Isolation and screening of antimicrobial biosurfactants obtained from mangrove plant root-associated bacteria.

The unique properties of biosurfactants obtained from microbes, including their activity at extreme temperatures, make them more attractive than synthetic alternatives. Henceforth, the principle objective is to isolate and detect the antibacterial and antifungal activities of the biosurfactants produced from bacteria of the economically competitive mangrove ecosystem. Using the serial dilution method, 53 bacterial strains were recovered from the Manakudy mangrove forest in Kanyakumari, India, for the investigation. Different biosurfactant screening methods and morphological and biochemical tests were opted to select the potential biosurfactant producer. After the initial screening, two strains were discovered by 16S rRNA gene sequencing followed by extraction using chloroform: methanol (2:1) by the precipitation method. The partially purified biosurfactants were then screened for antimicrobial properties against pathogens including Mucor sp., Trichoderma sp. Morphological, biochemical, and 16S rRNA gene sequencing identified the two strains to be gram-positive, rod-shaped bacteria namely Virgibacillus halodentrificans CMST-ZI (GenBank Accession No.: OL336402.1) and Pseudomonas pseudoalcaligenes CMST-ZI (GenBank Accession No (10 K): OL308085.1). The two extracted biosurfactants viz., 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, as well as cycloheptane efficiently inhibited human pathogens, including Enterococcus faecalis, and fungi, including Mucor sp., Trichoderma sp., indicated by the formation of a zone of inhibition in pharmacological screening. Thus, there is a growing interest in the prospective application of these biosurfactants isolated from marine microbes, exhibiting antimicrobial properties which can be further studied as a potential candidate in biomedical studies and eco-friendly novel drug development.

DNA vaccine incorporated poly (lactic-co-glycolic) acid (PLGA) microspheres offer enhanced protection against Aeromonas hydrophila infection.

Encapsulation of DNA vaccines onto carriers enhances the immunogenicity of an antigen. Specifically, biodegradable polymers offer sustained release of vaccines which is crucial for any targeted delivery approach. Poly (lactic-co-glycolic) acid (PLGA) microspheres were used to load a DNA vaccine having a targeted gene of outer membrane protein (OMP) of Aeromonas hydrophila to clone and construct a DNA vaccine using a eukaryotic expression vector system (pVAX1-OMP DNA) and delivery in Carassius auratus against A. hydrophila infection. PLGA microspheres were prepared by emulsion technique oil-in-water and characterized by a High-Resolution Scanning Electron Microscope (HR-SEM). The results of PLGA-pVAX1-OMP DNA microspheres shows that average of 100-150 µm particle size and a loading efficiency (LE) of 68.8 %. Results indicate that C. auratus fed with PLGA-pVAX1-OMP DNA microspheres revealed a significant improvement in innate immune response, which includes, myeloperoxidase activity, respiratory burst and total immunoglobulin level compared with control group fish. The immune-related gene, IL1ß, IL10, TGF, c-type, and g-type lysozyme also showed significantly higher expression after immunization. Furthermore, dietary supplementation of the PLGA-pVAX1-OMP DNA (G III) group exhibited a significantly higher survival rate (78 %) than the control group of fish. These results help us to understand the of mechanism of DNA vaccine administrated feed through PLGA nanoparticles resistance to infection by regulating systemic and innate immunity in Carassius auratus.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E. coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L. rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A. hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGF-ß), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A. hydrophila.

Oral delivery of pVAX-OMP and pVAX-hly DNA vaccine using chitosan-tripolyphosphate (Cs-TPP) nanoparticles in Rohu, (Labeo rohita) for protection against Aeromonas hydrophila infection.

