Kondabattula Ganesh

Nucleotide sequence of L1 and part of P1 of hexon gene of fowl adenovirus associated with hydropericardium hepatitis syndrome differs with the corresponding region of other fowl adenoviruses

In order to study the serotypic variations in hydropericardium hepatitis syndrome (HHS) causing virus, the DNA was extracted from the purified virus, a 0.7 kb variable region of hexon gene encoding L1 and part of P1 amplified and sequenced. Both nucleotide and derived amino acid sequences, corresponding to the variable region, were compared with the published fowl adenovirus sequences (FAV serotypes 10, 1 and 8). As expected the 0.7 kb sequence showed single open reading frame (ORF). There was a nucleotide sequence variation of 8.2, 28.1 and 40.3%, respectively, with FAV serotypes 10, 1 and 8. The dendrogram constructed with the nucleotide sequences showed that HHS virus and FAV10 are closer to each other and are away to FAV1 and FAV8. However, the derived amino acid sequence showed variations as high as 28.8, 38 and 45.1% with FAV serotypes 10, 1 and 8, respectively. Such high degree variation has been found due to the shift in the reading frame caused by deletions indicating that the FAV4 associated with HHS is unique and different from FAV10. To the best of our knowledge this is the first report on nucleotide sequence analysis of hexon gene fragment of FAV4 associated with HHS.

Detection of fowl adenovirus associated with hydropericardium hepatitis syndrome by a polymerase chain reaction.

Hydropericardium hepatitis syndrome HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction PCR) was developed to detect the fowl adenovirus FAV) associated with HHS. The virus from infectedl ivers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs.

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.

Co-administration of flagellin augments immune responses to inactivated foot-and-mouth disease virus (FMDV) antigen.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal virus known that affects livestock health and production. This study aimed to investigate the effect of flagellin, a toll-like receptor 5 agonist, on the immune responses to inactivated FMDV antigen in guinea pig model. Our results showed that the co-administration of flagellin with FMDV antigen through intradermal route induces earlier and higher anti-FMDV neutralizing antibody responses as compared to FMDV antigen alone. Both IgG1 and IgG2 antibody-isotype responses were enhanced, but the IgG1/IgG2 ratios were relatively low, indicative of TH1 type of immune activation. On live viral challenge, flagellin+FMDV immunized guinea pigs showed 70% (7 out of 10) protection rate as compared to 40% (4 out of 10) in FMDV alone immunized guinea pigs. The results demonstrate that the co-administration of flagellin augments immune responses (preferably TH1 type) and protective efficacy against FMDV in guinea pigs.

Purification and characterization of the aetiological agent of hydropericardium hepatitis syndrome from infected liver tissues of broiler chickens.

Hydropericardium hepatitis syndrome in broiler chickens is an acute, infectious disease characterized by high mortality, excess pericardial fluid and multifocal hepatic necrosis. The aetiological agent was purified to homogeneity from infected liver tissues from field outbreaks. Electron-microscopic and serological confirmation of the virus were undertaken and the disease was reproduced experimentally in broiler chicks. The results indicated that an adenovirus, fowl adenovirus serotype 4, was alone responsible for the disease in the materials studied.

Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.

An effective mannosylated chitosan nanoparticle DNA vaccine for FMD virus.

We are reporting you an effective DNA vaccine for FMD virus, and it was prepared using mannosylated chitosan (MC) nanoparticles to form the FMDV-pVAC-VP1-OmpA complex particles. These particles were characterized for their physical properties like morphology, size and charge prior evaluating the actual vaccine delivery effect of MC-nanoparticles. The immunological evaluation indicated that the 20 μg of DNA vaccine complexed with MC nano-particles was found optimum in inducing the immune response in G pigs as measured by FMDV specific neutralising antibodies and Th1/Th2 responses using micro SNT and ELISA, respectively.

Sindbis virus replicase-based DNA vaccine construct encoding FMDV-specific multivalent epitope gene: studies on its immune responses in guinea pigs.

Foot‐and‐mouth disease (FMD) is still a perennial global menace affecting livestock health and production. It is imperative to figure out new ways to curb this disease. In this study, a sindbis virus replicase‐based DNA vaccine, pSinCMV‐Vac‐MEG990, encoding a multivalent epitope gene (representing tandemly linked VP1 C‐terminal halves of three foot‐and‐mouth disease virus (FMDV) serotypes) was constructed. In vitro transfection studies in BHK‐21 cells revealed that the construct was able to express FMDV‐specific antigen but does not overproduce the antigen. Immunization of guinea pigs with the construct at dose rate of 10, 5, 2 and 1 μg per animal through intramuscular route showed significant neutralizing antibody induction at all doses against all serotype tested as compared to non‐immunized controls. On viral challenge of guinea pigs 4 week post‐immunization with 1000 GPID50 of FMDV serotype A, it was observed that the immunization not only delayed the appearance and reduced the severity of FMD lesions significantly (P < 0.05) but also provided complete protection in several guinea pigs. In fact, two of six and one of six guinea pigs were completely protected in 10 and 5 μg immunized groups, respectively. These results suggest that the development of the replicase‐based DNA vaccine may provide a promising approach as an alternative vaccine strategy for controlling FMD.

Improved immune response by ID-pVAC: A secretory DNA vaccine construct delivered by PLG micro particles against foot and mouth disease in guinea pigs

Foot and mouth disease (FMD) outbreaks usually have devastating effects on the economy of countries were disease is endemic due to direct and indirect cost; most of them related to international trade embargoes of animals and animal products. Although currently used inactivated vaccine provides protection, it has several drawbacks like short duration of immunity, and the requirement for containment facilities. A DNA vaccine construct which expresses the secretary antigens, delivered through micro particles could be one of the alternate approaches to overcome these limitations. Present study is envisaged to prepare a DNA vaccine construct containing the VP1 sequence of FMDV serotype O in pVAC vector. DNA vaccine was formulated by adsorbing plasmid DNA construct on cationic micro particles and administered in guinea pigs @25 μg DNA vaccine construct per animal intramuscularly. Sera samples collected were analyzed by sandwich ELISA and SNT, shown enhanced immune response in PLG adjuvanted DNA vaccine. MTT and 3H Thymidine incorporation have shown good CMI responses to PLG adjuvanted DNA. When challenged with 100 gpid50 of homologous virus 5 of the six animals were protected.

DNA vaccine (P1-2A-3C-pCDNA) co-administered with Bovine IL-18 gives protective immune response against Foot and Mouth Disease in cattle

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals causing considerable economic loss in the affected countries. Presently used tissue culture inactivated vaccine protects the vaccinated animals for a short duration. DNA vaccines along with appropriate adjutants is one of the approach for the development of alternative vaccine. In the present study, we constructed P1-2A-3CpCDNA (containing P1-2A-3C coding sequences of FMDV Asia-1 Ind 63/72) and bovine IL-18 pCDNA plasmids and evaluated in cattle. Four groups of calves each group containing six calves were vaccinated with 200μg of plasmid DNA vaccine P1-2A-3CpCDNA, P1-2A-3CpCDNA+ bIL-18pCDNA and inactivated vaccine respectively where as fourth group was unvaccinated. P1-2A-3CpCDNA+bIL-18pCDNA vaccinated animals have shown higher levels of neutralizing antibodies and specific T-cell proliferation responses. Higher levels of CD4(+) and CD8(+) cells were observed in these animals. Similarly, IL-18 adjuvanted group has shown increased Th1 and Th2 cytokine responses. All the vaccinated animals were challenged with cattle adapted FMD homologous Asia1 virus two weeks after the booster dose. IL18 co administered DNA vaccine construct has protected four out of six animals challenged with homologous virus.