ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 58 (4) 2003
COMPARATIVE PLASMA BIOCHEMICAL CHANGES AND
SUSCEPTIBILITY OF ERYTHROCYTES TO IN VITRO PEROXI
DURING EXPERIMENTAL TRYPANOSOMA CONGOLENSE A
BRUCEI INFECTIONS IN SHEEP
V.O. Taiwo1, M.O. Olaniyi1 and A.O. Ogunsanmi2
1. Departments of Veterinary Pathology and
2. Department Wildlife and Fisheries Management, University of Ibadan, Ibadan. Nigeria
Abstract
Comparative plasma biochemical changes and the degree of erythrocyte susceptibility to in
peroxidation were studied in sheep experimentally infected with Trypanosoma congolense and
Both groups of infected sheep showed significant alterations (P<0.05; P<0.01) in the plasma b
parameters studied, while there was significantly increased (P<0.05) erythrocyte susceptibility
peroxidation using 1.5% hydrogen peroxide during the course of trypanosome infection. Plasm
biochemical changes include elevation of total plasma protein and globulin levels with resultan
in albulin:globulin ratio, hypocholesterolaemia and hypoglycaemia in both groups of infected s
These changes were more severe (P<0.05) in T. brucei-infected than in T. congolense-infecte
While there were no significant changes in plasma alkaline phosphatase (ALP) and alanine
aminotransferase (ALT) in T. congolense-infected sheep, there were significant increases (P<
plasma levels of these enzymes in T. brucei-infected sheep. There was increased erythrocyte
susceptibility to in vitro peroxidation in both groups of animals infected with the two species of
trypanosomes, but it was more pronounced in T. brucei-infected sheep.
It is concluded that peroxidative injury to erythrocytes, which could have been due to loss o
erythrocyte membrane integrity, and hypocholesterolaemia with resultant increased red cell de
is one of the major causes of anaemia during trypanosome infection in domestic animals. This
shows that sheep appear to be more susceptible to the pathogenic effects of T. brucei than of
congolense.
Introduction
Small ruminants are fully susceptible to trypanosomosis (1, 2) and the
economic impact of the disease on these animas has been shown to be
substantial (3). Serum biochemical changes during trypanosomosis have
been reported in cattle (4), sheep and goats (5). These changes have been
extensively reviewed (6).
Disruption of erythrocyte membrane integrity has been reported to be caused
directly by the trypanosomes (7, 8, 9) or indirectly by secreted products of
trypanosomes (10, 11, 12, 13, 14). Significant reduction in erythrocyte
membrane sialoglycoproteins, due to increased activity of circulating
neuraminidases (sialidases) has also been reported to play a significant role
in the development of anaemia in African animal trypanosomosis (15, 16,
17). These phenomena have been reported to be responsible for early
sequestration and destruction of erythrocytes by cells of the mononuclear
phagocytic series and subsequent anaemia during trypanosomiasis (18).
Erythrocyte peroxidation has been observed to be one of the factors, which
play an important role in the pathogenesis of anaemia in acute
trypamosomosis in mice infected with T. brucei (19). Trypanosomes and
activated phagocytes (macrophages and neutrophils) are known to elaborate
sialidases (15, 20), proteases (14, 21), reactive oxygen radicals such as
O2=, OH- an erythrocyte membranes leading to their rapid destruction during
infection (16, 17, 18).
This study was designed to investigate some plasma biochemical changes
in sheep and the dynamics of in vitro erythrocyte peroxidation during T.
congolense and T brucei infections in order to determine the possible
correlation with the degree of susceptibility of sheep to the infections.
Materials and Methods
Infection of Animals with Trypanosomes
Twenty, (10 male and 10 female) healthy West African dwarf (WAD) sheep
aged between 1-11/2 years and weighing between 14.5 and 16kg purchased
at a local market in Ibadan, Nigeria were used for this experiment. They were
housed in the Small Animal Unit of the Teaching and Research Farm,
University of Ibadan, Nigeria. The animals were fed a combination of grass
and legume hay supplemented with commercial sheep concentrates. All had
access to fresh clean water ad libitum.
