A family of toxins, referred to as the Repeats in Toxins (RTX), have been associated with infections caused by gram-negative organisms including E. coli, Proteus, Morganella, Pasteurelia, Actinobacillus, and Bordetella spp. (Menestrina G., et al, Toxicology 87 (1994) 249-267, and references referred to therein). The prototype toxin is Escherichia coli .alpha.-hemolysin which is a primary virulence factor of urinary infections, peritonitis, meningitis, and septicemia caused by virulent strains of E. coli. The RTX toxins share several common features which are discussed in the review by Welch, Mol. Microbiol. 5, 521, 1991.
The RTX toxins of Actinobacillus pleuropneumoniae play an important role in infections cause by strains of Actinobacillus pleuropneumoniae (J. Frey et al., in Bacterial Protein Toxins, Zbl.Bakt. Suppl. 24, Freer et al. (Eds)., Gustav Fischer, Stuttgart, Jena, N.Y., 1994). Actinobacillus pleuropneumoniae is the agent responsible for swine pleuropneumonia, a severe contagious disease which causes great economic losses (J. I. MacInnes and N. L Smart, Actinobacillus and Haemophilus in Pathogenesis of Bacterial Infections in Animals, 2nd ed. C. L. Gyles and C. O. Thoen, Iowa State University Press, Ames, Chapter 16). A. pleuropneumoniae is a gram-negative bacterium of the family Pasteurellaceae J. I. MacInnes and N. L Smart, supra). Twelve serotypes of the organism have been described by serotyping based on capsular polysaccharides (Nicolet, J. Can. Vet. J. 29:578-580, 1988 and Nielsen, R., cta. Vet. Scand. 27:453-455, 1986). Differences in virulence have been observed among the serotypes and a number of virulence factors have been considered responsible for the differences. These factors include capsular polysaccharides, liposaccharides, exotoxins and adhesion factors (J. I. MacInnes and N. L Smart, supra).
Two hemolytic RTX toxins, ApxI and ApXII, and one non-hemolytic RTX toxin, ApxIII have been identified in different A. pleuropneumoniae serotype strains J. Frey et al., supra) The toxins are secreted into the growth medium by A. pleuropneumoniae, they have different molecular masses, and they can be distinguished serologically by polyclonal and monoclonal antibodies (J. Frey et al., supra). It has been reported that A. pleuropneumoniae serotypes which produce a combination of two Apx toxins are more virulent than serotypes with one toxin alone (J. Frey et al., supra). The serotypes producing ApxI and ApxII are the most virulent (J. Frey et al., supra). No A. pleuropneumoniae strains have been identified which produce all three toxins J. Frey et al., supra).
RTX toxins have also been reported to be secreted in other bacterial strains including Bordetella pertussis (Betsou et al.1993 and CA 1,189,790) E. coli, Listeria, Moraxella, Pseudomonas, Staphylococcus, Vibrio (Nakai et al. 1983) and Neisseria meningitidis (Thompson et al.1993).
Vaccines which have been developed for preventing infections by A. pleuropneumoniae have been based on whole live cells, attenuated cells, lysates, culture supernatants, and extracts of A. pleuropneumoniae. Canadian Patent 1,189,790 describes a vaccine containing A. pleuropneumoniae cells, cell fragments, extracts and/or metabolites, and an adjuvant derived from Bordetella pertussis. Other proposed vaccines contain: (a) inactivated toxin of serotype I and optionally a toxin of another serotype (EP-A-420.743); (b) whole cell or sonicated whole cell components of a virulent strain obtained after passage in a host (WO9321951-A); (c) transferring binding protein, cytolysin and/or APP4 (WO9308283-A); (d) at least one immunogenic part of at least one cytolytic A. pleuropneumoniae protein prepared by recombinant DNA methods (Canadian Patent Application 2,045,950); (e) an iron-repressible outer membrane protein of molecular weight 105 kD (Canadian Patent Application 2,045,950); (f) outer membrane proteins having a major dominant antigenic protein component of 42 kD and a haemolysin of 105 kD and/or macrophage toxin of 120 kD (Canadian Patent Application No. 2,040,544); (g) inactivated toxin of serotype 1 of A. pleuropneumoniae which is obtained from culture supernatant (EP-420743); (h) extracellular proteinaceous materials from the culture medium of strains of at least two different serotypes of A. pleuropneumoniae. (EP-420743); or, (i) hemolysin antigen produced by recombinant techniques. Many of the known vaccines have limited effectiveness particularly against infection by heterologous serotypes.
Conventional formalin-killed bacterins provide limited protection against challenge with homologous serotypes of A. pleuropneumoniae and poor protection against heterologous serotypes (Neilson, 1984). In contrast, convalescent pigs are completely protected from challenge with homologous serotypes and significantly protected from disease from heterologous serotypes (Neilson, 1984).
Many of the antigenic components of A. pleuropneumoniae which have been investigated as potential vaccine candidates all fall short of affording complete cross-protection. Devenish et al. (1990) demonstrated homologous protection from challenge when gel-purified ApxI and ApxII cytolysins were used as a vaccine. However, others have reported less than complete protection with partially purified toxin vaccines, although the protection afforded is increased when these vaccines are enriched with other cellular components such as outer membrane proteins (OMPs) (Van den Bosch et al., 1992).
Live attenuated vaccines made from strains of A. pleuropneumoniae lacking in capsule production have been reported to protect against homologous challenge (Rosendal et al., 1990; Inzana et al., 1988). Results have not been reported with heterologous challenge. Live strains deficient in RTX toxin production have been reported to afford no protection against disease (Inzana et al., 1991). Jansen (1994) has concluded that both opsonization of A. pleuropneumoniae to enhance phagocytosis and neutralization of the RTX toxins are necessary for immune protection against disease.