Cytokine mRNA profiling of peripheral blood

mononuclear cells from trypanotolerant and

trypanosusceptible cattle infected with Trypanosoma
congolense
Grace M. O'Gorman, Stephen D. E. Park, Emmeline W. Hill, Kieran G. Meade, Laura C.
Mitchell, Morris Agaba, John P. Gibson, Olivier Hanotte, Jan Naessens, Stephen J. Kemp
and David E. MacHugh
Physiol. Genomics 28:53-61, 2007. First published 19 September 2006;
doi: 10.1152/physiolgenomics.00100.2006

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 Physiol Genomics 28: 53– 61, 2006.
First published September 19, 2006; doi:10.1152/physiolgenomics.00100.2006.

CALL FOR PAPERS 2nd International Symposium on Animal Functional Genomics

Cytokine mRNA profiling of peripheral blood mononuclear cells from

trypanotolerant and trypanosusceptible cattle infected with
Trypanosoma congolense
Grace M. O’Gorman,1 Stephen D. E. Park,1 Emmeline W. Hill,1 Kieran G. Meade,1
Laura C. Mitchell,2 Morris Agaba,3 John P. Gibson,4 Olivier Hanotte,3
Jan Naessens,3 Stephen J. Kemp,2 and David E. MacHugh1
1
Animal Genomics Laboratory, School of Agriculture, Food Science and Veterinary Medicine, College of Life Sciences,

Downloaded from http://physiolgenomics.physiology.org/ by guest on April 19, 2013

University College Dublin, Belfield, Dublin, Ireland; 2School of Biological Sciences, University
of Liverpool, Liverpool, United Kingdom; 3International Livestock Research Institute, Nairobi, Kenya;
and 4The Institute of Genomics and Bioinformatics, University of New England, Armidale, Australia
Submitted 31 May 2006; accepted in final form 22 August 2006

O’Gorman GM, Park SD, Hill EW, Meade KG, Mitchell LC,
Agaba M, Gibson JP, Hanotte O, Naessens J, Kemp SJ,
MacHugh DE. Cytokine mRNA profiling of peripheral blood
mononuclear cells from trypanotolerant and trypanosusceptible
cattle infected with Trypanosoma congolense. Physiol Genomics
28: 53– 61, 2006. First published September 19, 2006;
doi:10.1152/physiolgenomics.00100.2006.—To examine differences
in cytokine profiles that may confer tolerance/susceptibility to bovine
African trypanosomiasis, N’Dama (trypanotolerant, n ⫽ 8) and Boran
(trypanosusceptible, n ⫽ 8) cattle were experimentally challenged
with Trypanosoma congolense. Blood samples were collected over a
34-day period, and RNA was extracted from peripheral blood mono-
nuclear cells. The expression levels of a panel of 14 cytokines were
profiled over the time course of infection and between breeds. Mes-
senger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN
genes were significantly downregulated across the time course of
infection in both breeds. There was an early increase in transcripts for
genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and
IL12) in N’Dama by 14 days postinfection (dpi) compared with
preinfection levels that was not detected in the susceptible Boran
breed. By the time of peak parasitemia, a type 2 helper T cells
(TH2)-like cytokine environment was prevalent that was particularly
evident in the Boran. Increases in transcripts for the IL6 (29 and 34
dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were
higher in the Boran compared with N’Dama. These findings highlight
the implications for using murine models to study the bovine immune
response to trypanosomiasis, where in some cases cytokine expression
patterns differ. Overall, these data suggest that the trypanotolerant
N’Dama are more capable of responding very early in infection with
proinflammatory and TH1 type cytokines than the trypanosusceptible
Boran and may explain why N’Dama control parasitemia more
efficiently than Boran during the early stages of infection.
trypanosomiasis; gene expression; quantitative real-time RT-PCR
BOVINE AFRICAN TRYPANOSOMIASIS, or “nagana,” is a wasting
disease that affects cattle populations across much of central
Article published online before print. See web site for date of publication
(http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: D. E. MacHugh,
Animal Genomics Laboratory, School of Agriculture, Food Science and
Veterinary Medicine, College of Life Sciences, Univ. College Dublin, Belfield,
2Dublin 4, Ireland, UK (e-mail: david.machugh@ucd.ie.).
Africa. The disease is caused by the protozoan parasites
Trypanosoma congolense, T. vivax, and T. brucei brucei and is
transmitted in the saliva of biting tsetse flies (Glossina spp.).
When an infected tseste fly bites a mammalian host and
deposits metacyclic forms of the parasite at the site of inocu-
lation, a raised inflammatory swelling called a chancre devel-
ops within a few days that precedes parasitemia and other
clinical signs (1). Thereafter, metacyclics differentiate into
bloodstream forms, migrate to the bloodstream, and cause
systemic infection (34). Parasitemia, detected in the peripheral
blood, is usually apparent 1–3 wk later and may persist in
waves for many months with fever occurring intermittently
(14). From the period when parasitemia is first detected, there
is a rapid decrease in hematological parameters. Major clinical
signs in ruminants include anemia (6, 30), lymphoid enlarge-
ment, lethargy, and loss of condition with progression of the
disease. Marked immunosuppression is usually observed with
reduced host resistance to secondary infections (7). Typically,
this disease is chronic, extending over many months, and fatal
if untreated (14).1
The scale of this problem is immense; indeed, the tsetse belt
of sub-Saharan Africa, where the disease is endemic and a
major constraint to livestock production, covers 37 countries
and an area of ⬃10 million km2. In many parts of Africa, cattle
are a vital source of income, particularly for smallholder
farmers, and the annual cost of bovine trypanosomiasis to
African agriculture has been estimated at 1 billion US dollars
per annum (21). However, over thousands of years, and pre-
sumably under high tsetse challenge, certain West African Bos
taurus cattle populations have evolved tolerance to trypanoso-
miasis (31). One of the best-characterized trypanotolerant
breeds is the N’Dama, and this cattle population represents
both a unique genetic resource for African cattle agriculture
and an important model to understand mechanisms of trypano-
tolerance (29). In contrast, zebu populations (B. indicus) tend
1
The 2nd International Symposium on Animal Functional Genomics was
held May 16 –19, 2006, at Michigan State University in East Lansing, MI, and
was organized by Jeanne Burton of Michigan State University and Guilherme
J. M. Rosa of University of Wisconsin, Madison, WI (see meeting report by
Drs. Burton and Rosa, Physiol Genomics 28: 1-4, 2006).

