The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.
ABSTRACT TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.
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ABSTRACT: Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.Veterinary Immunology and Immunopathology 10/2004; 101(3-4):191-202. · 1.88 Impact Factor
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ABSTRACT: The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24h elicited a marked increase in mRNA expression for IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway, although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components.Cytokine 08/2005; 31(1):72-86. · 2.52 Impact Factor
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ABSTRACT: Interleukin-12 (IL-12) and interleukin-23 (IL-23) are proinflammatory cytokines produced by macrophages and dendritic cells in response to infection with intracellular pathogens. The IL-12 receptor (IL-12R) is a heterodimer composed of 2 subunits, β1 and β2. The IL-23 receptor (IL-23R) is a heterodimer composed of the IL-12Rβ1 subunit and a unique IL-23R subunit. Given the importance of IL-12 and IL-23 for modulating inflammation and the host immune response, the IL-12 and IL-23 receptor genes may be suitable candidate genes for studying disease resistance in dairy cattle. We hypothesize that single nucleotide polymorphisms (SNP) exist within these genes and that they contribute to variation in health and production traits in dairy cattle. To investigate this, a selective DNA pool was constructed using bull semen based on the estimated breeding values for somatic cell score (SCS), an indicator trait used to achieve genetic improvement for resistance to mastitis. Gene segments were amplified from this pool by PCR and the amplicons were sequenced to reveal SNP. A total of 10 SNP, including 2 in IL-12Rβ1, 5 in IL-12Rβ2, and 3 in IL-23R were identified. The SNP (n=5) were found in the 5' untranslated region (UTR) putative promoter regions of the genes, and SNP IL-23R c.1714A>C was a nonsynonymous SNP. Canadian Holstein bulls (n=492) were genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA). No association was found with SCS based on bull deregressed estimated breeding values for SCS; however, associations of SNP in the IL-12Rβ2 gene (c.-511A>G, c.87A>G, c.2957A>C) were found with milk and protein yield. Further investigation will be required to elucidate the biological and practical relevance of these SNP.Journal of Dairy Science 10/2010; 93(10):4860-71. · 2.57 Impact Factor
Department of Microbiology and Parasitology, College of Veterinary Medicine and Animal Resources, King Faisal University,
The Cytokine Markers in Staphylococcus aureus Mastitis of Bovine Mammary
A. M. Alluwaimi1,5, C. M. Leutenegger2, T. B. Farver3, P. V. Rossitto4, W. L. Smith4and J. S. Cullor4
Addresses of authors:1Department of Microbiology and Parasitology, College of Veterinary Medicine and Animal Resources,
King Faisal University, PO Box 35252, Al-AHSAA, 31982 Saudi Arabia;2Departments of Medicine and Epidemiology,
3Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis 95616, USA;
4Department of Population Health and Reproduction, Veterinary Medicine Teaching and Research Center, University of
California, Davis, Tulare, CA 93274, USA;5Corresponding author: Tel.: 966-55935964; fax: 966-3-5816635; e-mail:
With 3 figures and 3 tablesReceived for publication August 13, 2002
TaqMan?real time PCR was used to study the transcrip-
tional activity of the bovine IL-2, IL-6, IL-12p40, IFN-c,
TNF-a and granulocyte-monocyte colony stimulating factor
of whole milk cells in bovine mammary gland experimentally
infected with Staphylococcus aureus. Cytokine transcriptional
activity was monitored at 7, 24 and 32 h Post-infection (Pi).
