Evaluation of the immunogenic properties of the recombinant Histophilus somni outer membrane protein 40 kDa (rOMP40).

BACKGROUND: Gram-negative bacterial infections are a serious problem in beef and dairy cattle. Bacterial outer membrane proteins (OMPs) play a pivotal role in cellular survival and the host-bacterium interaction. Histophilus somni OMP40 was identified as a porin with homology between its N-terminal amino acid sequence and the sequences of porins of other gram-negative bacteria The aim of this study was to produce recombinant H. somni OMP40 (rOMP40), optimize its production and evaluate its immunogenic properties in calves. The cross-reactivity of anti-rOMP40 antibodies were also checked. RESULTS: The highest overexpression of rOMP40 was demonstrated by Escherichia coli C41 using the autoinduction process. Double immunization of calves (20 µg rOMP40 per animal) induced a significant increase of anti-rOMP40 antibodies in the IgG1 (P ≤ 0.01) and IgG2 (P ≤ 0.01, after first immunization only) subclasses, but not IgM. ELISA revealed increased reactivity of the IgG against surface antigens of E. coli and Pasteurella multocida after the second immunization (P < 0.01). Cross reactivity of anti-rOMP40 antibodies with ~ 40 kDa antigens of most common gram-negative pathogens was shown by Western blotting. CONCLUSION: Immunization with H. somni rOMP40 induced a humoral response in cattle with broad cross-reactivity with similar antigens of other species of Pasteurellaceae and Enterobacteriaceae families and the delayed-type hypersensitivity reaction. The obtained results encourage further study to evaluate the protective effect of the produced protein as a subunit vaccine in cattle.

Whole-cell Proteus mirabilis urease inhibition by aminophosphinates for the control of struvite formation.

The study evaluated the in vitro impact of a series of aminophosphinic urease inhibitors on Proteusmirabilis. The group of compounds comprised structurally diverse analogues of diamidophosphate built on an N-C-P scaffold. The influence of urease inhibition on urea-splitting activity was assessed by whole-cell pH-static kinetic measurements. The potential to prevent struvite formation was determined by monitoring changes in pH and ionic composition of artificial urine medium during P. mirabilis growth. The most active compounds exhibited stronger positive effect on urine stability than the acknowledged inhibitor acetohydroxamic acid. The high anti-ureolytic and pH-stabilizing effect of urease inhibitors 4 and 14 was well correlated with their reported kinetic properties against pure urease from P. mirabilis (Ki values of 0.62±0.09 and 0.202±0.057 µM, respectively, compared to 5.7±0.4 µM for acetohydroxamic acid). The effect of repressed ureolysis upon the viability of Proteus cells was studied using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] metabolic efficiency assay and LIVE/DEAD fluorescent staining. Most of the compounds caused whole-cell dehydrogenase activity loss; four structures (1, 2, 4 and 14) reduced the culture viability by nearly 70 % at 1 mM concentration. Results of dual fluorescent staining suggested that besides urea-splitting prevention, the structures additionally exerted an outer-membrane-destabilizing effect.