Mortality event involving larvae of the carpet shell clam Ruditapes decussatus in a hatchery: isolation of the pathogen Vibrio tubiashii subsp. europaeus.

Diseases caused by bacteria belonging to the genus Vibrio are a common, as yet unresolved, cause of mortality in shellfish hatcheries. In this study, we report the results of routine microbiological monitoring of larval cultures of the carpet shell clam Ruditapes decussatus in a hatchery in Galicia (NW Spain). Previous episodes of mortality with signs similar to those of vibriosis affecting other species in the installation indicated the possibility of bacterial infection and led to division of the culture at the early D-veliger larval stage. One batch was cultured under routine conditions, and the other was experimentally treated with antibiotic (chloramphenicol). Differences in larval survival were assessed, and culturable bacterial population in clams and sea water was evaluated, with particular attention given to vibrios. Severe mortalities were recorded from the first stages of culture onwards. The pathogen Vibrio tubiashii subsp. europaeus was detected in both batches, mainly associated with larvae. Moreover, initial detection of the pathogen in the eggs suggested the vertical transmission from broodstock as a possible source. Experimental use of antibiotic reduced the presence and diversity of vibrios in sea water, but proved inefficient in controlling vibrios associated with larvae from early stages and it did not stop mortalities.

Phytoplankton production systems in a shellfish hatchery: variations of the bacterial load and diversity of vibrios.

AIMS: Outbreaks of disease caused by some Vibrio species represent the main production bottleneck in shellfish hatcheries. Although the phytoplankton used as food is one of the main sources of bacteria, studies of the associated bacterial populations, specifically vibrios, are scarce. The aim of the study was the microbiological monitoring of the microalgae as the first step in assessing the risk disease for bivalve cultures. METHODS AND RESULTS: Two phytoplankton production systems were sampled weekly throughout 1-year period in a bivalve hatchery. Quantitative analysis revealed high levels of marine heterotrophic bacteria in both systems throughout the study. Presumptive vibrios were detected occasionally and at low concentrations. In most of the cases, they belonged to the Splendidus and Harveyi clades. CONCLUSIONS: The early detection of vibrios in the microalgae may be the key for a successful bivalve culture. Their abundance and diversity were affected by factors related to the hatchery environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first long study where the presence of vibrios was evaluated rigorously in phytoplankton production systems and provides a suitable microbiological protocol to control and guarantee the quality of the algal cultures to avoid the risk of transferring potential pathogens to shellfish larvae and/or broodstock.

Vibrios in hatchery cultures of the razor clam, Solen marginatus (Pulteney).

Hatchery culture of the razor clam, Solen marginatus (Pulteney), has recently been developed in Galicia (NW Spain). However, recurrent episodes of mortalities of larval and post-larval cultures have been recorded during the course of various studies. The disease signs were similar to those described for other bivalve species in outbreaks caused by bacteria of the genus Vibrio. In this article, we present the results of microbiological monitoring of two batches of razor clams with different survival rates. All fermentative isolates were identified as members of the Splendidus clade within the genus Vibrio. Some of these isolates, identified as Vibrio splendidus-like, were clearly associated with the batch suffering mortalities, indicating their possible role as pathogens. Similar strains were found in the broodstock, suggesting vertical transmission of these bacteria. This is the first study of the microbiota associated with hatchery culture of S. marginatus, and the results will provide useful information for the optimization of a protocol for hatchery culture of this bivalve species.

Independent estimates of marine population connectivity are more concordant when accounting for uncertainties in larval origins.

Marine larval dispersal is a complex biophysical process that depends on the effects of species biology and oceanography, leading to logistical difficulties in estimating connectivity among populations of marine animals with biphasic life cycles. To address this challenge, the application of multiple methodological approaches has been advocated, in order to increase confidence in estimates of population connectivity. However, studies seldom account for sources of uncertainty associated with each method, which undermines a direct comparative approach. In the present study we explicitly account for the statistical uncertainty in observed connectivity matrices derived from elemental chemistry of larval mussel shells, and compare these to predictions from a biophysical model of dispersal. To do this we manipulate the observed connectivity matrix by applying different confidence levels to the assignment of recruits to source populations, while concurrently modelling the intrinsic misclassification rate of larvae to known sources. We demonstrate that the correlation between the observed and modelled matrices increases as the number of observed recruits classified as unknowns approximates the observed larval misclassification rate. Using this approach, we show that unprecedented levels of concordance in connectivity estimates (r = 0.96) can be achieved, and at spatial scales (20-40 km) that are ecologically relevant.

