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Active Immunization with Pasteurella multocida Lysate Elicits Antibody that Protects Rabbits against Virulent Pasteurella multocida and Protects Mice by Passive Immunization
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First Online 12-05-2021|
Methods: A phage lysate vaccine of Pasteurella multocida was prepared using isolated lytic phage and subjected to vaccine response study in rabbits. In this experiment, two mouse fixed-dose of lysate was used and the prophylactic efficacies of lysates were evaluated in rabbits by passive mouse protection test (PMPT), western blotting and direct virulent challenge.
Result: Passive transfer of antibodies to mice using post-immunization sera of rabbits protected the animals against the challenge with A: 1 strain (75%) and B: 2 strain (50%). In western blotting, a total number of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The study suggested the significance increase in humoral immunity by phage lysate in murine Pasteurellosis.
MATERIALS AND METHODS
The study was conducted during July 2010 to June 2012 at Indian Veterinary Research Institute (Deemed University), Izatnagar, Bareilly, Uttar Pradesh, India. The lyophilized culture of Pasteurella multocida P52 and A: 1 strain were revived by standard methods. Bulk stocks of phages were prepared by conventional liquid culture methods as described by Rawat and Verma (2007). 1 liter sterilized NZCYM broth in a flat bottom flask (3-liter capacity Hafkins flask) was inoculated with 50 µl of 18-hour pure broth culture of Pasteurella multocida serotype A: 1 and incubated at 37oC for 2 hours. To each flask, which contains the young culture sufficient quantity of a previously available stock suspension of phage was inoculated to attain a 1:100 final phage bacterial ratio. The phage bacteria mixture was incubated at 37oC with vigorous intermittent shaking until complete lysis obtained (approximately 6 hours was observed). The bacterial lysate was filtered through a 0.22-micron membrane filter and collected aseptically in sterile bottles. The stocks were stored at 40oC for 1 month to eliminate residual lytic activity attributed to phage-induced enzymes. The sterility of phage lysate stock was tested by standard methods (Indian Pharmacophoea, 2010).
All the experimental protocols carried out on laboratory animals were approved by (Letter no:25 dated 2 Dec 2011, IVRI IAEC proceedings) the Institute Animals Ethics Committee (IAEC) of Indian Veterinary Research Institute (IVRI), Izatnagar-243122 (India). Animals were kept in IAEC-approved facilities and provided water and food ad-lib. Blood was collected through cardiac puncture of animal.
Virulence and protection tests
Six rabbits (1-1.5 kg) have been tested for anti-P. multocida antibodies by Passive Mouse Protection Test (PMPT) and free of antibodies were selected for further study. Four rabbits, were immunized subcutaneously with 250 µl of phage lysate and two rabbits were kept as unvaccinated control. On days 21 post immunization four vaccinated and two unvaccinated rabbits were given s/c injection of P. multocida P52 (106 CFU/ml).
Confirmation of P. multocida by PCR tests
Serogroup-specific primers used in PM-PCR and Cap-PCR assay for confirmation of P. multocida (Townsend et al., 2001; Harshit et al., 2014). The organism was revived in NZCYM broth by 18-24 h incubation at 37oC and plated subsequently onto blood agar to study cultural characteristics. The culture was then tested for purity by biochemical tests as per standard techniques. The genomic DNA of the isolate was extracted by CTAB method and the isolate was reconfirmed as P. multocida by PM-PCR followed by determination of capsular type by multiplex PCR.
Passive mouse protection test (PMPT)
Serum samples were collected from all the vaccinated rabbits on 0, 7th, 21st, 28th, 35th, 42nd days of post-vaccination. Serum samples were also collected from controls rabbits. Two groups of six mice with 6 weeks old were given 0.3 ml of pooled sera via the intraperitoneal route. After 24 hrs they were a challenge with 0.25 ml of 10-6 dilution of P52 and A: 1 strain that had been grown to early log phase in BHI broth. The infected mice were observed for 5 days and deaths were recorded twice daily.
