G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes.

G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

Genetic and phylogenetic characterization of polycistronic dsRNA segment-10 of bluetongue virus isolates from India between 1985 and 2011.

The smallest polycistronic dsRNA segment-10 (S10) of bluetongue virus (BTV) encodes NS3/3A and putative NS5. The S10 sequence data of 46 Indian BTV field isolates obtained between 1985 and 2011 were determined and compared with the cognate sequences of global BTV strains. The largest ORF on S10 encodes NS3 (229 aa) and an amino-terminal truncated form of the protein (NS3A) and a putative NS5 (50-59 aa) due to alternate translation initiation site. The overall mean distance of the global NS3 was 0.1106 and 0.0269 at nt and deduced aa sequence, respectively. The global BTV strains formed four major clusters. The major cluster of Indian BTV strains was closely related to the viruses reported from Australia and China. A minor sub-cluster of Indian BTV strains were closely related to the USA strains and a few of the Indian strains were similar to the South African reference and vaccine strains. The global trait association of phylogenetic structure indicates the evolution of the global BTV S10 was not homogenous but rather represents a moderate level of geographical divergence. There was no evidence of an association between the virus and the host species, suggesting a random spread of the viruses. Conflicting selection pressure on the alternate coding sequences of the S10 was evident where NS3/3A might have evolved through strong purifying (negative) selection and NS5 through a positive selection. The presence of multiple positively selected codons on the putative NS5 may be advantageous for adaptation of the virus though their precise role is unknown.

Bifunctional 3-Hydroxy-4-Pyridinones as Potential Selective Iron(III) Chelators: Solution Studies and Comparison with Other Metals of Biological and Environmental Relevance.

The binding ability of five bifunctional 3-hydroxy-4-pyridinones towards Cu2+ and Fe3+ was studied by means of potentiometric and UV-Vis spectrophotometric measurements carried out at I = 0.15 mol L-1 in NaCl(aq),T = 298.15 K and 310.15 K. The data treatments allowed us to determine speciation schemes featured by metal-ligand species with different stoichiometry and stability, owing to the various functional groups present in the 3-hydroxy-4-pyridinones structures, which could potentially participate in the metal complexation, and in the Cu2+ and Fe3+ behaviour in aqueous solution. Furthermore, the sequestering ability and metal chelating affinity of the ligands were investigated by the determination of pL0.5 and pM parameters at different pH conditions. Finally, a comparison between the Cu2+ and Fe3+/3-hydroxy-4-pyridinones data herein presented with those already reported in the literature on the interaction of Zn2+ and Al3+ with the same ligands showed that, from the thermodynamic point of view, the 3-hydroxy-4-pyridinones are particularly selective towards Fe3+ and could therefore be considered promising iron-chelating agents, also avoiding the possibility of competition, and eventually the depletion, of essential metal cations of biological and environmental relevance, such as Cu2+ and Zn2+.

Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization.

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

Novel tacrine-benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer's Disease.

Pursuing the widespread interest on multi-target drugs to combat Alzheimer´s disease (AD), a new series of hybrids was designed and developed based on the repositioning of the well-known acetylcholinesterase (AChE) inhibitor, tacrine (TAC), by its coupling to benzofuran (BF) derivatives. The BF framework aims to endow the conjugate molecules with ability for inhibition of AChE (bimodal way) and of amyloid-beta peptide aggregation, besides providing metal (Fe, Cu) chelating ability and concomitant extra anti-oxidant activity, for the hybrids with hydroxyl substitution. The new TAC-BF conjugates showed very good activity for AChE inhibition (sub-micromolar range) and good capacity for the inhibition of self- and Cu-mediated Aß aggregation, with dependence on the linker size and substituent groups of each main moiety. Neuroprotective effects were also found for the compounds through viability assays of neuroblastoma cells, after Aß1-42 induced toxicity. Structure-activity relationship analysis provides insights on the best structural parameters, to take in consideration for future studies in view of potential applications in AD therapy.

Deciphering type-specific neutralizing antibodies to bluetongue virus in goats of Andaman and Nicobar Islands, India.

