Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp.

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus.

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these âˆ¼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

A Biofloc-Based Aquaculture System Bio-augmented with Probiotic Bacteria Bacillus tequilensis AP BFT3 Improves Culture Environment, Production Performances, and Proteomic Changes in Penaeus vannamei.

Experiments were conducted to evaluate the probiotic effect of bio-augmented Bacillus tequilensis AP BFT3 on improving production, immune response, and proteomic changes of Penaeus vannamei reared in a biofloc system. Penaeus vannamei larvae (PL13) were stocked in 100-L tanks at a rate of 100 no per tank to study the effect of B. tequilensis AP BFT3 with and without biofloc (BFT-PRO and PRO). Control tanks devoid of probiotic strain were maintained in a clear water system. The growth and survival considerably increased in probiotic added biofloc reared shrimp than probiotic added clear water reared ones and control. Water quality significantly improved in probiotic added (PRO) and biofloc-probiotics (BFT-PRO) system than control. Microbiological investigations indicate increased heterotrophic bacterial load in BFT-PRO compared to the PRO and control. The quality of the isolated microbes was analyzed in terms of enzyme production, and an abundance of enzyme-producing bacterial population was observed in BFT-PRO shrimp. Immune-related genes were significantly upregulated in BFT-PRO shrimp, followed by the PRO and control. The proteomic data (2D gel electrophoresis and MALDI-TOF) of muscle tissue from the experimental animals identified 11 differentially expressed proteins. The Daxx OS and Lit v 1 tropomyosin was found upregulated in BFT-PRO shrimps. Downregulation of Na+/K+ATPase was observed in biofloc with probiotic-supplied groups. The findings revealed that the BFT system's efficacy could be improved through the addition of probiotics. The addition of B. tequilensis AP BFT3 as a probiotic in biofloc induced the expression of essential proteins, reducing contracting diseases during culture.

Marine actinomycetes Nocardiopsis alba MCCB 110 has immunomodulatory property in the tiger shrimp Penaeus monodon.

Shrimp farming constitutes an important source of revenue and employment in many developing countries. However, the shrimp industry has always been plagued with infectious diseases having varied aetiologies. Dominated by non - specific immune mechanism, preventive health care strategy is the most appropriate approach to protect the crop. The present study evaluated the efficacy of an actinomycete, Nocardiopsis alba MCCB 110 in eliciting non - specific immune mechanism in Penaeus monodon having Vibrio harveyi as the challenge organism. Haemocyte count, total protein, phenoloxidase, reactive oxygen intermediates, acid and alkaline phosphatase as well as the gene expression of proPO, peroxinectin, transglutaminase, alpha 2-macroglobulin, astakine, crustin, and penaeidin-3 were evaluated. The results demonstrated that the phenoloxidase, respiratory burst, total protein, acid and alkaline phosphatases were higher in the haemolymph of shrimps fed with Nocardiopsis alba MCCB 110 incorporated feed before and after challenge with Vibrio harveyi, compared to those of placebo fed animals. Up-regulation of six immune genes (alpha 2 macroglobulin, penaeidin -3, transglutaminase, proPO, crustin and peroxinectin) during the post-challenge were recorded. Survival of shrimp among the Nocardiopsis alba administered ones was 83% while it was 50% in placebo fed group. The elevated levels of nonspecific immune gene transcripts and concurrent increase in non specific immunity besides the higher survival rate in the Nocardiopsis alba administered group demonstrated the immunomodulatory property of the marine actinomycete Nocardiopsis alba MCCB 110 in the tiger shrimp Penaeus monodon, and on administering it through diet shrimp could be protected from vibriosis especially of V. harveyi.

Bio-augmentation of heterotrophic bacteria in biofloc system improves growth, survival, and immunity of Indian white shrimp Penaeus indicus.

