Study On The Pathogenicity Of Actinobacillus Pleuropneumoniae ApxIV And Gene Deleted Attenuated Vaccine

Actinobacillus pleuropneumoniae (A. pleuropneumoniae; APP) is the etiological agent of porcine contagious pleuropneumonia (PCP), a severe and often fatal respiratory disease of swine, which is associated with significant economic losses in industrialized pigs production worldwide.The virulence of A. pleuropneumoniae is associated with several factors which involved in the pathogenesis of A. pleuropneumoniae of PCP. However, the virulence of A. pleuropneumoniae has been found to be strongly but not exclusively correlated with the presence of Apx toxins. Four different Apx toxins have been found to be produced by the 15 serotypes: Apxâ… , Apxâ…¡, Apxâ…¢and Apxâ…£. The Apx toxins Apxâ… , Apxâ…¡and Apxâ…¢have been demonstrated to play a predominant role in pathogenesis, and are strongly immunogenic and involved in the induction of protective immunity. However, whether Apxâ…£contributes to the pathogenesis of A. pleuropneumoniae infection is still unknown. To assess the pathogenicity of Apxâ…£and development of a potential marker vaccine that could be used to differentiate vaccinated animals from infected animals, an Apxâ…£A inactivated mutant QDS01 was constructed based on a single apxâ…¡C gene deletion mutant HB04C^-, and the apxâ…£gene complemention strain was also constructed based on an APP-Escherichia. coli shuttle vector pJFF224-XN. Experimental infection in mice and piglets were carried out to determine the role of the Apxâ…£toxin in pathogenesis, the immunogenicity of QDS01 was assessed in this study. Below is more detailed information on this study:1. Construction of recombination suicide plasmidsA 2745 bp fragment of the N-terminal of apxâ…£A was amplified from the genomic DNA of A. pleuropneumoniae single mutant HB04C^- according to the reference sequence stated in the Genbank (assession no. AF021919), and cloned into Salâ… -Notâ… sites of pBluescript SK(+), resulting in plasmid pSK-â…£A. Then, the 605 bp fragment from apxâ…£A gene in pSK-â…£A was digested by BamHâ… -Smaâ… and removed, and the stick end of BamHI was blunt-ended by Klenow fragment and ligated again, resulting in plasmid pSKΔⅣA. The transconjugation plasmid pEΔⅣA was constructed by ligation of the Salâ… -Notâ… fragment containing the truncated N-terminal of apxâ…£A into pEMOC2.2. Construction of apxâ…£deleted mutant and complementation strainPlasmid pEΔⅣA was transformed into E. coliβ2155 to form donor cells. Donor cells and acceptor cells (A. pleuropneumoniae single deleted mutant HB04C^-) were mixed for 6 h. Then bacteria were plated onto chloramphenicol selective TSA, the Cm^R colonies were picked and inoculated into TSB without any antibiotics. Bacteria were pelleted, plated on sucrose-containing plates and incubated overnight. Sucrose-resistant colonies were tested for chloramphenicol resistence. Finally, the sucrose-resistant (Suc^R) and chloramphenicol sensitive (Cm^S) colonies were further confirmed by PCR. The colonies with the correct PCR profile were confirmed by Southern blot and sequencing assays. The apxâ…¡C/apxâ…£A double deleted mutant was named QDS01. For the trans-complementation study, the intact apxâ…£gene was cloned from A. pleuropneumoniae field isolate HB04 with primers P7 and P8, and ligated into E. coli-APP shuttle vector pJFF224-XN, resulted in complementation plasmid pVC3. pVC3 was introduced into QDS01 by electroporation. A chloramphenicol-resistant (Cm^R) transformant QA01 was selected. Heredity stability of the shuttle vector and expression of Apxâ…£in QA01 were evaluated.3. Characterization of the APP mutantsHeredity stability of these A. pleuropneumoniae mutants were confirmed by series passages and PCR. In order to confirm whether the deletion in apxâ…£gene affected the growth ability of A. pleuropneumoniae, the growth curves were drawn, result suggested no difference between parent strain and mutants. The degree of attenuation of the A. pleuropneumoniae double deleted mutant in mice was investigated by comparing with the single mutant HB04C^- of A. pleuropneumoniae using a murine infection model. Data indicated that deletion in apxâ…£caused a 3-fold attenuation of A. pleuropneumoniae.4. Virulence in pigsThe virulence of the mutant