1094-8341/06 $8.00 Copyright © 2006 the American Physiological Society 53

54 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE
to be susceptible and generally can only be maintained through
the use of costly trypanocidal drugs. The well-characterized
trypanosusceptible East African Boran breed has been used as
a model of trypanosusceptibility for many previous studies
(44).
The profound susceptibility of many breeds of cattle to
trypanosomiasis is easily understood when the complexity of
the host-parasite relationship itself is considered. T. congolense
is a unicellular, extracellular, motile parasite with a specific
preference for the peripheral vasculature, where it is found
either free swimming or attached via its anterior end to endo-
thelial cells. The parasite is completely exposed to the host’s
immune system and yet, in many instances, survives to prolif-
erate, resulting in characteristic waves of parasitemia (for
review, see Ref. 26). The array of host evasion mechanisms are
complex (for review, see Ref. 9) and include the well-known
phenomenon of antigenic variation of the variable surface
glycoprotein (VSG) (2, 4, 8). In this regard, successful switch-
ing of the antigenic coat of the trypanosome plays a key role in
avoidance of the host immune system. Despite the battery of
tactics utilized by the parasite to evade detection and destruc-
tion, the superior response of N’Dama cattle has allowed these
animals to survive and be productive in areas of tsetse chal-
lenge without the use of trypanocidal drugs (33). It is impor-
tant, however, to acknowledge that N’Dama and Boran cattle
are equally susceptible to the initial infection; however, once
infected, the N’Dama have a superior capacity to control
parasite proliferation, control anemia, and maintain body
weight (43).
Although the mechanisms of trypanotolerance employed by
trypanotolerant N’Dama cattle are still poorly understood,
research efforts have focused on elucidating the enhanced
control of parasitemia and anemia. For example, a hemopoietic
chimera twin study reported that control of parasitemia was
independent of the hemopoietic system, while control of ane-
mia was dependent on the same system (36). In addition, T cell
depletion studies suggested that both parasitemia and anemia
were independent of T cells and antibody (38, 47). Overall,
there appears to be two independent mechanisms, an innate
control of parasite growth and control of anemia involving the
hemopoietic system.
The cytokine environment and changes in the type 1/type 2
helper T cell (TH1/TH2) cytokine balance can influence disease
progression in infected animals. Indeed, responses have been
measured in an effort to understand their role in trypanotoler-
ance and trypanosusceptibility (23, 27, 52). In particular, a
study by Mertens et al. (27) examined the profiles of a number
of cytokines in peripheral blood mononuclear cells (PBMC)
from infected N’Dama and Boran cattle and suggested a
possible protective role for the IL-4 protein. Due to the acces-
sibility of mouse models and the prohibitive cost of bovine
challenge experiments, a large number of these studies in
recent years have been based on the murine immune response
to trypanosome infection (20, 40, 50). It has been shown in the
mouse model that survival requires a TH1 cytokine environ-
ment and classical macrophage activation in the early stage of
infection, allowing the mice to control the first peak of para-
sitemia. Subsequently, a switch to the TH2 cytokine environ-
ment and alternatively activated macrophages during the
chronic/late stages of the disease appears to be beneficial for
Physiol Genomics • VOL
survival, probably because the inflammatory parasite-control-
ling response is harmful to the host (2). However, caution
should be exercised when extrapolating findings from these
studies and applying them to the bovine model (34), where
more work is needed to determine whether mechanisms similar
to those in the murine model are at play. In terms of work
carried out in cattle, some studies have focused specifically on
lymph node cells (25). However, the immune response to
bovine trypanosomiasis is highly compartmentalized, and the
response in lymph node cells can be considerably different
from that in PBMC. It is difficult to generalize cytokine
profiles based on a small number of studies, considering the
inherent variability in experimental design in terms of animals,
parasite clone, and infection protocol including the dosage and
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time points examined. Consequently, the present study aims to
improve our understanding of the bovine immune response,
and in particular the cytokine response of PBMC, to T. con-
golense infection, building on previous transcriptional profiling
work carried out with susceptible Boran (17). A panel of
cytokines, chosen to characterize the presence of either a TH1
or TH2 type cytokine environment, was used for this purpose.
MATERIALS AND METHODS
Animals and experimental challenge. All the animal procedures
were approved by the Institutional Animal Care and Use Committee
of the International Livestock Research Institute (ILRI), in concor-
dance with the Animals (Scientific Procedures) Act (United Kingdom)
1986. Animals were reared in a trypanosomiasis-free research farm at
Kapiti Plains Estate, Kenya, and were therefore never infected with
trypanosomes before the experimental challenge. Eight N’Dama and
eight Boran (19- to 28-mo-old females) were experimentally infected
with T. congolense clone IL1180 (15, 41) via the bite of eight infected
tsetse flies (Glossina morsitans morsitans) per animal (1, 10). Packed
cell volume (PCV) was measured routinely throughout the course of
infection (56), and animals with PCVs that dropped to 15% were
removed from the experiment and treated with a trypanocide (Berenil,
7 mg/kg body wt). Parasitemia scores were also taken at the same time
points as those used for the PCV measurements and monitored using
the darkground/phase contrast buffy coat method (32, 45).
Blood collection, PBMC isolation, and RNA extraction and puri-
fication. Peripheral blood (200 ml) was collected before infection and
at 14, 21, 25, 29, and 34 days postinfection (dpi) in heparanized
syringes. These time points were chosen to coincide with the first
wave of parasitemia. PBMC were isolated, using Percoll gradients
(GE Healthcare, Buckinghamshire, UK) and previously described
methods (53). The isolated PBMC were washed with sterile PBS and
stored immediately at ⫺80°C in Tri-reagent (Medical Research Cen-
ter, Cincinnati, OH) for further processing. Total RNA was extracted
with chloroform and precipitated with isopropanol. Each sample was
further DNase treated with RQ1 RNase-free DNase (Promega,
Southampton, UK) and purified using the RNeasy mini kit (Qiagen,
Crawley, UK). RNA quality and quantity were subsequently assessed
using the 18S/28S ratio and RNA integrity number (RIN) on an
Agilent Bioanalyzer with the RNA 6000 Nano LabChip kit (Agilent
Technologies, Dublin, Ireland).
cDNA synthesis and real-time quantitative RT-PCR. Four micro-
grams of total RNA from each sample were reverse transcribed into
cDNA with oligo(dT) primers, using a SuperScript III first-strand
synthesis SuperMix kit according to the manufacturer’s instructions
(Invitrogen, Paisley, UK). The converted cDNA was quantified using
a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,
Wilmington, DE), diluted to 20 ng/␮l working stocks, and stored at
⫺20°C for subsequent analyses. Primers for real-time quantitative
28 • www.physiolgenomics.org
 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE 55
Table 1. Bovine oligonucleotide primers used for real-time qRT-PCR
Amplicon Primer
Gene Symbol Gene Name Primer Sequence (5⬘-3⬘) Size, bp Concentration, nM

