Pasteurella multocida B:2 is an important veterinary pathogen causing fatal and acute haemorrhagic septicaemia (HS) in bovine. A live vaccine candidate, P. multocida B:2 GDH7 was reported to enable protection in cattle and buffaloes via intranasal (i. n.) administration. This potential vaccine was also reported to be self-transmitted from the vaccinated animal to the free-ranging animals allowing wider vaccination coverage. Prior to commercialisation, this potential vaccine requires further characterisation in accordance with the authoritative guidelines from the World Organisation for Animal Health (OIE). Hence, in this study, the potential vaccine strain, P. multocida B:2 GDH7 and the virulent parent strain were characterised through genomic and proteomic profiling. A crucial first step was to develop a sensitive yet simple and robust identification test to differentiate both strains which has been achieved by the development of a precise yet straightforward PCR method. In genomic profiling, Repetitive Extragenic Palindromic sequence-PCR (REP-PCR) was manipulated and both strains have a different display of genomic DNA band patterns. Some of the major OMPs were observed and prominent immunogens of P. multocida, OmpA and OmpH were observed to be expressed differently between these strains through SDS-PAGE analysis. In conclusion, a reproducible PCR detection method has enabled differentiation of both strains. Further characterisation of these strains shows a significantly different profile through genomic and proteomic profiling.

• Abubakar, M. S., & Zamri-Saad, M. (2011). Clinico-pathological changes in buffalo calves following oral exposure to Pasteurella multocida B:2. Basic and Applied Pathology, 4(4), 130–135. https://doi.org/10.1111/j.1755-9294.2011.01113.x

• Aiswarya, V., Mathakiya, R. A., Bhanderi, B. B., & Roy, A. (2017). Characterization of Pasteurella multocida isolates of buffalo origin from Gujarat state of India by outer membrane protein profile analysis. Buffalo Bulletin, 36(2), 313-322.

• Ataei, S., Burchmore, R., Christopher Hodgson, J., Finucane, A., Parton, R., & Coote, J. G. (2009). Identification of immunogenic proteins associated with protection against haemorrhagic septicaemia after vaccination of calves with a live-attenuated aroA derivative of Pasteurella multocida B:2. Research in Veterinary Science, 87(2), 207–210. https://doi.org/10.1016/j.rvsc.2009.01.007

• Bernardini, M. L., Sanna, M. G., Fontaine, A., & Sansonetti, P. J. (1993). OmpC is involved in invasion of epithelial cells by Shigella flexneri. Infection and Immunity, 61(9), 3625–3635. https://doi.org/10.1128/IAI.61.9.3625-3635.1993

• Biot-Pelletier, D., Pinel, D., Larue, K., & Martin, V. J. J. (2018). Determinants of selection in yeast evolved by genome shuffling. Biotechnology for Biofuels, 11(1), 282. https://doi.org/10.1186/s13068-018-1283-9

• Biswas, A., Shivachandra, S. B., Saxena, M. K., Kumar, A. A., Singh, V. P., & Srivastava, S. K. (2004). Molecular variability among strains of Pasteurella multocida isolated from an outbreak of haemorrhagic septicaemia in India. Veterinary Research Communications, 28(4), 287–298. https://doi.org/10.1023/B:VERC.0000026656.77847.5b

• Bochner, B. R. (2009). Global phenotypic characterization of bacteria. FEMS Microbiology Reviews, 33(1), 191–205. https://doi.org/10.1111/j.1574-6976.2008.00149.x

• Boyce, J. D., Cullen, P. A., Nguyen, V., Wilkie, I., & Adler, B. (2006). Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host. Proteomics, 6(3), 870–880. https://doi.org/10.1002/pmic.200401342

• Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3

• Chaudhuri, P., & Goswami, P. P. (2001). Cloning of 87 kDa outer membrane protein gene of Pasteurella multocida P52. Research in Veterinary Science, 70(3), 255–256. https://doi.org/10.1053/rvsc.2001.0470

• Darmon, E., & Leach, D. R. F. (2014). Bacterial genome instability. Microbiology and Molecular Biology Reviews, 78(1), 1–39. https://doi.org/10.1128/MMBR.00035-13

• Davies, R. L. (2004). Genetic diversity among Pasteurella multocida strains of avian, bovine, ovine and porcine origin from England and Wales by comparative sequence analysis of the 16S rRNA gene. Microbiology, 150(12), 4199–4210. https://doi.org/10.1099/mic.0.27409-0

• Davies, R. L., MacCorquodale, R., & Caffrey, B. (2003). Diversity of avian Pasteurella multocida strains based on capsular PCR typing and variation of the OmpA and OmpH outer membrane proteins. Veterinary Microbiology, 91(2–3), 169–182. https://doi.org/10.1016/S0378-1135(02)00300-0

• Davies, R. L., Parton, R., Coote, J. G., Gibbs, H. A., & Freer, J. H. (1992). Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype-A1 under different growth conditions. Journal of General Microbiology, 138(5), 909–922.

