Growth Performance, Metabolic Efficiency and Nutrient Utilization of BALB/C Mice Infected with Leishmania major Fed with Standard Rat Pellets or Annonaceae Fruit Pulp Pellets

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*Corresponding author: Email: clennyson@gmail.com;
International Journal of TROPICAL DISEASE
& Health
30(2): 1-14, 2018; Article no.IJTDH.39784
ISSN: 2278–1005, NLM ID: 101632866
Growth Performance, Metabolic Efficiency and
Nutrient Utilization of BALB/C Mice Infected with
Leishmania major Fed with Standard Rat Pellets or
Annonaceae Fruit Pulp Pellets
Lenny Mwagandi Chimbevo
1,2*
, Simon Muturi Karanja
2
, Jennifer A. Orwa
3
,
Christopher Omukhango Anjili
4
and Suliman Essuman
5
1
Department of Biomedical Sciences, School of Health Sciences, Kirinyaga University, Kerugoya,
Kenya.
2
Institute of Tropical Medicine and Infectious Disease (ITROMID), School of Public Health, Jomo
Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya.
3
Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute (KEMRI),
Nairobi, Kenya.
4
Centre for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI),
Nairobi, Kenya.
5
Department of Medical Microbiology, Medical School, Mount Kenya University, Thika, Kenya.
Authors’ contributions
This work was carried out in collaboration between all authors. Authors LMC, SMK, JAO and COA
designed the study, developed the protocol and managed the literature searches the study. Author
LMC performed the experimental analyses of the study. Authors LMC and SE performed the statistical
analysis and participated in the development of the first draft of the manuscript. All authors read and
approved the final manuscript.
Article Information
DOI: 10.9734/IJTDH/2018/39784
Editor(s):
(1) Giuseppe Murdaca, Clinical Immunology Unit, Department of Internal Medicine, University of Genoa, Italy.
Reviewers:
(1) Marjorie A. Jones, Illinois State University, USA.
(2) Nwambo Joshua Chidiebere, American University of Nigeria, Nigeria.
Complete Peer review History:
http://www.sciencedomain.org/review-history/24325
Received 13
th
December 2017
Accepted 21
st
February 2018
Published 25
th
April 2018
ABSTRACT
This study was aimed at evaluating the clinical, biochemical, and hematological changes in male
BALB/C mice infected with Leishmania major fed with standard rat pellets (RP) and Annonaceae
Fruit Pulp pellets (AFPP) in different experimental exposures. The results of the study showed good
Original Research Article

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
2
palatability, acceptability and normal behaviour of the mice during the whole experimental period
except in the infected groups. Furthermore, the results achieved with low levels of Annonaceae fruit
pulp pellets crude proteins (3.81±0.14) and lipids (2.95±0.18) were comparable with that of RP at a
level of 25.28±0.04 and 18.60±0.06 respectively. The growth parameter, metabolic efficiency and
feed utilization parameters between the rat pellets and Annonaceae fruit pulp pellets in non-
infected, infected non-treated and infected treated groups of mice did not differ. At the same time
haematological changes, liver and kidney function parameters and lipid profile of the mice that were
feed with rat pellets and Annonaceae fruit pulp pellets in non-infected, infected non-treated and
infected treated were also comparable same. Other parameter measured in the study such as
weight of organs (liver and spleen) parasite burden (LDU) followed the same trend. However, some
of the infected non-treated groups had parameters beyond the normal ranges than the non-infected
and infected treated groups at the end of the experimental period. It can be concluded that
Annonaceae fruit pulp pellets can be utilized as source of raw material in the manufacture of animal
feeds and neutraceuticals upon further research in higher animal model.
Keywords: BALB/C; leishmania major; Annonaceae; growth performance; metabolic efficiency; feed
utilization.
1. INTRODUCTION
One of the world’s most devastating neglected
tropical diseases of documented epidemiological;
and experimental public health importance is
Leishmaniasis [1]. In developing countries where
it is manifested, it often co-exists with chronic
malnutrition, one of the main risk factors for its
development [2,3,4]. In most of the study
conducted, only a few emphasized on the
relationship between leishmaniasis progression
and malnutrition. Malnutrition is a serious health
problem that remains common in many parts of
the world [5,6].
In the developing world, Leishmaniasis combined
with malnutrition is a public health problem. The
most frequent form of malnutrition is Protein
energy malnutrition (PEM). Globally, about 800
million people, including over 150 million children
under 5 years of age, most of them in developing
countries, are affected by PEM [7]. The excess
morbidity and mortality associated with
malnutrition is due to the impairment of sufferers’
defense mechanisms, which predisposes them to
infectious diseases [8]. Further, the poor
understanding of the impact of PEM on immune
response against Leishmania infection coupled
with neglected nutritional status of the host [9,10]
worsen the situation. Studies have revealed that
good protein energy (PE) promote adaptive
immune response in mice infected with L.
chagasi [11]. The BALB/C mice provide a unique
opportunity to study human cutaneous
leishmaniasis (CL) in its active form due to their
susceptibility to L. major infection. As a model, it
reproduces clinical and pathological features of
human CL.
Food insecurity, especially in the developing
world, has necessitated research in alternative
source of food substances. Therefore, natural
products, notably those derived from plants, have
been used to help mankind sustain its nutrition
and health since the dawn of medicine. The
importance of plants in agriculture and medicine
has stimulated significant scientific interest [12].
However, a restricted range of plant species, with
regard to their medicinal importance, has
experienced detailed scientific inspection,
resulting into comparatively insufficient
knowledge concerning their potential role in
nutraceutical application [13]. Therefore, to attain
a reasonable perception of medicinal
values/benefits, comprehensive investigations on
the role of plants in the management of human
ailments are needed.