Two species of trypanosomes used in this study, Trypanosoma
congolense (Binchi Bassa Strain) and T. brucei (Lafia Strain) were obtained
from Nigerian Institute for Trypanosomasis Research (NITR), Vom. The
animals were divided into two groups, A and B, each consisting of 5 males
and 5 females. Groups A animals were each given 3.5x106 T. congolense,
while group B animals were also given 3.5x106 T. brucei all in 1ml of sterile
normal saline by intraperitoneal (i.p.) inoculation.
Blood Collection and Plasma Biochemistry
Ten millilitres (ml) of blood was collected from each animal on days 0, 7,
14, 21, 28, 35, 42, 49 and 56 days post-infection (p.i.) by jugular venapucture
into tubes containing disodium ethylene diamine tetraacetic acid (Na-EDTA)
anticoagulant. Plasma was obtained from the blood sample by centrifugation
at 2,000g for 10 minutes at 27oC. Total plasma protein, albumin and globulin
levels were determined as described (2); plasma glucose level was
determined by glucose oxidase method (24). Total plasma cholesterol level
was measured using the method described (25). The concentrations of
alanine aninotransferase (ALT) and aspartate aminotransferase (AST) in the
plasma samples were determined spectrophotometrically (26). The level of
alkaline phosphatase (ALP) in the plasma samples was determined as
described (27).
In vitro Erythrocyte Peroxidation:
One trillion (109) washed erythrocytes per animal, obtained from the
middle third of packed red cells after centrifugation, were used for this assay.
The in vitro erythrocyte peroxidation assay was carried out on days 0, 7, 14,
21, 28, 35, 42, 49 and 56 p.i. as described (28). Briefly, the washed
erythrocytes were suspended in 2ml of 0.9% saline solution in labeled
sample tubes.
To each of these was added 0.5ml 1.5% hydrogen peroxide. Washed
erythrocytes suspended in 2.5ml of 0.9% saline solution served as the
control. The samples were incubated at 37oC for 90 minutes after which the
reaction was stopped with 0.5ml of 10% trichloroacetic acid. The suspension
was centrifuged at 1,500g for 10 minutes followed by filtration through
Whatman No. 1 filter paper. To each filtrate was added 0.75ml of 0.67%
thiobarbituric acid and then re-heated at 100oC for 20 minutes. The samples
were cooled to 20oC and the by-products of peroxidation (thiobarbituric acid
reactive substances) were measured spectrophotometrically at 535nm.
Statistical analysis of data
The data were subjected to statistical analysis to test any significant
differences between the two groups using analysis of variance (ANOVA)
(29).
Results
Changes in levels of plasma proteins and enzymes AST, ALT and ALP in
the T. congolense and T. brucei-infected sheep are shown on Table 1. The
levels of total plasma protein levels remained unchanged from the preinfected levels for the first three weeks post-infection (p.i.). On the 28th day
p.i., the plasma protein levels showed significant increases (p<0.05) from
5.85±0.24g/dl and 6.75±0.19g/dl at pre-infection to 8.90±0.12g/dl and
8.13±0.18g/dl in groups A and B, respectively (Table 1). The levels of plasma
globulin showed significant (p<0.05) increases above pre-infection levels in
both groups A and B from 28 dpi and 21 dpi, respectively till the experiment
was terminated on 56 days p.i. (Table 1). No significant changes were
observed in the levels of plasma albumin in both groups throughout the
experiment.
While the plasma levels of ALP and AST showed no significant changes
(p>0.05) from the pre-infection levels in group B animals, those in group A
had significant increases (p<0.05) in both ALP and AST levels in plasma
from 14th day p.i. and thereafter remained above the pre-infection level till
the termination of the experiment. There were no significant changes
(p>0.05) in the levels of ALT in all the infected animals as these remained
consistently within the pre-infection ranges throughout the course of the
experiment (Table 1).
