Construction, Characterization And Immunogencity Of A △apxIC/△apxIIC Double Mutant Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia (PCP) caused by Actinobacilluspleuropneumoniae (APP) is a severe, contagious pulmonary disease of pigs that causesimportant economic losses in industrialized pigs production worldwide.The first observation of this disease was made by Pattison et al. (1957), now occursin most pig-keeping countries. Immunization is the main method to prevent and control it.The current dominant commercial vaccines are still killed whole cell bacterins andsubunit vaccines, which generally reduce the mortality of APP infection. However thosevaccines frequently failed to prevent severer morbidity and economic losses, due to thechronic effects of the disease, on growth rate and feed efficiency.In contrast, natural and experimental infection can induce protection against anyheterologous serotype. The attenuated live vaccines could re-present protective antigensand stimulate a lasting immune response that may be more efficacious in preventing thedisease. Therefore, studies of A. pleuropneumoniae vaccines tend to focus on liveattenuated vaccines constructed by inactivating virulence-associated genes. The methodsdescribed previously for the construction of mutant strains of A. pleuropneuminiae havesome disadvantage. For example, mutagenesis using a chemical or a transposon mediatedapproaches present a random approach and allow for the selection of mutants with adistinct phenotype only; muttions not resulting in a distinct phenotype can not beconstructed by these means. All these mutants, which are achieved by systems allowingtargeted mutagenesis by homologous recombination, however, carry a permanentantibiotic resistance marker and, therefore, are not suitable as vaccine strains. Because theApx toxins are confirmed to be particularly important in the virulence and induction ofcross-protective immunity, a live attenuated vaccine strain with avirulent RTX toxinsmust be the optimal candidate for an A. pleuropneumoniae vaccine.Based on above considerations, we developed an attenuated A. pleuropneumoniaeserovar 1 strain, SLW03, which expressed and secreted inactive Apxâ… A and Apxâ…¡A, bydeleting the apxâ… C and apxâ…¡C genes of the SLW01 strain in this study. A potentialvaccine candidate was produced without an antibiotic resistance marker by a sucrosecounter-selection strategy. Actinobacillus pleuropneumoniae serovar 1 is the predominantserovar in China, and a strain that produces Apxâ… and Apxâ…¡toxin was chosen. Here wedescribe the construction and characterization of the double mutant, SLW03, testing of itsvirulence in mice, and demonstration of its cross-protective efficacy against challengewith virulent A. pleuropneumoniae in pigs. The main research was described as follows:1. Cloning of genes and construction of recombination suicide plasmids According to the GenBank sequences, the two flanks of both apxâ… C and apxâ…¡Cgenes were amplified and cloned from SLW01 genome. Those genes were highlyconserved compared with the sequences published. The upstream and downstream ofapxâ… C and apxâ…¡C gene were respectively subcloned into suicide plasmid pEMOC2,which contained a sucrose sensitive (sacB) gene. The recombination suicide plasmidswere designated as pEM△ⅠC and pEM△ⅡC which contained 385bp-deleted apxâ… C and340 bp-deleted apxâ…¡C respectively.2. Transconjugation and identification of the mutants SLW02 and SLW03The single mutant strain SLW02, from which only apxâ… C was deleted, wasconstructed first. The E. coil donor strainβ2155 transformed with plasmid, pEM△ⅠC, wasconjugated with the recipient, SLW01. After transconjugation, Chloramphenicol-resistant(Cm^R) transconjugants were analyzed for the presence of a first crossover event by PCR.This first step selected for clones in which the whole plasmid had been incorporated intothe recipient chromosome. Colonies with the correct PCR profile were incubated in TNB.Aliquots were then plated on to TNA plates containing 10% (w/v) sucrose. Afterincubation for 24 h, sucrose-resistant (Sue^R) colonies exhibiting a non-mucoid phenotypewere tested for the chloramphenicol sensitivity (Cm^s) phenotype, which was indicative ofloss of plasmid vector sequences. Following this, Sue^R and Cm^s colonies were identifiedusing PCR to determine the presence of the second crossover. The double mutant strainSLW03 was constructed using the same method based on the single mutant strain SLW02.The single mutant strain SLW02 was used as the recipient strain, and the E. coilβ2155transformed with plasmid pEM△ⅡC was used as the donor strain.3. Characterization of the mutantsTo investigate hemolytic activity, colonies of the mutant or parent strains wereinoculated on to TSA plates containing fresh, defibrinated sheep erythrocytes and 10μg/ml NAD. Plates were incubated for 24 h at 37℃and hemolysis was assessed visually.Clear zones around the colony indicated hemolysis activity. After 24 h culture, the parentstrain SLW01 showed strong hemolytic activity in an approximately 2-3 mm zone. Thesingle mutant strain, SLW02, showed weak hemolytic activity, giving a 0.5-1 mm zone,but the double mutant strain SLW03 produced no hemolysis.The expression of Apxâ… and Apxâ…¡proteins in the double mutant strain SLW03 wasexamined by two separate Westem blot analyses (using the anti-Apxâ… A monoclonalantibody and using the anti-Apxâ…¡A monoclonal antibody) with TE buffer as the negativecontrol. In the Western blot using the anti-ApxlA monoclonal antibody, a unique proteinband with a molecular mass of 110 kDa was shown in both the supematant of the double mutant strain SLW03 and the supernatant of the parent strain SLW01. In the Western blotusing the anti-Apxâ…¡A monoclonal antibody, a band of the expected 105 kDa was seen inthe supernatants of both the double mutant strain SLW03 and the parent strain SLW01.The results of the Western blot analysis indicate that the double mutant