Abstract
Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and H9N2 influenza infection are two economically important diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant (JOL967) to deliver highly conserved extracellular domains of H9N2 M2 (M2e) to induce protective immunity against both H9N2 infection and FT. To increase the immunogenicity of M2e, we physically linked it with CD40L and cloned the fusion gene into either prokaryotic constitutive expression vector pJHL65 or mammalian expression vector pcDNA3.1+. Then pJHL65-M2eCD40L or pcDNA-M2eCD40L recombinant plasmid was electroporated into JOL967 strain and the resultant clones were designated as JOL2074 and JOL2076, respectively. We demonstrated that the chickens vaccinated once orally with a co-mix of JOL2074 and JOL2076 strains elicited significantly (p < 0.05) higher M2e-specific humoral and cell-mediated immunity compared to JOL2074 alone vaccinated group. However, SG-specific immune responses were comparable in both the vaccination groups. On challenge with the virulent H9N2 virus (105 TCID50) at 28th day post-vaccination, chickens that received a co-mix of JOL2074 plus JOL2076 strains exhibited significantly (p < 0.05) lower lung inflammation and viral load in both lungs and cloacal samples than JOL2074 alone vaccinated group. Against challenge with the lethal wild-type SG, both the vaccination groups exhibited only 12.5% mortality compared to 75% mortality observed in the control group. In conclusion, we show that SG delivering M2eCD40L can act as a bivalent vaccine against FT and H9N2 infection and further studies are warranted to develop this SG-M2eCD40L vaccine as a broadly protective vaccine against avian influenza virus subtypes.
Highlights
Avian influenza (AI) and fowl typhoid are two highly contagious infectious diseases that cause huge economic losses in poultry industry worldwide [1, 2]
The presence of M2eCD40L gene in pJHL65 and pcDNA3.1 recombinant plasmids was confirmed by digestion of the recombinant clones with restriction-specific enzymes to release a fragment of 123 bp size
Our IF results indicated that recombinant Salmonella Gallinarum (SG) bacteria (JOL2074) efficiently displayed surface M2e expression as evidenced by the reactivity of the M2-specific polyclonal antibody with the expressed M2eCD40L protein, which was lacking in the negative bacterial control, JOL2068 strain (Figures 1A and B)
Summary
Avian influenza (AI) and fowl typhoid are two highly contagious infectious diseases that cause huge economic losses in poultry industry worldwide [1, 2]. In South Korea, H9N2 subtype has been endemic since 2000 and the infection is mainly controlled through the use of an oil adjuvanted inactivated H9N2 vaccine, which has dramatically decreased the incidence of H9N2 infection in chicken farms [14]. These conventional vaccines require a large supply of specific-pathogen-free (SPF) embryonated eggs and vaccine production requires a long timeline that could be threatened during pandemic situations. Novel approaches should be devised that are egg independent and cost-effective, but easy to amplify and broadly protective against LPAI viruses, H9N2
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