The presence of antibodies to bluetongue virus (BTV) and the viral antigen is reported recently from the Andaman and Nicobar Islands, a group of islands at the juncture of the Bay of Bengal and the Andaman Sea. A retrospective study was conducted to investigate the presence of neutralizing antibodies to different BTV serotypes in the seroconverted goats of the Islands. Thirty six samples out of 186 serum samples tested were selected on the basis of high antibody titre as predicted in an indirect ELISA. Each of the selected serum samples was used for neutralization of six BTV serotypes (BTV-1, BTV-2, BTV-9, BTV-10, BTV-16 and BTV-23), the most commonly reported serotypes in India. Out of 36 serum samples used in the neutralization study, neutralizing antibodies could be determined in 15 samples. The neutralizing antibodies to BTV-10 were found in more number of the serum samples followed by BTV-1, BTV-2 and BTV-23 and BTV-9 and BTV-16. Many of the serum samples could neutralize more than one BTV serotypes indicating possible widespread superinfections by multiple BTV serotypes in goats in the Islands. Majority of the serum samples used in the neutralization study could not neutralize any of the six BTV serotypes commonly reported from India indicating possible circulation of other BTV serotypes yet to confirm. The present study reveals circulation of multiple BTV serotypes in Andaman and Nicobar Islands where there was no such report available earlier. The findings are laudable as the baseline information for further investigations to identify and characterize the virus and competent vectors and for implementing appropriate suitable control strategies for bluetongue in the Islands and the nearby territories.

A Light-up Logic Platform for Selective Recognition of Parallel G-Quadruplex Structures via Disaggregation-Induced Emission.

The design of turn-on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G-quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition-driven disaggregation (on-signal) of an ultrabright coumarin-quinazoline conjugate. The synthesized probe selectively lights-up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label-free molecular logic system. Finally, our molecule preferentially stains the G4-rich nucleoli of cancer cells.

New Multitarget Hybrids Bearing Tacrine and Phenylbenzothiazole Motifs as Potential Drug Candidates for Alzheimer's Disease.

Research on neurodegenerative brain disorders, namely the age-dependent Alzheimer's disease (AD), has been intensified in the last decade due to the absence of a cure and the recognized increasing of life expectancy for populations. To address the multifactorial nature and complexity of AD, a multi-target-directed ligand approach was herein employed, by designing a set of six selected hybrids (14⁻19) that combine in the same entity two pharmacophores: tacrine (TAC) and 2-phenylbenzothiazole (PhBTA). The compounds contain a methoxy substituent at the PhBTA moiety and have a variable length linker between that and the TAC moiety. The docking studies showed that all the compounds assure a dual-binding mode of acetylcholinesterase (AChE) inhibition, establishing π-stacking and H-bond interactions with aminoacid residues at both active binding sites of the enzyme (CAS and PAS). The bioassays revealed that the designed compounds display excellent AChE inhibitory activity in the sub-micromolar range (0.06⁻0.27 µM) and moderate inhibition values for amyloid-ß (Aß) self-aggregation (27⁻44.6%), compounds 14 and 15 being the lead compounds. Regarding neuroprotective effects in neuroblastoma cells, compounds 15, 16 and 19 revealed the capacity to prevent Aß-induced toxicity, but compound 16 showed the highest neuroprotective effect. Overall these hybrid compounds, in particular 15 and 16, with promising multitarget anti-AD ability, encourage further pursuing studies on this type of TAC-PhBTA derivatives for potential AD therapy.

Speciation Studies of Bifunctional 3-Hydroxy-4-Pyridinone Ligands in the Presence of Zn2+ at Different Ionic Strengths and Temperatures.

The acid-base properties of two bifunctional 3-hydroxy-4-pyridinone ligands and their chelating capacity towards Zn2+, an essential bio-metal cation, were investigated in NaCl aqueous solutions by potentiometric, UV-Vis spectrophotometric, and 1H NMR spectroscopic titrations, carried out at 0.15 ≤ I/mol -1 ≤ 1.00 and 288.15 ≤ T/K ≤ 310.15. A study at I = 0.15 mol L-1 and T = 298.15 K was also performed for other three Zn2+/Lz- systems, with ligands belonging to the same family of compounds. The processing of experimental data allowed the determination of protonation and stability constants, which showed accordance with the data obtained from the different analytical techniques used, and with those reported in the literature for the same class of compounds. ESI-MS spectrometric measurements provided support for the formation of the different Zn2+/ligand species, while computational molecular simulations allowed information to be gained on the metal-ligand coordination. The dependence on ionic strength and the temperature of equilibrium constants were investigated by means of the extended Debye-Hückel model, the classical specific ion interaction theory, and the van't Hoff equations, respectively.