IL-12 and TNF-a levels were significantly elevated at 24 h Pi
followed by sharp decrease at 32 h pi. IL-2 level was de-
creased at 32 h pi. IL-12 and IFN-c showed a significant
interaction at 24 h pi. The significant elevations of the IL-12
and TNF-a transcriptional level most likely indicate their
important role in regulation of the immune responses of
bovine mammary gland in S. aureus infection. Depression of
IL-2 could reflect the suppressive nature of the S. aureus
Mastitis is inflammation of mammary gland mainly as a result
of invasion of pathogenic microorganisms (Bramley, 1991;
Radostits et al., 1994). Staphylococcus aureus (S. aureus)
mastitis is a contagious disease, characterized by congestion,
hardness of udder and formation of milk clots (Sutra and
Poutrel, 1994). The pathological feature of S. aureus infection
is formation of abscesses in deep tissue. These occluded areas
become foci for bacterial shedding in chronically infected
animals and refractory to antibiotic therapy (Sutra and
Poutrel, 1994). The significant diagnostic feature of S. aureus
mastitis is the presence of high number of viable bacteria in
milk and high number of somatic cells, especially polymor-
phonuclear cells in the mammary gland tissue and milk (Daley
et al., 1991b).
The nature and potency of the host immune responses are
critical in defining the outcome of the bacterial infection
(Riollet et al., 2000a). The immune responses in S. aureus
infected mammary glands are defined by the lymphocyte
subpopulations, and other recruited leucocytes and cytokines
released by them (Yokomizo et al., 1994; Ferens et al., 1998;
Persson Waller and Golditz, 1999). However, the virulent
arsenal of S. aureus enables the organism to deceive and
suppress the immune system (Sutra and Poutrel, 1994).
Enterotoxin and toxic shock syndrome toxins are the most
important virulent factors that mediate S. aureus pathogen-
icity. Numerous reports indicated that S. aureus enterotoxin A
(SEA), B (SEB), C (SEC) and toxic shock syndrome toxin-1
(TSST-1), act as superantigens (SAg) by activating a wide
range of T-lymphocytes and mononuclear cells and stimulating
the release of various types of cytokines (Bjork et al., 1992;
Yokomizo et al., 1994; Brunmark and O’Rourke, 1997; Ferens
et al., 1998). SEA, SEB, SEC and TSST-1 were shown to
induce polyclonal activation of bovine T-cells, CD4+and
CD8+subsets, and monocytes with release of wide range of
cytokines like, interleukin-2 (IL-2), interferon-c (IFN-c) and
tumor necrosis factor-a (TNF-a) (Yokomizo et al., 1994).
Furthermore, it was reported that these SAgs stimulate high
level of interleukin-10 (IL-10) and interleukin-4 (IL-4) (Ferens
et al., 1998). Excessive production of IL-10 and IL-4 contri-
buted to the development of immunosuppression in S. aureus
infection by temporary inhibition of CD4+cells and recruit-
ment of high level of CD8+T suppressor cells (Ferens et al.,
1998). Riollet et al. (2000b) examined the pathophysiological
changes in bovine mammary gland infused with S. aureus
a-toxin. In addition to the early recruitment of neutrophils, an
increase in CD8+ T cells of cd phenotype was observed.
Expression of interleukin-12 (IL-12) mRNA in milk cells was
also detected in the early stages of post-challenge. Changes in
cellular subsets and cytokine synthesis were considered crucial
in orchestrating the inflammatory responses to S. aureus
In this study, the pattern of IL-2, interleukin-6 (IL-6), IL-12
subunit P40, IFN-c, TNF-a and granulocyte-monocyte colony
stimulating factor (GM-CSF) transcription in the bovine
mammary gland experimentally infected with S. aureus was
pursued. Early cytokine transcriptional changes could be
useful as a forecasting tool in detection of the subclinical
S. aureus mastitis. Transcriptional pattern of these cytokines in
the early stages of S. aureus infection could help to unravel
their role in the pathophysiology of S. aureus mastitis. Finally,
important key cytokine marker(s) could emerge that provide
efficient diagnostic and therapeutic mean in the early detection
of S. aureus mastitis.
U. S. Copyright Clearance Center Code Statement: 0931–1793/2003/5003–0105$15.00/0www.blackwell.de/synergy
J. Vet. Med. B 50, 105–111 (2003)
? 2003 Blackwell Verlag, Berlin
Materials and Methods
Six Holstein dairy cows in their third lactation or higher were
purchased from a local dairy producer. Individual quarters
were considered healthy after 3 months of routine microbio-
logy screening, sampled twice 21 days apart, until the day of
experiment (Milk Quality lab, School of Veterinary Medicine,
University of California at Davis) and somatic cell count
(SCC) [Diary Herd Improvement Association (DHIA), Tulare,
Preparation of S. aureus inoculum
Staphylococcus aureus, Strain Newbould 305 (Parsad and
Newbould, 1968 ) was obtained from Dr William Owens, Hill
Farm Research Station, Louisiana State University.
