Actinobacillus pleuropneumoniae (A. pleuropneumoniae), the causative agent of porcine contagious pleuropneumonia (PCP), is a significant pathogen of the world pig industry, vaccination is potentially an effective tool for the prevention of PCP. The purpose of present study was to enhance the immunogenicity of A. pleuropneumoniae live vaccine strain HB04C- (serovar 7), which was unable to express ApxIA, and to develop effective multivalent vaccines for the respiratory pathogens based on the attenuated A. pleuropneumoniae. We introduced a shuttle vector containing intact apxIA gene into HB04C-, generating HB04C2, an A. pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA. Then we investigated the biological characteristics of HB04C2. We found that the shuttle vector expressing ApxIA was stable in HB04C2, and the growth ability of HB04C2 was not affected by the shuttle vector. We observed that HB04C2 elicited detectable antibodies against ApxIA and ApxIIA when it was administrated intratracheally as a live vaccine in pigs, and all immunized pigs were protected from heterologous virulent A. pleuropneumoniae (serovar 1) challenge. In conclusion, we demonstrated that A. pleuropneumoniae live vaccine could be used as a vector for expression of heterologous antigens.
Actinobacillus Infections ; prevention & control ; veterinary ; Actinobacillus pleuropneumoniae ; classification ; immunology ; Animals ; Bacterial Proteins ; biosynthesis ; genetics ; Bacterial Vaccines ; biosynthesis ; immunology ; Hemolysin Proteins ; biosynthesis ; genetics ; Pleuropneumonia ; microbiology ; prevention & control ; Swine ; Swine Diseases ; microbiology ; prevention & control ; Vaccines, Attenuated ; biosynthesis ; immunology

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.
Actinobacillus Infections/microbiology/*prevention & control ; Actinobacillus pleuropneumoniae ; Animals ; Antigens, Bacterial/immunology ; Bacterial Proteins/*immunology ; Bacterial Vaccines/*immunology ; Cholera Toxin/*chemistry ; Female ; Hemolysin Proteins/*immunology ; Immunization, Secondary ; Mice ; Mice, Inbred ICR ; Plants, Genetically Modified ; Zea mays/*genetics

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; metabolism ; Animals ; Bacterial Proteins ; genetics ; immunology ; Bacterial Vaccines ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Hemolysin Proteins ; genetics ; immunology ; Immunization ; Male ; Mice ; Random Allocation ; Recombinant Proteins ; genetics ; immunology ; Toxicity Tests, Acute

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.
Actinobacillus Infections/prevention & control ; Actinobacillus pleuropneumoniae/genetics/*immunology ; Animals ; Antibodies, Bacterial/blood ; Bacterial Proteins/analysis/*immunology ; Cytokines/analysis/blood ; Disease Models, Animal ; Female ; Hemolysin Proteins/analysis/*immunology ; Immunoglobulin A/blood/immunology ; Intestines/immunology ; Lung/cytology/immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/*immunology ; Saccharomyces cerevisiae/*genetics/immunology ; Survival Analysis ; Time Factors ; Vaccination ; Vaccines, Synthetic/administration & dosage/*immunology