Escherichia coli (E.coli), a gram-negative bacterium in the family Enterobacteriaceae, induces multi-species bacterial diseases and is widespread all over the world (1). Infections with E.coli are common in avian, humans and other mammals, posing a threat to food safety and public health. Infected animals and contaminated water can severely affect human health, causing neonatal sepsis, bacterial meningitis, diarrhoeal disease, and urinary tract infection (2–4). Compared with humans, E. coli infection in animals is often limited to clinical monitoring result in recessive infection state, which exhibit long-lasting consequences for the environment (5, 6).

Bovine colibacillosis represents an ongoing and major challenge to the livestock industry due to the role of E.coli as the commonest bacteriological pathogen in several growth and development stages with substantial economic consequences. Although most E. coli, which is found ubiquitously in the environment, are not pathogenic for animals in healthy conditions, postpartum cows and newborn calves are more susceptible to bacterial infections due to immunocompromised state. In calves, enterotoxigenic E. coli infection can cause severe diarrhea; in dry period, intramammary E. coli infection causes mastitis, resulting in decreased milk production and poor milk quality (7, 8). Infection during each stage of pregnancy and labor may lead to uterine inflammation and even fatal bacteremia and septicemia. Currently, antibiotics are still the mainstay of treatment for E. coli-caused infections. For example, on some cattle farms, antibiotics were added into the daily feed to prevent colibacillosis. Meanwhile, use and abuse of antibiotics led to antibiotics resistance getting more and more serious over time (9, 10). A study in the USA reported that resistance to extended-spectrum cephalosporins increased from 5.46% to 12.97% and fluoroquinolones resistance reached 28.22% during the 8-year study period (11). In England, there were 616 multidrug-resistant (MDR) out of 1434 E. coli isolates from Oxfordshire (12). In a recent study, a bad trend was discovered that may be causing more severe concerns about antibiotic resistance. It was reported that even without using quinolones, using other antibiotics also contributed to acquiring quinolone resistance (13). Instead of treatment with antibiotics, vaccines provide long-lasting protection from bacterial infections for animals through periodic immunization in the livestock industry. The diversity of serotypes and cross-protection between them pose a significant challenge in vaccine research and development.

As the main immunological feature of E. coli organisms, the bacteriophage antigen, O antigen, is still used as the main basis for E. coli serotype classification and also influences its virulence and pathogenicity (14). Enormous structural diversity of O antigens lies in the sugar composition and the linkages between monosaccharides (15). Currently, as many as 185 O antigens are known. The poor cross-protection between serotypes has been demonstrated in practice, which means that it is difficult to cover multiple serotypes of E. coli infection with immunization against a few antigens (16). From the perspective of pathogenicity, immunization against colonization factors and adhesins in specific pathogenic E. coli can prevent their infection (14). However, virulence factors are often transferred horizontally between bacteria via plasmids, islands, phages, etc., which is making widespread prevention of E. coli by immunization difficult. Even though, attempts using new strategies for vaccine development have shown promising results (17, 18).

During the past several decades, novel vaccines have gained increasing attention to achieve better immune protection. Among them, bacterial ghosts (BGs) have been attracting significant interest among researchers due to their potent adjuvant effects and favorable immunologic effects (19, 20). BGs are cell envelopes originated from gram-negative bacteria losing cytoplasmic contents but maintaining morphology and surface structure such as lipopolysaccharides (LPS), outer membrane proteins, adhesins, etc. The formation mechanism for BGs is that channels in the bacterial cell membrane by genetic engineering methods or chemical approaches leads to cytoplasmic contents flowing to the surrounding medium, sequentially leaving an empty bacterial shell without nucleic acid. BGs are also considered as an intruder by the immunity system because of antigenic determinant on the surface of bacteria cells, which can be recognized by macrophages and in turn eliciting systemic humoral and cellular immune responses. As vaccine, BGs can trigger elevated levels of antibodies (21); as an adjuvant, they have proven to improve the protective efficacy of Chlamydia abortus and Newcastle disease virus vaccines (22–24).