Days post-infection
Parameter
0
7
14
21
28
35
42
49
5.9±0.2d*
6.3±0.2f
6.1±0.1d
6.2±0.2f
6.1±0.1d
6.4±0.2f
6.6±0.1c
6.9±0.1e
8.9±0.1b
8.1±0.2c
8.6±0.2b
8.4±0.2c
8.8±0.2b
8.2±0.1c
9.3±0.2a
9.0±0.3a
3.5±0.2ab
3.1±0.1b
3.2±0.1a
3.2±0.3ab
3.4±0.3ab
3.0±0.2b
3.2±0.3ab
3.3±0.2ab
3.5±0.1a
3.6±0.1a
3.2±0.2ab
3.3±0.3ab
3.2±0.2ab
3.1±0.2b
3.0±0.1b
3.3±0.3ab
Globulin (g/dl)
Group A
Group B
2.4±0.2d
3.2±0.1e
2.9±0.1c
3.0±0.2e
2.7±0.3c
3.4±0.1d
3.4±0.2b
3.6±0.3d
5.4±0.3b
4.5±0.2c
5.4±0.3b
5.1±0.2b
5.6±0.3b
5.1±0.3b
6.3±0.4a
5.7±0.2a
ALP (IU/l)
Group A
Group B
68.7±4.2c
69.5±1.5a
68.1±1.3c
66.9±1.7ab
74.8±1.5b
67.9±1.5ab
75.7±3.8ab
68.0±1.8ab
78.4±1.2a
65.7±1.5b
76.4±4.1ab
69.8±1.5a
77.4±2.9a
67.6±1.2ab
78.3±2.7
68.1±1.6
5.5±0.3d
4.9±0.7a
5.4±0.1d
5.3±0.3a
8.4±0.5a
4.8±0.6a
8.2±0.4a
5.2±0.6a
7.2±0.3c
5.0±0.8a
8.4±0.9a
5.3±0.6a
7.6±0.2b
5.2±0.5a
7.8±0.6b
5.1±0.6a
5.4±0.3a
5.3±0.2a
5.2±0.1a
5.4±0.3a
5.2±0.2a
5.3±0.3a
5.6±0.4a
5.3±0.2a
5.6±0.4a
5.4±0.4a
5.5±0.2a
5.3±0.2a
5.3±0.5a
5.5±0.5a
5.3±0.3a
5.2±0.3a
Total plasma protein (g/dl)
Group A
Group B
Albumin (g/dl)
Group A
Group B
AST (IU/l)
Group A
Group B
ALT (IU/l)
Group A
Group B
*Values on the same row with different superscripts differ significantly (P<0.05).
The results of changes in plasma cholesterol, glucose and in vitro erythrocyte
peroxidation are shown in Figs. 1, 2 and 3, respectively. Animals in both
groups developed hypocholesterolaemia from the 7th day p.i., which became
more severe with progression of infection till the experiment was terminated.
However, animals in group B suffered more severe hypocholesterolaemia
(p<0.01) than those in group A (Fig. 1). Similarly, animals in both groups
developed hypoglycaemia from 14 days p.i. onwards. Hypoglycaemia was
however, more pronounced in group B animals on 14, 21and 35 days p.i.
(Fig. 2).
Fig 1: Changes in plasma cholesterol levels in WAD Fig 2: Changes in plasma glucose levels in WAD sheep
sheep experimentally infected with trypanosomes. experimentally
infected
with
trypanosomes.
The mean pre-infection in vitro erythrocyte
peroxidation value was 0.14±0.01 absorbance units
(at 1.5% H2O2) for both groups of animals (Fig. 3).
In vitro erythrocyte peroxidation rose significantly
(p<0.05; p<0.01) to 0.27±0.01 and 0.32±0.01
absorbance units in groups A and B animals,
respectively on day 28 p.i. and was maintained at
these high levels throughout the experiment in both
groups, and especially significantly more so
(P<0.05) in group B than in group A animals (Fig.
3).
Fig 3: Kinetics of in vitro erythrocyte peroxidation during
experimental infection of WAD sheep with
trypanosomes.
Discussion
The results of this experiment indicated significant alterations in several
biochemical parameters in the Trypanosoma congolense and T. bruceiinfected sheep. The results indicated that there was hyperproteinaemia as a
result of hypergammaglobulinaemia (P<0.05) and a decrease in
albumin:globulin ratio. This observation, resulting from trypanosome antigen
stimulation and antibody production, notably IgM (30, 31) in infected sheep,
is consistent with the reports of others (5, 32, 33).
Hypoglycaemia was observed in both groups of infected sheep and this
became apparent during the period of first wave of parasitaemia (14 days
p.i.). Hypoglycaemia, which has been shown to occur during trypanosomosis
(34, 35), is reported to be due to excessive utilization of blood glucose by
trypanosomes for their metabolism (31). The result of enzyme assays
showed elevation of both ALP and AST only in T. brucei- infection sheep,
while these enzymes remained unchanged in T. congolense-infected sheep.
