Gram-negative bacteria of veterinary significance that are known to encode RTX (repeats in the structural toxin) toxins include Actinobacillus equuli,1 Actinobacillus pleuropneumoniae (A. pleuropneumoniae),2 Actinobacillus porcitonsillarum,3 Actinobacillus suis,4 [Pasteurella] mairii and [Actinobacillus] rossii,5 Mannheimia haemolytica (M. haemolytica),6 Moraxella bovis (M. bovis),7 [Pasteurella] aerogenes,8 and Bibersteinia (Pasteurella) trehalosi.9 Recent studies also reported indirect evidence suggesting the presence of RTX toxins in Avibacterium [Haemophilus] paragallinarum (Mena-Rojas et al., 2004) and Moraxella ovis (M. ovis).10 Recently we identified complete classical RTX operons in Moraxella bovoculi (M. bovoculi) isolated from infectious bovine keratoconjunctivitis (IBK; "pinkeye") affected dairy and beef calves in northern California, as well as in Moraxella ovis (M. ovis) originally isolated from sheep with conjunctivitis.11
Based on the results of studies with M. bovis in cattle, the presence of the M. bovis cytotoxin (RTX A protein; MbxA) is known to be necessary for pathogenesis of IBK.12 The M. haemolytica RTX toxin LktA (leukotoxin) is involved in the pathogenesis of bovine shipping fever pneumonia. In cattle, leukotoxin induced neutrophil destruction in the lung causes release of inflammatory mediators that gives rise to the lung pathology associated with shipping fever pneumonia. Instillation via endobronchial inoculation of A. pleuropneumoniae Apx toxins (RTX toxins) into porcine lungs induces lung lesions that are typical for porcine pleuropneumonia.13 A role for an ~110-kDa protein presumed to be an RTX toxin has been suggested in Haemophilus paragallinarum, the causal agent of infectious coryza.14
Most of our understanding of RTX toxin genes and their expression/activation comes from studies with uropathogenic Escherichia coli (E. coli) that express an RTX toxin HlyA. The gene encoding HlyA, hlyA, is contained within a 4 gene operon organized 5'-C-A-B-D-3'. The product of the RTX A gene is a structural toxin that requires activation by the RTX C gene product to become hemolytic.15-17 This activation occurs via fatty acylation of specific lysines in the protein.18,19 Following activation, HlyA is secreted by membrane transport proteins encoded by the B and D genes and a third protein, TolC.20,21 The regulation of transcription through the RTX operon in E.coli involves the protein RfaH and JUMPStart DNA sequences.22
Experimental Vaccines Against RTX Toxin Producing Agents
Different approaches to prevent diseases associated with RTX toxin producing bacteria have been attempted experimentally. One of the more commonly utilized methods has been the use of recombinant RTX toxin vaccines; this approach has been used in vaccines to prevent M. haemolytica associated shipping fever in cattle,23 A. pleuropneumoniae associated pleuropneumonia in swine,24 and M. bovis associated infectious bovine keratoconjunctivitis in cattle.25,26
A vaccine trial that examined the use of an M. bovis pilin-cytotoxin recombinant vaccine against IBK reported that pilin-MbxA vaccinated calves had the lowest overall cumulative proportion of ulcerations.25 Ocular culture result data suggested that vaccination with an M. bovis antigen had an effect upon the particular Moraxella species isolated from an ulcerated cornea. Moraxella bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with cytotoxin (recombinant carboxy terminus of cytotoxin [MbxA]) and pilin-MbxA.25 Moreover, the vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of non M. bovis species of Moraxella (Moraxella bovoculi) in ocular cultures.25 Studies are currently underway to evaluate the efficacy of both subunit and whole M. bovoculi and M. bovis vaccines to prevent IBK.
One recent discovery is that M. bovoculi sp. nov encodes an RTX that has 82% deduced amino acid sequence identity with the cytotoxin of M. bovis.11 Studies are currently underway to determine whether vaccinating cattle with recombinant M. bovoculi cytotoxin alone or in conjunction with recombinant M. bovis pilin-cytotoxin is effective at preventing IBK.
An alternative approach to vaccination against RTX toxin producing bacteria has focused less on the toxins, and more on additional antigens that are present in/on the bacteria. Colonization of the nasopharynx by wild-type M. haemolytica was reduced by intranasal exposure of feedlot calves to leukotoxin-deficient M. haemolytica.27 Antibody responses against outer membrane proteins of M. haemolytica were associated with resistance to experimental challenge.28,29 Vaccination of cattle with recombinant M. haemolytica outer membrane protein (OMP) PlpE enhanced resistance against experimental challenge with virulent M. haemolytica.30 The addition of this major immunogenic outer membrane lipoprotein antigen to commercial M. haemolytica vaccines enhanced the level of protection afforded by these vaccines against experimental challenge.30,31
Commercial Veterinary Vaccines Against RTX Toxin Producing Bacteria
Despite the advances in experimental vaccine development to RTX toxin producing veterinary pathogens, most commercially available vaccines are bacterin based. Currently, veterinary vaccines are manufactured against the following RTX toxin producing bacteria: A. pleuropneumoniae, M. haemolytica, and M. bovis.
Commercial vaccines against swine pleuropneumonia include bacterins (Emulsibac® APP (MVP); Pneu Pac® (Schering-Plough); Suvaxyn® RespiFend® APP (Fort Dodge); Haemo Shield® P (Novartis (Farm Animal)); Parapleuro Shield® P (Novartis (Farm Animal)); Pneu Pac®-ER (Schering-Plough); Pneu Parapac®+ER (Schering-Plough); DurVacTM Appear-HP (Durvet); Parapleuro Shield® P+BE (Novartis (Farm Animal)) and modified live cultures (Ingelvac® APP-ALC (Boehringer Ingelheim)) (Source: Compendium of Veterinary Products database, January 31, 2008).