• De Alwis, M. C. L. (1999). Haemorrhagic septicaemia. Australian Centre for International Agricultural Research.

• El-Brolosy, & M. A, Stainier D. Y. R. (2017). Genetic compensation: A phenomenon in search of mechanisms. PLoS Genetics, 13(7), e1006780. https://doi.org/10.1371/journal.pgen.1006780

• Galdiero, M., De Martino, L., Pagnini, U., Pisciotta, M. G., & Galdiero, E. (2001). Interactions between bovine endothelial cells and Pasteurella multocida: Association and invasion. Research in Microbiology, 152(1), 57–65. https://doi.org/10.1016/S0923-2508(00)01168-2

• Godlewska, R., Wiśniewska, K., Pietras, Z., & Jagusztyn-Krynicka, E. K. (2009). Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: Function, structure, role in pathogenesis and potential application in immunoprophylaxis: Minireview. FEMS Microbiology Letters, 298(1), 1–11. https://doi.org/10.1111/j.1574-6968.2009.01659.x

• González-Torres, P., Rodríguez-Mateos, F., Antón, J., & Gabaldón, T. (2019). Impact of homologous recombination on the evolution of prokaryotic core genomes. MBio, 10(1), e02494-18. https://doi.org/10.1128/mBio.02494-18

• Hazwani-Oslan, S. N., Tan, J. S., Saad, M. Z., Halim, M., & Ariff, A. B. (2017). Improved cultivation of gdhA derivative Pasteurella multocida B:2 for high density of viable cells through in situ ammonium removal using cation-exchange resin for use as animal vaccine. Process Biochemistry, 56, 1–7. https://doi.org/10.1016/j.procbio.2017.02.016

• Ishii, S., & Sadowsky, M. J. (2009). Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution: Minireview. Environmental Microbiology, 11(4), 733–740. https://doi.org/10.1111/j.1462-2920.2008.01856.x

• Kamal, N. M., Zamri-Saad, M., Masarudin, M. J., & Othman, S. (2017). Interaction between Pasteurella multocida B:2 and its derivatives with bovine aortic endothelial cell (BAEC). BMC Veterinary Research, 13(1), 1-8. https://doi.org/10.1186/s12917-017-1109-1

• Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. https://doi.org/10.1038/227680a0

• Martorana, A. M., Motta, S., Di Silvestre, D., Falchi, F., Dehò, G., Mauri, P., Sperandeo, P., & Polissi, A. (2014). Dissecting Escherichia coli outer membrane biogenesis using differential proteomics. PLOS One, 9(6), 100941. https://doi.org/10.1371/journal.pone.0100941

• Munir, R., Shahwar, D., Farooq, U., Nawaz, I., Shahzad, I., & Khanum, A. A. (2007). Outer membrane protein profiling of Pasteurella multocida. Pakistan Veterinary Journal, 27(1), 1–4.

• Oliveira, P. H., Prazeres, D. M. F., Monteiro, G. A., & Pedro H., Oliveira, G. A. M. (2015). DNA instability in bacterial genomes: Causes and consequences. In M. Poptsova (Ed.), Genome analysis: Current procedures and applications (pp. 261-284). Caister Academic Press.

• Oslan, S. N. H., Tan, J. S., Saad, M. Z., Halim, M., Mohamed, M. S., & Ariff, A. B. (2018). Influence of amino acids and vitamins on the growth of gdhA derivative Pasteurella multocida B:2 for use as an animal vaccine. Bioprocess and Biosystems Engineering, 42(3), 355–365. https://doi.org/10.1007/s00449-018-2040-y

• Othman, S., Parton, R., & Coote, J. (2012). Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival. Microbial Pathogenesis, 52(6), 353–358. https://doi.org/10.1016/j.micpath.2012.03.005

• Othman, S. S., Zamri-Saad, M., Abdul Rahim, R., & Zunita, Z. (2017). Pasteurella multocida B:2 GDH7 (Malaysian Patent No. MY-168662-A). IP UPM Malaysia. http://www.upmip.upm.edu.my/action.do?view=ipdetail&cluster=1&ipid=887&ipdetailid=870&projectleaderid=47&page=search&search=pasteurella&searchkey=1

• Othman, S. S. (2007). Contstruction of an attenuated Pasteurella multocida B:2 by mutation in the gdhA gene [Master’s thesis, University Putra Malaysia]. Universiti Putra Malaysia Institutional Repository. http://psasir.upm.edu.my/id/eprint/11652/1/FPV_2007_11_A.pdf

