Simultaneous quantitative analysis of multiple protein species within a single sample using standard scanning densitometry.

It is often desirable, when conducting Western blot analyses, to accurately quantify the relative expression of multiple target proteins in a single sample. A common problem occurs: however, when the target proteins vary beyond the linear range of the detection system; thus precluding accurate densitometric analysis for all samples. For example, analysis of teleost immunoglobulin structure under non-reducing but denaturing conditions, yields multiple, differentially polymerized forms (redox forms) within a single sample, which can exceed single log differences in concentration, as visualized by chemiluminescent and X-ray film development. To resolve this difficulty an efficient technique has been developed that uses dilutions of a single sample, allowing accurate quantification of target proteins within their potentially unique and varied linear range of detection. Upon consideration of the respective dilution factor that yields an appropriate estimate, the multiple targets can be quantified. When the results from this technique are compared to other systems possessing more expansive linear ranges, the results obtained are comparable to within 1%. Thus, laboratories without access to more sensitive and costly densitometric instrumentation can still employ standard densitometric analysis to accurately quantify multiple targets in a single sample.

The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments.

This paper describes the development of a highly sensitive TNT immunosensor consisting of a highly specific monoclonal antibody coupled with a prototype fluorescence-based detector system (KinExA Inline Biosensor, Sapidyne Instrument Inc). The antibody developed possesses a high affinity for TNT (association constant, aK 8.2) with minimal cross reactivity with other compounds such as tetryl, 2,4-dinitrotoluene and 2-amino-4,6-dinitrotoluene. This system provides sample assessment within 160 s from source acquisition and possesses sensitivity for TNT of 0.05 microg/L in ground water. The sensor can be regenerated in 8 min, allows a minimum of 40 repeated readings, and has a standard error of 0.1-0.4% between repeat readings. The fluidics and software allow samples to be obtained from up to eight different sources allowing the user to examine the stratification of the pollutant in the water column. We believe that this immunosensor can be used to rapidly assess trace levels of TNT in environmental water samples.

Vibrio anguillarum antigen stimulates mitogenesis and polyclonal activation of salmonid lymphocytes.

An antigen preparation of Vibrio anguillarum, a salmonid pathogen, acts as a potent in vitro mitogenic stimulator of splenic and pronephric (anterior kidney) lymphocytes from coho salmon (Oncorhynchus kisutch), chinook salmon (O. tshawytscha) and rainbow trout (Salmo gairdneri). This antigen (VA) is comparable in its mitogenic activity to Concanavalin A (Con A), Escherichia coli lipopolysaccharide (LPS), and phytohemagglutinin (PHA). VA gives peak mitogenic responses in coho five days after initiation of cell culture. VA also appears to be a nonspecific polyclonal activator as determined by the generation of plaque forming cells to trinitrophenyl (TNP) and fluorescein (FI) haptenic determinants. Chemical characterization is limited, but it appears that Vibrio LPS could be responsible for these activities.

Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires.

An O-antigen extract of the fish pathogen Vibrio anguillarum was used to stimulate plaque-forming cells (PFC) in coho salmon. The kinetics of the response demonstrates peak PFC per organ on day 16 post immunization for the spleen and anterior kidney. Significant PFC were also seen in thymic tissue. PFC responses were shown to be immunoglobulin mediated and antigen specific. Inhibition profiles of responding PFC populations demonstrate that the cells of the anterior kidney are more restricted in their recognition of antigen than those of the spleen. These data lend functional support to the concept of the hematopoietic nature of the anterior kidney of teleosts.

Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemical and cellular analysis of the B cell response.

In vivo and in vitro analyses of the antibody responses of rainbow trout (Oncorhynchus mykiss) confirmed the existence of immunological memory in this species. An enhanced in vitro secondary antibody response was found to be due strictly to an expansion of the antigen-sensitive precursor pool without a concomitant increase in clone size. In contrast to the development of immunological memory in mammalian species, there was no evidence for affinity maturation during the primary or secondary response. A distinct shift in the fine specificity profiles of the antibodies, however, did occur during the generation of the secondary response. Additionally, more than a single injection of the priming antigen, TNP-KLH was required to produce an enhanced in vitro response to this T-dependent antigen. However, a second priming injection was not required to produce an enhanced secondary response to the T-independent form of antigen, TNP-LPS. These results indicate that memory in trout may be due to a simple expansion of the antigen-specific precursor pool without many of the qualitative changes in antibody or B cell function associated with the expression of memory in mammals.

