Developing an inactive vaccine against field-isolated Pasteurella multocida serogroup B

Pasteurella multocida, a Gram-negative coccobacillus from the Pasteurellaceae family, causes severe infections in farm animals, especially cattle and sheep. It is isolated in approximately 20% of cattle deaths. This situation adversely affects animal production, leading to significant economic losses in many countries and posing an important problem. It is an opportunistic pathogen and can be found as a saprophyte in the respiratory tract of healthy animals. When the host's immune system is weakened, it causes severe conditions such as hemorrhagic septicemia, fowl cholera, atrophic rhinitis, enzootic calf pneumonia, and shipping fever. This study was conducted to characterize P. multocida and its virulence genes and to evaluate the immunogenicity of an inactivated vaccine belonging to serogroup B via different administration routes. A total of 250 samples were collected from cattle (n=130), sheep (n=70), goats (n=45), and pet birds (n=5) showing respiratory symptoms, which were sent or brought to the laboratory from different regions of Turkey. Among these, 167 samples were from the lungs of cattle (n=97), sheep (n=40), and goats (n=30); the remaining 83 were nasal swabs from cattle (n=33), sheep (n=30), goats (n=15), and pet birds (n=5). In the primary isolation procedure, 5% blood agar was used to isolate the pathogen from the samples. As a result of bacteriological culture, 27 (8.4%) P. multocida strains were confirmed by PCR. PCR-positive P. multocida strains were analyzed and typed for 19 different virulence genes (outer membrane, fimbrial antigen genes, somatic antigen genes, and iron-binding genes). Suspected P. multocida isolates were tested for the kmt1 gene by PCR. Among strains isolated from different sources, 57.12% were from cattle (12), 23.8% from sheep (5), 14.28% from goats (3), and 4.8% from pet birds (1). For typing, capsule genes (cap B and cap E) were first examined. Subsequently, plpB, tadD, gatG, hgbA, ompA, toxA, pcgD, latB, nctB, ppgB, natG, hgbB, and exbB genes were analyzed. None of the isolates carried ompA, toxA, pcgD, latB, nctB, ppgB, natG, hgbB, or exbB. Two cap B samples isolated from cattle were similar in terms of virulence gene profiles. The candidate vaccine bacterial strain (CapB+, plpB+, tadD+, gatG+, hgbA+) was produced in BHI broth at 37 °C for 48 hours to approximately 1×10⁹ cfu/ml. Purity was checked by plating on blood agar. After inactivation with formalin (0.05%), the bacterial suspension was centrifuged (20 °C, 2000 × g, 20 minutes) and washed three times with PBS. The bacterial pellet was resuspended in PBS to 1×10⁹ cfu/ml using McFarland standard No. 4. The candidate vaccine was prepared by homogenizing the bacterial suspension (20 ml) with Montanide ISA 206VG adjuvant (20 ml, Seppic-France). The prepared candidate vaccine was administered to mice via two different routes (subcutaneous-SC and intramuscular-IM) at a dose of 0.3 ml. Mice were re-vaccinated 21 days later. Blood samples were collected from vaccinated and unvaccinated animals on days 0, 21, and 35. The seropotency of the animal groups was measured by indirect ELISA (450 nm), and OD₄₅₀ data were compared with the control group. The mean OD₄₅₀ value was 0.575 in the SC vaccination group and 0.503 in the IM group. In both groups, the cut-off value was approximately 0.323 (Figure 3.8). Five weeks later, the SC group maintained a high antibody level (OD₄₅₀: 1.0961), while the IM group showed a generally faster increase in antibody titers (OD₄₅₀: 1.042). Data were analyzed using variance analysis and Duncan's multiple range test (p<0.05). Mice were challenged via intraperitoneal injection with live P. multocida serotype B and E strains containing 1×108 LD₅₀ (0.3 ml injection = 3×107 cfu). All mice challenged with serotype B survived, whereas all mice challenged with serotype E died within 48 hours. In this study, P. multocida was found to be more frequently isolated from cattle compared to other species, and serogroup B strains carried fewer virulence genes. The inactivated P. multocida serogroup B candidate vaccine elicited good seropotency but did not protect against serogroup E, indicating the absence of cross-protection. These results suggest that inclusion of multiple serogroups in vaccine formulations is required for broader protection.