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Trained immunity refers to the memory acquired by innate immune cells, leading to cross-protection and non-specific responses to subsequent infection, thereby improving host survival. Trained immunity induction is a combined effect of immune signaling, metabolic changes, and epigenetic modifications. The present study evaluated the induction of markers of the phenomenon of trained immunity in common carp, which is trained using β-glucan. The mammalian target of rapamycin (mtor) and hypoxia-inducible factor (hif1α), the metabolic basis of trained immunity; the histone deacetylase (hdac7), one of the markers of epigenetic modifications, metabolic activity of activated cells and expression profiles of proinflammatory cytokines viz. il6a, tnfαa2, and ifnγ were targeted in the study and analyzed in vivo. Besides in vivo analysis, in vitro analysis of mtorc2, hif1α, hdac7, and ifnγ were analyzed. In vitro analyses were performed on head kidney macrophages isolated and maintained in L-15 media and double trained with β-glucan at 100μg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with β-glucan at 20 mg/kg body weight for training followed by a resting phase for 6 days and were restimulated with the same dose. Head kidney was collected from the fish post-induction as well as post-restimulation. The expression profile of mtorc2, hdac7, and hif1α were found elevated post-stimulation of β-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-β-glucan stimulation was confirmed by the high lactate and LDH production detected in the cell culture supernatant. Overall, the study revealed the expression profile of the trained immunity markers and the increased metabolic activity in cells induced with β-glucan, which further validates that the action of trained immunity is indispensable in fish on encounter with a potential ligand. The study supports the existing reports on trained immunity in teleost fish with evidence at the genomic level. However, further studies are required to understand the responses and actions of trained immune cells during infection in detail.

Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde-3-phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic-co-glycolic acid) - Chitosan (PLGA-Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated. Two treatment groups [T1 - (PLGA-Chit-NPs-pDNA), T2 - (PLGA-NPs-pDNA) and one control group (T0 - 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT-PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.

Tilapia lake virus (TiLV) is a serious pathogen of farmed Nile tilapia (Oreochromis niloticus) responsible for significant mortalities. In this study, we investigated a disease outbreak in cage-farmed Nile tilapia in India. The infected fish exhibited clinical signs such as severe scale loss, haemorrhage, exophthalmia, and fin and tail rot. The samples were screened for Tilapia lake virus (TiLV) by reverse-transcriptase PCR and also subjected to detailed bacteriological investigation to understand the association between TiLV and co-infecting bacterial pathogens. Bacteria were isolated from TiLV-infected and apparently healthy fish, and identified by conventional microbiological methods, followed by 16SrRNA gene sequencing. TiLV was detected by PCR in all the samples exhibiting clinical signs, while apparently healthy fish were negative for the virus. A total of 34 bacterial isolates belonging to the genera Aeromonas, Staphylococcus, Enterococcus, Plesiomonas, Enterobacter, Bacillus, Lysinibacillus, Solibacillus and Exiguobacterium were isolated from the virus-infected tilapia. However, Aeromonas veronii was found to be the most dominant bacterium isolated from the surface lesions and the internal organs of all infected fish. Antibiotic susceptibility testing revealed that A. veronii, by far, was susceptible to cephalosporins (ceftriaxone, cefotaxime, cefpodoxime), chloramphenicol, aminoglycosides (amikacin, gentamicin), ciprofloxacin and chloramphenicol. Experimental infection using intraperitoneally injected A. veronii reproduced the clinical signs of naturally infected Nile tilapia, and a lethal dose 50 (LD50) mortality was observed by day 7 post-challenge. Furthermore, A. veronii could be re-isolated from the experimentally infected fish. Based on this evidence, we propose that virulent A. veronii as a co-infecting bacterium can have an important role in the severity and outcome of the disease in Nile tilapia infected by TiLV.

Amphiprion ocellaris (ocellaris clownfish) is one of the most commercially important marine ornamental fish. A cell line designated as OCF was developed for the first time from the caudal fin of this fish species. The cell line was maintained in Leibovitz’s—15 medium supplemented with 15% FBS (Fetal Bovine Serum) and was successfully subcultured up to 34 passages. The cell line was authenticated by sequencing mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes. The growth rate of the OCF cell line was maximum in medium containing 20% FBS and 1% of 0.2 M NaCl at 28 °C. Chromosome analysis revealed 48 diploid chromosomes. The OCF cell line was transfected with the pMaxGFP plasmid vector with 7% efficiency and GFP expression was observed. The OCF cell line was used for testing nervous necrosis virus (NNV) susceptibility. Cytopathic effect (CPE) was observed in terms of plaque formation after virus inoculation. Nested PCR confirmed the susceptibility of the OCF cell line to NNV. The cell line was successfully cryopreserved by a slow freezing procedure at − 80 °C with a revival efficiency of 70–75%. The study revealed that the OCF cell line would be useful for virological studies. In addition, the cell line would play an important role as an in vitro tool for carrying out toxicological and biotechnological studies.

Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is an emerging fish virus that primarily affects tilapines. However, the virus has also been detected in a few non-tilapines. As tilapia is generally farmed in polyculture systems along with carps in South Asian countries, there is a likelihood that TiLV-infected tilapia can transmit the virus to the co-cultured species. In view of the above, the susceptibility of three carp species, namely catla (Catla catla), mrigal (Cirrhinus mrigala) and silver carp (Hypophthalmichthys molitrix) was evaluated vis-à-vis tilapia, following experimental infection with TiLV. No clinical signs and histopathological alterations could be observed in carps. RT-qPCR revealed that TiLV copy numbers in liver and brain of all the three carps were almost negligible and did not show any increase with time, suggesting that the virus did not replicate in liver and brain, the target organs of TiLV. Further, TiLV could not be isolated from pooled liver and brain tissues of carps using permissive CFF cell line. On the contrary, in tilapia, typical clinical signs and histopathological lesions were observed and there was significant increase in TiLV copy number up to 6 days post-injection. Furthermore, the virus was successfully isolated from pooled liver and brain tissue of infected tilapia. From the above findings, it could be concluded that C. catla, C. mrigala and H. molitrix are resistant to TiLV infection and unlikely to be carriers for this virus.

White spot syndrome virus (WSSV) is a pathogen causing significant economic losses to shrimp aquaculture worldwide. Previously, five genome sequences of the virus from farmed shrimp ( Penaeus vannamei and Penaeus monodon ) in India were reported, all originating from farms located on the east coast of the country. Here, we report three new and distinct WSSV genome sequences, two from shrimp ( P. vannamei ) farmed on the west coast of India and the third from the east coast.

Histamine poisoning occurs when temperature-abused marine fish containing elevated levels of histamine are consumed. Histamine-producing bacteria found in fish can colonize processing surfaces and form biofilms. In this study, the biofilm-forming abilities of histamine-producing bacteria from Indian mackerel (Rastrelliger kanagurta) and the effect of hypochlorite treatment on biofilm formation were studied. The isolates of this study produced histamine in the range of 471 to 2,126 ppm. The histidine decarboxylase gene hdc was detected in all isolates producing histamine except in one strain each of Psychrobacter pulmonis and Proteus vulgaris. All isolates tested in this study produced moderate biofilms under control conditions, whereas exposure to 1 and 3 ppm of sodium hypochlorite significantly enhanced biofilm formation. However, exposure to 5 ppm of sodium hypochlorite showed an inhibitory effect on biofilm formation by all the isolates except Klebsiella variicola. The results of this study suggest that histamine-producing bacteria can form stable biofilms and that this activity may be enhanced by the application of low levels of sodium hypochlorite, a phenomenon that might influence the persistence of histamine-producing bacteria in fish processing areas.

Polymeric immunoglobulin receptor (pIgR) is a protein that transports Immunoglobulins (Igs) from epithelial cells into the external secretion system of the animal. In the present study, we characterized the partial pIgR gene from Labeo rohita and analyzed its expression in response to the pDNA (pGPD-IFN) vaccine, and studied its correlation with the expression of IgM, in the mucosal-associated lymphoid tissues (MALT). The significant (p

Hypoxia in aquatic environment is often a recurrent stressor which can affect growth and health of fishes. However, nutritional intervention can help aquatic animals to cope up with environmental stressors so as to alleviate the adverse effects. Here, we examined the impact of L-Arginine (Arg) supplementation on hypoxia-induced changes in immune responses and resistance to Aeromonas hydrophila infection in Indian Major Carp, Cirrhinus mrigala. The fish were fed with graded levels of Arg for 60 days and exposed to 72 h sublethal level of hypoxia (0.50 ± 0.16 mg/L dissolved oxygen [DO]). The six treatment groups with three replicates having different levels of Arginine such as N0.0 (0% Arg + Normoxia), H0.0 (0% Arg + Hypoxia), N0.7 (0.70% Arg + Normoxia), H0.7 (0.70% Arg + Hypoxia), N1.4 (1.40% Arg + Normoxia), and H1.4 (1.40% Arg + Hypoxia) were used in the study. Subsequently, the fish of different treatment groups were challenged with A. hydrophila. The hypoxia exposed fish exhibited significantly higher (P

DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 degrees C, when 2 mu g of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.