Recently, different approaches have been used to develop human and veterinarian vaccines. Such approaches either utilize the cellular surface displaying properties (e.g., Protein A in Gram-positive bacteria and specific outer-membrane proteins in Gram-negative bacteria) (Wernérus & Ståhl, 2004), or biotechnology-based techniques (Motin & Torres, 2009). The novel biotechnology approaches involve genetically engineered vaccines, inverted pathogenicity (utilizing virulence factors to prevent or treat a disease), and bacterial ghosts system (Motin & Torres, 2009; Paukner et al., 2006). The preserved surface structures and components of the BG can induce both innate and adaptive immune response.
In the current study, the highest serum bactericidal activity (100% eradication) was achieved in the BG-adjuvant subcutaneously immunized animals. Salmonella cells disappeared totally in the faeces of the immunized rats by subcutaneous injection of both BG and BG-adjuvant vaccine after virulence challenge test. In previous studies, the sodium hydroxide (MIC) induced BG of S. aureus showed significant lowering of the total bacterial load within the internal organs (liver, spleen, lungs, and kidneys) of all vaccinated rats groups (orally, subcutaneously, and intravenously) (Park et al., 2016).
In a related study, Salmonella enteritidis ghosts (SEG) that were also prepared by the same agent (MIC of NaOH) showed a comparable immune response. The highest serum bactericidal effect was shown in the intramuscular (SEG) with complete Freund’s adjuvant followed by intramuscular then finally orally vaccinated rats group at the sixth week. Similarly, the intramuscular (SEG) vaccine with adjuvant gave the highest IgG titers and showed the least bacterial load in the internal organs’ homogenates, followed by intramuscular, then orally vaccinated group in week 8 and week 10. All vaccinated groups exhibited significant humoral and cellular immune responses in comparison to the non-vaccinated rat group (Vinod et al., 2014).
In this study, the highest IgG titers were at the maximum level in the subcutaneously vaccinated group with BG-adjuvant followed by the BG subcutaneously vaccinated group at the last day (day 42) in the last period of immunization program. The bacterial load in liver homogenate significantly reduced in the subcutaneously vaccinated rats by BG only after virulence challenge and disappeared in the vaccinated group by BG-alum. In a previous study, the highest IgG antibody activity and the serum bactericidal activity was elicited in subcutaneously vaccinated group at week 9 followed by intravenously then finally orally vaccinated group. Likewise, CD4 + and CD8 + T-cell populations were produced in the largest percentage in the subcutaneously intravenously then orally vaccinated group. The survival rate was 100% in the intravenously immunized, while about 60% in non-immunized group (Vinod et al., 2015).
In another previous study, fish that were immunized intraperitoneally by genetically produced Edwardsiella tarda showed stronger serum agglutination titer than those immunized by traditional (formalin-killed pathogen) vaccine (Kwon, Yoon, Sung, & Kim, 2006). The immunized cattle by genetically prepared BGs of Brucella suis S2 showed the same titers of IgG, interleukin 4, INF-ᵧ and T-cells as that showed in the conventional formalin-killed Brucella) immunized cattle (Liu et al., 2015).
In extraordinary route of immunization which was through aerosol of the genetically induced BG of Actinobacillus pleuropneumoniae elicited higher antigen-specific IgM and IgA in the bronchoalveolar lavage and plasma cells influx in comparison to the traditionally (irradiated-killed bacteria) immunized pigs. Additionally the survival rate was 100% in the BG vaccinated animals (Hensel et al., 2000; Katinger et al., 1999). The antibody titer reached to the maximum in the immunized rats at the third week with neglected titers in the immunized ones (Wu et al., 2017). In the current trial, the survival rate in the BG subcutaneously vaccinated rats’ group was 100%. This was followed by the BG-adjuvant subcutaneously vaccinated group (75%), while the lowest survival rate was in the orally vaccinated group. In a corresponding results in another study, the immunization of rats by subcutaneous injection of BGs of Listeria monocytogenes that were prepared by sponge-like protocol (a chemically induced method) (Amro, Salem-Bekhit, & Alanazi, 2014) protected the immunized rats by 100% in the opposite of 0% percent survival in non-immunized animals. By using the same protocol, sponge-like protocol, Salmonella typhimurium ATCC 14028 was turned into BGs then orally administered to a rat group. The serum of the orally vaccinated group showed agglutination reactions between antigen O and H against antibodies that prove the correct Salmonella envelop structure that may protect against live Salmonella infection (Sheweita, 2014).
