Development Of A Live Attenuated Vaccine Against Lawsonia Intracellularis And The Mechanism Of Intracellular Survival

Porcine proliferative Enteropathy(PPE)is an important intestinal disease caused by the obligate intracellular bacterium Lawsonia intracellularis and is endemic to pig production worldwide.It characterized by acute hemorrhagic diarrhea,intermittent diarrhea,loss of appetite and slow growth among growing-finishing pigs aged 6 to 20 weeks,which causes highly economic impact in swine worldwide due to its negative impacts on daily wight gain and feed conversion rate and its increased mortality effects.At present,pig producers have mainly relied on available commercial vaccines and antimicrobials to prevent and control PPE.Current commercial vaccines have been demonstrated effectively in pigs,however,vaccination has not been widely used in China due to its high price,and PE is easily ignored by producers and veterinarians.L.intracellularis is an obligate intracellular bacterium that requires a specific microaerophilic environment and permissive cell cultures to be cultivated in vitro.Moreover,the extraction and in vitro maintenance of L.intracellularis is extremely difficult,only a few laboratories around the world are capable of isolation and cultivation of L.intracellularis in vitro.To date,there is no report regarding the successful isolation and maintenance of L.intracellularis from infected intestinal samples in China.Therefore,no commercial PPE vaccine with independent intellectual property rights has been developed in China,and the intracellular survival mechanism of L.intracellularis is remaining unclear.In this study,L.intracellularis from China was successfully isolated from ileum samples of infected pigs in Jiangsu province.Further,PPE attenuated vaccine with independent intellectual property rights was developed and the intracellular survival mechanism of L.intracellularis was explored.Our results will provide valuable information for the effective prevention and control of PPE.Experiment 1: Preparation and characterization of a new monoclonal antibody specific against Lawsonia intracellularis and its application In this study,the purified membrane protein 2(Omp2)of L.intracellularis was used as an immunogen,and monoclonal antibodies against Omp2 were prepared by lymphocytic hybridoma technology.Then the monoclonal antibodies were applied to detect L.intracellularis in infected cells and tissues.The results showed that three hybridoma cell lines,named 4D9,3G2 and 7G5,could stably secrete monoclonal antibodies against Omp2 protein were obtained.Ascites was prepared from BALB/c mice,the titers of ascitic fluids were1:20,48,000,1:512000 and 1:256,000,respectively.An indirect immunofluorescence analysis revealed that the 4D9,3G2 and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs,such as Salmonella cholerae,Salmonella typhimurium,Escherichia coli,Brachyspira hyodysenteriae.Further studies showed that the monoclonal antibodies could combine specifically to L.intracellularis in monolayer infected cells and in the crypt of the ileum from infected tissues of PPE.Our founding suggested that the novel monoclonal antibody specific to L.intracellularis will be useful for the evaluation of L.intracellularis infection in vivo and in vitro.Experiment 2.Isolation and in vitro cultivation of L.intracellularis from China and establishment of a mouse infection modelIn this study,an epidemiological investigation of the seroprevalence of L.intracellularis antibodies in eight major pig-producing provinces in China during 2019-2020 was conducted,and isolated L.intracellularis from infected intestines and then established an infection model of L.intracellularis in mice.Our results showed that of the 3586 serum samples,2837(79.1%,95%CI : 77.7%,80.4%)were positive for L.intracellularis antibody,and the positive rate of L.intracellularis antibody in pig farms was 100%,indicating the widespread existence of L.intracellularis in pig farms in major pig producing provinces in China.Subsequently,the L.intracellularis strain LJS19051 from the ileum samples of pig infected with PPE in China was successfully isolated and established in cell culture.Furthermore,L.intracellularis DNA and antibodies could be detected in the feces and serum samples of infected mice,respectively.Moreover,infected crypts showed typical PE lesions and L.intracellularis antigen was detected in infected mice by immunofluorescence at 28 days post inoculation.Our founding indicated that the new L.intracellularis strain LJS19051 was obtained and could successfully proliferate in ICR mice.Experiment 3.Development of a novel live attenuated vaccine for porcine proliferative enteropathy and evaluation of its immune effectIn this study,LJS19051 strain was attenuated through multiple passages in McCoy cells,and then assessed the virulence in mice of LJS19051 after passage at 10,20,40,60,and 80 in vitro.Finally,passage 80 was chosen to develop a live attenuated vaccine and to evaluate its efficacy in mice.The results demonstrated that passage 80 of LJS19051 had no effect on growth rate,had lower fecal shedding and had