Currently, genetic engineering is the most common method used to produce BGs. Many studies have shown that protein E, which is translated from the lysis gene E of the ΦX174 bacteriophage, has a lytic function on bacteria. However, the mechanism of the effect has not been fully elucidated. Initial recognition of the E gene cleavage function was based largely on morphology. The cell envelope breaks down more gradually in two distinct stages: the cell wall rolls back, then the plasma membrane disintegrates. However, as the authors state, it is not possible to draw any detailed conclusions regarding the mechanism of breakdown of the cell envelope at a macro-molecular level in either case from micrographs (25). In the following study, other groups attributed the function of the E gene to the activation of unspecified autolytic functions or the formation of polymeric “transmembrane tunnels” that opened the cytoplasm directly to the medium based on physiological and scanning-EM studies (26). In the two-stage model, the assumption of “transmembrane tunnels” was further demonstrated and widely accepted: Protein E induces the generation of transmembrane channels (40~200nm) via (a) protein E integration into the inner membrane; (b) protein conformational change transferring the C-terminus across the inner membrane, and(c)fusion of the inner and outer membranes. Cytoplasmic contents such as nucleic acid, ribosomes, etc. flowed out via these transmembrane channels because of osmotic pressure between the internal and external environments of the cell, resulting in a complete bacterial envelope (27). Starting with gene E-induced vibrio cholerae ghosts by Werner Lubitz in 1994 (28), constant modification of gene E as well as vectors enhances cleavage efficiency in some bacteria (29). Researches have shown that fusion genes of mutant gene E and staphylococcal nuclease A (SNA) gene not only induce the genetic inactivation of E.coli cultures efficiently but also prevent horizontal gene transfer which contribute to the propagation of antibiotic resistance (30). Thus, in this study, the tandem two genes (E+SNA) were utilized for bacteria BG production.

As a novel vaccine strategy, it is important to optimize the immune conditions for BGs to achieve a better protective effect. Considering the special structure and infection routes, BGs can be administered via subcutaneous (SC), intramuscular (IM), intraperitoneal (IP), oral (O), respiratory (RP), rectal (R), etc. (31, 32). Salmonella enterica ghosts robustly stimulate humoral and cell-mediated immune responses in chickens via the intramuscular, subcutaneous, and oral routes. No significant differences in immune markers were found between immune routes, except for significantly higher IgA levels in the oral group compared to SC and IM groups (33). Acinetobacter ghosts have also been shown to be effective by leukocyte counts, assays of immune cell activity, and phagocytosis when administered subcutaneously, intramuscularly, intraperitoneally, and orally, but the differences between the various immunization routes remain unknown (34). In addition, as a novel vaccine, the differences in immunization effect of BGs compared with traditional inactivated vaccine are of particular interest. Currently, there are no relevant studies about bovine pathogenic E. coli ghosts. Therefore, in this study, we evaluated the immune effects of traditional inactive vaccines with aluminium adjuvants via IM and E. coli ghosts through subcutaneous (SC), intramuscular (IM), and oral (O) to contribute to the application of BGs as a novel vaccine candidate in the future.

The expression vector for the lysis plasmid in this study was pBV220 with the temperature-sensitive regulatory system λpL/pR-CIts857, which initiates the translation of the fragment inserted in its polyclonal site (35). Compared to vectors using inducers such as IPTG for induction, pBV220 was more convenient. However, this plasmid also had some limitations for this research. Since positive clones containing the pBV220 plasmid exhibit ampicillin resistance, the plasmid could only be applied to those Gram-negative bacteria that are ampicillin-susceptible. Unfortunately, ampicillin resistance was extremely common in gram-negative bacteria, which would result in many bacteria not being able to prepare BGs with this plasmid. To solve this problem, we could change the screening conditions by modifying the vector. For example, the blue-white screening element on the pUC19 plasmid might sidestep the problem by inserting the entire temperature-controlled lysis cassette into the polyclonal site of pUC19 on the basis of the plasmid constructed in this experiment (31).

Although the plasmid constructed in this study could be expressed efficiently in wild-type E. coli CE9 strain, the lysis efficiency decreases with increasing passages, probably because of the segregational instability and structural instability (36). Therefore, unless this problem is solved, the passage times of positive clones should be minimized to achieve effective cleavage.

Complete inactivation of pathogenic E.coli CE9 could not be achieved by E gene lysis alone. Following induction, residual live bacteria needed to be further inactivated further. β-propanolactone interacts with the guanine bases of nucleic acids to inactivate bacteria, viruses, and other microorganisms, and was widely used in the preparation of virus vaccines. β-propanolactone was hydrolyzed completely to β-hydroxypropionic acid without any toxicity by reaction with water (37). β-propanolactone has been studied for the inactivation of residual bacteria after BGs induction. The remaining viable bacteria with appropriate concentrations of β-propanolactone at 60 min at 42°C could be complete inactivated. Although β-propanolactone provides good inactivation, it was not suitable for mass production due to its high price. Several studies have demonstrated that gentamicin inactivates bacteria due to its effects of methylation, inhibition of protein synthesis, and increased cell permeability (38). In the present study, no live bacteria were found in the BGs after lyophilisation, perhaps because the CE9 ghost strain did not tolerate low temperatures, so no additional inactivation reagents are needed to ensure vaccine safety. Inactivation seemed like an easy problem to solve due to the many inactivators available, but how to ensure the antigenic structure of the bacterial surface while killing residual bacteria still needs to be proven by rigorous experimental data.

