Isolation and characterization of an antigen from the fish pathogen Moritella viscosa

Aims: Moritella viscosa is a Gram‐negative psychrophilic bacterium that causes winter ulcer disease in farmed fish. The aim of the study was to describe an outer membrane protein of roughly 20 kDa in pathogenic M. viscosa and to compare the coincident protein of strains isolated from different fish species and geographical locations. Methods and Results: The protein was isolated from a pathogenic strain of M. viscosa. An oligopeptide sequence obtained with MS/MS analysis showed homology to Escherichia coli OmpA and Neisseria surface protein A. The protein was named Moritella viscosa outer membrane protein 1 (MvOmp1), and sequence analysis confirmed that it is an integral membrane protein consisting of eight antiparallel β‐strands, three short periplasmic turns and four long hydrophilic extracellular loops. The encoding gene, mvomp1, was fully sequenced in nine strains representing different serotypes and phenotypes. The results revealed some differences in the extracellular loops between strains. The mvomp1 gene was cloned and expressed in E. coli, and the recombinant product was recognized by anti‐M. viscosa polyclonal antisera. Conclusions: The results indicate that MvOmp1 is a major protective antigen of M. viscosa. Significance and Impact of the Study: The results open up possibilities for use of the protein as a part of a subunit vaccine in the future.     

Atlantic salmon bath challenged with Moritella viscosa – Pathogen invasion and host response

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80–110 g) were exposed to a bath challenge dose of 7 × 10⁵ cfu ml^-1 for 1 h at 8.9 °C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis.Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1β, C3, ISG15 and CD83 were studied. Increased expression of IL1β and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the host's immune system by suppressing relevant immune responses.     

Effects of water-level regulation on the nearshore fish community in boreal lakes

The fish community in the littoral areas of eight regulated lakes and five reference lakes in Finland was sampled by electrofishing. No significant effect of winter drawdown on species richness was recorded across lakes. Total fish density for stony bottoms of the regulated and reference lakes averaged 19.3 and 32.7 individuals per 100 m², respectively, but this difference was not statistically significant. The combined proportion of littoral fish species, including minnow (Phoxinus phoxinus), bullhead (Cottus gobio), alpine bullhead (Cottus poecilopus), nine-spined stickleback (Pungitius pungitius), and stone loach (Barbatula barbatula), supplemented with zoobenthos feeders ruffe (Gymnocephalus cernuus) and young burbot (Lota lota), was much lower in the regulated lakes than in the reference lakes. Besides winter drawdown, other variables, such as nutrient level and lake size, affected the fish community. Guest editors: K. M. Wantzen, K.-O. Rothhaupt, M. M?rtl, M. Cantonati, L. G.-Tóth & P. Fischer Ecological Effects of Water-Level Fluctuations in Lakes     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Typing of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> of fish and human diarrhoeal origin by outer membrane proteins and lipopolysaccharides

A study was undertaken to discriminate the strains of Aeromonas hydrophila isolated from fish and diarrhoeal samples by SDS-PAGE analysis of outer membrane proteins (OMPs) and lipopolysaccharides (LPSs). Common bands at 47 kDa positions for OMPs and at 31–38 kDa for LPSs were observed. No strain of A. hydrophila from clinical or fish samples was found identical in either OMPs or LPSs profile.     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Eco-morphological patterns of the sagitta of Antarctic fish

Morphology and morphometry of the sagittae otolith were studied in pelagic and mesopelagic fish. The shape, margins and rostrum of four groups of otoliths from several species were analyzed: group 1 (pelagic fish associated with the under ice cover N = 42), group 2 (pelagic fish associated with water offshore N = 9), group 3 (mesopelagic fish associated with extensive vertical migration N = 57) and group 4 (mesopelagic fish associated with short vertical migration N = 54). E (maximum width of the sagitta /maximum length of the sagitta %), R (rostrum length (RL)/maximum length of the sagitta %) and S (sulcus area (SS)/otolith area (OS) %) indexes were calculated for each species. Sagittae of pelagic groups (1 and 2) showed the smallest sagitta dimensions in relation to the total length of the fish, in this group the sagitta shape is variable. Sagittae of mesopelagic fish (groups 3 and 4) showed variable shape and edges. The shape in group 4 was polygonal and these species have more width than length. Statistical analysis showed significant differences in the E, R and S indexes. These results were compared with other 19 species, belonging to six families, taken from a publisher-edited literature. E, R and S-values could be used to characterize the sagittae of the Antarctic fish and could be considered as a useful tool for fish ecology studies.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.     

