Chitosan-carboxymethyl tamarind gum in situ polyelectrolyte complex-based floating capsules of ofloxacin: In vitro-in vivo studies.

The current research attempted to design and evaluate sustained stomach-specific ofloxacin delivery by single-unit hydrodynamically balanced system (HBS)-based floating capsules. These HBS-based floating capsules of ofloxacin were prepared using two oppositely ionic polymers, namely cationic-natured low molecular mass chitosan (LMMCH) and anionic-natured carboxymethyl tamarind gum (CMTG). FTIR results indicated the in situ formation of a polyelectrolyte complex in-between two oppositely charged polymers (i.e., in-between -NH2 group of the cationic natured LMMCH and -COOH groups of the anionic natured CMTG) and the nonexistence of any drug-polymer interaction(s) within these formulated ofloxacin HBS capsules. All these LMMCH-CMTG ofloxacin HBS capsules exhibited drug content uniformity, a sustained in vitro drug-releasing profile over 10 h. The ofloxacin HBS capsules (formulated with 75 mg LMMCH and 25 mg CMTG), which was selected as best formulation (for further studies), exhibited excellent in vitro floatation behaviour in SGF (pH 1.2) over 6 h without any floating lag-time, whereas the same formulation containing barium sulfate (100 mg) instead of drug demonstrated prolonged stomach-specific gastroretention in an in vivo X-ray imaging study using rabbits. Therefore, these types of HBS floating capsules can be useful for stomach-specific gastroretentive floating delivery of other drugs.

In vivo immunostimulatory effect of the amoebocyte lysate and plasma of Asian horseshoe crab, Tachypleus gigas in a piscine model.

Antimicrobial proteins/peptides are becoming a new generation of immunostimulants for prevention and disease control in human and animals, including aquatic animals. As the haemolymph of horseshoe crabs (Tachypleus) contains broad ranges of bioactive compounds, we have explored the in vivo immunostimulating potential of amoebocyte lysate and plasma using a fish model. Indian major carp, Labeo rohita, yearlings were injected intraperitoneally with two doses of lysate and plasma at 50 and 100 µg protein per fish. No abnormalities and/or mortalities were recorded in any group. L. rohita injected with 50 µg lysate and 100 µg plasma protein showed significant enhancement (P < 0.01) of various haematological and immunological parameters. There was a significant rise in the total protein and globulin content, myeloperoxidase and respiratory burst activity following injection with 50 µg lysate and 100 µg plasma protein. The agglutinating and haemagglutinating activities were increased albeit not significantly (P > 0.01) in any groups. On the contrary, a significantly high hemolysin titre was recorded in fish that received 100 µg plasma protein. Following challenge with Aeromonas hydrophila, both lysate and plasma protein(s) cross protected the fish after 30 days. The highest survival (50%) was recorded in group injected with 50 µg lysate protein, followed by 45% in both 100 µg lysate and plasma protein injected groups.

Current prospects and challenges in fish vaccine development in India with special reference to Aeromonas hydrophila vaccine.

Infectious diseases are adversely affecting aquaculture practices throughout world and Asian countries are no exception. Indian aquaculture practices are facing serious setback due to a variety of infectious agent's which are responsible for severe mortality and morbidity of all the cultured freshwater fish species leading to severe economic losses. The emergence of antibiotic resistant pattern, residual effect and environmental degradation due to indiscriminate use of antibiotics has necessitates the development of suitable alternate prophylaxis measures for better protection. In this regard, vaccine(s) has proved to be an effective strategy against pathogens to improve the fish production. Over the years numerous studies have been conducted to develop vaccine(s) against different pathogens. While most of the efforts are made to develop vaccine against bacterial pathogens especially against Aeromoniasis and Edwardsiellosis, few attempts have also been made against certain other bacterial, parasitic and fungal pathogens as well. Despite various successful experimental attempts, till date no vaccines against any of the pathogens are commercially available for Indian aquaculture. This review principally focuses on the current state of art in the development of vaccine against different microbial pathogens in general and Aeromonas hydrophila in particular since the bacterium is a major pathogen which is involved in a number of disease conditions in all the cultured fish species in India. Herein in this review, details of various experimental approaches made to find out a potential vaccine candidate which in turn can induce protective immune responses in host alongwith the constraints associated with it in developing a suitable vaccine against this bacterium and its market potential have been illustrated from an Indian perspective.

