Background: Pneumonic pasteurellosis is an emerging disease cause gigantic losses among calves. Objective: The present study was designed to study the different methods for diagnosis of the disease with special reference to the role of vitamin D in occurrence of the disease. Materials and Methods: Ninety seven buffalo calves were used in this study, 10 calves were clinically healthy (control group), other 87 calves were diseased. All calves were subjected to thorough clinical and ultrasonographic examination. In addition two blood samples were taken for hematological studies and all sera were tested by commercial kits for biochemical analysis. Bacteriological studies and detection of virulence-associated genes by PCR were applied. Results: Hematobiochemical revealed leucocytosis with neutrophilia. A significant decrease in serum glucose, total proteins, albumin and vitamin D. Pulmonary lesions were diagnosed ultrasonographically. Pulmonary parenchyma appeared homogenous hypoechoic structure "Hepatinization" with pleurisy as there is an accumulation of anechoic fluid between the parietal and visceral pleura. Nineteen isolates of Pasteurella multocida were isolated from nasal swabs and pulmonary tissues with antigenic typing belonging to type B2 and the pathogenicity test revealed that 100% mortality of injected mice with different time intervals ranged from 24-72 h. The high prevalence of Pasteurella filamentous haemagglutinin A (PfhA) and transferrin binding protein encoding (tbpA) genes with prevalence rate (46.7 and 94.9%), respectively, among the isolates point toward the positive association of these virulence genes with the occurrence of the disease in buffalo calves. Postmortem examination of the dead animals showed pulmonary congestion, edema, consolidation and hepatization of some pulmonary lobes. Fibrino broncopneumonia and edema with infiltration of inflammatory cells are the most prominent finding histopathologically. Conclusion: This study suggests that decrease of vitamin D has a role in occurence of the diseases. In addition Pasteurella multocida is highly susceptible to enrofloxacin, sulphamethoxazole and nitrofurantoin than other antimicrobials.