Trophic interactions between native and introduced fish species in a littoral fish community

The trophic interactions between 15 native and two introduced fish species, silverside Odontesthes bonariensis and rainbow trout Oncorhynchus mykiss, collected in a major fishery area at Lake Titicaca were explored by integrating traditional ecological knowledge and stable‐isotope analyses (SIA). SIA suggested the existence of six trophic groups in this fish community based on δ¹³C and δ¹⁵N signatures. This was supported by ecological evidence illustrating marked spatial segregation between groups, but a similar trophic level for most of the native groups. Based on Bayesian ellipse analyses, niche overlap appeared to occur between small O. bonariensis (<90 mm) and benthopelagic native species (31·6%), and between the native pelagic killifish Orestias ispi and large O. bonariensis (39%) or O. mykiss (19·7%). In addition, Bayesian mixing models suggested that O. ispi and epipelagic species are likely to be the main prey items for the two introduced fish species. This study reveals a trophic link between native and introduced fish species, and demonstrates the utility of combining both SIA and traditional ecological knowledge to understand trophic relationships between fish species with similar feeding habits.     

Serological Reactivity of Humans to a Vibrio cholerae Common Antigen

Over the course of seven pandemics, Vibrio cholerae serotypes have varied. In 1992 the appearance of a new serotype, O139 Bengal, began the eighth cholera pandemic. Several new O139 antigens have been identified, yet a common V. cholerae antigen has not been described. In this study, a monoclonal antibody specific against an 18.7-kDa outer membrane antigen reacted in dotblot analysis with 292 epidemiologically diverse V. cholerae isolates including O1, non-O1, and O139 serotypes. Serum collected from volunteers experimentally challenged with V. cholerae O139, and rabbit antisera to V. cholerae O1, were reactive with the 18.7-kDa antigen by Western immunoblot. This is the first report that the 18.7-kDa antigen is present in V. cholerae O139. Received: 11 August 1997 / Accepted: 22 September 1997     

Impact of fish predation on cladoceran body weight distribution and zooplankton grazing in lakes during winter

1. It is well accepted that fish, if abundant, can have a major impact on the zooplankton community structure during summer, which, particularly in eutrophic lakes, may cascade to phytoplankton and ultimately influence water clarity. Fish predation affects mean size of cladocerans and the zooplankton grazing pressure on phytoplankton. Little is, however, known about the role of fish during winter. 2. We analysed data from 34 lakes studied for 8–9 years divided into three seasons: summer, autumn/spring and winter, and four lake classes: all lakes, shallow lakes without submerged plants, shallow lakes with submerged plants and deep lakes. We recorded how body weight of Daphnia and then cladocerans varied among the three seasons. For all lake types there was a significant positive correlation in the mean body weight of Daphnia and all cladocerans between the different seasons, and only in lakes with macrophytes did the slope differ significantly from one (winter versus summer for Daphnia). 3. These results suggest that the fish predation pressure during autumn/spring and winter is as high as during summer, and maybe even higher during winter in macrophyte‐rich lakes. It could be argued that the winter zooplankton community structure resembles that of the summer community because of low specimen turnover during winter mediated by low fecundity, which, in turn, reflects food shortage, low temperatures and low winter hatching from resting eggs. However, we found frequent major changes in mean body weight of Daphnia and cladocerans in three fish‐biomanipulated lakes during the winter season. 4. The seasonal pattern of zooplankton : phytoplankton biomass ratio showed no correlation between summer and winter for shallow lakes with abundant vegetation or for deep lakes. For the shallow lakes, the ratio was substantially higher during summer than in winter and autumn/spring, suggesting a higher zooplankton grazing potential during summer, while the ratio was often higher in winter in deep lakes. Direct and indirect effects of macrophytes, and internal P loading and mixing, all varying over the season, might weaken the fish signal on this ratio. 5. Overall, our data indicate that release of fish predation may have strong cascading effects on zooplankton grazing on phytoplankton and water clarity in temperate, coastal situated eutrophic lakes, not only during summer but also during winter.     

Intranasal immunization of the combined lipooligosaccharide conjugates protects mice from the challenges with three serotypes of Moraxella catarrhalis

Background

There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection.

Methods

Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations.

Results

Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy.

Conclusions

The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses.     

Bactericidal Monoclonal Antibody Against <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis> Lipooligosaccharide Cross-Reacts with <Emphasis Type="Italic">Haemophilus Spp.</Emphasis>

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

Effects of Moritella viscosa antigens on pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) cell line (SHK-1)

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1β (IL-1β) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1β and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1β expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1β and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-β and IL-8 expression.     

Identification and characterization of myeloma-associated antigens in Trichinella spiralis

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4⁺, CD8⁺ T lymphocyte, and CD19⁺ B lymphocyte were significantly increased (P < 0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P < 0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P > 0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.