Therapeutic Efficacy of Phage PIZ SAE-01E2 against Abortion Caused by Salmonella enterica Serovar Abortusequi in Mice

Abstract

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage P_IZ SAE-01E2 against S Abortusequi in a mouse model of abortion were investigated. Phage P_IZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of P_IZ SAE-01E2. All of these characteristics indicate that P_IZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 10³ CFU/mouse of S Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of P_IZ SAE-01E2 (10⁸ PFU/mouse, multiplicity of infection = 10⁵) 1 h before or after S Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage P_IZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (<10² CFU/g) compared to those in the placenta and the uterus of the mice in the control group (>10⁶ CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P < 0.05) compared to those in the placenta and blood of the mice in the control group. Altogether, these findings indicate that P_IZ SAE-01E2 shows the potential to block abortions induced by S Abortusequi in vivoIMPORTANCES Abortusequi is an important pathogen that can induce abortions in mares. Although S Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S Abortusequi infection is essential. In this study, a new lytic phage, P_IZ SAE-01E2, infecting S Abortusequi was isolated, and the characteristics of P_IZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of P_IZ SAE-01E2 before or after S Abortusequi challenge provided effective protection to all pregnant mice. Thus, P_IZ SAE-01E2 showed the potential to block abortions induced by S Abortusequi in vivo.

Keywords: Salmonella enterica subsp. enterica serovar Abortusequi; abortion; mares; mice; phage therapy.

FIG 1

Biological characteristics of phage P_IZ SAE-01E2. (A) Transmission electron micrographs of P_IZ SAE-01E2. Phage P_IZ SAE-01E2 belongs to the Siphoviridae family. The diameter of its head is approximately 60 ± 5 nm, and the length of its noncontractile tails is approximately 115 ± 5 nm. (B) Temperature sensitivity of P_IZ SAE-01E2. The phage titers showed no significant differences from 4°C to 40°C and decreased less than 2 log units from 50°C to 70°C. (C) pH stability of P_IZ SAE-01E2. The phage titers showed no significant differences at pH values of 4 to 10; the phage titers significantly decreased at a pH value of 12 (P < 0.01). (D) One-step growth curve of P_IZ SAE-01E2. The initial 5 and 10 min are an eclipse period and a latent period of P_IZ SAE-01E2, respectively, and the average burst size of P_IZ SAE-01E2 is approximately 215 PFU/cell. The values indicate the means and standard deviations (SD) (n = 3).

FIG 2

The genome of phage P_IZ SAE-01E2. The CLC Genomics Workbench (version 8.1) program was employed to visualize the putative ORFs and their direction of transcription. The direction of the arrows represents the direction of gene transcription. The different colors represent different functional modules. The putative functions and names of the genes are listed.

FIG 3

Bactericidal activity of phage P_IZ SAE-01E2 in vitro. S. Abortusequi bacteria were cocultured with phage P_IZ SAE-01E2 in LB medium at different MOIs. S. Abortusequi bacteria not infected with phage were used as a negative control. The bacterial numbers were counted at the indicated time points. The values represent means and SD (n = 3).

FIG 4

Bacterial load in abortive mice. Mice on gestational days (GD) 3.5, 4,5, 6.5, 9.5, and 14.5 were selected and challenged with 2 × 10³, 2 × 10⁴, or 2 × 10⁵ CFU/mouse of S. Abortusequi ATCC 9842. The dying mice and all abortive mice were immediately euthanized. Different organs were homogenized, and a suspension was used to count the load of S. Abortusequi ATCC 9842. When challenged with 2 × 10³ CFU/mouse of S. Abortusequi ATCC 9842, the bacterial load in the uterus was significantly higher than that in the fetus (P < 0.001) and the bacterial loads in the placenta and spleen were significantly higher than the bacterial load in the fetus (P < 0.01). When challenged with 2 × 10⁴ CFU/mouse of S. Abortusequi ATCC 9842, the bacterial loads in the uterus, placenta, and spleen were significantly higher than the bacterial load in the fetus (P < 0.01). The values represent the means and SD (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Bacterial loads in the organs. (A) Bacterial loads in the placenta of phage P_IZ SAE-01E2-treated or S. Abortusequi ATCC 9842-infected mice. (B) Bacterial loads in the uterus of phage P_IZ SAE-01E2-treated or S. Abortusequi ATCC 9842-infected mice. The black line represents the dynamic changes in the bacterial loads in the placenta of bacterium-infected mice; the green line represents dynamic changes in the bacterial loads in the placenta of mice that were treated with phage P_IZ SAE-01E2; the red line represents the dynamic changes in the bacterial loads in the placenta of mice that were pretreated with phage P_IZ SAE-01E2. All experiments were repeated three times. The values represent the means and SD (n = 3). The values on the x axis are times (in hours).

FIG 6

Inflammatory cytokine expression in the placenta. The levels of the inflammatory cytokines IFN-γ, TNF-α, and IL-10 and the anti-inflammatory cytokine IL-17 in the placenta were determined at 12 h, 24 h, and 48 h after bacterial infection. The values represent the means and SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.