ACTB Actin, beta F-CAGCAGATGTGGATCAGCAAGC 91 300

R-AACGCAGCTAACAGTCCGCC
GAPDH Glyceraldehyde-3-phosphate dehydrogenase F-TTCTACTGGCGCTGCCAAGG 107 300
R-GATCCACAACAGACACGTTGGG
YWHAZ Tyrosine 3-monooxygenase/tryptophan 5- F-GCATCCCACAGACTATTTCC 120 300
monooxygenase activation protein, zeta polypeptide R-GCAAAGACAATGACAGACCA
HPRT1 Hypoxanthine phosphoribosyltransferase-1 F-TGCTGAGGATTTGGAGAAGG 154 300
R-CAACAGGTCGGCAAAGAACT
SDHA Succinate dehydrogenase complex, subunit A, F-GCAGAACCTGATGCTTTGTG 185 300
flavoprotein R-CGTAGGAGAGCGTGTGCTT
PPIA Peptidylprolyl isomerase A F-CATACAGGTCCTGGCATCTTGTCC 108 300
R-CACGTGCTTGCCATCCAACC
IFNG Interferon, gamma F-TCAAATTCCGGTGGATGATCTGC 150 300

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R-GACCATTACGTTGATGCTCTCCG
LTA Lymphotoxin alpha (TNF superfamily, member 1) F-ACGCCTCTTCTTTCTTCGCCT 111 900
R-CCGAGGAGGACTCAGAAACTGA
IL1RN Interleukin-1 receptor antagonist F-CGAACCCCATACTATGTTCC 111 300
R-AAGTCAGTGATGTTCACGGC
CCL2 Chemokine (C-C motif) ligand 2 F-GCTGAAACTTGAATCCTCTCG 147 300
R-CTGTTGAATGTATAGCAGCAGG
IL1A Interleukin-1, alpha F-TTCGAGATATGTCAGGTCCATACC 116 300
R-AGTCACAGGAAGCTGAGAATCC
IL2 Interleukin-2 F-CCAGAGAGATCAAGGATTCAATGG 108 300
R-CAGCGTTTACTGTTGCATCATCA
IL4 Interleukin-4 F-ACGCTGAACATCCTCACAACG 125 900
R-CGCCTAAGCTCAATTCCAACC
IL6 Interleukin-6 F-ATCAGAACACTGATCCAGATCC 145 100
R-CAAGGTTTCTCAGGATGAGG
IL8 Interleukin-8 F-GAAGAGAGCTGAGAAGCAAGATCC 142 300
R-ACCCACACAGAACATGAGGC
IL10 Interleukin-10 F-AAGGTGAAGAGAGTCTTCAGTGAGC 110 100
R-TGCATCTTCGTTGTCATGTAGG
IL12 Interleukin-12 F-AGATAAAACCAGCACAGTGG 154 100
R-GATACTTCTAAGGCACAGGG
TNF Tumor necrosis factor (TNF superfamily, member 2) F-GCTCCAGAAGTTGCTTGTGC 149 900
R-AACCAGAGGGCTGTTGATGG
TGFB Transforming growth factor, beta 1 F-TGCTTCAGCTCCACAGAAAAGA 116 300
R-AGGCAGAAATTGGCGTGGT
NFKB1 Nuclear factor of kappa light polypeptide gene F-ATACTGAACAATGCCTTCCGG 135 300
enhancer in B-cells 1 R-CACGTCAATGGCCTCAGTGTAG

qRT-PCR, quantitative RT-PCR; F, forward; R, reverse.

RT-PCR (qRT-PCR) were designed, where possible, to span two
exons, using the Vector NTI Advance software package (Invitrogen),
and commercially synthesized (Invitrogen). Details for these primer
sets are provided in Table 1. Each reaction was carried out in a total
volume of 25 ␮l with 2 ␮l of cDNA (20 ng/␮l), 12.5 ␮l of 2⫻ PCR
master mix (BioGene, Cambridgeshire, UK), 1.25 ␮l of SYBR Green
I (a 1/40,000 dilution with DMSO; BioGene, Cambridgeshire, UK),
and 9.25 ␮l of primer-H2O. Optimal primer concentrations were
determined by titrating 100, 300, and 900 nM final concentrations, and
disassociation curves were examined for the presence of a single
product. Real-time qRT-PCR was performed using an MX3000P
quantitative PCR system (Stratagene, La Jolla, CA) with the following
cycling parameters: 95°C for 10 min (PCR hot start), followed by 45
cycles of 95°C for 15 s and 60°C for 1 min, followed by amplicon
dissociation (95°C for 1 min, 55°C for 30 s, increasing 0.5°/cycle for
81 cycles).
Flow cytometry of PBMC samples. To quantify any changes in the
percentages of different cell subsets in the PBMC samples across
the time course, cell surface markers for CD4⫹, CD8⫹, ␥␦ T cells,
and B cells were examined using flow cytometry. These analyses
were carried out using the PBMC samples from each animal before
infection and at 14, 21, 25, 29, and 34 dpi. Briefly, 1 ⫻ 106 PBMC
in PBS were added to 96-well microtiter plates, primary monoclo-
nal antibody was added, and these samples were incubated on ice
for 30 min. Cells were then washed three times in fluorescence-
activated cell sorting (FACS) medium [RPMI 1640, no HEPES,
5% horse serum (Sigma-Aldrich), and 0.01% sodium azide]. Sec-
ondary FITC-conjugated antibody was then added, and cells were
incubated on ice in the dark for 30 min and fixed with 2%
formaldehyde. The monoclonal antibodies used were as follows:
ILA11 [bovine CD4⫹ T cells (3)], ILA51 [bovine CD8⫹ T cells
(11, 49)], CC15 [bovine ␥␦ T cells (5)], and ILA30 [bovine B cells,
IgM (37) (from J. Naessens, ILRI)]. Data were collected using a
BD FACscan instrument and analyzed with the CellQuest version
3.3 software package (BD Biosciences, San Jose, CA).
Data analysis. Analyses of putative reference, or “housekeep-
ing,” genes were carried out using the geNorm version 3.4 Mi-
crosoft Excel add-in (54). Briefly, geNorm calculates a gene
expression stability measure “M” for a control gene as the pairwise
variation for that gene with all other tested control genes. The

Physiol Genomics • VOL 28 • www.physiolgenomics.org

56 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE

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Fig. 1. Cytokine mRNA expression profiles from real-time quantitative RT-PCR analyses of peripheral blood mononuclear cells (PBMC) of N’Dama and Boran
cattle infected with T. congolense. Data analysis was carried out using the standard 2⫺⌬⌬CT method, where CT is cycle threshold (22). All CT values were
corrected using the housekeeping gene PPIA, and time point 0 was used as the calibrator. Each point on the graph represents the mean fold change in gene
expression relative to preinfection levels ⫾ SE.

