Trypanosome non-specific antibody responses during trypanosoma congolense infection of cattle
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Date
1998
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Publisher
Sokoine University of Agriculture
Abstract
Trypanosome infections of cattle are characterized by concomitant increase in
serum IgM, development of antibodies reacting with non-trypanosome antigens and
an increase in the proportion of CD5+ B cells in peripheral blood and spleen. It is
not known whether the three events are related. In mice and humans, CD5+ B
cells have been shown to predominantly produce IgM antibodies that are
polyreactive in nature. This experiment was initiated first to confirm whether
trypanosome non-specific antibodies develop during the course of Trypanosoma
congolense infections of susceptible Boran or resistant N’Dama cattle. In addition,
to investigate whether a different trypanosome species, Trypanosoma vivax, can
also induce these antibodies. Secondly, to investigate whether the CD5+ B cells,
which increase during trypanosome infections of cattle, are the source of the
trypanosome non-specific antibodies observed.
Experimental infections were initiated Trypanosoma congolense by tsetsefly bite in
13 susceptible Boran and 6 resistant N’Dama cattle. A separate group of 4 Boran
cattle was also infected with a different trypanosome species T. vivax. Serum
samples were collected from infected cattle at different time points and tested in
ELISA for trypanosome-specific and trypanosome non-specific antibodies. Seven
Boran cattle from the T. congolense-vai&ci&d group were killed between 31-51 after infection and mononuclear cells prepared from spleen tissue. The cells were
immunoglobulins using monoclonal antibody IL-A58. Separate populations of
CD5+ and CD5' B cells were obtained by sorting using a flow-cytometer. Equal
numbers of CD5+ and CD5' B cells were tested in the Silver Immunogold (SIG)
blot assay for enumeration of number of cells secreting IgM, IgG and antibodies
reacting with non-trypanosome antigens B-galactosidase, ovalbumin and lysozyme.
ELISA tests on sera from both Boran and N’Dama cattle infected with
Trypanosoma congolense revealed an increase in antibodies which react with a
cytochrome, but less or no reactivity was found for antigens such as ssDNA and
TNP. A similar development of trypanosome non-specific antibodies reacting with
B-galactosidase was found in T. vivax infections of Boran cattle. The trypanosome
non-specific antibodies were exclusively IgM, while the trypanosome-specific
antibodies were both IgM and IgG. Results from the SIG blot assay revealed that
numbers of IgM- and IgG-secreting cells were not different between CD5+ and
CD5’ populations (P>0.05). However, significantly more cells in the CD5+
population secreted antibodies reacting with non-trypanosome antigens than in the
CD5’ population (p<0.05).
number of non-trypanosome antigens such as B-galactosidase, ferritin and
double-stained for CD5 using monoclonal antibody IL-A67 and surface It is concluded from these studies that trypanosome non-specific antibodies develop
during trypanosome infections of both in Boran and N’Dama cattle, they can be
induced by different trypanosome species, are exclusively IgM and mainly secreted
by the CD5+ B cells. In the first experiment, IgM antibodies reacting with a number of unrelated nontrypanosome
antigens were detected in serum of trypanosome infected cattle.
These antibodies were mainly secreted by the CD5+ B cells. However, the
specificity of these antibodies is not known. Two alternative hypotheses have been
put forward to explain the reactivity to unrelated antigens observed in serum of
trypanosome infected cattle. The first one ascribes reactivity to unrelated antigens
due to presence of different antibody clones, each one possessing different specifity
to unrelated antigens due to presence of antibody molecules, each one capable of
binding more than one unrelated antigens, such as the poly reactive antibodies
secreted by murine and human CD5+ B cells. This experiment was initiated to
investigate whether the trypanosome non-specific antibodies are polyclonal or
poly reactive.
A pool of serum was made from samples of 6 Boran cattle on 30 days after
infection, when trypanosome non-specific antibody levels were highest. The serum
as observed in cases of polyclonal activation. The second one attribute reactivity pool was passed through immunoaffinity colums conjugated with either
trypanosome antigens or non-trypanosome antigen B-galactosidase. Antibody
fractions that bound to the column and those which did not bind were collected and
tested in ELISA for their reactivity to trypanosome and non-trypanosome antigens.
