A total of 37 different inactivated P. multocida vaccines from different sources either locally prepared or imported from different sources were comparatively tested for relative potency following both single dose and booster dose vaccination assays. The study objective was to minimize the time factor exhausted in the evaluation processes of the inactivated fowl cholera vaccines. So it is planned to compare between single and booster dose vaccinations and their related potency. Correlation between protection associated with the single dose and booster dose vaccination were evaluated and average requirement for protection was 43.7% in single dose vaccination assay compared to 76.2 % associated with booster dose vaccination assay. In the same concern, the correlation between both assays for the seroconversion was estimated using ELISA and the minimum requirement was 1.8X cut off value in the single dose vaccination assay compared to 2.25X cut off value in the booster dose vaccination assay. In conclusion, single dose vaccination assay could be valuable in the evaluation of inactivated fowl cholera vaccines through determination of protection indices and/or estimation of humoral immune response if the above mentioned data is considered

Respiratory diseases are one of the major causes of economic losses to poultry industry. Fowl cholera is a septicemic respiratory complex where it is highly common and widely distributed disease of poultry and other avian species.¹ Fowl cholera is a wide and commonly distributed disease of poultry and of major economic importance.² The disease can express itself in an acute or a chronic form. In the acute form, the clinical signs are seen only in the few hours before death as fever, ruffled feathers, and mucus discharge from mouth, diarrhea and increased breathing rate. The chronic form of the disease can follow an acute stage or may be the only form of the disease present in the flock. Signs of this form generally linked to localized infection at wattles, sinuses, leg or wing joints, swollen eyes, twisted neck, rales and pin headed necrotic foci in the liver with a septicemic picture.²

Fowl cholera can be prevented by eliminating all reservoirs of infections and then preventing the re-entry of the organism into the property. Implementation of standard good management practices, effective sanitation regime and good biosecurity program will help prevention of fowl cholera.³

P. multocida vaccines are used to help control of Fowl cholera. P. multocida exists in 16different serovars and the most common serovars associated with Fowl cholera outbreaks are serovars 1, 3 and 4. P. multocida vaccines based mainly on inactivated cells of P. multocida.² Evaluation and quality control of the efficacy of this vaccine are based mainly on vaccination challenge test by which the protective indices are estimated.⁴

The immune system defends the organisms against infectious diseases and one of the major immunological defense mechanism is the humoral immune response, which is mediated by serum antibodies secreted by B cell.⁵ Serological testing is a useful tool in explanation of immune status of the birds and the Enzyme Linked Immunosorbent Assay (ELISA) is a useful tool for determination of antibody response against certain pathogen infection of vaccine inoculated.

The objective of the present work was to study the availability of using single dose vaccination assay in through determination of protection percent and humoral immune response in comparison to the booster dose vaccination assay of P. multocida inactivated vaccine in chickens.

Pasteurella multocida vaccines

A total of 37different inactivated P. multocida vaccine batches yearling 2012 up to 2016, from different manufacturers sources either locally prepared or imported from abroad were tested by vaccination challenge assay method using virulent P. multocida in parallel to serological evaluation using ELISA.

Pasteurella multocida strains

Virulent Pasteurella multocida serovars 1, 3 and 4 were used to perform challenge test. These serovars were supplied from the reference strain bank, CLEVB (Central Laboratory for The Evaluation of Veterinary Biologics).

Chickens

A total of 120Specific Pathogen Free (SPF) chickens aging 6-8 weeks were used for each fowl cholera vaccine batch tested to perform this study which starting from 2012 up to 2016. This birds were divided into 3groups, the 1stone comprised 45birds and received only one dose then challenged and serologically tested, the 2nd group comprised also 45birds and received both primary dose and 3weeks later received a booster dose then challenged and serologically tested and finally the 3rd group were 30birds kept as negative unvaccinated group. All birds were vaccinated with the corresponding Fowl cholera vaccine batch (0.5ml/dose/chickens) subcutaneously.

Swiss mice

Six Swiss mice weighed about 20-25 gm, 2 for each P. multocida serovar were inoculated with the stock culture of P. multocida. This was done before every challenge test to rebuild the virulence of P. multocida serovars in a dose of 100 – 500 CFU/ mouse intraperitoneally.

Blood samples

Twenty blood samples were collected from each group per each tested batch of vaccines 3weeks post vaccination in case of single dose vaccination assay or 3weeks post the second dose of vaccination in case of booster dose vaccination assay, then sera were separated to be tested using ELISA.

