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Aims To investigate the antibacterial activity of three (palmarosa, basil and rosemary) essential oils (EOs) on Aeromonas veronii and Aeromonas caviae, and determine minimum inhibitory concentration (MIC) of potent EO against tetracycline and sulfonamide resistant strains. Methods and Results Palmarosa oil (PMO) showed significantly (p < 0.05) higher inhibition zones against both A. veronii and A. caviae (n = 30) than basil and rosemary in the disk diffusion assay. The MIC (% v/v) of PMO ranged from 0.008% to 1.00%. The mean MIC was significantly higher for A. caviae (0.48 ± 0.24%) than A. veronii (0.21 ± 0.15%). Further, the MIC of PMO was compared in six groups: Group 1: Tetracycline Resistant A. veronii (TRV); Group 2: Tetracycline Resistant A. caviae (TRC); Group 3: Sulfonamide Resistant A. veronii (SRV); Group 4: Sulfonamide Resistant A. caviae (SRC); Group 5: Susceptible A. veronii (SV) and Group 6: Susceptible A. caviae (SC). No significant differences were observed between overall resistant (TRV+ SRV) and susceptible A. veronii (SV). However, in A. caviae, the resistant group had a lower MIC than the susceptible group. Moreover, the MIC was significantly lower for TRC (0.31 ± 0.11%) as compared to SRC (0.46 ± 0.10%). The time of kill of PMO for both the species of Aeromonas was 20–30 min. Conclusion Palmarosa oil exhibited significantly higher activity on A. veronii than A. caviae. The resistant strains of A. caviae were inhibited at a lower concentration than susceptible strains. Significance and Impact of the Study Palmarosa oil could be explored as an alternative antimicrobial agent for mitigating antimicrobial resistance and managing Aeromonas infection in fish and their risks to public health.

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin (LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.

Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant bacterial disease responsible for mortality in Indian catfish, Clarias magur, a potential aquaculture species in the Indian subcontinent. In fish, innate immunity elicited by pathogen recognition receptors (PRRs) plays an important role in providing protection against bacterial infection. Information on PRRs including Toll-like receptors (tlrs) and their response to bacterial pathogens remains unexplored in magur. Toll-like receptor 2 (tlr2), a phylogenetically conserved germ-line encoded PRR recognizes specific microbial structure and trigger MyD88-dependent signaling pathway to induce release of various cytokines responsible for innate immune response. In the present study, tlr2 gene of magur was characterized and downstream signaling was studied following challenge with A. hydrophila. The full-length cDNA of magur tlr2 (mtlr2) comprised of 3,066 bp with a single open reading frame of 2,373 bp encoding 790 amino acids having a theoretical pI value of 6.11 and molecular weight of 90 kDa. Structurally, it comprised of signal peptide (1–42aa), one leucine-rich repeat region (LRR) at N-terminal (LRR1-NT: 50–73 aa) and C-terminal (LRR-CT: 588–608 aa), twenty LRRs in between, one trans-membrane (Tm) domain (609–631aa) followed by cytoplasmic TIR domain (670–783aa). Phylogenetically, mtlr2 is closely related to pangasius and channel catfish. Highest basal expression of mtlr2, myd88 and il-1β in spleen, nf-kb in anterior kidney was observed. Lowest basal expression of mtlr2 in skin and myd88, nf-kb and il-1β in muscle was detected. Significant up-regulation of mtlr2 and downstream expression occurred at 3, 8, 24 h post infection to A. hydrophila in important immune organs such as liver, spleen, intestine and kidney. These findings highlight the vital role of tlr2 in eliciting innate immune defence against A. hydrophila infection.

Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant problem among the bacterial diseases responsible for causing mortality in catfishes. The objective this study was to understand the modulation of innate immune parameters and histopathological alteration in Indian catfish Clarias magur (Hamilton, 1822), following intraperitoneal injection with A. hydrophila. At 3 to 8 h post-infection (hpi), respiratory burst activity, myeloperoxidase, protease, total anti-protease and α2 macroglobulin increased significantly (p

The study evaluates optimum hydraulic loading rate (HLR) based on the fish and plant production performance and nutrient removal percentage in aquaponics. The growth of Gotukola (Centella asiatica (L.))—a leafy vegetable as well as traditional medicinal plant—and koi carp (Cyprinus carpio var. koi.)—a high demand ornamental fish—were evaluated at three different HLRs, viz. 2.6 m day−1 (T1), 7.8 m day−1 (T2), and 13.0 m day−1 (T3). Treated aquaculture wastewater was utilized in a combination of the freshwater in 1:1 ratio with 2.1 kg m−3 fish stocking density. The highest fish mean length; weight; feed conversion ratio; specific growth rate; NH3, NO2−, and NO3− removal percentage; and plant growth parameters were observed. Considering fish (koi carp) and plants (Gotukola) growth parameters as well as nutrient removal percentages, a HLR of 7.8 m day−1 was found to be the optimum in aquaponics.

Biofloc technology degrades waste into useful resources exploiting microbes and can be used in zero‐water exchange systems. To study the effect of different biofloc systems on haematological and metabolic response of Labeo rohita fingerlings, a 60‐days experiment was conducted using four long lasting carbon sources. Seven hundred and fifty fingerlings having mean weight of 4.80 ± 0.12 g were randomly distributed into 15 tanks (n = 50 per tank). Five experimental groups were set in triplicate; T1 (Tapioca), T2 (Wheat), T3 (Corn) T4 (Sugar bagasse) and control (clear water). In‐situ biofloc was developed in 300 L fibre‐reinforced plastic (FRP) tanks and a C/N ratio of 15 was maintained. Water quality variables indicated ammonia immobilization by heterotrophic bacteria, as the dominant mechanism for the removal of toxic‐nitrogenous compounds in the biofloc systems. Results exhibited significantly higher floc volume (53.33 ± 7.88 ml/L), haemoglobin content (6.61 ± 0.03 g/dl) and total leucocyte count (109.66 ± 0.06 thousand cells/mm³) in tapioca biofloc system. Furthermore, the digestive and anti‐oxidative enzymes activities were also significantly higher in tapioca biofloc system. The lactate dehydrogenase and malate dehydrogenase enzyme assays showed a decreased level in tapioca biofloc system as compared with other biofloc systems and control group. Our observations indicate that tapioca biofloc system could improve the water quality, haematological and anti‐stress responses of L. rohita fingerlings in biofloc systems and thus can effectively replace other carbohydrate sources for the biofloc system.

An experiment was carried out for 60 days to utilize the phytoremediated aquaculture wastewater for production of Gotukola (Centellaasiatica (L.)), a leafy vegetable as well as a traditional medicinal plant; and koi carp (Cyprinuscarpiovar. koi), a popular ornamental fish with high market demand. Treated water and borewell water were mixed in different combinations namely 100:0 (T1), 75:25 (T2), 50:50 (T3) and 25:75 (T4) to produce four treatments. Additionally three controls were also maintained to compare the efficiency of the treatments. These were Control 1 consisting of hydroponic component with plant (C1), Control 2 consisting of hydroponic component without plant (C2) and Control 3 with only borewell water (C3). Plants in Control 3 were used to compare the efficiency of plant growth in field condition. On the basis of plant growth parameters viz., leaf width, number of leaves, number of plant-lets, number of runners, length of runners and the final yield, T1 showed the best performances. However, it was not significantly different from T2 and T3 except the final yield. Best growth performance of fish viz., weight gain, length gain, feed conversion ratio (FCR), feed efficiency ratio (FER), protein efficiency ratio (PER), and specific growth rate (SGR) was observed in T3. Considering the fish colour, there was no significant difference between the treatments for chroma and hue values, but best chroma values were obtained in T1 followed by T2 > T3. This denotes that the treated wastewater positively affects the fish colour by pigment enhancement. The efficiency of the aquaponics was judged by percentage nutrient removal and biofilter performance. The highest removal of nitrite-N (92.34%), nitrate-N(70.55%) and ammonia-N (71.36%) were observed in T1 and it was positively related with the plant growth. The highest phosphate-P removal percentage (59.78%) and best biofilter performance (3.24 g−3 day−1) were observed in T3 and also, comparatively a higher plant growth was obtained in T3 than T4 and C1. Considering the overall parameters viz., fish growth, fish health, plant growth and water quality parameters, equal proportion of treated wastewater and borewell water (T3) was found to be the most effective combination. The results revealed that the phytoremediated aquaculture wastewater can be sustainably reused in aquaponics for fish and plant production.

