Автореферат и диссертация по ветеринарии (16.00.00) на тему:Создание первой карты генома Aktinobacillus pleuropneumonie и сравнительное макрорестрикционное картирование 12 референтных штаммов

1 ВВЕДЕНИЕ.

2 ОБЗОР ЛИТЕРАТУРЫ.

2.1 Actinobacillus pleuropneumoniae.

2.1.1 Общая характеристика и таксономическое положение.

2.1.2 Распространение и значение.

2.1.3 Факторы вирулентности.

2.1.3.1 Капсула.

2.1.3.2 Липополисахариды.

2.1.3.3 RTX-токсины.

2.1.3.4 Протеины наружной мембраны (ОМР).

2.1.3.5 Другие потенциальные факторы вирулентности.

2.2 Исследование генома.

2.2.1 Рестрикционный анализ.

2.2.2 ДНК - гибридизация.

2.2.3 Создание карт генома.

3 МАТЕРИАЛЫ И МЕТОДЫ.

3.1 Бактерии и плазмиды.

3.1.1 Условия культивирования бактерий.

3.1.2 Определение оптической плотности культуры.

3.2 Выделение и очистка нуклеиновых кислот.

3.2.1 Получение общей ДНК бактерий.

3.2.2 Выделение плазмидной ДНК.

3.2.3 Определение концентрации ДНК.

3.2.4 Получение высокомолекулярной общей ДНК

A. pleuropneumoniae.

3.3 Расщепление ДНК рестрикционными эндонуклеазами.

3.3.1 Расщепление ДНК, заключенной в агарозную матрицу.

3.3.2 Неполное расщепление ДНК.

3.3.3 Расщепление ДНК во фрагментах, вырезанных из агарозного геля.

3.4 Полимеразная цепная реакция (ПЦР).

3.5 Агарозные гели.

3.5.1 Гелевый пульс-электрофорез.

3.5.2 Определение величины фрагментов ДНК.

3.6 Выделение ДНК из агарозного геля.

3.7 Блоттинг по Саузерн.

3.8 Гибридизация по Саузерн.

3.8.1 Гены.

3.8.1.1 Мечение ДНК-зондов биотином.

3.8.1.2 Радиоактивное мечение ДНК-зондов изотопом фосфора 32Р.

3.8.1.3 Гибридизация с биотинилированными ДНК-зондами.

3.8.1.4 Гибридизация с радиоактивными ДНК-зондами.

4 РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЙ.

4.1 Физическая карта A. pleuropneumoniae АР76.

4.2 Генетическая карта A. pleuropneumoniae АР76.

4.3 Макрорестрикционный анализ референтных штаммов.

5 ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ.

6 РЕЗЮМЕ.

1. Akoopyants, N. S., S. W. Clifton, D. Kersulyte, J. E. Crabtree, В. E. Youree, C. A. Reece, N. O. Bukanov, E. S. Drazek, B. A. Roe and D. E. Berg (1998):

2. Analyses of the cag pathogenicity island of Helicobacter pylori. Mol. Microbiol. 28, 37-53.

3. Allardet-Servent A., Michaux-Charachon S., Jumas-Bilak E., Karayan L. and M. Ramuz (1993):

4. Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. Bacteriol. 175, 7869-7874

5. Altmann, E., J. R. Brisson and M. B. Perry (1986):

6. Structural studies of the capsular polysaccharides from Haemophilus pleuropneumoniae serotype 1. Biochem. Cell. Biol. 64, 707-716

7. Anagnostopoulos, C., P. J. Piggot and J. А. Носн (1993): The genetic map of Bacillus subtilis.in: Sonenshei, A. L., J. А. Носн und R. Losick (Hrsg.) Bacillus subtilis and other Gram-positive Bacteria. ASM, Washington, D. C., S. 425-461

8. Anderson, C., A. A. Potter and G.-F. Gerlach (1991):1.olation and molecular characterization of spontanously occuring cytolysinnegative mutants of Actinobacillus pleuropneumoniae serotype 7.1.fect. Immun. 59, 4110-4116

9. Baarsch, M. J., R. W. Scamurra, K. Burger, D. L. Foss, S. K. Maheswaran and M. P. Murtaugh (1995):1.flammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae. Infect Immun. 63, 3587-35941. Bachmann, P. (1972):

10. Beitrag zur Epidemiologie der kontagiosen Pleuropneumonie beim Schwein. Schweiz. Arch. Tierheilk. 114, 362-3821. Bautsch, W. (1988):

11. Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional field inversion gel electrophoresis techniques. Nucleic Acids Res. 16, 11461-11467

12. Beck, M., J. F. van den Bosch, I. M. Jongenelen, P. L. Loeffen, R. Nielsen, J. Nicolet and J. Frey (1994):

13. RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains.

14. Bihlmaier, A., U. Romling, T. F. Meyer, B. Tummler and C. P. Gibbs (1991): Physical and genetic map of the Neisseria gonorrhoeae strain MS11-N198 chromosome.

