| Chicken coccidiosis is a parasitic disease with a high infection rate and mortality rate in chickens,with a mortality rate of up to 80% in severe cases.Coccidiosis live vaccine can reduce the intestinal lesions,relieve the weight gain inhibition on chickens to a certain extent,and reduce the death caused by chicken coccidiosis.However,it is still unable to completely control the intestinal damage caused by coccidiosis in chickens,and the weight gain of chickens slows down during immunization.The Ch IL-4-Ch IL-2 fusion gene was introduced into the food-grade foreign protein expression system to construct a novel recombinant L.lactis NZ3900/p NZ8149-Ch IL-4-Ch IL-2(abbreviated as RLIL4/2)that could express and secrete the Ch IL-4-Ch IL-2 fusion protein to further improve the immune effect and reduce the side effects of coccidiosis live vaccine.PCR,cell culture in vitro,western blot,enzyme linked immunosorbent assay,RT-q PCR,hematoxylin-eosin staining,α-Naphthyl acetate esterase staining,methyl green pyronine staining,immunofluorescence staining,16 S r DNA sequencing and animal immune challenge tests to study the bioactivity of the protein product secreted by recombinant L.lactis NZ3900/p NZ8149-Ch IL-4-Ch IL-2 and its synergistic effect and mechanism on coccidiasis live vaccine,and the laboratory safety.The results showed that:(1)The recombinant L.lactis NZ3900/p NZ8149-Ch IL-4-Ch IL-2 was constructed,and the target protein could be detected in both the supernatant and the bacteria.The induced RLIL4/2 culture supernatant significantly enhanced the proliferative activity of primary chicken splenic lymphocytes activated by Con A or LPS(P < 0.05).The results indicated that RLIL4/2 could express and secrete the Ch IL-4-Ch IL-2 fusion protein,the Ch IL-4-Ch IL-2 fusion protein could promote the proliferation of chicken spleen lymphocytes in vitro.(2)Tissue homogenate enrichment culture,fecal enrichment culture and PCR were used to detect the colonization and evacuation of RLIL4/2 in the chicken intestine.It was found that RLIL4/2 could be colonized in all intestinal tracts of chicks from 1 to 7 days after oral administration,and the small intestinewas dominant(duodenum > jejunum > cecum).(3)The SPF chickens were given with different doses of RLIL4/2 combined with the trivalent live vaccine with four precocious strains at 4 days of age and 14 days of age,and challeng with coccidiosis virulent strains at 28 days of age.The intestinal lesions scores of duodenal,jejunal,and cecal of 10 chickens in each vaccination group were ≤ 1,the weight gain in 1×109 and 1×1010 CFU RLIL4/2 + vaccine co-inoculation groups were significantly higher than that in vaccine-only group(p < 0.05),the relative reduction rate of oocysts was greater than 95%,the intestinal lesions scores in duodenum,jejunum and cecum were significantly lower than those in vaccine-only group(P<0.05),and the anti-coccidial indexes(ACI)were 187.85 and 193.33,while the ACI in vaccine-only group was 174.61.The results showed that the combined inoculation with RLIL4/2 could improve the anticoccidiosis efficacy of coccidiosis live vaccine from medium effect to high efficiency,among which 1×109 CFU/ feather was the lowest effective dose of RLIL4/2 to play the immune synergistic effect on coccidiosis live vaccine.(4)SPF chickens were first co-inoculation with RLIL4/2 and the trivalent live vaccine with four precocious strains at 4 days of age,followed by a second immunization at an interval of 5 to 7 days,and then challenged at the age of 25 days.When the interval between two immunizations was 6 to 7 days,the intestinal lesions scores of duodenal,jejunal and cecal in at least 8 experimental chickens in all vaccination groups were ≤ 1.The average weight gain of experimental chickens in the RLIL4/2+vaccine groups were significantly higher than that in the vaccine-only groups with the same immunization interval(P<0.05),the relative reduction rate of oocysts was greater than 97%.When the immunization interval was 7 days,the ACI of vaccine-only group reached 160,which can effectively resist chicken coccidiosis;At the interval of 6 and 7 days,the ACI of the RLIL4/2+vaccine groups were 171.78 and 183.64,respectively,achieving medium and high efficacy against coccidia.The results showed that co immunization with RLIL4/2 could shorten the immunization interval of chicken coccidiosis live vaccine by 1 day.