Epitheliocystis, an emerging aquaculture disease, has been reported in >90 fish species from 14 countries. In the present study, we report epitheliocystis from cultured rohu Labeo rohita, an Indian major carp, which is the major cultured species in the Indian subcontinent. The affected fish exhibited lethargy, swimming near the water surface followed by mortality. In wet mounts of gills, infestation with dactylogyrids, trichodinids and myxosporeans was observed. Subsequently, following euthanasia, the affected fish were dissected and subjected to routine bacteriological, virological and histopathological investigations. The bacteria could not be isolated from internal organs, namely kidney and liver. In addition, cytopathic effects were not observed in the Cyprinus carpio koi fin (CCKF) cell line following inoculation of filtered tissue homogenate from pooled kidney samples. In histology of gill sections, amorphous intracytoplasmic inclusions typical of epitheliocystis were observed in epithelial cells, and the presence of chlamydial DNA in gills was confirmed by amplification of 16S rRNA gene sequence of Chlamydiales. The sequencing of amplicons (774 bp) revealed that the epitheliocystis agents from rohu belong to family Candidatus Parilichlamydiaceae in the order Chlamydiales. Since the sequences of epitheliocystis agent from rohu showed

In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (Labeo rohita), was identified as Bacillus amyloliquefaciens using 16S rRNA gene sequence analysis combined with amplification of species-specific BamHI and barnase genes. The probiotic potential of the strain was evaluated using an array of in vitro tests along with safety and genetic analyses. The isolate showed potent antimicrobial response against several fish pathogenic bacteria, survived a wide pH range (2–9), and was resistant up to 10% bile salt concentration. With regard to the in vitro adhesion properties, the strain showed significantly high in vitro adhesion to mucus, auto and co-aggregation capacity, and cell surface hydrophobicity. The strain was non-haemolytic, able to produce extracellular enzymes, viz., proteinase, amylase, lipase, and cellulase, and showed significant free radical scavenging activity. A challenge study in rohu revealed the strain COFCAU_P1 as non-pathogenic. The presence of putative probiotic marker genes including 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase, arginine/ornithine antiporter ArcD, choloylglycine hydrolase, LuxS, and E1 β-subunit of the pyruvate dehydrogenase complex was confirmed by PCR, suggesting the molecular basis of the probiotic-specific functional attributes of the isolate. In conclusion, the in vitro and genetic approaches enabled the identification of a potential probiotic from autochthonous source with a potential of its utilization in the aquaculture industry.

In the present study, we have developed a continuous cell line from the heart tissue of the Oreochromis niloticus and used for studying susceptibility to tilapia lake virus (TiLV). The cell line, designated as OnH, has been subcultured up to 82 passages. The optimal growth of OnH cells was observed at 28-32 °C in iL-15 medium supplemented with 20 % fetal bovine serum. Karyotype analysis revealed that the modal chromosome number of OnH cells was 44. Partial amplification and sequencing of 16S rRNA gene confirmed the origin of OnH cell line from O. niloticus. Immunophenotyping revealed that OnH cells were of epithelial origin. These cells were successfully transfected with pAcGFP1-N1 mammalian expression vector. OnH cells showed cytopathic effects following inoculation with TiLV. The virus titration study indicated that the cells were highly susceptible to TiLV with TCID50 value of 105.3/mL. The qRT-PCR studies revealed that the optimal temperature for TiLV replication in OnH cells was 28 °C. Further, transmission electron microscopy of TiLV-infected OnH cells showed a number of electron-dense virus particles measuring 60-90 nm diameter, which were enclosed in the vesicles in the cytoplasm. Therefore, the newly established OnH cell line provides a valuable tool for isolation of viruses from disease cases suspected to be of viral etiology in this candidate species' and also for transgenic and genetic manipulation studies.

