Cells in Milk

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at Unilever.com
Thu Jul 19 09:21:28 EST 2001


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 at 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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