In a nutraceutical landscape, plants with a long
history of use in ethno medicine are a rich source
of active phyto-constituents that provide
medicinal or health benefits against various
ailments and diseases [14]. One such plant
family with extensive traditional use in Kenya and
mostly grown in the coast region is Annonaceae
family which include; A. cherimola; Mill
(cherimoya), A muricata; L (soursop), A.
squamosa; L (sugar apple) A. senegalensis; L
(wild soursop) and A. reticulata; L (custard
apple). The fruits of these plants contain
numerous bioactive substances [15]. These fruits
are also possible contributors of the carotenoids,
vitamins, mineral salts, fibres and bioactive
compounds [16,14]. Thus they can be
incorporated as components of diets to provide a
delicate balance of food security. The fact that
they are used as food by other parts of the world

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3
[16] with Kenyans having similar gastronomic
habits [17] and also exhibit medicinal potency is
a significant aspect of modern trends in
research focusing on food-medical interface
(Neutraceuticals).
Nonetheless, these plants are neglected
especially as sources of alternative foods for
reasons not well understood. The present paper
reports the influence of diet made from
Annonaceae fruit pulp on growth performances,
metabolic efficiency and nutrients utilization on
BALB/C mice infected with L. major. This might
reflect on proper utilization of these neglected
crops for development of nutraceutical products.
It might also contribute towards curbing food
insecurity in the region where they are grown.
2. MATERIALS AND METHODS
2.1 Plant Material
The ripe fresh fruits of Annonaceae (A. muricata
and A. squamosa) were collected from farms in
coast province (Kilifi and Kwale Counties) of
Kenya. The harvested fruits collected were then
transported to the Jomo Kenyatta University of
Agriculture and Technology (JKUAT) in the
Department of Food Science Technology, Food
Biochemistry laboratories. In the laboratory, the
fruits pulps were dried using a constant
temperature and humidity chamber (Tokyo
Thermo Tech Co. Ltd, Japan) set at 25ºC.
Thereafter, feed pellets were made from the
dried fruit pulp. The pellets made were taken to
the Centre for Biotechnology Research and
Development (CBRD), Kenya Medical Research
Institute (KEMRI) leishmaniasis laboratories for
in vivo trials.
2.2 Animal Feeds
Standard rat pellets (Rate pellets®, Unga Feeds
Ltd, Kenya) and Annonaceae fruit pulp (AFP)
were used in the study. To ensure palatability of
the AFP formulated feed, cubes were made from
the powdered AFP by mixing with wheat flour as
a binder after addition of water using a pellet-
making machine. Proximate composition analysis
of the feeds was performed as described in the
Association of Official Analytical Chemists
(AOAC) manual [18].
2.3 Parasites
Metacyclic promastigotes of L. major (strain
IDUB/KE/83=NLB-144) maintained by
cryopreservation and in vitro culture, and periodic
passage in BALB/c mice at KEMRI were cultured
in Schneider’s insect medium (Gibco, Invitrogen),
supplemented with 20% fetal calf serum (FBS)
(Life Technologies), glutamine (2 mM), penicillin
G (100 U/mL), and streptomycin (100 µg/mL).
The parasites were harvested in the stationary
phase after 8–10 days of culture, centrifuged (at
1000rpm for 5 min), washed twice with
Schneider’s insect medium, counted, and used to
inoculate mice.
2.4 Animals
Male BALB/c mice (3-4 weeks old) obtained from
Kenya Medical Research (KEMRI) animal house
were fed with standard rat pellets (Rate pellets®,
Unga Feeds Ltd, Kenya), Annonaceae fruit pulp
(AFP) and water ad libitum and kept under
standard temperature (26ºC and 60% humidity)
and a natural light-darkness cycle. All in vivo
experiments with mice were performed according
to the established bioethical standards of the
KEMRI’s Animal Care and Use Committee
(ACUC), Scientific and Ethics Review Unit
(SERU).
2.5 Experimental Design
The mice were equally and randomly allocated
into experiment groups assigned to groups of six
mice per group as follows; Non-Infected fed with
Rat pellets (NI-RP), Infected Non-Treated fed
with Rat pellets (INT-RP), Infected Treated fed
with Rat pellets (IT-RP), Non-Infected fed with
Annonaceae fruit pulp (NI-AFP), Infected Treated
fed with Annonaceae fruit pulp (IT-AFP), Infected
Non-Treated fed with Annonaceae fruit pulp
(INT-AFP). After the different experimental
exposures, the mice were closely monitored on a
daily basis for agility, hair ruffling, appetite,
vomiting, urine colour, skin turgor, ocular tension,
limb paralysis, convulsions and roll-over
movements [19]. Body weight and temperature
were measured on a weekly basis. After lesion
have developed, density of parasites was
determined by counting the number of
amastigotes form of parasite from smears made
from ulcer stained with Leishman’s stain [20].
The infected footpads were measured using a
direct reading vernier caliper and lesion size
calculated [21]. Means of weekly readings were
calculated to facilitate comparison of lesion
progression. Growth performance and nutrient
utilization was assessed at the end of the
experimental period (12 weeks) described by as
follows; Body mass gain (BMG) = [(Final Body

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
4
Mass [FBM]) - (Initial Body Mass [IBM])]/IBM] x
100, Specific growth rate (SGR% per day) =
[(FBM) - (IBM)/number of trial days] x 100,
Metabolic growth rate (MGR g/kg
0.8
/day) =
(BMG)/{[(IBM/1000)
0.8
+ (FBM/1000)
0.8
]/2}/
duration of the trial days, Feed conversion ratio
(FCR) = Dry feed fed (g)/BMG (g), Protein
efficiency ratio (PER) = fresh BMG (g)/Crude
Protein (CP) fed (g), Protein productive value
(PPV)% = [(final mice body protein in g – initial
mice body protein in g)/total protein consumed in
g] x 100 and Apparent lipid conversion (ALC)% =
[(final mice body lipid in g - initial mice body lipid,
g)/total crude lipid consumed in g] x 100 [22]. At
the end of the experimental period (12 weeks),
the animals were fasted for about 4 hours with
free access to water and sacrificed by inoculation
with 100µL of pentabarbitone sodium (Sagatal®).
2 mL of blood sample was collected for
determination of haematological and biochemical
parameters, renal and liver functions and blood
metabolites using a Retroflon® Plus
automated analyzer. Liver and spleen impression
smears were used to quantitate the parasite
loads [23].
