Progress 12/15/01 to 12/14/06
Outputs
OUTPUTS: We evaluated whether transposon-generated mutants of A. pleuropneumoniae (Apl) UWP36N, a strain that only produces ApxI, are reduced in virulence or can prevent disease in pigs challenged with a virulent strain. These mutants produce altered levels of ApxI, an RTX hemolysin that is responsible for much of the damage to lung tissue and mortality associated porcine pleuropneumonia. We compared the ability of a hlyX mutant, which hyperproduces ApxI, to cause disease in an intranasal infection model with its immediate parent. 8 week old pigs were administered 10 9 CFU of either strain. 1 of 5 pigs in the hlyX group and 3 of 7 pigs in the parental groups showed labored breathing and/or cough. Hemorrhagic lesions were observed on the lungs in both groups. Fibrinous exudate and adhesions were only seen in pigs infected with the parental strain suggesting that hlyX controls expression of an unknown factor that affects the production of a fibrinous exudate. Approx. 10 7 CFU of each
strain were recovered per 100 ml of lung lavage fluid from pigs with significant hemorrhagic lesions. A mutation in the hlyX gene does not significantly reduce the disease causing ability of Apl strains that only produce ApxI and that HlyX may affect other genes that specifically elicit the accumulation of a fibrinous exudate. We evaluated whether mutations in napA which encodes a periplasmic nitoreductase or fur which encodes an iron-responsive transcriptional regulator reduce the virulence of the parental strain. Both of these mutants were found to be avirulent. We evaluated whether napA, hlyX, tatA, and fur mutants could prevent disease in pigs challenged with a fully virulent serotype 1 wild type strain. The napA, tatA, and fur mutants produce reduced or no ApxI. Pigs were vaccinated intranasally with 10 6 CFU and 10 days later with an intramuscular boost of the same mutant. After intranasal challenge with 10 9 CFU of the virulent strain, the vaccinated pigs were monitored for
symptoms of porcine pleuropneumonia. Pigs vaccinated with the hlyX and tatA mutants had no signs of clinical disease and had no or minimal lung lesions. Pigs vaccinated with the fur mutant showed only depression/lethargy and minimal lung lesions. All unvaccinated pigs had clinical signs ranging from lethargy and depression to labored breathing, cough and frothy (often blood-tinged) discharge from the nose and mouth, their lungs were covered with a fibrinous exudate that adhered to the pleural lining and hemorrhagic lesions were present in most lobes of the lungs. From 10 4 to >10 8 CFU of the challenge strain was isolated per 100 ml of lung lavage fluid from the unvaccinated pigs. Pigs vaccinated with the napA mutant showed signs of labored breathing and cough and significant areas of hemorrhage and fibrinous pleuritis. We recovered <10 4 CFU of the virulent strain/100 ml lung lavage fluid from pigs vaccinated with the hlyX, tatA or fur mutants. The vaccine strains were isolated from
the tonsils of less than 50% of the pigs at necropsy. The hlyX and tatA mutants protected the pigs from clinical disease when challenged intranasally with a virulent serotype 1 strain.
PARTICIPANTS: Susan E. H. West was Principal Investigator and directed all aspects of the project and also participated in the infection and vaccine studies. Jennifer Kisilewski, Beth Fredricks, Chris Murphy, Karen Horvath, and Cary Pierson all were research technicians who carried out experiments. Bridget Tallman was a M.S. graduate student who worked on the vaccine studies. Mindy Sue Wesely was an undergraduate student who worked on the virulence studies. There were no partner organizations or collaborators. Bridget Tallman, a M.S. graduate student, and Mindy Sue Wesely, an undergraduate pre-veterinary student, both received academic training.
TARGET AUDIENCES: This project is of interest to the swine industry. It is also of interest to scientists studying respiratory diseases.
PROJECT MODIFICATIONS: The one major change was our inability to isolate a mutant that was mutated in the gene which directly regualtes production of the ApxI hemolysin. A. pleuropneumoniae is an organism that is difficult to manipulate genetically.
Impacts
The results from this study may lead to the development of a vaccine against A. pleuropneumoniae. We have found that several mutant strains are avirulent when administered intra-nasally to pigs and that these intra-nasal administration of these strains can prevent clinical disease in pigs when challenged with a fully-virulent serotype 1 strain.