tested genes are then ranked in order of decreasing M values or RESULTS

increasing mRNA expression stability. The optimal number of
reference genes can also be estimated by geNorm by examining the
pairwise variation “V” between two sequential normalization fac-
tors. It is recommended that the inclusion of additional reference
genes is not required below V values of 0.15 (54). The 2⫺⌬⌬CT
method (where CT is cycle threshold) was used to determine mean
fold changes in gene expression between breeds at a particular time
point and between time points for each breed (22). On the basis of
the geNorm analyses, the peptidylprolyl isomerase A gene (PPIA)
alone was used as the reference gene (additional reference genes
were not required based on geNorm criteria), and preinfection
values were used as calibrators to generate the graphs shown in
Fig. 1. Student’s t-test was used to identify significant differences
in gene expression between breeds and time points.
Physiol Genomics • VOL
Parasitemia and PCV. Trypanosomes were detected in the
peripheral blood of all animals experimentally infected in this
study by 12–15 dpi, with peaks of the first wave of parasitemia
occurring at 15–22 dpi. The mean PCVs for both the N’Dama
and Boran groups fell from 38.1 and 37.8%, respectively, at 8
dpi to 28.7 and 28.4%, respectively, at 22 dpi with a marked
reduction from 12 dpi onward, when trypanosomes first ap-
peared in peripheral blood. Between 12 and 22 dpi, there were
similar rates of PCV decline in both breeds. In the period
between 22 and 35 dpi, the rate of PCV decline in N’Dama was
slower than for Boran, with a mean minimum PCV measure-
28 • www.physiolgenomics.org
 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE 57
Table 2. Significant mean fold differences in cytokine mRNA expression between N’Dama
and Boran animals at each time point
Gene 0 dpi 14 dpi 21 dpi 25 dpi 29 dpi 34 dpi

LTA 1.7-fold 1 ND NS NS 4.5-fold 1 ND NS NS

P ⫽ 0.046 P ⫽ 0.006
IL1RN NS NS NS NS NS 1.7-fold 1 BN
P ⫽ 0.028
CCL2 NS NS NS NS NS 2.7-fold 1 BN
P ⫽ 0.030
IL2 1.6-fold 1 BN NS NS NS NS NS
P ⫽ 0.045
IL10 NS 3.5-fold 1 ND 4.3-fold 1 BN 2.1-fold 1 BN 1.7-fold 1 BN NS
P ⫽ 0.014 P ⫽ 0.001 P ⫽ 0.008 P ⫽ 0.047
IL8 NS NS NS NS NS 4.0-fold 1 BN
P ⫽ 0.040
IL6 NS NS NS NS 5.4-fold 1 BN 7.1-fold 1 BN

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P ⫽ 0.03 P ⫽ 0.010
TGFB1 NS NS NS 5.9-fold 1 ND 2.1-fold 1 BN NS
P ⫽ 0.037 P ⫽ 0.056
TNF 1.9-fold 1 BN 1.5-fold 1 ND NS NS NS NS
P ⫽ 0.045 P ⫽ 0.047
IL4 NS NS NS NS 3.6-fold 1 BN NS
P ⫽ 0.020
ND, N’Dama; BN, Boran; 1, increase; NS, not significant at P ⱕ 0.05; dpi, days postinfection.