15. Mol. Microbiol. 5, 2529-2539 Birnboim, H. C. and J. Doly(1979):

16. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1517

17. Birren, В. andE. Lai (1993): Pulsed field gel elektrophoresis. Academic Press, Cambrige

18. Bisping W. und G. Amtsberg (1988):

19. Farbatlas zur Diagnose bakterieller Infektionserreger der Tiere. Verlag Paul Parey, Berlin und Hamburg1. Blaha, T. (1992):

20. Zur Pravalenz der respiratorischen Erkrankungen des Schweines in den wichtigen schweinefleischproduzierenden Landern. Colleg. Vet. 23, 64-67

21. Blanchard, P. C., R. L. Walker and I. Gardner (1993): Pleuropneumoniae in swine associated with an urease-negative variant of Actinobacillus pleuropneumoniae. J. Vet. Diagn. Invest. 5, 279-282

22. Bolivar, F. and K. Backman (1979): Plasmids of Escherichia со//as cloning vectors. Methods Enzymol. 68, 245

23. Borr, J. D., D. A. Ryan and J. I. MacInnes (1991):

24. Analysis of Actinobacillus pleuropneumoniae and related organisms by DNA-DNA hybridization and restriction endonuclease fingerprinting. Int. J. Syst. Bacteriol. 41, 121-129

25. Bosse, J. Т., and J. I. MacInnes (1997):

26. Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease.1.fect. Immun. 65, 4389-4394

27. Boyer, H. W. and D. Roulland-Dussoix (1969):

28. A complementation analysis of the restriction and modification of DNA in1. Escherichia coli.1. J. Mol. Biol. 41, 459

29. Brade, H., L. Brade and E. T. Rietschel (1988):

30. Structure-activity relationships of bacterial lipopolysaccharides (endotoxins).

31. Current and future aspects.

32. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. 268, 151-179

33. Bunka, S., C. Christensen, A. A. Potter, P. J. Willson and G. F. Gerlach (1995):

34. Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5. Infect. Immun. 63, 2797-2800

35. Bunka, S., S. Jeckstadt, A. Oltrogge, P. Schoss und K. Petzold (1990): Vorkommen von Antikorpern gegen Actinobacillus pleuropneumoniae in Schweineseren Haufigkeit und Verteilung der Serovare 2, 3, 7 und 9. Tierarztl. Umschau 45, 397-402

36. Carle, G. F. and M. V. Olson (1984):

37. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12, 5647-5664

38. Carle, G. F., M. Frank and M. V. Olson (1986):

39. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232, 65-68

40. Cheng, H. P. and T. G. Lessie (1994):

41. Multiple replicons constituting the genome of Pseudomonas cepacia 17616. J. Bacteriol. 176, 4034-4042

42. Chevallier, В., D. Dugourd, К. Tarasiuk, J. Harel, M. Gottschalk, M. Kobisch and J. Frey (1998)

43. Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae. FEMS Microbiol. Lett. 160, 209-216

44. Chu G., Vollrath D. and R. W. Davis (1986):

45. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582-5

46. Clark, S. M., E. Lai, B. W. Birren and L. Hood (1988): A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields. Science 241, 1203-1205

47. Cole, S. T. and I. Saint Girons (1994):1. Bacterial genomics.

48. FEMS Microbiol. Rev. 14, 139-160

49. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-544

50. Cruijsen, Т. L., L. A. Van Leengoed, Т. C. Dekker-Nooren, E. J. Schoevers and J. H. Verheijden (1992):

51. Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs. Infect. Immun. 60, 4867-4871

52. Davidson, В. E., J. MacDougall and I. Saint Girons (1992): Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes. J. Bacterid. 174, 3766-3774

53. Deneer, H. G. and A. A. Potter (1989 a):

54. Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae. Infect. Immun. 57, 798-804

55. Deneer, H. G. and A. A. Potter (1989 b):1.entification of a maltose-induced major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae. Mirob. Pathog. 6, 425-432