Differences in cell surface proteins of drug resistant and sensitive cell lines.

Lactoperoxidase catalyzed iodination of cell surface proteins has been used to compare theophylline and ouabain resistant variants with the drug sensitive parental cell lines. In cells showing contact inhibition a high molecular weight protein was observed to be preferentially labelled. In contrast this was not observed for non contact inhibited cells. These results support a previously suggested correlation between growth regulation and the exposure of this cell surface protein.

A study of some molecular and kinetic properties of two tRNA methyltransferases from mouse plasmocytoma.

A tRNA(adenine-1)methyltransferase and a tRNA(cytosine-5)methyltransferase have been partially purified from mouse plasmocytoma MOPC 173. Their apparent Mr are 200000-230000 and 110000-140000, respectively, as determined by gel filtration and density gradient centrifugation. Both enzymes exhibit maximum activity in the presence of high concentrations of monovalent cations (0.175 M and 0.25 M KCl, respectively) and in the absence of magnesium. Their kinetic constants have been determined at various KCl concentrations, with several tRNA species as substrates. These constants may differ by more than one order of magnitude, depending upon the substrate used, and they are strongly dependent upon the ionic concentration as well. The possibility that the tRNA(adenine-1)methyltransferase from mouse plasmocytoma is different from the homologous enzyme purified from a normal rat tissue [Glick, J. M. and Leboy, P. S. (1977) J. Biol. Chem. 252, 4790-4795] is discussed.

Intracellular distribution of antibodies in rat lymph node cells during primary response. A kinetic study.

The kinetics of appearance of immunoglobulin- and antibody-containing cells in rat lymph nodes was studied by immunoenzymatic techniques between days 12 and 90 after a single injection of horse-radish peroxidase. Three distinct phases appeared during the primary repsonse. Between days 8 and 17, essentially no antibody-containing cells were detectable, while non-specific immunoglobulin-containing cells amounted to up to 9% of the total cell population. The peak of antibody-forming cells was observed between days 18 and 24, and their number decreased slowly during the last phase. Days 18-20 were marked by a sharp peak of a distinct category of cells containing antibodies in a limited area of their cytoplasm. One day before this event, the percentage of immunoglobulin-forming cells decreased drastically, and remained very low throughout the response. These results are discussed on the basis of a previously published model, which proposes that non-specific immunoglobulin-containing cells are precursors to antibody-containing cells, and that the transformation between these two categories of cells involves a stage where antibody synthesis is restricted to a few endoplasmic reticulum vesicles.

Genetic control of igG2a production in the response to sheep erythrocytes.

A low IgG2a response in B10 mice during the primary response to sheep red blood cells (SRBC) is described. Analysis of the response in B10 × BALB/c hybrid progenies and in congenic strains indicates that this low response is a dominant phenotype placed under the control of a single Mendelian gene or a group of closely linked genes. This gene(s) is neither linked to CH allotypes orH2 haplotypes, nor is it sex-linked. It can be considered as an isotype- and antigenspecific regulatory gene of the immune response.

Differences de specificite entre les methylases de myelome et de foie vis-a-vis du tRNA d'E. coli K 12.

This paper describes an attempt to find a difference between the patterns of methylation of E. coli tRNA by extracts of two mouse tissues. Two samples of tRNAs, methylated in two separate experiments with extracts of myeloma and of liver in presence of either 14C or 3H S-Adenosyl-L-Methionine, were pooled and fractionated together on a RPC column. The results show a difference in the specificities of the two extracts. Chromatography on DEAE Sephadex suggests that the tRNA Met is methylated by the enzymes on the myeloma, while enzymes from liver react very little, if at all, with that particular tRNA species. Studies have been undertaken in order to find out whether similar differences can also be demonstrated in homologous systems.

Immune response to the p-azobenzenearsonate (ABA)-GAT conjugate: role of I region genes in the selective activation of ABA-specific or GAT-specific T helper cells.