ment of 24.8 ⫾ 3.2% at 29 dpi in N’Dama and 19.9 ⫾ 4% at
32 dpi in Boran.
PBMC cell subset analyses. The fluctuations in cell subsets
that were measured within breeds used preinfection values (and
not uninfected controls) to determine “unchallenged variation,”
and this must be taken into account when assessing the signif-
icance of the variation measured after infection. Overall, there
was a decrease in the percentage of CD8⫹ and ␥␦ T cells in
both groups across the time course with the exception of a
slight increase in CD8⫹ and ␥␦ T cells in Boran at 25 dpi. The
percentage of CD4⫹ T cells in N’Dama decreased until 25 dpi,
after which the numbers stabilized. There was no significant
decrease in the percentage of CD4⫹ T cells in Boran over the
time course. The percentage of B cells fell in both N’Dama and
Boran from 0 to 14 dpi and then increased until the end of the
time course. There was no significant difference between the
two groups in CD4⫹ T cells at 0, 14, 29, and 34 dpi. There
were modest differences in CD4⫹ T cells in Boran compared
with N’Dama at 21 and 25 dpi (P ⫽ 0.045 and P ⫽ 0.035,
respectively; higher in Boran). There were no significant dif-
ferences in CD8⫹ T cells between the two groups at 0, 14, and
29 dpi; however, there were significant differences at 21, 25,
and 34 dpi (P ⫽ 0.015, P ⫽ 0.024, and P ⫽ 0.028, respec-
tively; higher in Boran). There were no significant differences
in either ␥␦ T cells or B cells between the two groups at any of
the time points.
qRT-PCR reference gene analyses. Because the effect of
various experimental treatments on the expression of tradi-
tional qRT-PCR reference, or housekeeping, genes is largely
unknown, it was important to identify a stable reference gene
for the particular physiological conditions inherent in the
present study. Six genes were chosen to represent a panel of
potential qRT-PCR reference genes for this study [␤-actin
(ACTB), GAPDH, tyrosine-3-monooxygenase/tryptophan-5-
monooxygenase activation protein, ␨-polypeptide (YWHAZ),
hypoxanthine phosphoribosyltransferase-1 (HPRT1), succinate
dehydrogenase complex, subunit A, flavoprotein (SDHA), and
Physiol Genomics • VOL
PPIA]. Real-time qRT-PCR was carried out with primers for
all six genes on all animals over the entire time course (0 –34
dpi). The data were then analyzed using the geNorm package,
and M stability values were generated for specific comparisons
between the N’Dama and Boran groups at each time point and
also across the time course for each breed (i.e., 0 vs. 14 dpi, 0
vs. 21 dpi, 0 vs. 25 dpi, etc.). The recommended M value cutoff
is 0.5, with higher M values representing more variable gene
expression. The gene that exhibited the greatest stability
throughout the time course was PPIA, with M values ranging
from 0.13 to 0.28. In contrast to this, the most variable of the
putative qRT-PCR reference genes was SDHA, with M values
ranging from 0.51 to 0.88. In addition, geNorm was used to
determine the optimal number of reference genes for normal-
ization, based on a recommended cutoff V value of 0.15;
additional reference genes would not have contributed to a
more accurate normalization factor. Therefore, PPIA was used
as a single standard reference gene for these experiments.
Cytokine mRNA profiles of PBMC from N’Dama and Boran.
The mRNA profiles of 14 cytokine genes were examined by
real-time qRT-PCR. The graphs in Fig. 1 represent mean fold
changes relative to preinfection values for both the N’Dama
and Boran groups. Because of differences in expression before
infection, these graphs represent changes within a particular
breed over time, while significant differences between the
breeds are represented in Table 2. The list of cytokines repre-
sented in Table 2 excludes cytokines with no significant dif-
ference between the breeds at any time point measured. With
the 2⫺⌬⌬CT method of data analysis, the mean fold change at
time 0 should be very close to 1 (since 20 ⫽ 1) (22). Minor
deviations from a value of 1 can be attributed to experimental
variation. An advantage of the 2⫺⌬⌬CT method is that verifi-
cation of the time 0 fold change is a convenient way to check
for variation among samples. Importantly, in the present ex-
periment, only minor deviations were observed.
By 14 dpi, when parasites were detected in the peripheral
blood of all animals, the mRNA expression of a number of
28 • www.physiolgenomics.org
58 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE
proinflammatory mediators increased in N’Dama but not Boran
compared with preinfection levels. The expression of the IFNG
gene increased by 4.3-fold (P ⫽ 0.037), that of IL1A by
5.5-fold (P ⫽ 0.011), that of IL12 by 2.0-fold (P ⫽ 0.041), that
of NFKB1 by 1.6-fold (P ⫽ 0.021), and that of TNF by 2.1-fold
(P ⫽ 0.009). There was a 1.5-fold higher expression level (P ⫽
0.047) of the TNF gene in N’Dama compared with Boran at 14
dpi. At the same time, the expression of the IL2 gene was
downregulated in Boran by 3.1-fold (P ⫽ 0.000).
At 21 dpi, there was a significant 4.3-fold higher level of
IL10 gene expression in Boran compared with N’Dama (P ⫽
0.001). A number of proinflammatory mediators remained
higher than preinfection levels in N’Dama including the IL1A
gene (4.2-fold, P ⫽ 0.034) and TNF (2.7-fold, P ⫽ 0.025),
whereas IL2 [6.3-fold in N’Dama (P ⫽ 0.000) and 9.0-fold in
Boran (P ⫽ 0.000)], IL8 [5.0-fold in N’Dama (P ⫽ 0.009) and
3.0-fold in Boran (P ⫽ 0.014)], and IL1RN [3.0-fold in
N’Dama (P ⫽ 0.000) and 2.0-fold in Boran (P ⫽ 0.000)] were
significantly downregulated at this time compared with prein-
fection.
A continued trend of IL2, IL8, and IL1RN downregulation at
25 dpi compared with preinfection values was observed. There
were significant differences between N’Dama and Boran at 25
dpi in the expression levels of the LTA, IL10, and TGFB1
genes. LTA was 4.5-fold higher in N’Dama compared with
Boran (P ⫽ 0.006), and TGFB1 was also higher in N’Dama
compared with Boran (5.9-fold, P ⫽ 0.037), while Boran had
higher levels of the IL10 transcript (2.1-fold, P ⫽ 0.008).
Differences in the expression levels of the IL10, IL6,
TGFB1, and IL4 genes were detected at 29 dpi between
N’Dama and Boran. In each case, the levels of expression were
higher in the susceptible Boran (IL10, 1.7-fold, P ⫽ 0.047; IL6,
5.4-fold, P ⫽ 0.030; TGFB1, 2.1-fold, P ⫽ 0.056; and IL4,
3.6-fold, P ⫽ 0.020). Again, there was a continued trend of
downregulation for the IL1RN, IL2, and IL8 genes that contin-
ued through 34 dpi. The largest significant difference between
N’Dama and Boran at 34 dpi was in IL6 gene expression, with
a 7.1-fold higher level in Boran compared with N’Dama (P ⫽
0.010).
DISCUSSION
The study of the bovine immune response to T. congolense
infection is more meaningfully described in the context of the
clinical data collected during the infection time course. The
parasitemia observations are largely in agreement with a pre-
vious study that reported similar prepatent times and time to
first peak of parasitemia (43). In addition to becoming para-
sitemic, both the N’Dama and Boran animals developed ane-
mia, and the kinetics of this anemia, as measured by PCV, were
also comparable with the study by Paling et al. (43). Cumula-