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

Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ± 12 g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P

DNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression of GAPDH and IFN-γ genes in the transfected cell were confirmed by Western blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita) fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1 × 105 CFU/fish) at 35 day post-initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recorded highest RPS of 63.16% while the pGPD vaccinated group showed 47.37% when compared with 63.33% cumulative mortality of control group. The results regarding respiratory burst activity, myeloperoxidase activity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore, the current study displayed pGPD+IFN group having significant (p

The purpose of this study was to evaluate the dietary effects of Bacillus amyloliquefaciens COFCAU_P1 on immune-biochemical response, immune-related gene expression and resistance of Labeo rohita against Aeromonas hydrophila infection. Fingerlings of L. rohita were fed with three B. amyloliquefaciens-supplemented diets (1 × 107, 1 × 108 and 1 × 109 cells g−1 of diet) for 30 days. Different innate immune parameters, biochemical responses and immune-related gene expression were measured at the end of the 10, 20 and 30 days of feeding. Control and test fish were challenged intraperitoneally by A. hydrophila after 30 days of experimental feeding. Mortality was observed and cumulative mortality percentage was calculated. The immunological (superoxide anion, myeloperoxidase and antiprotease) and biochemical (total protein, albumin and globulin) responses were significantly (p

A study evaluating water quality and nutrient dynamics in inland saline water was carried out using biofloc technology with different C/N ratios employed to raise Litopenaeus vannamei juveniles. The study was carried out for 60 days in FRP with no water exchange. Salinity, temperature, Dissolved oxygen, pH, nutrients, Biofloc Volume and Total Suspended Solids were monitored. All biofloc units indicated reduction in alkalinity at 40th day except the treatment with highest (25:1) C/N ratio, pH and alkalinity was not significant different among various treatments. Dissolved Oxygen was found to be significantly decreasing with increasing C/N ratios. The least Dissolved Oxygen was recorded at higher C/N ratios of 20:1 and 25:1 at the end of experiment. The Total Ammonical Nitrogen (TAN) and NO2–Nin C/N ratios 15:1 and 20:1 were found to have a significantly decreasing in trend after 20th and 18th day respectively. The NO3–N, PO43-–P, BVF and TSS in biofloc were shown an increasing trend in all the experiments. The present study elucidates the suitability of optimum C/N ratios in biofloc for maintaining the water quality to raise L. vannamei in inland ground saline water. The finding could help in reducing the environmental concern saline waste water discharge from the shrimp pond to the land.

Columnaris disease, caused by Flavobacterium columnare, is one of the important bacterial diseases responsible for large-scale mortalities in numerous freshwater fishes globally. This disease can cause up to 100% mortality within 24 h of infection and is considered to be a cause of concern for aquaculture industry. Despite being a serious disease, scarce information is available regarding host-pathogen interaction, particularly the modulation of different immune genes in response to F. columnare infection. Therefore, in the present study, an attempt has been made to study expression of important immune regulatory genes, namely IL-1β, iNOS, INF-γ, IL-10, TGF-β, C3, MHC-I and MHC-II in gills and kidney of Catla catla following experimental infection with F. columnare. The expression analysis of immune genes revealed that transcript levels of IL-1β, iNOS, IL-10, TGF-β, C3 and MHC-I were significantly up-regulated (p

An experimental trial was conducted for 90 days to evaluate the growth performance, immunophysiological response of GIFT strain of Tilapia in biofloc-based rearing system and to assess the relative percentage survival in 3 days after challenging with the virulent strain of Aeromonas hydrophila. Fingerlings with an average body weight 0.98 ± 0.06 g were stocked in triplicate at different stocking densities of 200 (SD1), 250 (SD2), 300 (SD3) and 350 (SD4) m−3 in biofloc-based treatments and 150 (C) m−3 in control (clear water). Biofloc-based units (SD1 and SD2) obtained significantly better (P

Porous copper was electrodeposited using hydrogen bubble dynamic template. Various electrolytes having CuSO4 and H2SO4 in fixed molar ratio approximately equal to 0.3 and at temperature 18 °C were utilized for potentiostatic electrodeposition at various cathodic potentials. After electrodeposition, electrochemical impedance spectroscopy studies were conducted in 2.5 M NaCl solution at room temperature for determining electrochemically active true surface area. The surface morphologies of the deposits were analyzed using scanning electron microscope. Electrolyte with composition 0.12 M CuSO4 and 0.4 M H2SO4 resulted in maximum electrochemically active true surface area due to more open structure with thick dendrite branches which was correlated to high hydrogen evolution rate.