Immunization of GAT non-responders with ABA-GAT leads to the activation of ABA-specific T cells. These hapten specific T cells are Lyt-1+2- helper cells capable of inducing anti-ABA antibody responses in vivo or B cell activation in vitro. However, their activation does not modify the GAT non-responder phenotype. Immunization of GAT responder mice with ABA-GAT activates GAT-specific T cells, which can help anti-ABA and anti-GAT antibody responses. Since the responder and non-responder strains used in these experiments differ only in the alleles present in the I region, the results suggest that the selective activation of hapten- or carrier-specific T cells is controlled by I region genes. Yet sensitization of the two strains with ABA-KLH or ABA-Tyr induces KLH-specific or ABA-specific T cells, respectively. This provides further evidence that the use of an immunogenic carrier prevents the expression of the hapten-specific T cell clones present in the repertoire of both responder and non-responder animals. Macrophages from responder animals pulsed with ABA-GAT can present ABA and GAT determinants to T cells. Thus, the absence of ABA-specific T cells in responders primed with ABA-GAT and their presence in GAT non-responders reflects a competition between hapten- and carrier-specific T cells and not an epitope selection by macrophages. We discuss the significance of the results in terms of Ir genes determining the self-plus-antigen-specific T cell repertoire rather than controlling antigen presentation by macrophages.

[An hypothesis for the process of selection at the subcellular level leading to the appearance of new clones]. / Une hypothèse sur un processus de sélection au niveau subcellulaire conduisant à l'émergence de nouveaux clones.

According to the hypothesis we propose, the stimulation of one lymphocyte by an antigen induces the simultaneous expression of a great diversity of immunoglobulins of different specificities. Each molecular species is associated with the corresponding mRNA within a subcellular structure: the ergastoplasmic cisterna. It has been shown that in some responding lymphocytes at an early stage of the immune response a few such cisternae are loaded with antibodies while most of the cisternae are synthesizing non specific immunoglobulins. The main point of our proposal is that the selective action of antigen bears on these cisternae and that the mRNA corresponding to the immunoglobulins fitting best to the antigen is transcribed to DNA which is then inserted into the genome. This cell and its progeny become thereafter a monospecific clone submitted to regulation as an element of the network.

Inhibition of murine plasmocytoma tumours with antibody activity by their respective specific antigens.

Three murine plasmocytoma tumour which secrete specific antibodies have been studied for the effect of specific antigens (pneumococcal C polysaccharide and dinitrophenylated bovine gamma globulin) on the growth of these tumours in vivo. In each case, the effect of the specific antigen was to inhibit the growth of that tumour which synthesized the specific antibody. Low molecular weight haptens had no effect on tumour growth. We suggest that this antigen specific growth inhibitory effect is a function of the antigen's binding to membrane bound antibody resulting in defective membrane function.

Mechanism of immune stimulation and tolerance based on an hypothesis on the cell division mechanism directed by the cell membrane.

The binding of the antigen to membrane receptors is suggested to modify the structure of the membrane and consequently the activity of some of its enzymes and the flow of metabolites. The nature of the effects may be a function of the quantity of the interacting antigen.

Immune response to the p-azobenzenearsonate-L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) conjugate. III. Mechanisms of Ir gene-controlled phenotype conversion.

Immunization of GT (random copolymer of L-glutamic acid51-L-tyrosine49) nonresponder animals with p-azobenzenearsonate (ABA) GT conjugates elicits an antibody response to both ABA and GT epitopes which is induced by ABA-specific T helper cells. Expression of these hapten-specific helpers is under the control of an I region gene which also regulates the proliferative T cell response to ABA. Conversion of the unresponsive phenotype to GT is, therefore, dependent on the ABA Ir gene and escapes the influence of the GT-specific I region-controlled suppressive pathway. Studies on the influence of ABA/polymer coupling ratio on T and B cell responses suggest that ABA-specific T cells, like conventional carrier-specific helpers, require linked interactions with B lymphocytes to provide helper signals. GAT (terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10) nonresponder animals immunized with ABA-GAT conjugates also develop an antibody response to ABA which is induced by ABA-specific T helper cells. Comparison of antibody affinity, specificity, isotypes and idiotypes in different mouse strains demonstrates that hapten-specific helper cells stimulate antibody responses to ABA which are qualitatively similar to those induced by GAT-specific helpers. However, ABA-specific helper cells do not permit the conversion of the I region gene-controlled nonresponder phenotype to GAT. The data suggests that high ABA density, which is required for optimal ABA help expression, extinguishes the immunogenicity of GAT determinants at both T and B cell levels.

