Using In Vitro Immunomodulatory Properties Of Lactic Acid Bacteria For Selection Of Probiotics Against Salmonella Infection In Broiler Chicks

Poultry is known to be a major reservoir of Salmonella. Lactic acid bacteria can competitively exclude pathogens by e adherence to the host intestinal epithelium and enhancement of the immunomodulatory activity of host. Furthermore, different studies have described the successful use of recombinant lactic acid bacteria(recLAB) to deliver anti-inflammatory molecules at the mucosal level to treat inflammatory diseases caused by pathogens or chemical reagents. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties(in addition to conventional indicators such as resistance to acid and bile, antibacterial activity, its own adhesion and anti-adhesion of pathogens to intestinal epithelial cells) for potential use as probiotics against Salmonella infection in broiler chicks, and investigate the ability of Lactobacillus to express biologically active chick anti-inflammation cytokine interleukin-10(cIL-10). In addition, polyclonal antibodies of poultry cytokines LITAF, IL-1β, IL-6, IL-10 and IL-12 were prepared for studies on the poultry immune responses.1. Prokaryotic expression and production of polyclonal antibodies against cytokinesThe full length of LITAF, IL-1β, IL-6, IL-10 and IL-12 were cloned from genome of broiler chick. In this study, antigenic determinants encoded by 100, 268, 186, 156 and 141 amino acids of LITAF, IL-1β, IL-6, IL-10 and IL-12, respectively were cloned into plasmid pET-32a(+) or pET-21a(+) vectors which had double restriction sites of Kpn I and Hind III or BamH I and Hind III, and then the positive recombinant plasmids pET-32a(+)-LITAF, pET-32a(+)-IL-1β, pET-32a(+)-IL-6, pET-32a(+)-IL-10 and pET-21a(+)-IL-12 were transformed into the host E.coli BL21(DE3) or Transetta(DE3) as expression bacteria(engineered bacteria of LITAF, IL-1β, IL-6, IL-10 and IL-12). After IPTG induction, target proteins were obtained. Then purified target proteins were used for immunizing rabbits to prepared polyclonal antibodies against poultry cytokines LITAF, IL-1β, IL-6, IL-10 and IL-12. These antibodies can be used to detect cytokines at protein level.2. Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetablesIn this study, 16 randomly selected LAB strains from fermented sauerkraut, and these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of 6 tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of LITAF and IL-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection.3. Selection of chicken intestinal derived lactic acid bacteria and protection against Salmonella infection in broiler chicksAmong 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic(pH 2.5) and bile salt(ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties(Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities(Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of LITAF, IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.4. Engineering construction of lactic acid bacteria and functional verificationAmong the 5 recLAB strains, L. casei P4-cIL-10 had the highest ability to express cIL-10, and with the 163.8 ng/ml of production, then followed by L. casei LP1-cIL-10, L. rhamnosus P5-cIL-10 and L. plantarum PZ01-1-cIL-10. L. reuteri LY01-cIL-10 had no detected cIL-10 expression. In addition, a clear band was detected in the cell(C) fraction from cultured L. plantarum PZ01-1-cIL-10, L. casei LP1-cIL-10, L. casei P4-cIL-10 and L. rhamnosus P5-cIL-10 at the expected size for mature cIL-10. Furthermore, L. casei P4-cIL-10 strain had a stronger ability to decrease the production of LITAF, IL-1β, IL-6 and IL-12 from mononuclear cells from chick spleens than L. casei P4 strain at 24 h and 48 h post-treatment with LPS, it showed that L. casei P4-c IL-10 strain induced a stronger protective effect than its wild type strain. Finally, like L. casei P4, L. casei P4-cIL-10 strain also had probiotic properties, including survival under conditions simulating the GI tract, antimicrobial activity, adhesion, anti-adhesion and anti-invasion abilities and immunomodulatory property. These results suggest that recombinant strain L. casei P4-cIL-10 could be used as a probiotic candidate.In conclusion, this study enriched screening parameters of lactic acid bacteria in vitro and validated the importance of immunomodulatory activities of lactic acid bacteria in vivo. In addition, we explored potential applications of recombinant lactic acid bacteria as probiotic candidates in poultry.