Effect of bio-augmentation of Bacillus spp in biofloc on growth, survival and immunity in Indian white shrimp Penaeus indicus was evaluated. Nine Bacillus strains were isolated and screened individually as well as in the form of a consortia. To maintain a C:N ratio of 12:1 a blend of carbohydrate sources was used. Bio-augmentation with bacterial consortium and Virgibacillus sp. produced improved growth and immunity. Shrimp survival ranged from 80 to 95% among treatments. Production was higher (35%) in the biofloc tanks with an average body weight (ABW) of 10.89 ± 1.2 g. On evaluating the immune responses, it was found that trypsin significantly (P < 0.05) enhanced Prophenoloxidase (PO) activity in Lysinibacillus, Bacillus cereus, Bacillus licheniformis and Bacillus subtilis bio-augmented groups. Laminarin induced PO activity was observed in groups supplemented with Oceanobacillus sp., Bacillus sp.and Bacillus megaterium. The lysozyme (LZ) activity was significantly (P < 0.05) higher in B. cereus and Microbial Consortia (MC), while other treatments were less effective. Total hemocyte count (THC) significantly (P < 0.05) increased in all treatment groups compared to the control. Hyaline hemocyte (HH) count was significantly (P < 0.05) higher in the control group (14.43%). Semi granular hemocytes (SGH) was higher in groups treated with Lysinibacillus, Bacillus sp., B. licheniformis and B. subtilis. The granular hemocyte (GH) count was significantly (P < 0.05) higher in Virgibacillus sp., B. cereus, B.megaterium and Oceanobacillus sp. The biofloc alone (BF), treated and augmented with B. megaterium significantly (P < 0.05) increased phagocytic activity. Highly significant phagocytic index (PI) was observed in bio-augmented groups, BF and MC. The relative expression levels of immune genes were found to be significantly up-regulated in shrimps grown in bio-augmented groups. Enhanced immunological parameters implies that bio-augmentation of biofloc with Bacillus spp. improved immunity in shrimps. Hence, bio-augmentation of probiotics in biofloc may be useful in improving culture conditions to produce P. indicus.

Epitope mapping of the White Spot Syndrome Virus (WSSV) VP28 monoclonal antibody through combined in silico and in vitro analysis reveals the potential antibody binding site.

White Spot Syndrome Virus (WSSV) infecting shrimp is an enveloped double-stranded DNA virus. The WSSV is a member of the genus Whispovirus. The envelope protein VP28 is the most investigated protein of WSSV. In the present study, the epitope mapping of the monoclonal antibody (MAb) C-33 was carried out. Based on the epitope mapping results, an antigen-antibody interaction model was derived. Peptide scanning and confirmation of epitopes of MAb C-33 were carried out using the sequence data. The MAb was reactive to the epitope of both recombinant VP28 and the whole virus. The results of the study indicated the presence of an epitope region. The epitope region is found positioned within two peptides, covering 13 amino acids. Framework and CDR (complementarity determining regions) of heavy and light chain (VH & VL) sequences showed identity to germline immunoglobulin sequences. The Web Antibody Modelling (WAM) selected for further evaluation based on a comparative analysis of WAM and Rosetta server-generated models of the Fv region. The docking study using WAM generated model revealed that the residues from LEU98 to GLY105 are active in antibody binding. The findings of this study could form a structural basis for further research in VP28 based diagnostics and therapeutics or vaccine discovery.

Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine.

White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×10(9) DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×10(6) DNA copies of inactivated virus preparation (IVP) day(-1) for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.

Prosthodontic perspective to all-on-4® concept for dental implants.

The clinical success and longevity of endosteal dental implants as load bearing abutments are controlled largely by the mechanical setting in which they function. The treatment plan is responsible for the design, number and position of the implants. In biomechanically compromised environment such as poor quality bone, strain to the crestal bone can be reduced by increasing the anterioposterior spread of implants, placement of longer implants and maximizing the number of implants. The All-on-4(®) concept is one such treatment procedure which enlightens us for its use in the completely edentulous patients and which also leaves behind the routine treatment alternative of conventional dentures with successful outcome in the short term, long term and the retrospective studies that have been done in the past. The area of concern for any treatment alternative lies in the success of the prosthesis and its prosthodontic perspective involving the principles of occlusion. This article reviews the All-on-4(®) concept and its prosthodontic aspects.

