International Research Journal of Biochemistry and Bioinformatics (ISSN-2250-9941) Vol. 4(3) pp. 24-29, May, 2014
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Copyright © 2014 International Research Journals
Full Length Research Paper
Influence of high dietary vitamin C and E oral
administration on anemia and organ damage in wistar
rat infected with Trypanosoma brucei brucei
(Federe strain)
Ajakaiye Joachim Joseph1, Kugu Bashir Adamu*1, Shu’aibu Yahya1, Bizi Ramatu Lawan2,
Benjamin Sunday Martina2, Muhammad Asma’u Asabe1, Mohammad Bintu1 and
Mazadu Melemi Richard
Extension Services Unit, Consultancy and Extension Services Division, Nigerian Institute for Trypanosomiasis
Research, No. 1 Surame Road, U/Rimi, P. M. B. 2077, Kaduna, Nigeria.
Trypanosomiasis Research Department, Nigerian Institute for Trypanosomiasis Research, No. 1 Surame Road, U/Rimi,
P. M. B. 2077, Kaduna, Nigeria.
*Corresponding author: Mobile Phone: +2348067029512; E-mail: [email protected]
The influence of vitamin C and E in experimental Trypanosoma brucei brucei (Federe strain) infected
Wistar rats was investigated. The rats were infected intraperitoneally with the same parasite load. All
infected animals developed terminal Parasitemia and percentage PCV, its severity in the untreated
infected animals was significantly (P<0.05) higher than the three infected groups treated with the
vitamins. The group given vitamin C and E (combined) developed a significantly (P<0.05) less severe
Terminal Parasitemia and percentage PCV than those given vitamin C and E (separately). The positive
control showed higher increase in Alanine transaminase, aspartate transaminase, alkaline phosphatase,
urea and creatinine. The Vitamins significantly (P<0.05) prevented the disease-induced increase in these
parameters. The two vitamins combination prevented, to a significant degree, the disease-induced
elevation of serum urea and creatinine. The organ-body weight ratio significantly (P<0.05) increased in
all the infected groups. Treatment with the vitamins however, significantly (P<0.05) decreased the
weight ratio with higher effect in the group treated with combined vitamins. It was concluded that oral
administration of both vitamins C and E ameliorated the degenerative changes in tissues, blood and
organs associated with Trypanosoma brucei brucei (Federe strain) infection in Wistar rats.
Keywords: Trypanosoma brucei brucei, vitamin C, vitamin E, anemia, organ damage, Wistar rat.
African trypanosomiasis is one of the most neglected
Tropical diseases, consisting of a number of important
human and animal pathologies caused by parasitic
protista of the order Kinetoplastida. Human African
Trypanosomiasis (HAT), or sleeping sickness, and
Animal African Trypanosomiasis (AAT), or nagana, are
vector-borne diseases, which are primarily cyclically
transmitted by tsetse fly. The animal trypanosomiasis
challenge, caused by several species of trypanosome,
e.g. Trypanosoma vivax, Trypanosoma congolense and
Trypanosoma brucei brucei cause about 3 million deaths
annually in cattle and has a marked impact on African
agriculture (Kalu et al., 2001; Njiokou et al., 2004). There
is considerable variation in the pathogenicity of different
strains and the susceptibility of different host species. T.
brucei brucei, like other pathogenic trypanosomes is
covered by a dense protein layer consisting of a single
protein called the variable surface glycoprotein (VSG),
which acts as a major immunogen and elicits the
formation of specific antibodies. The parasites are able to
evade the consequences of these immune reactions by
switching the VSG, a phenomenon known as antigenic
Ajakaiye et al. 25
variation (Damian, 1997). The hematological and
biochemical abnormalities induced by trypanosomes
arose from their direct effect via their products on host
cells such as red blood cell (RBC), white blood cell
(WBC), platelets and tissues such as liver, kidney, bone
marrow and lymphoid organs, resulting in cell destruction
and organ malfunction as well as extractions from and
additions to host chemistry associated with parasite
metabolism (Anosa, 1988; Ekanem and Yusuf 2008;
Akanji et al., 2009). The oxidative stress which occurs in
trypanosomiasis host is as a result of systematic ascorbic
acid depletion due to increased ascorbic acid
consumption in infected animals; this oxidative stress
leads to peroxidative tissue damage, which elevates
erythrocyte free radicals, oxidative haemolysis and
depletion of erythrocyte and liver glutathione by free
radicals generated by the trypanosome. As a result
membrane Phospholipids and Proteins are attacked
leading to alteration in membrane structure, which also
affects the membrane fluidity. Vitamin C (Ascorbic acid)
is a water-soluble antioxidant capable of protecting
against oxidative injuries in the aqueous compartments of
cell membrane while Vitamin E is a fat-soluble compound
comprising tocopherols and tocotrienols (Brigelius-Flohe,
1999). As a fat-soluble antioxidant, it stops the production
of reactive oxygen species (e.g. Oxygen ion and
peroxides) formed when fat undergoes oxidation (Packer,
2001; Devasagayam et al; 2004). This work was carried
out to investigate the Influence of High Dietary Vitamin C
and E Oral administration on Anemia and Organ damage
in Wistar Rats infected with Trypanosoma brucei brucei
(Federe strain).
