| Clostridium perfringens(C.perfringens),a Gram-positive,spore-forming anaerobic bacillus that is widely distributed in natural environments and can usually be found in the gastrointestinal tract of livestock and poultry as an opportunistic pathogen,is associated with human food-borne diseases as well as animal diseases and exotoxins are its main virulence factors.Animals suffer from rapid disease and high mortality,causing serious economic losses to the animal husbandry industry.The exotoxins secreted by C.perfringens are its main pathogenic factors.The pathogenicity of C.perfringens is the result of the combined action of multiple exotoxins,in which?,?and?exotoxins are the most common and highly toxic toxins.In addition,C.perfringens is a typical intestinal pathogen,which causes animal disease by secreting exotoxin through intestinal infection.According to the traditional C.perfringens toxin typing scheme,C.perfringens is classified into five toxin types(A,B,C,D,and E)based on the major toxins?,?,?,and?produced by each toxin type,while a recently updated study proposed that C.perfringens strains should be classified into seven toxinotypes,A to G.The seven serotypes of the bacteria A,B,C,D,E,F,and G can secrete?toxin,this toxin is one of the most important exotoxin secreted by these bacteria,its effect is cytotoxicity,hemolytic activity,increased vascular permeability,and platelet aggregation.?1 toxin is one of the exotoxin secreted by type B and C bacteria that can cause cell necrosis and body death.It causes infection of intestinal necrotizing inflammation and intestinal toxemia in newborn animals and poultry.The effect of?2 toxin is lethality and cytotoxicity;it can cause necrotizing enteritis and hemolytic necrosis in infected animals,and can also cause enteritis and enterotoxemia in many animals and poultry.Another toxin is?toxin,it is mainly an exotoxin secreted by type B and D bacteria,and it can cause muscular nephropathy(Nephrotic syndrome)in infected livestock.The course of the disease is very short.Animals generally die within a short time after the onset of disease.C.perfringens disease has severely restricted the development of animal husbandry.The most effective way to prevent this disease is to vaccinate animals.Vaccination plays an extremely important role in the prevention and control of diseases and is an indispensable means to fight against infectious diseases.From the perspective of the safety of vaccines in practice,inactivated vaccines and subunit vaccines are more constant choices.However,inactivated vaccines have too weak antigenicity,more important epitopes may be lost,and fire-fighting vaccines cannot be replicated.One vaccination cannot make the body secrete enough antibodies to persuade immunity.Thus,multiple vaccinations will necessary which will cause multiple stresses in animals.Therefore,it is necessary to seek antigen substances with a broad spectrum of immune protection against the pathogenic strains and stimulate its natural immunity.Based on the pathogenic characteristics of C.perfringens through intestinal infection,effective intestinal mucosal immunity is an ideal way to prevent C.perfringens infection.As the carrier of oral vaccine for intestinal mucosa immunity,Lactic acid bacteria have apparent benefits in carrying exogenous antigen into the intestinal tract through oral rout,which can ensure the integrity of antigen and persuade active mucosal immune response.Particularly,the construction of Lactobacillus constitutive expression vectors does not need to be induced.At the same time,using Lactobacillus isolated from animal intestine with the ability to colonize in the host as the host bacteria expressing exogenous genes,it will be more operative to induce the local mucosal immune response against the antigen.Based on,this study used Lactobacillus isolated from the host animal chicken,the constitutive expressio n of C.Perfringens quadruple major toxoids carrier of plasmid electoporate into chicken source Lactobacillus.After that,the study analyzed the features and induced mucosal immune response ability in chicken.The main research contents and results are as follows:1.Identification and analysis the adhesion,colonization,and probiotic characteristics of Chicken derived L.crispatus strainsIn order to obtain a live bacterial carrier that initiates mucosal immunity and delivers specific antigenic substances,this experiment evaluated and observed the Lactobacillus strains isolated from the intestinal tract of healthy chickens in our laboratory.In this study,for screening Lactobacilli that can cause mucosal immunity,deliver specific antigen and analyzing their probiotic characteristics;the isolated strains plated onto MRS-Ca CO3 medium plate,anaerobically cultured at 42°C and then the gram positive,non-movement and rod-shaped bacteria were selected and subjected to analysis via catalase,oxidase and nitrate reduction activity tests,and sugar fermentation reaction-based phenotypic identification,and sequence analysis of 16S r RNA amplified by PCR assay.Results showed that there were 3Lactobacillus strains obtained from the intestinal tract of chicken,including Lactobacillus crispatus(L.crispatus)N-11,5-1-1,and 5-3-2 strains.Moreover,this study evaluated the prospective probiotics characteristics of Lactobacillus strains,by in-vitro and in-vivo experimental analysis.Results indicating all three strains were able to grow well at 37°C and42°C,and showed certain anti-Gram positive and Gram negative bacteria activity.Among three strains,L.crispatus N-11 showed significantly high inhibition zone agains pathogenic bacteria,especially E.coli and C.perfringens.Adhesion experiments showed that all of the isolated Lactobacillus strains had adhesion ability and could colonize in intestinal tract.In addition,to evaluate its probiotics characteristics chickens were selected as a target animal.And isolated Lactobacillus strains were used as a feed additive;their mixture concentration at the rate of 1g/kg-g-1 of diet fed to chicks,produces significantly effect on increase body weight,FCR,and improved intestinal histomorphology of chickens,especially villus height and villus height:crypt depth ratio compared with control group.2.Identification of constructed recombinant L.crispatus strainsFurthermore,we preceded our study further and constructed recombinant chicken-derived L.crispatus(p PG-E-?