Short peptidoglycan recognition protein 5 modulates immune response to bacteria in Indian major carp, Cirrhinusmrigala.

Peptidoglycan recognition proteins (PGRPs) function in host antibacterial responses by recognizing bacterial peptidoglycan (PGN). In the present study, a short pgrp5 (named mpgrp5) was identified in Cirrhinus mrigala (mrigal). The full-length cDNA of the mpgrp5 gene was 1255 bp, containing an open reading frame of 746 bp encoding a protein of 248 amino acids. The predicted protein contained the typical Pgrp/amidase domain, conserved Zn2+, and PGN binding residues. The phylogenetic analysis revealed that the mpgrp5 is closely related to Pgrps reported in Labeo rohita, Cyrinus carpio, and Ctenopharyngodon idella. The ontogenetic expression of mpgrp5 was highest at 7 days post-hatching (dph) and its possible maternal transfer. mpgrp5 was constitutively expressed in all tissues examined, with the highest expression observed in the intestine. Furthermore, mpgrp5 was found upregulated in mrigal post-challenge in a time-dependent manner at 6hpi in the liver (3.16 folds, p < 0.05) and kidney (2.79 folds, p < 0.05) and at 12hpi in gill (1.90 folds, p < 0.01), skin (1.93 folds, p < 0.01), and intestine, (2.71 folds, p < 0.05) whereas at 24hpi in spleen (4.0 folds, p < 0.01). Our results suggest that mpgrp5 may play an important role in antibacterial immune response from early life stages in mrigal.

Βeta-glucan stimulation induces trained immunity markers in common carp, Cyprinus carpio.

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 ß-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 ß-glucan at 100µg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with ß-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 ß-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-ß-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 ß-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.

Multi-drug resistance, integron and transposon-mediated gene transfer in heterotrophic bacteria from Penaeus vannamei and its culture environment.

Multi-drug resistance (MDR) in bacteria is regarded as an emerging pollutant in different food production avenues including aquaculture. One hundred and sixty out of 2304 bacterial isolates from shrimp farm samples (n = 192) of Andhra Pradesh, India, were MDR. Based on biochemical identification and 16S rRNA sequencing, they were grouped into 35 bacterial species with the predominance of Vibrio parahaemolyticus (12.5%). The MDR isolates showed highest resistance toward oxytetracycline (89%) with more than 0.2 MAR (multiple antibiotic resistance), demonstrates a high-risk source. The most prevalent antibiotic-resistance gene (ARG) and mobile genetic element (MGE) detected were tetA (47.5%) and int1 (46.2%), respectively. In conjugation experiments, overall transfer frequency was found to be in the range of 1.1 × 10-9 to 1.8 × 10-3 with the transconjugants harbouring ARGs and MGEs. This study exposed the wide distribution of MDR bacteria in shrimp and its environment, which can further aggravate the already raised concerns of antibiotic residues in the absence of proper mitigation measures.

Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming.

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.

Bicistronic DNA vaccine macromolecule complexed with poly lactic-co-glycolic acid-chitosan nanoparticles enhanced the mucosal immunity of Labeo rohita against Edwardsiella tarda infection.

DNA vaccine is an important tool to elicit both humoral and cellular immunity. Present study investigates mucosal immune response of Labeo rohita (15 ± 04 g) to plasmid DNA (pDNA) vaccine macromolecule complexed with nanoparticles (NPs). Poly lactic-co-glycolic acid (PLGA), Chitosan (Chit) and PLGA-Chit-NPs were synthesized by double emulsion solvent evaporation method. Synthesized NPs were complexed with pDNA (pGPD + IFN) vaccine construct. Size and zeta potential of PLGA-NPs, Chit-NPs and PLGA-Chit-NPs-pDNA complex were recorded to be 120 nm and +0.5 mV, 117 nm and +32 mV, 189 nm and +11 mV, respectively. Immunization by immersion was carried out in two groups receiving PLGA-Chit-NPs-pDNA (T1) and PLGA-NPs-pDNA (T2) respectively. After immersion, samples were collected on 0, 2, 4, 7, 15 and 30 days from mucosa-associated lymphoid tissues (MALT) for mRNA expression studies of IgM, IgD and IgZ using qRT-PCR. Significant up-regulation of the mRNA expression of IgM, IgD, and IgT were observed in MALT in immunized fish compared to control. After 30 days post-immunization fish were infected with a virulent strain of Edwardsiella tarda. The highest relative percentage survival was observed in T1 (64.7%) compared to T2. The study showed better efficiency of pDNA-PLGA-Chit-NPs compared to pDNA-PLGA-NPs for inducing adaptive mucosal immunity in fish.

