In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Mouse monoclonal antibodies (MAbs) were produced by using yellow grouper nervous necrosis virus (YGNNV) as an immunogen, isolated from infected yellow grouper, Epinephelus awoara (Temminck & Schlegel), and propagated in GB cells. In enzyme linked immunosorbent assay (ELISA), 43 hybridoma clones secreting MAbs strongly reacted with the purified virus. Ten of them showed a higher neutralization index (NI) value between 6.5 and 4.5 (log₁₀ NI) than the other 33 MAbs against YGNNV infection in cell culture. All 10 MAbs belonged to the IgG isotype with a κ light chain and recognized the 42 kDa coat protein of YGNNV by Western blot analysis. Immunohistochemical results demonstrated that the viral signals co-located with pathological lesions observed in retina, brain and spinal cord. These results indicate that the MAbs are useful for confirmative diagnosis of YGNNV infection.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

Production and characterization of monoclonal antibodies to leukocytes and serum immunoglobulin in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Ig⁺ leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5–7.4, 23.7–50.1, 25.2–26.1 and 5.2–8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5–36.3, 1.9–2.8, 6.4–8.3 and 1.9–3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.     

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.     

Preparation of monoclonal antibody against Macrobrachium rosenbergii Nodavirus and application of TAS-ELISA for virus diagnosis in post-larvae hatcheries in east China during 2000-2004

“Whitish muscle disease” of Macrobrachium rosenbergii, also called “whitish disease” or “white tail disease”, is a new serious epizootic disease that has occurred in recent years in giant freshwater prawn culture regions, mainly in southern China. This disease occurred in post-larvae 3-5 days to 3 weeks after desalting. Clinical signs include the development of white spot in muscles or milky muscles throughout the body, causing serious loss in few days, with a mortality rate of 40-90%. A 26-27 nm icosahedral non-enveloped virus, identified as M. rosenbergii Nodavirus (MrNV), was confirmed as the aetiological agent. Twelve hybridomas strongly secreting monoclonal antibodies (Mabs) against MrNV were shown to be specific for MrNV and reacted with MrNV 42 kDa coat protein by Western blot. A triple antibody enzyme-linked immunosorbent assay (TAS-ELISA) was developed and shown to be a useful diagnostic tool for MrNV infection.     

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.     

Purification and characterization of carbon monoxide dehydrogenase from the aerobic hyperthermophilic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis>

The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses carbon monoxide (CO) oxidation activity under heterotrophic growth conditions. Using activity stain gel analysis, CO oxidation activity was detected in a protein with a molecular mass of 210 kDa. The 210 kDa CODH protein was purified to homogeneity from A. pernix. Aeropyrum Mo-CODH catalyzed the oxidation of CO with a specific activity of 2.1 μmol CO min⁻¹ mg⁻¹ at 95°C, pH 8.0 using methyl viologen as the electron acceptor. The CODH protein showed high oxygen and thermo stability. The protein contains three subunits: L (86.6 kDa), M (34.5 kDa), and S (12.6 kDa), which form the LM₂S complex. The molecular mass of the complex was calculated by gel filtration and found to be 163.7 kDa. N-terminal amino acid sequencing and peptide mass fingerprinting analysis of the subunits indicated that they corresponded to NP_148462.1, NP_148464.2, and NP_148465.1, and their genes annotated the molybdo iron-sulfur flavoprotein carbon monoxide dehydrogenase S, L, and M subunits, respectively. Phylogenetic analysis revealed that CODH belongs to a novel clade of diverse CODHs.     

Induction,purification and partial characterization of vitellogenin in an Indian major carp Catla catla (Ham.)

Vitellogenin was purified from the serum of 17‐β estradiol (E₂)‐induced juvenile Catla catla using a simple two‐step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS‐PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone‐induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.     

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS‐PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross‐reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody‐based assays.     