56. Ely В., Ely Т. W., GerardotC. J. and A. Dingwall (1990):

57. Circularity of the Caulobacter crescentus chromosome determined by pulsedfield gel electrophoresis.

58. J. Bacteriol. 172, 1262-1266

59. Feinberg, A. P. and B. Vogelstein (1983):

60. A technique for radiolabeling DNA restriction endonuclease fragments to highspecific activity.1. Anal. Biochem. 132, 6-13

61. Fenwick В. W. and В. I. Osburn (1986)

62. Vaccine potential of Haemophilus pleuropneumoniae oligosaccharide-tetanustoxoid conjugates.1.fect. Immun. 54, 583-586

63. Fenwick, B. and S. Henry (1994):1. Porcine pleuropneumonia.

64. J. Am. Vet. Med. Assoc. 204, 1334-1340

65. Fenwick, B. W., В. I. Osburn, J. S. Cullor, S. C. Henry and H. I. Olander (1986):

66. Mortality in swine herds endemically infected with Haemophilus pleuropneumoniae: effect of immunization with cross-reacting lipopolysaccharide core antigens of Escherichia coli. Am. J. Vet. Res. 47, 1888-1891

67. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.1. Science 269, 496-512

68. Fonstein, M. and R. Haselkorn (1995) Physical mapping of bacterial genomes. J. Bacterid. 177, 3361-3369

69. Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends in Microbiol. 3, 257-261

70. Frey, J. and J. Nicolet (1988):

71. Regulation of hemolysin expression in Actinobacillus pleuropneumoniaeserotype 1 by Ca2+.1.fect. Immun. 56, 2570-2575

72. Frey, J., H. van den Bosch, R. Segers and J. Nicolet (1992): Identification of a second hemolysin (Hlyll) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli. Infect. Immun. 60, 1671-1676

73. Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. J. Gen. Genet. 139, 1723-1728

74. Frey, J., M. Beck, J. F. van den Bosch, R. P. A. M. Segers and J. Nicolet (1995):

75. Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains. Mol. Cell Probes 9, 277-282

76. Frey, J., R. Kuhn and J. Nicolet (1994)

77. Association of the CAMP phenomenon in Actinobacillus pleuropneumoniae with the RTX toxins Apxl, Apxll and Apxlll. FEMS Microbiol. Lett. 124, 245-2511. Fussing V. (1998):

78. Genomic relationships of Actinobacillus pleuropneumoniae serotype 2 strains evaluated by ribotyping, sequence analysis of ribosomal intergenic regions, and pulsed-field gel electrophoresis. Lett. Appl. Microbiol. 27, 211-215

79. Fussing, V., K. Barfod, R. Nielsen, K. Moller, J. P. Nielsen, H. C. Wegener and M. Bisgaard М (1998)

80. Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark. Vet. Microbiol. 62, 145-162

81. Gardiner, K., W. Laas and D. Patterson (1986):

82. Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Somat. Cell. Mol. Genet. 12, 185-195

83. Gerlach, G. F., C. Anderson, A. A. Potter, S. Klashinsky and P. J. Willson (1992 a):

84. Cloning and expression of a transferrin-binding protein from Actinobacilluspleuropneumoniae.1.fect. Immun. 60, 892-898

85. Gerlach, G. F., C. Anderson, S. Klashinsky, A. Rossi-Campos, A. A. Potter and P. J. Willson (1993):

86. Molecular characterization of a protective outer membrane lipoprotien (OmIA) from Actinobacillus pleuropneumoniae serotype 1. Infect. Immun. 61, 565-572

87. Gonzalez, G. C., D. L. Caamano and A. B. Schryvers (1990): Identification and characterisation of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae. Mol. Microbiol. 4, 1173-1179

88. Gonzalez, G. C., R. Yu, P. R. Rosteck and A. B. Schryvers (1995): Sequence, genetic analysis, and expression of Actinbacillus pleuropneumoniae transferrin receptor genes.

89. Microbiology 141, 2405-2416

90. GramT. and P. Ahrens (1998):1.proved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein. J. Clin. Microbiol. 36, 443-448

91. Grimont, F. and P. A. Grimont(1986):

92. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools.