Publications
- S. E. H. West, K. Murphy, K. Horvat, C. Pierson, L. B. Regassa**, and J. L. Buss. 2002. Investigations into the Regulation of ApxI Production by A. pleuropneumoniae. International Pasteurellaceae Society Meeting 2002.
- West, S. E. H., H. Steinberg, M. Wesley, and B. Fredricks. 2005. An A. pleuropneumoniae hlyX mutant is not reduced in virulence in a serotype 1 strain. Pasteurellaceae 2005 sponsored by the American Society for Microbiology and the International Pasteurellaceae Society.
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Progress 01/01/04 to 12/31/04
Outputs
Actinobacillus pleuropneumoniae(Apl) is the causative agent of porcine pleuropneumonia, a highly contagious, and frequently fatal respiratory tract disease in swine. We have evaluated whether four transposon-generated mutants of A. pleuropneumoniae (Apl) UWP36N, a strain that contains a deletion in the ApxII gene, can prevent disease in pigs that are challenged with the wild type strain. These four mutants produce no or reduced levels of ApxI, an RTX hemolysin/cytotoxin that is responsible for much of the damage to lung tissue and mortality associated with this disease, and contain mutations in one of the following genes: napA, hlyX, tatA, and fur. The pigs were vaccinated intranasally with 10 6 CFU of one of the mutants followed 10 days later with an intramuscular boost with 10 6 CFU of the same mutant. After intra-nasal challenge with 10 9 CFU of A. pleuropneumoniae 4074mm-ap, the vaccinated pigs were monitored for symptoms of porcine pleuropneumoniae. At necropsy,
the lungs were evaluated for pathological lesions, samples were obtained for histopathological analysis, and the number of bacteria present in the tonsils and in lung lavage fluid were determined. Pigs vaccinated with mutants UWP36N (apxIIA), UWP36N-#60 (apxIIA hlyx) and UWP36N-#206 (apxIIA tatA) did not show any signs of clinical disease and no or minimal lung lesions upon both gross and histopathological examination, while pigs vaccinated with mutant UWP36N-#215 (apxIIA fur) showed only depression/lethargy and minimal lung lesions. All pigs in the unvaccinated control groups had clinical signs ranging from lethargy and depression to labored breathing, cough and frothy (often blood-tinged) discharge from the nose and mouth. The lungs of these pigs were covered with a fibrinous exudate and were often adhered to the pleural lining. Large (1-3 cm)hemorrhagic lesions were present in most lobes of the lungs. From 10 4 to greater than 10 8 CFU of the challenge strain was isolated per 100
mls of lung lavage fluid from the unvaccinated pigs. Pigs vaccinated with UWP36N-#1 (apxIIA napA) , which does not produce either ApxI or ApxII, showed signs of labored breathing and cough and significant areas of hemmorrhage and fibrinous pleuritis. We recovered 104 or fewer CFU/100 ml of lavage fluid of the wild type Apl strain from the lungs of pigs vaccinated with UWP36N, UWP36N-#60, UWP36N-#206, and UWP36N-#215. The vaccine strains were isolated from the tonsils of less than 50% of the pigs at necropsy, indicating that these strains are unable to persist for more than a few weeks in the tonsils. In summary, mutants UWP36N, UWP36N-#60, and UWP36N-#206 protected the pigs from clinical disease when challenged intra-nasally with a virulent serotype 1 strain.
