P. multocida strains A1, A3, A12, and D2, along with E. coli serotypes O125, O151, and O157 were used in the preparation of inactivated polyvalent P. multocida, inactivated polyvalent E. coli, and combined inactivated P. multocida and E. coli vaccines, as well as challenge test. The strains were supplied by the Aerobic Bacterial Vaccines Department, Veterinary Serum and Vaccine Research Institute (VSVRI), Abbassia, Cairo.

A total of 170 Swiss white mice, weighing 25 - 30 g, were used. Twenty mice were used to assess the safety of the produced vaccinates, while the remaining 150 mice were used to determine the LD50 for both the prepared inactivated P. multocida vaccine and the combined inactivated P. multocida and E. coli vaccine.

A total of 380 P. multocida seronegative rabbits, about 2 - 3 weeks of age and weighing 1.5 - 2 Kg, were used. Ten rabbits were used for the passage of P. multocida and E. coli strains while the remaining 370 rabbits were used for evaluation of prepared vaccines. The rabbits had not been previously vaccinated or treated with antibiotics and were reared in accordance with biosafety and biosecurity guidelines.

Montanide ISA 70 (Seppic, Paris, France) is an oil/surfactant-based adjuvant. It was developed for the production of water-in-oil (W/O) emulsions.

P. multocida strains A1, A3, A12, and D2 were grown separately in tryptic soy broth (TSB) for 24 hours at 37˚C with continuous shaking. Each strain was standardized at 4 × 10⁹ CFU/ml. The bacteria were inactivated using 0.5% formalin (37% - 41% concentration) and cultured for 24 hours at 37˚C. After complete inactivation, each culture was assessed for purity, safety, and sterility according to [12] guidelines. Cultures were then mixed evenly, preserved with 0.01% thiomersal, and stored at 4˚C until needed.

E. coli serotypes O157, O151, and O125 were cultured in brain heart infusion broth and adjusted to a concentration of 1.2 × 10⁸ CFU/ml. The cultures were then inactivated using 0.5% formalin (37% - 41% concentration) and incubated at 37˚C for 24 hours. After complete inactivation, each culture was assessed for purity, safety, and sterility according to [12] guidelines. The cultures were combined in equal amounts, preserved with 0.01% thiomersal, and stored at 4˚C until needed.

Inactivated P. multocida, inactivated E. coli, and combined inactivated P. multocida and E. coli vaccines were prepared using the formulation method recommended by Montanide™ manufacturer. Montanide™ ISA 70 was used to create a water-in-oil (W/O) emulsion vaccine with a 30/70 (W/W) aqueous/oil ratio. For the combined inactivated P. multocida and E. coli vaccine, equal amounts of P. multocida and E. coli antigens were added to ensure that the antigenic content for both antigens was at least 10⁸ EID50. Stable preparations were achieved by combining the aqueous medium with Montanide™ ISA 70 at room temperature or lower and vigorously stirring for 15 - 30 minutes, as advised by the manufacturer’s instructions.

The prepared vaccines were tested for sterility, safety, and purity using the established worldwide standards outlined [13] .

The potency of the produced inactivated P. multocida and combined inactivated P. multocida and E. coli vaccines was initially assessed by determining the LD50 in mice, as outlined by [12] . The mice were divided into three groups: the first group (50 mice) was vaccinated with the prepared inactivated P. multocida vaccine, the second group (50 mice) was vaccinated with the combined inactivated P. multocida and E. coli vaccine, and the third group (50 mice) served as an unvaccinated control. The vaccinated groups received a booster dose three weeks later and were subsequently challenged three weeks after the booster to determine the lethal dose fifty (LD50). The vaccinated mice were separated into 10 groups and challenged with a tenfold serial dilution of mixed virulent P. multocida strains (5 vaccinated and 5 unvaccinated animals per dilution). Mortality rates were monitored and recorded for one week. Reference [14] formula was used to compute the lethal dose (LD50).

$\text{LD}50\%=\text{above}50-50/\text{above}50-\text{below}50$

Depending on the satisfactory results of the protective value of the prepared vaccines in mice, the experimental design was then applied to the target animal, rabbits.