2.6 Data Analysis
Haematology and biochemical parameters, blood
metabolites, growth performance parameters and
body weights data were analyzed using mean
separation by GenStat program [24]. Mean
separation was done through Fischer least
significance difference. Comparisons between
two treatments were done by means of unpaired
Student’s t-test and significance established by
ANOVA. A difference of P < 0.05 was considered
statistically significant.
3. RESULTS
3.1 Proximate Composition Analysis and
Palatability of the Experimental Feeds
Good palatability, acceptability and normal
behaviour were observed during the whole
experimental period except in the infected group.
In the non-infected group, minimal residual feeds
were observed in the cages. The feed
composition parameters in % Dry Weight Basis
(DWB) of the two experimental feeds (Table 1)
differ significantly (p < 0.05). The two
experimental feeds were analyzed for proximate
feed composition parameters in % Dry Weight
Basis (DWB); carbohydrates, crude lipids, crude
proteins, moisture content, ash content, dry
matter and fibre content (Table 1). The analyzed
proximate composition parameter of the two
feeds; RP and AFPP differed significantly (P <
0.05). The RP had a higher value of crude
protein and crude lipids, 25.28±0.04 and
18.60±0.06 respectively against the AFPP;
3.81±0.14 and 2.95±0.18 respectively. However,
the AFPP had higher values of carbohydrates,
moisture content, ash content, dry matter and
fibre content of 40.12±0.38, 6.20±0.12,
5.93±0.50, 93.80±0.12 and 59.56±2.64 against
the RP of 27.14±2.05, 2.17±0.98, 3.51±0.04,
1.42±0.24 and 25.67±1.28 respectively. Good
palatability, acceptability and normal behaviors
were observed during the whole experimental
period in all groups of BALB/C mice except in the
infected groups.
Table 1. Proximate composition analysis of
diets used in feeding BALB/C mice
Parameter Experimental diets
Rat pellets
(RP)
Annonaceae fruit
pulp pellets
(AFPP)
Crude Proteins 25.28
a
±0.04 3.81
b
±0.14
Crude Lipids 18.60
a
±0.06 2.95
b
±0.18
Carbohydrate 27.24
a
±2.05 40.12
b
±0.38
Moisture content 2.17
a
±0.98 6.20
b
±0.12
Ash content 3.51
a
±0.04 5.93
b
±0.50
Dry matter 1.42
a
±0.24 93.80
b
±0.12
Fibre content 25.67
a
±1.28 59.56
b
±2.64
Mean values (n=3) ± SEM. Values appended by different
small letters within a row are significantly different (P < 0.05).
3.2 Growth Performance and Nutrient
Utilization
The body mass changes in the different groups
of BALB/C mice measured weekly. Generally,
the BMG was observed in the two NI groups of
BALB/C mice; NI-RP and NI-AFPP. However, the
BMG in the NI-RP group was higher
(56.10±1.49) compared to that of NI-AFPP
(49.11±1.39). A significant decrease in body
weight was observed in both the IT-RP and IT-
AFPP before treatment (Figure 1). However, this
reversed after treatment with Pentostam in week
4 with the IT-RP and IT-AFPP groups having
almost the same BMG values of 27.31±0.66 and
27.56±1.37 respectively at the end of the
experimental period. There was also a significant
decrease in BMG in both INT-RP and INT-AFPP
groups (P < 0.05) (Fig. 1) with the INT-AFPP
group having greater decrease in BMG
(-40.71±0.68) than the INT-RP group
(-34.62±0.79).

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
5
Other growth performance, metabolic efficiency
and nutrient utilization parameters such as IBM,
FBM, SGR, FCR, PER, PPV and ALC followed
the same trend. Higher values of these growth
performance, metabolic efficiency and nutrient
utilization parameters were observed in the RP
groups as compared to the AFPP groups except
in the INT where low values were observe in the
RP groups than in the AFPP groups. Statistically,
there was a significant difference in most of the
parameters (p < 0.05) among the different
groups in different experimental treatment in the
growth performance, metabolic efficiency and
nutrient utilization parameters. The onset of the
diseases decreased all the growth performance,
metabolic efficiency and nutrient utilization
parameters investigated (Table 2). Slightly high
SGR was observed in the NI-RP group
(13.24±0.11) than in the NI-AFPP group
(11.60±0.23). However, in the infected treated
(IT) groups, both the RP and AFPP groups had
almost the same SGR. Although both the INT
groups had a negative SGR, the
negativity was more in the AFPP group than the
RP group.
3.3 Haematological Changes
Hematological changes are presented in Table 3.
A decrease in Hemoglobin (Hb) below the normal
range was observed in all experimental groups
except in NI groups. Red Blood Cells (RBC),
Hematocrit (HCT) or Packed Cell Volume (PCV),
Mean Capsular Volume (MCV), Mean
Corpuscular Haemoglobin (MCH) and Mean
Corpuscular Hemoglobin Concentration (MCHC)
decrease in all the experimental groups.
However, a significant decrease (p < 0.05) in Hb
was observed in the INT groups as compared to
the NI groups. After treatment with pentostam in
the IT groups, Hb increased was significant (p <
0.05), although it was still below the physiological
range. The RBC, HCT, MCV, MCH and MCHC
followed the same trend as Hb. Total white blood
cells (WBC) or total leucocytes count (TLC)
increased significantly (p<0.05) in infected
groups compared to NI groups. Differentially,
Leukocytes, platelets (PLT) and leukocyte
populations (neutrophils, lymphocytes,
eosinophils, monocytes and basophils) increased
significantly in the INT groups compared to the
NI groups. The increase in these parameters was
within normal range in all the experimental
groups. However after treatment in the IT groups,
these parameters increased returning almost to
the values in the NI groups. On comparison, the
groups showed at least one significant difference
among the averages of Hb (p=0.001), HCT
(p=0.05), MCV (p=0.001), MCH (p=0.001), RBC
(p=0.001), leukocytes (p=0.008), lymphocytes
(p=0.013), neutrophils (p=0.029), monocytes
(p=0.002) and basophils (p=0.005).