The inoculum was prepared by growing the challenge
organism three times on bovine blood agar (BBA) medium
and then inoculated into brain heart infusion (BHI) broth the
day before inoculation. The overnight stock culture was
diluted to 1 · 105CFU/ml by measuring adsorbance at 620
nm (0.025 OD ¼ 1 · 107). The culture density of the bacter-
ium in BHI broth was then diluted to 150 CFU/ml in 10 mm
NaPO4 (pH 7.3) before inoculation. The CFU/ml in the
infusion inocula was confirmed by viable counting of direct
plating on BBA.
The randomly designated quarters of each cow were milked out
and the teat end was swabbed with 70% alcohol. Then 2 ml of
the inoculum was injected through the teat end with a syringe
fitted with a 11=3-inch teat infusion cannula. No control quar-
ters were designated; however, the somatic cells were moni-
tored in healthy quarters in parallel to the infected quarters.*
Sampling and testing for S. aureus
Milk samples were collected from the infected and non-infect-
ed quarters at the time points immediately before inoculation,
7, 24 and 32 h post-infection (pi). Individual quarters were
predipped with Teatguard (Ecolab, St Paul, MN, USA)
cleaned, and dried. Individual teats were stripped and the first
2–3 streams of milk was discarded, then 40 ml of milk was
collected for cytokine analysis and bacterial count (Milk
Quality Lab, School of Veterinary Medicine, University of
California, Davis CA, USA).
Staphylococcus aureus was identified according to the
microbiological procedure stated by National mastitis council
(National Mastitis Council, 1990). Briefly, milk samples were
swabbed onto BBA agar and incubated overnight at 37?C, for
bacterial identification the following tests were performed,
positive catalase reaction, hemolysis pattern, and positive test
for coagulase on rabbit plasma (National Mastitis Council,
1990). Duplicate milk samples were also submitted to DHIA
Preparation of milk cells
Each milk sample was centrifuged at 700 g for 20 min and the
pellet washed twice with 50 ml phosphate buffered saline.
Using commercial kit (RNeasy mini kit, Qiagen, Valencia, CA)
5 · 106cells were lysed with 350 ll of the lysis buffer according
to the manufacturer’s recommendations and kept at )80?C
until extraction of RNA and synthesis of complementary
The total RNA (tRNA) was extracted from the frozen lysed
milk cells using a commercial RNeasy?total RNA extraction
kit (RNeasy mini kit, Qiagen). To remove contaminating
genomic DNA, the extracted tRNA was treated with 10 U/ll
of RNase free DNase I (DNase, Amersham Pharmacia
Biotech Inc, Piscataway, NJ, USA) at 37?C for 10 min, heat
inactivating at 95?C for 5 min and chilling on ice.
Reverse transcription of total RNA
For the synthesis of cDNA, 20 ll reverse transcription (RT)
mix containing 50 U MuLVreverse transcriptase, 5 mm MgCl2,
10 mm Tris–HCl (pH 8.3), 50 mm KCl, 1.25 lm random
hexadeoxyribonucleotide [pd (N)6] primers, 0.5 U/ll RNase
inhibitor (GeneAmp?RNA PCR kit; PE-Biosystems, Foster
City, CA, USA) and 1 mm dNTPs (Amersham Pharmacia
Biotech), was heated to 42?C for 60 min and then inacti-
vated at 95?C for 5 min. The final volume of the mix was
adjusted to 100 ll with RNase free water. The cDNA was
analysed immediately or stored at )30?C until use.