93. Ann. Inst. Pasteur. Microbiol. 137B, 165-1751. Gulig, P. A. (1990):

94. Virulence plasmids of Salmonella typhimurium and other salmonellae. Microb. Pathog. 8, 3-11

95. Gunnarsson, A., B. Hurvell and E. L. Biberstein (1978): Serologic studies of porcine strains of Haemophilus parahaemolyticus (pleuropneumoniae): antigenic specificity and relationship between serotypes. Am. J. Vet. Res. 39, 1286-1292

96. Gunnarsson, A., E. L. Biberstein and B. Hurvell (1977)

97. Serologic studies on porcine strains of Haemophilus parahaemolyticuspleuropneumoniae): agglutination reactions.

98. Am. J. Vet. Res. 38, 1111-1114

99. Hanish J. and M. McClelland (1990):

100. Methylase-limited partial Notl cleavage for physical mapping of genomic DNA.

101. Nucleic Acids Res. 18, 3287-3291

102. Heue,M.-C., M. Sirois, C. Ouellet und M. Boissinot (1996): Cloning, sequencing and overproduction of Mn-SOD and Cu,Zn-SOD EMBL-Datenbank, Accession number U51441

103. Hobbs, M. M., M. J. Leonardi, F. R. Zaretzky, T.-H. Wang and Т. H. Kawula (1996):

104. Organization of Haemophilus ducreyi 35000 chromosome. Microbiology 142, 2587-2594

105. Honeycutt, R. J., M. McClelland and B. W. Sobral (1993): Physical map of the genome of Rhizobium meliloti 1021. J. Bacterid. 175, 6945-69521.zana, T. J. (1987):

106. Purification and partial characterization of the capsular polymer of Haemophilus pleuropneumoniae serotype 5. Infect. Immun. 55, 1573-15791.zana, T. J. (1991):

107. Virulence properties of Actinobacillus pleuropneumoniae. Microb. Pathog. Ц, 305-3161.zana, T. J. and B. Mathison (1987):

108. Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5. Infect. Immun. 55, 1580-15871.zana, T. J., J. Ma, T. Workman, R. P. Gogolewski and P. Anderson (1988):

109. Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5. Infect. Immun. 56, 1880-18891.zana, T. J., J. Todd, C. Koch and J. Nicolet (1992):

110. Serotype specificity of immunological assay for the capsular polymer of

111. Actinobacillus pleuropneumoniae serotypes 1 and 9.

112. Vet. Microbiol. 31, 351-3621.zana, Т. J., J. Todd, J. N. Ma and H. Veit(1991):

113. Characterization of a non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5: role of the 110 kilodalton hemolysin in virulence and immunoprotection. Microb. Pathog. 10, 281-296

114. Jacques, M., M. Belanger, G. Roy and В. Foiry(1991):

115. Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelialcells and frozen lung sections.

116. Vet. Microbiol. 27, 133-143

117. Jacques, M., S. Rioux, B. Foiry and M. Belanger (1992):

118. Affinity for porcine respiratory tract mucus is found in isolates of

119. Actinobacillus pleuropneumoniae.1.: 12th Congress of the International Pig Veteriary Society, the Hague 1992. Proceedings S. 207

120. Jansen R., J. Briaire, E. M. Kamp and M. A. Smits (1992): Comparison of the cytolysin II genetic determinants of Actinobacillus pleuropneumoniae serotypes. Infect. Immun. 60, 630-636

121. Jansen, R., J. Briaire, H. E. Smith, P. Dom, F. Haesebrouck, E. M. Kamp, A. L. J. Gielkens and M. A. Smits (1995):

122. Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils. Infect. Immun. 63, 27-37

123. Jensen, A. E. and T. A. Bertram (1986):

124. Morphological and biochemical comparison of virulent and avirulent isolates of Haemophilus pleuropneumoniae serotype 5. Infect. Immun. 51, 419-424

125. Kamp, E. M., J. К. Popma, J. Anakotta and M. A. Smits(1991): Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by using monoclonal antibodies. Infect. Immun. 59, 3079-3085

126. Kamp, E. M., N. Stockhofe-Zurwieden, L. A. Van Leengoed, M. A. Smits (1997):

127. Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs. Infect. Immun. 65, 4350-4354

128. Kamp, E. M., Т. M. Vermeulen, M. A. Smits and J. Haagsma (1994): Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis. Infect. Immun. 62, 4063-4065

129. Karaolis, D. K., J. A. Johnson, С. C. Bailey, E. C. Boedeker, J. B. Kaper and P. R. Reeves (1998):

130. A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.

131. Proc. Natl. Acad. Sci. U.S.A. 95, 3134-3139. Kauc L. and S. H. Goodgal (1989):

132. The size and a physical map of the chromosome of Haemophilusparainfluenzae.1. Gene 83, 377-380

133. Khetawat, G., R. K. Bhadra, S. Kar and J. Das (1998):

134. Vibrio cholerae 0139 Bengal: combined physical and genetic map andcomparative analysis with the genome of V. cholerae 01.

