S. K. Bandyopadhyay

Production and characterization of monoclonal antibodies to peste des petits ruminants (PPR) virus.

Peste des petits ruminants (PPR) is an acute, febrile viral disease of small ruminants, caused by a virus of the genus Morbillivirus. PPR and rinderpest viruses are antigenically related and need to be differentiated serologically. In the present study, 23 mouse monoclonal antibodies were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma cells. Among these, two belong to the IgM class and the remaining 21 to various subclasses of IgG. The MAbs from the IgG class designated 4B6 and 4B11 neutralized PPR virus in vitro. In radioimmunoprecipitation assay, 10 MAbs recognized nucleoprotein, 4 recognized the matrix protein and one each haemagglutinin and phosphoprotein. The remaining 7 MAbs failed to precipitate any defined viral protein. The reactivity pattern of the monoclonal antibodies in indirect ELISA indicated a close antigenic relationship within three Indian PPR (lineage 4) virus isolates and also within two rinderpest vaccine strains. All PPR virus isolates could be distinguished from rinderpest vaccine viruses on the basis of the reactivity pattern of all MAbs and anti-N protein MAbs. A set of six monoclonal antibodies specific to PPR virus could also be identified from the panel. From the panel of MAbs available, two MAbs were selected for diagnostic applications, one each for the detection of antigens and antibodies to PPR virus.

One-step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical samples.

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7–17 days post infection (p.i.). and in oral swabs 7–15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea

OBJECTIVE To evaluate the antimicrobial efficacy of berberine, a plant alkaloid. METHODS Five multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. RESULTS For both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC(50) of berberine chloride for STEC/EPEC isolates varied from 2.07 μM to 3.6 μM with a mean of (2.95 ± 0.33) μM where as for ETEC strains it varied from 1.75 to 1.96 μM with a mean of (1.87 ± 0.03) μM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. CONCLUSIONS The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Characterization of an Indian bluetongue virus isolate by RT-PCR and restriction enzyme analysis of the VP-7 gene sequence.

The reverse transcription–polymerase chain reaction (RT-PCR) was standardized to amplify the VP-7 gene sequences of an Indian isolate of bluetongue virus serotype 23. Using two different sets of primers, a sequence of 1156 bp comprising the complete coding sequence of the VP-7 gene and its 770 bp internal sequence were amplified. The sensitivity of RT-PCR, using these two sets of primers individually was 40 pg and 4 pg, with the external and internal primers, respectively, whereas the nested PCR was 100-fold more sensitive than the single PCR with the external primers. Further, by restriction enzyme digestion of the 1156 bp amplicon, using CfoI, PstI and TaqI enzymes, the Indian isolate was found to be genetically different from isolates from the United States and Australia. RT-PCR and restriction enzyme digestion were applied to detect virus directly in blood samples taken from sheep suspected of bluetongue virus infection.

Use of zinc chloride as alternative stimulant for in vitro study of nitric oxide production pathway in avian splenocyte culture.

Nitric Oxide (NO) plays an important role for regulation of cellular and vascular response of inflammation and initiates killing mechanism in the host-defense reactions. NO production is regulated through inducible nitric oxide synthase (iNOS) pathway in response to infections and injurious agents besides pro-inflammatory cytokines secreted by the host. Evaluation of NO pathway is one of the major targets which can evaluate various immunomodulators for their therapeutic interaction on innate immune system. Lipopolysaccharide (LPS) and concavalin A (ConA) are used as standard mitogen for peripheral blood mononuclear cells and splenocyte of mammalian and avian cell culture. During the present investigation ZnCl2 has been explored as standard mitogen and result was comparable with standard mitogen. The result has been evaluated on the basis of relative mRNA expression of iNOS and interferon gamma and nitrite assay. Observation indicated significantly higher expression of both biomolecules in comparison to control, LPS, ConA treated group. This study indicated that, ZnCl2 can also be used as standard stimulant for molecular mining of innate immunity.

Sequence analysis of the nucleoprotein gene of Asian lineage peste des petits ruminants vaccine virus.