Quantification of cytokine cDNA by real-time TaqMan? ?PCR
The details of the real-time TaqMan?PCR system of bovine
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and,
the design of cytokine primers and probes were previously
described (Leutenegger et al., 2000). The sequence of the
primers and probes are listed in Table 1. Briefly each bovine
cDNA was assayed for the cytokine profile and for GAPDH
as an endogenous control in separate wells in 25 ll PCR
mixtures containing final concentrations of 400 nm primer,
80 nm probe and commercially available PCR mastermix
(TaqMan?Universal PCR Mastermix, PE-Biosystems) con-
taining 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 5 mm MgCl2,
2.5 mm deoxynucleotide triphosphates, 0.625 U of AmpliTaq
Gold DNA polymerase per reaction, 0.25 U AmpErase UNG
per reaction and 10 ll of the diluted cDNA sample. The
samples were placed in 96 well plates and amplified in an
automated fluorometer (ABI Prism 7700 Sequence Detection
System, PE-Biosystems). Amplification conditions were 2 min
at 50?C, 10 min at 95?C, 40 cycles of 15 s at 95?C and 60 s at
The cytokine transcripts were calculated according to the
comparative threshold cycle method (Leutenegger et al.,
All statistical analyses were performed using BMDP statis-
tical software (BMDP Statistical Software, Inc., Statistical
*Our previous study on the normal mammary gland was
considered as reference to the cytokine transcriptional activity
in the healthy mammary gland (Alluwaimi and Cullor, 2002).
106 A. M. Alluwaimi et al.
Solutions, Limited, Crosse’s Green, Cork, Ireland). The
statistical methods used were one- and two-factor analysis
Descriptive statistics revealed variance instability, highly
significant violations of the normality assumption for para-
metric comparison of two independent groups, and highly
significant skewness and kurtosis coefficients for all cytokine
responses eliminated or dramatically reduced the distribution-
al problems noted above.
All infected quarters developed clinical mastitis by 24 h pi.
Infected quarters manifested gradual decrease in milk produc-
tion, changes in milk appearance, particularly appearance of
milk clots, and heat.
Bacterial count indicated a sharp rise at 24 h pi (8000 CFU/
ml ± 4381). No clinical sign was noticed in normal quarters.
Somatic cell count
A continuous rise of SCC at all time points pi was recorded
(Fig. 1). Whereas, in normal quarters SCC was far below the
level of that of infected quarters (Table 2).
One-factor analysis of variance with time being within
subject (repeated measures) factor revealed significant time
effect for SCC (P < 0.0001). The SCC time effect had a highly
significant (positive) linear component. Finally, SCC indicated
a significant difference between pre-infection, 7 and 24 h pi
The pattern of cytokine transcription at different time points pi
The mRNA transcription of all cytokines was detected at all
time points. At all times, TNF-a had the highest level of
transcription whereas, IL-6 and IL-2 had the lowest level.
However, IL-12 had the second highest transcriptional level at
24 and 32 h pi. Figure 2 shows the uniform pattern of cytokine
transcription seen for TNF-a, GM-CSF, IL-6 and IL-12. The
transcriptional level of these cytokines displayed a sharp drop
at 7 h followed by a dramatic rise at 24 h and a sharp decrease
at 32 h. In contrast, IFN-c and IL-2 transcription recorded a
continuous reduction over the time (Fig. 3).
As the level of each of the six cytokines was determined at
pre-infection, 7, 24 and 32 h, a total of 24 responses were
available from each experimentally infected quarter, one
infected quarter from each of the six studied cows. The
statistical method used to analyze these data was the two-
factor repeated measures analysis of variance with type of
cytokine (?cytokine?) and time of response (?time?) being the
repeated measures factors. The initial analysis showed a highly
significant (P < 0.0001) cytokine by time interaction. This
means that the nature of the responses among the cytokines
was not parallel for all times and the nature of the responses
recorded at pre-infection, and 7, 24 and 32 h was not the
parallel for all cytokines. Therefore, four one-factor repeated
Table 1. Sequence of PCR primers and TaqMan?probes specific bovine GAPDH, cytokines and growth factorsa
Probe sequence (5¢-3¢)
aIntron–exon junctions are italicized.