135. J. Bacteriol. 180, 4516-4522.

136. KlLIAN, M., J. MESTECKYand R. E. Schrohenlohner (1979): Pathogenic species of the genus Haemophilus and Streptococcus pneumoniae produce immunoglobulin A1 protease. Infect. Immun. 26, 143-149

137. Kilian, M., J. NicoLETand E. L. Biberstein (1978):

138. Biochemical and serological characterization of Haemophilus pleuropneumoniae (Matthews and Pattison 1961) Shope 1964 and proposal of a neotype strain.1.t. J. Syst. Bacteriol. 28, 20-26

139. Kohara, Y., K. AKiYAMAand K. Isono (1987):

140. The physical map of E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genogenomic library. Cell 50, 495-508

141. Kolble, K. and R. B. Sim (1991)

142. An angle-variable three-dimensional pulsed field gel electrophoresis system. Anal. Biochem. 192, 32-38

143. Current Microbiol. 15, 141-1441.ngford, P. R., В. M. Loynds and J. S. Kroll(1996):

144. Cloning and molecular characterization of Cu, Zn superoxide dismutase from

145. Actinobacillus pleuropneumoniae.1.fect. Immun. 64, 5035-50411.blond, P., M. Redenbach and J. Cullum (1993):

146. MacDonald, J. and A. N. Rycroft (1992):

147. Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2. Infect. Immun. 60, 2726-2732

148. MacInnes, J. I. and J. D. Borr (1990):

149. The family Pasteurellaceae: modern approaches to taxonomy. Can. J. Vet. Res. 54 Suppl:S6-11

150. MacInnes, J. I. and S. Rosendal (1987):

151. Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms. Infect. Immun. 55, 1626-1634

152. MacInnes, J.I., J. D. Borr, M. Massoudi and S. Rosendal (1990): Analysis of southern Ontario Actinobacillus (Haemophilus) pleuropneumoniae isolates by restriction endonuclease fingerprinting. Can. J. Vet. Res. 54,244-50

153. Mahan, M. J., J. M. Slauch and J. J. Mekalanos (1996):

154. Environmental regulation of virulence gene expresion in Escherichia,

155. Salmonella, and Shigella spp.in: Neidhardt, F. C. (Hrsg.),: Escherichia coli and Salmonella. Cellular and Molecular Biology.

156. Aufl. ASM Press, Washigton D. C., S. 2803-2815

157. Mannheim, W. (1984): Family III Pasteurellaceae.in: N. R. Krieg and J. G. Holt (Hrsg.): Bergey's Manual of Systematic Bacteriology.

158. Wiliams and Wilkins, Baltimore, U. S. A., 550-575 Matschullat, G. (1982):

159. Uber die Haemophilus-Pleuropneumonie des Schweines Prakt. Tierarzt 63, 1047-1055

160. McClelland М., Jones R., Patel Y. and М. Nelson (1987): Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucleic Acids Res. 15, 5985-6005

161. Meinkoth, J. and G. Wahl (1984):

162. Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267-2841. Mekalanos, J. J. (1992):

163. Environmental signals controlling expression of virulence determinants in bacteria.1. J. Bacterid. 174, 1-7

164. Michel, E. and P. Cossart (1992):

165. Physical map of the Listeria monocytogenes chromosome.

166. J. Bacterid. 174, 7098-7103

167. Mittal, K. R., R. Higgins and S. Lariviere (1988):

168. Moller К., Nielsen R., Andersen L. V. and M. Kilian (1992): Clonal analysis of the Actinobacillus pleuropneumoniae population in a geographically restricted area by multilocus enzyme electrophoresis. J. Clin. Microbiol. 30, 623-627

169. Morschhauser, J., V. Vetter, L. Emody and J. Hacker (1994): Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross-talk between different adhesin gene clusters. Mol. Microbiol. Ц, 555-566

170. Mullis, К. B. and F. A. Faloona (1987):

171. Specific syntesis of DNA in vitro via a polymerase catalised chain reaktion. Methods Enzymol. 155, 335-350

172. Musser, J. M., V. J. Rapp and R. K. Selander (1987): Clonal diversity in Haemophilus pleuropneumoniae. Infect. Immun. 55, 1207-1215