Seroprevalence of bluetongue and presence of viral antigen and type-specific neutralizing antibodies in goats in Tripura, a state at Indo-Bangladesh border of northeastern India.

Bluetongue (BT) is a notifiable multiple species transboundary viral disease of domestic and wild ruminants. Though the disease is enzootic in India, little is known of the disease burden and prevalent serotypes in Tripura, a hilly state of northeastern India sharing a vast porous border with Bangladesh. A surveillance study was conducted to understand the disease burden in goats in Tripura. Serum (n = 1240) and blood (n = 194) samples were collected during the year 2014 to 2017 from all the eight districts of Tripura. The overall prevalence of BT seroconversion was 47.58% whereas the presence of viral antigen was 20.61% at the individual level. Percent seroconversion was found more (50.47 ± 4.00, CI 41.31 to 49.47) in adult goats in comparison to the younger animals where it was 45.39 ± 2.08, CI 42.63 to 58.31. Presence of neutralizing antibodies in selected serum samples (n = 72) was investigated by serum neutralization test (SNT) against six bluetongue virus (BTV) serotypes and BTV-1 was found as most predominant (65.27%) followed by BTV-16 (26.38%), BTV-10 (20.83%), BTV-9 and 23 (13.88%), and BTV-2 (6.94%). To the best of our knowledge, this is the first study conducted in Tripura to investigate the presence of BTV antigen and type-specific neutralizing antibodies in apparently healthy goats.

Isolation and Characterization of Bluetongue Virus Recovered from Blood Samples by Immunoaffinity Purification.

An immunoaffinity chromatography (IAC) method was optimized for the selective capture of bluetongue virus (BTV) from blood samples and isolation of the virus in cell culture. The antibody against BTV core particles (lacking the outer capsid proteins VP2 and VP5) was used for the optimization of IAC technique. The antibody against BTV core particle was conjugated with Protein A-virus complex and the complex was dissociated using elution buffer (4 M MgCl2 with 75 mM HEPES, pH 6.5). The optimized IAC method specifically purified the BTV without capturing other commonly infecting small ruminant's viruses like gaotpox virus (GTPV), sheeppox virus (SPPV), Peste des petits ruminants virus (PPRV) and Foot and mouth disease virus (FMDV). The blood samples (n = 22), positive for BTV antigen in sandwich-ELISA were attempted for virus isolation in the BHK-21 cell using the optimized IAC method. A total of seven BTV were isolated by selective capturing of the virion particles. The isolated viruses were characterized by RNA-PAGE, sequence analysis and serum neutralization test (SNT). Electropherotypic analysis of viral dsRNA in the RNA-PAGE revealed the presence of ten dsRNA segments characteristic of BTV. Out of seven isolates, four isolates were identified as BTV-1 and three isolates were identified as BTV-16 based on nucleotide sequences of segment-2. Phylogenetic analysis of segment-2 nucleotide sequence segregated BTV-1 and BTV-16 isolates to monophyletic cluster at close proximity to other eastern topotype. In SNT, hyperimmune serum (HIS) against BTV-1 neutralized the four BTV-1 isolates up to a titer > 256 and HIS against BTV-16 neutralized the three BTV-16 isolates up to a titer > 128. The IAC technique will be useful for the selective capture of BTV from mixed infection (BTV with other small ruminant's viruses) and isolation from blood sample having low viral load by enrichment.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody.

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Acetamide Derivatives of Chromen-2-ones as Potent Cholinesterase Inhibitors.