The main driver for National Surveillance Programme for Aquatic Animal Diseases (NSPAAD) in India is the threat of diseases to the growth of aquaculture. Keeping this in consideration, the Department of Fisheries (DoF), Government of India (GoI) is implementing NSPAAD involving 31 collaborating centres in 19 states, and this programme is being coordinated by ICAR-NBFGR, Lucknow. The programme has been successful in developing a network of aquatic animal health laboratories; diagnostic capability for detection of OIE/NACA-listed and emerging pathogens; mechanism for first time confirmation of new diseases; sending alerts/advisories to stakeholders; and providing scientific advice to the farmers. NSPAAD has helped in strengthening passive disease surveillance, which is evident by detection of seven new pathogens. It has enabled DoF in better understanding of disease situation in the country, and meeting international disease reporting obligations. Currently, DoF is contemplating to further strengthen the disease surveillance programme.

Infection with Aphanomyces invadans is a serious disease affecting freshwater and brackishwater fish. Even after 50 years of its first occurrence, to date, there is no effective treatment for this disease. To develop an effective treatment against this disease, in the current study, we have evaluated the potential of six commonly used chemicals and drugs with known antifungal properties against different life-stages (zoospore production, germination of zoospores and growth of hyphae) of A. invadans. The chemicals namely formalin, potassium permanganate (KMnO4), hydrogen peroxide (H2O2), sodium thiosulphate (Na2S2O3) and drugs including amphotericin B and fluconazole were evaluated in a dose-dependent manner (0.1, 1.0, 5, 10 and 100 ppm) under in vitro conditions. Out of the chemicals tested, formalin could completely inhibit zoospore production and significantly inhibit germination of zoospores and growth of hyphae at 10 ppm, however, complete inhibition of zoospore germination and growth of hyphae could only be observed at 100 ppm. In the case of H2O2, there was significant inhibition of germination of zoospores and growth of hyphae at the highest concentration of 100 ppm, but it could not completely inhibit zoospore production, germination of zoospores and growth of hyphae. KMnO4 at the highest tested concentration (100 ppm), completely inhibited zoospore production and significantly inhibited germination of zoospores and growth of hyphae. Na2S2O3 was found to be ineffective against all the three life-stages of A. invadans even at the highest tested concentration of 100 ppm. Out of the two antifungal drugs, fluconazole could reduce the germination of zoospores and growth of hyphae significantly at a dose of 1 ppm. On the other hand, in the case of amphotericin B, significant inhibition of germination of zoospores and growth of hypahe was observed only at 100 ppm. Both drugs completely inhibited zoospore production only at 100 ppm. From the findings of the present study, it can be inferred that out of the six compounds, formalin and fluconazole have better anti-oomycete effects under in vitro conditions. The information generated would serve as a basis for developing treatment measures against infection with A. invadans in aquaculture ponds.

Motile aeromonads are significant pathogens of freshwater ornamental fish, responsible for diverse types of infections. Among motile aeromonads, Aeromonas veronii is an important fish pathogenic bacterial species consisting of two biovars, veronii and sobria. In the present study,the pathogenicity of Aeromonas veronii isolated from infected goldfish (Carassius auratus) was assessed. A total of 68 bacterial isolates from 123 infected samples were presumptively identified as genus Aeromonas based on six biochemical tests. These isolates were recognized at the species level as A. veronii bv.sobria (5) and A. veronii bv.veronii (6) strains, and validated using the Moeller decarboxylase and dihydrolase test. In-vivo pathogenicity of A. veronii to C. auratus resulted in 100% morality in challenged host within one week of post experimental injection for both biovars. The isolates showed significant phenotypical activities such as the haemolytic, proteolytic, caseinase, nuclease, crystal violet binding activities and biofilm formation which are indicative of their virulence potential. Virulence genes such as the enterotoxin genes (act, alt and ast), haemolytic toxin genes (hlyA and aerA) and the gene of type IV pilus (tapA) were detected in both the biovars of A. veronii species, but ast and tapA genes were absent in A.veronii bv.sobria and A.veronii bv.veronii, respectively. All the isolates were resistant to penicillin-G, ampicillin, ticarcillin, amoxyclav, carbencillin, carbapenem, tetracycline, steroids, fluroquinolone, and trimethoprim. Although both the biovars were virulent, in- vivo study showed that A. veronii bv. sobria was more virulent than A. veronii bv. veronii in goldfish. Statement of relevance Aeromonas veronii can cause significant mortality in freshwater ornamental fish resulting in economic losses. Understanding the processes of A. veronii pathogenicity and virulence will assist in controlling this pathogen and the spread of disease.