173. Negrete Abascal, E., V. R. Tenorio, J. J. Serrano, C. Garcia and M. De La Garza (1994):

174. Secreted proteases from Actinobacillus pleuropneumoniae serotype 1 degrade porcine gelatin, hemoglobin and immunoglobulin A. Can. J. Vet. Res. 58, 83-861. Nicolet, J. (1968):

175. On haemophilosis of swine. I. Identification of a frequent agent: Haemophilus parahaemolyticus.

176. Pathol. Microbiol. Basel, 31, 215-225 Nicolet, J. (1988):

177. Taxonomy and serological identification of Actinobacillus pleuropneumoniae. Can. Vet. J. 29, 578-580

178. Nicolet, J. (1992): Actinobacillus pleuropneumoniae.in: A. D. Leman, В. E. Straw (Hrsg.): Diseases of Swine. 7. Aufl. Iowa State University Press, Ames, Iowa, U. S. A.

179. Nicolet, J., H. Konig und E. Scholl (1969):

180. Zur /-/aemopMvs-Pleuropneumonie beim Schwein: II. Eine kontagiose Krankheit von wirtschaftlicher Bedeutung. Schweiz. Arch. Tierheilk. 1Ц, 166-1741. Nielsen, R. (1984):

181. Haemophilus pleuropneumoniae serotypes cross protection experiments. Nord. Veterinaermed. 36, 221-2341. Nielsen, R. (1985 a):

182. Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae). Serotypes 8, 3 and 6. Serological response and cross immunity in pigs. Nord. Veterinaermed. 37, 217-2271. Nielsen, R. (1985 b):

183. Serological characterization of Haemophilus pleuropneumoniae {Actinobacillus pleuropneumoniae) strains and proposal af a new serotype: serotype 9.

184. Acta Vet. Scand. 26, 501-5121. Nielsen, R. (1986 a):

185. Serological characterization of Actinobacillus pleuropneumoniae strains and proposal of a new serotype: serotype 12. Acta Vet. Scand. 27, 453-4551. Nielsen, R. (1986 b):

186. Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes a and b. Acta Vet. Scand. 27, 49-581. Nielsen, R. (1988):

187. Seroepidemiology of Actinobacillus pleuropneumoniae. Can. Vet. J. 29, 580-5821. Nielsen, R. (1990):

188. New diagnostic techniques: a review of the HAP-group of bacteria. Can. J. Vet. Res. 54, 68-72

189. Nielsen, R. and P. J. O'Connor (1984):

190. Serological characterization of 8 Haemophilus pleuropneumoniae strains and proposal of a new serotype: serotype 8. Acta Vet. Scand. 25, 96-106

191. Nielsen R., L. O. Andresen andT. Plambeck(1996):

192. Serological characterization of Actinobacillus pleuropneumoniae biotype 1 strains antigenically related to both serotypes 2 and 7. Acta Vet. Scand. 37, 327-336

193. Nielsen R., L. O. Andresen, T. Plambeck, J. P. Nielsen, L. T. Krarup and S. E. Jorsal (1997):

194. Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds. Vet. Microbiol. 54, 35-46

195. Niven, D. F., J. Donga and F. S. Archibald (1989):

196. Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin.

197. Mol. Microbiol. 3, 1083-1089

198. Ochman, H., F. C. Soncini, F. Solomon and E. A. Groisman (1996): Identification of a pathogenicity island required for Salmonella survival in host cells.

199. Proc. Natl. Acad. Sci. U.S.A. 93, 7800-7804

200. Paradis S. E., Dubreuil D., Rioux S., Gottschalk M. and M. Jacques (1994):

201. High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells. Proceedings, S. 193.

202. Pattison, I. Н., D. G. Howell and J. Elliott (1957):

203. Haemophilus-Wke organism isolated from pig lungs and associated pneumonic lesions. J. Сотр. Pathol. 67, 320-329

204. Perry, M. В., E. Altmann, J. R. Brisson, L. M. Beynon and J. C. Richards (1990):

205. Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus (Haemophilus) pleuropneumoniae strains. Serodiagn. Immunother. In Infect. Dis. 4, 299-308

206. PullingerG. D., G. D. Baird, С. M. Williamson and A. J. Lax (1989): Nucleotide sequence of a plasmid gene involved in the virulence of salmonellas.