For various pathogens, there were differences in the immunization routes for BGs vaccines. In this study, E. coli CE9, which causes diarrhea in calves, was the intestinal infection. Intramuscular and subcutaneous injections were two of the most common immunization routes, so three routes were set for the comparison of BGs immunization: oral, subcutaneous, and intramuscular. For IgA and IgG levels, all groups were immunized with a two-week interval after the first immunization, and the antibody levels were significantly increased in all experimental groups after 2 weeks of booster immunization. The SC group had higher levels of specific IgG antibodies at all times and also significantly elevated IgA levels, while the IM group had significantly higher levels of IgA antibodies at day 28 and also elicited high levels of IgG, indicating that both subcutaneous and intramuscular injections induced robust humoral immune responses. Salmonella ghosts in chickens have been shown to result in the highest serum-specific antibody levels by intramuscular injection compared to other immunization routes (39). These findings are in agreement with earlier reports: higher levels of IgG elicited by Salmonella ghosts intramuscularly than orally in chicken (40). Another study showed close antibody levels for intramuscular and subcutaneous injections of Salmonella ghosts, with slightly higher antibody levels in the IM group than in the SC group, which were similar to the results in this study (33). Subcutaneous immunization of BGs against Haemophilus parvum in swine and Bordetella bronchiseptica in dogs has shown good results. From these available references, subcutaneous and intramuscular immunizations were the most versatile and effective immunization routes against various BGs (31, 41, 42).

The IgG and IgA antibody levels in the oral group remained suboptimal. Taking the immune protection rate together, the antibody levels induced by the 2 immunizations were not sufficient to resist infection. Oral immunization was more convenient than injection, but it is susceptible to loss and can lead to unstable immunological effects. Previous studies have demonstrated that outer membrane vesicles (OMVs) from E. coli encapsulated into Zein Nanoparticles coated with a Gantrez^® AN–Mannosamine Polymer Conjugate elicited high levels of IgG antibodies, while oral administration of the outer membrane protein alone did not elicit an antibody response (43). In contrast, displaying CD0873 on liposomes without encapsulation elicited significantly different IgG and IgA titers from the control group after three oral immunizations (44). Oral exposure to stx+ eae+ intimin-positive E. coli O157: H7 as an oral vaccine twice weekly for 6 weeks would increase resistance of cattle to colonization by wild-type E. coli O157: H7 following challenge (45). Our findings also showed a trend of higher antibody levels for IgG and IgA with the immunization duration. These findings suggested that we may achieve better immunologic effects with oral E. coli ghost vaccines, but rather need to find more appropriate immune-related conditions, such as increasing the time of immunization and appropriate encapsulants.

In general, two immunizations were more effective than a single immunization. The prime-boost immunization strategy of Salmonella Gallinarum (SG) ghosts had an advantage over single immunization in chickens because it induces a robust immune response for optimum protection against fowl typhoid (46). EHEC O157:H7 BGs, on the other hand, have been shown to significantly stimulate efficient humoral and cellular immune responses and to be as protective as two immunizations (47). It illustrated that different strains, administration routes, and experimental animals need to choose different immunization protocols. In the present study, the IM group showed a significant increase in IgA-specific antibodies after the second immunization compared to the first immunization, which facilitated defense against bacterial infection. Considering that there was no significant difference in IgG antibody levels between 14 and 28 days, it remains uncertain whether the improved protective efficiency due to increased lgA levels based on mucosal immunity. Therefore, it is necessary to explore the role of booster vaccination through single and multiple immunizations of BGs via the intramuscular route.

It has been shown that the immunologic effects of BGs vaccines were superior to those of inactivated vaccines (21), but the adjuvants have a significant impact on the immunization effects. In this study, the inactivated vaccine containing aluminium adjuvant elicited high antibody levels and cytokine levels, but the antibody and cytokine levels and protection rate remained low relative to the intramuscular and subcutaneous injections with BGs. The local metabolism of aluminium adjuvant was found to be hard (unpublished observations). In contrast, no injection site swelling was observed after 2 intramuscular injections of BGs.

