Elsevier

Microbial Pathogenesis

Volume 128, March 2019, Pages 90-96
Microbial Pathogenesis

The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model

https://doi.org/10.1016/j.micpath.2018.12.042Get rights and content

Highlights

  • •

    The protein vaccine ABA392/pET30a is a good immunogen to elicit cellular and humoral immunogenicity.

  • •

    Intranasal administration of this novel protein vaccine able to provoke mucosal immunity by the formation of BALT.

  • •

    This purified protein vaccine has no toxicity to the liver or kidney.

Abstract

Haemorrhagic septicaemia (HS) is a well-known high fatality septicaemic disease happening among bovines. The disease is caused by the Pasteurella multocida serotype B:2 bacteria. P. multocida B:2 has high mortality and morbidity rates and is spread through the intranasal and oral routes in bovines. In this study, our aim was to investigate the efficacy of the recombinant protein vaccine, ABA392/pET30a via intranasal inoculation by targeting the mucosal immunity. The constructed recombinant protein vaccine ABA392/pET30a was subjected to an animal study using Sprague Dawley rats. The study was divided into two parts: active and passive immunization studies. Both studies were carried out through the determination of immunogenicity (using Total White Blood Cell (TWBC) Count with Indirect ELISA) and histopathogenicity, analyzing (Bronchus Associated Lymphoid Tissue (BALT) formation) in lungs. As a result, the IgA and IgG development of both tested groups: group 1 (50μg/mL protein vaccine) and group 2 (100μg/mL protein vaccine) showed equivalent with the positive control group 4 (formalin-killed P. multocida B:2). However, there was a significant difference when compared with the negative control group 3 (normal saline). These results demonstrate that both the protein vaccine at the concentration 50μg/mL and 100μg/mL have the same efficacy as the commercially available positive control vaccine. From the studies, higher concentration of protein vaccine at 100μg/mL showed higher development of both IgA and IgG compared to 50μg/mL protein vaccine. Higher and rapid development of IgA compared to IgG showed that mucosal immunity has been induced through the intranasal administration of the protein vaccine. In addition, leucocytosis was observed at each dose of vaccination showed that the protein vaccine is capable to induce the immune responses of the host. Histopathogenicity studies of the vaccinated groups showed more BALT formation and no severe lesions after challenge compared to the negative control group. Besides, no inflammatory onsite or anaphylactic responses were observed after the intranasal inoculation which proved to be safer and provided longer lasting immunity. Therefore, recombinant protein vaccine ABA392/pET30a could be a potential candidate for intranasal administration which can provoke mucosal immunity against HS disease.

Introduction

Office International des Epizooties (OIE) has classified haemorrhagic septicaemia (HS) in the List B category of disease due to its high mortality rate in infected cattle especially in Africa and Asia regions [1]. The Pasteurella multocida serotype B:2 is a gram negative bacteria that causes HS in bovines [2,3]. The disease resulted to low production of meat and milk from healthy bovines which will cause serious economic loss and destructive welfare problems around the world [4].

Commercial vaccines that are currently available for HS, unable to provide a long-lasting immunity and their field of efficacy is unknown [5]. Improper vaccine administration techniques, poor potent of vaccine usage and improper vaccine storage condition are the contributing factors for the failure of an effective HS vaccines in cattle [5,6]. ABA392 clone was isolated from P. multocida B:2 and it harbors the virulence factor of the bacteria which caused HS [7]. The expression and potentials of the recombinant clone ABA392 as an expressed protein vaccine candidate against HS has been described and explored in previous studies which proved to be immunogenic [8,9]. The use of ABA392 clone which harbour only the sequence that code for a virulent factor of P. multocida B:2 is much safer, precise and efficient than taking the whole genome and even the whole bacteria [10]. Thus, it is expected that the expressed recombinant clone of ABA392/pET30a protein vaccine could elicit proper antibody titer production and able to protect the domestic bovines against HS attack through mucosal immunization.

HS attack is acute and therefore prevention is preferable rather than treatment of the disease. Mucosal vaccination, is the most preferable defense for shielding the host from P. multocida B:2 infection in bovines, since this method reduces the susceptibility of bovines to the pathogen [11]. Transmission of disease of P. multocida B:2 bacteria is through intranasal and oral routes of bovines [12]. Thus, intranasal administration of mucosal protein vaccine is chosen since it mimics the oronasal route of infection and able to provoke both systemic and mucosal immunity. This type of administration also prevents the induction of unfavorable immune reactions against allergens and self-antigens [13].

