Janhavi R Rao

Molecular characterisation of paraflagellar rod protein gene (PFR) of Trypanosoma evansi

Paraflagellar rod (PFR), the major constituent proteins of flagellum, is restricted to kinetoplastids, euglenoids and dinoflagellates. Owing to their strategic location and invariable nature the proteins are considered as prospective vaccine targets. The present communication reports molecular cloning of the coding sequences of PFR1 and PFR2 genes, the two important constituent proteins of PFR of Trypanosoma evansi, Izatnagar isolate. The cloned nucleotide sequence of PFR1 revealed 99.8% sequence homology between the Izatnagar and China isolates with a single nucleotide change at position 867 bp of PFR1 open reading frame (ORF). The nucleotide sequencing data also revealed 99.8, 82.1, 79.9 and 72.9% sequence homology with Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Crithidia daenei, respectively. The cloned nucleotide sequence of PFR2 gene revealed 99.9% sequence homology between the Izatnagar and China isolates with a single nucleotide change at position 928 bp of PFR2 ORF of Izatnagar isolate. The PFR2 nucleotide sequence also showed 99.9, 82.4, 75.3 and 74.8% sequence homology with the published sequence of T. brucei, T. cruzi, L. infantum and C. fasciculata, respectively. The conserved nature of various PFR genes present in kinetoplastids could be exploited for development of a protective vaccine against multiple Trypanosoma species.

Application of a stability-indicating HPTLC method for quantitative analysis of sertraline hydrochloride in pharmaceutical dosage forms

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of sertraline hydrochloride in the bulk drug and in formulations has been established and validated. Aluminum foil HPTLC plates precoated with silica gel 60F254 as stationary phase were used with toluene-ethyl acetate-ethanol-ammonia 8:2:0.5:0.1 (v/v). A compact spot was obtained for sertraline hydrochloride (RF 0.33 ± 0.02). Densitometric analysis was performed in absorbance mode at 273 nm. Regression analysis of calibration data revealed a good linear relationship (r2 = 0.9996 ± 0.02) between response and concentration over the range 2000–12000 ng per band. The method was validated for accuracy, precision, linearity, limits of detection and quantification, and robustness. Sertraline hydrochloride was subjected to acidic and alkaline hydrolysis, oxidation, dry heat treatment, and photodegradation. Peaks of degradation products were well resolved from that of the pure drug and had significantly different RF values. Statistical analysis showed the method enabled repeatable and selective analysis of sertraline hydrochloride. The method can be used for routine quantitative analysis of sertraline hydrochloride in the bulk drug and in formulations.

β-Tubulin gene based PCR-RFLP method for specific detection of Babesia bigemina and Theileria annulata isolates.

Abstract Singh, H., Cheema, P.S., Mishra, A.K., Tewari, A.K., Rao, J.R., Ravindran, R., Ray, D. and Bansal, G.C. 2010. β-tubulin gene based PCR-RFLP method for specific detection of Babesia bigemina and Theileria annulata isolates. J. Appl. Anim. Res., 37: 233–238. For early detection of Babesia bigemina and Theileria annulata β-tubulin gene was targeted based on PCR assays. Subsequently, on the basis of sequence analysis of β-tubulin gene fragment of various isolates, a new PCR-RFLP method based on enzymes EcoRI and HindIII has been developed for species identification of B. bigemina and T. annulata isolates.

Development of de novo PCR Primers Generated from Arbitrary PCR for the Detection of Babesia bigemina in Bovines

Abstract Ravindran, R., Rao, J.R. and Mishra, A.K. 2010. Development of de novo PCR primers generated from arbitrary PCR for the detection of Babesia bigemina in bovines. J. Appl. Anim. Res., 38: 109–112. A set of de novo oligonucleotide polymerase chain reaction (PCR) primers yielding a 480 bp product were developed with an analytical sensitivity of 0.5 ng using the sequence information of a B. bigemina specific monomorphic RAPD fragment. The primers did not amplify any products from DNA templates of bovine and bubaline hosts as well as other common protozoans like Theileria annulata, Trypanosoma evansi and Toxoplasma gondii. These primers will help in detection of this pathogen.

Analytical sensitivity of 35 copy B1 PCR assay in detecting Toxoplasma gondii infection in mouse.

Abstract Kumar, M.U., Mishra, A.K., Rao, J.R. and Tewari, A.K. 2010. Analytical sensitivity of 35 copy B1 PCR assay in detecting Toxoplasma gondii infection in mouse. J. Appl. Anim. Res., 38: 65–67. A 35 copy PCR assay was tested for its efficacy and in amplifying the DNA of theoretically lowest number of tachyzoites of Toxoplasma gondii, R.H. strain in Swiss albino mice. Two μl (20 ng) of tachyzoite DNA with F 5′-GGAACTGCATCCGTTCATGAG3′ (BG1) and R 5′-TCTTTAAAGCGTTCGTGGTC3′ (BG100) primers yielded an 194 bp product on agarose gel. The PCR assay was found sensitive in detecting as low as 1.21 pg of DNA of T. gondii, R.H. strain. Hence, the assay may be used for the diagnosis of toxoplasmosis in clinical samples.

Lymphocyte Migration Inhibition Response to Babesia bigemina Live and Dead Antigens

Abstract Tewari, A.K., Sharma, N.N., Rao, J.R. and Mishra, A.K. 1996. Lymphocyte migration inhibition response to Babesia bigemina live and dead antigens. J. Appl. Anim. Res., 10: 187–193. In bovine babesiosis caused by Babesia bigemina, the role of cell mediated immune response (CMIR) was assessed by lymphocyte migration inhibition test (LMIT) following immunisation with babesial dead antigens administered alone or in combination with immunomodulators and upon challenge exposure to live virulent B. bigemina. Parasite-specific LMIT response was detectable in components. The migration inhibition (MI) index peaked at 14 days following immunisation and the index was significantly high from day 7 to 28 upon virulent challenge.