Fig. 1. Body weight changes of BALB/c mice infected with L. Major fed with the experimental
diets for 12 weeks
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
Body Weight (g)
Time (We eks)
NI-RP INT-RP IT-RP NI - AFP INT - AFP IT - AFP

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6
3.4 Liver Function Tests
The function test changes are presented in Table
4. Alanine aminotransferase (ALT) also called
Serum glutamic pyruvic transaminase (SGTP),
aspartate aminotransferase (AST) also called
Serum glutamic oxaloacetate transaminase
(SGOT), lactate dehydrogenase (LDH), Alkaline
phosphatase (ALkP) sorbitol dehydrogenase
(SDH) creatine kinase (CK) and bilirubin activity
increased significantly in the INT groups
compared to the NI groups. After treatment, the
activity ALT (SGTP), AST (SGOT), LDH, SDH,
CK and bilirubin decreased almost returning to
that of NI groups. The total protein, albumin,
globulin and ALkP decreased significantly in INT
animals compare to the NI animals. Most of the
liver function tests were found to be within
normal range in all IT and NI groups. A
significant increase in ALT (SGTP) beyond the
physiological range (183.49±3.18) was observed
in INT-AFPP group. Also a higher activity of AST
(SGOT) beyond the physiological range
(218.20±3.56 and 184.87±5.99) was observed in
INT-RP and INT-AFPP groups respectively. The
activity of amylase increased beyond the
physiological range whereas that of LDH
decreases below the range in NI-RP
(24.33±0.67), NI-AFPP (17.90±0.69) groups but
increased significantly beyond the physiological
range (40.17±0.29) in the INT-AFFP group. After
comparison, there was at least one significant
difference among the averages of all biochemical
parameters used to ascertain liver function of the
mice.
3.5 Kidney Function Tests and Lipid
Profiles
The kidney function tests and lipid profile
changes are presented in Table 5. Creatinine,
urea and blood urea nitrogen (BUN) increased
significantly (p<0.05) in the INT group compared
to NI groups. In all the IT groups, creatinine,
blood Urea and BUN levels decreased
significantly (p<0.05) compared to the INT
groups. Creatinine levels beyond the
physiological range were observed in all BALB/C
mice groups while BUN decreased in NI-AFPP
(25.16±0.11) and IT-AFPP (23.82±0.10) groups.
However, the increased urea levels in all the
BALB/C mice groups were found to be within
normal range. Serum sodium, chloride,
potassium and phosphorous increased
significantly in the INT groups compared to the
NI groups although values remained within the
physiological ranges. After treatment, a decrease
in these ions was observed in the IT groups.
Cholesterol, triacylglycerides (TGs), high-density
lipoproteins (HDL), low density lipoprotein (LDL)
and cholesterol/HDL ratio increased significantly
(p<0.05) in the INT groups in NI groups. This is
an indicative of liver damage and risk of
development of cardiovascular diseases. All lipid
profile values were within the physiological range
except HDL in INT-RP (2.73±0.11), IT-RP
(1.23±0.07), INT-AFPP (3.03±0.06) and IT-AFPP
(1.90±0.13) groups suggesting a relationship
between leishmaniasis and cardiovascular
diseases. Statistically, there was no significant
difference between lipid profiles of NI-RP and NI-
AFPP (0.09) and INT-RP and INT-AFPP
(p=0.08) but there was significant differences
between NI and INT (p=0.003) and INT and IT
(p=0.001) groups.
3.6 Lesion Size and Liver and Splenic
Length, Size and Parasite Burden
The lesion sizes (mm
2
) for the INT and IT groups
were determined and are presented in Fig. 2.
Generally, there was an increase in the size of
the lesions with the progression of the disease.
After the initiation of treatment on the IT groups,
the lesion sizes decreased. However, the rate of
decrease of the size of the lesions was higher in
the IT-RP group than in the IT-AFPP group (Fig.
2). The lesion sizes of the INT groups increased
significantly until the end of the experiment, with
the INT-AFPP having larger lesion sizes than the
INT-RP group. The hepatic and splenic length
(mm) and size (mg) for the NI, INT and IT groups
(Table 6) determined at the end of the
experiment varied significantly (p<0.05). The
relative mean weight and length of the liver and
the spleen increased with increasing the time of
infection. A significant differences (p<0.05) was
observed between the weight and length of
infected (both IT and INT) and non-infected (NI)
BALB/C mice groups. However, no significant
difference (P>0.05) was observed between NI-
RP and NI-AFPP. The same trend was observed
between ITC-RP and IT-AFPP.
The parasite density of L. major amastigotes in
liver and spleen for infected not treated (INT) and
IT groups of BALB/C mice (Table 6) determined
after the experimental period (12 weeks) showed
significant variations. In all the IT groups of
BALB/C mice, parasite load decreased
significantly (p<0.05) as compared to the INT
groups. The reduction in parasite load was
significantly (p<0.05) more in the IT-RP group as
compared to the IT-AFPP group. After the

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7
experimental period (12 weeks), hepatomegaly
and splenomegaly was observed on the INT
groups of BALB/C mice. Impression smears of
liver and spleen on the INT groups of BALB/C
determined at the end of the experimental period
(12 weeks) showed dissemination of L. major
amastigotes inside and outside macrophages.
4. DISCUSSION
The results of the present investigation
demonstrate that AFPP may be a good dietary
protein source for BALB/C mice feed because
growth performance and feed utilization of
Annonaceae group was similar to control group
(RP fed group). No significant difference in
growth performance (BMG, SGR and MGR) and
feed utilization parameters (FCR, PER and ALC)
show that digestion and absorption of nutrients
from RP and AFPP were similar. Nutrient
utilization values observed in this study signify
excellent utilization of the diets. Growth
performance of RP fed group was similar to
AFPP group indicating that nutrients and energy
availability from the AFPP for the protein
synthesis were similar to animal protein (RP).
Further, the growth performance and feed
utilization parameter of the infected and treated
groups feed with RP and AFPP returned to
normal. This shows that AFPP can be used as
an alternative source of feed for BALB/C mice.
