P. Swain

Biological effects of bacterial lipopolysaccharide (endotoxin) in fish: A review

Bacterial lipopolysaccharides (LPS), also termed endotoxins, considered to be a major virulence factor, are responsible for the lethal effects and clinical manifestations of diseases in humans and animals. Higher animals are extremely sensitive to endotoxin even at low doses but lower vertebrates like fish are often resistant to endotoxic shock. Toll-like receptor (TLR)-4 is mainly involved in the activation of the immune system by LPS through the specific recognition of its endotoxin (Lipid A) moiety. Although several Toll-like receptors are present in fish, those molecules specifically involved in TLR-4 mediated endotoxin recognition have not been fully established in different fish species. Despite this, LPS has the potency to express cytokines, acute-phase proteins and also exerts immunological, pathological, physiological, immuno-endocrinological and neuro-immunological effects in several fish species. The immunostimulating effects of endotoxin by triggering various immune parameters such as T and B lymphocytes, macrophages, and complement systems have been established in teleosts. This article reviews the multiple biological effects of endotoxin which will further strengthen the knowledge among researchers on various aspects of endotoxin in lower vertebrates, particularly in the piscine system.

Role of maternally derived immunity in fish

Maternal immunity is of paramount importance for protection of young ones at early stage of life since the immune factors of an immunocompetent female are transferred transplacentally or through colostrum, milk or yolk to an immunologically naive neonate. Both innate and adaptive type of immunity are transferred of from mother to offspring in fishes. These factors include immunoglobulin (Ig)/antibody, complement factors, lysozymes, protease inhibitors like alpha macroglobulin, different types of lectins and serine proteases like molecules. Among different types of Ig viz. IgM, IgD, IgT/IgZ and IgM-IgZ chimera types, IgM is present in most of the teleostean fishes. In teleosts, IgM either as a reduced/breakdown product or monomeric form is usually transferred to the offsprings. The maternally derived IgM usually persists for a limited duration, exhausts within the completion of yolk absorption process, and completely disappears thereafter during larval stages. Maternal transfer of immunity which provides defense to embryo and larvae depends upon the health as well as the immune status of brood fish. The overall health status of brood fish can affect breeding performances, quality seed production and protection of offsprings. However, factors such as age, maturation, reproductive behaviour and nutrition (micro and macro-nutrients) may affect the immunity in brood fishes. Besides these, seasonal changes such as photoperiods, temperature, adverse environmental conditions, and stress conditions like handling, crowding, and water pollution/contamination can also affect the immunity of brood fishes. The maintenance of the brood stock immunity at high level during vitellogenesis and oogenesis, is utmost important for reducing mortalities at larval/post larval stages through maximum/optimum transfer of maternal immunity. Brood stock immunization prior to breeding as well as selective breeding among the disease resistant families might be the ideal criteria for producing quality seed.

Parenteral immunization of fish, Labeo rohita with Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses.

Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas hydrophila such as Poly d, l-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and 42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier for parenteral immunization in fish, Labeo rohita.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita.

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture.

The emergence of diseases and mortalities in aquaculture and development of antibiotics resistance in aquatic microbes, has renewed a great interest towards alternative methods of prevention and control of diseases. Nanoparticles have enormous potential in controlling human and animal pathogens and have scope of application in aquaculture. The present investigation was carried out to find out suitable nanoparticles having antimicrobial effect against aquatic microbes. Different commercial as well as laboratory synthesized metal and metal oxide nanoparticles were screened for their antimicrobial activities against a wide range of bacterial and fungal agents including certain freshwater cyanobacteria. Among different nanoparticles, synthesized copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver doped titanium dioxide (Ag–TiO2) showed broad spectrum antibacterial activity. On the contrary, nanoparticles like Zn and ZnO showed antifungal activity against fungi like Penicillium and Mucor species. Since CuO, ZnO and Ag nanoparticles showed higher antimicrobial activity, they may be explored for aquaculture use.

Comparative sensitivity of different serological tests for seromonitoring and surveillance of Edwardsiella tarda infection of Indian major carps.