The present study examines the effectiveness of DNA vaccine against Aeromonas hydrophila through oral route using chitosan-tripolyphosphate (Cs-TPP) nanoparticles encapsulation. The virulent gene of outer membrane protein (OMP) and hemolysin (hly) related to pathogenicity of A. hydrophila was used to construct a DNA vaccine using pVAX1, and the construct was named as pVAX-OMP and pVAX-hly DNA vaccines. The pVAX-OMP and pVAX-hly DNA vaccines were encapsulated by Cs-TPP nanoparticles and size measured by field emission scanning electron microscopy (FE-SEM). The encapsulation efficiency of Cs-TPP nanoparticles was found to be 79.6% for pVAX-OMP DNA and 82.3% for pVAX-hly DNA binding with Cs-TPP nanoparticles. The stability and invitro release profile of plasmid DNA was also determined after encapsulation using DNase and chitosanase. DNA vaccines distribution in tissues was investigated in fish fed with the pVAX-OMP, pVAX-hly and pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles and confirmed by PCR and multiplex PCR. The results suggest that Cs-TPP nanoparticles encapsulated DNA vaccine delivered into fish by feeding. After oral vaccination of Labeo rohita were challenged with A. hydrophila by intraperitoneal injection. Relatively, gene expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (Interlukin-10 and Tumor Growth Factor ß) was up-regulated in heart and kidney for pVAX-OMP+pVAX-hly vaccinated group. Moreover, fish fed with pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles had a significantly higher survival rate (76.2%) against A. hydrophila. This study concludes that pVAX-OMP and pVAX-hly DNA vaccines can be delivered orally using Cs-TPP nanoparticles for protection against A. hydrophilainfection.

Macrobrachium rosenbergii nodavirus (MrNV)-CP-RNA-2 DNA vaccine confers protective immunity in giant freshwater prawn Macrobrachium rosenbergii against MrNV infection.

Macrobrachium rosenbergii Nodavirus (MrNV) causes white tail disease (WTD) in Giant freshwater prawn Macrobrachium rosenbergii which leads to immense economic losses in hatcheries and farms. In the present study, we cloned the capsid protein gene of MrNV-CP-RNA-2 (1146 bp) into a DNA vaccine vector pVAX1 to form MrNV-CP-RNA-2- pVAX1. The bacterial transformant, containing the MrNV-CP gene, was coated on the fish diet pellets and fed to juvenile M. rosenbergii for 40 days. After the vaccine delivery, group of M. rosenbergii were challenged with virulent MrNV on 20 and 40th days post-vaccination (dpv) respectively and monitored for the survival. The non-vaccinated M. rosenbergii succumbed to death (100%) within 5 days, whereas the MrNV-CP-RNA-2- pVAX1 treated groups had the survivals of 60 and 80% in 20 and 40 dpv respectively (P ≤ 0.001). To study the MrNV infection level, double step PCR was performed at different dpv. The results revealed that in 20 dpv group, the infection was decreased to 65% and in 40 dpv group the infection decreased to 69% from control diet fed prawns (P < 0.001). Haematological parameters like coagulation time, total haemocyte count (THC) and oxyhaemocyanin levels were performed for the control and vaccinated prawns. The vaccination helped to decrease the time of coagulation, improved THC and oxyhaemocyanin levels at a significant level (p < 0.001) when compared to the non-vaccinated group. The immunological parameters like prophenol oxidase (ProPO), superoxide anion and intra-agar lysozyme activity were also performed and the results revealed that the level of proPO, superoxide anion and lysozyme activities were significantly (P ≤ 0.05) increased in 20 and 40 dpv groups respectively, when compared with the non-vaccinated groups. Based on the vaccination trials, the DNA vaccine construct MrNV-CP-RNA-2-pVAX1 effectively improved the survival against MrNV challenge, helped to decrease viral load and enhanced the immune system to protect the prawn from MrNV infection. This vaccine construct is highly useful to protect the M. rosenbergii from MrNV infection.

Effect of Argemone mexicana active principles on inhibiting viral multiplication and stimulating immune system in Pacific white leg shrimp Litopenaeus vannamei against white spot syndrome virus.