Mucosal immunity in primary glomerulonephritis: II. Study of the serum IgA subclass repertoire to food and airborne antigens.

IgA specific for 7 food and 6 airborne antigens were sought in the serum of 30 adult patients with IgA mesangial nephropathy (IgA GN), 23 with membranous nephropathy (MGN), 20 with idiopathic nephrotic syndrome (INS), 11 with membranoproliferative GN (MPGN) and 22 healthy controls by means of an enzyme-linked immunoassay. The IgA subclass was determined using monoclonal antibodies. Increased levels of IgA specific for gliadin, bovine serum albumin (BSA), ovalbumin, lysozyme and alpha-lactalbumin were found in IgA GN, while increased levels of IgA to BSA, ovalbumin, lysozyme and alpha-lactalbumin were observed in MGN; IgA specific for alpha-lactalbumin were increased in INS, and MPGN patients had reduced levels of IgA to BSA and increased levels of IgA to beta-lactoglobulin and alpha-lactalbumin. These specific IgA to food antigens were restricted to the IgA1 subclass. Patients with IgA GN had significantly increased levels of IgA specific for Dermatophagoides pteronyssinus (DP) and Dactil while the MGN group showed increased levels of IgA specific for DP, feathers, Dactil and mold. INS patients had increased levels of IgA specific for DP, feathers, Dactil, mold and dog hairs, while MPGN patients had increased levels of IgA specific for feathers, Dactil, dog hairs and mold. All these specific IgA to airborne antigens were restricted to the IgA1 subclass. Patients with the four types of primary glomerulonephritis had decreased IgA specific for cat hairs which were of both the IgA1 and IgA2 subclasses. We conclude that anomalies of the IgA repertoire to environmental antigens are also encountered in primary glomerulonephritis other than IgA GN.

Secretory IgA are elevated in both saliva and serum of patients with various types of primary glomerulonephritis.

Secretory immunoglobulin A (IgA) was determined by means of an enzyme-linked immunosorbent assay (using as capture antibody an MoAb specific for secretory component) in saliva and serum from 46 patients with IgA mesangial nephritis (IgAGN), 36 with an idiopathic nephrotic syndrome (INS), 30 with an idiopathic membranous nephropathy (MGN) and 40 healthy controls. Secretory IgA levels were elevated in both saliva and serum of patients with primary glomerulonephritis (P < 0.05; Mann-Whitney test) regardless of the histological type of the primary glomerulonephritis. Salivary IgA1 and IgA2 levels were increased in the saliva of patients with IgAGN, INS and MGN (P < 0.05; Mann-Whitney test). The monomeric/total IgA ratio, and interferon-gamma and soluble IL-2 receptor levels, in saliva did not differ between the patients and controls (P > 0.05; Mann-Whitney test). We conclude that the mucosal immune system is activated in forms of glomerulonephritis other than IgAGN.

Mucosal immunity in primary glomerulonephritis. III. Study of intestinal permeability.

To study the specificity of gut hyperpermeability in primary glomerulonephritis, we investigated intestinal permeability by means of 51Cr-EDTA testing in 20 healthy individuals and in 30 patients with Immunoglobulin A nephropathy (IgA GN), 25 with idiopathic nephrotic syndrome (INS) and 20 with immune complex glomerulonephritis (IC-GN; membranous+membranoproliferative glomerulonephritis). Gut permeability was statistically increased in each patient group versus the controls [controls: 2% (0.4-3.9); IgA GN: 3.25% (0.7-17.70); INS: 3.71% (0.82-10); IC-GN: 3.40% (0.30-16); results are median (range); p < 0.005, nonparametric Mann-Whitney test]. An increase in intestinal permeability exceeding the upper limit of control values (95th percentile) was observed in 36% of IgA GN, 60% of INS and 50% of IC-GN patients. We conclude that intestinal permeability is frequently increased in primary glomerulonephritis and may also be increased in types of glomerulonephritis other than IgA GN.