The elevation of these enzymes is in consonance with earlier reports (36, 37,
38) in various trypanosome-infected animals. This has been reported to be
due to tissue breakdown (necrosis) and inflammation in the host, particularly
of the liver, heart, muscle and kidney (39). Another possibility is the increase
induced by the lysed trypanosomes (34, 40) at different stages of the
infection. It is possible to suggest that in this case, increases seen only in T.
brucei-infected sheep were due to the fact that T brucei has the ability to
invade solid tissue, especially the liver, kidney and heart, thereby localizing
and causing tissue damage. This leads to the release of these enzymes from
the damaged tissues and are measurable in the plasma.
There are reports that blood lipids play important roles in the pathogenesis
of trypanosomosis (11, 41). Previous studies have indicated that plasma
cholesterol levels decrease during trypanosome infection in sheep (42).
White Fulani (trypanosusceptible) cattle had significantly higher plasma lipid
levels than N’Dama (trypanotolerant) cattle (43), implying that the former
would support more parasite growth and proliferation, hence more pathology
than the latter. In the present study, both groups of infected sheep developed
hypocholesterolaemia, which was more severe in the T. brucei-infected than
in T. congolense-infected sheep. Trypanosomes have been reported to
cleave sialic acids from the glycophorins on the erythrocyte membranes
through their secretion of sialidase (15). They are also known to utilize
erythrocyte membrane sialoglycoproteins for their proliferation and
differentiation through the use of trans-sialidase (44). One possible outcome
of the above is hypocholesterolaemia and a loss in the integrity of the
erythrocyte membrane and resultant destruction by the mononuclear
phagocyte system in vivo and their susceptibility to in vivo and in vitro
peroxidative damage.
In this study, the erythrocytes of both groups of infected sheep showed
increased susceptibility to in vitro peroxidation, suggesting that erythrocytes
of both groups of sheep had undergone some adverse surface membrane
changes, which made them prone to in vitro peroxidation. However, T.
brucei-infected sheep showed higher susceptibility (P<0.05) to in vitro
peroxidation than T. congolense-infected sheep. Similar observations were
reported in mice infected with T. brucei (19). It has been shown that
reduction in capacity of the erythrocytes of trypanosome-infected mice to
prevent free-radical-mediated membrane damage may predispose
erythrocytes to peroxidation as this will accelerate their aging and increase
their deformability, hence susceptibility to fragementation and destruction
(46). The most important substances in cellular or parasite cytotoxicity are
the products of oxidative burst and nitrogen oxide metabolism (22, 47, 48).
Reactive oxygen derived from free radicals such as superoxide hydrogen
peroxide, singlet oxygen, hydroxyl radicals and hydrochloric acid are also
produced from neutrophils and activated macrophages during trypanosome
infections (49, 50). In addition to this, macrophages also synthesize nitrogen
oxides such as nitric oxide and nitrate from the terminal guanido-nitrogen
atom of L-arginine (23, 51, 52). Both reactive oxygen products and nitrogen
oxide have been shown to be very important in tumoricidal, bactericidal and
killing of parasites such as in T. cruzi (53). They are also capable of initiating
a self-propagating reaction of oxidative damage to the polyunsaturated fatty
acids component of erythrocyte plasma membrane, leading to its destruction.
In the present study, the degree of in vitro erythrocyte peroxidation at
1.5% H2O2 was observed to be more marked (P<0.05) in T. brucei-infected
than in T. congolense-infected sheep. This can be attributed to the fact T.
brucei caused a more pronounced reduction in erythrocyte membrane sialic
acid concentration (17), hence more pronounced in vivo erythrocyte
membrane damage rendering them more susceptible to in vitro peroxidation
than in T. congolense-infected sheep.
It can be concluded from this study that erythrocyte surface membrane
damage induced by both infecting trypanosomes and host-derived
substances render erythrocytes of infected animals more prone to in vitro
peroxidative damage and this may account, in part, for the development of
anaemia in vivo during trypanosomosis. Trypanosoma brucei appears to be
more pathogenic to sheep than T. congolense.
Acknowledgement
This study was partly funded through the University of Ibadan Senate
Research Grant Code No. SRG/FVM/1995/7A awarded to the first author.
LINKS TO OTHER ARTICLES IN THIS ISSUE
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