Cytokine Markers in Staphylococcus aureus Mastitis107
measures analyses of variance were performed to determine the
significance of the cytokine effect at each time the cytokine
levels were recorded. To determine the nature of significant
cytokine effects, pairwise multiple comparisons of mean values
were performed using the Bonferroni confidence interval
method. Also, six one-factor repeated measures analyses of
variance were performed to determine the significance of the
time effect for each cytokine. Time effects were further
examined by the linear, quadratic, and cubic functions of
time. All effects were determined to be statistically significant
when P < 0.05.
Analysis of time-effect on the level of each cytokine
For each cytokine, a one-factor analysis of variance with time
being within subject (repeated measured) factor revealed
(P ¼ 0.010) TNF-a (P ¼ 0.030). The IL-2 time effect had a
significant (negative) linear component (P ¼ 0.005). However,
the TNF-a time effect had significant cubic component
(P ¼ 0.002). Whereas, the IL-12 time effect had significant
(positive) linear (P ¼ 0.03) and cubic (P ¼ 0.02) components.
Table 3 shows the significance of the linear, quadratic and
cubic components of the observed time effects.
The overall analysis revealed that IL-12 and TNF-a were the
only cytokines that showed a significant positive difference in
their transcriptional level between time points, 7 and 24 h pi.
IL-2 however, showed a significant negative difference in its
transcriptional activity between 7 and 24 h pi.
IL-2(P ¼ 0.008), IL-12
Comparison of the cytokine transcriptional pattern
One-factor analysis of variance revealed highly significant
(P < 0.0001) cytokine effect at each time period when data
were recorded. The comparison analysis of cytokine showed
that transcription of TNF-a was significantly higher than any
other cytokines at pre-infection and 7 h pi. Moreover, at the
same time points, IL-6 was significantly lower than that of all
cytokines, except IL-2 at 7 h pi. At 24 h pi the TNF-a and IL-
12 transcription was not significantly different from each other
but both was significantly higher than the levels of IFN-c , IL-2
and IL-6. At 24 and 32 h pi, IL-2 and IL-6 transcription was
not significantly different but were both significantly lower
than the levels of all other cytokines. Table 3 summarizes the
results of comparisons between cytokines.
Early diagnosis of S. aureus mastitis is vital in its successful
treatment and eradication (Bramley, 1991, Radostits et al.,
1994). Monitoring cytokines involved in the regulation of
immune responses during the infection could be useful in
determining cytokine marker(s) that could be utilized as a
forecasting tool in the early diagnosis of S. aureus infection. In
this study the transcription of six bovine cytokines, IL-2, IL-6,
IL-12, IFN-c, TNF-a and GM-CSF were monitored in the
bovine mammary gland experimentally infected with S. aureus
at pre-infection and 7, 24 and 32 h pi.
Fig. 1. Geometric mean of somatic cells count (SCC) in Staphylococ-
cus aureus infected bovine mammary gland at different time points.
The SCC showed a continuous increase in its level at all time points
post-infection. The significant differences over time are indicated by
Table 3. Comparison among mean cytokine responses at four time points (numbers) data were recorded and summary of the time effect
(alphabet) for each cytokine1
P-values for time effect components
Pre-infection 7 h pi2
24 h pi32 h pi LinearQuad Cubic
1For each cytokine, geometric mean values with a superscript in common are not significantly different from each other with a level of
significance of 5% over all comparisons.2Post-infection.