Characterization and production of polyclonal antisera against pangasius (Pangasianodon hypophthalmus) serum immunoglobulin IgM derived from DEAE cellulose based ion exchange chromatography

Pangasius (Pangasianodon hypophthalmus) is a commercially important candidate species in freshwater aquaculture and it is important to understand the immune system of pangasius against infectious disease. The present study was aimed at the purification, characterization and quantification of serum IgM in pangasius (P. hypophthalmus). Serum IgM was purified by diethylaminoethyl (DEAE) cellulose based ion exchange chromatography. The molecular weight of native immunoglobulin was found to be 798 kDa. Heavy (H) and light chains were found to possess molecular weight of 70.1 and 26 kDa respectively. A 248 bp segment of IgM H chain gene of pangasius was amplified and sequenced (partial). The antisera raised against pangasius immunoglobulin cross‐reacted with the immunoglobulin H chain of catfish such as Clarias gariepinus and Clarias batrachus, but not with their light chain indicating epitope sharing among IgM H chains in these catfish. The produced polyclonal antisera were used to develop an enzyme linked immuosorbent assay to quantify IgM levels in pangasius. In conclusion, the present study provides its future implications for epidemiology and immunology studies in pangasius.     

Production of monoclonal antibodies against yellowtail ascites virus

A total of 31 antibody-secreting hybridoma cells against the yellowtail ascites virus (YAV) were established. These monoclonal antibodies (MAbs) reacted with the cytoplasm of YAV-infected CHSE-214 cells, but not with uninfected CHSE-214 cells in an immunofluorescence test. Using these MAbs, two classes of polypeptides (VP2 and VP3) were characterized by immunoprecipitation followed by SDS-PAGE, although one MAb did not react with either polypeptide. Fifteen out of the 17 MAbs that were reactive with VP2 polypeptides neutralized virus infectivity, but all 13 MAbs that were reactive with VP3 did not neutralize infectivity. In the immunofluorescence test, 29 out of the 31 MAbs obtained showed the same reaction pattern to 12 YAV isolates from yellowtail, Seriola quinqueradiata, goldstriped amberjack, Seriola aureovittata, and threeline grunt, Parapristipoma trilineatum, from different geographical regions. The remaining two MAbs showed slightly different reaction patterns to the YAV isolates. The reaction patterns of the MAbs to the VR-299, Sp and Ab strains of IPNV were also investigated. Fourteen MAbs reacted to all three IPNV strains. The other 17 MAbs showed a negative reaction with at least one strain.     

Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana)

Three forms of female-specific plasma protein (FSPP 1-3) were purified from blood plasma of estrogen-treated white perch (Morone americana) by combining several types of ion-exchange chromatography including a novel, fast flow, strong anion exchanger (POROS media), followed by gel filtration. Native FSPP 1, FSPP 2 and FSPP 3 had molecular masses of 532 kDa, 532 kDa and 426 kDa, respectively. The apparent mass of purified FSPP 1 and FSPP 2 after SDS-PAGE under reducing conditions was ∼ 180 kDa, while FSPP 3 appeared as a major ∼ 148 kDa band. All of the FSPPs resembled one another with respect to amino acid composition but each appeared to be immunologically distinct. In double immunodiffusion using anti-total FSPP (antiserum raised against vitellogenic female plasma pre-absorbed by male plasma), each FSPP formed one precipitin line that crossed those produced by both others. A rabbit antiserum was raised against each FSPP and absorbed with combinations of the other two FSPPs to ensure specificity. Using the antisera, each FSPP was detected by immuno-electrophoresis in plasma from vitellogenic females or estrogen-treated male or immature fish, but no FSPP was detected in normal male plasma. Endoprotease (Asp-N) digests of the FSPPs were subjected to HPLC separation for N-terminal sequencing and mapping of isolated peptides to published vitellogenin (Vg) sequences. Results of these analyses indicate that white perch FSPP 1, FSPP 2, and FSPP 3 can be classified into three Vg groups identified in previous studies: VgA, VgB, and VgC-like protein, respectively. This is the first report, of which we are aware, on isolation of more than two Vg proteins from any species of vertebrate except the chicken. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and partial characterization of vitellogenin from shorthead redhorse (<Emphasis Type="Italic">Moxostoma macrolepidotum</Emphasis>) and copper redhorse (<Emphasis Type="Italic">Moxostoma hubbsi</Emphasis>) and detection in plasma and mucus with a heterologous antibody

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of ∼425 kDa (copper redhorse) and ∼450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of ∼150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.     