• Prasannavadhana, A., Kumar, S., Thomas, P., Sarangi, L. N., Gupta, S. K., Priyadarshini, A., Nagaleekar, V. K., & Singh, V. P. (2014). Outer membrane proteome analysis of Indian strain of Pasteurella multocida serotype B:2 by MALDI-TOF / MS analysis. Scientific World Journal, 2014, 617034. https://doi.org/10.1155/2014/617034

• Rafidah, O., & Zamri-Saad, M. (2013). Effect of dexamethasone on protective efficacy of live gdhA derivative Pasteurella multocida B:2 vaccine. Asian Journal of Animal and Veterinary Advances, 8(3), 548–554. https://doi.org/10.3923/ajava.2013.548.554

• Rafidah, O., Zamri-Saad, M., Shahirudin, S., & Nasip, E. (2012). Efficacy of intranasal vaccination of field buffaloes against haemorrhagic septicaemia with a live gdhA derivative Pasteurella multocida B:2. Veterinary Record, 171(7), 175. https://doi.org/10.1136/vr.100403

• Sarwar, N., Muhammad, K., Rabbani, M., Younus, M., Sarwar, M., Ali, M. A., Hanif, K., & Kamran, M. (2013). Optimization of physico-chemical factors augmenting in vitro biomass production of Pasteurella multocida. Journal of Animal and Plant Sciences, 23(4), 1085–1088.

• Saxena, M. K., Singh, V. P., Kumar, A. A., Chaudhuri, P., Singh, V. P., Shivachandra, S. B., Biswas, A., & Sharma, B. (2006). REP-PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India. Veterinary Research Communnications, 30(8), 851–861. https://doi.org/10.1007/s11259-006-3321-5

• Somshekhar, S. H., Veeregowda, B. M., Suryanarayana, V. V. S., Leena, G., Dhama, K., & Chakraborty, S. (2014). Outer membrane protein (OMP) profiles of Pasteurella multocida isolates associated with haemorrhagic septicaemia by SDS-PAGE and Western blot analysis. Asian Journal of Animal and Veterinary Advances, 9(8), 513-518. https://doi.org/10.3923/ajava.2014.513.518

• Tabatabaei, M., Liu, Z., Finucane, A., Parton, R., & Coote, J. (2002). Protective immunity conferred by attenuated aroA derivatives of Pasteurella multocida B:2 strains in a mouse model. Infection and Immunity, 70(7), 3355–3362. https://doi.org/10.1128/IAI.70.7.3355

• Tan, H. Y., Nagoor, N. H., & Sekaran, S. D. (2010). Cloning, expression and protective capacity of 37 kDa outer membrane protein gene (ompH) of Pasteurella multocida serotype B:2. Tropical Biomedicine, 27(3), 430–441.

• Townsend, K. M., Dawkins, H. J., & Papadimitriou, J. M. (1997). REP-PCR analysis of Pasteurella multocida isolates that cause haemorrhagic septicaemia. Research Veterinary Science, 63(2),151-155. https://doi.org/10.1016/s0034-5288(97)90009-6

• Townsend, K. M., Frost, A. J., Lee, C. W., Papadimitriou, J. M., & Dawkins, H. J. S. (1998). Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates. Journal of Clinical Microbiology, 36(4), 1096–1100.

• Turni, C., Singh, R., & Blackall, P. J. (2018). Genotypic diversity of Pasteurella multocida isolates from pigs and poultry in Australia. Australian Veterinary Journal, 96(10), 390–394. https://doi.org/10.1111/avj.12748

• Wheeler, R. (2009). Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants. Bioscience Horizons, 2(1), 1–12. https://doi.org/10.1093/biohorizons/hzp002

• World Organisation for Animal Health. (2008). Principle of veterinary vaccine production: OIE terrestrial manual. OIE. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2008/pdf/1.01.06_VACCINE_PRODUCTION.pdf

• World Organisation for Animal Health. (2016). 1.1.8. Principles of veterinary vaccine production: Manual of diagnostic tests and vaccines for terrestrial animals 2016. OIE. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/1.01.08_VACCINE_PRODUCTION.pdf

• Zamri-Saad, M. (2013). Haemorrhagic septicaemia of cattle and buffaloes in Asia. UPM Press.

• Zamri-Saad, M., & Annas, S. (2016). Vaccination against haemorrhagic septicaemia of bovines: A review. Pakistan Veterinary Journal, 36(1), 1–5. https://doi.org/10.1097/QCO.0b013e3283638104

• Zheng, H., Lu, L., Wang, B., Pu, S., Zhang, X., Zhu, G., Shi, W., Lu, Z., Wang, H., Wang, S., Zhao, G.,& Zhang, Y. (2008). Genetic basis of virulence attenuation revealed by comparative genomic analysis of mycobacterium tuberculosis strain H37Ra versus H37Rv. PLOS One, 3(6), 2375. https://doi.org/10.1371/journal.pone.0002375