Interferon-regulatory factors, IRF3 and IRF7 in Asian seabass, Lates calcarifer: Characterization, ontogeny and transcriptional modulation upon challenge with nervous necrosis virus.

Interferon regulatory factor (IRF) 3 and IRF7 are key regulators of type I interferon (IFN) gene expression for the antiviral immune response. In the present study, interferon regulatory factor 3 and 7 from Asian seabass, namely AsIRF3 and AsIRF7 were cloned and characterized. The full-length cDNA sequence of IRF3 and IRF7 consisted of 2965 and 2343 bp respectively. AsIRF3 and AsIRF7 were true orthologes of vertebrate IRF3/7 and showed similar domain organization, with an N-terminal DBD which consisted five tryptophan residues in IRF3 and four in IRF7, a C-terminal IRF3 domain and a serine rich region. Both IRF3 and 7 constitutively expressed during the ontogenesis and in all tissues of healthy fish. The expression of both genes was up-regulated following NNV challenge with obvious transcript abundance in brain heart and kidney. Ectopic expression of AsIRF3 and AsIRF7 displayed activation of ISRE/NF-κB promoters and modulation of interferon, ISGs and pro-inflammatory cytokine gene expression. These observations indicated that IRF3 and IRF7 play an important role in Asian seabass's antiviral defense and the RIG-IRF-IFN axis is conserved in the species.

Antiviral activity of transiently expressed mitochondrial antiviral signaling adapter, MAVS orthologue from Asian seabass.

The innate immune signaling adapter, Mitochondrial antiviral signaling protein (MAVS) coordinates the signals received from two independent RLRs (RIG-1 and MDA5) to induce IFN & interferon stimulatory genes (ISGs). In the present study, we report identification of an orthologue of MAVS from Lates calcarifer (LcMAVS) and its functional role in piscine RLR signaling. The LcMAVS-cDNA was cloned into pcDNA and transfected into SISS cells. LcMAVS was detected to be a 61KDa protein in western blot. Confocal microscopy demonstrated the mitochondrial localization of LcMAVS. In addition, pcDNA-MAVS transfected cells were protected against Nervous Necrosis Virus (NNV) infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral transcript levels. Ectopic expression of LcMAVS resulted in activation of an ISRE-containing promoter (52 folds over control cells) as well as transcriptional expression of IRF-3, IFN-1 and IFN-inducible genes including Mx and ISG15 (p<0.05). These results suggest that LcMAVS is involved in the antiviral immunity as one of the adaptors in fish IFN-activation pathway.

Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (∼207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish.

Expression Profile of Penaeus monodon Ubiquitin Conjugating Enzyme (PmUbc) at Protein Level in White spot syndrome virus Challenged Shrimp.

White spot syndrome virus (WSSV) is one of the major pathogens in shrimp aquaculture. Four proteins of WSSV are predicted to encode a RING H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimps can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, expression profile of Penaeus monodon Ubiquitin conjugating enzyme (PmUbc) was studied at protein level in WSSV challenged shrimp. A time point analysis of the expression of PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge in P. monodon. Recombinant PmUbc (rPmUbc) was produced in prokaryotic expression vector, BL21 (DE3) pLys S. The PmUbc expression pattern was studied by ELISA with rPmUbc antibodies raised in rabbit. A significant increase in PmUbc expression at 24 h post infection (hpi) was observed followed by a decline till 72 hpi. Since the up-regulation and a tremendous decline of PmUbc protein expression was observed at 24 and in 72 hpi respectively in ELISA, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. Many findings have shown that viral infection can up-regulate expression of ubiquitin and that the ubiquitin system plays a key role in the course of viral infection. The present study reveals the expression patterns of PmUbc at protein level in WSSV infected P. monodon. However, further studies are to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this major culprit in shrimp culture industry.