Evaluation of interferon gamma (IFN-γ) of <Emphasis Type="Italic">Labeo rohita</Emphasis> as an immunomodulator: in vitro expression model

Interferon gamma (IFN-γ) or type II interferon is a cytokine that is critical for innate and adaptive immunity against viral and some bacterial and protozoal infections. The importance of IFN-γ in the immune system lies in its ability to inhibit viral replication directly and most importantly from its immunomodulatory effects. Previously, we successfully co-administered IFN-γ along with GAPDH gene of Edwardsiella tarda as bicistronic DNA vaccine in Labeo rohita. In order to ascertain the individual role of IFN-γ, the present study involves cloning and expression of 552-bp IFN-γ open-reading frame (ORF) of L. rohita in striped snakehead (SSN-1) cell line using eukaryotic expression vector system (pQE-TriSystem) followed by transfection in peripheral blood lymphocytes (PBMCs) to evaluate its immunomodulatory ability in comparison to polyinosinic-polycytidylic acid (Poly I:C)-treated PBMCs. The 18.7-kDa protein, expressed in the pQE-IFNγ-transfected SSN-1 cells, reacted with anti-His antibody in Western blot confirming it to be recombinant IFN-γ, whereas the relative expression of IFN-γ, iNOS, Mx, and IL-1β genes in PBMCs was quantified at 24 h and 48 h post treatment by qPCR. The comparative kinetics of all four genes showed significantly (p?<?0.05) high upregulation pattern in both pQE-IFNγ-transfected cell group and Poly I:C-treated cell group demonstrating recombinant IFN-γ as an equally efficient inducer like Poly I:C. Thus, our in vitro experiment results highlight the immunomodulatory potential of recombinant IFN-γ as an analogue to synthetic Poly I:C which warranted future studies to further explore the potential of recombinant IFN-γ as an effective vaccine adjuvant against different microbial invasion.     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

Assessment of the new generation aeration systems efficiency and water current flow rate,its relation to the cost economics at varying salinities for Penaeus vannamei culture

The selection of aerators and correct numbers can play an essential role in reducing the cost of production in aquaculture. The new generation aerators, namely spiral leaf, air‐jet, submersible and impeller, used in aquaculture, were assessed for its aeration efficiency and energy cost compared with the commonly used paddle wheel aerator. Of the aerators tested, the impeller had the highest aeration efficiency of 2.098 kg O₂/kW hr, followed by paddle wheel with 1.436 kg O₂/kW hr at 20‰ salinity. The spiral and air‐jet aerators had maximum aeration efficiency of 1.326 and 1.419 kg O₂/kW hr, respectively, at 35‰ salinity. The submersible aerator was not efficient as its maximum efficiency was 0.380 kg O₂/kW hr. The water flow by paddle wheel was 3 ft/sec and also provides better coverage than other types of aerators. The efficiency of aerators was high in optimum salinities (20‰ and 35‰) than the low or high saline condition. The average energy cost of shrimp pond aeration per hectare was lowest for impeller, followed by paddle wheel aerator. The study provided economic comparisons of vannamei culture using different aeration systems by keeping a uniform set of economic assumptions. Shrimp farms with impellors can give 14%–25% high returns across salinities, whereas spiral leaf can provide 5% high returns at 35‰ salinity. The combination of the type of aerators and the calculated use based on the salinity of the culture systems can result in energy‐saving and also a reduction in the production cost.     

Specific immune response in mucosal and systemic compartments of Cirrhinus mrigala vaccinated against Edwardsiella tarda: In vivo kinetics using different antigen delivery routes

Mucosal immune barriers confer protection against invading fish pathogens. Here, we conducted an experiment for 60 days to assess the mucosal and systemic immune response in Mrigal (Cirrhinus mrigala), an Indian major carp. Fish were immunized with inactivated Edwardsiella tarda by four different routes, namely, oral, immersion, injection, and anal intubation. An indirect enzyme‐linked immunosorbent assay (ELISA) was used to measure the specific immune response (antibody) in serum and mucus (collected from skin, gill, and gut) of the fish on 0, 15, 30, 45, and 60 days postimmunization. For specific immune response in the serum, significantly higher (p < 0.05) optical density (OD) values were obtained in the anal group (0.52 ± 0.03) and in the oral group (0.48 ± 0.03). In the skin mucus, significantly higher OD values were obtained in the oral group (0.48 ± 0.04) and immersion group (0.32 ± 0.03). In the gill mucus, significantly higher OD values were obtained in the oral group (0.82 ± 0.08) and the immersion group (0.73 ± 0.03). In the gut mucus, significantly higher OD values were obtained in the immersion group (0.080 ± 0.007) compared to the rest of the treatments. Fish from all the groups were challenged with LD₅₀ dose of E. tarda at the end of the experiment. We conclude that oral and immersion immunization routes offer better protection of C. mrigala compared to other antigen delivery routes.     

