The live attenuated vaccine strain, SG9R, has been used against fowl typhoid worldwide, but it can revert to the pathogenic smooth strain owing to single nucleotide changes such as nonsense mutations in the rfaJ gene. As SG9R possesses an intact Salmonella plasmid with virulence genes, it exhibits dormant pathogenicity and can cause fowl typhoid in young chicks and stressed or immunocompromised brown egg-laying hens. To tackle these issues, we knocked out the rfaJ gene of SG9R (named Safe-9R) to eliminate the reversion risk and generated detoxified strains of Safe-9R by knocking out lpxL, lpxM, pagP, and phoP/phoQ genes to attenuate the virulence. Among the knockout strains, live ΔlpxL- (Dtx-9RL) and ΔlpxM-9R (Dtx-9RM) strains induced remarkably less expression of inflammatory cytokines in chicken macrophage cells, and oil emulsion (OE) Dtx-9RL did not cause body weight loss in chicks. Live Dtx-9RM exhibited efficacy against field strain challenge in one week without any bacterial re-isolation, while the un-detoxified strains showed the development of severe liver lesions and re-isolation of challenged strains. Thus, SG9R was optimally detoxified by knockout of lpxL and lpxM, and Dtx-9RL and Dtx-9RM might be applicable as OE and live vaccines, respectively, to prevent fowl typhoid irrespective of the age of chickens.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens’ tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNβ, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

Hong SM, et al. J Vet Sci. 2023 Jun;24:e51. https://doi.org/10.4142/jvs.23039

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry industry, and O78 serogroup APEC strains are prevalent in chickens. In this study, we aimed to understand the evolutionary pathways and relationships between O78 APEC and other E. coli strains. To trace these evolutionary pathways, we classified 3101 E. coli strains into 306 subgenotypes according to the numbers and types of single nucleotide polymorphisms (RST0 to RST63-1) relative to the consensus sequence (RST0) of the RNA polymerase beta subunit gene and performed network analysis. The E. coli strains showed four apparently different evolutionary pathways (I-1, I-2, I-3, and II). The thirty-two Korean O78 APEC strains tested in this study were classified into RST4-4 (45.2%), RST3-1 (32.3%), RST21-1 (12.9%), RST4-5 (3.2%), RST5-1 (3.2%), and RST12-6 (3.2%), and all RSTs except RST21-1 (I-2) may have evolved through the same evolutionary pathway (I-1). A comparative genomic study revealed the highest relatedness between O78 strains of the same RST in terms of genome sequence coverage/identity and the spacer sequences of CRISPRs. The early-appearing RST3-1 and RST4-4 prevalence among O78 APEC strains may reflect the early settlement of O78 E. coli in chickens, after which these bacteria accumulated virulence and antibiotic resistance genes to become APEC strains. The zoonotic risk of the conventional O78 APEC strains is low at present, but the appearance of genetically distinct and multiple virulence gene-bearing RST21-1 O78 APEC strains may alert us to a need to evaluate their virulence in chickens as well as their zoonotic risk.

Avian pathogenic Escherichia coli (APEC) causes enormous economic losses and is a primary contributor to the emergence of multidrug resistance (MDR)-related problems in the poultry industry. Bacteriophage (phage) therapy has been successful in controlling MDR, but phage-resistant variants have rapidly emerged through the horizontal transmission of diverse phage defense systems carried on mobile genetic elements. Consequently, while multiple phage cocktails are recommended for phage therapy, there is a growing need to explore simpler and more cost-effective phage treatment alternatives. In this study, we characterized two novel O78-specific APEC phages, vWAO78-1 and vHAO78-1, in terms of their morphology, genome, physicochemical stability and growth kinetics. Additionally, we assessed the susceptibility of thirty-two O78 APEC strains to these phages. We analyzed the roles of highly susceptible cells in intestinal settlement and fecal shedding (susceptible cellassisted intestinal settlement and shedding, SAIS) of phages in chickens via coinoculation with phages. Furthermore, we evaluated a new strategy, susceptible cell-assisted resistant cell killing (SARK), by comparing phage susceptibility between resistant cells alone and a mixture of resistant and highly susceptible cells in vitro. As expected, high proportions of O78 APEC strains had already acquired multiple phage defense systems, exhibiting considerable resistance to vWAO78-1 and vHAO78-1. Coinoculation of highly susceptible cells with phages prolonged phage shedding in feces, and the coexistence of susceptible cells markedly increased the phage susceptibility of resistant cells. Therefore, the SAIS and SARK strategies were demonstrated to be promising both in vivo and in vitro.