Cloning and expression of Bos indicus interleukin-4 in mammalian cells.

Dendritic cells (DC) which are located at the interface of innate and adaptive immunity are targets of infection by many RNA and DNA viruses. Advances in the ex vivo generation of monocyte derived non proliferating dendritic cells have been used for clinical application like immunotherapy. IL-4 cytokine plays essential role in the maturation and generation of DCs. Bos indicus interleukin 4 (boIL-4) 408 bp was amplified from PBMC's and cloned in pBSIIKS+ vector. The sequence analysis showed N terminal 69 bp signal sequence and one N-glycosylation site. The phylogenetic tree analysis showed that Bos indicus IL-4 is closely related to the ruminant IL-4 and least sharing of genetic line of human and mouse IL-4. The recombinant bolL-4 protein was expressed in CHO cells which secreted a 16 kDa protein which was confirmed by SDS PAGE and western blotting. The rec-boIL-4 protein proliferated the bovine PBMC's, decreased production of nitric oxide in antigen stimulated macrophages, and phagocytosed the micro particles confirming its activity on dendritic cells.

In vitro assessment of praziquantel and a novel nanomaterial against protoscoleces of Echinococcus granulosus.

The present study describes the activity of a nanomaterial on protoscoleces of Echinococcus granulosus, which exhibited morphological changes and apoptosis. Apoptotic changes were deduced on the basis of effector caspase activation and nucleosomal laddering. Invaginated protoscoleces maintained in vitro became evaginated and had hooks, presumptive suckers and stalks. Degenerative changes of protoscoleces were evidenced after treatment with praziquantel and nano-combination. Protoscoleces treated with praziquantel had distinct attestation of necrosis and nano-combination-treated protoscoleces had signatures of apoptosis.

Molecular and biochemical mining of heat-shock and 14-3-3 proteins in drug-induced protoscolices of Echinococcus granulosus and the detection of a candidate gene for anthelmintic resistance.

Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a disease that affects both humans and animals. In humans the disease is treated by surgery with a supplementary option of chemotherapy with a benzimidazole compound. During the present study heat-shock protein 60 (HSP 60) was identified as one of the most frequently expressed biomolecules by E. granulosus after albendazole treatment. Data were correlated with 14-3-3 protein signature, and overexpression of this molecule after albendazole induction was an indicator of cell survival and signal transduction during in vitro maintenance of E. granulosus for up to 72 h. This observation was further correlated with a uniform expression pattern of a housekeeping gene (actin II). Out of three ß-tubulin gene isoforms of E. granulosus, ß-tubulin gene isoform 2 showed a conserved point mutation indicative of benzimidazole resistance.

PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo.

Trypanosoma evansi, the causative organism of 'surra' expresses its variable surface glycoprotein (VSG) at early, middle and late stages of infection in animals. The variable antigenic nature of VSG caused by switching its expression type favours evasion from the host immune response and leads to chronic and persistent infection. Developing a polymerase chain reaction (PCR)-based diagnostic tool targeting the VSG gene is expected to be highly specific and sensitive for diagnosis of surra. Hence, in the present study, we have designed EXP3F/4R primer pair and amplified the 1.4 kb of VSG gene of T. evansi and studied the phylogenetic relationship by in silico analysis. The PCR method was standardised using another set of primer, DITRYF/R, and 400 bp was amplified from blood and tissue samples of experimentally infected animals. Applying the PCR method, we were able to detect as low as 0.15 trypanosomeml(-1). Considering the number of parasite-to-DNA concentration, the PCR method has a sensitivity of 0.015 pg ml(-1). The PCR could detect the presence of the parasite as early as 24h post-infection (p.i.) and 72 h p.i., respectively, in experimentally infected rats and buffalo. No amplification was observed with DNA of Babesia bigemina and Theileria annulata, indicating the primers are specific for T. evansi. The PCR method could detect the dog, lion and leopard isolates of T. evansi. Similarly, amplifying the DNA from the experimentally infected tissues was also found to be sensitive. Thus, the findings of this study favour the application of PCR over the parasitological methods for the detection of the early and/or chronic stage of surra in domestic and wild animals.