Security is the major concern in mobile ad hoc network (MANETs) due to its dynamic behavior. Mobile ad hoc network is the collection of nodes and it is infrastructure fewer networks which means a node can join or leave in network simultaneously. Because of vibrant topology it is more susceptible to different kind of security attack such as Denial of service (DoS), wormhole, replay, masquerade, black hole etc. Such network uses routing protocol to transmit the packet from one end to another and each node behaves as host or router which can select suitable path for transmission of packet. In this, we determine the black hole attack in AODV routing protocol. Black hole is a network attack in which the malicious node advertises itself that it has freshest route to deliver the packet and then discard or drop it. This paper propose black hole attack detection using security algorithm and the simulation of this is perform on NS-2.34 network simulator.

Yellow pigmented, filamentous, Gram-negative bacteria belonging to genus Flavobacterium are commonly associated with infections in stressed fish. In this study, inter-species diversity of Flavobacterium was studied in apparently healthy freshwater farmed fishes. For this, ninety one yellow pigmented bacteria were isolated from skin and gill samples (n = 38) of three farmed fish species i.e. Labeo rohita, Catla catla and Cyprinus carpio. Among them, only twelve bacterial isolates (13.18%) were identified as Flavobacterium spp. on the basis of morphological, biochemical tests, partial 16S rDNA gene sequencing and phylogenetic analysis. On the basis of 16S rDNA gene sequencing, all the 12 isolates were 97.6-100% similar to six different formally described species of genus Flavobacterium. The 16S rDNA based phylogenetic analysis grouped these strains into six different clades. Of the 12 isolates, six strains (Fl9S1-6) grouped with F. suncheonense, two strains (Fl6I2, Fl6I3) with F. indicum and the rest four strains (Fl1A1, Fl2G1, Fl3H1 and Fl10T1) clustered with F. aquaticum, F. granuli, F. hercynium and F. terrae, respectively. None of these species except, F. hercynium were previously reported from fish. All the isolated Flavobacterium species possessed the ability of adhesion and biofilm formation to colonize the external surface of healthy fish. The present study is the first record of tropical freshwater farmed fishes as hosts to five environmentally associated species of the Flavobacterium.

In this study, we have described the development and characterization of monoclonal antibodies (MAbs) directed against thymocytes of rohu, Labeo rohita. MAbs were obtained by immunizing BALB/c mice with freshly isolated and nylon wool column enriched mononuclear cells of thymus. Positive clones against thymocytes were screened by cellular ELISA. The hybridoma showing strong reactivity with nylon wool enriched mononuclear cells, and non-reactivity with a rohu thymus macrophage cell line and rohu serum was selected and subjected to single cell cloning by limiting dilution. The MAbs secreted by a positive clone were designated as E6 MAb. Western blotting of reduced protein from enriched thymocytes showed that E6 reacted with a 166.2 kDa polypeptide and belongs to the IgG1 subclass. Flow cytometric analysis of gated lymphocytes, revealed that the percentage of E6 positive (E6+) cells in thymus (n = 5, 720.4 ± 79.70 g) was 89.7 %. Similarly, the percentage of E6+ cells in kidney, spleen and blood (n = 5) was 6.71, 1.71 and 1.88 %, respectively. In indirect immunoperoxidase test, E6+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio and were densely packed in the central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. E6 MAb also reacted with a small population of lymphocytes in blood smear. This MAb appears to be a suitable marker for T lymphocytes and can be a valuable tool in studying immune response and ontogeny of L. rohita immune system.