Alzheimer's disease (AD), a neurodegenerative disorder, is a serious medical issue worldwide with drastic social consequences. Inhibition of cholinesterase is one of the rational and effective approaches to retard the symptoms of AD and, hence, consistent efforts are being made to develop efficient anti-cholinesterase agents. In pursuit of this, a series of 19 acetamide derivatives of chromen-2-ones were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential. All the synthesized compounds exhibited significant anti-AChE and anti-BChE activity, with IC50 values in the range of 0.24-10.19 µM and 0.64-30.08 µM, respectively, using donepezil hydrochloride as the standard. Out of 19 compounds screened, 3 compounds, viz. 22, 40, and 43, caused 50% inhibition of AChE at 0.24, 0.25, and 0.25 µM, respectively. A kinetic study revealed them to be mixed-type inhibitors, binding with both the CAS and PAS sites of AChE. The above-selected compounds were found to be effective inhibitors of AChE-induced and self-mediated Aß1-42 aggregation. ADMET predictions demonstrated that these compounds may possess suitable blood-brain barrier (BBB) permeability. Hemolytic assay results revealed that these compounds did not lyse human RBCs up to a thousand times of their IC50 value. MTT assays performed for the shortlisted compounds showed them to be negligibly toxic after 24 h of treatment with the SH-SY5Y neuroblastoma cells. These results provide insights for further optimization of the scaffolds for designing the next generation of compounds as lead cholinesterase inhibitors.

Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent.

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.

Recent progress on pyrazole scaffold-based antimycobacterial agents.

New and reemerging infectious diseases will continue to pose serious global health threats well into the 21st century and according to the World Health Organization report, these are still the leading cause of death among humans worldwide. Among infectious diseases, tuberculosis claims approximately 2 million deaths per year worldwide. Also, agents that reduce the duration and complexity of the current therapy would have a major impact on the overall cure rate. Due to the development of resistance to conventional antibiotics there is a need for new therapeutic strategies to combat Mycobacterium tuberculosis. Subsequently, there is an urgent need for the development of new drug candidates with newer targets and alternative mechanism of action. In this perspective, pyrazole, one of the most important classes of heterocycles, has been the topic of research for thousands of researchers all over the world because of its wide spectrum of biological activities. To pave the way for future research, there is a need to collect the latest information in this promising area. In the present review, we have collated published reports on the pyrazole core to provide an insight so that its full therapeutic potential can be utilized for the treatment of tuberculosis. In this article, the possible structure-activity relationship of pyrazole analogs for designing better antituberculosis (anti-TB) agents has been discussed and is also helpful for new thoughts in the quest for rational designs of more active and less toxic pyrazole-based anti-TB drugs.

Synthesis and Sensing Applications of Fluorescent 3-Cinnamoyl Coumarins.

We have synthesized two novel fluorescent 3-(4-diethylaminocinnamoyl) coumarins that exhibit fluorescence quenching upon exposure to a nerve agent simulant, diethylchlorophosphate (DCP), providing a basis for rapid and sensitive DCP chemosensing. Furthermore, these coumarin derivatives display two-photon fluorescence upon illumination with near-infrared laser pulses and their two-photon (TP) absorption cross-section was evaluated. The potential for TP bio-imaging of these compounds was investigated by their cellular uptake in HeLa cells by TP confocal microscopy.

Detection of Babesia bigemina infection in cattle from north-eastern India by polymerase chain reaction and its genetic relatedness with other isolates.

A total of 333 blood samples were collected from cattle suspected for haemoprotozoan infections from three states of north-eastern part of India. All the samples were examined for diagnosis of Babesia bigemina infection using PCR for detection of specific DNA. Out of these, 12 (3.60%) samples were found positive for B. bigemina DNA on PCR using the organism-specific primers derived from 18S ribosomal RNA (rRNA) gene of B. bigemina. An expected size of 1124-bp PCR product was visualized on agarose gel electrophoresis with all the 12 samples, and four of the products was further cloned and sequenced. Basic Local Alignment Search Tool (BLAST) analysis of B. bigemina sequences generated in the present study share 99.2 to 99.7% identity at 18S rRNA gene nucleotide sequence level. These Indian B. bigemina sequences were found to be closely related with the cognate gene nucleotide sequences of B. bigemina from Argentina and Kenya where 99.1 to 99.9% and 99.0 to 99.7% nucleotide identities were observed, respectively. Distant relationship of these Indian organisms was observed with few cognate gene sequences from China where more than 7% divergence was observed in the distance matrix.