Humoral and cellular immunity in response to an in silico-designed multi-epitope recombinant protein of Theileria annulata.

Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.

Humoral and Cell-Mediated Immune Response Validation in Calves after a Live Attenuated Vaccine of Babesia bigemina.

The current vaccines to control bovine Babesia bigemina (B. bigemina) infection are not fully protective and vaccination failures incur heavy losses to the cattle industry around the world. Using modified micro-aerophilous stationary phase, we developed a culture-derived attenuated live vaccine against B. bigemina and tested a single subcutaneous inoculation of 2 × 108 infected erythrocytes in calves. The protection was measured after a lethal intravenous challenge with 5 × 108 virulent calf-derived B. bigemina. Our results demonstrated that a single shot of attenuated vaccine was capable of inducing robust humoral and cell-mediated immune responses in calves. We found a significant increase in the IgG antibody titers post-challenge and a strong proliferation of both CD4+ and CD8+ T cells contributing towards the protection. Our vaccine provided complete protection and parasitic clearance, which was followed for more than 100 days post-challenge. This immunity against babesiosis was directly linked to strong humoral responses; however, the parasitic clearance was attributed to significant T cells effector responses in vaccinated calves as compared to the infected control calves. We anticipate that these results will be helpful in the development of more efficient culture-derived vaccines against Babesia infections, thus reducing significant global economic losses to farmers and the cattle industry.

Expiry of a completely splenectomised calf in post-operative period due to mixed piroplasm infection: a case report.

Bovine babesiosis is an infectious protozoan disease and causes significant economic losses in terms of production loss and mortality. The genus Babesia belongs to the family Babesiidae order piroplasmida and is transmitted by ticks globally. The signs of disease are particularly prominent in old or immuno-compromised animals. The spleen plays a vital role in defence against hemoparasites like Babesia. A young cross-bred cow calf of about 3 months of age was splenectomised to propagate Babesia in vivo experimentally. Prior to splenectomy, the calf was examined through microscopy and PCR analysis and was found negative for any kind of piroplasms including Babesia. The calf was completely splenectomised, but the calf was naturally infected during its postoperative period. The calf expired after naturally acquiring Babesia bigemina and Theileria annulata during the 11 th day of postoperative period owing to increased parasitaemia, exhibiting typical mixed parasitic infection stigmata e.g. reddish urine, elevated temperature up to 41.38°C. This study concluded that complete splenectomy along with dexamethasone administration in the postop period caused exceptional increase in parasitaemia. This parasitaemia couldn't be countered by any symptomatic treatment because of the absence of spleen and greatly reduced immunity of the animal.