strain SLW03 isstill capable of secreting Apxâ… and Apxâ…¡after deletion of apxâ… C and apxâ…¡C, as is theparent strain, SLW01.For growth of SLW03, cultures of the parent strain SLW01 and mutant strain SLW03were grown with shaking at 37℃in TSB (supplemented with NAD and sterile bovineserum) overnight. The cultures were sub-inoculated into fresh supplemented TSB at a1:1,000 dilution. The OD₆₀₀ of the bacterial cultures was determined at intervals of 1 h.No obvious difference was observed in the in vitro growth curves of SLW01 and SLW03,indicating that deletion of the apxâ… C and apxâ…¡C genes had no significant influence on thegrowth ofA. pleuropneumoniae SLW01.4. The immune and protective assay of the double mutant strain SLW03 in miceEighty female Balb/C mice were divided into three big groups. Group 1 of 32 micewere vaccinated 2x10⁸ CFU SLW03. Group 2 of 32 mice were injected with 0.2 mLkilled vaccine. Group 3 of 16 mice were injected with TSB as control. Two weeks afterthe first vaccination, mice were vaccinated again at the same dose. Two weeks otter thebooster immunization, mice were challenged with difference dose of SLW01 (serovar 1)and XFNZ (serovar 7).Mice in control group were all death within 12 h after challenge. Mice in bothSLW03 vaccinated group and killed cell vaccinated group were gained 100% protectionagainst the low dose challenge. With the hige dose challeng, SLW01 still offered mice100% protection against homologous serovar and 75% protection against heterogenousserovar. While the killed cell vaccine only offered respectively 25%and 37.5%protection against challenge with homologous and heterogenous serover.Sera from unvaccinated control mice did not display ELISA or IHA antibodiesthroughout the experiment. A significant difference was observed between the SLW03vaccinated group and the control group (P＜0.01). There was no significant differences inELISA-Apxâ… or -Apxâ…¡titers were observed between killed cell vaccinated group and thecontrol group (P＞0.05), the antibody level in the IN immunization group was higher thanthat in the IM group on day 28 and the difference was statistically significant (P＜0.05).The results suggest that the attenuated live vaccine SLW03 is better than the killed cellvaccine in mice.5. The immune and protective assay of the double mutant strain SLW03 in pigs Thirty-six 6-week-old pigs were randomly divided into six experimental groups ofsix. Groups 1 and 4 were vaccinated intramuscularly (IM) twice with 1x10⁹ CFU ofSLW03 in 1 ml TSB. Groups 2 and 5 were inoculated twice intranasally (IN) with thesame dose of SLW03 at an interval of 2 weeks. Groups 3 and 6 served as unvaccinatedcontrol groups inoculated with TSB. Two weeks after the booster immunization, groups 1,2 and 3 were challenged intratracheally with SLW01 (serovar 1). Groups 4, 5 and 6 werechallenged with SHB09 (serovar 9). Groups 3 and 6 therefore served asunvaccinated/challenged control groups.One hundred percent (6/6) protection against challenge with homologous (SLW01)and heterologous (SHB09) A. pleuropneumoniae was conferred on the pigs immunizedwith SLW03 by the IM and IN routines, compared with no protection in unvaccinatedpigs. After challenge, pigs in the IM and IN vaccinated groups showed slight depressionand decreased appetite for 2 days. These signs were not observed after the third daypost-challenge. All pigs in unvaccinated groups 3 and 6, challenged with serovars 1 and 9,respectively, showed severe respiratory distress and were euthanized (except one in group6) within 72 h after challenge. Postmortem examination showed severe lung lesions,pleural effusion and adhesive pleuritis in the unvaccinated/challenged pigs. Both the IMvaccinated/challenged group (1 and 4) and the IN vaccinated/challenged groupindividually showed significantly lower clinical scores compared with theunvaccinated/challenged group (P＜0.01). However, there was no significant differencebetween the IM vaccinated/challenged group and the IN vaccinated/challenged group(P＞0.05).All unvaccinated pigs challenged with serovars 1 and 9 showed adhesive pleuritisand focal lung abscesses. In group 1, one of the six pigs showed disseminated pulmonaryhemorrhage, and two pigs showed localized hemorrhagic foci in the lungs; the other threepigs had no detectable lung lesions. In group 2, only one pig showed localized pulmonaryhemorrhage; the other five had no observable lung lesions. In group 4, two of six pigswere detected disseminated pulmonary hemorrhage, and three pigs showed localizedhemorrhagic foci in the lungs. In group 5, two pigs were observed localized pulmonaryhemorrhage; the others had no detectable lung lesions. The lung lesion scores in the IMand IN vaccinated/challenged groups (groups 1, 4 and groups 2, 5) were significantlylower than those of the unvaccinated/challenge pigs (groups 3 and 6) (P＜0.01). The lunglesion scores of the IN vaccinated/challenged groups, 2 and 5, was lower than those of theIM vaccinated/challenge groups, 1 and 4, and the difference was statistically significant(P＜0.05). The result indicates that IN immunization is more effective than the IM route. A large increase in IHA and ELISA antibody titers in pigs given IM or INimmunization was observed alter the initial (day 14) or second (day 28) vaccination withlive vaccine SLW03. A significant difference was observed between IM or IN vaccinatedgroups and unvaccinated groups (P＜0.01). Although no significant differences inELISA-Apxâ… or -Apxâ…¡titers were observed between IN-and IM-immunized groups onday 14 (P＞0.05), the antibody level in the IN immunization group was higher than that inthe IM group on day 28 and the difference was statistically significant (P＜0.05). Inaddition, no significant difference in IHA titers was observed between the IN and IMimmunization groups (P＞0.05) following either the initial or second vaccination. Serafrom unvaccinated control pigs did not display ELISA or IHA antibodies throughout theexperiment.