Argemone mexicana called as Mexican prickly poppy is a species of poppy found in Mexico and now widely naturalized in many parts of the world with broad range of bioactivities including anthelmintic, cures lepsory, skin-diseases, inflammations and bilious fevers. Plant parts of A. mexicana were serially extracted with hexane, ethyl acetate, methanol and performed antiviral and immunostimulant screening against WSSV and Vibrio harveyi respectively. The control groups succumbed to death 100% within three days, whereas the mortality was significantly (P < 0.5) reduced to 17.43 and 7.11 in the ethyl acetate extracts of stem and root treated shrimp group respectively. The same trend was reflected in the immunostimulant screening also. Different diets were prepared by the concentrations of 100 (AD-1), 200 (AD-2), 300 (AD-3) and 400 (AD-4) mg kg-1 using A. mexicana stem and root ethyl acetate extracts and fed to Pacific white leg shrimp Litopenaeus vannamei weighed about 9.0 ±â€¯0.5 g for 30 days. The control groups fed with the normal diets devoid of A. mexicana extracts. The antiviral screening results revealed that, the ethyl acetate extract of the stem and root were effectively suppressed the WSSV and it reflected in the lowest cumulative mortality of treated shrimps. After termination of feeding trials, group of shrimps from control and each experimental group were challenged with virulent WSSV by intramuscular (IM) injection and studied cumulative mortality, molecular diagnosis by quantitative real time PCR (qRT-PCR), biochemical, haematological and immunological parameters. Control group succumbed to 100% death within four days, whereas the survival was significantly (P < 0.001) increased to 30, 45, 75 and 79% in AD1, AD-2, AD-4 and AD-5 diets fed shrimp groups respectively. qRT PCR results with positive correlation analysis revealed that, the WSSV copies were gradually decreased when increasing the A. mexicana extracts in the diets. The highest concentrations (300 and 400 mg g-1) of A. mexicana extracts in the diets helped to reduce the protein level significantly (P < 0.05) after WSSV challenge. The diets AD-3 and AD-4 also helped to decrease the coagulation time of maximum 64-67% from control groups and maintained the normal level of total haemocyte, oxyhaemocyanin level after WSSV challenge. The proPO level was significantly increased (Column: F = 35.93; P ≤ 0.001 and Row: F = 37.14; P ≤ 0.001) in the AD1-AD-4 diet fed groups from the control diet fed groups. The lowest intra-agar lysozyme activity of 1.63 mm found in control diet fed group and the activity were significantly (P < 0.05) increased to 4.86, 7.89, 9.12 and 10.45 mm of zone of inhibition respectively in AD1 to AD4 diet fed groups.

Physicochemical properties of anti Vibrio harveyi egg yolk antibody (IgY) and its immunological influence in Indian white shrimp Fenneropenaeus indicus.

Edible antibodies specific to host pathogens is an attractive approach to establish protective immunity, especially against gastrointestinal pathogens both in humans and animals. The edible antibody of anti-Vibrio harveyi IgY (anti-V. h IgY) was produced by antigen mixed with immunoadjuvant Asparagus racemosus and Glycine max. Hens were immunized and eggs were collected five weeks after the immunization. Anti-V. harveyi IgY stability in different digestive enzymes such as trypsin and chymotrypsin were evaluated to determine its ability to withstand in the gastrointestinal tract of F. indicus. Specific binding activity and concentration (average 9.5% of total IgY content) of the anti-V. h IgY were determined by the ELISA using V. harveyi antigen. Further the anti-V. h IgY diets including V.h wo, V.h A, V.h G and control diets were fed to F. indicus for 60 days. After 30 and 60 of feeding, group of shrimps were challenged with virulent V. harveyi. After the respective days of feeding, haematological and immunological changes were studied. The parameters including total haemocyte count (THC), coagulase activity, oxyhaemocyanin level, prophenoloxidase, intracellular superoxide anion production, lysozyme, phagocytosis and bacterial agglutinin had significantly (P ≤ .001) increased in the experimental groups in comparission with the control diet fed shrimps. The anti-V. h IgY coated diets helped to reduce the Vibrio load and boosted the immune system in F. indicus's against V. harveyi challenge. The research work shows the potential applications of egg yolk antibodies as anti-bacterial prophylactic uses for infectious diseases and suggests an edible antibody concept as an alternative to conventional antibiotics.