In terms of haematological changes, lesion size
and organ length, size functioning and parasite
burden, Leishmania major is an aetiological
agent of cutaneous leishmaniasis, which is a
parasite of the skin on humans. However, in
BALB/C mice, it attacks visceral organs in
addition to the local lesion at the point of
inoculation [25]. The parasites can be deposited
in major visceral organs involved in the synthesis
of major macromolecule after the digestion,
absorption and transport. Nonetheless, BALB/C
mice model was chosen for this study as a
suitable model for the investigation of interaction
between malnutrition and leishmaniasis. In
earlier studies, malnourishment contributed to
higher parasite loads found in the blood, skin,
bone marrow, lymph node, liver and spleen and
favoured the development of leishmaniasis [6].
Based on these observations, this model
provides an excellent opportunity to elucidate the
factors implicated in severe malnutrition.
Moreover, as it has been shown in this work, the
model can be used for further investigation on
the relationship between severe malnutrition and
leishmaniasis
Fig. 2. Lesion sizes of BALB/C mice infected with L. major and feed with RP and AFPP on 12
weeks

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8
Table 2. Growth performance and nutrient utilization in BALB/C mice infected with L. major fed with experimental diets of 12 weeks
Parameter
Rat pellets (RP)
Annonaceae fruit pulp pellets (AFPP)
NI-RP INT-RP IT-RP NI-AFPP INT-AFPP IT-AFPP
IBM 19.89
a
±0.11 20.18
b
±0.11 20.18
b
±0.11 19.99
a
±0.19 19.76
a
±0.07 19.93
a
±0.13
FBM 31.02
b
±0.21 13.18
d
±0.13 25.68
a
±0.10 29.74
b
±0.12 11.71
c
±0.13 25.38
a
±0.19
BMG 56.10
c
±1.49 -34.62
a
±0.79 27.31
b
±0.66 49.11
e
±1.39 -40.71
d
±0.68 27.56
b
±1.37
SGR 13.24
c
±0.30 -8.34
a
±0.22 6.54
b
±0.14 11.60
d
±0.23 -9.58
a
±0.17 6.49
b
±0.29
FCR 3.05
b
±0.07 -4.93
c
±0.11 6.28
a
±0.18 3.51
b
±0.10 -4.16
c
±0.07 6.56
a
±0.32
PER 0.33
a
±0.01 -0.21
b
±0.01 0.16
c
±0.04 0.29
a
±0.01 -0.24
b
±0.004 0.16
c
±0.01
PPV 0.66
a
±0.06 0.02
b
±0.01 0.46
a
±0.16 0.46±0.01 0.01
b
±0.04 0.46±0.01
ALC 52.67
a
±3.31 22.17
b
±2.60 40.17
c
±6.60 49.36
a
±4.49 18.7
d
±6.60 38.70
e
±6.60
Mean values (n=3) ± SEM. Values appended by different small letters within a row are significantly different (P < 0.05)
Table 3. Hematological changes of BALB/C mice infected with L. Major fed with RP and AFPP for 12 weeks
Parameter Rat pellets (RP) Annonaceae fruit pulp pellets (AFPP) Ref. Range
NI-RP INT-RP IT-RP NI-AFPP INT-AFPP IT-AFPP
HB (g/dL) 12.76
a
±0.67 8.07
b
±0.10 10.40
a
±0.10 12.09
a
±0.14 8.58
b
±0.08 9.89
ab
±0.10 12.60 - 20.50
WBC x 10
9
/L 6.11
a
±0.12 9.08
b
±0.09 6.58
a
±0.16 5.91
a
±0.05 11.97
c
±0.12 9.91
b
±0.05 3.48 - 14.03
RBC x 10
12
/L 8.39
b
±0.06 5.96
a
±0.12 8.05
b
±0.12 7.04
c
±0.09 6.10
ac
±0.11 8.08
b
±0.05 6.93 – 12.24
HCT/PCV (%) 48.30
a
±0.88 43.13
c
±0.79 46.80
b
±0.08 46.97
b
±0.88 44.60
c
±0.63 44.80
c
±0.47 42.10 - 68.30
MCV (FL) 54.28
c
±0.97 46.28
d
±0.53 51.44
a
±0.62 51.28
a
±0.66 42.30
e
±0.53 49.61
b
±0.38 50.70 – 64.40
MCH (FL) 30.98
b
±0.44 23.22
a
±0.19 26.38
c
±0.38 26.82
c
±0.54 22.03
a
±0.19 26.72
c
±0.42 13.20 – 17.60
MCHC (pg)
31.04
c
±0.58
23.20
b
±0.31
28.37
ad
±0.19
27.54
a
±0.16
25.30
c
±0.12
27.70
a
±0.97
23.30 – 32.70
PLT x 10
12
/L
511.98
a
±19.54
695.32
b
±7.39
611.98
c
±19.54
449.58
d
±23.47
727.40
e
±19.54
357.66
f
±12.23
420.00 – 1698.00
Neutrophils (%) 24.89
b
±0.51 30.06
a
±1.15 23.39
ac
±0.62 23.73
c
±0.37 36.50
d
±0.79 26.06
e
±0.40 9.86 – 39.11
Lymphocytes (%) 74.62
a
±0.35 83.95
b
±0.61 73.57
a
±0.39 61.95
c
±0.65 78.20
d
±0.95 65.62
e
±1.41 50.00 – 96.00
Monocytes (%) 3.72
c
±0.13 4.05
b
±0.13 3.72
c
±0.06 3.97
c
±0.08 4.00
b
±0.17 5.38
a
±0.33 3.29 – 12.48
Eosinophils (%) 0.72
a
±0.06 0.85
a
±0.08 0.63
c
±0.05 0.58
d
±0.05 0.60
b
±0.02 0.61
b
±0.07 0.11 – 4.91
Basophils (%) 0.10±0.00 0.07
a
±0.00 0.08
ab
±0.00 0.09
b
±0.01 0.05
c
±0.01 0.09
b
±0.00 0.00 – 1.84
Abs Neutrophils 3.15±0.24 4.77±0.19 4.75±0.17 3.85±0.17 7.05±0.29 4.07±0.19 0.00 – 3.83
Abs Lymphocytes 3.13±0.11 5.09±0.17 3.79±0.07 4.10±0.07 6.14±0.05 4.84±0.09 2.22 – 9.83
Abs Monocytes 0.90±0.03 1.27±0.06 0.63±0.04 0.79±0.04 1.78±0.04 0.68±0.07 0.21 – 1.25
Abs Eosinophils 0.24±0.04 0.43±0.02 0.13±0.02 0.17±0.02 0.38±0.15 0.28±0.05 0.01 – 0.49
Abs Basophiles 0.01±0.0 0.07±0.00 0.13±0.00 0.02±0.00 0.15±0.02 0.05±0.01 0.00 - 0.18
Mean values (n=3) ± SEM. Values appended by different small letters within a row are significantly different (P < 0.05)

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9