Experimental site
The study was conducted at the Nigerian Institute for
Trypanosomiasis and (Onchocerciasis) Research (NITR),
and located in Kaduna North Local Government Area of
Kaduna State, at latitude 10° 30´ 00´´ N and longitude 7°
25´ 50´´ E of Nigeria.
Experimental animals
Twenty five Albino Wistar rats purchased from the rat
colony of NITR, Kaduna, were used as subjects for the
experiment. They were randomly divided into five groups
(A, B, C, D and E) of five rats each, in well ventilated
plastic cages with a 15×22×10 m3 dimension equipped
with wire mesh lids. The rats were acclimatised for two
weeks and duly dewormed with standard drugs before
commencement of the experiment. Group A was neither
treated nor infected (positive control), group B was
intraperitoneally infected with 1 × 106 innoculum
containing T. brucei brucei (Federe strain) parasites only
(negative control), while groups C, D and E were given
the same dose of innoculum and in addition they were
treated orally with 150 mg/kg body weight of vitamin C;
150 mg/kg body weight of vitamin E and the combination
of 150 mg/kg body weight each of vitamins C and E,
respectively. Vitamins C and E were products of a
commercial company (VMD, n.v./S.A, Arendonk,
Belgium) and were obtained from a Veterinary
commercial outlet in Kaduna, Nigeria. The animals were
fed with a basal diet obtained from a commercial feed
outlet (Vital Feeds Plc., Kaduna, Nigeria) and water was
given ad libitum. The rats had average weight of 200 –
240 g at the commencement of the experiment. Feed
constituents and calculated bromatological analyses of
the basal diet are as shown in Table 1. The basal diet
contain 11.50 MJ/Kg of metabolisable energy (ME), 16.50
g of crude protein (CP), 5.50 g of calcium and 1.45 g of
available phosphorus, calculated to be slightly above the
nutrient requirement recommended for laboratory animals
(NRC, 1995).
Vitamin supplement per (kg) diet: Vitamin A, 6000
IU, vitamin D3, 5000 IU, vitamin E; 23.0 mg; vitamin k3,
4.0 mg; thymine, 11.0 mg; riboflavin, 4.0 mg; vitamin B12,
0.005 mg; pyridoxine, 1.8 mg; pantothenic acid, 20,0 mg;
nicotinic acid, 35 mg; folic acid, 2.5 mg; choline chloride,
Mineral supplement (mg/kg diet): Cobalt, 0.40 mg;
iron, 130 mg; copper, 5 mg; zinc, 18 mg; iodine, 1.55 mg.
Inoculation of rats with parasite
The parasites T. brucei brucei (Federe strain) was
obtained from the stabilates kept in Vector and
Parasitology Department of NITR, Kaduna, Nigeria. The
parasite was inoculated into a clean rat which serves as
donor rat. Infected blood from a donor rat at peak
parasitaemia, that is, 4 days post infection (DPI) was
collected by means of tail picking and diluted with cold
physiological saline. The number of parasite in the diluted
blood was determined through the method described by
Herbert and Lumsden (1976), and a volume containing
approximately 1× 106 parasites was injected
intraperitoneally into each rat in the infected groups.
Blood Sample collection, organs collection and
Serum Analysis
Tail blood was collected daily for monitoring parasitemia
as described by Herbert and Lumsden (1976) and PCV
by the micro-haematocrit method. On 28 DPI, the rats
were sacrificed by humane decapitation prior anesthesia
with sterile cotton impregnated chloroform, and blood
was collected in plain vacutainers, serum was harvested
and used for estimation of alanine amino-transferase
26 Int. Res. J. Biochem. Bioinform.
Table 1. Composition and calculated bromatological analysis of basal diet
Soya cake
Wheat offal
Brewer’s dried grain
Vegetables oil
Monocalcium phosphate
Dry molasses
Sodium chloride
Pre-mix Vitamins(a) and Minerals(b)
Quantity in g/kg diet
Calculated analysis /Kg
ME, MJ /kg
CP, g
Methionine +Cystine, g
Tryptophan, g
Threonine, g
Ca, g
P (a), g
Na, g
CI, g
Source: Dale and Batal (2006).
