Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax.

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics.

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

Immunocompetence of juvenile chinook salmon against Listonella anguillarum following dietary exposure to polycyclic aromatic hydrocarbons.

Juvenile chinook salmon (Oncorhynchus tschawytscha) were fed a mixture of 14 polycyclic aromatic hydrocarbon (PAH) compounds that reflected the PAH composition of salmon stomach contents in an urban estuary of Puget Sound, Washington (USA). Following a 28-d dietary exposure, a standardized Listonella anguillarum challenge model was used to determine whether PAH exposure (16, 64, and 252 mg/kg wet wt feed) causes reduced disease resistance under the conditions examined in this study. To assess innate immunity, five replicate groups of fish per dose were acclimated for one week, exposed to a lethal concentration 60 of bacteria, and monitored for 14 d. In a parallel experiment, the effects of PAH exposure on the acquired immune response were examined by immersion vaccinating fish against L. anguillarum and allowing specific immunity to develop for three weeks prior to challenge. All mortalities were aseptically sampled to confirm L. anguillarum infections. No significant differences in fish length, weight, or coefficient of condition were observed. These controlled laboratory experiments suggest that dietary exposures to an environmentally relevant mixture of PAH compounds do not alter the immunocompetence or growth of juvenile chinook salmon.

Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L).

Data are presented that demonstrate the application of the neutral red retention assay (NRR) to monitor the effects of a bacterial inoculation on the haemocyte lysosomes of the European flat oyster Ostrea edulis, Pacific oyster Crassostrea gigas and scallop Pecten maximus. Bivalves were acclimated to three temperature regimes (5, 15 and 25 degrees C), at constant salinity for 7 days in the laboratory. Once baseline responses to acclimation temperature had been established, the effects of an in vivo inoculation on haemocyte lysosomal stability were assessed using the NRR assay. Lysosomal membrane stability was reduced in the presence of bacteria for all three species of bivalve, but destabilisation of C. gigas haemocyte lysosomes appeared to be most sensitive to the presence of the bacterium Listonella anguillarum. For all three bivalve species, the reduction in lysosomal stability appeared to be proportional to the growth of the bacterial inoculate. Using appropriate controls, the NRR assay was demonstrated to have great potential as a tool with which to make rapid initial assessments of the immune status of bivalve molluscs.

[Agglutination of hen egg-yolk immunoglobulins (IgY) against Salmonella enterica, serovar enteritidis]. / Aglutinación de inmunoglobulinas de yema de huevo de gallina (IgY) contra Salmonella enterica serovariedad enteritidis.

Two groups of 6 laying hens were used to produce IgY. In the vaccinated group (V), hens were injected by intramuscular route with two doses of a Salmonella enterica serovar Enteritidis bacterin at 20-day interval. In the control group (T) hens remained unvaccinated. Four IgY extractions were performed on the egg production of both groups. The first two extractions were carried out using the yolks obtained from the eggs produced during the 4th and 5th post-vaccination week (extracts 1V and 1T) and the other two using the ones from the 6th, 7th and 8th week (2V and 2T). Starting from the extracts 1V and 1T other products were obtained by freezing-thawing (1V-A and 1T-A) and simple (1V-B and 1T-B) or double (1V-C and 1T-C) flow capillary dialysis concentration. All these products were compared using an ELISA test specific for the detection of chicken antibodies against flagellar antigens of S. Enteritidis. In this test, V extracts were positive whereas T extracts were negative. The extract 1V was more positive than the extract 2V. The extract 1V-C was the most positive and was therefore selected to be used as an antiserum in the agglutination tests. This extract contained 1.9 g/dl of total proteins, 0.028 g/dl of triglycerides and 0.012 g/dl of cholesterol and showed an electrophoretic pattern characteristic of IgY. The 1T-C extract was used as a negative control in the agglutination tests. Slide somatic and tube flagellar agglutination tests were simultaneously carried out using both IgY extracts and a standard rabbit anti-Salmonella (IgG) sera. Overall 367 strains from the Enterobacteriaceae family were tested together with two other strains belonging to the Vibrionaceae family. The 1V-C extract specifically agglutinated S. Enteritidis strains in the same way as the rabbit sera. This extract also agglutinated other Salmonella strains antigenically related to S. Enteritidis. Salmonella which did not share somatic or flagellar antigens with S. Enteritidis, other different species of the Enterobacteriaceae family and the two strains of the Vibrionaceae family were all negative. None of the strains tested was agglutinated by the 1T-C extract. This paper show that it is possible to use specific IgY to identify S. enterica serovars. The more extended use of IgY for diagnostic purposes may be a convenient way to complement the current use of mammal polyclonal antibodies.