Table 4. Liver functions in BALB/C mice infected with L. major fed with RP and AFPP for 12 weeks
Parameter Rat pellets (RP) Annonaceae fruit pulp pellets (AFPP) Ref. Range
NI-RP INTC-RP IT-RP NI-AFPP INT-AFPP IT-AFPP
Glucose (mg/L) 122.71±2.12 123.87±1.95 124.37±1.91 126.17±0.45 124.27±0.65 125.20±0.41 106.00 – 278.00
T-Bilirubin (g/dL) 9.02±0.17 11.18±0.10 9.52±0.29 10.20±0.06 12.68±0.35 9.68±0.07 7.12 -12.05
C-Bilirubin (g/dL) 2.57±0.11 3.43±0.05 2.57±0.11 3.07±0.26 4.23±0.35 2.80±0.08 0.00 – 2.00
U-Bilirubin (g/dL) 3.67±0.17 4.83±0.13 3.17±0.13 3.83±0.16 4.33±0.20 3.00±0.11 0.00 – 3.00
ALT (IU/L) 77.49±2.40 115.99±1.02 98.66±1.31 83.49±3.18 183.49±3.18 81.82±2.86 41.00 – 131.00
AST (IU/L) 119.87±2.16 218.20±3.56 111.53±0.69 134.87±5.86 184.87±5.99 129.87±5.66 28.00 – 191.00
GGT (IU/L) 25.43±0.55 28.77±0.48 24.60±0.46 23.77±0.60 31.43±0.56 23.77±0.60 20.00 – 40.00
ALKP (IU/L) 132.36±1.53 127.36±1.33 134.03±1.21 157.36±4.40 140.70±1.70 155.70±3.03 118.00 – 187.00
T-Protein (g/dL) 51.77±0.90 45.94±0.04 47.12±0.33 42.95±0.51 35.02±0.51 45.27±0.51 43.00 – 70.00
Albumin (g/dL) 26.46±0.49 23.34±0.58 35.27±0.51 24.50±0.32 22.27±0.40 26.35±0.14 27.00 – 46.00
Globulin (g/dL) 34.56±0.64 25.22±0.47 35.34±0.35 26.34±0.38 23.18±0.42 26.68±0.10 27.00 – 42.00
Alb/Glob ratio 0.77
d
±0.02 0.93
d
±0.03 1.98
bc
±0.05 1.10
a
±0.02 1.06
a
±0.02 2.12
b
±0.04 0.00 – 1.09
CPK (IU/L) 3.48
b
±0.22 4.02
c
±0.08 1.79
a
±6.86 1.79
a
±0.07 2.46
d
±0.05 1.60
a
±0.04 2.50 – 3.70
Amylase (U/L) 796.97
b
±17.47 1580.31
a
±54.65 896.97
d
±7.90 813.64
e
±23.68 1413.64
e
±36.61 896.97
f
±10.69 210.43 -323.57
LDH (IU/L) 24.33a±0.67 34.66b±0.39 27.17c±0.68 17.90d±0.69 40.17e±0.29 24.83a±0.52 26.8 – 34.00
SDH (IU/L) 29.15
a
±0.90 34.50
c
±0.53 31.16
ab
±0.54 23.83
d
±0.39 43.67
e
±0.44 29.17
a
±0.25 27.00 - 37.00
Mean values (n=3) ± SEM. Values appended by different small letters within a row are significantly different (P < 0.05)

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
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Table 5. Kidney functions and lipid profile in BALB/C mice infected with L. Major fed with RP and AFPP for 12 weeks
Parameter
Rat pellets (RP)
Annonaceae fruit pulp pellets (AFPP)
Ref. range
NI-RP INT-RP IT-RP NI-AFPP INT-AFPP IT-AFPP
Creatinine (mg/L) 2.57±0.16 3.32±0.15 2.49±0.19 2.12±0.11 2.87±0.25 2.48±0.10 0.50 - 0.80
Blood Urea Nitrogen 26.66±0.16 31.32±0.15 27.49±0.19 25.16±0.11 27.66±0.25 23.82±0.10 7.00 – 26.00
Urea (mg/L) 7.88±0.11 9.11±0.11 7.38±0.18 6.88±0.22 10.03±0.06 6.88±0.15 7.00 – 26.00
Sodium (meq/L) 143.48±0.74 147.13±1.04 142.96±0.46 135.63±1.33 143.63±0.93 137.29±0.78 125.30 – 187.40
Chloride (meq/L) 116.23±0.82 121.90±1.49 117.40±0.64 121.23±1.10 131.23±1.56 122.90±1.91 109.6 – 138.80
Potassium (meq/L) 6.16±0.13 6.49±0.17 5.49±0.19 7.14±0.06 6.82±0.17 6.32±0.21 7.30 – 12.07
K/Na Ratio 0.04±0.00 0.04±0.00 0.04±0.00 0.05±0.00 0.05±0.00 0.05±0.00 0.06 – 0.09
Phosphorus (meq/L) 3.77±0.09 3.72±0.09 3.65±0.08 4.22±0.16 5.22±0.19 4.22±0.02 8.20 – 14.70
Calcium (mg/dL) 2.59±0.06 2.30±0.03 2.74±0.04 2.76±0.11 2.68±0.09 3.43±0.06 9.40 – 12.70
Cholesterol (g/dL) 3.75±0.18 7.13±0.16 4.81±0.16 4.22±0.14 7.13±0.16 5.31±0.26 11.10 – 24.60
Triglycerides (g/dL) 1.16±0.04 2.48±0.05 1.68±0.05 1.34±0.06 3.11±0.08 1.81±0.03 1.050 – 5.35
HDL Cholesterol (g/dL) 0.77±0.02 2.73±0.11 1.23±0.07 1.00±0.09 3.03±0.06 1.90±0.13 0.40 – 1.00
LDL Cholesterol (g/dL) 1.34±0.11 3.10±0.09 2.12±0.15 1.51±0.11 3.10±0.09 2.12±0.15
Cholesterol/ HDL Ratio 4.23±0.08 2.97±0.39 4.37±0.07 4.65±0.07 2.97±0.14 4.30±0.13
Mean values (n=6) ± SEM. Values appended by different small letters within a row are significantly different (P < 0.05)
Table 6. Body weight (g) and relative weight (mg) and length (mm) of the liver and spleen in BALB/C mice infected with L. major and feed with RP
and AFPP on 12 weeks after infection
Parameter Organ NI-RP INT-RP IT-RP NI-AFP INT-AFP IT-AFP
Body Weight Day 0 19.89
a
±0.11 20.18
a
±0.11 20.18
a
±0.11 19.99
a
±0.19 19.76
a
±0.07 19.93
a
±0.13
Day 84 31.02
b
±0.21 13.18
c
±0.13 25.68
d
±0.10 29.74
ab
±0.12 11.71
c
±0.13 25.38
d
±0.19
Relative length (mm) Liver 22.49
c
±0.14 47.50
b
±0.24 26.07
a
±0.53 22.08
c
±0.11 51.10
d
±0.38 28.15
ae
±0.28
Spleen 15.83
b
±0.44 40.33
a
±0.64 19.50
c
±0.31 18.83
d
±0.32 43.17a±0.44 20.17
c
±0.19
Relative weight (mg) Liver 1.36
b
±0.02 2.66
d
±0.04 1.59
a
±0.02 1.44
ab
±0.06 3.16
c
±0.06 1.61
a
±0.04
Spleen 0.14
a
±0.00 2.48
c
±0.07 0.15
a
±0.00 0.14
a
±0.00 2.34
c
±0.03 0.21
b
±0.01
LDU (10
6
) Liver ND 564.59
a
±13.24 ND ND 645.44
