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Calcium oxalate urolithiasis in dogs is a common medical condition that is increasing in prevalence. From 1981 to 1985, approximately 6.8% of uroliths analyzed at the Minnesota Urolith Center were composed of calcium oxalate, compared with 42% of

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in American Journal of Veterinary Research

Abstract

Objective

To determine the effect of milk and blood serum constituents on cytotoxicity of Staphylococcus aureus on mammary epithelial cells.

Design

In vitro incubation of cells with cytotoxic agents and milk and serum constituents.

Sample Population

Mammary cells, milk, and blood obtained from 3 cows.

Procedure

Staphylococcal α-toxin and culture supernatants from S aureus M60 and an α-toxin-negative mutant of M60 were incubated with bovine mammary epithelial cells in the presence of milk fractions, serum, and divalent cations. Propidium iodide fluorescence was used as a measure of cell damage.

Results

Skim milk and milk whey inhibited S aureus cytotoxic agents. Skim milk protected against α-toxin damage to a greater extent than milk whey. Serum from an adult animal was more protective than was fetal serum. Milk fat and serum albumin had no protective effect. Divalent calcium and Mg2+ were more effective inhibitors of mammary epithelial cell damage caused by α-toxin than of damage attributable to M60 culture supernatant. Divalent calcium and Mg2+ at concentrations similar to those of free Ca2+ and Mg2+ in normal bovine milk decreased cytotoxic damage attributable to α-toxin. However, concentrations similar to those of total Ca2+ and Mg2+ in normal milk were required to decrease cell damage caused by M60 culture supernatant. The α-toxin-negative mutant was less cytotoxic than the M60 parent strain.

Conclusions

Casein, as well as Ca2+ and Mg2+ in bovine milk, inhibit the cytotoxic effect of S aureus on mammary epithelial cells.(Am J Vet Res 1996;57:308-312)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To develop semi-defined media that support growth of the bovine pathogen, Pasteurella haemolytica, and use them to examine production of leukotoxin and an arginine-binding protein by this organism.

Sample Population

10 P haemolytica A1 strains and 1 P multocida strain.

Procedure

Bacterial strains were cultivated at 37 C in media containing various amino acids, carbon sources, vitamins, and cofactors, and absorbance (OD600) was measured. Leukotoxin and arginine-binding protein production were assessed by immunoblot analysis.

Results

Optimal growth required supplementation with 0.1 % fetal bovine serum, gelatin, or purified bovine serum albumin. Calcium pantothenate and thiamine were essential for growth, and a variety of carbon sources could be utilized. In the complete medium, 15 amino acids were included; however, in the minimal medium, no amino acids were required. All strains (except P multocida) grew in the complete medium and 7 grew well in the minimal medium. Leukotoxin was not produced when amino acids were limiting, but could be enhanced by addition of 0.2% NH4SO4. Production of the arginine-binding protein was not affected by nitrogen availability or by presence of L-arginine.

Conclusions

Two media that support good growth of P haemolytica strains were developed. The minimal medium is simple to prepare and manipulate and its use revealed a potential role of nitrogen availability in the regulation of leukotoxin expression.

Clinical Relevance

Creation of these media will permit continued studies of the response of P haemolytica to environmental conditions that may mimic those encountered in the bovine respiratory tract during shipping. (Am J Vet Res 1997;58:749–754)

Free access
in American Journal of Veterinary Research

SUMMARY

The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr.

Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

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in American Journal of Veterinary Research

as described elsewhere. 16,23 Briefly, CRECs and ERECs were isolated from fresh canine and equine trachea, respectively, by enzymatic digestion with 1.4% pronase k and 0.1% DNase I l in calcium- and magnesium-free MEM for 48 hours. After digestion

Full access
in American Journal of Veterinary Research