Rapid and effective method for separation of Staphylococcus aureus from somatic cells in mastitis milk.

Quantitative PCR can be an effective method for identifying the bacteria causing mastitis. However, PCR detection is hampered by the presence of inflammatory somatic cells. To eliminate this problem, we attempted to establish methods that allow the effective separation of bacterial cells from somatic cells in mastitis milk with amino-silica. Somatic cells and Staphylococcus aureus cells have different sizes, surface structures, and overall electrical charges; therefore, their adsorption and desorption behavior on amino-silica was also different. We found that although amino-silica could efficiently adsorb both somatic cells and Staph. aureus, somatic cells were adsorbed much more strongly than bacterial cells. We identified conditions under which most of the somatic cells adsorbed and only Staph. aureus desorbed from amino-silica upon addition of a desorption solution. We demonstrated that this procedure effectively eliminated somatic cells in heavily contaminated milk samples, which resulted in improved clarity of the PCR band. These results indicate that pretreatment of the samples with amino-silica made the PCR-based strategy for identifying and quantifying disease-causing bacteria applicable for all milk samples.

Technical note: measurement of ferritin in bovine milk and its clinical significance.

A quantitative ELISA was developed for bovine milk ferritin with an assay limit of 0.16 ng/mL of bovine spleen ferritin. Ferritin-binding activity was detected in bovine milk samples, and this binding activity was inhibited by increasing ionic strength with the addition of 0.5 M (NH4)2SO4. Heat treatment (60 degrees C, 20 min) of bovine milk in the presence of 0.5 M (NH4)2SO4 resulted in a 15 to 58% increase in ferritin concentrations compared with untreated samples. Although the recovery of bovine spleen ferritin added to milk was still low (55 to 90%), even in the presence of increased ionic strength with 0.5 M (NH4)2SO4, recovery was improved by heat treatment at 60 degrees C for 20 min (92 to 95%). Milk ferritin concentrations in 30 milk samples from quarters of 25 cows with mastitis (mean +/- SE: 134.2 +/- 28.7 ng/mL) were significantly higher than those in 17 quarter milk samples from 17 noninfected lactating cows (7.2 +/- 1.2 ng/mL), suggesting that bovine milk contains putative ferritin-binding proteins that inhibit immunoassay for milk ferritin and that bovine milk ferritin is an indicator of IMI.

Well-differentiated biphasic synovial sarcoma in the atlanto-occipital joint of a Holstein cow.

A 7-year-old Holstein cow developed a large cystic mass in the region between the atlantoaxis and larynx. The mass extended to the synovium in the atlanto-occipital joint. Many villous projections were present on the inner surface of the tumor tissue, and irregular clefts were formed in the inside. Two cell types, epithelioid-like synovioblasts and spindle cells, were present. Immunohistochemical analysis showed that the cells stained positively for cytokeratin (AE1/AE3) and vimentin. Both cells had similar fine structures ultra-structurally. Vacuoles present in the cytoplasm were full of an acid mucous substance. The tumor was diagnosed as a well-differentiated biphasic synovial sarcoma. This is the first report of a rare case of synovial sarcoma, from the viewpoint of its origin.