tively, the clinical data suggest that the animals in this exper-
imental challenge exhibited typical phenotypic responses to T.
congolense experimental infection.
Normal responses to infection in the N’Dama and Boran
animals also include well-documented fluctuations in circulat-
ing leukocyte populations (12, 39, 55). The development of
bovine monoclonal antibodies has facilitated a more accurate
description of these changes in cell subset populations in both
tolerant and susceptible breeds (for review, see Ref. 35). An
early leukopenia has been well documented during experimen-
Physiol Genomics • VOL
tal trypanosomiasis infection (12, 36, 42). Also, after parasites
are detected in the blood, the proportion of B cells increases,
mainly because of the expansion of the CD5⫹ B cell subpopu-
lation (39). An expansion of B cells was also observed in this
study after 14 dpi, but there were no significant differences
between the N’Dama and Boran animals in the proportion of
circulating B cells. There were also no significant differences
in the proportions of ␥␦ T cells, marginal differences in CD4⫹
T cell proportions, and only ⬃6% higher CD8⫹ T cell propor-
tion in Boran at three time points. Fluctuations detected during
the course of infection in this study would therefore not be
expected to mask accurate interpretation of cytokine expres-
sion profiles between the N’Dama and Boran to any great
extent.
The majority of studies in recent years that have profiled the
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host cytokine response to trypanosome infection have been
based on the mouse model of the disease (2, 19, 20, 40, 50).
Murine models have been used to a greater extent primarily
because of the large expense and difficulty associated with
bovine trypanosome challenge experiments. Indeed, murine
models of tolerance and susceptibility to trypanosomiasis are
well developed and offer an accessible means by which to
study the underlying mechanisms involved. A recent review of
the role of mouse models in African trypanosomiasis discusses
their great potential but also highlights that there are clear
differences between the bovine and murine systems (13).
Furthermore, many of the bovine studies published to date
have focused on the immune response in specific cell types,
primarily lymph node cells, spleen cells, and isolated macro-
phages, or in vitro stimulation experiments (24, 25, 46, 48, 52).
Overall, there are relatively few studies where profiling of
cytokines in PBMC has been carried out in cattle (27, 52).
Therefore, the aim of the present study was to enhance the
understanding of cytokine responses in the bovine model of T.
congolense infection and shed light on differences in cytokine
profiles between N’Dama and Boran cattle that may be due to
the mechanisms underlying trypanotolerance and trypanosus-
ceptibility.
The relative mRNA expression levels for 14 cytokine genes
were determined for the trypanosusceptible Boran and trypano-
tolerant N’Dama before infection and at 14, 21, 25, 29, and 34
dpi. By the time trypanosomes were observed circulating in
blood, at 14 dpi, N’Dama were producing higher levels of
proinflammatory and TH1 type cytokines than Boran, including
the products of IFNG, IL12, TNF, and IL1A genes. Addition-
ally, NFKB1 gene expression increased consistently over time,
with greater increases in N’Dama. In the murine model, mice
deficient in TNF protein have been found to be highly suscep-
tible to T. congolense infection, and the TNF gene is thought to
play a major role in resistance to infection in mice (18).
However, the role of TNF in host tolerance/susceptibility to
bovine trypanosomiasis is not fully understood, and in this
study, the TNF transcript generally increased in both breeds up
to a maximum at 25 dpi, with N’Dama showing significant
increases at 14, 21, and 25 dpi compared with preinfection; it
is therefore possible that TNF plays a role in disease progres-
sion in both N’Dama and Boran. Overall, these data suggest
that, during the very early stages of systemic infection,
N’Dama cattle are better able to induce classically activated
macrophages and a TH1 type response. Classically activated
macrophages are effective in combating parasites by secreting
28 • www.physiolgenomics.org
 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE
nitric oxide (NO) and oxidative radicals; therefore, this could
explain why N’Dama are able to control trypanosome numbers
more efficiently during the first peak of parasitemia (36). The
difference in peak parasitemia between N’Dama and Boran
may have important consequences: lowering the amount of
available trypanosomes may allow N’Dama to keep pathology
under control and to maintain effective responses. Indeed, the
highly polarized TH1 type response that is observed when mice
are infected with African trypanosomes has recently been
reviewed (26, 50), with IFN-␥ secretion regulating key aspects
of immunity (16).
The expression levels of the IL2, IL4, IL8, and IL1RN genes
were all significantly downregulated over the time course, with
decreases in expression particularly noticeable after 14 dpi
(when parasites are detectable in the bloodstream). Suppres-
sion of cellular immune responses is a feature of trypanoso-
miasis in the bovine and murine host. The decreased synthesis
of the IL2 transcript and a depressed T cell proliferative ability
(based on in vitro stimulation of cells from infected animals)
have previously been documented in mice and bovine studies
using lymph node cells, spleen cells, and mononuclear blood
cells (24, 27, 28, 46, 48, 51). From real-time qRT-PCR data on
PBMC of T. congolense-infected N’Dama and Boran animals,
a decrease in the IL2 transcript, particularly at 21 dpi, was
reported in both groups, and no significant difference was
detected between the breeds (27). These data are in agreement
with our observations in the present study, although we ob-
served a slightly larger decrease in the Boran group. However,
the same study also reported an increase in IL4 production in
N’Dama at 32 dpi, and it was hypothesized that this might play
a protective role in infection (27). In the present study, we
initially observed a decrease in IL4 gene expression at 21 dpi
in both breeds, which is in concordance with the previous
work; however, we observed no increase in IL4 expression in
either breed at 29 or 34 dpi. This may be due to subtle
differences in the experimental setup or may even reflect
slightly different time courses, such that we may have observed
an upregulation of IL4 gene expression if PBMC samples had
been collected after 34 dpi. The decrease in IL8 gene expres-
sion represented the most profound change in gene expression
of any of the cytokines in this study and was similar in both
breeds until 34 dpi. The decrease may reflect a switch to a more
predominant TH2 type response or have a more specific unde-
fined role (to the best of our knowledge, there are no published
reports of the suppression of IL-8 production in trypanosome-
infected cattle).
The gene expression profiles of the IL6 and IL10 genes
showed a pattern of upregulation relative to preinfection be-
tween 14 and 21 dpi, followed thereafter by a decrease in