Out of three hundred and seventy rabbits aged 2 - 3 weeks, all were reared under proper management conditions, including appropriate food, biosafety and biosecurity measures with no history of previous infection or vaccination. The rabbits were divided into four groups for the experiment. Group 1 (85 rabbits) was vaccinated with the polyvalent inactivated P. multocida vaccine, Group 2 (65 rabbits) was vaccinated with the polyvalent inactivated E. coli vaccine, and Group 3 (145 rabbits) received the combined inactivated polyvalent P. multocida and E. coli vaccine. Group 4 (75 rabbits) was maintained as a non-vaccinated control group Table 1 .

All rabbit groups received two doses of 0.5 ml subcutaneously of the prepared vaccines, with one-month interval between doses. Rabbits in all groups (except for 5 rabbits from each group, which were kept for blood sample collection) were challenged with virulent strains of P. multocida or E. coli depending on the vaccine type, three weeks after booster vaccination.

Random blood samples were collected every two weeks post-vaccination and the humoral immune response was evaluated serologically.

1) Indirect haemagglutination test (IHA)

The IHA test was used to assess the humoral immune response to P. multocida. The glutraldehyde treated RBCs and capsular antigens of P. multocida strains were produced in accordance with [15] . The vaccine was considered effective if it induced seroconversion in the sera of vaccinated rabbits.

2) Enzyme linked immunosorbent assay (ELISA)

The antibody titers against P. multocida and E. coli in the serum of vaccinated rabbits were determined using the ELISA assay described by [16] .

Rabbits vaccinated with the inactivated P. multocida and the combined inactivated P. multocida and E. coli vaccines (Groups 1 and 3), along with the control group, were challenged with 0.1ml of 1 - 2 × 10² CFU of P. multocida strains A1, A3, A12, and D2 (20 vaccinated and 10 control rabbits per strain). Similarly, rabbits vaccinated with the inactivated E. coli and combined inactivated P. multocida and E. coli vaccines (Groups 2 and 3), along with the control group, were challenged with 0.1 ml of 1.2 × 10⁶ CFU of E. coli strains O157, O151, and O125 (20 vaccinated and 10 control rabbits per strain) to estimate the protection percentage of the prepared vaccines. Following the challenge, rabbits were monitored daily for a week for any mortality or clinical symptoms. The clinical findings of both vaccinated and control rabbits were evaluated and documented.

The statistical analysis of the obtained data was performed using SPSS software, version 21 (IBM Corp., Armonk, NY, USA, 2012). A t-test was employed to compare the means between groups and assess the significance of differences in the outcomes of interest. The data were first checked for normality using the Shapiro-Wilk test to ensure that they met the assumptions of the t-test. A two-sample independent t-test was used to compare the differences between the vaccinated groups in terms of humoral immune response. For comparisons between groups, a paired t-test was applied. The level of significance was set at p < 0.05. Results were presented as mean ± standard deviation (SD), and 95% confidence intervals (CIs) were calculated to interpret the magnitude of the effects.

The project was approved by the Institutional Animal Care and Use Committee (ARC-IACUC) of the Agricultural Research Center, under the approval code ARC CLEVB 122-24.

The cultures of P. multocida and E. coli used in prepared vaccines were confirmed to be pure.

The prepared vaccines were free from bacterial, fungal, and Mycoplasma contamination as described by [12] .

The prepared vaccines were found to be safe, producing no clinical, local, systemic or necropsy findings during a two-week observation period after injection of a double field dose of each vaccine.

The potency of the prepared inactivated P. multocida and the combined inactivated P. multocida and E. coli vaccines was initially evaluated by determining the LD50 in mice. The vaccinated mice exhibited LD50 values that were 2.00 and 2.12 log units higher than those of the control mice for the single and combined vaccines, respectively.

According to Table 2 , the IHA test on serum samples from rabbits vaccinated with inactivated P. multocida and the combined inactivated P. multocida and E. coli vaccines demonstrated detectable P. multocida antibodies as early as the second week after vaccination. The antibody titers reached 32, 64, 32, and 16 for strains A1, A3, A12, and D2, respectively, in the inactivated P. multocida vaccine, while they reached 64, 64, 64, and 32 for the same strains in the combined inactivated P. multocida and E. coli vaccine. By the 4th week post vaccination, the antibody titers increased to 64, 128, 64, and 32 for strains A1, A3, A12, and D2, respectively, in the inactivated P. multocida vaccine, while they increased to 128, 128, 128, and 64 for the same strains in the combined inactivated vaccine. The booster dose administered four weeks after the primary vaccination led to a rapid increase in antibody titers, peaking at the 4th week post-boost and persisting until the 6th week, after which they decreased at the 8th post-boost as shown in Table 2 . Data from this test indicate that the combined inactivated P. multocida and E. coli vaccine showed slightly higher antibody titers compared to the inactivated P. multocida vaccine alone. Statistical analysis of the data revealed a p value of 0.014 between the single and combined vaccines, indicating a statistically significant difference.

