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.
Emerging and re-emerging diseases in fish cause drastic economic losses in the aquaculture sector. To combat the impact of disease outbreaks and prevent the emergence of infections in culture ...systems, understanding the advanced strategies for protecting fish against infections is inevitable in fish health research. Therefore, the present study aimed to evaluate the induction of trained immunity and its protective efficacy against Streptococcus agalactiae in tilapia. For this, Nile tilapia and the Tilapia head kidney macrophage primary culture were primed using β-glucan @200 μg/10 g body weight and 10 μg/mL respectively. Expression profiles of the markers of trained immunity and production of metabolites were monitored at different time points, post-priming and training, which depicted enhanced responsiveness. Higher lactate and lactate dehydrogenase (LDH) production in vitro suggests heightened glycolysis induced by priming of the cells using β-glucan. A survival rate of 60% was observed in β-glucan trained fish post challenge with virulent S. agalactiae at an LD50 of 2.6 × 107 cfu/ml, providing valuable insights into promising strategies of trained immunity for combating infections in fish.
•Innate immune training using β-glucan can elevate cytokine transcript levels.•Expression of trained immunity markers, mTOR and HIF1α elevated post immune training with β-glucan in tilapia.•Higher lactate and LDH production post β-glucan induction in the supernatant of tilapia head kidney macrophage culture.•Survival rate of 60% in Streptococcus agalactiae-challenged immune trained tilapia.
Consumption of temperature-abused marine fish containing elevated levels of histamine results in histamine poisoning. Histamine is a biogenic amine produced in fish by the action of certain groups of ...bacteria which are capable of producing an exogenous enzyme called histidine decarboxylase (HDC). Morganella morganii is one of the major causative organisms of histamine poisoning. In this study, the histamine forming potential of M. morganii (BSS142) was evaluated when it was co-incubated with proteolytic as well as polyamine forming bacteria. This experiment was designed to examine whether biotic factors such as proteolysis and the presence of other amines influenced histamine forming ability of BSS142. The study showed that the proteolytic activity of Aeromonas hydrophila as well as Pseudomonas aeruginosa greatly enhanced the histamine forming ability of M. morganii. Psychrobacter sangunis, a non proteolytic polyamine producer, negatively influenced histamine production by M. morganii.
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.
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 °C, when 2 µg 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.
Noroviruses are significant seafood-borne pathogens, commonly associated with the consumption of filter feeding bivalve molluscs. Here, we report the development of a reverse transcription polymerase ...chain reaction (RT-PCR) method using primers based on the RNA-dependent RNA polymerase gene of norovirus genogroup II (NoV GII). Samples of bivalves were processed for the concentration of virus and extraction of RNA, followed by reverse transcription PCR. A total of 50 molluscan shellfish samples were analyzed, of which 16 samples yielded positive amplifications of norovirus nucleic acid. The PCR method described here, involving a single set of primers, is useful for rapid screening of shellfish for NoV GII.
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 (LD
50
) 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.
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.
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.
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•Expression profiles of trained immunity markers in common carp post immune training with β-glucan.•Elevated expression profiles of trained immunity markers and proinflammatory cytokines were observed post-training.•Increase in metabolic activity and production of metabolites revalidates the phenomenon of the metabolic shift in trained immune cells.
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 < 0.05) up-regulation of pIgR mRNA was observed in the mucosal tissues of vaccinated fish by qRT-PCR. The highest expression of the gene was detected in gill tissue at 15 days post-vaccination (dpv) followed by skin and gut at 30 dpv. The expression of IgM also showed a similar pattern and indicated a direct correlation of pIgR expression in all the tested tissues. The protective immune response of the DNA vaccine was measured as the relative percentage of survival (RPS) by challenging vaccinated fish with live Edwardsiella tarda (1 × 105 CFU/Fish). The result showed a significantly high relative percentage survival (RPS) in the vaccinated group (47.05%) compared to the control group. Many factors contributing to the immune response of the vaccine. One of the most critical aspects is the rise in IgM level in local tissues. In other higher vertebrates, pIgR is reported to act as the transporter of IgM. The positive correlation in the expression of pIgR and IgM observed in the present study demonstrated the possible role of pIgR as the transporter of IgM in L. rohita. The study suggests the possibility of the secretory nature of IgM in fish.
•Characterization of polymeric immunoglobulin receptor gene (pIgR) in L. rohita•mRNA expression of pIgR and IgM gene in mucosal associated lymphoid tissues in response to DNA vaccine and E. tarda infection•Vaccine efficacy study against E. tarda infection in terms of relative percentage survival (RPS).
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