Pooled sera of each subgroup were blotted against the P52 and P. multocida (A: 1) strain. The proteins were prepared by scrapping continuous lawn culture of P52 into 3 ml PBS. This suspension, as well as phage lysate components, were mixed with an appropriate proportion of sample buffer and placed in boiling water bath for 5 min., centrifuged and 20 µl of supernatant loaded per well on 1 mm, 12% polyacrylamide gel transferred to nitrocellulose membrane using 250 mAmp for 1.5 hrs in a standard transblot apparatus. Primary sera was applied at a dilution of 1/200 and incubated overnight at room temp. Rabbit anti-Goat IgG was applied at 1/1000 dilution and incubated for 2 hrs at 37oC blot were developed with 4-chloro 1-naphthol.
RESULTS AND DISCUSSION
Immunoblotting of the Pasteurella multocida antigen was done with pooled sera from 21 and 35 days PI of the rabbit. Total numbers of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The molecular weight of those 4 bands was 25 kDa, 35 kDa, 55 kDa and 70 kDa respectively (Fig 3 and 4).
Current vaccines are required to administer parentally, booster dose and are less efficacious (Proceeding of FAO/APHCA workshop on HS 1991). Earlier studies showed that vaccination of buffaloes and poultry with killed bacteria generally results in serotype-specific immunity (Carpenter et al., 1991; Qiang et al., 2020; Shukla et al., 2021) whereas, heterologous protection is conferred only with the attenuated strain (Elena Garrido et al., 2008; Waffa et al., 2014; Myint. 2000). Experiments using anti-Pasteurella sera suggested the significance of humoral immunity in murine pasteurellosis. Passive transfer of sera containing anti-lipopolysaccharides, outer membrane proteins and lipid antibodies could protect mice against direct challenge with virulent P. multocida (A: 1 and P52). Vaccines based on purified extracts or recombinant antigens will not lend themselves to large-scale production in countries where the disease exists. Though, these techniques support large-scale production in disease outbreak conditions. We found heterologous protection using phage inactivates cells with an only single administration. These results were supported by the finding of Wang and Glisson (1994). Western blot and PMPT of pooled sera indicate that antibody (IgG) to identifiable protein in phage lysate were associated with protection. Esber et al. (1985) revealed that staphylococcal phage lysate elicit a significant increase in anti-staphylococcal IgG1, IgG2a and IgG2b level in experimented animals. Many attempts have been tried to explore specific immunogenic proteins of P. multocida isolates from HS and FC.
No single component of P. multocida is solely responsible for protective immunity. Bacterial cell wall fractions, LPS, proteins and polysaccharide contribute towards solid protective immunity. It can be opined from the present investigation that 27 kDa, 35 kDa, 55 kDa and 70 kDa proteins detected in phage lysates may be associated with a protective response against virulent challenge but, their specific role remains to be elucidated. By reviewing the current status and options for developing better immunizing preparations for Pasteurellosis, phage-lysate appears to be a methodology with great potential. Such preparations are expected not only to circumvent most limitations of live vaccines but also to have an additional advantage. Being inactivated preparations, their field use at the time of outbreak or during young age will be possible without any danger of death. Nevertheless, our results suggest that the use of phage inactivated vaccine is a good candidate for a new vaccine for Pasteurellosis and phage lysed cells of P. multocida provide umbrella-like protection against different serogroups of the organism. Hence, further investigations are needed for exploring the potential use of lower dose of phage lysate vaccine without booster vaccination regimen.
- Ahmad, T.A., Rammah, S.S., Sheweita, S.A., Haroun, M., El-Sayed, L.H (2014). Development of immunization trials against Pasteurella multocida. Vaccine. 32: 909-917.
- Ashraf, A., Mahboob, S., Al-Ghanim, K., Huma, T., Shah, M. (2014). Immunogenic Activity of Lipopolysaccharides from Pasteurella multocida in Rabbits. Journal of Animal Plant Science. 24: 1780-1785.
- Carpenter, T.E., Snipes, K.P., Kasten, R.W., Hird, D., Hirsh, D.C (1991). Molecular epidemiology of Pasteurella multocida in turkeys. American Journal Veterinary Research. 52: 1345
- Durairajan, R. (2012). Development and characterization of bacteriophage-lysed bacterin of Pasteurella multocida., PhD Thesis submitted to (Deemed University) Indian Veterinary Research Institute, Izatnagar (UP), India.