In this study, ABA392/pET30a protein was subjected on Sprague dawley (SD) rats to determine the efficacy of the purified recombinant ABA392/pET30a vaccine against HS disease via intranasal administration. The findings were further analysed using bioinformatics strategies via software and programs on allergenicity.

Section snippets

Bacterial strains

The P. multocida subsp.multocida serotype B:2 (PMB2) (ATCC®43137™) was employed as a live challenged strain bacteria. Isolates of bacterium P. multocida serotype B:2 were reactivated in blood agar at 37 °C for 24 h from a glycerol stock which were kept at −20 °C from the Molecular Bacteriology and Toxicology Laboratory, Institute of Biological Sciences, Faculty of Science, University of Malaya.

Preparation of vaccine and challenge strains

The recombinant protein vaccines used were expressed and purified by Ref. [14]. To prepare the

Protein allergenicity

The expression of His-tag ABA392/pET30a protein showed a molecular weight of 32 kDa which is similar to the one compute using Expasy MW bioinformatics tools using sequence obtained from sequencing report. Allergen prediction result from AlgPred tool predicated that the expressed purified protein ABA392/pET30a was non-allergen, with the score value of −1.816611 [Threshold = −0.4]. No experimentally proven IgE epitope was found in the protein sequence. In addition, the positive predictive value

Discussion

Haemorrhagic septicaemia is highly fatal disease in bovines that is caused by P. multocida B:2. Although, several vaccines have been developed, it has been observed at field level that the cases reported for haemorrhagic septicaemia is still ongoing among cattle and buffalos [20,21]. Recently 133 clinically diagnosed HS affected farms were happened in Karnataka State, India which caused huge loss to the livestock farm community estimated mortality loss ($415 per animal) during the HS outbreak [

Conclusion

In conclusion, this study showed that recombinant protein vaccine ABA392/pET30a could be a promising candidate in the prevention of haemorrhagic septicaemia disease. It is also able to act as an immunogen to provoke the mucosal immunity against the P. multocida B:2 antigen by developing the high immunoglobulins IgA and IgG level and inducing the formation of bronchus associated lymphoid tissues with no toxicity to the rats.

Conflicts of interest

The authors have no conflict of interests. All authors attest that they meet the ICMJE criteria for authorship.

Acknowledgements

KTL performed the research and wrote the manuscript. SI designed the research study and revised the manuscript. NK analysed and interpreted the data and revised the manuscript. JM analysed and revised the manuscript. NNR revised the manuscript. All authors approved the final version of the manuscript. This paper was supported by University of Malaya under the grant numbers: [PG066-2015A].

References (44)

  • A. Moustafa et al.

    A case–control study of haemorrhagic septicaemia in buffaloes and cattle in Karachi, Pakistan, in 2012

    Transboundary Emerg. Dis.

    (2017)
  • I. Salmah

    Molecular Studies of Pasteurella Multocida Animal Isolates

    (1997)
  • J. Hussaini et al.

    Recombinant clone ABA392 protects laboratory animals from pasteurella multocida serotype B

    J. Vet. Adv.

    (2012)
  • I. Salmah et al.

    HS Vac(PMB): a Recombinant Vaccine against HS Caused by P. Multocida Serotype B. Innovation and Creativity EXPO 2010

    (2010)
  • J. Gray et al.

    Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection

    Sci. Transl. Med.

    (2017)
  • J. Holmgren et al.

    Mucosal immunity and vaccines

    Nat. Med.

    (2005)
  • D.V. Rita et al.

    A Recombinant Subunit HSABA392 as a potential Vaccine for Haemorrhagic septicaemia disease in livestock

    Trop. Biomed.

    (2018)
  • N.M. Jumahat et al.

    Identification of immunogenic soluble protein of Pasteurella multocida

    J. Adv. Biomed. Patobiol. Res.

    (2015)
  • S. Saha et al.

    AlgPred: prediction of allergenic proteins and mapping of IgE epitopes

    Nucleic Acids Res.

    (2006)
  • J. Hussaini et al.

    Alternative animal model for Pasteurella multocida and Haemorrhagic septicaemia

    Biomed. Res.

    (2013)
  • S.A. Nassar et al.

    Protective effect of Egyptian propolis against rabbit pasteurellosis

    BioMed Res. Int.

    (2013)
  • C.A. Johnson-Delaney

    Exotic Companion Medicine Handbook for Veterinarians

    (1996)
    • View full text
    © 2018 Elsevier Ltd. All rights reserved.