Disease surveillance programmes provide information on diseases that mitigate aquaculture production. Infectious myonecrosis virus (IMNV) has emerged recently as one of the OIE (Office International des Epizooties), the World Organisation for Animal Health listed viral diseases in penaeid shrimps, caused by dsRNA virus. The present study was conducted to investigate the prevalence of IMNV along the Indian coastal states. Samples of the Pacific whiteleg shrimp Penaeus vannamei and the green tiger shrimp Penaeus monodon were collected from 21 randomly selected active shrimp farms located along the coast using a risk-based two stage random sampling survey. Screening of the sampled shrimps based on nested RT-PCR diagnostic tool, did not detect IMNV in any of the samples examined indicating absence of IMN or IMNV incidence in shrimp aquaculture along Indian coastal states.

Infection with tilapia lake virus (TiLV) has been reported to cause large-scale mortalities in tilapines, the second most cultured species in the world. Interestingly, in polyculture system, although tilapia is reported to suffer high mortalities due to infection with TiLV, the disease has not been reported in co-cultured species. Nevertheless, natural host switch is a frequent phenomenon among viruses of lower vertebrates, especially fish viruses. To analyse the risk of spread of virus to new host species, it is essential to study the susceptibility of other fish species to the virus. In India, tilapia is generally cultured along with Indian major carps (IMC) in polyculture systems. Keeping this in consideration, in the present study, the susceptibility of rohu Labeo rohita, an important major carp, to TiLV was evaluated following experimental infection and it was compared with a susceptible host, i.e. Nile tilapia Oreochromis niloticus. The results revealed that the experimentally-infected rohu did not exhibit any clinical signs and mortality. On the other hand, in tilapia, a cumulative mortality of 80% was observed during the experimental period of 12 days with clinical signs typical of TiLV disease. In histopathological examination of liver, one of the main target organ of TiLV, typical syncytial cells were observed in tilapia whereas, no alterations were observed in rohu. Importantly, in rohu, the virus copy numbers in liver and intestine were almost negligible (

Fish waste water nutrient recycling in an aquaponic system was studied under different stocking densities of Koi Carp, Cyprinus carpio var. koi, along with spinach, Beta vulgaris var. bengalensis. Fish growth performance, plant growth, nutrient dynamics, and nutrient removal and their dependence on different stocking densities, namely 1.4, 2.1, and 2.8, were observed, of the different combinations, fish stocked at 1.4 kg/m3 had the best growth. Percent nutrient removal (NO3–N, PO4–P, and K) was significantly higher at 1.4 kg/m3. Thus, 1.4 kg/m3 stocking density can be suggested as optimum for Koi Carp production in spinach aquaponic systems.

The experiment was aimed at standardization of water flow rate in aquaponic system in order to correlate nutrient removal and water quality with growth of Cyprinus carpio var. koi (koi carp fingerlings) and Beta vulgaris var. bengalensis (spinach). Different flow rates, i.e., 3.2, 1.5, and 1.0 l min−1, were assigned as treatments T1, T2, and T3, respectively, with spinach plants (28 plant m−2), whereas S1 and S2 were the treatments having flow rates of 1.5 and 1.0 l min−1, respectively, without plants. Control (C) was set at flow rates of 3.2 l min−1 without plants. Treatment T2 (1.5 l min−1) showed highest weight gain of koi carp fingerlings and also height gain of spinach plants as compared to other treatments. There was no significant difference in length gain, percentage weight gain, specific growth rate, feed conversion ratio, feed efficiency ratio, and protein efficiency ratio as compared to other treatments and control. All the treatments, T1, T2, and T3, effectively remove nitrate (77–78 %), phosphate (47.04–55.06 %), and potassium (22.85–29.16 %) from fish effluent tanks. These results suggest that flow rates 3.2, 1.5, and 1.0 l min−1 were effective under aquaponic system. Of which, 1.5 l min−1 can be suggested as optimum water flow rate for the growth of spinach and koi carp in aquaponic system as percentage weight gain in fish, percentage height gain, and yield of plants were higher compared to flow rates 3.2 and 1.0 l min−1.