-?2-?-?1/L.crispatus N-11,5-1-1,and 5-3-2)expressing C.Perfringens?-?2-?-?1 quadruple main toxoids,using an e GFP marker.According to the result of polymerase chain reaction(PCR),restriction enzyme analysis digestion,and sequencing analysis of p PG-E-?-?2-?-?,successfully showed a target band of the plasmid p PG-E-?-?2-?-?1,with the expected size at 3450 bp and EGFP target band with a size of about 762 bp were obtained.This study analyzed the effect of recombinant plasmid transformation on the reproductive ability of Lactobacillus strains.The results showed that the reproductive ability of recombinant L.crispatus N-11,5-1-1,and 5-3-2 was not significantly different from that of parental bacteria,indicating that the inserted foreign gene did not affect the growth of L.crispatus N-11,5-1-1,and 5-3-2.Additionally,this study systematically evaluated the ability of recombinant L.crispatus strains to tolerate the digestive tract environment and the ability of recombinant L.crispatus to colonize the intestine.The experimental results of digestive tract environmental tolerance showed that recombinant Lactobacillus can tolerate simulated gastric environment,and its growth tendency is obviously increased with p H increase.Meanwhile,recombinant Lactobacillus tolerance was observed in 0.05%,0.1%,0.2%,and 0.3%bile salt.The results showed good tolerability to intestinal fluid environment and its survivability rate was high in 0.2%bile salt,while showed slowdown survivabiliy maintenance,when the concentration was reached to0.3%.In order,to detect the constitutive expression of toxoids proteins in recombinant L.crispatus p PG-E-?-?2-?-?1/L.crispatus N-11,5-1-1,and 5-3-2,the recombinant L.crispatus N-11,5-3-2,and 5-1-1 were cultured with conventional MRS medium to collect the bacteria.The expression of the e GFP screening tag protein was observed under ultra-high resolution microscope and performed flow cytometery.The results showed obvious green fluorescence in recombinant strains.In addition,the recombinant strains were cultured on MRS medium and the expression of the fusion toxoids protein were identified successfully by western blot.Results showed the C.perfringens fusion toxoids protein?,?,?1 and?2 better constitutive expression in recombinant Lactobacillus stains.Moreover,on the basis of significant characteristics among L.crispatus N-11,5-1-1,and 5-3-2,the recombinant L.crispatus N-11strain was selected and further proceeded to evaluate the intestinal colonization ability and immunogenicity analysis,experiment results show that recombinant L.crispatus can be colonized in the chicken's intestine,and distributed in the jejunum,ileum,colon and other parts.After 1 day of oral administration,the colonization rate in the jejunum,ileum and colon on the first day after oral administration was high is 71.9%,57.3%,and 63.0%respectively,with the metabolism,the colonization rate of recombinant L.crispatus in the chickens intestine gradually decreased,but showed still maintenance a colonization level on the 15th day.The strong intestinal colonization ability of recombinant L.crispatus can effectively stimulating the body's mucosal immune response to provide protection.3.Model of experimental induction of necrotic enteritis in chickens by C.PerfringensA total of 20,one day old chickens were divided into two groups,among them 1 group was further divided into 4 groups;each group was included 4 chickens.To in duce infection and to determine the lethal dose(LD100)of toxins in chicken,inoculation of C.perfringens crude extract toxins was started at day 10,at a dose rate of 60 mg/kg,80 mg/kg,100 mg/kg,and 120 mg/kg,respectively.(Toxin amount/chicken body weight),for 7 consecutive days,while control group of chickens were inoculated 1 ml of PBS.Taking into account bird mortality,daily base monitored all chickens during trial period any mortality(day 10 to 25)individually undertook necropsy to achieve the actual cause of death is due to NE infection or any other disease attack or stress.Those chickens who's died by the administration of absolute lethal dose was preceded for further observation,included performances,gross pathological observation,and histopathological changes in gastrointestinal tract.The performance of chickens after orally inoculated of C.perfringens was significantly reduced as compared to control.The Gastrointestinal tract of control chickens showed well developed normal flora of the intestine with prominent folds of duodenum,jejunum,ileum and cecum,and the chickens inoculated by crude extract toxins of C.perfringens,the intestines showed sever inflammation and diphtheritic pseudo-membrane were seen.Upon Histological examination of necrotic enteritis,the characteristics microscopic lesions were found in all died chickens'samples,while no signs found in control chickens.The microscopic lesions of field cases during NE outbreak in chickens were highly related with seen in this study.It can be seen that the experimental infection mod el of necrotizing enteritis in chickens caused by the toxin infection of C.perfringens is consistent with the clinical case.The determined LD100 was measured 120 mg/kg that killed all infected chicks.4.Analysis immunogenicity of recombinant p PG-E-?