Table 2. The SCC (cells/ml) of normal and infected quarters post-
Mean SCC of normal
SCC of infected
7 h pi
24 h pi
32 h pi
91 913 ± 21 598
93 928 ± 56 777
136 697 ± 114 233
121 546 ± 114 512
58 815 ± 69, 749
6 230 000
108 A. M. Alluwaimi et al.
A peculiar picture in the mode of mRNA transcription of
IL-12, TNF-a and GM-CSF emerged. These cytokines
expressed a sharp elevation and reduction in their transcription
at 24 and 32 h pi, respectively. Although, analysis of cytokine
interaction at different time points did not disclose significant
interaction between IL-12 and TNF-a, both cytokines
expressed very similar pattern in their rate of transcription
and mode of elevation at the 24 h pi. The significant sharp
increase and decrease in the transcriptional activity of IL-12
and TNF-a at 24 h pi could be explained in the view of the
previous S. aureus experimental infection studies (Daley et al.,
1991a,b). It was shown that experimental infection of mam-
mary gland with S. aureus was associated with cyclic increase
and decrease in the quantity of viable bacteria with inverse
concomitant cycling of neutrophils cells. The cycling was also
associated with great phagocytosis and superoxide anion
impairment (Daley et al., 1991a,b). On the other hand, Riollet
et al. (2001) noticed that neutrophils from quarters infected
with S. aureus were not in a highly activated state. Although in
this study the level of somatic cells in the infected quarters
showed a continuous increase, the duration of the experiment
and time intervals probably were not long enough to exploit
the cycling phenomenon. Therefore, the sharp increase and
decrease in the transcriptional level of TNF-a and IL-12 could
be the result of the functional impairment of somatic cells in
the infected mammary gland.
The significant increase in the activity of IL-12 and TNF-a
transcription at 24 h pi could be attributed to their important
mechanism that these cytokines may perform in S. aureus
There is compelling evidence that neutrophils play an
important role in maintaining the inflammatory responses
(Riollet et al., 2000a). Activated neutrophils were shown to
secret a wide range of pro- and/or anti-inflammatory cytokines
like interleukin-1b, IL-6, GM-CSF, interleukin-8 (IL-8), IL-12
and TNF-a (Cassatella, 1995; Bliss et al., 1999, 2000). Hence,
augmentation of TNF-a transcription was most probably
because of the activation of the newly recruited neutrophils
through up-regulation of IL-8 (Riollet et al., 2000a). Monit-
oring the IL-8 transcription in S. aureus infected mammary
glands exhibited a transcriptional pattern similar to that of
TNF-a (unpublished data). Interestingly, infusion of mam-
mary glands with TNF-a induced large neutrophils influx,
whereas infusion of a known chemo-attractant-like comple-
ment component 5a or IL-8 recruited small amounts of
neutrophils (Persson et al., 1993). Therefore, TNF-a elevation
in the mammary gland could reflect its important role in
aggravating neutrophil chemo-attraction to the mammary
gland. TNF-a could act as neutrophil chemo-attractant via
enhancing neutrophil attachment to the endothelium or
through induction of chemo-attractant cytokines (Larrick
and Kunchel, 1988).
The capability of neutrophils to produce IL-12 in mice and
human was seen as an important mechanism in the inflamma-
tory responses by polarizing CD4+ T cells towards type-1 T
cell phenotypes (Bliss et al., 1999, 2000). In the bovine
Fig. 2. Geometric mean of relative
quantification of TNF-a, GM-CS-
F, IL-12 and IL-6 at different time
points. Despite the differences in
their rate of transcription,
expressed a similar pattern of
transcription were the time data
were recorded. The significant
differences over time are indicated
by broken lines.
Cytokine Markers in Staphylococcus aureus Mastitis109
mammary gland, Riollet et al., (2000b, 2001) detected IL-12
mRNA in neutrophils from glands injected with S. aureus
a-toxin and S. aureus. IL-12 is an important mediator that
links the innate with the specific immunity (Trinchieri, 1995).
This cytokine appears to have a central role in maintaining
antibody production (Trinchieri, 1995). Antibodies play a
crucial role in S. aureus infection by interferring with bacterial
attachment, neutralizing the bacterial toxins and enhancing
phagocytosis via opsonization (Sutra and Poutrel, 1994).
Interleukin-2 and IFN-c transcription continuously declined
with time pi. Decrease of IFN-c transcription was not
significant at all time points pi. It is well documented that
bovine IL-12 has a synergistic effect on IFN-c (Collins et al.,
1998, 1999). Nevertheless, impairment of IFN-c to respond to
IL-12 synergistic effect could be attributed to the continuous
reduction of IL-2. It was shown that synergism of IL-2 with
IL-12 increased the IFN-c mRNA half-life but did not increase
the IFN-c transcriptional rate above that induced by IL-12
alone (Chan et al., 1992). Alternatively, transcription of
IL-2 mRNA level was significantly dropped between 24 and
32 h pi. The significant drop in the IL-2 transcription is
difficult to be justified in view of the available data. Neverthe-
less, evidence is available that might explain the suppressive
nature of S. aureus mastitis. Ferens et al. (1998) reported that
S. aureus enterotoxins stimulated a high level of IL-4 and
IL-10 mRNA in peripheral blood mononuclear cells (PBMC).