Challenge test

The vaccinated birds were challenged with 2x10² to 3x10² CFU/ challenge dose from the different regained virulent P. multocida strains(15vaccinated and 5unvaccinated birds/each serovar) 3weeks post vaccination in case of single dose vaccination assay or 3weeks post the second dose of vaccination in case of booster dose vaccination assay. Mortalities were observed, recorded and re-isolation of the challenge strain were done from the internal organs (Liver and heart blood) of dead cases and the protective indices (PI) were calculated using the following formula described by.⁶

Where PI is the protective indices, M is the mortality and PML is the post-mortem lesions

ELISA

ELISA was conducted on serum samples collected from all groups in different tested batches and the test performed according to standard procedures of the two different commercial kits used. The first one is Pasteurella multocida antibody test kit (Synbiotics Corporation, Cat. No.96-6527) referred in this study as kit 1 while the second kit is Pasteurella multocida antibody test kit (IDEXX Laboratories. Inc., Cat. No 99-09251) which referred in this study as kit 2 ELISA was performed and interpreted as directed by the manufacturers.

Generally, Fowl cholera vaccines are evaluated by sterility, safety and potency tests. Potency testing depends mainly on challenge test and determination of humoral immune response by ELISA as shown in Table 1. A total of 32out of 37 Fowl cholera vaccine batches were tested and gave satisfactory results for approval to be used in the poultry farms according to the Egyptian standards for evaluation of veterinary biologics (2004). According to the protection level obtained, the tested fowl cholera vaccine batches was grouped into 7categories. The 1st group comprises 6 batches out of 32 and achieved protection of 41% in case of single dose vaccination assay compared with 70% in case of booster dose vaccination assay. As regards to 2nd group comprises 3 batches out of 32 and gave a protection of 42 % and 72 %, the 3rd group comprises 11batches (the highest average number of tested batches) out of 32 and gave a protection of 43% and 75%, the 4th group comprises 2 batches out of 32 and gave a protection of 44 % and 76%, the 5th group comprises 7batches out of 32 and obtained a protection 44% and 78%, the 6th group comprises 2 batches out of 32 and gave a protection of 45% and 80%. And the last 7th group comprises only one batch out of 32 and gave a protection of 47% and 82% in case of single and booster dose assays for each group respectively.

On the other hand, Table 1 also showed a comparison between the humoral immune response expressed ELISA mean titer for the same batch group at the same protection level. It was noticed that , the antibody titers at the protection level of 41% was parallel to 272 and 717ELISA antibody titer for both types of ELISA kits respectively in case of single dose vaccination assay while it was 341 and 896at the protection level of 70% in case of booster dose vaccination assay. Also it is clear that the antibody titer was increased as the protection level increased in a harmonious manner for both assays at all level of protections.

By the same manner, Table 2 illustrated that 5 fowl cholera vaccine batches out of 37 are evaluated as unsatisfactory (According to Egyptian standards for evaluation of veterinary biologics, 2004) where it got a protection level lower than 70% which is the minimum requirement for protection starting with 60% protection in 2vaccine batches, 48%, 47% and 45% protection with one vaccine batch for each. Meanwhile, the protection achieved by the same batches when used only in single dose were 32%, 29%, 27% and 25% respectively.

As regards to the ELISA antibody titer of such unsatisfactory resulted batches, the corresponding antibody titers were 292 and 767with the protection rate 60% compared to 234 and 614with protection rate 32% in case of booster dose and single dose vaccination assays respectively. Also antibody titer decreased as the protection percent decreased in a parallel manner matched the immune status of the tested vaccine and birds in the rest unsatisfactory results of the tested batches.

Table 3, showed the average responses of birds vaccinated with either single or booster dose vaccination assays regarding both humoral responses and protection obtained. The average protection percent of all tested satisfactory batches was 76.2 with the booster dose compared to 43.7 with the single dose vaccination assay. Meanwhile, the average of measured ELISA antibody titer was 387 and 1053 compared to 309 and 843for both kits and both assays respectively. On the other hand, the average protection percent of all tested unsatisfactory batches was 50with the booster dose compared to 29with the single dose vaccination assay. At the same time, the average of measured antibody titer was 242 and 619compared to 194 and 495for both kits and both assays respectively.