Many times heavy and continuous rains give rise to flooding in the rivers. Devastating floods occurred in rivers Ganga and Kosi in Bihar (India) and in Damodar and Hooghly rivers in West Bengal (India) during 2011. In the present paper, passive microwave remote sensing data is utilized for detection and monitoring of floods that occurred in Darbhanga (Bihar) and Midnapore (West Bengal) India, in the year 2011during monsoon season. Special Sensor Microwave Imager Sounder (SSMIS) brightness temperature (Tb) data at 19 GHz & 91 GHz in both vertical and horizontal polarisations and Soil Moisture and Ocean Salinity (SMOS) Tb data at 1.4 GHz are used to detect and monitor the flood occurrences. An attempt has been made to detect and monitor the flood events using 19 GHz Tb, difference of 19 and 91 GHz Tbs, and 1.4 GHz Tb, with both horizontal and vertical polarizations. Highest sensitivity to flood occurrence is observed for (19 GHz - 91 GHz) Tbs in horizontal polarization. Flood affected areas are mapped using (19 GHz - 91 GHz) Tbhs (Brightness temperatures with horizontal polarisation) values and compared with the Radarsat-1 images to show a general agreement between passive and active microwave remote sensing data. The comparison also shows an over-estimation of flooded area from passive microwave data.

India is the second largest fish producing country in the world with production of 12.6 million tonnes (mt) in 2017-18, and Indian major carps (IMCs) contribute bulk of this fish production. Catla, Catla catla is the fastest growing species among IMCs. However, the survival rate of catla during larval rearing is normally lower than the other IMCs i.e rohu Labeo rohita, and mrigal Cirrhinus mrigala. Continuous efforts are devoted for the identification of nutritional and environmental requirements of fish larvae in order to reduce hatchery mortalities. However, very little information is available regarding physiology of the immune system, especially during the late larval and juvenile stages. Hence, understanding the ontogenetic development of immune-relevant genes in the larval stages of catla will serve as the markers for the development of immune competence and thereby, will be beneficial in developing effective immune intervention strategies. In the present study, expression profiles of some of the important innate (IL-1β, IL-10, iNOS and C3) and adaptive immune (RAG-1, Ikaros, IgM and IgZ) genes during ontogenetic developmental stages and in different tissues of catla were investigated using quantitative real-time PCR. The results revealed that immune genes IL-1β, C3, IgM and IgZ were detected in the unfertilized eggs indicating their maternal inheritance. Immune genes, IL-1β, IL-10 and iNOS were expressed significantly during initial larval developmental stages whereas C3, RAG-1, Ikaros, IgM and IgZ showed significant expression during advanced stages of larval development in catla i.e. from 23 days post hatch (dph). Study of tissue distribution pattern of the genes indicated that innate immune genes were ubiquitously expressed in different tissues with varying degree of expression, whereas adaptive immune genes were predominantly expressed in lymphoid organs of catla. The information thus generated will improve knowledge on the maturation of the immune system in catla and will aid in deciding the appropriate age for vaccination in this teleost species.

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco's modified Eagle's medium at 37°C with 5% CO2 and showed 85+-% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28°C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.

Flavobacterium columnare is currently one of the important bacterial pathogens causing columnaris disease in several farmed fish species across diverse geographies. A presumptive colum naris disease outbreak in farmed catla, Catla catla (Hamilton), was investigated with the aim of isolating and identifying the causative pathogen. F. columnare (strain RDC-1) was isolated from gills of infected fish and identified by conventional biochemical methods, and through species specific polymerase chain reaction and sequencing of the 16S rDNA for molecular identification. Strain RDC-1 belonged to genomovar II with 99% similarity to available 16S rDNA sequences of F. columnare, and also shared 70% DNA-DNA relatedness with known strains of F. columnare. Bath immersion studies of RDC-1 showed development of columnaris disease in catla fin gerlings within 7 days, with a cumulative mortality of 83.3%. This is the first molecular confirma tion of Flavobacterium columnare as a fish pathogen of farmed Catla catla in India.