Comparison of immunogenic properties of native and inactivated Mannheimia haemolytica Lkt in calves.

The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorbance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MTT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.

Evaluation of Mannheimia haemolytica leukotoxin prepared in nonsupplemented and BSA or FBS supplemented RPMI 1640 medium.

Mannheimia haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Supernatant preparations containing Lkt produced by M. haemolytica serotype 1, grown in RPMI 1640 medium supplemented with BSA or FBS and without supplements were evaluated during this study. Analysis of obtained Lkt showed presence of 105 kDa antigen (SDS-PAGE electrophoresis). The obtained bacterial protein fraction estimated as Lkt was detected by Western blotting with mouse monoclonal (Mab 605 and Mab 601) anti-Lkt antibodies. No significant differences were found in obtained leukotoxin between wildtype and reference M. haemolytica strains. Our studies showed that growth in media supplemented with BSA or FBS had no significant influence on leukotoxin production. When BSA or FBS supplements were used, additional protein fractions in electrophoregrams SDS-PAGE were observed. These protein bands did not react with Mab 605 and/or Mab 601 in Western blotting analysis. Lkt immunogenicity was detected by immunoblotting with sera from Lkt immunized rabbits and calves.

The influence of transport stress on the humoral immunological response of calves induced by Mannheimia haemolytica leukotoxin.

In the contemporary systems of cattle production, transport stress is the most essential poly-etiological factor responsible for inducing unfavourable reactions in the animals. The main reason for this phenomenon is the immunosuppressive effect of steroid hormones on cellular and humoral protective mechanisms. The purpose of the present study was to establish the relationship between the cortisol concentration as an indicator of the stress reaction occuring directly after the transportation of calves and the specific humoral immune response to the leukotoxin (Lkt) antigen produced by the M. haemolytica strain. The experiment was carried out on 19 clinically healthy calves, weighing about 100 kg and transported by track for about 2 hours. After the delivery of the animals for feeding to the traditional cattle-house, the calves were immunized s.c.: group I with 1 ml Lkt (in conc.--10 microg/ml) with 1 ml of adjuvant on the 1st and 14th day after the transportation, group II with the same Lkt doses on the 3rd and 16th day after the transportation. The animals of the control group were vaccinated on the 1st and 14th day after the transportation with the twice diluted adjuvant. In examined sera the cortisol concentrations and the level of Lkt antibodies were measured by ELISA test. The cytotoxin neutralizing (CN) antibody level (cytotoxity assay) was determined with a simple visual assay. The study revealed, significant differences (P < or = 0.01) in serum cortisol levels between the control and experimental animals. The analysis of the absorbance of the sera in both groups immunized with Lkt showed substantial differences (P < or = 0.05) from the 6th through to 22nd day of the experiment compared with the control group. The analysis of the results of CN antibody titers showed no differences between the sera from group I and II. Based on the results obtained in this experiment it can be assumed, that a short transportation stress has no important influence on the level of specific humoral anti-Lkt response.