Title page for ETD etd-10132010-020243

Type of Document Master's Thesis
Author Russell, Laura René
URN etd-10132010-020243
Title Treponema hyodysenteriae :growth and production of hemolysin
Degree Master of Science
Department Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Wilkins, Tracy D. Committee Chair
Gregory, Eugene M. Committee Member
Johnson, John L. Committee Member
Smibert, Robert M. Committee Member
  • Hemolysis and hemolysins
Date of Defense 1988-03-08
Availability restricted
Treponema hyodysenteriae is the causative agent of swine dysentery, a mucohemorrhagic disease of the intestines. T. hyodysenteriae requires phospholipids and cholesterol (or cholestanol) for growth, and it produces a a-hemolysin (TH) which is induced (300 fold) by the addition of RNA core to cultures. TH is bound to the RNA core which acts as a carrier or stabilizer. I\lyobjectives were to (i) obtain successful continuous growth of T. hyodysenteriae; (ii) study the production of the hemolysin produced by T. hyodysenteriae; (Ui) study the effects of different oligonucleotide carriers on the induction of hemolysin, (iv) examine various purification procedures for nipid, efficient partial purification of the hemolysin, (v) separate the hemolysin from the RNA core carrier, and (vi) to detennme whether T. hyodysenteriae can use coprostanol, the most common intestinal sterol, to satisfy its sterol requirement. I found that optimal growth of T. hyodysenteriae could be achieved by using BHI- glucose broth supplemented with calf serum (I O~/o). serum replacement (100/0), or phosphatidylcholine liposomes which contained cholesterol or cholestanol. Optimal growth required 1 % 02 and stirring of the culture. l\laximal hemolytic titers were obtained during late-log to early-stationary phase by cultures grown in the presence of RNA core. Hemolysin production was induced as soon as 5 minutes after addition of RNA core to cultures. This production was inhibited by chloramphenicol. Polyguanylic acid and RNase treated RNA core did not significantly increase hemolytic titers of cultures grown in their presence. Partial purification of hemolysin was achieved by acetic acid clarification followed by ammonium sulfate precipitation (65% saturation). With these procedures > 90% of the hemolytic activity was recovered. Although, hydroxylapatite adsorption and polyethyleneimine precipitation completely adsorbed or precipitated hemolytic activity I I was unable to efficiently recover the activity. Partially purified hemolysin was c}10toxic to CHO cells, and caused lysis rather than the rounding effect caused by many cytotoxins.
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