Impacts
The results from this study may lead to the development of a vaccine against A. pleuropneumoniae.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Actinobacillus pleuropneumoniae (Apl) causes a severe, contagious, and often fatal respiratory disease of pigs, known as porcine pleuropneumonia. Both acute and chronic forms of the disease exist. Elimination of chronic infection and the carrier state could have a significant impact on the control of this disease which is transmitted from pig to pig by aerosols. Apl produces multiple distinct RTX-like hemolysins/cytotoxins that are involved in the pathogenesis of porcine pleuro-pneumonia. Of the various Apl hemolysins/cytotoxins, ApxI is considered to be the most damaging. Our goal is to identify the environmental signals and define the molecular mechanisms which regulate ApxI production. Previously, we identified 5 genes involved in nitrate reduction which have an effect on ApxI production. We hypothesized that ApxI production is repressed by a regulatory protein that responds to signals generated as a result of the napA mutation. We have tested the napA mutant and
its parent in a swine infection model. We found no difference in virulence between the napA mutant and its immediate parent which produces only ApxII. Both strains were able to colonize the tonsils of infected pigs, but did not cause lung disease. Both of these strains did prevent colonization of the tonsils by the wild type parental strain which produces both hemolysins. These results suggest that these strains could serve as potential live vaccine strains that could prevent colonization by the wild type strain. Additional mutants are being tested in this model and include fur and tatA mutants. Understanding the regulatory mechanism and environmental stimuli that influence production of the ApxI hemolysin will lead to applications in immunoprophylaxis, diagnosis, chemotherapy, and control of this important swine disease.
Impacts
The results from this study may lead to the development of a vaccine against A. pleuropneumoniae.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Actinobacillus pleuropneumoniae (Apl) causes a severe, contagious, and often fatal respiratory tract disease of pigs, known as porcine pleuropneumonia. Both acute and chronic forms of the disease exist. Apl produces multiple distinct RTX-like hemolysins/cytotoxins that are involved in the pathogenesis of porcine pleuropneumonia. Of the various Apl hemolysins/cytotoxins, ApxI is considered to be the most damaging. Our goal is to define the molecular mechanism involved in production of the ApxI hemolysin by Apl and to identify the environmental factor(s) which induce or repress its production. Using transposon mutagenesis, we have identified several genes that affect production of the ApxI hemolysin. These genes are involved in reduction of nitrate to nitrite and include napA, fdxGH, tatA, and moaA. We have also found that mutations in the genes encoding hlyX (fnr), fur, dnaJ, and in two ORFs of unknown function also reduce ApxI production. The identification of 5 genes
involved in nitrate reduction suggests that ApxI is produced in response to the signals associated with this complex metabolic process. We hypothesize that a primary (unidentified) regulatory gene responds to environmental signals produced as a result of the reduction of nitrate to nitrite by the NapA periplasmic nitrate reductase to activate or repress ApxI production. As, mutations in hlyX and fur do not abolish ApxI production but do affect the levels of ApxI produced, we propose that HlyX and Fur act as secondary regulators which regulate genes involved in nitrate reduction. To test this hypothesis, we plan to: (i) Identify the in vitro growth conditions which affect production of ApxI, (ii) Determine the infection sites and stages of disease at which ApxI is produced, (iii) Determine whether the ApxI primary regulatory mutant(s) are reduced in virulence, (iv) Isolate a mutant of the primary ApxI regulatory gene that controls ApxI expression in response to an unidentified signal
generated as a result of the napA mutation. We have determined that ApxI is not produced under anaerobic growth conditions. We are constructing a reporter fusion of the ApxICABD promoter to the E. coli lacZ gene so that we may quantitate the effect of anaerobic growth on ApxI production. We have cloned and sequenced the A. pleuropneumoniae napfdaghbc operon and genes flanking this operon. We are constructing a reporter fusion of the napfdaghbc operon to the E. coli lacZ gene so that we may quantitate the expression of the napfdaghbc operon. We are reconstructing the mini-Tn10 transposon so that it contains a selectable marker other than kanamycin resistance that will function in A. pleuropneumoniae. This min-Tn10 transposon will be used to mutagenize the A. pleuropneumoniae napA mutant to identify the putative repressor of ApxI production.
Impacts
Understanding these regulatory mechanisms may lead to applications in vaccine development and/or chemotherapy of Apl infections that will help protect against the mortality of the acute form of the disease and also prevent development of a carrier state or the chronic form of the disease. This work may also elucidate the mechanism by which Actinobacillus pleuropneumoniae causes acute and chronic disease in pigs. Elimination of chronic infection and the carrier state could have a significant impact on the control of this disease.
Publications
- S. E. H. West, K. Murphy, K. Horvat, C. Pierson, L. B. Regassa, and J. L. Buss. 2002. Investigations into the Regulation of ApxI Production by A. pleuropneumoniae. Proceedings of the International Pasteurellaceae Society Meeting 2002, Banff, Canada.
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