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens.

Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria (Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.

Oral cholera vaccines containing B-subunit-killed whole cells and killed whole cells only. I. Cross-reacting antigens of members of family Vibrionaceae and the vaccines.

Antigenic similarity between the oral cholera B subunit-whole cell (BS-WC) and whole-cell only (WC) vaccines and strains of the family Vibrionaceae was studied by crossed immunoelectrophoresis. A reference system consisting of Vibrio cholerae Inaba E1 Tor antigenic extract and homologous rabbit antiserum was applied in the study. The system was represented by 30 anodically migrating antigens forming distinct precipitation bands. Antigenic extracts of other members of the family Vibrionaceae showed the following numbers of cellular antigens shared in common with the reference system: Vibrio cholerae non-01-30, V. mimicus-23, V. fluvialis-15, V. parahaemolyticus-10, Aeromonas hydrophila-7, A. sobria-5, A. caviae-4 and Plesiomonas shigelloides-5 antigens. Homologous rabbit antiserum reacted with 11 antigens of BS-WC vaccine and 10 antigens of WC vaccine. The number of antigens which members of the family Vibrionaceae shared in common with those preserved in the WC component of the vaccines were as follows: Vibrio cholerae non-01, 7; V. mimicus, 5; V. fluvialis, 3; V. parahaemolyticus, 3; Aeromonas hydrophila, 2; A. sobria, 2; and A. caviae, 2; Plesiomonas shigelloides, 1. None of the strains produced an antigen reacting with anti-cholera toxin antibodies. The presence of common antigens in the vaccine and among members of family Vibrionaceae indicates that the oral cholera vaccine could stimulate immunity effectively against other members of the family.

Serotyping of Plesiomonas shigelloides strains with our own antigenic scheme. An attempted epidemiological study.

Thirty-four O and 19 H sera were used to test 169 strains of Plesiomonas shigelloides from several countries of three continents. The most frequent O serovar was O17 (O antigen identical with Shigella sonnei phase I) which occurred in the OH serovar combinations: O17:c, O17:d and O17:f. Some OH serovars were represented by a single strain; others, despite the small number of strains in hand, were evidently ubiquitous, having been isolated in mutually distant countries or at long time intervals. The source of our strains was most often human stools, diarrhoeal or collected at preventive examinations during epidemiological and ecological investigations, the droppings of animals (dogs, cats, pigs, sewer rats, water birds) and surface, sewer or aquarium water. The usefulness of serotyping in epidemiological investigation in the patients' environment was confirmed by a finding of two different serovars in one of our patients and her cat.

Comparison of enterobacterial common antigen from different species by serological techniques.

Enterobacterial common antigen (ECA) was isolated from a number of selected species (including Salmonella montevideo, Shigella sonnei and Plesiomonas shigelloides) using the extraction method described by Männel and Mayer [Eur. J. Biochem. 86, 361-370 (1978)]. ECA of all these species behaved identically in enzyme-linked immunosorption assay (ELISA) and in its inhibition using monoclonal anti-ECA antibodies. Immunoblotting showed a ladder-like pattern of at least 20 bands for all preparations tested. ECA modified at its lipid moiety (e.g. by phospholipases A2 and D or by mild acid hydrolysis) lost its coating capacity leaving, however, the serological reactivity as detected by inhibition assays intact. In contrast, reduction of the carboxylic groups of 2-acetamido-2-deoxy-D-mannopyranosyluronic acid destroyed the serological reactivity. Deacylated ECA was also not detectable in immunoblotting. Chemical reacylation restored the reactivity of deacylated ECA in ELISA and in immunoblot and thus proved the essential function of fatty acids for the physicochemical properties of the molecule. 2-Acetamido-2-deoxy-D-glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029.

