There was a guy called Doug Redelman who had some data on milk on the Purdue CD-ROM and an abstract on the ISAC 96 and 98 DETECTION OF SUBCLINICAL BOVINE MASTITIS BY THE DIFFERENTIAL INFLAMMATORY CELL COUNT (DICC) OF MILK Doug Redelman Sierra Cytometry Mastitis caused by Staphylococcus aureus is one of the most difficult and expensive problems for the dairy industry. Prompt detection of S. aureus mastitis is important because the organism is contagious and because chronic infections become increasingly refractory to treatment. S. aureus mastitis causes an influx of inflammatory cells into the milk but is generally difficult to detect since it causes few if any overt symptoms. The standard procedure for detecting cells in milk is the somatic cell count (SCC) that enumerates ethidium bromide stained nuclei in detergent treated milk. Unfortunately, although parturition is a high-risk time for mastitis, bovine milk produced early in lactation normally contains large numbers of cells so that the SCC is not very informative. The Differential Inflammatory Cell Count (DICC) is a flow cytometric procedure that identifies and enumerates the principal categories of cells in milk. The DICC is performed by diluting milk with a solution containing a dye that labels all cells (e.g., Hoechst 33342 or SYBR Green I) plus a second dye that labels only dead cells (propidium iodide). The live cells distinguished by the dye combination are then further classified by their light scatter properties. Several hundred normal-appearing milk samples collected at the beginning of lactation were examined via the DICC and by standard milk culture procedures. DICC analyses detected subclinical mastitis as effectively as the culture procedures illustrating that the DICC is a useful and informative test to detect subclinical mastitis including cases occurring early in lactation. Otherwise see Milk leucocyte population patterns in bovine udder infection of different aetiology Leitner G, Shoshani E, Krifucks O, Chaffer M, Saran A JOURNAL OF VETERINARY MEDICINE SERIES B-INFECTIOUS DISEASES AND VETERINARY PUBLIC HEALTH 47: (8) 581-589 OCT 2000 Abstract: This study compared the different leucocyte populations in milk from udders infected with different mastitic pathogens and in different stages of infection. Milk samples were collected from quarters free of intramammary infection, acutely infected with Escherichia coli or Staphylococcus aureus and chronically infected with S. aureus, coagulase-negative staphylococci (CNS) or Streptococcus dysgalactiae. Udder bacteriological status was confirmed after three consecutive bacteriological examinations from weekly quarter milk samples. At the time of the trial, milk samples were tested for somatic cell count (SCC) and differential cell count by both light microscopy (LM) and flow cytometry. Monoclonal antibody (mAb) CD11a/CD18 was used in order to differentiate between leucocytes and epithelial cells when tested by flow cytometry. Udder quarters free of intramammary infection had a mean SCC lower than 10(7) x 10(3) cells/ml in which the epithelial cells were the main cell type followed by polymorphonuclear cells (PMNs), while macrophages and lymphocytes had a lower concentration. Only 56% of the cells were labelled with the mAb anti-CD11a/CD18. In either acute E. coli- or S. aureus-infected quarters, SCC were significantly higher (P < 0.0001) than in samples from the time of inoculation, with over 90% of the cells labelled with the mAb anti-CD11a/CD18. The main cell type was neutrophils. In chronically infected cows, differences in SCC and in leucocyte patterns were found between infecting pathogens as well as between quarters harbouring the same pathogen. In all the chronically infected quarters, SCC was significantly higher (P < 0.05) than in uninfected ones. The distribution of the leucocyte patterns in the quarters infected with S. dysgalactiae did not differ from that in quarters with acute infection with both E. coli and S. aureus. In the cows chronically infected with S. aureus or CNS, the proportion of PMN was higher but not significantly different from quarters free of intramammary infection, while epithelial cells were significantly lower (P < 0.05). The T lymphocytes bearing CD4(+) or CD8(+) were significantly higher in quarters chronically infected with S. aureus than in quarters free of intramammary infection and in quarters acutely infected with either E. coli or S. aureus. In all samples B cells were negligible. and A STUDY BY FLOW-CYTOMETRY OF LYMPHOCYTES IN SOW COLOSTRUM LEJAN C RESEARCH IN VETERINARY SCIENCE 57: (3) 300-304 NOV 1994 Abstract: Mammary secretions contain leucocytes which may be of value to the neonate. The cells obtained from sow colostrum (1 to 2.5 x 10(6) ml(-1)) are mainly lymphocytes (10 to 25 per cent) and epithelial cells (more than 20 per cent). In milk, there are few lymphocytes (0.5 to 2 per cent) and mostly alveolar epithelial cells. The study of lymphocytes in the mammary secretions of sows has been made difficult by the high proportion of epithelial cells, which could not be separated from lymphocytes, and by a high background in membrane immunofluorescence labelling. This paper describes a method for the study of the cells in the mammary secretions of sows by flow cytometry. It showed that 70 to 90 per cent of colostral lymphocytes were T lymphocytes, with T8 lymphocytes predominating over T4, and that the ratio T4/T8 was significantly lower in colostrum (0.57) than in blood (0.80). There were no lymphocytes expressing interleukin-2-receptors in the colostrum of sows. If you want to look at human milk try to get hold of the thesis of Kirsi-Marjut Järvinen from the Department of Dermatology, Skin and Allergy Hospital University of Helsinki, Finland on HUMAN MILK IMMUNOLOGY IN RELATION TO THE DEVELOPMENT OF COW’S MILK ALLERGY IN THE BREAST-FED who used microscopy for analysis or look at the Determination and Study on Immune Active Cell and TNF-a in Colostrums FU Wenping, CHANG Guizhen, LI Yarui. CHINESE JOURNAL OF PERINATAL MEDICINE 3: (3) 169-171 2000, Abstract? Objective To compare immune active cell and TNF-a’s contents between preterm colostrums and full term colostrums to suport the preterm infants to feed their own mothers' breast milk early. Methods Fifty samples of preterm and full term colostrums were obtained from mothers who are 2 to 5 days after delivery. These samples were measured by percentages of differentiation's cell expessing CD3?CD4?CD8?CD19 clusters by direct or indirect immunofluorescence using a flow cytometre and Tumor nectosis factor-alpha's contents by ELISA method. Results The percentages of T3?T4?T8 lymphocytes and ratio of T4 to T8 in preterm clolstrums were lower than those of full term colostrums, respectively. The percentage of B lymphocyte in preterm colostrums was a little higher than full term. But data were not significantly difference(P>0.05).While the level of Tumor nectosis factor-alpha in preterm colostrums was higher significantly than that of full term.(P<0.05) Conclusion Preterm colostrums are rich in immune active substances. It is vital to feed preterm colostrum as early as possible. However, it might need translation Foss Electric in Daenmanrk was doing cytometry in milk if I remember rightly. They might also have some appropriate protocols, I would have thought investigations into beer would be much more inspiring if you are in Munich Regards Gerhard -----Original Message----- From: Dr. Wolfgang Beisker [SMTP:beisker@gsf.de] Sent: Wednesday, July 18, 2001 5:00 PM To: Cytometry Mailing List Subject: Cells in Milk Hi, flowers I am looking for Infos about measuring cells in milk. There has been an article in Cytometry, in 1988, which I have. It should be possible to extract the cells (lymphocytes, granulocytes etc) with lightscatter, what I have heard so far, but we not really successful. Perhaps there are more outside there, who may be interested in... Best regards from Munich Wolfgang Beisker Flow is Fun -- Dr. Wolfgang Beisker GSF - National Research Center of Environment and Health Flow Cytometry Group Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany
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