(5)SPF chickens were co-inoculation with RLIL4/2 and the trivalent live vaccine with four precocious strains at 4 and 14 days of age,and challenged at 4,5 and 6 days after the second immunization,respectively.The average weight gain of experimental chickens in each immune group increased with the delay of the challenge time(P<0.05),but the oocysts output was on the contrary.When attacked on the 6th day after the second immunization,the intestinal lesion scores of 10 experimental chickens in the vaccine-only group were ≤ 1,and the ACI reached 160,which could effectively resist chicken coccidiosis.When attacked on the 5th and 6th day after the second immunization,the intestinal lesion scores of 10 experimental chickens in the RLIL4/2+vaccine groups were ≤ 1,and the ACI was 166.48 and 184.72,respectively,achieving the medium-effective and efficient coccidius-resistant effect.The results indicated that co inoculation with RLIL4/2 could advance the immune generation period of coccidiosis live vaccine 1d.(6)SPF chickens were co-inoculation with 0.4 to 1.0 feather/chicken of coccidiosis live vaccine and RLIL4/2 at the age of 4 days,and were immunized once at the age of 14 days(double the first immunization vaccine),and challenged at the age of 25 days.The chickens average weight gain of the RLIL4/2+vaccine groups decreased with the decrease of vaccine dose(P<0.05),while the oocysts output increased(P < 0.05).When the vaccination dose was within the range of 0.6 to 1.0 feather/chicken of coccidiosis live vaccine,the duodenum,jejunum and cecum intestinal lesions scores of at least 8 chickens in the inoculation groups were ≤1,and the ACI were greater than 160.The results showed that when co-inoculation with RLIL4/2,reducing the dose of chicken coccidiosis live vaccine by 40% can still effectively resist chicken coccidiosis.(7)SPF chickens were immunized with RLIL4/2 and coccidiosis live vaccine at 4 and 14 days of age,respectively.RLIL4/2 upregulated the expression of Th1 cytokines(IL-2,IFN-γ,TNF-α)and Th2cytokines(IL-4)in immune-related organs and intestinal tissues from the transcriptional and protein levels compared with the vaccine-only group on the 7th,14 th,and 28 th days after the first immunization(P<0.05),and upregulated the expression of SIg A protein in intestinal fluid(P<0.05).The transformation numbers of T lymphocytes,B lymphocytes and APCs in the main immune organs and intestines were up-regulated on the 7th and 14 th day after the first immunization(P < 0.05),and the transformation numbers of T lymphocytes and B lymphocytes in the intestinal tissues were still up-regulated on the 28 th day(P < 0.05).In addition,it was found that the type of intestinal flora in the vaccination group remained stable,and oral administration of RLIL4/2 increased the relative abundance of beneficial intestinal bacteria(P<0.05),while decreased the relative abundance of opportunistic bacteria on the 7th day after the second immunization(P<0.05).The results showed that RLIL4/2 could enhance the immune synergistic effect on coccidiosis live vaccine by enhancing cellular immunity,humoral immunity,intestinal mucosal immune response and regulating intestinal probiotic flora.(8)SPF chickens were inoculated with RLIL4/2 at 4 and 14 days of age with once and twice single doses(1×109 CFU/ feather),and once overdose(1×1011 CFU/ feather),respectively,no clinical abnormality or death was observed in chicks from 0 to 14 days after inoculation.Oral administration of single dose RLIL4/2 twice increased the weight gain of chickens(P < 0.05),did not cause histopathological changes in vital tissues and organs,and increased the intestinal probiotic flora(P<0.05).The translocation of RLIL4/2 in chickens after inoculation was detected by tissue homogenate enrichment culture and PCR,it was found that RLIL4/2 was not distributed to the heart,liver,spleen,lungs,kidneys,and blood.RLIL4/2was only detected in the intestinal feces of chickens raised in the same cage on the 4th day after oral administration,and RLIL4/2 was not detected in environmental samples.It shows that RLIL4/2 is safe for chickens,does not translocate to tissues outside the intestinal tract,and has poor horizontal and environmental transmission capabilities.To sum up: the food-grade recombinant L.lactis NZ3900/p NZ8149-Ch IL-4-Ch IL-2 was successfully constructed,the co-inoculation of RLIL4/2 with chicken coccidiosis live vaccine in chickens could enhance cellular,humoral,and intestinal mucosal immunity,promote the proliferation of APCs,and increase the number of probiotics.RLIL4/2 chicken coccidiosis vaccine immune time interval between shortened the 1d,immune period ahead of the 1 d,reduce the dosage of 40% of the vaccine,but also has good security. |
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