Prediction, Synthesis and Evaluation of a Synthetic Peptide as an Enzyme-Linked Immunosorbent Assay (ELISA) Candidate for Screening of Bovine Antibodies against Theileria annulata.

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen's kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata.

Growth and proliferation of caprine bone marrow mesenchymal stem cells on different culture media.

The growth and proliferation of mesenchymal stem cells are very sensitive in in vitro and a number of factors like media play a significant role in that context. In this study we assessed effect of different media on growth and proliferation of bone marrow derived mesenchymal stem cells (BMMSCs). The BMMSCs were isolated from caprine bone marrow and were subjected to magnetic activated cell sorting against CD90+, CD105+, CD271+and CD34- along with FC blocker. After characterisation, 2 × 104 cells were seeded in 12 well culture plates in four different media viz. MesenCult, MesenPRO, StemPro and complete DMEM (15 % FBS) to study their growth kinetic for 6 days from passage 0 (P0) to passage 3 (P3). The population doubling time (PDT) was derived from growth curve using logarithmic formula. The results showed that the BMMSCs growth and proliferation was highest in MesenCult media in P0 which varied significantly (p < 0.05) from rest of media and from P1 to P3, it was MesenPRO which yielded maximum cells (p < 0.05). The PDT was also in line with growth curve findings. In conclusion, the MesenPRO media had higher growth and proliferation rate from P1 to P3 although MesenCult had higher cell numbers in P0. In conclusion, the use of MesenPRO media could be a better option than conventional media when mesenchymal stem cells are used in clinical applications and other therapeutic purposes taking consideration to its higher growth and proliferation rate. And MesenCult would be a great option to harvest MSCs from P0.

Certain hormonal profiles of postpartum anestrus jersey crossbred cows treated with controlled internal drug release and ovsynch protocol.

AIM: The study was conducted to determine the serum levels of certain hormones in post-partum anestrus cows following treatment with controlled internal drug release (CIDR) and Ovsynch protocol. MATERIALS AND METHODS: A total of 30 postpartum anestrus cows were divided into three equal groups after thorough gynecoclinical examination. The Group 1 animals received an intravaginal progesterone device on day 0 and 2 ml of prostaglandin F2α (PGF2α) on day of CIDR removal (7th day), Group 2 cows were treated with ovsynch protocol (gonadotropin-releasing hormone [GnRH]-PGF2α-GnRH) on day 0, 7 and 9, respectively, and Group 3 cows were supplemented with mineral mixture and treated as control. The serum estrogen, progesterone, triiodothyronine, and thyroxine concentration were estimated using enzyme-linked immunosorbent assay kit and absorbance was read at 450 nm with Perkin Elmer Wallac 1420 Microplate Reader. RESULTS: There was a significant increase in progesterone level in Group 1 after withdrawal of CIDR as compared to other two groups. However, the estrogen assay revealed a greater concentration in Group 2 against Group 1 on day 7 of sampling. However, there was no significant difference for serum triiodothyronine (T3) and thyroxine (T4) irrespective of treatment protocols and days of sampling. CONCLUSION: Treatment with CIDR based progesterone therapy and drug combinations may affect the reproductive hormonal balance like estrogen and progesterone, which is inevitable for successful return to cyclicity and subsequent fertilization and conception. However, as far as serum T3 and T4 concentration concerned it may not give an astounding result.