The complete nucleotide sequence of the nucleocapsid (N) protein of the peste-des-petits ruminants vaccine virus (PPRV Sungri/96) belonging to the Asian lineage was determined. The gene was 1692 nucleotides in length and encoded a polypeptide of 525 amino acids. The PPRV Sungri/96 N gene has a nucleotide homology of 92% for PPRV Nigeria 75/1 to 55.5% for canine distemper virus. At amino acid level the homology was 94.1% with PPRV Nigeria 75/1, while with other morbilliviruses, PPRV Sungri/96 had only 71.4–64.9% amino acid identity. The phosphorylation prediction reveals eight conserved sites across morbilliviruses, whereas in the C-terminal portion of the protein the sites are not conserved. Phylogenetic analysis of different N proteins of morbilliviruses revealed five well-defined clusters as observed previously. To the best of our knowledge this is the first report describing the nucleocapsid gene sequence of PPRV Indian isolate.

An evaluation of antigen B family of Echinococcus granulosus, its conformational propensity and elucidation of the agretope.

The present communication evaluates the antigen B (AgB) family of bubaline isolates of Echinococcus granulosus with respect to their conformational propensity and also discusses the stretches of agretope. AgB, which is abundantly present in hydatid cyst fluid, is encoded by a gene family, AgB1-AgB5. Hydatidosis is of zoonotic and economic importance in India. Buffaloes serve as the intermediate host. However, to date the AgB family has not been fully analysed. During the present study two different primers used for amplification of AgB1 revealed homology to Echinococcus canadensis (G8) as well as E. granulosus sensu stricto (G1/G2). The sequence of AgB3 is homologous to that of the well-defined species, Echinococcus ortleppi (G5), and the predicted amino acid sequence of AgB4 is homologous to bovine isolates identified earlier. alpha- and beta-amphipathic structures were recorded in all the antigens designated as T-cell receptor sites. The antigenic index of different stretches correlated with hydrophilicity because the hydrophobic residues are not accessible to the cells. In this study, we investigated the binding propensity of AgB to MHC II in order to determine stretches of agretope. Agretopes began with four hydrophilic residues. Two to three additional hydrophilic residues were present in the internal motif. This comparison of AgB and its family of bubaline isolates, with respect to their sequence information, alpha- and beta- amphipathic regions, antigenic index and stretches of agretope is the first such report from India.

Prevalence of Deg Nala disease in eastern India and its reproduction in buffaloes by feeding Fusarium oxysporum infested rice straw

OBJECTIVE To undertake a study on prevalence of Deg Nala disease in eastern states of India and to reproduce the disease in buffaloes by the Fusarium spp., isolated from the affected region. METHODS During this investigation, a survey was conducted covering four states of eastern region to identify the Deg Nala cases as well as to isolate and characterize the causative agent(s). An experimental study was carried out to reproduce the disease in healthy male buffaloes (2-3 years age) by randomly dividing them into five groups (four in each group). Each individual group was fed with rice straw artificially infested with either of the two representative isolates of Fusarium oxysporum (F. oxysporum) (F01, F02) or representative reference strains of Fusarium equiseti (F. equiseti) (ITCCF-2470) and Fusarium moniliforme (F. moniliforme) (ITCCF-4821) for 30 days, whereas the control group was fed with normal rice straw only. RESULTS A total of 658 Deg Nala cases were recorded and 12 Fusarium isolates were identified from the mouldy rice straw collected from these affected areas. The characterization of the isolates revealed three species viz., F. oxysporum, F. equiseti and F. moniliforme, among which F. oxysporum was predominant. The disease was artificially reproduced in three buffaloes in F01 group and one in F02 group within 20-23 days by feeding F. oxysporum infested rice straw which resembled the clinical symptoms and gross lesions of natural Deg Nala cases. CONCLUSIONS The field investigation and laboratory studies, including experimental production of Deg Nala disease suggest the possible involvement of mycotoxins. However, further investigations needs to be done to understand nature of the toxic factors involved in production of the Deg Nala disease.

Detection of canine echinococcosis by coproantigen ELISA

Objective: To study the canine echinococcosis by coproantigen ELISA method. Methods: During the present investigation experimental infection was established using evaginated worms of Echinococcus granulosus (E. granulosus). To check cross reactivity two pups were infected with Taenia hydatigena (T. hydatigena). In order to detect the presence of antigen, hyperimmune sera were raised against excretory-secretory products of adult worms E. chinococcus gronulosus. Faecal sample collected either from experimentally infected pups or from other sources were heated at 70 ℃ to detect heat stable soluble antigen. Results: Pups harbouring less than 104 worms showed negative results. Samples collected from 14 days onwards from experimentally infected animals harbouring more than 104 worms showed positive value. The maximum positive samples were detected in samples collected from in and around slaughter house and the least number of samples were detected positive maintained by dog squad. Conclusions: The affinity purified IgG exhibited promising results for detection of canine echinococcosis by indirect ELISA.