207. Nucleic Acids Res. 17, 7983

208. Rapp, V. J. and R. F. Ross (1986):

209. Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection. Infect. Immun. 54, 751-760

210. Rapp, V. J., R. F. Ross and B. Z. Erckson (1985):

211. Serotyping of Haemophilus pleuropneumoniae by rapid slide agglutination and inderect fluorescent antibody tests in swine. Am. J. Vet. Res. 46, 185-192

212. Rapp, V. J., R. S. Munson and R, F. Ross (1986):

213. Outer membrane protein profiles of Haemophilus pleuropneumoniae.1.fect. Immun. 52, 414-420

214. Rioux, S., C. Girard, D. Dubreuil and M. Jacques (1996):

215. Evaluation of the protective efficacy of Actinobacillus pleuropneumoniaeserotype 1 detoxified lipopolysaccharides in pigs.1. 14th Congress of the International Pig Veterinary Society, Bologna 1996. Proceedengs, S. 205

216. Romling, U. and B. Tummler (1991):

217. The impact of two-dimensional pulsed-field gel electrophoresis techniques for the consistent and complete mapping of bacterial genomes: refined physical map of Pseudomonas aeruginosa РАО. Nucleic. Acids. Res. 25, 3199-3206

218. ROmling, U., D. Grothues, T. Heuer and B. Tummler (1992): Physical genom analysis of bacteria. Electrophoresis, 13, 626-631

219. ROmling, U., D. Grothues, W. Bautsch and B. Tummler (1989): A physical genome map of Pseudomonas aeruginosa РАО. EMBO J. 8, 4081-4089

220. ROmling, U., К. D. Schmidt and В. Tummler (1997):1.rge genome rearrangements discovered by the detailed analysis of 21 Pseudomonas aeruginosa clone С isolates found in environment and disease habitats.

221. J. Mol. Biol. 271, 386-404.

222. Romling, U., R. Fislage und B. TOmmler (1995):

223. Theorie und Anwendung der Makrorestriktionsanlyse fur die klonale Analysevon Erregern.1.fect. Immun. 23, 4-8

224. Rosendal, S. and D. A. Boyd (1982): Haemophilus pleuropneumoniae serotyping. J. Clin. Microbiol. 16, 840-843

225. Rossi-Campos, A., C. Anderson, G. F. Geralch, S. Klashinsky, A. A. Potter and P. J. Willson (1992):1.munization of pigs against Actinobacillus pleuropneumoniae with two pecombinant protein preparations Vaccine 10, 512-518

226. Rycroft, A. N., D. Williams, J. M. Cullen and J. Macdonald (1991): The cytotoxin of Actinobacillus pleuropneumoniae (pleurotoxin) is distinct from the haemolysin and is associated with a 120 kDa polypeptide. J. Gen. Microbiol. 137, 561-568

227. Saiki, R. K., S. Scharf, F. A. Faloona, К. B. Mullis, G. T. Horn, H. A. Erlich and N. Arnheim (1985):

228. Enzymatic amplification of p-Globin genomic sequences and restriction site analysis for diagnostic of sickl cell anemia. Science 230, 1350-1354

229. Sambrook, J., E. F. Fritsch and T: Maniatis (1989):in: C. Nolan (Hrsg.): Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, NY, USA

230. Sanderson К. E., A. Hessel, S.-L. Liu and К. E. Rudd(1996):

231. The Gentic Map of Salmonella typhimurium. Edition VIII.1.: F. C. Neidhardt (Hrsg.): Escherichia coli and Salmonella. Cellular andmolecular biology.

232. Aufl. ASM Press, Washington D. C., S. 1903-1999

233. Schubert, S., A. Rakin, H. Karch, E. Carniel and J. Heesemann (1998): Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans. Infect. Immun. 66, 480-485

234. Schwartz, D. S. and C. R. Cantor (1984):

235. Separation of yaest chromosome-sized DNAs by pulsed-field gradient gelelectrophoresis.1. Cell 37, 67-65

236. Schwartz, D. S., W. Saffran, J. Welsh, R. Haas, M. Goldenberg and C. R. Cantor (1982):

237. New techniques for purifying large DNAs and studying their properties and packaging.

238. Cold Spring Harbor Symp. Quant. Biol. 47, 189-195

239. Sebunya, K. and J. R. Saunders (1983):

240. Haemophilus pleuropneumoniae infection in swine: a review.

241. J. Am. Med. Ass. 182, 1331-1337

242. Selander, R. К., D. A. Caugant, H. Ochman, J. M. Musser, M. N. Gilmour and T. S. Whittam (1986):

243. Methods of multilocus enzyme electrophoresis for bacterial populationgenetics and systematics.

244. Appl. Environ. Microbiol. 51, 873-8841. Serwer, P. (1987):

245. Gel electrophoresis with discontinuous rotation of the gel: An alternativ to gel electrophoresis with changing direction of the elecrtric field. Electrophoresis 10, 301-304