b
±12.58 ND
Spleen ND 13.28
a
±0.42 ND ND 19.02
a
±0.24 ND
Mean values (n=6) ± SEM. Values appended by different small letters within a row are significantly different (P < 0.05). ND – not done

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11
Malnutrition resulting from dietary protein
deficiency decreases weight as age progresses
[11]. Further, cure of diseases depends upon the
development of an effective immune response
that activates macrophages [26] which can be
promoted and enhanced by good protein nutrition
in diets. During Leishmaniasis, there is a
profound immunosuppression in the host that
promotes the survival of parasites [1]. In this
study, it is revealed that treatment of infected
animals coupled with good nutrition brought the
levels of most biological parameters such as Hb
and total leucocytes count (TLC) to normal range
as compared to the abnormal levels in infected
animals.
Kidney function tests include estimation of urea,
blood urea nitrogen and creatinine. Renal
abnormalities caused by Leishmania have been
well documented in experimental animal studies
and are comprised of interstitial and glomerular
abnormalities [27]. Sodium, chloride and
potassium and K/Na ratio values give an
indication of electrolyte/water balance, whereas
high levels of calcium implies thyroid or
parathyroid, intestine, pancreas, kidney and
borne metastasis. Although in the current study
there were elevated levels of sodium, chloride
and potassium and K/Na ratio, the role of
infection caused by L. major on damage of these
organs causing the elevated levels of these
parameter is yet to be established. The reason
may be that these parameters can vary with
mouse strain/stock, age, sex; blood sampling
method, environmental conditions pathogen
status and the laboratory as well as nutrition [28].
The task of establishing a range of reference
values for rodents is very difficult. This is
because of very many variables, such as gender,
age, genetic variation, diet and environmental
conditions to which these animals are subjected,
must be considered [29]. In this study, mice of
the same sex and age infected with the same
strain of L. major were used. Therefore, they may
provide a useful starting point to investigate the
effect of leishmaniasis and nutrition on
hematological parameters. Since significant
variation of biological parameters may occur
between individual mice strain, stock,
laboratories and method of sampling, individual
laboratories should establish normal reference
values for their facility [30].
The most important organ concerned with
majority of biochemical activities in the human
body is the liver. Since it has a great capacity to
detoxify toxic substances and synthesize useful
biological molecule, damage inflicted by
hepatotoxic agents is of grave consequence. The
increased level of ALT, AST, ALP, and bilirubin is
conventional indicator of liver injury. In
Leishmaniasis liver damage due to high parasitic
load in infected groups resulting in elevated
levels of ALT (SGTP), AST (SGOT), ALP and
SDH. An increased level of bilirubin was
observed in this study suggesting loss of
functionality of the liver in Leishmaniasis.
Albumin is used to detect liver damages whereas
globulin and total protein content is used to
detect and immunoglobulin status, a key
indicator in fighting of infections in organisms
which is affected by nutrition. The stabilization of
serum bilirubin, ALT (SGPT), AST (SGOT), and
ALP levels is a clear indication of the
improvement in the functional status of the liver
cells [31]. The increase in AST (SGOT) and ALT
(SGPT) levels has been reported in the visceral
leishmaniasis (VL) patients [32] which is depicted
in this study for cutaneous leishmaniasis (CL)
using mice model. In the current study, there was
no significant elevation in the levels of liver
function tests in all the treated groups as
compared to infected controls.
The mononuclear phagocyte system (spleen,
liver, bone marrow, intestinal mucosa and
mesenteric lymph nodes) is the normal habitat of
Leishmania parasite [33]. However, the parasite
may be found in endothelial cells of the kidneys,
suprarenal capsules, lungs, meninges and in
cerebrospinal fluid [34]. In leishmaniasis there
can be possibilities of multiple organ damage as
indicated by elevated levels of non-specific
markers such as lactate dehydrogenase (LDH)
indicating possible damage of liver, heart,
skeletal muscles and lungs and creatine kinase
(CK) commonly elevated in heart and skeletal
muscle damage and muscular dystrophies.