In a previous study, the prepared Klebsiella pneumonia by the same protocol (Sponge-like chemical protocol) gave both cellular and humoral immune responses in form of subcutaneous (the highest activity), inhalation, intraperitoneal, and intramuscular routes. The produced phagocytic activity, INF- ᵧ levels, and bactericidal actions were much higher in the vaccinated rats (MM et al., 2017). Different strains of Vibrio cholerae were evacuated using the E-lysis gene –genetic approach- and exploited in vaccination of the experimental animals via different routes of administration. Vibrio cholerae El Tor, Ogawa strains ghosts intraperitoneal vaccination of rats were compared to the conventional vibrio vaccination and produced more IgG titers and vibriocidal activity (Francis O Eko et al., 1994).
In contrast to the result of the current study, the oral vaccines of salmonella’s BGs failed to protect rats, another study showed that orally vaccinated animals by BGs of E. coli O157:H7 (Mayr et al., 2005b) and H. pylori (Talebkhan et al., 2010) were survived by 93% and 100% respectively. Additionally, the bacterial colonization in intestine (E. coli BG vaccine) and stomach (H. pylori BG vaccine) was reduced. Also, there was significant existence of anti H. pylori and Omp-specific antibodies in H. pylori BG vaccinated animal group (Talebkhan et al., 2010). Furthermore, the oral vaccine of V. cholerae O1 or O139 strains protected about 50% of the immunized infant mice cholera models. It also produced anti-LPS and antitoxin coregulated pilli (F. O. Eko, Mayr, Attridge, & Lubitz, 2000) and vibriocidal antibodies (Francis O Eko et al., 2003) in the vaccinated group of rabbits. Further, it could reduce- dose correlated- the duodenal mucosal colonization by vibrio after vibrio challenge local injection (Jalava, Eko, Riedmann, & Lubitz, 2003).
In this study, the highest percentage of granulocytes was raised in the BG-adjuvant subcutaneously immunized group. Significant induction of dendritic cells antigen presentation and release of different interleukins and anaphlytoxins were followed the subcutaneous immunization of rabbits by BG of V. cholerae H1 strains (Francis O Eko et al., 2015). Significant increase in antigen-specific T-cells proliferation (Jawale, Chaudhari, & Lee, 2014), plasma antigen-specific IgA and IgG, INF-ᵧ, interleukin 2, T-cells as well as bacterial colonization reduction in the immunized chicken internal organs (Jawale & Lee, 2016). It was compatible with the results of the current study that 100% protection was accomplished by the immunization of both rabbits and mice by genetically induced Pasturella multocida and Pasturella haemolytica BGs by subcutaneous route (Marchart et al., 2003). In another report, both species of salmonella, S. gallinarum and S. typhimurium BGs that were genetically synthesized, significantly reduced the mortality rates in immunized chicken when they were administered intramuscularly (Jawale & Lee, 2016) or even orally (Jawale et al., 2014).
The chemically prepared (tween 80 protocol) Salmonella enterica serovar typhimurium BGs vaccines were able to protect all the immunized rats (survival rate 100%) without adjuvant. It also offered both humoral and cellular immune responses in case of subcutaneous route of administration. The minor immune response of the oral BG vaccine may refer to the gastric intestinal digestion as well as possible intentional reflux by the animal itself.