246. Shope, R. E., D. C. White and G. Leidy (1964):

247. Porcine contagious pleuropneumonia. 1. Experimental transmission, etiologyand pathology.1. J. Exp. Med. 119, 357-368

248. Smith, C. L. and C. R. Cantor (1986):

249. Approaches to physical mapping of the human genome.

250. Cold Spring Harbor Symp. Quant Biol. 51, 115-122

251. Smith, H. O. and M. L. Birnstiel (1976): A simple method for DNA restriction mapping. Nucleic Acids Res. 3, 2387-2398

252. Smith, H. O. and R. D. Kolodner(1988):

253. Mapping of Escherichia coli chromosomal Tn5 and F insertions by pulsed field gel elektrophoresis. Genetcs 119, 227-236

254. Southern, E. M., R. Anand, W. R. Brown and D. S. Fletcher (1987): A model for the separation of large DNA molecules by crossed field gel electrophoresis.

255. Nucleic Acids Res. 15, 5925-5943

256. Straw, В. E., V. K. Tuovinen and M. Bigras Poulin (1989): Estimation of the cost of pneumonia in swine herds. J. Am. Vet. Med. Assoc. 195, 1702-1706

257. Suwanto A. and S. Kaplan (1989):

258. Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J. Bacteriol. 171, 5850-5859

259. Tascon, R. I., J. A. Vazquez-Boland, С. B. Gutierrez-Martin, J. I.

260. Rodriguez-Barbosa, and E. F. Rodriguez-Ferri (1994):

261. The RTX haemolysins Apxl and Apxll are major virulence factors of the swinepathogen Actinobacillus pleuropneumoniae: evidence from mutationalanalysis.

262. Mol. Microbiol. 14, 207-216

263. Molekulare Mechanismen der durch Eisenmangel induzierten Expression transferrinbindender Proteine von Actinobacillus pleuropneumoniae. Dissertation, Tierarztliche Hochschule Hannover

264. Toremorrell, M., C. Pijoan , K. Janni, R. Walker and H. S. Joo (1997): Airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus in nursery pigs. Am. J. Vet. Res. 58, 828-832

265. Trucksis M., Michalski J., Deng Y. K. and Kaper J. B. (1998):

266. The Vibrio cholerae genome contains two unique circular chromosomes.

267. Proc. Natl. Acad. Sci. U S A 95, 14464-14469

268. Turmel, С., E. Brassard, R. Forsyth, K. Hood, G. W. Slater and J. Noolandi (1990):

269. High resolution zero integrated field electrophoresis of DNA. in: Lai, E. und B. Birren (Hrsg.): Electrophoresis of large DNA-Molecules: Theory and Applications. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, S. 101-132

270. Valcaret, J., A. Allardet-Servent, G. Bourg, D. O'Callaghan, P. Michailesco and M. Ramuz (1997):1.vestigation of the Actinbacillus actinomycetemcomitans genome by pulsedfield gel elctrophoresis.

271. Van Leengoed, L. A. M. G. and E. M. Kamp (1989):

272. Endobronchial inoculation of various doses of Haemophilus (Actinobacillus) pleuropneumoniae in pigs. Am. J. Vet. Res. 50, 2054-2059

273. Vogelstein, B. and D. Gillespie (1979):

274. Preparative and analytical purification of DNA from agarose.

275. Proc. Natl. Acad. Sci. U S A 76, 615-619

276. Wahl, G. M., M. Stern and G. R. Stark (1979):

277. Efficient transfer of large DNA fragments from agarose gels to diazobenzyl-oxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76, 3683-3687

278. Wards, B. J., M. A. Joyce, M. Carman, F. Hilbink and G. W. deLisle (1998): Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains. Vet. Microbiol. 59, 175-1811. Welch R. A. (1991):

279. Pore-forming cytolysins of gram-negative bacteria. Mol. Microbiol. 5, 521-8100

280. Wilke, M., В. Franz and G.-F. Gerlach (1997):

281. Characterization of a large transferrin-binding protien from Actinobacillus pleuropneumoniae serotype 7. J. Vet. Med. 44, 73-86

282. Willems, H., C. Jager and G. Baljer (1998):

283. Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii.

284. J. Bacteriol. 180, 3816-3822