluri , the cause of enteric septicaemia in channel catfish ( Ictalurus punctatus ), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments from a λ-ZAP phage genomic library of E. ictaluri . Four flagellin genes ( fliC1, fliC2, fliC3 and fliC4 ) are arranged in tandem within 6 kb in the E. ictaluri genome. Each flagellin-coding sequence is preceded by a σ²⁸ recognition site consensus sequence. The predicted amino acid sequences of all four flagellin proteins (between 36 and 37.5 kDa) are similar in the N-terminal (1–160 aa) and C-terminal (last 74 aa) portions and are divergent in the central portion of the proteins. Proteins encoded by flC1, fliC2 and fliC3 are more similar to each other (88–90% aa identity) than to the protein encoded by fliC4 (76–78% aa identity). basic local alignment search tool analysis of GenBank sequences showed that all flagellin aa sequences are more similar to those of Serratia marcescens (72–74% identity) than to those of Edwardsiella tarda (≤64% identity). Primary determination of E. ictaluri flagellin gene sequences facilitate advanced studies on the role of flagella in host–pathogen interaction.     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.     

黄颡鱼甘露糖受体基因的克隆和功能分析

甘露糖受体(MR)隶属凝集素超家族,主要表达于巨噬细胞和未成熟的树突状细胞表面。MR不仅在先天免疫防御中发挥重要作用,还通过参与抗原呈递,激活T淋巴细胞,启动获得性免疫应答过程。本研究采用同源克隆技术获得黄颡鱼甘露糖受体(pf MR)基因,采用荧光定量PCR技术检测MR在正常黄颡鱼体内的分布情况,采用鲖爱德华菌感染黄颡鱼头肾巨噬细胞和甘露聚糖封闭MR方法研究黄颡鱼MR在抗细菌感染中的作用。结果显示,pf MR同团头鲂、草鱼、斑马鱼和尼罗罗非鱼的MR聚为一支。pf MR在所检测的12个组织中均有分布,其在肾脏、脾脏和肌肉组织中表达量较高,在血液中表达量较少。鲖爱德华菌感染黄颡鱼头肾巨噬细胞后,pf MR、IL-1β和TNF-a均被细菌诱导表达,超氧阴离子和一氧化氮的含量也上升,超氧阴离子在感染30 min后即显著上升,一氧化氮在感染12 h后才显著上升。甘露聚糖竞争结合MR,显著抑制巨噬细胞内化GFP标签的鲖爱德华菌,加入EDTA减少内化的荧光强度,加入Ca~(2+)使内化的荧光强度回升。研究表明,黄颡鱼头肾巨噬细胞MR参与鲖爱德华菌的识别和内吞过程,而且依赖Ca~(2+)。