Evaluation of shear bond strength of three different types of artificial teeth to heat cure denture base resin: an in vitro study.

CONTEXT: Bonding of artificial teeth and denture base material remained a significant problem for successful prosthetic treatment. AIM: The purpose of this in vitro study was to evaluate shear bond strength of three different artificial teeth with heat cure denture base material after various surface conditioning methods and thermocycling. MATERIALS AND METHODS: Ninety mandibular first molar denture teeth were selected. They were divided into three groups, Group I: Nanocomposite resin (Veracia), Group II: Composite teeth (Endura), and Group III: Cross-linked acrylic denture teeth (Acry rock). All groups were further subdivided into subgroups based on surface conditioning methods. Subgroup A: No surface conditioning (control group), Subgroup B: Surface conditioning with methyl methacrylate-based bonding agent (Vitacoll), and Subgroup C: Air abrasion with 50 µm aluminum oxide powder particles plus silane-coupling agent (Monobond-S) and Vitacoll bonding agent. Evaluation of shear bond strength of all the specimens was done using universal testing machine. RESULTS: Mean shear bond strength of Group I, Subgroup B (6.87 ± 0.934) showed higher value when compared with Group II, Subgroup B (6.76 ± 1.84) and Group III, Subgroup B (5.66 ± 2.18). The control group (untreated surface) of all three types of artificial teeth used in this study showed significantly lower shear bond strength values than experimental groups (surface conditioning methods; P < 0.05). CONCLUSION: The results of this study suggest that there were significant differences in shear bond strength among control group and surface treatment groups of denture teeth bonded to heat cure denture base resin.

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon.

Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

Anti-white spot syndrome virus activity of Ceriops tagal aqueous extract in giant tiger shrimp Penaeus monodon.

White spot syndrome virus (WSSV), the most contagious pathogen of cultured shrimp, causes mass mortality, leading to huge economic loss to the shrimp industry. The lack of effective therapeutic or prophylactic measures has aggravated the situation, necessitating the development of antiviral agents. With this objective, the antiviral activity in the aqueous extract of a mangrove plant Ceriops tagal in Penaeus monodon was evaluated. The Ceriops tagal aqueous extract (CTAE) was non-toxic to shrimps at 50 mg/ml when injected intramuscularly at a dosage of 10 µL/animal (0.5 mg/animal) and showed a protective effect against WSSV at 30 mg/ml when mixed with WSSV suspension at a 1:1 ratio. When the extract was administered along with the diet and the animals were challenged orally, there was a dose-dependent increase in survival, culminating in 100 % survival at a concentration of 500 mg/kg body weight/day. Neither hypertrophied nuclei nor the viral envelope protein VP28 could be demonstrated in surviving shrimps using histology and indirect immunofluorescence histochemistry (IIFH), respectively. To elucidate the mode of action, the temporal expression of WSSV genes and shrimp immune genes, including antimicrobial peptides, was attempted. None of the viral genes were found to be expressed in shrimps that were fed with the extract and challenged or in those that were administered CTAE-exposed WSSV. The overall results suggest that the aqueous extract from C. tagal can protect P. monodon from white spot syndrome virus infection.

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression.

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

Molecular characterization of a crustin-like antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV.

A crustin-like antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustin-like AMP from other shrimp crustins. The differential expression of the crustin-like AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two ß-glucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two gram-positive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded pre- and post-challenge white spot syndrome virus (WSSV) by semi-quantitative RT-PCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustin-like AMP was found to be constitutively expressed in the animal and a significant down-regulation could be noted post-challenge WSSV. Remarkable down-regulation of the gene was observed in the immunostimulant fed animals pre-challenge followed by a significant up-regulation post-challenge WSSV. Tissue-wise expression of crustin-like AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustin-like AMP and confer significant protection to P. monodon against WSSV infection.