Table 2. Serum chemistry of Wistar rats infected with T. brucei brucei (Federe strain) and administered with vitamins C and E (Means ±
SEM, n = 5)
Not infected not
42.90± 0.94
36.90± 1.01
36.70± 1.13
35.40± 1.10
59.40± 0.56 D
106.00± 1.9A
63.80± 0.63C
67.60± 1.02B
Infected not
32.20± 0.70A
Infected + Vit. C
Infected + Vit. E
31.40± 0.31A
Infected + Vit. C
and E
28.50± 0.85B
Values with different superscripts within a row are statistically different (P<0.05)
(ALT), aspartate amino-transferase (AST) and alkaline
phosphatase (ALP) activities using the method described
by Bergmeyer et al. (1978) with the aid of commercial
reagent kit (Gasellch aft fur Biochemica und Diagnostica,
Wiesbgden, Germany).The serum samples were also
used for the estimation of Urea and Creatinine by the
Diacetylmonoxime and Jaffe’s reactions, respectively as
described by Kaplan et al. (1988). Organs were removed
aseptically from all the groups and kept in 10 % buffered
Statistical Analysis
from this experiment are
presented as mean ± SEM. Data were analyzed by the
one-way analysis of variance (ANOVA) and the
significance of differences
between mean values
computed for particular levels of experimental factors was
determined by (Duncan, 1955) post-hoc test and means
that differs at p < 0.05 were considered significant.
Table 2 presents the result of the serum biochemical
indicators in this experiment. Infected groups showed
significant (P<0.05) increase in the levels of ALT, AST,
ALP and creatinine when compared to the uninfected
group. Group II showed significant (P<0.05) increase in
Ajakaiye et al. 27
Table 3. Organ: body weight ratios of Wistar rats infected with T. brucei brucei (Federe strain) and administered with vitamins
C and E (Means ± SEM, n = 5)
Not infected not
1.47± 0.24C
Infected not
3.18± 0.23 A
Infected + Vit. C
Infected + Vit. E
1.87± 0.08 B
1.79± 0.06 BC
Infected + Vit. C
and E
1.63± 0.05 BC
3.10± 0.00
4.82± 0.11
3.43± 0.08
3.44± 0.10
0.59± 0.04
2.13± 0.04
1.71± 0.02
1.83± 0.02
1.27± 0.20
0.83± 0.01
1.06± 0.03
0.7± 0.02
0.73± 0.01
0.80± 0.01
3.30± 0.06
Values with different superscripts within a row are statistically different (P<0.05)
Table 4. Terminal Parasitemia and % change in PCV of Wistar rats infected with T. brucei brucei (Federe strain)
administered with vitamins C and E (Means ± SEM, n = 5)
Not infected
not treated
Infected not
Infected + Vit. C
Infected + Vit. E
Infected + Vit. C
and E
Initial PCV (%)
48.90± 1.03
48.60± 1.2
48.70± 1.13
49.00± 1.22
Final PCV (%)
29.20± 0.95
40.50± 1.60
39.70± 1.97
44.10± 1.27
(-)8.10± 1.35
(-)9.00± 2.04
% change
Values with different superscripts within a row are statistically different (P<0.05). Positive signs (+) indicates increases and
negative signs (-) indicates decreases.
urea level and significant (P<0.05) lower level in treated
groups in comparison with uninfected untreated group.
Values recorded for AST, ALT, Urea and Creatinine were
significantly (P<0.05) higher in group II when compared
to the vitamins treated groups, while ALP showed related
pattern but in an opposite direction.
Table 3 present the result of the organ body weight
ratio, which significantly (P<0.05) shows increase in
organ-body weight ratio of the heart, liver and spleen in
all the infected groups when compared to the negative
control. However, there was no significant (P>0.05)
difference observed in the organ body weight ratio of the
kidney in group IV whereas a significant decrease
was observed in groups III and V when
compared to the negative control.
In Table 4, The Parasitemia of groups III, IV and V
were significantly (P<0.05) lower than group II and
among the infected and treated groups, group V showed
least level of parasitemia. In the same Table, infected
groups developed anaemia as observed in the downward
displacement of their PCV profiles in comparison to the
uninfected group. Nevertheless, there was a significant
(P<0.05) increase in final PCV of the treated groups
when compared to infected untreated group.
