Evaluation of Adjuvanticity and Protective Efficacy of Recombinant ORFV B2L Protein Adjuvanted FMD Vaccine in Guinea Pigs

The present study evaluates the adjuvant effect of rec-B2L protein of orf virus and widely used oil adjuvant with purified inactivated FMDV 146S trivalent antigens (O, A and Asia 1) in guinea pig model. Rec-B2L protein expressed in prokaryotic expression system was coadministered intradermally with FMDV trivalent antigens. The serum antibody titre, CMI response and protectiveefficacy against FMDV-type ‘O’ virulent virus challenge were assessed. The antibody titres of oil adjuvant group were found highest. However, rec-B2L group showed better antibody titre than the control group. The levels of IgG1 were found to be constant throughout the experiment except some fluctuations noticed on 14th and 28th dpv against serotype A and Asia1. Rec-B2L group showed higher levels of IgG2 against serotype A on 14th and 28th dpv than FMDV trivalent antigens alone. Further, the adjuvant effect of rec-B2L protein was also reflected by enhanced levels of IFN-c, TNF-b on 28th dpv; IL-15, IL10 on 7 dpv in the immunized serum samples. The oil adjuvant group and rec-B2L group showed a centumprotection following challenge on 28th dpv. These preliminary findings indicate the adjuvant effect of rec-B2L protein

Status of sheep sera to bluetongue, peste des petits ruminants and sheep pox in a few northern states of India

Bluetongue (BT), peste des petits ruminants (PPR) and sheep pox are the most economically important viral diseases of sheep in India. Serum samples obtained from sheep in five northern states of the country were screened for antibody against these agents to explore the extent of spread of these infections. A total of 516 serum samples were screened for the presence of antibodies against BT and PPR viruses. Of these, 155 samples were also tested for antibodies against sheep pox virus. BT antibodies were found in 293 (56.8%) animals, PPR virus antibodies in 215 (41.7%) and sheep pox virus antibodies in 106 (68.3%). Of the serum samples tested, 25.2% were positive for antibodies against all three viruses. These findings clearly demonstrated not only the enzootic nature of disease, but also the co-existence of antibodies to more than one of these viruses which would indicate that concurrent infections were common. Therefore, control measures should focus in combating all three diseases simultaneously by exploring the possibility of a trivalent vaccine or the use of multiple genes expressing vectored vaccine

Feeding behaviour of Foot and Mouth Disease (FMD) infected Holstein Friesian crossbred calves offered with therapeutic diet

Foot and Mouth Disease (FMD) affects dry matter intake (DMI) and performance due to mucosal erosions developed on the tongue and gums. Therapeutic diets (TD) in a physical form compatible with the inflammatory condition of the mouth ensures optimum intake with changed behavioural pattern. The present study aimed to record the feeding behaviour of FMD infected calves by offering a therapeutic diet. A total of 22 Holstein Friesian crossbred male calves (10–12 months) were used in which 4 calves were of Control (CON) and 18 calves of treatment groups viz. Therapeutic Diet- Mash (TDM), Therapeutic Diet-Cooked (TDC ), Therapeutic Diet-Customized Nutrient Supplement (TDCNS ) were infected with FMD virus in the bio-containment facility (BSL-III). The therapeutic diet (TD) was fed to calves in the forenoon session and ad libitum hybrid Napier in afternoon session. The diet had high protein and energy with varied physical forms (mash or cooked). The infected group calves experienced difficulty in eating viz. 0.00 ± 0.00, 19.57 ± 3.19, 25.48 ± 4.13 & 27.30 ± 4.38 min in CON, TDM, TDC , TDCNS respectively. The time values were highly significant between the groups (p < 0.01). Licking behaviour was noticed only in the morning session. Feed tossing, ruminating behaviour was noticed in the afternoon session in all the animals irrespective of groups. The frequency of urination was more in the morning and the defecation was comparable between the groups. The lethargy score was high for 3 days post-infection in the infected groups and subsequently declined. Non-nutritive behaviour is not seen either in the forenoon or in the afternoon session. In conclusion, tailor made therapeutic diets with changed physical forms (mash or cooked) was compatible with the inflammatory condition of the mouth

Quantitative polymerase chain reaction: another tool to evaluate viable virus content in live attenuated orf vaccine

A probe-based real-time polymerase chain reaction (PCR) assay based on the highly conserved DNA polymerase gene of orf virus (ORFV) for the quality control of attenuated orf vaccine is reported. Primary lamb testis (PLT) cells were infected with orf vaccine virus and harvested at a critical time point to obtain maximum viable virus content as determined by real-time PCR. DNA extracted from these harvests was subjected to real-time PCR. A critical time point for the harvesting of PLT cells infected with various log10 dilutions of vaccine virus was found to be 42 h (highest slope of 3.335), which was obtained by comparing the slopes of standard curves of different time intervals. The assay was employed to evaluate viable virus content in different batches of orf vaccine. The titres estimated by real-time PCR and conventional TCID50 were comparable with a correlation of 0.8169. Thus, the real-time PCR assay could provide an alternative method or supplementary tool to estimate live ORFV particles in attenuated orf vaccine