The IgM fraction purified on B-galactosidase reacted with B-galactosidase,
cytochrome, ferritin and the trypanosome lysate. Similar results were obtained for
IgM fraction purified on a trypanosome lysate column. The IgM fraction that
exhibited reactivity to different antigens was present in both pre-and post-infection
only with trypanosome lysate but not with the non-trypanosome antigens. The
trypanosome-specific IgG fraction was only found in post-infection sera.
polyreactive. Their presence in pre-infection sera indicates that the infection does
trypanosome-specific IgG antibodies are monoreactive and specifically induced by
trypanosome infection.
EXPERIMENT THREE
Trypanosome infections in cattle induce production of both trypanosome-specific
and the trypanosome non-specific antibodies. It is known that specific antibodies
sera. In contrast, the IgG fraction purified on trypanosome lysate column reacted These results conclude that trypanosome non-specific IgM antibodies are polyreactive. Their presence in pre-infection sera indicates that the infection does
trypanosome-specific IgG antibodies are monoreactive and specifically induced by
trypanosome infection.
EXPERIMENT THREE
Trypanosome infections in cattle induce production of both trypanosome-specific
and the trypanosome non-specific antibodies. It is known that specific antibodies
sera. In contrast, the IgG fraction purified on trypanosome lysate column reacted
These results conclude that trypanosome non-specific IgM antibodies are
not specifically induce them, but helps to amplify their production. In contrast that are directed at the exposed determinants of the variable surface glycoprotein
coat play a role of destruction of trypanosomes and eventual elimination of
infection. However, the significance of specific antibodies, which recognize
trypanosome non-specific antibodies are not known. Some workers suggested that
antibodies recognizing products of lysed trypanosomes contribute to
immunopathological processes such as development of anaemia. However, others
suggested that some of these antibodies, such as those binding to trypanosome
enzyme cystein protease may play a protective role to the host by neutralizing the
enzymatic function of the enzyme. The polyreactive trypanosome non-specific
antibodies may potentially bind to host or trypanosome antigens; and both
pathogenic and protective consequences are possible. It is therefore, important to
study regulation of antibody responses which takes place during trypanosome
infections in cattle. Information obtained may help in designing means by which
protective antibody responses can be selectively upregulated at the expense of
pathogenetic responses. T lymphocytes play an important regulatory role on
antibody responses. CD4+ T cells provide helper function to antibody production
by B cells during T-cell dependent antibody responses. CD8+ T cells sometimes
antibody responses during trypanosome infections of cattle is not known. The aim
antibody response during T. congolense infection of Boran cattle.
of this experiment was to investigate the role played by CD4+ or CD8+ on
various antigens released after the destruction of trypanosomes, and the Cattle were depleted of CD4+ or CD8+-T cells subpopulations by intravenous
injection of specific monoclonal antibodies IL-A11 or IL-A105 respectively, before
infection. The levels of the two cell subsets in peripheral blood were monitored by
flow-cytometric analyses. Serum samples collected at various time points were
tested in ELISA for determination of levels of trypanosome-specific and
trypanosome non-specific antibodies.
Flow-cytometric analyses of peripheral blood mononuclear cells revealed a
complete depletion of these T cells subpopulations over a period of two weeks.
Serum samples collected at various time points were tested in ELISA assay for
specific antibodies reacting with whole lysate of trypanosomes, non-specific
antibodies reacting with a non-trypanosome antigen fl-galactosidase, and total IgM.
Trypanosome-specific antibodies were detected in both IgM and IgG isotypes. In
contrast, non-specific antibodies reacting with B-galactosidase were exclusively
IgM. Depletion of CD4+-T cells significantly reduced levels of specific, nonspecific
and total IgM (p<0.05) while depletion of CD8+-T cells no effect on
these antibody types (p>0.05). These results show that CD4+-T cells play a crucial role in production of trypanosome-specific as well as the trypanosome non-specific antibody responses to T. congolense infection in susceptible Boran cattle. CD8 T cells have no effect onantibody responses to trypanosome infections in cattle.
Description
PhD Thesis
Keywords
Trypanosoma congolense, Trypanosoma congolense infections-cattle, Trypanosoma vivax, Trypanosome non-specific antibodies