These effects are consequences of the
stimulation of the immune system by L. major,
which promotes the inflammatory components of
atherosclerosis, which are primarily the parasite-
activated macrophages [35]. Several studies
suggest that pathogenic bacteria, viruses and
protozoa, contribute to the atherogenesis
process [36,37,38,39,40]. The increased
inflammation caused by these pathogens
promotes macrophage activation and migration
to the atheroprone site. Alternatively,
proatherogenic status may be attributed to the
systemic oxidative stress induced by infection,
which enhances lipoprotein or endothelium
oxidation. Pathogens involved in atherosclerosis
development usually induce a systemic infection

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
12
instead of a localized infection such as an L.
major infection. However, even localized
infections, such as odontologic ones, may be
associated with the development of
atherosclerosis [41].
In this study, microscopic examination of stained
impression smear of liver and spleen showed the
density of amastigotes in two organs that
demonstrated the pathological effect of parasite.
The results of this study have demonstrated that
the infected mice show the hepatosplenomegaly
sign of pathological effect of L. major
promastigote in the infected mice. The weight of
liver was increased with increasing days of
infection in the INT mice compared with NI mice
on 12 weeks. For both the liver and spleen, NT
mice had the highest LDU. Significantly (p <0.05)
higher parasite load in the liver and spleen
occurred in NT mice and the lowest LDU
occurred in the IT mice. There were no
significant difference between the following
groups, NI-RP and NI-AFPP (p > 0.05), IT-RP
and IT-AFPP (p > 0.05), INT-RP and INT-AFPP
(p > 0.05), indicating that there is no difference
between the two feeds.
5. CONCLUSION AND RECOMMENDA-
TION
From the study it can be demonstrated that there
was no difference in growth performance,
metabolic efficiency and nutrients utilization
parameters between RP and AFPP among the
same treatment of BALB/C mice. This suggests
that Annonaceae fruit pulp can be used as an
alternative source of raw material in the
manufacture of animal feeds. It has been
observed that the data obtained from present
investigation related to different growth
performance, metabolic efficiency and nutrients
utilization make Annonaceae fruit a good
candidate for manufacture of neutraceuticals.
The choice of animal model (BALB/C mice) may
be a major limiting factor that could have
influenced the applicability of the results of this
study since different laboratories have different
reference range values. Thus, there is a need to
study these parameters in another higher animal
model preferable primates such as monkey and
baboons to validate the findings of this study for
utilization of Annonaceae fruits in the
nutraceutical manufacturing industry.
CONSENT
It is not applicable.
ETHICAL APPROVAL
As per international standard or university
standard, written approval of Ethics committee
has been collected and preserved by the
authors.
ACKNOWLEDGEMENT
The Kenyan Government through National
Commission for Science, Technology and
Innovation (NACOSTI) supported the study.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
REFERENCES
1. Serafim TD, Malafaia G, Silva ME,
Pedrosa ML, Rezende SA. Immune
response to Leishmania (Leishmania)
chagasi infection is reduced in
malnourished BALB/c mice Mem Inst
Oswaldo Cruz, Rio de Janeiro. 2010;
105(6):811-817.
2. Badaró R, Jones TC, Lorenço R, Cerf BJ,
Sampaio D, Carvalho EM, Rocha H,
Teixeira R, Johnson WD. A prospective
study of visceral leishmaniasis in an
endemic area of Brazil. J Infect Dis. 1986;
154:639-649.
3. Cerf BJ, Jones TC, Badaró R, Sampaio D,
Teixeira R, Johnson WD JR. Malnutrition
as a risk factor for severe visceral
leishmaniasis. J Infect Dis. 1987;156:1030-
1033.
4. Maciel BL, Lacerda HG, Queiroz JW,
Galvão J, Pontes NN, Dimenstein R,
McGowan SE, Pedrosa LF, Jerônimo SM.
Association of nutritional status with the
response to infection with Leishmania
chagasi. Am J Trop Med Hyg. 2008;79:
591-598.
5. Blössner M, Onis M. Malnutrition:
Quantifying the health impact at national
and local levels, World Health
Organization, Geneva. 2005;51.
6. Carrillo E, Jimenez M, Sanchez C, Cunha
J, Martins CM, Seva AP, Moreno J. Protein
malnutrition impairs the immune response
and influences the severity of infection in a
hamster model of chronic visceral
leishmaniasis. PLoS ONE. 2014;9(2):
e89412.
DOI: 10.1371/journal.pone.0089412

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
13
7. WHO. Reducing risks, promoting healthy
life, The World Health Report. 2002;
(Chapter 4).
Available:http://www.who.int/whr/2002/en/
(Accessed 2017 Jan 27)
8. Gross RL, Newberne PM. Role of nutrition
in immunologic function. Physiol Rev.
1980;60:188–302.
9. Malafaia G. Protein-energy malnutrition as
a risk factor for visceral leishmaniasis: A
review. Parasite Immunol. 2009;31:587-
596.
10. Malafaia G, Serafim TD, Silva ME,
Pedrosa ML, Rezende SA. Protein-energy
malnutrition decreases immune
response to Leishmania chagasi vaccine in
BALB/c mice. Parasite Immunol. 2009;
31:41-49.
11. Anstead GM, Chandrasekar B, Zhao W,
Yang J, Perez LE, Melby PC. Malnutrition
alters the innate immune response and
increases early visceralization following
Leishmania donovani infection. Infect
Immun. 2001;69:4709-4718.
12. Moghadamtousi SZ, Goh BH, Chan CK,
Shabab T, Kadir HA. Biological activities
and phytochemicals of Swietenia
macrophylla king. Molecules. 2013;18:
10465–10483.
13. Moghadamtousi SZ, Kamarudin, MNA,
Cha, CK, Goh BH, Kadir HA.
Phytochemistry and biology of Loranthus
parasiticus merr, a commonly used herbal
medicine. Am. J. Chin. Med. 2014;42:23–
35.
14. Chimbevo ML, