K.V. Rajendran

RNA interference-based therapeutics for shrimp viral diseases.

RNA interference (RNAi) has emerged as a powerful tool to manipulate gene expression in the laboratory. The presence of a double-stranded RNA (dsRNA) in eukaryotic cells triggers this post-transcriptional gene-silencing mechanism, leading to a sequence-specific degradation of the target mRNA. Among its many potential biomedical applications, silencing of viral genes stands out as a promising therapeutic strategy. Marine shrimp viral diseases, especially white spot disease (WSD), represents one of the most attractive targets for the development of therapeutic RNAi owing to its widespread economic impact. This review summarizes the current knowledge in the therapeutic application of RNAi for combating viral diseases in shrimp. The basic principles of RNAi are described, focusing on features important for its therapeutic manipulation. Subsequently, a stepwise strategy for the development of therapeutic RNAi is presented.

Production and characterization of monoclonal antibodies to the hemocytes of mud crab, Scylla serrata

Monoclonal antibodies (MAbs) to hemocytes of mud crab, Scylla serrata, were produced by immunizing mice with formalin-fixed hemocytes. Of the six MAbs produced, two (MAb 1D11 and MAb 1A2) reacted specifically with hemocyte proteins in western blot. MAb 1A2 showed strong immunofluorescent reaction with granular hemocytes and a weak reaction with semigranular cells. However, hyaline cells did not show any reaction. The MAbs also showed strong cross-reactivity with the hemocytes of different crab species but not with other crustaceans. The MAbs produced can be used as a marker for granular hemocytes of crabs.

Tissue specific expression profile of some immune related genes in Labeo rohita to Edwardsiella tarda infection

Abstract Rohu (Labeo rohita), an Indian Major Carp (IMC) is an economically important aquaculture species in India. Inspite of the technological advances, infectious diseases caused by viruses, bacteria and parasites have been a major limiting factor in the development and profitability of fish farms. At present, information regarding the immune status of the Indian major carps is limited. This lack of knowledge is a major impediment for establishment of effective preventive measures against broad spectrum of infectious agents. The present study was undertaken to examine the modulation of few immune‐regulatory genes: IgHC, NOD 1, TLR 22, iNOS and IL‐1&bgr; during experimental infection of E. tarda in L. rohita to understand their role in pathogenesis. Rohu fingerlings were intra‐peritoneally injected with Edwardsiella tarda (LD50 dose of 8.7 × 104 CFU/fish) and sampled for three immunologically important organs (kidney, liver and spleen) at different time intervals (zero hour or pre‐challenge and 6 h, 12 h, 24 h, 48 h and 96 h post challenge). For absolute quantification of genes by real time RT‐PCR, all the genes transcript were amplified from Poly I:C induced rohu lymphocytes and cloned in pTZ57R/T plasmid. Standard curves for each gene was generated from serially diluted plasmid bearing respective genes. Evaluation of copy number of different genes present in the tissue showed that the expression of IgHC, iNOS and IL‐1&bgr; was highest in kidney followed by spleen and least in liver. While for NOD 1 and TLR 22 gene, liver showed higher expression than kidney and spleen. Further, the expression of IgHC, INOS, TLR 22, NOD 1 and IL‐1&bgr; genes significantly differed (P < 0.05) in the E. tarda challenged fish when compared with pre‐challenged control fish. Among the five genes we studied, the basal expression of TLR 22 gene was highest. The result also depicts that iNOS and NOD 1 are immediate responsive genes as their expression reached maximum level at 6–24 h post infection (hpi) after which the expression declined. In contrast, TLR 22 and IgHC gene transcript showed enhanced expression during the late phase of with maximum expression observed after 48 hpi and 96 hpi respectively. IL‐1&bgr;, being the exception, showed high expression both at 24 hpi and 96 hpi. From this study, we conclude that these five immune genes have a definite role to play in the defense mechanism of host (L. rohita) against E. tarda. HighlightsPathogenesis related to Edwardsiella tarda on Labeo rohita was studied.E. tarda exposure modulates TLR22, NOD1, IgHC, iNOS, IL‐1&bgr; expression in L. rohita.Involvement of various immune pathways in disease processes was determined.Expression patterns demonstrated orchestration of various organs in host defences.Absolute quantization method used can be a referendum for various gene transcripts.

Monoclonal antibodies against recombinant GAPDH of Edwardsiella tarda reveal the conserved nature of the protein

ABSTRACT The outer membrane protein, encoded by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, of Edwardsiella tarda is a highly conserved immunogenic protein. The GAPDH was cloned and expressed in Escherichia coli. The purified protein was used to produce mouse monoclonal antibodies (MAbs). Four stable hybridomas producing MAbs (3G12, 4E9, 5A11 and 9G1) against rGAPDH were obtained. The heavy chains of antibodies produced by the hybridomas were of the isotypes IgG1 and IgM. Cross reactivity of MAbs (3G12 and 9G1) was observed with GAPDH of Aeromonas hydrophila and Micrococcus luteus. MAbs 3G12 and 4E9 reacted with Vibrio cholerae, Salmonella enterica and Penaeus monodon tissues but not with vertebrate GAPDH. None of the MAbs reacted with Staphylococcus aureus. The results indicate that the level of conservation of GAPDH is high among evolutionarily close species. The MAbs developed will be a useful tool to study the evolutionarily conserved and functionally diverse GAPDH.

Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes

DNA-based immunization has proven to be an effective prophylactic measure to control aquatic animal diseases. In order to improve the efficiency of vaccine against fish pathogen, novel delivery mechanism needs to be adopted. In the present study we nanoconjugated the previously constructed DNA vaccine (pGPD + IFN) with chitosan nanoparticles (CNPs) by complex coacervation process. After construction of the vaccine, an in vivo vaccination trial was conducted in which 2 groups of rohu (L. rohita) fingerlings were vaccinated with CNPs-pGPD + IFN, one group by oral route (incorporated in feed for 14 days) and the other by immersion route (primary and booster immunised), whereas, a third group was intramuscularly (I/M) injected (initial and booster immunised) with naked pGPD + IFN and subsequently challenged with E. tarda (8.7 × 104 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, expression kinetics of immune-related genes and pathological manifestation. Evaluation of RPS analysis revealed that CNPs-pGPD + IFN groups recorded highest RPS (81.82% and 72.73% in oral and immersion vaccinated fish group respectively) while the naked pGPD + IFN injected group showed 63.62% RPS when compared with 55% cumulative mortality of control group. In addition, NBT, myeloperoxidase activity, serum lysozyme activity and specific antibody titre in case of CNPs-pGPD + IFN groups showed higher activities during all the time points. Furthermore, CNPs-pGPD + IFN groups showed significant (p < 0.05) upregulation of different immune gene transcripts (IgHC, iNOS, TLR22, NOD1 and IL-1β) in three immunologically important tissues post immunization (both primary and booster dose) as well as after challenge. Thus, from this study, we can conclude that oral or immersion vaccination with CNPs-pGPD + IFN can orchestrate an effective immunisation strategy in organizing a coordinative immune response against E. tarda in L. rohita exhibiting minimum stress to the host with maximum efficacy.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in Asian seabass, Lates calcarifer: Cloning, ontogeny and expression analysis following bacterial infection or ligand stimulation

ABSTRACT NOD1 (Nucleotide‐binding oligomerization domain‐containing protein 1) is one of the most prominent intracellular Nod‐like receptors (NLRs), responsible for detecting different microbial components and products arising from tissue injury. Here, we have identified and cloned NOD1 transcript in the Asian seabass, Lates calcarifer (AsNOD1), which consists of 3749 nucleotides and encodes for a predicted putative protein of 900 AA. The AsNOD1 possesses the typical structure of NLR family, consisting of N‐terminal CARD domain, centrally located NACHT domain and C‐terminal LRRs. The AsNOD1 showed ubiquitous tissue expression in 11 different tissues of healthy animals tested with high levels of expression in hindgut and gill. From the ontogenetic expression profile of AsNOD1, it is quite evident that this gene might follow a maternally‐transferred trend in euryhaline teleosts, as it is highly abundant in embryonic developmental stages. The constitutive immunomodulation of AsNOD1 in terms of expression level was clearly evident in the different tissues of Asian seabass‐injected either with Vibrio alginolyticus or poly I:C. However, injection with Staphylococcus aureus did not elicit similar immunomodulation except for the up‐regulation noticed at few time‐points in some tissues. SISK‐cell line induced with different ligands such as poly I:C, LPS and PGN also showed up‐regulation of AsNOD1 in certain time‐points in vitro. Based on the results obtained in the present study, it can be inferred that the AsNOD1 might play an immunoregulatory role upon exposure to different bacterial as well as viral PAMPs and also might be an important component of innate immune element during embryonic and larval development in the euryhaline teleost Asian seabass. HIGHLIGHTSAsian seabass NOD1 (AsNOD1) was identified, cloned and characterized.AsNOD1 was constitutively expressed in developmental stages.In healthy animals, AsNOD1 was highly expressed in hindgut and gill.V. aliginolyticus, poly I:C and bacterial ligands modulate the AsNOD1 expression both in vivo and in vitro.AsNOD1 might have both antiviral and antibacterial functions in marine euryhaline teleost.

Microscopic and cytochemical characterisation of haemocytes of the mud crab Scylla serrata (Forskål, 1775) (Decapoda, Portunidae)

Haemocytes of the mud crab Scylla serrata (Forskal, 1775) were characterised based on morphological features using light and electron microscopy, and cytochemistry. The cells were identified as hyaline, semigranular and granular haemocytes. Hyaline cells were the smallest haemocytes among the three types identified, having the highest nucleo-cytoplasmic ratio. The cells showed a number of cytoplasmic organelles and also contained a few small as well as large-sized granules. Semigranular haemocytes possessed moderate numbers of large-sized granules or numerous small-sized granules and comparatively less numbers of organelles. Granular haemocytes were the largest haemocytes with the lowest nucleo-cytoplasmic ratio and contained many large-sized granules. Cytoplasmic organelles were least observed in the granular haemocytes. These three haemocyte morphotypes constituted 60, 21 and 19%, respectively, of the total haemocyte population, while the total haemocyte count was 7.31 × 106 to 7.18 × 107 with a mean of 2.86 × 107 cells ml−1. In cytochemical studies performed to localize carbohydrates, lipids and prophenol oxidase, all the haemocyte types were positive for PAS and toluidine blue, indicating the presence of mucopolysaccharides, whereas semigranular and granular haemocytes were rich in carbohydrates and lipid moieties. Besides, prophenol oxidase was localised within the granules of semigranular and granular haemocytes. Hyaline haemocytes showed an abundance of well differentiated cytoplasmic organelles and granules, and there was a distinct differentiation between semigranular and granular haemocytes in terms of granules and organelles. This is the first report of the characterisation of haemocytes of the mud crab.