Comparative study of subcutaneous, intramuscular, and oral administration of bovine pathogenic Escherichia coli bacterial ghost vaccine in mice

Abstract

Escherichia coli is one of the most common bacterial pathogens in cattle. Prophylactic vaccines are considered promising strategies with the potential to reduce the incidence of colibacillosis. Some studies suggested that bacterial ghosts may serve as a novel approach for preventing bacterial infections. However, the roles of administration route on vaccine immunogenicity and efficacy have not been investigated. In this study, the efficacy of vaccination via different immune routes in generating humoral and cellular immune response was compared through subcutaneous (SC), intramuscular (IM), and oral (O) administration in female BALB/c mice with bacterial ghosts prepared using wild type Escherichia coli isolates CE9, while phosphate buffer saline (PBS) and inactivated vaccines containing aluminum adjuvants (Killed) were used as control. Our results showed that the plasmid pBV220-E-aa-SNA containing E. coli was efficiently cleaved at 42°C with 94.8% positive ratio as assessed by colony counts. Transmission electron microscopy (TEM) confirmed bacteria retained intact surface structure while devoid of cytoplasmic component. We found that total IgG titers in killed, IM and SC groups showed significant increase on 7, 14, 21 and 28 days post-immunization. The IgA level of the IM group was higher than that of all other groups on the 28th day. Meanwhile, four experimental groups showed a significant difference in IgA levels compared with PBS control. In the IM group, an increase in the relative percentages of CD3+CD4+ T cells was accompanied by an increase in the relative percentages of splenic CD3+CD8+ T cells. In comparison with the inactivated vaccine, intramuscular CE9 ghosts immunization elicited higher levels of IL-1β, IL-2, IL-6 and IL-12. Subcutaneous and intramuscular immunizations were significantly associated with improved survival in comparison with oral route, traditional vaccine and the control. Pathologic assessment revealed that less severe tissue damage and inflammation were found in lung, kidney, and intestine of IM group compared with other groups. The results above demonstrate that immunization of Escherichia coli CE9 ghosts via intramuscular injection elicits a more robust antigen-specific immune response in mice to prevent the Escherichia coli infection.

Keywords: Escherichia coli; bacterial ghosts; humoral immunity; lysis cassette; pBV220 vector.

Figure 1

Construction of the lysis plasmid and identification of bacterial ghosts (BGs) quality. (A) The lysis plasmid map showing the location of elements with different colors. (B) PCR identification of E-lysis gene, SNA gene, and tandem fragments E-aa-SNA. (C). The growth of CE9 strain and CE9 ghost strain (containing lysis plasmid) culturing at 42°C. (D) The lysis efficiency was indicated by the number of clones and DNA concentrations before and after lysis. (E) Transmission electron micrographs of the BGs. ****p<0.0001.

Figure 2

Specific IgG and IgA antibodies in mice immunized by E.coli CE9 ghosts. Specific antibody IgG levels in serum (A) and IgA level in intestinal lavage fluid (B) at 7, 14, 21 and 28 days after immunization. Significance was analyzed using 2-way ANOVA and Tukey’s multiple comparisons test. *p<0.05,**p<0.01,***p<0.001, ****p<0.0001.

Figure 3

Cellular immune responses of vaccinated mice. Percentage of CD3+/CD4+ (A) and CD3+/CD8+ (B) T cells in spleen detected by flow cytometry at day 28 post-immunization. The spleen coefficient (C) was calculated by spleen weight (g)/mice weight (g). Significance was analyzed using 1-way ANOVA and Tukey’s multiple comparisons test. *p<0.05,**p<0.01,***p<0.001.

Figure 4

Cytokine expression was assessed by RT-qPCR normalized to GAPDH at day 28 post-immunization. All groups were compared to the PBS group. The expression of IL-6 (A) and IL-1β (B) in the Killed group, IM group, and SC group were significantly elevated. The IL-4 expression levels (C) in the IM and O groups were significantly higher than the other groups. The IM group had the highest IL-2 expression level (D) among all groups. IFN-γ titers (E) were significantly higher in all immunization groups. The IM group had the highest IL-12 titer (F) among the groups. Significance was analyzed using 1-way ANOVA and Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

Survival rates of vaccinated mice after challenge with 5 LD₅₀ CE9 strain. Except for the PBS group, all experimental groups protected some mice from the CE9 strain-induced death.

Figure 6

Pathological histological evaluation of intestine (A–E), lung (F–J) and kidney (K–O) of mice in the PBS, Killed, IM, O and SC group at 7 days post-challenge. Intestinal villi (40×), alveoli (100×), and renal cortical structures (100×) were severely damaged in the PBS group. In the Killed group, Intestinal villi, alveoli structures were moderate damaged, while the renal cortical was mild damaged. Almost no damage appeared in the tissues of IM group. In the O group, intestinal villi showed mild injury, whereas moderate pulmonary and kidney lesions were observed. SC group showed engorged alveolar capillaries and peritubular capillaries.