PV: post vaccination; PB: post boostering.

Table 3 shows that the ELISA conducted on serum samples from rabbits vaccinated with the inactivated P. multocida and the combined inactivated P. multocida and E. coli vaccines revealed detectable P. multocida antibodies starting from the second week post-vaccination. The antibody titers reached 1279, 1078, 1688, and 965 for strains A1, A3, A12, and D2, respectively, in the inactivated P. multocida vaccine, while reached 1876, 1190, 1876, and 1039 for the same strains in the combined vaccine. At the 4th week post-vaccination, the antibody titers increased to 1714, 1636, 2254, and 1636 for strains A1, A3, A12, and D2, respectively, in the inactivated P. multocida vaccine, and to 1995, 2599, 2487, and 2166 for the same strains in the combined vaccine. Following the booster dose, the antibody titers in the inactivated P. multocida vaccine rapidly increased four weeks after the primary vaccination, peaking at 3164, 3286, 3254, and 2927 for A1, A3, A12, and D2, respectively. In the combined vaccine, the titers peaked at 3256, 3455, 3490, and 3288 for the same strains and persisted through the 6th week. By the 8th week post-booster, the titers had decreased to 1279, 1186, 1433, and 1487 for A1, A3, A12, and D2, respectively, in the inactivated P. multocida vaccine, and to 1354, 1251, 2250, and 2318 in the combined vaccine. Data from this test indicate that the combined inactivated P. multocida and E. coli vaccine produced slightly higher antibody titers compared to the inactivated P. multocida vaccine alone. Statistical analysis of the data revealed a p-value of 0.015 between the single and combined vaccines, indicating a statistically significant difference.

PV: post vaccination; PB: post boostering.

As indicated in Table 4 , the ELISA conducted on serum samples from rabbits vaccinated with the inactivated E. coli and the combined inactivated P. multocida and E. coli vaccines showed detectable E. coli antibodies as early as the second week after vaccination. The antibody titers reached 1612, 1587, and 1574 for strains O151, O157, and O125, respectively, in the inactivated polyvalent E. coli vaccine, while they reached 2013, 1938, and 1987 for the same strains in the combined vaccine. At the 4th week post-vaccination, the antibody titers increased to 2809, 2789, and 2768 for strains O151, O157, and O125, respectively, in the inactivated polyvalent E. coli vaccine, and to 2980, 2940, and 2801 for the same strains in the combined vaccine. Booster vaccination administered four weeks after the primary vaccination caused a rapid rise in antibody titers, which peaked at the 4th week post-booster, as detailed in Table 4 . These antibody levels persisted through the 6th week but then decreased gradually by the 8th week post-booster. The data indicate that the combined inactivated P. multocida and E. coli vaccine elicited slightly higher antibody titers compared to the inactivated polyvalent E. coli vaccine. Statistical analysis showed a p-value of 0.047 between the single and combined vaccines, indicating a statistically significant difference.

PV: post vaccination; PB: post boostering.

In the vaccinated rabbits, the protection rates observed in the challenge test with P. multocida strains (A1, A3, A12, and D2) showed the following results: for rabbits receiving the inactivated P. multocida vaccine (Group 1), the protection percentages were 80%, 85%, 85%, and 90% for strains A1, A3, A12, and D2, respectively. For rabbits receiving the combined inactivated P. multocida and E. coli vaccine (Group 3), the protection percentages were 85%, 90%, 90%, and 95% for the same strains. In contrast, control rabbits exhibited protection rates of 20%, 10%, 20%, and 20% for the respective strains, as shown in Table 5 . Conversely, the protection rates from the challenge test with E. coli strains (O151, O157, and O125) are presented in Table 6 . For rabbits receiving the inactivated E. coli vaccine (Group 2), the protection percentages were 80%, 80%, 85%, and 90% respectively, and 85%, 90%, and 95% for the same strains in the combined vaccine. The control rabbits exhibited protection rates of 20% for all three strains.