-?2-?-?1/L.crispatus N-11 in ChickensIn this study,to explore the oral immunity effect of recombinant L.crispatus p PG-E-?-?2-?-?1/L.crispatus N-11 strain was selected and chickens susceptible to C.perfringens were used as model animals to carry out the evaluation of the immunogenicity of recombinant Lactobacillus crispatus p PG-E-?-?2-?-?1/L.crispatus N-11.The experimental chickens were divided into five groups:Among them group I was orally immunized with recombinant L.crispatus p PG-E-?-?2-?-?1/L.crispatus N-11(recombinant vaccine group);Experimental group II/III/IV was respectively orally immunized with p PG-T7g10-PPT/L.crispatus N-11(empty vector control group)/L.crispatus N-11(non recombinant control group)/PBS(PBS control group);The experimental group V was kept as normal control group.The immunization procedure of recombinant L.crispatus was as follows:a total of three oral immunizations,the interval between each immunization were 2 weeks,each consecutive immunization for 3 days,and one immunization per day.Intestinal mucus and serum samples of each group of immunized chickens were collected at different time points after immunization,and ELISA method was used to detect the specific s Ig A antibody in intestinal mucus and the level of specific Ig Y antibody in serum.The test results showed that higher s Ig A antibody levels were detected in the intestinal mucus samples of chickens in the vaccine group immunized with recombinant p PG-E-?-?2-?-?1/L.crispatus N-11,and higher Ig Y antibody levels were also detected in the serum,compared with the control groups,the difference was significant.Furthermore,the number of Ig A in intestinal mucosa segments was observed via immunochemical analysis,results indicated that the numbers of Ig A in lamina propria of intestinal segments were higher compared with that of the control groups(p<0.05).The experimental results show that oral immunization of recombinant L.crispatus can not only induce local mucosal immune response in the animal body,but also stimulate the body to produce a systemic humoral immune response.Aditionally,The cytokines IFN-?,IL-2,IL-4,IL-10,IL-12,and IL-17 levels in the serum samples of immunized group showed significantly higher levels(p?0.01)in chickens immunized orally with p PG-?-?1-?-?2/L.crispatus N-11 as compared to empty vector and PBS groups.5.Evaluation of immunoprotective effection of recombinant p PG-E-?-?2-?-?1/L.crispatus N-11In order to verify the immune protection effect of oral recombinant p PG-E-?-?2-?-?1/L.crispatus N-11,this study conducted a challenge test on the 7th day after the third immunization,the experimental chickens in each group were challenged orally with the lethal dose(1×LD100)of mixed natural toxins of type A and type B C.perfringens and combined with oral viable bacteria to estimate the protection efficacy of engineered recombinant strain p PG-?-?1?-?2/L.crispatus N-11.The experimental results showed that the recombinant Lactobacillus group induced protective immunity by oral administration of recombinant p PG-E-?-?2-?-?1/L.crispatus N-11,and was able to resist the natural toxin of C.perfringens,with a protection rate was up to 100%(4/4),while p PG-T7g10-PPT/L.crispatus N-11,L.crispatus N-11,and PBS control chickens were died.At the same time,the results of histopathological observation showed that compared with the experimental chickens of the oral immunized recombinant L.crispatus group,the intestinal segments of the other groups had showed obvious pathological changes.The above results indicate that the recombinant p PG-E-?-?2-?-?1/L.crispatus N-11 has good oral immunogenicity against infection of C.perfringens.The research results also showed that the recombinant Lactobacillus crispatus has good probiotics for the target animal chickens.The indicators,liver and kidney function and the organ index of chickens were tested,and the results showed no significant difference with the PBS control group.Additionally,the effect of recombinant strain on chicken microbiota showed the relative abundance of Lactobacillus significantly increased while decreased the level of Eubacterium,Lachnoclostridium,romboutsia,and turicibactor at the genus level as compared to control group.In summary,this study analysed 3 strains of L.crispatus,isolated from the intestinal tract of chicken,and were investigated for probiotics and constructed the recombinant Lactobacillus constitutively expressing of C.perfringens?,?,?1 and?2 toxins.L.crispatus/p PG-E-?-?2-?-?1/L.crispatus N-11 was preceded to investigate immunogenicity,by using as an oral vaccine vector.On the basis of this study results,the oral administration of recombinant L.crispatus p PG-2-?-?2-?-?1/L.crispatus N-11 has good immunogenicity and can effectively induces the body to produce resistance to C.perfringens through oral immunization.It can effectively induce protective local mucosal immunity and systemic immune response against C.perfringens infection.The results showed that,recombinant p PG-E-?-?2-?-?1/L.crispatus N-11 can produce effective immunoprotection against the challenge of natural toxins mixture of C.perfringens type A and B to 1×LD100 dose.Additionally,it will be a prospective orally mucosal vaccine candidate for prevent infection of C.perfringens.The results of this study laid the foundation for the further development of the genetically engineered Lactic acid bacteria oral vaccine against C.perfringens. |
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