DNA polymorphisms and genetic relationship among populations of Acacia leucophloea using RAPD markers

RAPD(randomly amplified polymorphic DNA)markers were employed to characterize polymorphisms among 5 provenances of Acacia leucophloea and to detect genetic relatedness of the species with 6 other acacias(A.holosericea, A.auriculiformis, A.mangium, A.dealbata, A.ferruginea, and A.nilotica) widely grown in India.Of 194 markers scored for the provenances, 29.38% exhibited polymorphism.Also, 326 markers were generated among 7 species of Acacia, accounting for 55.82% of the polymorphisms.The fifteen 10-mer primers employed were capable of producing 1–8 polymorphic bands for the provenances, and 6–17 for all seven species of Acacia.The genetic similarity coefficient based on Jaccard's coefficient revealed that provenances Thirumangalam and Dharmapuri were closely related.The dendrogram based on a sequential agglomerative hierarchical non-overlapping(SAHN) clustering analysis grouped 4 provenances of A.leucophloea(Dharapuram, Thirumangalam, Pudukottai and Dharmapuri) into one cluster and the other provenance, Sendurai,into a separate cluster.The genetic similarity matrix for 7 Acacia species showed that A.nilotica and A.dealbata were distantly related, while A.holosericea and A.ferruginea were very closely related.Cluster analysis grouped the species of Acacias into 3 major groups of which A.dealbata alone formed a separate group.The RAPD markers generated 36 provenance-specific markers and 162 species-specific markers that could have strong applications for species identification and tree breeding programs for A.leucophloea and for other Acacia species included in this study.     

Characterization and production of polyclonal antisera against pangasius (Pangasianodon hypophthalmus) serum immunoglobulin IgM derived from DEAE cellulose based ion exchange chromatography

Pangasius (Pangasianodon hypophthalmus) is a commercially important candidate species in freshwater aquaculture and it is important to understand the immune system of pangasius against infectious disease. The present study was aimed at the purification, characterization and quantification of serum IgM in pangasius (P. hypophthalmus). Serum IgM was purified by diethylaminoethyl (DEAE) cellulose based ion exchange chromatography. The molecular weight of native immunoglobulin was found to be 798 kDa. Heavy (H) and light chains were found to possess molecular weight of 70.1 and 26 kDa respectively. A 248 bp segment of IgM H chain gene of pangasius was amplified and sequenced (partial). The antisera raised against pangasius immunoglobulin cross‐reacted with the immunoglobulin H chain of catfish such as Clarias gariepinus and Clarias batrachus, but not with their light chain indicating epitope sharing among IgM H chains in these catfish. The produced polyclonal antisera were used to develop an enzyme linked immuosorbent assay to quantify IgM levels in pangasius. In conclusion, the present study provides its future implications for epidemiology and immunology studies in pangasius.     

Evaluation of Lactobacillus plantarum as a water additive on host associated microflora,growth, feed efficiency and immune response of giant freshwater prawn,Macrobrachium rosenbergii (de Man, 1879)

