Genetic and antigenic characterization of Indian foot-and-mouth disease virus serotype O isolates collected during the period 2001 to 2012.

The phylogenetic analysis of VP1 sequences of the 39 type O foot and mouth virus (FMDV) isolates collected from different regions of India during the year of 2001-12 revealed that all isolates belonged to the Middle East - South Asia (ME-SA) topotype. Based on the amount of divergence among the isolates, the viruses were further classified into three distinct lineages namely Ind 2001, PanAsia and PanAsia-2 as well as a minor, unnamed group. Ind 2001 lineage viruses accounted for most of the current type O outbreaks. At the nucleotide level these isolates showed a divergence of 2% to 14% with an average sequence variation of ~9.9%. The serological spectrum of the current vaccine strain was studied by using bovine vaccinate serum (BVS) raised against O/IND/R2/75. All the current field isolates (n=24) were homologous ('r' value 0.4 to 1.0) to the vaccine strain. Examination of the amino acid sequences for selection pressure revealed the positive selection at amino acid sites 13 and 45.

Plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis.

Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates.

Typing of canine parvovirus isolates using mini-sequencing based single nucleotide polymorphism analysis.

The antigenic types of canine parvovirus (CPV) are defined based on differences in the amino acids of the major capsid protein VP2. Type specificity is conferred by a limited number of amino acid changes and in particular by few nucleotide substitutions. PCR based methods are not particularly suitable for typing circulating variants which differ in a few specific nucleotide substitutions. Assays for determining SNPs can detect efficiently nucleotide substitutions and can thus be adapted to identify CPV types. In the present study, CPV typing was performed by single nucleotide extension using the mini-sequencing technique. A mini-sequencing signature was established for all the four CPV types (CPV2, 2a, 2b and 2c) and feline panleukopenia virus. The CPV typing using the mini-sequencing reaction was performed for 13 CPV field isolates and the two vaccine strains available in our repository. All the isolates had been typed earlier by full-length sequencing of the VP2 gene. The typing results obtained from mini-sequencing matched completely with that of sequencing. Typing could be achieved with less than 100 copies of standard plasmid DNA constructs or ≤10¹ FAID50 of virus by mini-sequencing technique. The technique was also efficient for detecting multiple types in mixed infections.

Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis.

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.

Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity.

Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, while cross-protection against heterologous species is poor. As a first step toward the development of a broad specificity Eimeria vaccine, this study was designed to assess a purified recombinant protein from Eimeria maxima gametocytes (Gam82) in stimulating immunity against experimental infection with live parasites. Following Gam82 intramuscular immunization and oral parasite challenge, body weight gain, fecal oocyst output, lesion scores, serum antibody response, and cytokine production were assessed to evaluate vaccination efficacy. Animals vaccinated with Gam82 and challenged with E. maxima showed lower oocyst shedding and reduced intestinal pathology compared with non-vaccinated and parasite-challenged animals. Gam82 vaccination also stimulated the production of antigen-specific serum antibodies and induced greater levels of IL-2 and IL-15 mRNAs compared with non-vaccinated controls. These results demonstrate that the Gam82 recombinant protein protects against E. maxima and augments humoral and cell-mediated immunity.

Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds.

Eimeria infection in poultry is of significant economic interest worldwide. Development of a cost-effective sub-unit vaccine that provides cross-protection may help reduce loss in poultry industry. One approach explored by many investigators is to block the parasite invasion into gut epithelium. Use of microneme proteins to prevent parasite invasion is one of the most straightforward approaches in developing a preventive vaccine. Here we describe cloning and expression of microneme-1 protein of Eimeria tenella, obtained from an outbreak sample from India. We have evaluated the ability of the recombinant protein to elicit both cell mediated immune (CMI) and humoral immune responses. We also evaluated the efficacy of the recombinant protein in protecting against a homologous challenge. Our data indicate recombinant EtMIC1 is able to impart partial protection against homologous challenge in chicken. Inclusion of more invasion proteins may improve the efficacy of prophylactic vaccine against Coccidiosis.