Consequently, these cytokines induced a shift in T-cell
phenotypes from CD4+T-cells to CD8+T-cell phenotype.
Riollet et al. (2000b) confirmed the previous findings by
demonstrating a shift in cells expressing CD4+markers to
CD8+T cells. Moreover, Park et al. (1993) demonstrated that
CD4+T cells phenotype of bovine mammary gland infected
with S. aureus suffered a severe depression in their prolifer-
ation because of the activation of CD8+T cell which were
characterized with their suppressive effect. RT-PCR was also
failed to detect any IL-2 mRNA transcripts in cells of infected
quarters (Riollet et al., 2001). Hence, in the view of the above
evidence a dramatic decrease in the IL-2 and IFN-c transcrip-
tion was most probably as a result of anergy of CD4+T cells
and domination of CD8+T-cell phenotype. It is worthy to
note that treatment of S. aureus infected mammary gland with
rBoIL-2 enhanced the quality and quantity of neutrophils in
the infected quarters (Nickerson et al., 1989). Nevertheless, the
treatment had a transient effect on the activated neutrophils
because of the significant depression caused by the infection
(Daley et al., 1991b).
TNF-a, IL-6 and IFN-c were studied in a great detail in
coliform and endotoxin mastitis (Shuster et al., 1993, 1997;
Nakajima et al., 1997; Hoeben et al., 2000;
2001). The level of TNF-a in infected quarters and serum was
elevated only when the infection advanced to the severe stage.
However, in mild infection level of TNF-a was restricted to the
infected quarters (Shuster et al., 1993; Nakajima et al., 1997;
Hoeben et al., 2000). Contrary to the IL-6 transcriptional level
in S. aureus mastitis, level of IL-6 in coliform mastitis elevated
markedly. IL-6 reached its high level at 14 h pi (Shuster et al.,
1997). In contrast to the continuous decline in IFN-c
transcription in the present study, the level of secreted IFN-c
was dramatically increased in quarters with natural coliform
mastitis (Hisaeda et al., 2001).
Although S. aureus and coliform mastitis are associated with
high influx of neutrophils, their activation state and CD
markers expression are different. Shuster et al. (1997) showed
that recruited neutrophils expressed strong chemotactic activ-
ity with the ability to upregulate CD18. Whereas, neutrophils
form quarters chronically infected with S. aureus were less
active and with low tendency to express CD18 and CD11b
(Riollet et al., 2001).
In conclusion, IL-12 and TNF-a could play important
role in the immune responses to S. aureus infection in the
bovine mammary gland. Their significant difference between
7 and 24 h pi is a possible indication of their potential in
the pathobiology of S. aureus mastitis. Further study on
their role by reducing the time intervals at the early
infection most probably will help in defining their role in
the S. aureus mastitis. However, elucidation of the IL-4,
IL-10 and IL-8 transcriptional pattern in the S. aureus
mastitis is important, too. A significant depression of IL-2
detected in this study substantiates the need for immuno-
therapeutical intervention with IL-2 to restore the hampered
Hisaeda et al.,
Ahmed M. Alluwaimi was funded by Fullbright Fellowship.
C.M. Leutenegger is a recipient of a Swiss National Science
Foundation Grant no 823A-53569. Special thanks for Dr
Algailani A. Amin for reviewing this manuscript.
Fig. 3. Geometric mean of relative quantitation of IFN-c , IL-2 at
different time points. Both cytokines indicated a continuous decrease
in their transcriptional level were the time data were recorded. Only
IL-2 showed a significant fall in its transcriptional level at 24 and 32 h
post-infection. The significant differences over time are indicated by
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