A 90‐day experiment was conducted by rearing 1020 prawn juveniles (0.54 ± 0.03 g) in water supplemented with three different concentrations of probiotic bacteria viz. T1 (10⁷ cfu L^?1), T2 (10⁸ cfu L^?1), T3 (10⁹ cfu L^?1) and the control (C) (unsupplemented water), to evaluate probiotic effect of Lactobacillus plantarum. In the present study, the growth parameters (WG%, SGR) and feed utilization parameters (FCR, PER) significantly improved (P < 0.05) in T3. The growth and feed utilization parameters though improved marginally in T1 and T2, the difference was not significant (P > 0.05) compared to the control. The gastro‐intestinal Lactobacillus sp. count increased significantly (P < 0.05) in all the treatment groups, whereas the decrease in harmful bacteria was significant (P < 0.05) in T3 compared to the control. Similarly, the Lactobacillus sp. count in culture water increased significantly (P < 0.05) in all the experimental groups, whereas the decrease in harmful bacteria was significant (P < 0.05) in T2 and T3. The immune parameters (THC, PO and RB activity) and clearance efficiency significantly improved (P < 0.05) in T3 with concurrent decrease (P < 0.05) in cumulative mortality against Aeromonas hydrophila challenge. However, water quality did not improved (P > 0.05) in any of the treatment groups. The results indicate that Lactobacillus plantarum at a minimum concentration of 10⁹ cfu L^?1 could be used as water additive to confer its probiotic effect in prawn, Macrobrachium rosenbergii. Moreover, future studies with higher probiotic concentrations should be conducted for its efficient commercial scale field application.     

DNA polymorphisms and genetic relationship among populations of <Emphasis Type="Italic">Acacia leucophloea</Emphasis> using RAPD markers

RAPD (randomly amplified polymorphic DNA) markers were employed to characterize polymorphisms among 5 provenances of Acacia leucophloea and to detect genetic relatedness of the species with 6 other acacias (A. holosericea, A. auriculiformis, A. mangium, A. dealbata, A. ferruginea, and A. nilotica) widely grown in India. Of 194 markers scored for the provenances, 29.38% exhibited polymorphism. Also, 326 markers were generated among 7 species of Acacia, accounting for 55.82% of the polymorphisms. The fifteen 10-mer primers employed were capable of producing 1–8 polymorphic bands for the provenances, and 6–17 for all seven species of Acacia. The genetic similarity coefficient based on Jaccard’s coefficient revealed that provenances Thirumangalam and Dharmapuri were closely related. The dendrogram based on a sequential agglomerative hierarchical non-overlapping (SAHN) clustering analysis grouped 4 provenances of A. leucophloea (Dharapuram, Thirumangalam, Pudukottai and Dharmapuri) into one cluster and the other provenance, Sendurai, into a separate cluster. The genetic similarity matrix for 7 Acacia species showed that A. nilotica and A. dealbata were distantly related, while A. holosericea and A. ferruginea were very closely related. Cluster analysis grouped the species of Acacias into 3 major groups of which A. dealbata alone formed a separate group. The RAPD markers generated 36 provenance-specific markers and 162 species-specific markers that could have strong applications for species identification and tree breeding programs for A. leucophloea and for other Acacia species included in this study.     

Assessment of impact of aquaculture on Kolleru Lake (India) using remote sensing and Geographical Information System

Aquaculture is one of the fastest growing food industries and the rapid growth of aquaculture worldwide has resulted in growing concerns about its impact on important ecosystems. Kolleru Lake, India's largest fresh water body, and Ramsar site have undergone tremendous changes due to the development of aquaculture. To assess the impact of aquaculture on Kolleru, satellite data were found appropriate because of the synoptic‐detailed overview and accuracy. Satellite data of IRS 1D, LISS III from 2004 and Survey of India topographic maps from 1967 were processed using image processing techniques in erdas imagine and analysed in Geographical Information System (GIS) such as arc gis 9.1. Land use map prepared from the satellite data was verified in the field using Global Positioning System to check the land and water use classes and its areal extent. It was estimated from the topographic maps of 1967 that the total lake boundary area was 180.38 km², in which 70.70 km² had water throughout the year and 100.97 km² had water during the rainy season. The digital image processing of 2004 satellite data revealed that Kolleru was no longer a lake and the lake area of 62.65 km² (34.73%) only remained in a degraded state, extensively colonized by macrophytes. The total loss of lake area was 109.02 km² between 1967 and 2004, in which aquaculture was developed in 99.74 km², which represented 55.3% of the 1967 lake area. The maximum conversion to aquaculture occurred from the lake liable to be inundated during the 1967 rainy season. The area under agriculture was 16.62 km² in 2004, the increase in lake area for agriculture between 1967 and 2004 being 8.22 km² (4.55% of lake). If human induced degradation is allowed to continue, the lake will very soon disappear. Now, the Government of Andhra Pradesh, India, has initiated schemes and laws to restore the lake to its pre‐development state. Lessons learnt from the Kolleru Lake cautioned that there is a need for regular monitoring of important water resources throughout the world to protect the biodiversity of the earth.