Establishment Of IPMA Antigen Detection Method And Evaluation Of The Immunoprotective Efficacy Of Omp2 Protein Against Lawsonia Intracellularis

Lawsonia intracellularis is an enteric pathogenic bacteria that cause porcine proliferative enteropathy(PPE).L.intracellularis colonizes the enterocytes in the distal ileum,and can sometimes extend to the caecum;infection leads to delayed animal growth and reduced feed conversion ratios.The disease has seriously threatened the healthy development of the pig industries worldwide.In recent years,subclinical proliferative enteritis(PE)is prevalent in pig farms in China,which has become one of the intestinal diseases causing serious economic losses in intensive pig farming,but the mortality rate is not high.In addition,in pig production,antibiotics or chemical drugs are usually added to the feed to prevent bacterial infectious diseases,which also has a certain effect on the control of the disease.Therefore,PPE has received insufficient attention.Since 2020,China has implemented the policy of restricting and prohibiting swine hypertrophic enteritis in animal husbandry production.The harm of PPE has begun to receive widespread attention and it is urgent to research new prevention and control measures.1.Preparation of monoclonal antibodies of Sod C against Lawsonia intracellularisIn this study,the genome of the commercial live attenuated vaccine(Boehringer Ingelheim Vetmedica,Germany)was extracted as the template.The sodc gene was amplified by PCR and cloned into the prokaryotic expression vector p Gex-6p-1,and the recombinant plasmid p Gex-6p-1-sodc was confirmed to be constructed successfully.The recombinant protein Sod C was expressed by induction,and its reactivity was detected by Western Blot(WB).On this basis,BALB/c mice aged 6 weeks were immunized with the recombinant protein as immunogen.Two strains of positive hybridoma cells,named 1D6and 1F7 respectively,were screened by lymphocytic hybridoma technique,and ascites were prepared.The ascites titers of the two monoclonal antibodies were both 1:1024000 by ELISA.The subclass identification results of antibodies showed the subclass of 1D6 was Ig A/κ,and the subclass of 1F7 was Ig G3/κ.The identification of specificity showed that the two m Abs had good specificity in the reaction with L.intracellularis.2.Establishment and application of an immunoperoxidase monolayer assay(IPMA)for the detection of Lawsonia intracellularisIn this study,an immunoperoxidase monolayer assay(IPMA)method for detecting L.intracellularis was preliminarily established with monoclonal antibody 1D6.IPMA reaction conditions were optimized as follows:Mc Coy cells were inoculated with L.intracellularis for 24 h,the dilution ratio of primary antibody and HRP-goat anti-mouse Ig G secondary antibody were 1:800 and 1:2500 respectively,and incubation time was 45 min and 1 h respectively,IPMA detection effect was the best.The specificity and sensitivity test results showed that S.Cholerasuis,porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine circovirus 2(PCV2),and pseudorabies virus(PRV)were all negative,and the minimum detection limit of this method was 1×10~3L.intracellularis.The optimized IPMA method was used to detect the ileum tissue samples from pig farms in the surrounding areas of Jiangsu Province.A total of 92 positive samples were detected from 146 samples of ileal tissues.The positive rates of 3 pig farms in different areas were 62.3%,56.8%,and 46.3%respectively,and the overall positive rate was 63.0%.87 positive samples were detected by the PCR method.The positive coincidence rate of the two methods was 94.6%.The above results further prove that this method has a good clinical detection effect and can be used for the isolation and identification of L.intracellularis as well as epidemiological investigation.3.Evaluation of the immunoprotective efficacy of Omp2 protein against Lawsonia intracellularisIn order to develop a subunit vaccine against L.intracellularis,this study selected the Omp2 protein as a subunit vaccine antigen candidate from the five proteins(Hsp60,Omp2,Sod C,Lsa A and Slp)against L.intracellularis through a mouse immune protection test.A total of 32 6-week-old specific-pathogen-free(SPF)BALB/c mice were allocated into 4groups:PBS blank control group,L.intracellularis challenge control group,L.intracellularis attenuated live vaccine immunization control group,Omp2 subunit vaccine experimental group.The results of the challenge protection test showed that:mice immunized with Omp2 showed lower fecal shedding(p<0.001),fewer microscopic lesions,and colonization of the ileum compared with the L.intracellularis challenge control group.H&E staining results showed that in the challenge control group,7 mice(87.5%)were rated as L.intracellularis positive,in the attenuated vaccine control group,2 mice(25%)were rated as positive,and in the Omp2 group,1 mouse(12.5%)was rated as positive.Additionally,Omp2 protein can induce stronger serum Ig G(p<0.05)and IFN-γresponses after immunization,but no Ig G antibody was detected in the vaccine group before challenge.Compared with L.intracellularis challenge control group,the m RNA levels of zonula occludens-1(ZO-1)and Occludin(p<0.05)in intestinal mucosa of mice in Omp2test group were up-regulated(p<0.05),as well as the number of intestinal intraepithelial lymphocytes(IELS)(p<0.05).There was no significant difference in the m RNA levels of ZO-1 and Occludin between vaccine group and challenge control group.Compared with PBS control group,the levels of s Ig A in ileal mucosa in the remaining three treatment groups were significantly increased.The activity of LPS,α-AMS,and AKP in the Omp2group were significantly increased compared with the challenge control group(p<0.05).All the above-mentioned that the Omp2 protein can induce effective humoral immunity,decreased fecal shedding and bacteria colonization of the ileum,alleviated histopathological lesions,relieving dyspepsia caused by L.intracellularis infection,and provide better immune protection for immunized mice.On the one hand,the preparation of Sod C monoclonal antibody against L.intracellularis in this study was prepared,and the IPMA antigen detection method for L.intracellularis was successfully established,which has strong specificity,high sensitivity,and good repeatability.146 samples from abattoirs from three pig farms in Jiangsu province were tested using this method,which showed that L.intracellularis infection existed in the three pig farms.The results indicated that the method can be used for isolation and identification of L.intracellularis in the lab and detection of clinical samples.On the other hand,a new vaccine candidate against L.intracellularis was developed in this work,and the immunoprotective efficacy was evaluated by humoral immunity and cell-mediated immunity,fecal shedding,histopathological lesion mucosal immune barrier function,and capacity of digestibility and absorption.The novel subunit vaccine lays a good foundation for the prevention and control of PPE.