QDS01, HB04C^-, QA01 and w.t strain HB04 in pigs was assessed by an intratracheally (i.t.) infection model. Pigs were monitored carefully, clinical signs and lung lesion score were calculated according to admitted methods. Blood samples were taken, blood biochemical parameters such as blood glucose (BGlu), glutamic-oxal(o)acetic transaminase (GOT), glutamate-pyruvate transaminase (GPT) were determined. The results indicated that deletion of apxâ…£A led to the attenuation of the virulence of A. pleuropneumoniae, and virulence could be restored in QDS01 by complementation. Therefore, the data confirmed that Apxâ…£was essential for expression of the full virulence of A. pleuropneumoniae.5. Cloning, expressing of Apxâ…£AM and tentative development of differentiatail ELISATo develop an ELISA method for the differential diagnosis, the truncated fragment (apxâ…£AM), which was deleted in the apxâ…£A gene of the double mutant strain QDS01, was cloned from HB04C^- and ligated into the prokaryotic expression vector pGEX-KG, the recombinant protein glutathione 5-transferase(GST)-Apxâ…£AM was produced in E. coli strain BL21(DE3) containing the recombinant plasmids pKG-Apxâ…£AM, and purified using a glutathione-Sepharose 4B columns. The immunogenic activity of the fusion protein was confirmed by Western blot. Appropriate coating concentration and serum dilution ratio were determined by squared titration. The cut off threshold was determined by detection of A. pleuropneumoniae negative serums.6. Protection in miceForty-eight 6-week-old Balb/C mice were randomly divided into six experimental groups of eight mice to evaluate the efficacy of the apxâ…¡C and apxâ…£A double-deleted mutant QDS01 as attenuated live vaccine. Group 1 and 4 were vaccinated twice intraperitoneally (i.p.) with 1.0×10⁷ CFU of the QDS01 in 200μL TSB. As a positive control, Group 2 and 5 were vaccinated twice intraperitoneally with 1.0×10⁷ CFU of apxâ…¡C single-deleted mutant HB04C^- in 200μL TSB. Non-vaccinated control mice were inoculated with TSB. Two weeks after the second immunization, group 1, 2 and 3 were challenged intraperitoneally with 10×LD₅₀ (1×10⁷ CFU) of A. pleuropneumoniae WF83 (serovar 7) in 200μL TSB while groups 4, 5 and 6 were challenged with 10×LD₅₀ (1.8×10⁶ CFU) of A. pleuropneumoniae 4074 (serovar 1). The clinical signs were observed and number of the dead mice was recorded for 5 days post-challenge. Both QDS01 and HB04C^- have a protective efficiency of 100% against challenge of WF83, and 75% against challenge of 4074.Mice were blooded at day 0, 14 and 28, serum samples were detected by Apxâ…¡A, Apxâ…£A and Apxâ…£AM ELISA. Results suggested that QDS01 and HB04C^- can elicit antibodies against Apxâ…¡A and Apxâ…£A, but only HB04C^- immunized mice were positive in the Apxâ…£AM-ELISA, indicating that Apxâ…£AM-ELISA can be used to differentiate apxâ…£deleted mutant vaccinated animals from wild type strain infected animals.7. Protection in pigsFourteen 6-week-old pigs were randomly divided into three experimental groups. The five pigs in group 1 were vaccinated twice intratracheally (i.t.) with 1.3×10⁹ CFU of QDS01 in 5 mL TSB while the five pigs of group 2 were injected twice intratracheally (i.t) with the same dose of HB04C^- at an interval of 3 weeks. Group 3 comprising four pigs served as a negative control group injected with 5 mL TSB.Two weeks after the booster immunization, all ten pigs in groups 1 and 2 and three pigs in group 3 were challenged intratracheally with 5.0×10⁷ CFU of a heterologous A. pleuropneumoniae serovar 1 (4074) strain while the remaining pig in group 3 served as a control. The pigs were monitored daily for clinical signs of pleuropneumonia for 7 days after challenge. At day 7 post-challenge, all surviving pigs and the unchallenged control pig were euthanized and the scores of lung lesions were recorded. Bloods samples were collected via anterior vena cava venipuncture and examined by Apxâ…¡A, Apxâ…£A and Apxâ…£AM ELISA, results indicated that both QDS01 and HB04C^- vaccinated pigs elicited antibodies against Apxâ…¡A and Apxâ…£A, however, only HB04C^- immunized pigs were positive in the Apxâ…£AM-ELISA, suggesting that Apxâ…£AM-ELISA can be used in the differential diagnosis between QDS01 vaccinated animals and wild type strain infected animals.