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain. ECA of P. shigelloides showed the best-resolved 13C-NMR spectra, especially after the removal of non-stoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100 degrees C, 10 min). Subsequent 13C-NMR studies were therefore carried out with the mild-acid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA. The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide. The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatic and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides.

Experiences with serology of Plesiomonas shigelloides. 2. H-antigenic structure.

A total of 140 out of 144 motile Plesiomonas shigelloides strains were typable with the aid of a set of 15 H-antisera designated by letter symbols a through o. Commonly, strains of different O-serovars turned out to share the identical H-antigen and for some O-serovars it was typical to possess the certain type of H-antigen, as e.g. strains of serovar 017 which all have been to date characterized by the presence of H-antigens c, d or f. This set of 15 H-sera together with the previously described O-antigenic scheme of Plesiomonas may help to determine the frequency rates of OH serovars.

New O and H antigens and additional serovars of Plesiomonas shigelloides.

The antigenic scheme for Plesiomonas shigelloides described by Shimada and Sakazaki (1978) has been expanded to 107 serovars consisting of 50 O serogroups and 17 H antigens.

Experiences with serology of Plesiomonas shigelloides. 1. O-antigenic structure.

With a set of 30 O-antisera, O-antigens were identified in 80% of 158 Plesiomonas shigelloides strains. Only strains of one serovar (018) regularly contained capsular antigen that caused their inagglutinability in the live state. Two groups of serovars displayed some O-antigenic relationship: 03 and 029; 08, 011 and 012. Each serovar in either group possessed a specific O-antigen and "group-common" minor antigens, which were designated I, II and III. Serovar 017 possessed O-antigen identical with that of Shigella sonnei phase I; this serovar was the most frequent one. Some serovars seemed to be ubiquitous; this was indicated by their wide geographic distribution and findings in man, domestic and feral animals, and water.

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae.

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.

Common lipopolysaccharide specificity: new type of antigen residing in the inner core region of S- and R-form lipopolysaccharides from different families of gram-negative bacteria.

A new antigenic specificity, referred to here as common lipopolysaccharide (LPS) specificity, is described in the LPSs of gram-negative bacteria belonging to various families. The specificity is present in S- and R-form LPS but absent in Re mutants of different enterobacterial genera. By the use of purified LPS and monospecific antibodies obtained by immunoabsorption, the specificity is differentiated from the known core specificities of the genus Salmonella and the lipid A specificity by aid of the passive hemolysis and passive hemolysis inhibition test. In Salmonella minnesota R-form LPS, the specificity may be cryptic (R345, Rb2 mutant) or partly exposed in the intact molecule (R7, Rd1 mutant). The specificity is either demasked or completely exposed after mild acid hydrolysis for a short time, whereas it is destroyed after prolonged hydrolysis. Periodate oxidation, reduction, and hydrolysis under conditions that do not affect the ketosidic linkages of 2-keto-3-deoxyoctulosonic acid destroy the specificity in R4 (Rd2 mutant) LPS, but do not do so in R7 LPS. It is suggested that 2-keto-3-deoxyoctulosonic acid and a following neutral sugar are the compositional requirements for expressing the specificity.

On the serology of Plesiomonas shigelloides.

The serology of 87 strains of Plesiomonas shigelloides was studied. Thirty O antigenic groups and 11 H antigens were defined within the 87 strains, and an antigenic schema consisting of 40 serovars was established. Some O antigens of P. shigelloides were identical or closely related to those of some Shigella serovars.