Commercial vaccines against Mannheimia haemolytica include those that are described as bacterins (Triangle® 4 + PH-K (Fort Dodge); Triangle® 9 + PH-K (Fort Dodge); Triangle® 4 + PH/HS (Fort Dodge); Essential 2 + P (Colorado Serum); Bar Somnus 2PTM (Boehringer Ingelheim); Mannheimia haemolytica-Pasteurella multocida Bacterin (Colorado Serum)), toxoids (Pyramid® 4 + Presponse® SQ (Fort Dodge); Pyramid® 5 + Presponse® SQ (Fort Dodge); Presponse® SQ (Fort Dodge); Presponse® HM (Fort Dodge)), bacterin-toxoids (Covert® 5 + Antidote® PHM (AgriPharm); Express® 5-PHM (Boehringer Ingelheim); One Shot UltraTM 7 (Pfizer Animal Health); One Shot UltraTM 8 (Pfizer Animal Health); One Shot® (Pfizer Animal Health); Poly-Bac® B1 (Texas Vet Lab); Poly-Bac B® 3 (Texas Vet Lab); Poly-Bac B® Somnus (Texas Vet Lab); Super Poly-Bac B® Somnus (Texas Vet Lab); Super Poly-Bac B® 3 + IBR k (Texas Vet Lab); Antidote® PHM (AgriPharm); Pulmo-guardTM PH-M (Boehringer Ingelheim); Pulmo-guardTM PHM-1 (Boehringer Ingelheim); RESPISHIELDTM HM (Merial)), and avirulent live cultures (Vista® Once SQ (Intervet); Once PMH® SQ (Intervet)) (Source: Compendium of Veterinary Products database, January 31, 2008).
Commercial vaccines to prevent IBK that are directed against M. bovis include bacterins (Vision® 7 with Spur® (Intervet); 20/20 Vision® 7 with Spur® (Intervet); Alpha-7/MBTM (Boehringer Ingelheim); Alpha-7/MBTM-1 (Boehringer Ingelheim); Piliguard® Pinkeye + 7 (Schering-Plough); Cattle-VacTM Pinkeye 4 (Durvet); Maxi/Guard® Pinkeye Bacterin (Addison); Ocu-guard® MB (Boehringer Ingelheim); Ocu-guard® MB-1 (Boehringer Ingelheim); Piliguard® Pinkeye-1 (Durvet); Piliguard® Pinkeye-1 Trivalent (Schering-Plough); Pinkeye Shield® XT4 (Novartis (Farm Animal)); Resist® Pinkeye (AgriPharm)), and cell-free bacterins containing pili (Piliguard® Pinkeye TriView® (Schering-Plough)) (Source: Compendium of Veterinary Products database, January 31, 2008).
With recent advances in the development of experimental vaccines to RTX toxin-producing bacteria, it is possible that commercial vaccines will become available that can provide improved protection against RTX producing pathogens. The cost of production will likely effect marketability of these more recently developed experimental vaccines.
1. Berthoud H, et al. Vet Microbiol 2002;87:159.
2. Chang YF, et al. Dna 1989;8:635.
3. Kuhnert P, et al. Vet Microbiol 2005;107:225.
4. Burrows LL, et al. Infect Immun 1992;60:2166.
5. Mayor D, et al. Vet Microbiol 2006;116(1-3):194.
6. Lo RY, et al. Infect Immun 1987;55:1987.
7. Angelos JA, et al. AJVR 2001;62:1222.
8. Kuhnert P, et al. Infect Immun 2000;68:6.
9. Fisher MA, et al. AJVR 1999;60:1402.
10. Cerny HE, et al. J Clin Microbiol 2006;44:772.
11. Angelos JA, et al. Vet Microbiol 2007;125:73.
12. Beard MK, et al. Vet Microbiol 1994;42:15.
13. Kamp EM, et al. Infect Immun 1997;65:4350.
14. Mena-Rojas E, et al. FEMS Microbiol Lett 2004;232:83.
15. Hardie KR, et al. Mol Microbiol 1991;5:1669.
16. Hughes C, et al. FEMS Microbiol Immunol 1992;5:37.
17. Issartel JP, et al. Nature 1991;351:759.
18. Stanley P, et al. Mol Microbiol 1996;20:813.
19. Stanley P, et al. Microbiol Mol Biol Rev 1998;62:309.
20. Koronakis V, et al. FEMS Microbiol Immunol 1992;5:45.
21. Wandersman C, et al. Proc Natl Acad SciUSA 1990;87:4776.
22. Leeds JA, et al. J Bacteriol 1997;179:3519.
23. Conlon JA, et al. Infect Immun 1991;59:587.
24. Haga Y, et al. J Vet Med Sci 1997;59:115.
25. Angelos JA, et al. Vet Microbiol 2007;125:274.
26. Angelos JA, et al. Vaccine 2004;23:537.
27. Frank GH, et al. AJVR 2003;64:580.
28. Confer AW, et al. Vet Immunol Immunopathol 1995;47:101.
29. Morton RJ, et al. AJVR 1995;56:875.
30. Confer AW, et al. Vaccine 2003;21:2821.
31. Confer AW, et al. Vaccine 2006;24:2248.