expression. Maximal levels of IL6 transcript were reached at
21 dpi in both breeds, after which levels decreased but still
remained high relative to preinfection levels. Increased expres-
sion of the IL6 transcript from PBMC of infected cattle has
been reported for both N’Dama and Boran animals (27). In that
study, a disease-promoting role for IL-6 was suggested, and in
the present study, the significantly higher levels of IL6 tran-
script in Boran compared with N’Dama at 29 and 34 dpi would
support this hypothesis. Elevated levels of IL10 mRNA have
been reported in susceptible Boran and tolerant N’Dama (51,
52), coinciding with the first wave of parasitemia (52). The
same study reported that the NO response of monocytes to
Physiol Genomics • VOL
59
IFN-␥ was suppressed during infection and suggested a possi-
ble association with the upregulation of IL-10. Because immu-
nosuppression is a feature of infection in both N’Dama and
Boran, and elevated levels of IL-10 were detected in both
breeds, the study suggested that IL10 gene expression was not
associated with trypanotolerance. In our study, we have also
observed a parallel increase in IL10 mRNA transcription for
both the N’Dama and Boran animals; however, there are
differences, both quantitatively and temporally, between the
two breeds. After 14 dpi, the increase in IL10 gene expression
in Boran, reaching maximal levels at 21 dpi (4.3-fold higher in
Boran), was greater than that observed for the N’Dama ani-
mals.
By day 21, the time of peak parasitemia, we detected for
both breeds a trend toward TH2 responses, but this was much
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more obvious for the Boran, with increased levels of IL6 and
IL10 transcripts. This trend toward alternative macrophage
activation is not expected to facilitate clearance of the trypano-
somes in the Boran animals, particularly at a time when
parasite numbers reach a peak. Boran cattle are more depen-
dent on antibody responses to control the first peak of para-
sitemia than N’Dama cattle (35). It has been shown in a mouse
model that classically activated macrophages may be beneficial
for parasite clearance; however, at the same time, it was also
shown that they could contribute to pathology (2). A switch to
alternatively activated macrophages during chronic stages of
the disease appears to be beneficial for survival, probably
because the inflammatory parasite-controlling response is
harmful to the host (2). Further studies on isolated cell popu-
lations and knowledge of the parasite triggers and genetic
components that lead to particular activation patterns in mac-
rophages and T cells would allow us to understand, and
perhaps interfere with, an ineffective response in susceptible
animals.
ACKNOWLEDGMENTS
We thank the following for help and advice: Joel R. Mwakaya, Moses
Ogugo, Edith Authié, and all the staff at ILRI who assisted with animals and
laboratory work.
GRANTS
This work was supported by an Investigator Grant from Science Foundation
Ireland to D. E. MacHugh (grant no. 01/F.1/B028) and a Wellcome Trust
Functional Genomics Programme Grant (GR066764).
REFERENCES
1. Akol GW, Murray M. Early events following challenge of cattle with
tsetse infected with Trypanosoma congolense: development of the local
skin reaction. Vet Rec 110: 295–302, 1982.
2. Baetselier PD, Namangala B, Noel W, Brys L, Pays E, Beschin A.
Alternative versus classical macrophage activation during experimental
African trypanosomosis. Int J Parasitol 31: 575–587, 2001.
3. Baldwin CL, Teale AJ, Naessens JG, Goddeeris BM, MacHugh ND,
Morrison WI. Characterization of a subset of bovine T lymphocytes that
express BoT4 by monoclonal antibodies and function: similarity to lym-
phocytes defined by human T4 and murine L3T4. J Immunol 136:
4385– 4391, 1986.
4. Borst P, Cross GA. Molecular basis for trypanosome antigenic variation.
Cell 29: 291–303, 1982.
5. Clevers H, MacHugh ND, Bensaid A, Dunlap S, Baldwin CL, Kaushal
A, Iams K, Howard CJ, Morrison WI. Identification of a bovine surface
antigen uniquely expressed on CD4⫺CD8⫺ T cell receptor gamma/
delta⫹ T lymphocytes. Eur J Immunol 20: 809 – 817, 1990.
6. Dargie JD, Murray PK, Murray M, Grimshaw WR, McIntyre WI.
Bovine trypanosomiasis: the red cell kinetics of N’dama and zebu cattle
infected with Trypanosoma congolense. Parasitology 78: 271–286, 1979.
28 • www.physiolgenomics.org
60 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE
7. Darji A, Lucas R, Magez S, Torreele E, Palacios J, Sileghem M,
Bajyana Songa E, Hamers R, De Baetselier P. Mechanisms underlying
trypanosome-elicited immunosuppression. Ann Soc Belg Med Trop 72,
Suppl 1: 27–38, 1992.
8. Donelson JE. Antigenic variation and the African trypanosome genome.
Acta Trop 85: 391– 404, 2003.
9. Donelson JE, Hill KL, and El-Sayed NM. Multiple mechanisms of
immune evasion by African trypanosomes. Mol Biochem Parasitol 91:
51– 66, 1998.
10. Dwinger RH, Murray M, Moloo SK. Potential value of localized skin
reactions (chancres) induced by Trypanosoma congolense transmitted by
Glossina morsitans centralis for the analysis of metacyclic trypanosome
populations. Parasite Immunol 9: 353–362, 1987.
11. Ellis JA, Baldwin CL, MacHugh ND, Bensaid A, Teale AJ, Goddeeris
BM, Morrison WI. Characterization by a monoclonal antibody and
functional analysis of a subset of bovine T lymphocytes that express
BoT8, a molecule analogous to human CD8. Immunology 58: 351–358,
1986.
12. Ellis JA, Scott JR, Machugh ND, Gettinby G, Davis WC. Peripheral
blood leucocytes subpopulation dynamics during Trypanosoma congo-
lense infection in Boran and N’Dama cattle: an analysis using monoclonal
antibodies and flow cytometry. Parasite Immunol 9: 363–378, 1987.
13. Foote SJ, Iraqi F, Kemp SJ. Controlling malaria and African trypano-
somiasis: the role of the mouse. Brief Funct Genomic Proteomic 4:
214 –224, 2005.
14. Foreyt B. Veterinary Parasitology: Reference Manual. Ames, IA: Iowa
State Univ. Press, 2001.
15. Geigy R, Kauffmann M. Sleeping sickness survey in the Serengeti area
(Tanzania) 1971. I. Examination of large mammals for trypanosomes.
Acta Trop 30: 12–23, 1973.
16. Hertz CJ, Filutowicz H, Mansfield JM. Resistance to the African
trypanosomes is IFN-gamma dependent. J Immunol 161: 6775– 6783,
1998.
17. Hill EW, O’Gorman GM, Agaba M, Gibson JP, Hanotte O, Kemp SJ,
Naessens J, Coussens PM, MacHugh DE. Understanding bovine
trypanosomiasis and trypanotolerance: the promise of functional genom-
ics. Vet Immunol Immunopathol 105: 247–258, 2005.
18. Iraqi F, Sekikawa K, Rowlands J, Teale A. Susceptibility of tumour
necrosis factor-alpha genetically deficient mice to Trypanosoma congo-
lense infection. Parasite Immunol 23: 445– 451, 2001.
19. Kaushik RS, Uzonna JE, Zhang Y, Gordon JR, Tabel H. Innate
resistance to experimental African trypanosomiasis: differences in cyto-
kine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-
derived macrophages from resistant and susceptible mice. Cytokine 12:
1024 –1034, 2000.
20. Kitani H, Yagi Y, Naessens J, Sekikawa K, Iraqi F. The secretion of
acute phase proteins and inflammatory cytokines during Trypanosoma
congolense infection is not affected by the absence of the TNF-alpha gene.
Acta Trop 92: 35– 42, 2004.