Sequence and phylogenetic analyses of an Indian isolate of orf virus from sheep

The authors describe the isolation and identification of orf virus (ORFV) from an outbreak in a flock of sheep at Mukteswar, Uttarakhand, India, in 2009. The causative agent, ORFV was successfully isolated in primary lamb testes cells and identified using a semi-nested diagnostic polymerase chain reaction (PCR) and sequence and phylogenetic analyses of immunogenic envelope protein (B2L) coding gene. The affected animals showed characteristic proliferative skin lesions around the mouth and on nostrils and, in a few animals, lesions were also noticed on the tongue irrespective of age and sex. The morbidity, mortality and case fatality rates observed were 6%, 45% and 13%, respectively. Clinical samples were initially screened by counter immuno-electrophoresis and the serum neutralisation test; further positive skin scabs were tested with diagnostic PCR and virus isolation was performed on primary or secondary lamb testes cultures. Sequencing and phylogenetic analyses of the sheep isolate based on the B2L gene revealed that the isolate was closest to a goat isolate retrieved from an outbreak at the same geographic location in 2000. Furthermore, it also showed close genetic similarities with other Indian isolates reported earlier. Regular and systematic investigation of outbreaks is necessary to monitor the disease in susceptible populations. The development of rapid diagnostic methods as well as effective vaccine to control this infection not only from India but also other parts of the world is called for

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Not AvailableBluetongue (BT), peste des petits ruminants (PPR) and sheep pox are the most economically important viral diseases of sheep in India. Serum samples obtained from sheep in five northern states of the country were screened for antibody against these agents to explore the extent of spread of these infections. A total of 516 serum samples were screened for the presence of antibodies against BT and PPR viruses. Of these, 155 samples were also tested for antibodies against sheep pox virus. BT antibodies were found in 293 (56.8%) animals, PPR virus antibodies in 215 (41.7%) and sheep pox virus antibodies in 106 (68.3%). Of the serum samples tested, 25.2% were positive for antibodies against all three viruses. These findings clearly demonstrated not only the enzootic nature of disease, but also the co-existence of antibodies to more than one of these viruses which would indicate that concurrent infections were common. Therefore, control measures should focus in combating all three diseases simultaneously by exploring the possibility of a trivalent vaccine or the use of multiple genes expressing vectored vaccine.Not Availabl

Functional characterization of recombinant major envelope protein (rB2L) of orf virus

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa M-1-D-334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (similar to 60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as coadministration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations

Expression, purification, and functional characterisation of Flagellin, a TLR5-ligand

Flagellin, a Toll-like receptor 5 (TLR5)-ligand, is known for its activities like adjuvant, induction of pro-inflammatory cytokines and innate immunity. In this context, fliC gene of Salmonella Typhimurium was cloned into pET32a expression plasmid using in-house designed gene specific primers. The frame and orientation of the inserted fliC gene was confirmed upon colony PCR, restriction enzyme analysis and sequencing. Sequence analysis of fliC revealed proper orientation of the gene and had 1,485 nucleotides. Following transformation of pET-fliC plasmid into Escherichia coli BL21 (DE3) cells, the gene was expressed after inducing with IPTG (Isopropylβ-D-1-thiogalactopyranoside). The polyHis-tag-fliC was ~70kDa as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The identity/authenticity of the recombinant-fliC was confirmed by its specific reactivity with commercial anti-fliC MAb of S. Typhimurium. Further, the antigenic and functional properties of recombinant-fliC were determined espousing its ability to induce antigen specific antibodies in G pigs and increased m-RNA expression of certain pro-inflammatory mediators like TNF-α and GM-CSF in vitro

Morphological and phenotypic characterization of immature MoDCs.

<p>(A) Light microscopy (20X) of freshly isolated monocyte. (B) Light microscopy (20X) of 4^th day culture of presumably immature MoDCs showing clusters of veiled cells with pseudopodia. (C) Freshly isolated monocytes and 5 day culture of MoDCs were analysed for phenotypic changes by evaluating mRNA expression levels of various TLRs and costimulatory genes by qRT-PCR assay. Results are expressed as fold change (log10) in mRNA transcription of monocytes and MoDCs. Data presented are mean ± standard deviation of values obtained from three independent experiments involving two cattle of Hallikar breed. *<i>p</i>< 0.05.**<i>p</i>< 0.01.</p