Gp.(IV) revealed leukocytosis mainly due to rise in neutrophilic, lymphocytic and monocytic counts. This rise present in propolis treated rabbits suggests the immunomodulatory effect of propolis alone or its enhancing effect to immunity when used as adjuvant with killed vaccine (48,49).Propolis can act as an immune stimulant as abundant stimulation of the immune system of propolis that can increase the total leukocytes value (50).Also, this rise in WBCs may be due to multiplication of leukocyte precursors from pluripotent stem cells after treatment with Propolis (42).
Our findings agreed with Orsoli? and Basic (51,42), who reported that water-soluble derivative of propolis (WSDP) given to mice caused a significant elevation of leucocytes in peripheral blood.
In concern with phagocytic activity, gp.(IV) recorded the highest significant increase in the phagocytic activity followed by gp.(III) compared with other gps.(I and II). This result may be discussed due to the immunomodulatory effect of propolis and its components (52). Also, Possamai et al. (53) found that mononuclear phagocytes exposed to propolis adsorbed onto polyethelene glycol (PEG) microspheres showed high levels of superoxide release, phagocytosis, and microbicidal activity.
Propolis flavonoids were reported that it, as adjuvant, has immune stimulation, such as accelerate the transformation of T, B lymphocyte cell and augmenting neutralizing antibody titers (30).Gp.(III) showed a significant increase in the phagocytic activity compared with control, but this rise not as high as that of the propolis treated rabbits. This result may be attributed due to the effect of killed vaccine in induction of only humoral immune response without great effect on cell mediated immunity (45,54).
Regarding to the biochemical analysis for evaluation of liver functions, gp.(II) at the end of 6th week of the experiment showed a highly significant rise in serum activities of ALT, AST and ALP; that suggests the damaging effect of virulent P. multocida on the liver. Similar results previously obtained by Nassar et al. (15). These results disagreed with Thurston et al. (55) who found that rats injected a sublethal single subcutaneous dose (0.2 microgram/kg of body weight) of Pasteurella multocida D toxin showed non significant changes in ALT, AST and ALP serum activities. This difference with our work may be resulted from the difference in bacterial dose, duration of challenge or physiological difference in the animal species. Gp.III showed a significant increase in ALT and AST activities and total and indirect bilirubin at the end of the 2nd and 4th weeks. This could be interpreted due to mild hepatic irritating effect induced by formalized killed P. multocida vaccine injection. The same results previously obtained by Nassar et al. (15). Gp.IV showed no significant rise in ALT, AST and ALP activities and total, direct and indirect bilirubin when compared with the control (gp.I), that could discussed due to hepato-protective effect of propolis with its effect on improvement of liver functions (56).
Our results were confirmed by histopathological changes in gp.(II), where liver showed multiple focal areas of coagulative necrosis. While, the liver of vaccinated rabbits in gps.III and IV have less severe lesion represented by mononuclear cell infiltration in the portal areas and normal hepatocytes with mild portal congestion respectively. These results were in consistent with Mathy et al. (57) and Nassar et al. (37).
Serum urea and creatinine levels in bacterial challenged rabbits (gp.II) revealed a highly significant rise. In contrast, vaccinated rabbits either alone (gp.III) or with propolis (gp.IV) showed no significant changes in serum urea and creatinine levels, in comparison with control (gp.I) along the experimental periods.These results proved the renal damaging effect of virulent P. multocida which can be prevented by administration of killed vaccine alone or in combination with propolis.
Our obtained results were established by the histopathological findings in gp.(II), where kidneys showed vascular congestion, mononuclear cell infiltration with widening of the glomerular space. While, the kidneys of vaccinated rabbits (gp.III) have slight vacuolation in renal epithelium.
As evident from the current study, it it can be concluded that the use of propolis as adjuvant with formalized killed P. multocida vaccine is very effective in prolonging immune protection of rabbits against pasteurellosis and is better than the use of the vaccine alone.
We thanks and appreciates Dr. Mohamed Metwally M., assistant professor of pathology, Department of Pathology, Faculty of veterinary medicine, Zagazig University, Egypt for his help in performing the histopathology.
Conflict of interest
The authors declare no conflicts of interest.