- Elena Garrido, M., Bosch, M., Bigas, A., Badiola, I., Barbe, J., Llagostera, M. (2008). Heterologous protective immunization elicited in mice by Pasteurella multocida furoomph. International Journal of Microbiology. 11(1): 17-24.
- Esber, H.J., Ganfield, D., Rosenkrantz, H. (1985). Staphage lysate an immune modulator of primary immune response in mice. Immunopharmacology. 10: 77-82.
- Glisson, J.R., Contreras, M.D., Cheng, I.H., Wang, C. (1993). Sequence analysis of Pasteurella multocida major outer membrane protein (OMP) H and application of synthetic peptides in vaccination of chickens against homologous strain challenge. Avian Disease. 37: 1074.
- Harshit, V., Mayank, R., Ashabaree, S., Vikramaditya, U. (2014). Conventional and molecular characterization of Pasteurella multocida isolated from a case of swine septicaemic pasteuerllosis. Indian Journal of Animal Research. 48(6): 605-608.
- Hodgson, J.C., Finucane, A., Dagleish, M.P., Ataei, S., Parton, R., Coote, J.G. (2005). Efficacy of vaccination of calves against hemorrhagic septicemia with a live aroA derivative of Pasteurella multocida B: 2 by two different routes of administration. Infection and Immunity. 73: 1475-1481.
- Indian Pharmacopoeia. (2010). Government of India, Ministry of Health and Family Welfare. Indian Pharmacopoeia Commission. 3: ISBN 81-903436-9-6.
- Myint, A. (2000). Prevention of experimental haemorrhagic septicaemia with a live vaccine. A live vaccine against HS. Veterinary Record. 120: 500-501.
- Qiang, G., Zhenqi, D., Mingfu, N., Cuili, Q. (2020). The promotion of peony seed proteolysis product to the immune efficacy induced by ptfa gene dna vaccine of avian Pasteurella multocida. Indian Journal of Animal Research. 10.18805/ ijar.B-1268.
- Qureshi, S. and Saxena, H. M. (2014). Estimation of antibody titers in cattle vaccinated with Hemorrhagic Septicemia alum precipitated vaccine by MAT, IHA and Mab-ELISA. Veterinary World. 7: 224-228.
- Rawat, M. and Verma, R. (2007). Isolation, characterization, preservation and therapeutic use of bacteriophage against Staphylococcus aureus associated with ruminant mastitis. Progress Report of project BT/PR4194/AAQ/158/2003. Department of Biotechnology. New Delhi.
- Sarangi,L.N., Priyadarshini, A., Kumar, S., Thomas, P., Gupta, S.K., Nagaleekar, V.K., Singh, V.P. (2014). Virulence genotyping of Pasteurella multocida isolated from multiple hosts from India. Scientific World Journal. 2: 111-115.
- Shukla, S., Nayak, Anju., Sharma, R.K., Shukla, P.C., Singh, R.V. (2021). Bacteriophages: A potential new therapeutic alternate to antibiotics to treat chronic septic wounds in large animals. Indian Journal of Animal Research India. 10.18805/IJAR.B4317Journal.
- Sulakvelidze, A., Pasterneck, R., Brown, T. (2009). Method for vaccination of poultry by using Bacteriophage lysate bacterin. Patent-US 2009/0297561 A1.
- Townsend, K.M., Boyce, J.D., Chung, J.Y., Frost, A.J., Adler, B. (2001). Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. Journal of Clinical Microbiology. 39: 924-929.
- Waffa,A., Ahmed,N., Nagham, M., Al-Gburi, A., Hamoudi, S.R. (2014). An outbreak of hemorrhagic septicemia in a vaccinated herd of domestic water buffalo in Thi Qar province, Iraq. Mirror of Research in Veterinary Sciences and Animals. 3: 36-43.
- Wang, C. and Glisson, J.R. (1994). Passive cross-protection provided by antisera directed against in-vivo-expressed antigens of Pasteurella multocida. Avian Disease. 38: 506-514.
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