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.

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.

Host oyster tissue extracts modulate in vitro protease expression and cellular differentiation in the protozoan parasite, Perkinsus marinus.

Perkinsus marinus is responsible for a chronic disease (Dermo) of the Eastern oyster, Crassostrea virginica. In order to simulate the in vivo environment more closely, a chemically defined medium (JL-ODRP-3) was supplemented with tissue homogenate extracts or plasma from oysters possessing varying degrees of susceptibility to P. marinus infection. In media supplemented with extracts from highly susceptible oysters (C. virginica), P. marinus cells secreted elevated amounts of a set of low molecular weight serine proteases (LMP: 30-45 kDa) as assessed by enhanced digestion within gelatin-substrate SDS-PAGE gels. Oyster species of low susceptibility (C. gigas and C. ariakensis) did not exhibit this ability to upregulate P. marinus LMP expression. Oyster extract supplementation also led to pronounced changes in P. marinus cellular morphology, such that the cells were comparable to those observed within naturally infected oysters.

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.

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.

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.

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.

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.

Heuristic models of the intermonomeric disulfide bonding process.

The assembly of fully disulfide bonded trout Immunoglobulin M (IgM) was modeled heuristically. The basic mechanism was assumed to be the sequential formation of single disulfide bonds (DSB), linking two monomeric subunits. Biochemical details of the mechanisms of bond formation were not considered. The progression of bonding within each tetramer was described entirely in terms of the rates of bond formation. It was further assumed that the sequence of DSB formation from non-covalently associated tetramers to fully bonded tetramers progressed by the formation of one bond at a time. The experimentally observed ratios of covalently-linked subunits within the tetramers were compared with the values predicted by the models. The results of these analyses suggest that the final stages of trout Ig assembly (the DSB formation between monomeric subunits) must occur in intracellular compartments late in the secretory process itself, thereby yielding incompletely crosslinked tetramers. Further, it would appear that the rate of disulfide bond formation within a tetramer may be accelerated by the presence in that tetramer, of previously incurred, intermonomeric DSBs.

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.

The recombination activating gene 2 (RAG2) of the rainbow trout Oncorhynchus mykiss.

We have previously described the isolation and expression of RAG1 in trout to provide an initial understanding regarding the tissues involved in V(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical of RAG2 sequences. The proximity of RAG1 and -2 within this teleost is similar to that found in other vertebrates. The genes are convergently transcribed and share a 3' untranslated (UT) region [2. 8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6 - 8 kb). The entire 3' UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for both RAG genes. Northern blot analysis of one-year-old trout demonstrated strong expression of RAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of both RAG1 and -2 in the thymus followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both genes are expressed in embryos beginning at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based upon RAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of the RAG genes as well as that of the immune system itself.

The recombination activation gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis.

The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and -T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue- and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

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.

Effectiveness of an oral enteric coated vibrio vaccine for use in salmonid fish.

An oral enteric-coated Vibrio anguillarum vaccine was developed by initially coating lyophilized bacteria onto 0.9 mm diameter dextrose sugar beads followed by an Eudragit L-30D coat to serve as enteric protection. Vaccine efficacy was determined by both an in vivo challenge with live pathogen and measurements of serum and mucus antibody levels by ELISA. Survival rates after challenge were 80.3%, 83.3% and 70.3% among the vaccine group, positive and negative controls respectively. Serum and mucus antibody levels were found to be significantly greater in the vaccine group (p less than 0.01). In cases where equivalent survival among tested groups is observed, determination of antibody titer by ELISA may be a preferable indicator of vaccine efficacy.

Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol).

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.

Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57.

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.