21. Kristjanson PM, Swallow BM, Rowlands GJ, Kruska RL, de Leeuw
PN. Measuring the costs of African trypanosomosis, the potential benefits
of control and returns to research. Agric Syst 59: 79 –98, 1999.
22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2⫺delta delta C(T)) method.
Methods 25: 402– 408, 2001.
23. Lutje V, Mertens B, Boulange A, Williams DJ, Authie E. Trypanosoma
congolense: proliferative responses and interleukin production in lymph
node cells of infected cattle. Exp Parasitol 81: 154 –164, 1995.
24. Lutje V, Taylor KA, Boulange A, Authie E. Trypanosoma congolense:
tissue distribution of long-term T- and B-cell responses in cattle. Immunol
Lett 48: 29 –34, 1995.
25. Lutje V, Taylor KA, Kennedy D, Authie E, Boulange A, Gettinby G.
Trypanosoma congolense: a comparison of T-cell-mediated responses in
lymph nodes of trypanotolerant and trypanosusceptible cattle during pri-
mary infection. Exp Parasitol 84: 320 –329, 1996.
26. Mansfield JM, Paulnock DM. Regulation of innate and acquired immu-
nity in African trypanosomiasis. Parasite Immunol 27: 361–371, 2005.
27. Mertens B, Taylor K, Muriuki C, Rocchi M. Cytokine mRNA profiles
in trypanotolerant and trypanosusceptible cattle infected with the proto-
zoan parasite Trypanosoma congolense: protective role for interleukin-4?
J Interferon Cytokine Res 19: 59 – 65, 1999.
28. Mitchell LA, Pearson TW, Gauldie J. Interleukin-1 and interleukin-2
production in resistant and susceptible inbred mice infected with Trypano-
soma congolense. Immunology 57: 291–296, 1986.
Physiol Genomics • VOL 28 •
29. Murray M, Black SJ. African trypanosomiasis in cattle: working with
nature’s solution. Vet Parasitol 18: 167–182, 1985.
30. Murray M, Dexter TM. Anaemia in bovine African trypanosomiasis. A
review. Acta Trop 45: 389 – 432, 1988.
31. Murray M, Morrison WI, Whitelaw DD. Host susceptibility to African
trypanosomiasis: trypanotolerance. Adv Parasitol 21: 1– 68, 1982.
32. Murray M, Murray PK, McIntyre WI. An improved parasitological
technique for the diagnosis of African trypanosomiasis. Trans R Soc Trop
Med Hyg 71: 325–326, 1977.
33. Murray M, Trail JC, D’Ieteren GD. Trypanotolerance in cattle and
prospects for the control of trypanosomiasis by selective breeding. Rev Sci
Tech 9: 369 –386, 1990.
34. Naessens J. Bovine trypanotolerance: a natural ability to prevent severe
anaemia and haemophagocytic syndrome? Int J Parasitol 36: 521–528,
2006.
35. Naessens J, Howard CJ, Hopkins J. Nomenclature and characterization
of leukocyte differentiation antigens in ruminants. Immunol Today 18:
Downloaded from http://physiolgenomics.physiology.org/ by guest on April 19, 2013
365–368, 1997.
36. Naessens J, Leak SG, Kennedy DJ, Kemp SJ, Teale AJ. Responses of
bovine chimaeras combining trypanosomosis resistant and susceptible
genotypes to experimental infection with Trypanosoma congolense. Vet
Parasitol 111: 125–142, 2003.
37. Naessens J, Newson J, Williams DJ, Lutje V. Identification of isotypes
and allotypes of bovine immunoglobulin M with monoclonal antibodies.
Immunology 63: 569 –574, 1988.
38. Naessens J, Teale AJ, Sileghem M. Identification of mechanisms of
natural resistance to African trypanosomiasis in cattle. Vet Immunol
Immunopathol 87: 187–194, 2002.
39. Naessens J, Williams DJ. Characterization and measurement of CD5⫹ B
cells in normal and Trypanosoma congolense-infected cattle. Eur J Im-
munol 22: 1713–1718, 1992.
40. Namangala B, Noel W, De Baetselier P, Brys L, Beschin A. Relative
contribution of interferon-gamma and interleukin-10 to resistance to mu-
rine African trypanosomosis. J Infect Dis 183: 1794 –1800, 2001.
41. Nantulya VM, Musoke AJ, Rurangirwa FR, Moloo SK. Resistance of
cattle to tsetse-transmitted challenge with Trypanosoma brucei or
Trypanosoma congolense after spontaneous recovery from syringe-pas-
saged infections. Infect Immun 43: 735–738, 1984.
42. Naylor DC. The haematology and histopathology of Trypanosoma con-
golense infection in cattle. ii. Haematology (including symptoms). Trop
Anim Health Prod 3: 159 –168, 1971.
43. Paling RW, Moloo SK, Scott JR, Gettinby G, McOdimba FA, Murray
M. Susceptibility of N’Dama and Boran cattle to sequential challenges
with tsetse-transmitted clones of Trypanosoma congolense. Parasite Im-
munol 13: 427– 445, 1991.
44. Paling RW, Moloo SK, Scott JR, McOdimba FA, Logan-Henfrey LL,
Murray M, Williams DJ. Susceptibility of N’Dama and Boran cattle to
tsetse-transmitted primary and rechallenge infections with a homologous
serodeme of Trypanosoma congolense. Parasite Immunol 13: 413– 425,
1991.
45. Paris J, Murray M, McOdimba F. A comparative evaluation of the
parasitological techniques currently available for the diagnosis of African
trypanosomiasis in cattle. Acta Trop 39: 307–316, 1982.
46. Sileghem M, Flynn JN. Suppression of interleukin 2 secretion and
interleukin 2 receptor expression during tsetse-transmitted trypanosomia-
sis in cattle. Eur J Immunol 22: 767–773, 1992.
47. Sileghem M, Naessens J. Are CD8 T cells involved in control of African
trypanosomiasis in a natural host environment? Eur J Immunol 25:
1965–1971, 1995.
48. Sileghem MR, Flynn JN, Saya R, Williams DJ. Secretion of co-
stimulatory cytokines by monocytes and macrophages during infection
with Trypanosoma (Nannomonas) congolense in susceptible and tolerant
cattle. Vet Immunol Immunopathol 37: 123–134, 1993.
49. Smith RA, Kreeger JM, Alvarez AJ, Goin JC, Davis WC, Whipple
DL, Estes DM. Role of CD8⫹ and WC-1⫹ gamma/delta T cells in
resistance to Mycobacterium bovis infection in the SCID-bo mouse.
J Leukoc Biol 65: 28 –34, 1999.
50. Tabel H, Kaushik RS, Uzonna JE. Susceptibility and resistance to
Trypanosoma congolense infections. Microbes Infect 2: 1619 –1629, 2000.
51. Taylor K, Lutje V, Mertens B. Nitric oxide synthesis is depressed in Bos
indicus cattle infected with Trypanosoma congolense and Trypanosoma
vivax and does not mediate T-cell suppression. Infect Immun 64: 4115–
4122, 1996.
www.physiolgenomics.org
 BOVINE CYTOKINE PROFILES AFTER TRYPANOSOME CHALLENGE
52. Taylor K, Mertens B, Lutje V, Saya R. Trypanosoma congolense
infection of trypanotolerant N’Dama (Bos taurus) cattle is associated with
decreased secretion of nitric oxide by interferon-gamma-activated mono-
cytes and increased transcription of interleukin-10. Parasite Immunol 20:
421– 429, 1998.
53. Ulmer AJ, Scholz W, Ernst M, Brandt E, Flad HD. Isolation and
subfractionation of human peripheral blood mononuclear cells (PBMC) by
density gradient centrifugation on Percoll. Immunobiology 166: 238 –250,
1984.
Physiol Genomics • VOL 28 •
61
54. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De
Paepe A, Speleman F. Accurate normalization of real-time quantitative
RT-PCR data by geometric averaging of multiple internal control genes.
Genome Biol 3: RESEARCH0034, 2002.
55. Williams DJ, Naessens J, Scott JR, McOdimba FA. Analysis of peripheral
leucocyte populations in N’Dama and Boran cattle following a rechallenge
infection with Trypanosoma congolense. Parasite Immunol 13: 171–185, 1991.
56. Woo PT. The haematocrit centrifuge technique for the diagnosis of
African trypanosomiasis. Acta Trop 27: 384 –386, 1970.
Downloaded from http://physiolgenomics.physiology.org/ by guest on April 19, 2013
www.physiolgenomics.org