Protective effects of E. coli Nissle 1917 on chickens infected with Salmonella pullorum.

The probiotic E. coli Nissle 1917 (EcN) plays an important role in regulating the microbial components of the gut and preventing inflammation of the gastrointestinal tract. Currently, the long-term use of antibiotics for the treatment of lethal white diarrhea in chicks caused by Salmonella has led to increased morbidity and mutation rates. Therefore, we want to use EcN as an antibiotic alternative as an alternative approach to prevent Salmonella-induced white diarrhea in chickens. To date, there are no reports of EcN being used for the prevention and control of Salmonella pullorum (S. pullorum) in chickens. In vitro, pretreatment with EcN significantly decreased the cellular invasion of S. pullorum CVCC533 in a chicken fibroblast (DF-1) cell model. Then, 0-day-old egg-laying chickens were orally inoculated with EcN at a dose of 109 CFU/100 µL at either Day 1 (EcN1) or both Day 1 and Day 4 (EcN2). Then, S. pullorum CVCC533 was used to challenge the cells at a dose of 1.0 × 107 CFU/100 µL on Day 8. Next, the body weights and survival rates were recorded for 14 consecutive days, and the colonization of S. pullorum in the spleen and liver at 7 days post-challenge (dpc) was determined. Chicken feces were also collected at 2, 4, 6 and 8 dpc to evaluate the excretion of pathogenic bacteria in feces. The liver, duodenum and rectum samples were collected and analyzed by pathological histology at 7 dpc to evaluate the protective effect of EcN on the mucosa, villi and crypts of the small intestine. The spleen and bursa were collected, and the immune organ index was calculated. In addition, the contents of the cecum of chicks were collected at 7 dpc for 16S rRNA sequencing to detect the distribution of microbial communities in the intestine. The results showed that EcN was able to protect against CVCC533 challenge, as shown by decreased body weight loss, mortality and shedding of pathogenic bacteria in fecal samples in the EcN1 plus Salmonella challenge group (EcN1S) but not the EcN2 plus Salmonella challenge group (EcN2S). The pathogenic changes in the liver, duodenum and rectum also demonstrated that one dose but not two doses of EcN effectively prolonged the length of the pilus with decreased crypt depth, indicating its protective effects against S. pullorum. In addition, the 16S rRNA sequencing results suggested that EcN could enlarge the diversity of intestinal flora, decrease the abundance of pathogenic bacteria and increase the abundance of beneficial bacteria, such as Lactobacillus. In conclusion, EcN has shown moderate protection against S. pullorum challenge in chickens.

Protection against genotype VII Newcastle disease virus challenge by a minicircle DNA vaccine coexpressing F protein and chicken IL-18 adjuvant.

Genotype VII Newcastle disease virus (NDV) is still one of the most important virus threats severely affecting poultry production worldwide. Although inactivated vaccines are commercially available, there is still an urgent need to develop novel vaccine candidates for convenient and affordable vaccine application. Oral immunization using live attenuated bacteria such as Salmonella has recently attracted increasing interest, and in a previous study, we used a regulated delayed lysis Salmonella vector to deliver a DNA vaccine encoding the F protein and chicken IL-18 adjuvant together, named pYL23. To further improve its efficiency, we employed a novel in vivo minicircle DNA (mcDNA) platform to construct pYL58, which could maintain the complete plasmid during in vitro culture conditions and then transform into mcDNA in vivo whenever the plasmid was delivered by Salmonella into host cells. Compared with immunization with the parental strain harboring plasmid pYL23, immunization with Salmonella with pYL58 induced increased levels of serum IgY and mucosal sIgA in chickens, especially the intestinal and tracheal sIgA levels. Production of cytokines, including IL-4, IFN-γ, IL-18 and IFN-α, was also determined in serum and spleen cell culture supernatants after the 3rd immunization, and the results showed that the production of IFN-γ in the pYL58 group was significantly increased compared with that in the negative control group. Interestingly, compared with pYL23, significantly increased production of IFN-α in the cell supernatants from the pYL58 group was also observed. In addition, the CCK-8 assay results showed that the minicircle pYL58 significantly increased spleen cell proliferation. After virulent VII NDV challenge, pYL58 immunization could provide 70% protection compared with 50% protection in the pYL23 group, together with decreased virus titers in chicken lung samples at Day 5 and virus shedding at Days 3 and 5 post-challenge. This study demonstrated that the application of mcDNA technology dramatically increased the DNA vaccine efficiency, providing additional support for the use of our mcDNA platform in the veterinary field.

In Vivo Production of HN Protein Increases the Protection Rates of a Minicircle DNA Vaccine against Genotype VII Newcastle Disease Virus.

The Cre-recombinase mediated in vivo minicircle DNA vaccine platform (CRIM) provided a novel option to replace a traditional DNA vaccine. To further improve the immune response of our CRIM vaccine, we designed a dual promoter expression plasmid named pYL87 which could synthesize short HN protein under a prokaryotic in vivo promoter PpagC and full length HN protein of genotype VII Newcastle disease virus (NDV) under the previous eukaryotic CMV promoter at the same time. Making use of the self-lysed Salmonella strain as a delivery vesicle, chickens immunized with the pYL87 construction showed an increased serum haemagglutination inhibition antibody response, as well as an increased cell proliferation level and cellular IL-4 and IL-18 cytokines, compared with the previous CRIM vector pYL47. After the virus challenge, the pYL87 vector could provide 80% protection compared to 50% protection against genotype VII NDV in pYL47 immunized chickens, indicating a promising dual promoter strategy used in vaccine design.