In this experiment, there is Increase in levels of Alanine
aminotransaminase(AST), alkaline phosphatase(ALP), urea
and Creatinine due to infection. This agrees with the
findings of Hudson (1944), Kalu et al. (1989), Adah et al.
(1992), Ismaila et al. (2000) and Umar et al. (2008) who
reported increase in serum levels in experimental
trypanosomiasis. Increases in the levels of these
enzymes are indications of damage to liver, brain, and
cardiac muscles (Kaplan, 1988) and several workers
have reported hepatocellular damage and generalized
degenerative changes in other tissues and organs in
trypanosomiasis (Anosa et al., 1984; Bruijn, 1987). The
decrease observed in the levels of ALT, AST, ALP,
Creatine and Urea in the treated groups might be as a
result of Vitamins C and E supplementation.
The enlargement of the organs (Heart, Liver, Kidney
and Spleen) Otherwise known as cardiomegaly,
Hepatomegaly and Splenomegaly respectively as
observed in the result, is presumably due to membrane
damage caused by the large amount of free radicals and
other oxidative species being generated and the
28 Int. Res. J. Biochem. Bioinform.
concomitant reduction in systemic antioxidant reserves.
This agrees with the findings of Morrison et al. (1978)
who reported Hepatomegaly and Splenomegaly in
trypanosomiasis. The increase in size of liver and spleen
is caused by the activation of the immune system during
trypanosome infection. The prevention of organ damage
by vitamins C and E is predicated on its antioxidant
activity (Anderson and Theron, 1990). Spleen is involved
in producing antibodies that fight infection and as a result
of this intense activity the organ is enlarged. The kidney
serves many important functions, among are: Filtering out
wastes to be excreted in the urine and Stimulating red
blood cell production via the release of the hormone
erythropoietin. For this reason, it tends to increase in size
so as to meet the needs of the infected animals. It was
earlier reported that infection of trypanosome causes
anemia (Igbokwe and Nwosu 1997), this mean that there
is no enough red blood cell to carry adequate oxygen to
the tissues. If the anemia becomes chronic, it will lead to
rapid or irregular heartbeat, in this case, the heart must
pump more blood to make up for the lack of oxygen in the
blood. For this reason, the heart tends to enlarge above
normal, since the animal is untreated.
The change in PCV is an indicator which gives the
disease status and productive performance of T. brucei
brucei infection. Trypanosome infection caused anaemia
in both treated and untreated groups as a result of
massive erythrophagocytosis by an expanded and active
mononuclear phagocytic system (MPS) of the host
(Igbokwe and Nwosu, 1997). The low final-percentage
PCV observed in the infected groups may be as a result
of acute hemolysis due to growing infection, this result
agrees with earlier studies by Anosa (1988) and Igbokwe
et al. (1994). Unlike the untreated group, the groups
treated with vitamins C and E show significant (p<0.05)
percentage increase in the PCV, this virtually means the
vitamins have ameliorated the effect of the trypanosome
infection on the PCV values. Previous studies have
shown that infection with trypanosomes resulted in
increased susceptibility of red blood cell membrane to
oxidative damage probably as a result of depletion of
vitamins on the surface of the red blood cell (Igbokwe et
al., 1994, 1996; Taiwo et al., 2003; Akanji et al., 2009).
Severity of anaemia usually reflects the intensity and
duration of parasitaemia, this corroborates with several
reports by Ogunsanmi et al. (2001), Umar et al. (2007)
and Saleh et al. (2009) who attributed acute anaemia in
trypanosomiasis to rapidly increase Parasitemia.
The vitamins C and E aided in reducing the free radicals
being generated by the Trypanosoma brucei brucei
(Federe strain), ameliorated anemia and organ damage.
Nevertheless, the combined administration of vitamins
was more effective than single administration. The data
lend further support to the significant roles of oxidative
stress and depletion of endogenous antioxidant reserves
in the organ pathogenesis of African trypanosomiasis.
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How to cite this article: Ajakaiye J.J., Kugu B.A., Shu’aibu
Y., Bizi R.L., Benjamin S.M., Muhammad A.A., Mohammad
B. and Mazadu M.R. (2014). Influence of high dietary
vitamin C and E oral administration on anemia and organ
damage in wistar rat infected with Trypanosoma brucei
brucei (Federe strain). Int. Res. J. Biochem. Bioinform.
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