Polyclonal activation of salmonid B lymphocytes.

The process of in vitro polyclonal activation of coho salmon (Oncorhynchus kisutch) lymphocytes was examined with respect to the induction of mitogenesis, total immunoglobulin production, and the production of specific antibodies or plaque forming cells. These studies demonstrate that antigen specific stimulation of antibody production is not linked to mitogenic activity, or total immunoglobulin production, while the polyclonal activation of specific antibody production is closely linked to these functions. Stimulation of immunoglobulin production by phytohemagglutinin suggests that this mitogen may not be limited to T cell activation in salmonids or, alternatively, it may induce the production of lymphokines capable of polyclonally activating B cells. Further, fetal calf serum was found to cause production of large amounts of immunoglobulin in vitro without antigenic stimulation.

Ontogeny of IgM and IgM-bearing cells in rainbow trout.

We have studied the ontogenic development of immunoglobulin M (IgM) and of IgM-bearing cells in the rainbow trout, Oncorhynchus mykiss. Lymphocytes showing cytoplasmic IgM were first observed in embryos at 12 days before hatch (14 degrees C). At this stage, no cells positive for surface IgM were present. Lymphocytes bearing surface IgM were observed at 8 days before hatch (14 degrees C). Unfertilized trout eggs contained detectable amounts of IgM (11.2 +/- 2.6 micrograms/g of egg weight), indicating that transfer of IgM from mother to embryo can occur in salmonids. The levels of IgM from whole fish increase slowly after the appearance of intraembryonic cells that express surface IgM. The amount of IgM/g of tissue peaks around hatch, but this parameter shows lower values up to 2 months after hatch.

Cortisol mediated suppression of salmonid lymphocyte responses in vitro.

The suppressive activity of cortisol on the in vitro induction of coho salmon (Oncorhynchus kisutch) B cell activation was examined. Suppression was observed with splenic and pronephric (anterior kidney) derived lymphocytes. The kinetics of cortisol-induced suppression revealed distinct differences in the sensitivity of splenic and pronephric lymphocytes. Pronephric lymphocytes were only sensitive to cortisol early in the induction of the antibody response, whereas the splenic cells were sensitive to cortisol throughout the culture period. Addition of supernatants from antigen stimulated pronephric cultures completely restored the ability of pronephric lymphocytes to produce an antibody response, suggesting that this glucocorticoid-suppression may be mediated by inhibition of lymphokine production.

Coding sequences of the MHC II beta chain of homozygous rainbow trout (Oncorhynchus mykiss).

Six lines of homozygous rainbow trout (Oncorhynchus mikiss) from different genetic and geographical backgrounds have been produced as aquatic models for biomedical research by the chromosome set manipulation techniques of androgenesis and gynogenesis. Messenger RNA from spleens was extracted. and the MHC II B cDNA sequences, amplified by RT PCR, were cloned into plasmids. Sequences of the MHC II beta2 domains were highly conserved between the different plasmids from the same and different lines of trout. Most of the variability among sequences was found in the amino terminal half of the beta1 domain, which corresponds with the peptide binding region of the MHC II molecule. This diversity suggests that the different lines of trout may exhibit differences in immune response. Rainbow trout MHC II B sequences were similar to the MHC II B sequences of the Pacific salmon (O. gorbuscha, O. tshawytscha, O. nerka, O. miasou, O. kisutch). Southern blot analysis performed on the restricted DNA of the OSU and Hot Creek trout, and the doubled haploid progeny produced by androgenesis from OSU x Hot Creek hybrids indicates that two distinct genes encode the MHC II B sequences and that these genes are unlinked.

Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha).