Ontogenic changes in the contents of dopamine,norepinephrine and serotonin in larvae and postlarvae of the bivalve Pecten maximus

In the present study, norepinephrine (NE), epinephrine (E), dopamine (DA) and 5-hydroxy-tryptamine (5HT) have been quantified by high performance liquid chromatography (HPLC) in Pecten maximus during larval and post-larval development. With average values ranging from 2 to 5 pg μg^–1 of protein, NE remained low through the whole larval life, while epinephrine (E) was undetected. DA and 5HT were the most abundant monoamines with significant variations between larval, pre-metamorphic and post-larval stages. During the first 20 days, corresponding to D larval and umboned larval stages, levels of DA and 5HT increased from 15 to 30 and 10 to 15 pg μg^–1 of protein, respectively. Then during the last week of larval life, at the approach of metamorphosis, DA rose sharply from 30 to 50 pg μg^–1 of protein and 5HT from 15 to 50 pg μg^–1 of protein. Lastly during the first week of post-larval life (day 27 to day 34) DA and 5HT contents declined to levels similar to those detected in the first days of larval life reaching progressively 1 pg μg^–1 of protein during the second week of post-larval life (day 34 to day 55). These rapid and transient variations in monoamine contents (5HT and DA) around metamorphosis, present a great interest. However, this relation between neurochemical changes and metamorphosis must be confirmed with future studies in order to verify if such monoamines might be used as indicators of larval competence in P. maximus, a commercially important species.     

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.     

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.     

Feed Containing Novacq Improves Resilience of Black Tiger Shrimp,Penaeus Monodon,to Gill‐associated Virus‐induced Mortality

The ability of Novacq to improve resilience of black tiger shrimp, Penaeus monodon, to infection and mortality induced by gill‐associated virus (GAV) was investigated. Over a 26‐d period, shrimp were fed pellets with or without 10% Novacq. Following this, four replicate tanks, each containing 10 shrimp that had been fed either diet, were maintained as‐is, injected with saline or injected with GAV inoculum (i.e., 40 shrimp for each of the six groups). For shrimp (n = 20) in two of each group of four tanks, survival was monitored daily over 14 d and a pleopod was sampled from each shrimp on Days 0 and 14. For the other two tanks, a pleopod was sampled from each shrimp on Days 0, 3, 7, 10, and 14 to track changes in GAV loads over time. Survival was significantly higher (P < 0.05) from Day 7 onward among the group fed Novacq. GAV infection loads appeared to vary more between individuals in the Novacq diet cohort, but overall were not reduced significantly at any time points post‐challenge compared to shrimp tested from the Control diet cohort.     

Growth,body chemical composition and trypsin activity of South American catfish,surubim (Pseudoplatystoma sp.) juveniles fed different dietary protein and lipid levels

To evaluate protein and lipid requirement of South American catfish surubim (Pseudoplatystoma sp.) juveniles, nine semi‐purified diets containing three levels of protein (40%, 45% and 50%) and three levels of lipid (12%, 16% and 20%) were tested. After 8‐week feeding trial, body weight increase averaged 2124.3 ± 295.7%. Growth performance was significantly affected by dietary level of protein (P < 0.05). At the 40% protein level, increasing level of dietary lipid had a positive effect on final individual mean weight (protein sparing effect). Whole body protein and moisture contents were affected by the dietary level of lipid (P < 0.05). Whole body lipid content positively correlated with the level of dietary lipid (P < 0.05). Cannibalism related mortality was observed despite rearing fish in 24 h dark. Fatty acid composition of fish was affected by the dietary lipid level (P < 0.05). Polyunsaturated fatty acids increased with the increasing level of dietary lipid while saturated fatty acids and monounsaturated fatty acids decreased. Trypsin activity in the digestive tract of surubim was influenced by dietary levels of protein and lipid (P < 0.05). Our preliminary results suggest that the optimum protein/lipid ratio might be close to 45/16% for surubim juveniles.