In the present experiment, single nucleated cells collected from the spleen were examined by flow cytometry for the percentage of CD4+ and CD8+ T cells, and the results indicated that CE9 ghosts elevated CD4+ and CD8+ levels by intramuscular immunization. Other routes, however, revealed no significant differences in CD4+ and CD8+ T cell levels. On the one hand, intramuscular immunity could better activate CD4+ and CD8+ T cells compared to mucosal immunity (48, 49). On the other hand, although there was no significant difference in the proportion of CD4+ or CD8 T cells compared to the control group, the elevated cytokines such as IFN-γ, IL-4, and IL-6 also reflected the cellular immunity being activated. The expression level of IL-12 was significantly higher in the IM group compared to the other groups. CD4+ T cells differentiate into regulatory T cells (Treg) in response to antigen stimulation. In the presence of TGF-β, IL-12 promotes Treg cell differentiation into Foxp3⁺ IFN-γ⁺ Treg cells as well as IFN-γ expression (50). The increase in IL-6 also contributes to the conversion of CD4+ T to Th17 in the presence of TGF-β. Th17 cells promote a direct CXCL8-dependent chemotactic effect on neutrophils, which is important for antimicrobial immunity (51). Perhaps due to insufficient sample size, it was not sensitive enough to reflect a smaller difference.

The expression of IL-1β, IL-2, IL-4, IL-6, IL-12 and IFN-γ was increased differentially in groups. IFN-γ, a signature cytokine produced by TH1 cells, was elevated in all experimental groups. Th1 activated mononuclear phagocytes (MP) as well as produced IL-12 to promote Th1-type cellular immune responses (52). IL-12, a polarizing cytokine, induces CD4 T cells to differentiate into a Th1 subset by two-stage differentiation a) IL-12 induces the outgrowth of IFN-γ Th1 cells, b) about half of Th1 cells ultimately gain CX3CR1 expression and exhibit a cytotoxic effector profile depending on the transcription factor ZEB2, which, in turn, produces IL-2 and IFN-γ (53, 54). IFN-γ-dependent macrophage activation is important for bactericidal functions. In our study, the IL-12 level in the IM group was dramatically higher than all other groups, and the highest protection rate provided by the IM group may, at least partly, depend on this. It has been shown that IL-12 can be used as an adjuvant to improve the effect of tuberculosis treatment (55) IL-6 has been reported to induce B cells to differentiate into and enhance CD8+ T cells differentiation. Perhaps dependent on the significant increase in IL-6 levels, only significant differences in CD8 levels were detected in the IM group in this study. IL-2 and IL-6 have regulatory effects on the differentiation of plasma cells and B cells, respectively, implying that increased levels of both have an important role in the regulation of humoral immunity (56).

Pathogenic E. coli CE9 could cause death in mice, accompanied by blood in the stool and urine. So the lungs, intestines, and kidneys were observed pathologically after the attack. The differences in pathological changes in the three organs mentioned above were obvious. The mice in the PBS group had severe lung, intestine, and kidney damage. The survivors in the traditional inactivated vaccine group were saved from death, but they still had pathological changes in several organs. We were surprised to find that the O group seemed to have better intestinal protection than the lungs and kidneys after the attack. This may be due to the local immunity of oral administration in the intestine. The three organs in the IM were essentially identical to those in the physiological states. This may be due to the fact that the IM route stimulated cellular immunity and high levels of humoral immunity. Specific IgG antibodies prevented bacterial infection through humoral immunity, and increased levels of IgA prevented bacterial adhesion and colonization. The high levels of Th1 and Th2 cytokines secretion also indicated that cellular immunity was activated, clearing pathogenic bacteria and protecting organs from infection. IL-12 has been shown to promote cytotoxicity of pulmonary mucosal-associated invariant T cells and improve clearance of pathogenic bacteria by mucosal immunity (57). Overall, high survival rates are generally associated with better tissue protection. It was reasonable to speculate that this enhancement of organ protection would be important for the health status and productive performance of surviving animals.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Northeast Agricultural University (SRM-11).

JM designed and performed all the study. LL and YZ contributed to the experimental work. DL and JL contributed to the BGs vaccine and ELISA assays. JM and YF wrote the manuscript. YL and GW did corrections and critical review of this paper. All authors contributed to the article and approved the submitted version.

This research was funded by National Key Research and Development Program of China (2016YFD0501310).

The authors are thankful to Dongfang Shi at the College of Veterinary Medicine of Northeast Agricultural University for providing partial experimental equipment. Also, thanks to ZY and ZBB for their meaningful suggestions on the experimental design.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.