We examined the effects of acute stress on the immune system and disease resistance of juvenile chinook salmon (Oncorhynchus tshawytscha) in laboratory and clinical trials. Immune function, as measured by the ability of lymphocytes from the anterior kidney to generate specific antibody-producing cells (APC) in vitro, was depressed 4 h after stress, when plasma cortisol levels were highest. At the same time, resistance to the fish pathogen, Vibrio anguillarum, was also depressed. Compared with controls, plasma cortisol and APC of stressed fish were unchanged after 24 h, and disease resistance was enhanced as evidenced by higher survival rate and longer mean time to death of mortalities. After 7 days, even though numbers of APC were depressed, plasma cortisol concentration and disease resistance did not differ from controls. This pattern was generally the same, independent of the type of stress applied: i.e. being held out of water in a dipnet for 30 s, manipulation during hatchery operations for 4 h, or transportation for 9 h. These and earlier findings suggest that similar endocrine-immune interactions operate in the mammalian and salmonid systems during acute stress.

Salmonid B lymphocytes demonstrate organ dependent functional heterogeneity.

The passive hemolytic plaque assay was used to examine the functional heterogeneity of antibody producing cells in salmonid immune organs. In this study, the antibody response to Vibrio anguillarum antigens was induced by the injection of a somatic antigen extract. This antigen was also coated onto sheep red blood cells (SRBC) for plaque forming cell (PFC) determination. Previous studies have demonstrated that this response is antibody dependent and antigen specific (Kaattari and Irwin, 1985). The present study was focused upon the heterogeneity of antibody producing cells that arise in the spleen, anterior and posterior kidney of immunized coho salmon (Oncorhynchus kisutch). The functional heterogeneity of lymphocytes was assessed by histogram analysis of the antigen inhibition profiles of the plaque forming responses. These analyses have revealed that the anterior kidney lymphocytes possess a much more restricted profile of antibody specificities than do lymphocytes from the posterior kidney or spleen. These data suggest that B cell repetoires differ among the immune organs of salmonids.

Primary in vitro stimulation of antibody production by rainbow trout lymphocytes.

Trinitrophenylated (TNP) forms of E. coli lipopolysaccharide (LPS) and keyhole limpet hemocyanin (KLH) were used to produce antigen specific plaque-forming cell (PFC) responses with rainbow trout (Salmo gairdneri) splenocytes from unprimed fish in vitro. The culture system that was developed is described and characterized with respect to the kinetics and dose responses for both the haptenated and unhaptenated forms of the carriers. The induction of the PFC response to TNP-LPS was inhibited with TNP-lysine. Exposure to graded levels of gamma-radiation demonstrated a low dose augmentation of the PFC response with both antigens. Antigen addition experiments reveal that both antigens appear to stimulate the same population of antibody-producing B lymphocytes.

Elevated temperature treatment as a novel method for decreasing p57 on the cell surface of Renibacterium salmoninarum.

Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease.

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salmonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4C11, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a monomer that retains salmonid leucocyte agglutinating activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA, the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer. Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.

Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum.

A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.

Monoclonal antibodies against pili of serologically distinct Bacteroides nodosus.

Several monoclonal antibodies (McAbs) against pili of Bacteroides nodosus were examined to determine their reactivity with 11 different serotypes. One McAb was identified by enzyme-linked immunosorbent assay (ELISA) analysis that bound to nine of the 11 serotypes and another that bound to the remaining two serotypes tested. In addition, some McAbs demonstrated specificity for a single serotype, while others displayed specificities for up to five other serotypes. Comparison of immunoblot analysis with the ELISA revealed that the former method was not as sensitive in that all McAbs positive by the ELISA, were not positive by immunoblot. Possible explanations of these findings are discussed. There appear to be several antigenic determinants on B. nodosus pili and considerable sharing of these determinants between pili types. The 11 serotypes analyzed by the McAbs in this report are representative of all 20 US serotypes as well as the A-set and D-set categories of Australia. Therefore, the two epitopes recognized by two of the McAbs reported herein encompass all of the currently characterized B. nodosus serotypes and may provide a basis for bivalent vaccines efficacious for all types of B. nodosus induced footrot in sheep.

Induction of bovine plaque-forming cells in vitro.