no histopathological lesions when compared with passage10,20,40 and 60 of LJS19051.Therefore,passage 80 was selected to develop the attenuated vaccine against L.intracellularis(PPEV)and to evaluate its efficacy in mice.Our findings revealed that PPEV could increase the growth rate,elicit humoral and cellular immune responses,resist L.intracellularis infection,decrease significantly higher numbers of L.intracellularis in feces and ileum,increase the content of s Ig A in ileum mucosa,preserve normal gut barrier function,and increase the digestive,absorptive tract enzyme activity(LPS,ɑ-AMS,AKP)activities in ileum.Altogether,the results indicated that the attenuated vaccine of PPEV could induce humoral and cellular immune responses in mice,reduce intestinal barrier function injury,improve intestinal digestion and absorption function,and provided good protection to immunized mice.These findings provided valuable data for further development of commercial L.intracellularis vaccine.Experiment 4.L.intracellularis induced incomplete autophagy to promote intracellular survival via the AMPK signaling pathwayThe current study was aimed to explore the relationship between autophagy and intracellular survival of L.intracellularis.It was found that the dot aggregation of LC3 B could be observed in McCoy cells at 3,4 and 7 days after LJS19051 infection.Western-blot results showed that LJS19051 could promote the transformation of LC3-I to LC3-II.Further studies showed that LJS19051 infection could inhibit the fusion of autophagosome and lysosome,block the degradation of P62,and inhibit the formation of intact autophagic flow.To explore the effect of autophagy on intracellular survival of L.intracellularis,we detected the level of LI in cells treated with autophagy activator RAPA,and found that the content of L.intracellularis increased significantly in autophagy-activated cells.Inhibition of autophagy by 3-MA significantly reduced the viability of L.intracellularis in McCoy cells.These results suggested that autophagy plays a protective role in L.intracellularis intracellular survival.In vivo,it was found that L.intracellularis strains(LJS19051 and LJS19052)could also induce the transformation of LC3-II and block the degradation of p62 in the ileum of pigs infected with LJS19051 and LJS19052 strain for 21 days,suggesting that LJS19051 could also induce incomplete autophagy in intestinal epithelial cells in vivo.For further exploring the mechanism of incomplete autophagy induced by L.intracellularis,we detected the expression level of marker lysosome protein LAMP1,and the results showed that the LAMP1 expression level was significantly improved after LJS19051 infection.Further studies showed that AMPK signaling pathway played an important role in incomplete autophagy induced by L.intracellularis and its intracellular survival.LJS19051 infection could activate AMPK signaling pathway,and inhibition of this pathway by Compound C could significantly reduce autophagy level and the number of L.intracellularis in cells.Previous studies have shown that heat shock protein 60(Hsp60)plays an important role in protein folding,autophagy and apoptosis,while the relationship between Hsp60 of L.intracellularis and autophagy remains unclear.In current study,we found that L.intracellularis Hsp60 protein could also induce incomplete autophagy in McCoy cells,and Hsp60 may be induce incomplete autophagy by interacting with Lrpap1 and Capzb proteins.These results further explain the direct correlation between autophagy and intracellular survival of L.intracellularis and help to clarify the pathogenic mechanism of this bacterium.Experiment 5.L.intracellularis promotes intracellular survival by inhibiting ROS productionIn this study,McCoy cells were used as L.intracellularis infection model in vitro,and ROS level was detected in McCoy cells at 1-7 days after LJS19051 infection.Compared with the control group,LJS19051 could reduce ROS production in cells from the 3 day after infection.Further studies showed that LJS19051 could increase the antioxidant level of McCoy cells,characterized by MDA content decreasing,SOD activity increasing,NRF2,HO-1 gene and protein expression levels up-regulating in infected cells.Further studies showed that LJS19051 could increase mitochondrial membrane potential,increase ATP content in cells,inhibit the release of Cyt-C from mitochondria to cytoplasm,and inhibit mitochondrial function damage through the above methods,and thus inhibit apoptosis.Furthermore,LJS19051 could also activate NRF2 signaling pathway,and inhibition of this signaling pathway could increase ROS production level in infected cells,inducing oxidative stress and mitochondrial dysfunction,promoting cell apoptosis,and reducing the number of L.intracellularis in cells.These results suggest that L.intracellularis could reduce ROS production in cells via activating NRF2 signaling pathway after infection to improve antioxidant level of cells,inhibit mitochondrial function damage and cell apoptosis,and thus promote its survival in McCoy cells.Our results will provide a new perspective for effective prevention and control of PPE.