Fowl cholera is a highly contagious and economically important disease of poultry worldwide. It is extremely important for poultry producers to be able to get a good vaccine against all poultry pathogens especially that they have great effect on this industry like Fowl cholera. Evaluation of the efficacy of inactivated P. multocida or Fowl cholera vaccine depends mainly on testing of its potency using vaccination-challenge test prior to sale and distribution.⁴

Results of this study compared between two different vaccination assays either single dose or booster dose vaccination assays for the evaluation of inactivated Fowl cholera vaccine using vaccination–challenge test and monitoring the immune response through determining the antibody titer against the inoculated vaccine using ELISA.

Depending on the minimum requirement of protection (70%) which should be obtained after booster dose of vaccination of fowl cholera vaccine,⁷ the satisfactory protection obtained in this study varies from 70 up to 83% giving rise a protection of 76.2 as an average in case of booster dose vaccination assay compared to 41 up to 47% giving rise a protection of 43.7 as an average in case of single dose vaccination assay. Meanwhile the average correlated antibody titer at this protection level was 387 and 1053in case of booster dose vaccination assay compared to 309 and 843 ELISA antibody titer obtained with single dose vaccination assay using ELISA kit (1) and (2) respectively. Analytical view of these antibody titer revealed that, it is equal to or more than the 2.25x and 1.8x the calculated cut off value of both used kit (1) and kit (2). Also, these titers increased when the protection rate increased and decreased when the protection rate decreased as shown in Table 1 and Table 2. The same criteria was obtained by,⁸ who stated that fowl cholera vaccine consisted of serotypes 1, 3 and 4 P. multocida strains provided 70–100% protection against challenge with homologous strains. Also they found that the trivalent vaccine can induce immunogenic response in vaccinated chickens and so ELISA assay showed a considerable increase in antibody titer after twice vaccination of 6-8weeks aged chicken. In the same concern,⁹ assessed vaccine efficacy by measuring serum antibody titers 4 and 8weeks after a single vaccination with several adjuvants included in P. multocida vaccines in chickens. The study concluded that the vaccine formulated with oil adjuvant Mantonide ISA70, ISA774 and W/O emulsion based on tween/span induced a strong immune response against P. multocida. Also reported that,⁸ the antibody measured with ELISA highly correlated with protection against challenge with virulent organisms. In the same concept,¹⁰ reported that a blocking ELISA was developed and standardized for the detection of antibodies to P. multocida in vaccinated animal. Also used a commercial ELISA kit in a study to detect both IgA and IgG in vaccinated laying hens.¹¹

Regarding the average protection percent of all tested satisfactory batches, it was 76.2with the booster dose compared to 43.7with the single dose vaccination assay parallel to the average of measured antibody titer which was 378 and 1053compared to 309 and 843for the both used kits and both assays respectively. On the other hand, the average protection percent of all tested unsatisfactory batches was 50with the booster dose vaccination compared to 29 with the single dose vaccination assay parallel to the average antibody titer which was 242 and 619compared to 194 and 495for both kits and both assays respectively.¹² demonstrated the antibody titers following primary and booster vaccinations for 4groups of different breeds of commercial birds and the study concluded that all groups of vaccinated birds induced a significant immune response after primary and booster vaccination without significant difference in antibody titers between different breeds. On the other hand,¹³ Recorded that ELISA test did not appear to be adequate for the evaluation of the degree of protection induced in turkey flocks where turkey poults vaccinated at one day old with inactivated P. multocida bacterin showed low titers of antibody as measured by ELISA but turkeys vaccinated at 3 and 6weeks of age responded with a higher antibody titers and were resistant to virulent challenge with P. multocida. vaccinated birds with formalin killed fowl cholera bacterin, determined antibody titers using ELISA and challenged birds with virulent strain of P. multocida then concluded that the prepared formalin killed fowl cholera vaccine induce protective immune response and conferred protection against challenge protection infection caused by the virulent P. multocida strains.

By using a simple calculation regarding the finding of this study [Average protection with booster dose vaccination (76.2) and with single dose vaccination assay (43.7)] and according to the minimum requirement in the for veterinary vaccine evaluation which is 70% with the booster dose vaccination assay, the minimum requirement of protection associated with the single dose vaccination assay is 40.14%.⁷

It may be concluded that, the most important finding from the results of this study is the seroconversion of vaccinated birds with fowl cholera vaccine measured by ELISA concurrently with the protection obtained after challenge with the virulent P. multocida strains could be valuable and satisfactory in the evaluation of the efficacy of the fowl cholera vaccines using single dose vaccination assay and/or booster dose vaccination assay. Also, the minimum requirement of protection after challenge with the virulent P. multocida strains should be 40.14% or more in case of single dose vaccination assay.

None.

Authors declare that there is no conflicts of interest.

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