A culture system was developed for the in vitro inoculation of bovine splenic lymphocytes with sheep RBC and trinitrophenylated proteins. The addition of pokeweed mitogen to the cultures was necessary for the induction of plaque-forming cells to the antigen. This culture system required the use of autologous serum and the addition of mitogen.

The evolving story of Mycobacterium tuberculosis clade members detected in fish.

Advances in molecular analyses have permitted documentation of an increasing spectrum of mycobacteria infecting fish. Although some of these mycobacteria are not closely related, several species belong to the Mycobacterium tuberculosis clade. One member of the clade, M. marinum, is well known as an agent of piscine mycobacteriosis. Three other clade species, M. shottsii, M. pseudoshottsii and M. 'chesapeaki', have recently been identified as predominant disease agents in a widespread, continuing epizootic in wild striped bass of the Chesapeake Bay. A fifth clade member, M. ulcerans, has recently been indirectly detected in wild, African cichlid fish. As M. ulcerans is the third most common human mycobacterial infection worldwide, even such indirect evidence of M. ulcerans in fish must be more thoroughly investigated. Complicating the differentiation of these clade members is the growing recognition of intraspecies and interspecies variation in phenotypes, genes and virulence. Thus, researchers must be aware of the variety of piscine isolates within the M. tuberculosis clade. This review summarizes the methods of detection and differentiation for this important group of mycobacteria.

Immunization with bacterial antigens: bacterial kidney disease.

Bacterial kidney disease has consistently resisted attempts to control it by prophylactic immunisation. Although successful vaccines have been produced to a number of Gram-negative fish pathogens, the relatively simple method used in these cases have not been successful with Renibacterium salmoninarum. A more circumspect and thorough knowledge of the biological function of R. salmoninarum antigens must be obtained. Also required is a more precise understanding of the role of regional immunity in effective prophylaxis. Aspects of R. salmoninarum's biology provide a provocative challenge to the vaccinologist. Its residence in, and apparent commandeering of the macrophage, indicate that a vigorous cell-mediated response will probably be required to generate protective immunity. Its most biologically potent secreted product, p57, appears to be an aggressin. Further, p57 has the capability of frustrating immunoprophylaxis by either misdirecting the immune response, or by preventing its induction. Many immunization studies have used injection immunization and challenge protocols. It now appears that alternative routes of immunization which had been considered less protective (i.e. oral immunization) may not only be more efficacious, but may be the only route that does not lead to a misdirected and possibly pathological immune response. Also, the general reliance on serum antibodies as the only means to assess immunity is fraught with difficulties, particularly with pathogens such as R. salmoninarum. Recent advances in the analysis of cellular immunity will be a great aid in the design of future vaccines.

Concanavalin A supernatant recruits antigen-insensitive IgG memory B lymphocyte precursors into an antigen-sensitive precursor pool.

Spleen cells from mice primed to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) generate IgG anti-TNP memory responses when stimulated in vitro with either thymus-dependent (TD) or thymus-independent (TI) forms of the hapten. When supernatants from Con A-stimulated spleen cells (Con A Sup) were added to such secondary cultures the TI responses to DNP-dextran or TNP-T4 were augmented; the TD response to TNP-KLH was suppressed. Passage over Sephadex and addition of alpha-methyl-D-mannoside did not inhibit augmentation by Con A Sup, indicating that augmentation did not result from direct action of the lectin on the responding cells. Augmentation occurred equally well in cultures that had been depleted of T cells by treatment with anti-Thy-1.2 and complement. Limiting dilution analyses revealed that Con A Sup increased the frequency of TI-responding precursors approximately threefold while causing a concomitant decrease in TD-responding precursors. To determine the relationship of the additional TI precursors and those normally detected in the absence of Con A Sup, the TI-responding IgG precursors were first eliminated through selective suicide by using DNP-dextran plus BUdR and light treatment; subsequently no TI-responding IgG PFC could be detected to DNP-dextran unless Con A Sup was also added. The data suggest Con A Sup may augment the TI responses to DNP-dextran and TNP-T4 by recruiting additional precursors from a memory cell pool formerly insensitive to these forms of antigen.