Evaluation of immunogenic potential of 75kDa and 56kDa proteins of newcastle disease virus (NDV).

The R2B strain of virus of new castle disease virus (NDV) was propagated in 9-11 day old embryonated chicken eggs via allantoic cavity route and after seven serial passages virus was purified from allantoic fluid. Purified virus was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis which yielded six major polypeptides ranging from 38-200 kDa. Protein fractions, corresponding to 75 and 56kDa, resembling haemagglutinin-neuraminidase (HN) and fusion (F) proteins were used to ascertain their immunization potential. Immunization of viral proteins was compared with the whole virus vaccine. Among different group of birds, highest haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titers were obtained in birds immunized with whole virus vaccine followed by viral proteins, 75 and 56kDa in combination which was comparable with birds immunized with 56kDa protein alone. Despite lower values of HI and ELISA titers elicited by viral subunits in immunized birds, when challenged with virulent NDV strain, protection accorded by viral proteins in combination (75 +56kDa) or 56kDa alone was comparable with whole virus vaccine.

PCR based rapid detection of Salmonella from poultry samples.

Salmonella is very important from the zoonotic point of view, as it causes many diseases in animals and humans. This study was conducted during September 2005 to February 2006 to develop rapid detection system for Salmonella from poultry samples. In the present study 300 poultry samples were screened for Salmonella. Earlier, isolation and identification of Salmonella from clinical samples by traditional cultural techniques required laborious procedures which can last upto 7 days, whereas amplification of DNA sequences unique to an organism using the PCR improves both the speed of detection and the level of sensitivity at which organisms can be detected and has been increasingly used to identify several bacterial species from food and clinical samples. In this study Salmonella were rapidly detected by targeting invA gene, giving PCR product of 284 bp size. Therefore this technique can be used for the screening of Salmonella in the routine testing.

A competitive dipstick enzyme immunoassay for diagnosis of early pregnancy in bovine.

Early pregnancy diagnosis in bovines is one of the important aspects in efficient dairy farm management. In order to develop a competitive enzyme immunoassay (EIA) employing low cost reagents, anti-progesterone antiserum and progesterone-penicillinase enzyme conjugate were prepared. Using this anti-serum and conjugate along with pencillinV-starch-iodine substrate system, the competitive EIA was standardized. In the experiment, danazol, a weak androgen used to extract the progesterone bound to proteins in milk, was included after standardizing the optimum concentration. Incubation period and temperature and pH of the reaction mixture were also optimized. The developed test was validated with milk samples obtained from dairy farm and individual animal owners. Confirmation of the pregnancy was made by per rectum examination of the genital tract around 60 days post-insemination. The user friendly test procedure showed sensitivity and specificity of 83.3% and 87.5%, respectively as compared with residual binding method which was earlier developed in the laboratory with sensitivity and specificity of 100% and 87.5% respectively.

Partial purification and cytotoxic effects of Salmonella proteinous moieties on chick embryo fibroblasts.

Salmonella enterica serovars, viz., S. Weltevreden, S. Typhimurium, S. Gallinarum and S. Bareilly were treated with cephotaxime to release of intracellular proteins. The cephotaxime extract (CE) was salt precipitated with ammonium sulphate (45-70%) and dialyzed, and denoted as precipitated dialyzed proteins (PDP). Further, both CE and PDP of Salmonella Weltevreden and PDP of rest of the serovars were subjected to gel filtration using Sephacryl S-200HR. Different fractions along with CE and PDP were studied for their cytotoxicity using chicken embryo fibroblast (CEF). All the CE and PDP exerted cytotoxic effects, characterized by rounding, detachment, shrinkage and clumping of cells with syncytia formation. Also, the fractions eluted in the 2nd and 3rd peaks through Sephacryl S-200HR column invariably had cytotoxic activity. It was concluded that in place of Vero cell line, CEF cells could also be used to test cytotoxicity.

Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis).

Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH), and glycerol at different concentrations (3.5, 4, 5, 6, and 7 M each with 0.5 M sucrose and 0.4% BSA in DPBS) on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature buffalo oocytes. The COCs were harvested from the ovaries by aspirating the visible follicles. The recovery of post-thaw morphologically normal oocytes was lower in 3.5 and 4 M DMSO, EG, and PROH compared to 5, 6, and 7 M. In all the concentrations of glycerol, an overall lower numbers of oocytes recovered were normal compared to other cryoprotectants. Less number of oocytes reached metaphase-II (M-II) stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH, and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation was obtained in 7 M solutions of all the cryoprotectants. The cleavage rates of oocytes vitrified in different concentrations of DMSO, EG, PROH, and glycerol were lower than that of the fresh oocytes. The cleavage rates were higher in oocytes cryopreserved in 6 and 7 M DMSO, EG, PROH, and glycerol compared with oocytes cryopreserved in other concentrations. However, the percentage of morula and blastocyst formation from the cleaved embryos did not vary in fresh oocytes and vitrified oocytes. In conclusion, this report describes the first successful production of buffalo blastocysts from immature oocytes cryopreserved by vitrification.

Generation of anti-teliospores antibodies for immunolocalization and characterization of antigenic epitopes of teliospores of Karnal bunt (Tilletia indica) of wheat.

Polyclonal antibodies raised against intact teliospores of T. indica in New Zealand albino rabbits were used for the development of indirect immunofluorescence tests. Specificity of anti-teliospore antibodies was evaluated by cross reactivity studies on other bunt, smut and related pathogens. The characteristic reactivity pattern indicated that the antibodies reacted with Tilletia species only. Chemical modifications, heat and enzyme treatments followed by indirect immunofluorescence tests were employed to delineate the molecular nature of the surface antigens. There was partial or no loss in immunoreactivity by methanol, periodate, heat or trypsin treatments. Extensive periodate treatment altered the fluorescence pattern due to changes in configuration of carbohydrate antigen present in episporium. Sequential treatment of periodate and trypsin showed diminished fluorescence due to access of proteolytic enzyme into inner site of episporium thereby cleaving peptide epitope(s) after reorientation of carbohydrate moietiesby periodate treatment. It indicated glycoprotein nature or peptide nature of epitopes on the teliospore surface.

Anthocyanin, bisabolol and phenylammonialyase activity in cell cultures of Populus deltoides.

Phenolics, anthocyanin and alpha-bisabolol production from poplar (Populus delotides) in tissue culture was determined. A number of phenolic acids were identified by HPLC. PAL activity in response to phytohormones, cells growth and anthocyanin production showed a positive correlation. A component, alpha-bisabolol, was identified using gas chromatography and UV spectroscopy. In vitro production of said metabolites was influenced by phytohormones.

Differences in deoxycytidine metabolism in mouse and rat.

Bone-marrow macrophages from both rat and mouse release deoxycytidine derived from phagocytosed nuclei. Mouse plasma contains no detectable deoxycytidine (less than 0.1 microM), whereas the concentration in rat plasma is 18 microM. Enzyme assays of tissue extracts show that both mouse and rat spleen contain high deoxycytidine kinase activity. Mouse organs, including kidney, liver and lung, also have deoxycytidine deaminase activity. In contrast, rat tissues have virtually no deoxycytidine deaminase activity. Lack of deaminase provides an explanation for the presence of deoxycytidine in rat plasma. Cytotoxicity assays show that cultured mouse lymphoid cells grown in undialysed rat serum are more resistant to cytotoxic effects of deoxyadenosine than are those cells grown in dialysed rat serum. The results suggest that a major difference in deoxycytidine metabolism between mouse and rat may account for discrepancies in the pharmacological response of the two animals to certain nucleoside compounds.

Deoxycytidine excretion by mouse peritoneal macrophages: its implication in modulation of immunological functions.

Pyrimidine excretion by macrophages was studied in order to identify the potential immunoregulatory effector molecules. Deoxycytidine was found in the culture medium of thioglycollate-elicited mouse peritoneal macrophages, along with thymidine, which was shown by others to be a possible immunoregulatory substance. The identification of deoxycytidine was based on: (1) cochromatography with the authentic compound in four different solvents, (2) UV absorption spectral analysis, and (3) the enzymatic peak shift method. Phagocytosis of nucleated chicken erythrocytes, but not enucleated sheep erythrocytes, increased deoxycytidine excretion. The macrophages lacked both deoxycytidine kinase and deoxycytidine deaminase, which is consistent with their excretory pattern. Since it has been known that deoxycytidine can protect cells against cytotoxic effects of thymidine, we propose that deoxycytidine has a role in preventing immunosuppression by thimidine. In patients with adenosine deaminase deficiency, however, immunosuppression caused by combined toxicity of thymidine and deoxyadenosine may not be adequately prevented by deoxycytidine.