Evaluation of feed utilization, immune response and disease resistance in striped catfish, Pangasianodon hypophthalmus (Sauvage 1878) fed with a novel Aeromonas hydrophila biofilm vaccine.

Striped catfish, Pangasianodon hypophthalmus was immunized with Biofilm (BF) and Free cell (FC) of Aeromonas hydrophila vaccine at 1010 CFU g-1 for 20 days and monitored for growth parameters, immune responses and disease resistance up to 60 day post vaccination (dpv). Pangasius catfish in the BF vaccinated group had considerably higher growth and feed utilization than the FC vaccinated and unvaccinated groups (p < 0.05). Biofilm vaccinated group showed a significant increase (p < 0.05) in the mean weight gain (46.91 ± 0.59) than the FC (35.94 ± 0.21) and unvaccinated group (34.92 ± 0.35). The vaccinated fishes were challenged with A. hydrophila at 107 CFU/ml. Significant higher relative percentage survival (RPS) was recorded with BF (84.21 ± 1.49%) compared to that with FC (33.33 ± 1.21%). Polyclonal antibody-based ELISA was used to quantify the antibody titre. BF vaccinated group showed significantly higher antibody titer compared to other treatments (p < 0.05). Moreover, higher haematological parameters recorded in the present study were differentially stimulated by the oral administration of A. hydrophila biofilm vaccine. The mean total protein, albumin, and globulin levels of the BF vaccine groups were significantly higher (p < 0.05) than the mean total protein, albumin, and globulin contents of the unvaccinated group. Furthermore, biochemical stress parameters (SGPT, SGOT) in the vaccinated groups showed an incremental trend in the early days of the experimental period. However, the values were significantly lower (p < 0.05) in the biofilm group on 20 dpv onwards indicating improved health condition. Vaccinated BF fishes showed gut associated lymphoid tissues (GALT) within the laminar propria of mid gut. But in FC group fishes showed less aggregation of lymphoid cells. The unvaccinated control fish had no lymphoid cell aggregation in their intestines. The findings of the current research suggested that biofilm vaccine has the capability to be one of the potential oral vaccines in striped catfish against A. hydrophila infection.

Evaluation of biofilm of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer (BLOCH, 1790).

The present study evaluated the biofilm (BF) of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer. An 80-day experiment was carried out in circular fiber-reinforced plastic (FRP) tanks using free cell (FC) and BF of Vibrio anguillarum with triplicate in each. Heat-inactivated FC and BF cells at 107, 1010 and 1013 CFU/g fish/d were fed to fish for 20 days, agglutination antibody titer estimated at each 10 days interval up to 60-day post vaccination. As compared to FC and control there was a significant increase in agglutinating antibody titer in the biofilm vaccinated fishes. Among the 3 doses, BF at 1010 cfu/g fish/d was considered the ideal dose for vaccination. Relative percentage survival (RPS) was higher in biofilm vaccinated fish (85.4%) compared to that with free cells (27.0%). The study demonstrated the better performance of V. anguillarum biofilm oral vaccine compared that with free cell vaccine in L. calcarifer. The study further supports better performance of biofilm vaccine model with one more bacterial pathogen in a high carnivore fish.

Development of a sandwich vertical flow immunogold assay for rapid detection of oxytetracycline residue in fish tissues.

A rapid multiplex silver enhanced 'sandwich vertical flow immunogold assay (SVIA)' based on disposable porous filter-membrane was developed for on-site detection of oxytetracycline (OTC) residues in fish tissues. Artificial antigens were synthesized to raise selective anti-OTC rabbit antiserum and highly specific monoclonal antibody (mAb). The assay consists of three layers. The first layer was composed of immobilized anti-OTC rabbit antiserum (capture antibody) onto the membrane, the second layer was the molecule of interest (OTC) and the third layer was refined with detector immunogold labeled mAb. The sensitivity of the silver enhanced SVIA was as low as 2 ng mL-1 (within 4 min) representing a 125-fold increase in sensitivity over the HRP labeled sandwich vertical flow assay. The reliability of developed technique was co-evaluated with HPLC and validated using 115 field fish samples from different regions of southern India. The results demonstrated that SVIA has the high potentiality to be established as a semi-quantitative routine on-site screening tool.

Evaluation of the Sensitivity of the Flow Through Assay for detection of White Spot Syndrome Virus (WSSV) using a cocktail of monoclonal antibodies.

A panel of four monoclonal antibodies (C-05, C-14, C-38 and C-56) specific to VP28 of White spot syndrome virus (WSSV) were evaluated individually and in cocktail to increase sensitivity of the Flow Through Assay (FTA) for detection of the virus. Recombinant VP28 and semi purified WSSV was used as antigen for evaluation. Out of the total 11 cocktails and four individual of MAbs, 2 MAb cocktails C-05 + C-56 and C-14 + C-56 exhibited highest sensitivity in the FTA. The two MAb cocktail were 100 times more sensitive than 1-step PCR and nearly equivalent to 2-step PCR for the detection of WSSV. The detection limit of WSSV by MAb cocktail increased by two fold compared to the single MAb C-05 currently being used in (FTA).

Identification of putative genes involved in parasitism in the anchor worm, Lernaea cyprinacea by de novo transcriptome analysis.

There is little information on the genome sequence of Lernaea cyprinacea a major ectoparasite of freshwater fish throughout the world. We subjected the L. cyprinacea transcriptome (adult and free living stages) to Illumina HiSeq 2000 sequencing. We obtained a total of 31,671,751 (31.67 millions) reads for the adult parasitic stage and 33,840,446 (33.84 millions) for the free living stage. The reads were assembled into 50,792 contigs for the adult stage and 69,378 for the free living stage. Using the pfam database, 41.91% of the transcriptome was annotated. The transcriptome was mined for genes associated with parasitism. To examine gene expression changes associated with the parasitism of L. cyprinacea during the transit from the free living to parasitic stage, we studied the differentially expressed transcripts between the two stages. The microsatellite markers were also identified (9,843 for adult stage; 16,813 for free living stages) and this would facilitate population genetic studies in various geographical isolates of Lernaea. Our data provides the most comprehensive sequence resource available for L. cyprinacea and demonstrates that Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in a species lacking genome information. The data could open new avenues for a wide array of genetic, evolutionary, biological, ecological, epidemiological studies, and a solid foundation for the development of novel interventions against L. cyprinacea.

Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes.