Background: Foot-and-Mouth Disease (FMD) remains a serious threat to the Indian livestock sector due to significant economic loss associated with it. Systematic vaccination of large ruminants over the years has lead to a gradual reduction in the number of disease outbreaks in India. However exposure to FMDV infection in small ruminants has been recorded during the past few years (Rout et al., 2013). Sheep and goat population have not been vaccinated so far against FMD under the FMD-Control program (FMD-CP). The present study highlighted the outbreak of FMD in small ruminants in Karnataka, India. Methods: During the period 2018-19, seven suspected FMD outbreaks among sheep population in Bellary and Tumakuru districts of Karnataka state were investigated. Tongue epithelium (oral swabs) and foot lesions (n=23) from clinically affected sheep and tissues such as heart, lung, liver, spleen, lymph nodes and kidneys from lambs during post mortem (n=67) were collected. All the samples were processed in the laboratory for the detection of FMD virus antigen by employing Serotype differentiating antigen detection ELISA and by multiplex PCR. Heart tissue samples were also collected in buffered formalin for histopathology study and processed by routine paraffin embedding technique and stained with Hematoxylin and Eosin (H and E). Serum samples from the recovered animals were collected and screened by NSP-ELISA and LPB-ELISA to check the antibody status in the affected herd. Result: A total of seven suspected outbreaks of FMD involving 688 small ruminants was investigated. The outbreak of FMD due to FMDV serotype O was confirmed by ELISA and multiplex PCR assays. Clinically, the affected adult sheep showed typical signs of FMD, while mortality in young lambs was observed without apparent signs of disease. Histologically, heart tissues from FMD affected lambs showed myocardial necrosis with marked aggregations of lymphocytes and neutrophils in the myocardium and perivascular spaces. History of FMD outbreaks in cattle and common grazing land for the livestock, as well as sheep within the reach of these villages, may be the major contributing factors for the outbreaks in sheep populations.
Foot-and-mouth disease (FMD) is a significant threat to animal health globally. Prophylactic vaccination using inactivated FMD virus (FMDV) antigen is being practised for the control in endemic countries. A major limitation of the current vaccine is its susceptibility to high environmental temperature causing loss of immunogenicity, thus necessitating the cold chain for maintenance of its efficacy. Hence, the FMD vaccine with thermostable virus particles will be highly useful in sustaining the integrity of whole virus particle (146S) during storage at 4°C. In this study, 12 recombinant mutants of Indian vaccine strain of FMDV serotype O (O/IND/R2/1975) were generated through reverse genetics approach and evaluated for thermostability. One of the mutant viruses, VP2_Y98F was more thermostable than other mutants and the parent FMDV. The oil-adjuvanted vaccine formulated with the inactivated VP2_Y98F mutant FMDV was stable up to 8 months when stored at 4°C and induced protective antibody response till dpv 180 after primary vaccination. It is concluded that the VP2_Y98F mutant FMDV was thermostable and has the potential to replace the parent vaccine strain.
In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.
Ovine interferon-gamma (IFN-γ) cDNA was isolated from total mRNA of peripheral blood mononuclear cells from indigenous sheep by reverse transcription PCR. The cDNA was successfully cloned, sequenced and expressed in Escherichia coli as thioredoxin fusion protein. Sequence comparison of Indian sheep IFN-γ showed high nucleotide homology with published IFN-γ sequences of sheep and other species of ruminants. The cDNA codes for a mature polypeptide with a putative molecular weight of 17 kDa. The recombinant ovine IFN-γ, expressed in BL21 E. coli cells as His-tagged protein, was purified using Ni-chelation chromatography. Up to 250 µg of recombinant ovine IFN-γ could be obtained from 50 mL of bacterial culture. The production of this important cytokine has significant implications in therapy of major infectious diseases of small ruminants. It also has potential applications as adjuvant to enhance the protective efficacy of the existing vaccines.
Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use. The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2 TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its propagation and titration.
We determined complete nucleotide sequence of B5R gene homologue of Vaccinia virus (VACV) in five Buffalopox virus (BPXV) isolates of Indian origin. The obtained sequences were compared with themselves and with corresponding sequences of the other orthopoxviruses. Sequence analysis revealed 99.7-99.8% and 99.4-99.7% identities among the BPXV isolates for B5R gene at the nucleotide and amino acid levels, respectively. Sequence identities of B5R gene between BPXV and VACV isolates (98.1-99.7%) or other orthopoxviruses (95.6-99.2%) showed highly conserved nature of this protein and a closer relationship of BPXV isolates to VACV than to other orthopoxviruses.
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