Animal experiments

Table 15: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆ureC and A. pleuropneumoniae ∆∆∆∆exbB.

APPENDIX - ANIMAL EXPERIMENTS

Table 15 continued

APPENDIX - ANIMAL EXPERIMENT

192

Table 16: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆tbpA, A. pleuropneumoniae ∆∆∆∆tbpB and A. pleuropneumoniae

∆∆∆∆tbpBA.

APPENDIX - ANIMAL EXPERIMENTS

ble 16 continued

APPENDIX - ANIMAL EXPERIMENT

194

Table 17: Raw data for challenge experiment with A. pleuropneumoniae ∆∆∆∆dmsA and A. pleuropneumoniae ∆∆∆∆fhuA.

APPENDIX - ANIMAL EXPERIMENTS

ble 17 continued

APPENDIX

196

I.6 Index of figures

Fig. 1: Energy coupling mechanisms of high-affinity iron uptake systems. ... 33

Fig. 2: Humoral immune response of pigs. ... 78

Fig. 3: Local immune response of pigs ... 79

Fig. 4: Construction of pTF205EM302... 83

Fig. 5: Construction of pTF205mut404. ... 84

Fig. 6: Analysis of A. pleuropneumoniae tbp mutant strains.... 86

Fig. 7: Southern blotting of RDA fragments ... 89

Fig. 8: Amplification of RDA products from genomic A. pleuropneumoniae AP76 DNA ... 91

Fig. 9: Genomic map of A. pleuropneumoniae AP76 with localization of two RDA fragments ... 93

Fig. 10: Plasmid maps of plasmids pDM5-116, pFH29-4 and pFH29-II31... 94

Fig. 11: DmsA signal peptide and upstream nucleotide sequence... 96

Fig. 12: Alignment of DmsA TAT leader sequences from several bacteria... 96

Fig. 13: Western blot analysis of DmsA expression. ... 98

Fig. 14: Western blot analysis of anaerobic DmsA expression. ... 99

Fig. 15: PCR using primers oDMSAdel1 and oDMSAdel2 ... 99

Fig. 16: FhuA signal peptide and upstream nucleotide sequence... 101

Fig. 17: PCR using primers oFDEL1 and oFDEL2. ... 104

Fig. 18: Southern blot analysis of fhuA. ... 104

Fig. 19: Expression of FhuA in A. pleuropneumoniae serotype reference strains ... 105

Fig. 20: Western blot analysis of FhuA expression... 106

Fig. 21: Western blot analysis of anaerobic FhuA expression... 107

Fig. 22: Construction of pBMKD1 ... 109

Fig. 23: Construction of pECF2... 110

Fig. 24: PCR of A. pleuropneumoniae deletion mutants ... 111

Fig. 25: Southern blot analysis of deletion mutants... 112

Fig. 26: PFGE analysis of A. pleuropneumoniae mutants... 112

Fig. 27: Body temperatures of pigs... 114

Fig. 28: Endoscopical scores ... 114

Fig. 29: Humoral immune response of pigs ... 116

APPENDIX

I.7 Index of tables

Table 1: List of bacterial strains used in this study ... 42

Table 2: List of plasmids used in this study ... 46

Table 3: List of primers used in this study ... 50

Table 4: PCR conditions used in this study ... 55

Table 5: Components in the PCR reaction ... 55

Table 6: Dilutions of antisera used in this study ... 67

Table 7: Clinical findings during endoscopy of the porcine lung... 70

Table 8: Clinical scores as evaluated during endoscopy... 70

Table 9: Virulence of A. pleuropneumoniae parent and isogenic mutant strains following aerosol challenge ... 80

Table 10: RDA fragments identified via differential hybridization ... 90

Table 11: Large fragments containing original RDA fragments... 92

Table 12: A. pleuropneumoniae dmsA - similarities to known genes... 97

Table 13: A. pleuropneumoniae fhuA homologue - similarities to known genes... 102

Table 14: Virulence of A. pleuropneumoniae AP76 and isogenic mutant strains following aerosol challenge. ... 117

Table 15: Raw data for challenge experiment with A. pleuropneumoniae ∆ureC and A. pleuro-pneumoniae ∆exbB. ... 190

