Immuno-bioinformatic approach for designing of multi-epitope merozoite surface antigen of Babesia bigemina and evaluation of its immunogenicity in inoculated calves.

Babesiosis is a parasitic disease caused by intraerythrocytic parasites of the genus Babesia, which infect both wild and domestic animals. Merozoite surface antigens (MSAs) have been identified as efficient immunogens in Babesia-infected animals. MSAs play a key role in the invasion process and have been proposed as potential targets for vaccine development. Epitope-based vaccines offer several advantages over whole protein vaccines as the immunogenic proteins are small and can induce both Th1 and Th2 immune responses, which are desirable for protection. However, the MSA, particularly gp45, is polymorphic in Babesia bigemina, posing a challenge to vaccine development. The purpose of this study was to develop a recombinant gpME (gp45-multi-epitope) for a vaccine against Babesia bigemina. B-cell, T-cell, and HLA epitope predictions were used to synthesize the gpME sequence from the consensus sequence of gp45. The gpME sequence was synthesized and cloned in the pET28α vector through the commercial biotechnology company to get pET28-gpME. The plasmid cloned with the gpME sequence comprising 1068 bp was expressed in a bacterial expression system. A band of 39 kDa of rec-gpME was obtained via SDS-PAGE and Western blotting. Rec-gpME @200ng was injected in calves 3 times at 2 weeks interval. The humoral response was evaluated through the indirect ELISA method. The ELISA with rec-gp45 protein showed a significant value of optical density. The recombinant protein containing multiple epitopes from the MSA gp45 may represent a promising candidate for a vaccine against Babesia bigemina.

Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan.

The present study was focused on the incidence of ticks and tick-borne diseases (TTBD) in cross-bred cattle (Friesian x Sahiwal) of two farms (n = 2548) in district Lahore, Pakistan. We collected total of 572 ticks (adults and nymphs) and blood samples (10 ml) for microscopic i.e., blood smear test - Giemsa Stain (BST) and molecular analysis; Reverse Line Blot-General Primer-PCR (RLB-PCR) and Specie Specific Primer PCR (SP-PCR) from infested cattle (n = 100) from months of April to September. Results: The tick specie identified was Rhipicephalus microplus at both farms, with significant difference in infestations rate amongst both farms (p< 0.0001). The cross-bred cattle having higher ratio of Friesian blood and lower ratio of Sahiwal blood were mostly infested by ticks (p < 0.0458) and haemoparasites (p <0.474) and vice versa. The SP-PCR showed higher number of haemoparasites infection than BST, which revealed 16% T. annulata (p < 0.0001 and k value 0.485, 0.0001), 51% B. bigemina (p < 0.0001 and k value 0.485, 0.0001) and 15% A. marginale (p < 0.001 and k value 0.207, 0.001), respectively. The single infection with B. bigemina was 34% (n = 34/100) and A. marginale 6% (n = 6/100). The double infection with T. annulata/B. bigemina was 8% (n = 8/100) and B. bigemina/A. marginale 1% (n = 1/100). Whereas the triple infection with T. annulata/B. bigemina/A .marginale was 8% (n = 8/100). The phylogenetic study of isolated sequence of T. annulata revealed close homology to isolates from Iran (87%), B. bigemina to isolates from Cuba (94 to 100%) and A. marginale with isolates from Pakistan (99 to 98%).