BACKGROUND: The veterinary Rabies vaccine was produced using BHK-21 cells and PV strain. Although various protocols have been suggested for virus purification, they have an adverse effect on the final production and require further optimization.OBJECTIVES: The present study aimed to optimize the concentration and purification of the virus for rabies vaccine production.METHODS: First of all, the Pasteur virus strain (PV) was cultured by using BHK 21 cells with DMEM media contain bovine fetal serum (7 %) for five days. Subsequently, the virus purification was done via tangential flow filtration (TFF) system. The quality of purifying viruses was an assessment with titration and SDS-PAGE. Secondly, the virus inactivation was optimized using Minitab software based on three factors, namely time, temperature, and concentration. Afterwards, the inactivity of the samples was tested on mice. Finally, the virus potency was evaluated by the National Institute of Health (NIH) method.RESULTS: The viral titration test in TFF samples revealed that viral titer increased in comparison with the control group (P<0.05). The SDS-PAGE analysis of the purified and concentrated samples showed that the purified virus via TFF had a higher purity compared to the not-concentrated samples. Moreover, the NIH test indicated a 10-fold increase in potency result in the TFF group.CONCLUSIONS: The present study implied that the TFF method is highly suitable for condensation and purification of a high volume of viral fluid and could be applied on an industrial scale to increase the potency of the vaccine produced.

Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further purify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.

An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.

Conditions for purification of the ninth component of bovine complement (C9) were established. The conditions for binding and elution from diethylaminoethyl cellulose and hydroxylapatite were different than for human C9. Serum albumin, a frequent contaminant of bovine C9 preparations, was removed by chromatography on reactive-red agarose. The calculated molecular weight of bovine C9 was 66,000, and reduction with 2-mercaptoethanol affected its migration on polyacrylamide gel electrophoresis. Some preparations of bovine C9 migrated as 2 bands when partially reduced, but extensively reduced preparations had a single band.

C-reactive protein (CRP) was isolated from equine serum by use of calcium-dependent affinity chromatography conjugated pneumococcal C-polysaccharide, anion exchange chromatography, and gel filtration. It was identified as genuine CRP by its immunochemical cross-reactivity with anti-human CRP, its homology with human CRP in amino acid composition, and its pentameric structure as revealed by electron microscopy. Purified equine CRP had a molecular weight of approximately 118,000 and was composed of 5 identical, nonglycosylated and noncovalently associated subunits with molecular weight of approximately 23,000 each. Equine CRP migrated in the region between beta- and gamma-globulin by results of immunoelectrophoresis, and its isoelectric point was about 7.0. In horses, increased CRP concentration was associated with clinical pneumonitis, enteritis, and arthritis, compared with values obtained in clinically normal horses by use of single radial immunodiffusion method. After IM administration of turpentine oil or castration, serum CRP concentration increased to 6 times higher than baseline values. Results indicate that CRP may be an acute-phase reactant protein in horses.

Lipopolysaccharide (LPS) fractions were obtained from smooth cultures of Brucella abortus strains 2308 and S-19 by butanol extraction procedures. The LPS from the initial butanol extraction contained 10 to 15% protein and was reduced to less than 1% protein by treatment with proteinase K. The LPS fractions were identified and characterized on the basis of the chemical analysis, sodium dodecyl sulfate gel electrophoresis, cesium chloride gradients, electron microscopy, and gel immunodiffusion. Results indicated that the butanol procedure is a reliable method in the extraction of LPS from Brucella abortus cells. Proteinase K-treated LPS containing less than 1% protein from strain 2308 was used to vaccinate BALB/cByJ mice. Immune and protective criteria for vaccinated and nonvaccinated mice were increased immunoglobulin (IgG and IgM) titers in sera of prechallenge-exposed mice, reduced colony-forming units/spleen, and splenomegaly in post-challenge-exposed mice. Results indicated that proteinase K-treated LPS was immunuogenic as well as protective for mice.

Previously identified 39-, 50-, and 76-kd integral outer membrane proteins (IOMP) of Actinobacillus pleuropneumoniae, a respiratory tract pathogen, were separated by electroelution of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-obtained fragments and their role in opsonophagocytosis by porcine leukocytes was investigated by flow cytometry of fluorescein-labeled A pleuropneumoniae. Using specific antisera, immunoblot analysis indicated that the 3 proteins were antigenically distinct. Antibodies against each IOMP have an important role as opsonins for phagocytosis by porcine leukocytes. The effect of using a combination of an 3 of the specific antisera was minimal. Antiserum absorbed against intact A pleuropneumoniae and Escherichia coli organisms indicated that the antibodies to the 39-, 50-, and 76-kd IOMP were specific for A pleuropneumoniae antigens. Nonheat-treated antiserum did not increase phagocytosis, compared with heat-inactivated antiserum, indicating that complement may not have a major role in opsonization of A pleuropneumoniae.