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.

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.

The Asian Lions (Panthera leopersica) were widely present in Asian forests specially in subcontinent regions. However, they are now only found in the smaller pockets of captivity for rearing and wildlife conservation even in Asian region. The present study focused on the applied care and effectiveness of hand rearing protocol of lion cubs conducted in Lahore zoological garden. The study was also helpful in determining the nutritional effects of two different feeding protocols. For this, the lion cubs were separated in two different groups to evaluate the gained body weight and its growth. Three lion cubs were present in each single group followed by single feeding protocol. The feeding protocol was carried out by using two commercially available milk replacers i.e., KMR® Milk replacer and Royal Canin® for lion cubs initially. The diet was maintained using various mineral and vitamin supplements to check its correlation with growth and weight gained. Its evident from the study that cubs reared on KMR® Milk replacer showed better growth as compared to the Royal Canin® used in the study. It was also observed that more complications occurred during feeding and rearing protocol of lions in relation to health status. Instead of all complications, hand rearing of lion cubs is recommended for conservation in captivity. But complications must be studied that occurred due to various environmental and feeding factors.