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SUMMARY

Objective

To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification.

Sample Population

One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf.

Procedure

Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated.

Results

Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1 % glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium.

Conclusions

Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT. (Am J Vet Res 1998;59:851–855)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis.

Sample Population—Neutrophils isolated from blood samples obtained from healthy calves.

Procedure—Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)- N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis.

Results—Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin.

Conclusions and Clinical Relevance—The ability of LKT to cause apoptosis instead of oncosis is concentration- dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis. ( Am J Vet Res 2001;62:136–141)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT).

Sample Population—Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves.

Procedure—Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation.

Results—Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes.

Conclusions and Clinical Relevance—Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host. (Am J Vet Res 2000;61:51–56)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine effects of tylosin on ruminal concentrations of Fusobacterium necrophorum and fermentation products in cattle during rapid adaptation to a high-concentrate diet.

Animals

6 steers fitted with ruminal cannulas.

Procedure

Steers were assigned randomly to 2 treatment groups and switched from a 0 to an 85% concentrate diet during a 4-day period. Cattle received this diet, with or without tylosin (90 mg/steer/d), for 4 weeks. Samples of ruminal contents were collected daily beginning 2 days before the treatment protocol and in the first week of concentrate feeding. Four subsequent samples were collected at weekly intervals. Concentration of F necrophorum in samples was determined, using the most-probable-number technique. Ruminal pH and concentrations of volatile fatty acids (VFA), lactate, and ammonia also were determined. All steers received both treatments separated by 4 weeks (cross-over design), during which time they were fed alfalfa hay only.

Results

In control steers, concentration of F necrophorum increased in response to the high-concentrate diet. Tylosin-fed steers had lower concentrations of F necrophorum than control steers at all times during concentrate feeding. However, ruminal pH and concentrations of lactate, VFA, and ammonia did not differ between treatment groups.

Conclusions and Clinical Relevance

Tylosin caused a significant reduction in ruminal concentrations of F necrophorum during rapid adaptation to a high-concentrate diet but had no effect on fermentation products. The reduction in ruminal concentration of F necrophorum helps explain the reduction in prevalence of hepatic abscesses reported in tylosin-fed feedlot cattle. (Am J Vet Res 1999;60:1061-1065)

Free access
in American Journal of Veterinary Research