Different serological tests viz. indirect ELISA, indirect blocking ELISA, competitive ELISA and serum agglutination tests were evaluated to detect antibodies against Edwardsiella tarda in naturally infected fish sera for seromonitoring and epizootiological studies. Approximately 66.6, 62.5, 57.6 and 16.6% of the field sera samples were found to be positive by indirect ELISA, competitive ELISA, indirect blocking ELISA and serum agglutination test, respectively. The percentage of serum samples positive for E. tarda antibodies in serum agglutination, competitive ELISA and indirect blocking ELISA, when compared with indirect ELISA, were 33.3, 83.6 and 66.6%, respectively, but its use was restricted due to the requirement of several conjugates against different fish species and the difficulty in assaying large numbers of serum samples from different fish species in a limited time to enable seromonitoring of the disease prevalence. No significant difference (P<0.05) in the mean optical density value was found in indirect and competitive ELISA. Although the competitive ELISA was slightly less sensitive than the indirect ELISA, it could accommodate a large number of serum samples with one anti-rabbit conjugate, and the need for different fish conjugates as required in indirect ELISA was eliminated. As in medical and veterinary practices, these tests can now be used in aquaculture practices for seromonitoring and study of pre-exposure of Indian major carps to pathogens in enzootic areas.

Alginate–chitosan–PLGA composite microspheres induce both innate and adaptive immune response through parenteral immunization in fish

Alginate-chitosan-PLGA composite microspheres encapsulating outer membrane protein antigen of Aeromonas hydrophila as an antigen carrier was explored for the first time in a fish model. This composite microsphere showed distinct advantages over the conventional PLGA microparticles in aspects of the high encapsulation efficiency due to the protein-friendly microenvironment created by the hydrophilic alginate-chitosan cores of the composite microspheres, preventing initial burst release and the elimination of lyophilizing process. The antibody responses significantly increased and persist up to 9 weeks in composite microspheres unlike that PLGA microsphere, native OMP and FIA adjuvant. Moreover, several innate immune parameters as respiratory burst, lysozyme and complement activity were significantly increased in both composite and PLGA microspheres up to 9 weeks than other treated groups. It also gives protection from A. hydrophila infection and brought some hope, for its application in replacement with conventional PLGA microparticle for antigen delivery in fish.

Antigen adsorbed calcium phosphate nanoparticles stimulate both innate and adaptive immune response in fish, Labeo rohita H.

Calcium phosphate nanoparticles as an antigen/protein delivery was explored in a fish model Labeo rohita H. S-layer protein (of Aeromonas hydrophila) adsorbed on nano sized calcium phosphate particles elicited both innate and adaptive immune parameters which persisted up to 63 days of post immunization through parenteral immunization and gave cross protections.

Derivation of rough attenuated variants from smooth virulent Aeromonas hydrophila and their immunogenicity in fish.

Aeromonas hydrophila is a Gram-negative bacteria associated with many disease conditions of aquatic animals. Two rough attenuated variants were derived from two smooth virulent types of this bacterium by continuous passage separately in laboratory over a period of 8 years in brain-heart infusion agar. These variants were checked to be rough lipopolysaccharides (LPS) types and attenuated since they neither produced any disease nor mortality in the injected fishes. The immunogenic potential of these variants remained unaltered as compared to parent smooth types and fish immunized with these variants resisted the infection and mortality upon cross challenge. Because of their intact immunogenic potential and loss of virulence, they may be used as candidates for fish immunization.

Antigen adsorbed surface modified poly-ɛ-caprolactone microspheres stimulates both adaptive and innate immune response in fish.

Surface modified poly-ɛ-caprolactone microspheres as an antigen carrier was explored in a fish model. Outer membrane vesicles of Edwardsiella tarda adsorbed on to surface modified poly-ɛ-caprolactone microspheres with chitosan and alginate induces both innate and adaptive immune responses which persist up to 63 days of post immunization through parenteral immunization unlike that of free and FIA adjuvented antigens. These results highlight the role of these microspheres as an adjuvant/antigen carrier in fish.