De Long Xu

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

The effect of Bcg gene on antigen presentation of spleen adherent cells and peritoneal macrophages from Mycobacterium bovis BCG-infected Bcgs and Bcgr mice

To determine the role of the Bcg gene in the resistance and susceptibility of BCG-infected C57BL/6 (Bcg(s)) and its Bcg(r) congenic mice, the antigen presenting ability of spleen adherent cells and peritoneal macrophages, delayed type hypersensitivity (DTH) and lymphocyte blastogenic responses were investigated. The results obtained indicate that the DTH and lymphocyte blastogenic responses in Bcg(r) congenic mice were higher than in the Bcg(s) mice. Stimulation of spleen adherent cells with live Mycobacterium bovis BCG or PPD-BCG resulted in a higher antigen presenting ability in Bcg(r) than in Bcg(s) mice. However, comparatively low responses were associated with M. avium stimulation, with those in Bcg(r) being higher than in Bcg(s). I-A expression was also comparatively higher in Bcg(r) than in Bcg(s) mice. This study demonstrates that the Bcg gene seems to exhibit some effect on the antigen presenting ability of macrophages in immune responses of the congenic mice.

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Establishment of Bcgr congenic mice and their susceptibility/resistance to mycobacterial infection.

Bcg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Bcgr marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Bcgr congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all confirmed that the newly developed mice were highly congenic. These congenic mice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis BCG.