Development of Vaccines Against Nocardiosis in Fishes.

Nocardiosis, one of the most systemic and devastating diseases, is currently emerging as an important disease of cultured marine and freshwater fishes. The causative agent of this disease is Nocardia seriolae, a Gram-positive acid-fast bacterium. An effective vaccine/vaccination strategy against this pathogen is necessary to control the significant loss in aquaculture practices. In this chapter, we present the vaccination/immunization protocol in fish against both live (sublethal) and inactivated form of N. seriolae using ginbuna crucian carp (Carassius auratus langsdorfii) as a model. N. seriolae either in live (sublethal) form or inactivated antigenic form is found to elevate immunity in ginbuna and also can induce protective immunity upon challenge. In order to develop live vaccine, determination of sublethal dose is critical and needs to be established in the host fish species through pathogenicity and persistence studies. Herein for ginbuna, a sublethal dose of 10(6) CFU/mL was determined by pathogenicity study through a series of challenge doses followed by pathogen persistence study by microbiological and molecular techniques. On the other hand, for inactivated antigenic form, the concentration of the N. seriolae was approximately 10(8) CFU/mL. Although this study showed significant potential of both the forms of N. seriolae as candidate for vaccination, factors such as dose, duration and form need to be optimized in individual fish species.

Immune responses to live and inactivated Nocardia seriolae and protective effect of recombinant interferon gamma (rIFN γ) against nocardiosis in ginbuna crucian carp, Carassius auratus langsdorfii.

Looking into the fact that substantial mortality and morbidity is associated with intracellular Gram +ve bacterium, Nocardia seriolae infection, an effective vaccine against this pathogen is necessary to control the significant losses in aquaculture practices. Therefore, an attempt was made to evaluate the effect of live (sub-lethal) and inactivated (antigenic form) N. seriolae on cellular and humoral immunity in ginbuna crucian carp, Carassius auratus langsdorfii as well as the therapeutic potency of recombinant interferon gamma (rIFN γ) against N. seriolae infection. Effect of live and inactivated N. seriolae immunisation on the proliferation of CD4(+) T cells, CD8α(+) T cells and surface Ig M(+) cells in peripheral blood leucocytes, spleen, head kidney and trunk kidney of ginbuna was studied after 1st, 3rd, 7th, 15th and 30th day post immunisation. The percentage of CD8α(+) T cells in spleen and head kidney of ginbuna was significantly higher at 3rd day post immunisation. Similarly, surface Ig M(+) cells level was found to increase in both live and inactivated N. seriolae immunised groups. On the contrary, high percentage of CD4(+) T cells was observed in live N. seriolae immunised group in both the head and trunk kidneys at 30th day post immunisation. The humoral immune response to live and inactivated N. seriolae immunised ginbuna showed high antibody titre at 15th day post immunisation but the level declined subsequently in both the immunised groups. On challenge with virulent N. seriolae (1.2 × 10(8) CFU/ml), the relative percent survival was 62.5 and 75 in live and inactivated N. seriolae immunised groups, respectively. Furthermore, we have also studied the therapeutic potency of rIFN γ and found the possible involvement of IFN γ in resistance mechanism in fish. Administration of rIFN γ into ginbuna (at 10 µg/fish) one day before challenge study was found to protect ginbuna. The relative percent survival of ginbuna was 43.75 and 60 when challenged with 2 different doses of N. seriolae i.e., 1.2 × 10(8) CFU/ml and 5 × 10(7) CFU/ml, respectively. In summary, this study indicates that both forms of N. seriolae immunisation as well as rIFN γ indeed elicit an effective protective immunity which will help in designing suitable vaccine and/or adjunct therapy against N. seriolae infection in fish.

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp, Carassius auratus langsdorfii.

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α(+) T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α(+) T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1-2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α(+) T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α(+) T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4(+) T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM(+) cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α(+), CD4(+) T-cells and surface IgM(+) cells in fish.