Table 16: Raw data for challenge experiment with A. pleuropneumoniae ∆tbpA, A. pleuropneu-moniae ∆tbpB and A. pleuropneupleuropneu-moniae ∆tbpBA. ... 192

Table 17: Raw data for challenge experiment with A. pleuropneumoniae ∆dmsA and A. pleuro-pneumoniae ∆fhuA. ... 194

198

Acknowledgements

I wish to thank

Prof. Dr. G.-F. Gerlach for the idea for this work, for his constant guidance and motivation, for being there for all of his doctoral students, and for making sure that the cookie jar is never empty for long.

The members of the PhD committee entrusted with my supervision, Prof. Dr. L. Haas and Prof. Dr. B. Tümmler, for their interest in my work and their valuable discussions, and Dr. P. Langford.for the evaluation of this thesis.

Dr. I. Hennig-Pauka, Prof. Dr. M. Ganter and the Clinic for Pigs, for doing the bronchoalveolar lavages.

Prof. Dr. H.-J. Rothkötter and Dr. Astrid Hoffmann-Moujahid (Dept. of Functional and Applied Anatomy, Medical School Hannover) for performing FACS ad ELI spot analyses as well as for their help with necropsy duties.

My past and present coworkers at the Institute for Microbiology, in particular: Birgit Strommenger, Janin Stratmann, Anja Rothkamp (a.k.a. the Daltons), Svenja Thiede, Walaiporn Tonpitak, Denis Konine, Corinna Winterhoff, Achim Allgaier, Kathrin Radecker, Ilse Jacobsen, Sabine Jeckstadt, Silvia Kursawe, Kyaw Sunn, Karen Dohmann and all the other people in this institute who make this place the great workplace that it is.

Jörg Matusch for never letting the computers gain the upper hand.

Corine Bohan for the Pasteur quote.

Reinhild for the funding of my veterinary studies, which otherwise would not have been possible. My thanks extend to my grandmother Rosemarie.

My parents for their general relaxedness.

Steffen, for everything.

Danksagung

Ich möchte mich bedanken bei

Prof. Dr. G.-F. Gerlach für die Idee zu dieser Arbeit, seine ständige Führung und Motivation, dafür, dass er jederzeit für alle seine Doktoranden da ist und dafür, dass er immer dafür sorgt, dass die Keksdose nie lange leer ist.

Meinen Betreuern Prof. Dr. L. Haas und Prof. Dr. B. Tümmler, für ihr Interesse an meiner Arbeit und die wertvollen Hinweise sowie Dr. P. Langford für die Evaluierung meiner These.

Dr. I. Hennig-Pauka, Prof. Dr. M. Ganter und der Klinik für Kleine Klauentiere für die Durchführung der bronchoalveolären Lavage.

Prof. Dr. H.-J. Rothkötter und Dr. Astrid Hoffmann-Moujahid (Abteilung Funktionelle und Angewandte Anatomie, Medizinische Hochschule Hannover) für die Durchführung der FACS- und ELI-Spot-Analysen und für die tatkräftige Unterstützung bei der Sektion.

Meinen jetzigen und ehemaligen Mitarbeitern im Institut für Mikrobiologie insbesondere: Birgit Strommenger, Janin Stratmann, Anja Rothkamp -auch bekannt als 'die Daltons'-, Svenja Thiede, Walaiporn Tonpitak, Denis Konine, Corinna Winterhoff, Achim Allgaier, Kathrin Radecker, Ilse Jacobsen, Sabine Jeckstadt, Silvia Kursawe, Kyaw Sunn und all den anderen guten Geistern, die dieses Institut zu einem so schönen Arbeitsplatz machen.

Corine Bohan für das Pasteurzitat.

Jörg Matusch dafür, dass er die Computer jederzeit im Griff hat.

Reinhild für die Finanzierung meines Tiermedizinstudiums, ohne die dieses nicht möglich gewesen wäre. Auch meiner Oma Rosemarie vielen Dank.

Meinen Eltern für ihre allgemeine Gelassenheit.

Im Dokument The role of iron in Actinobacillus pleuropneumoniae infection (Seite 190-0)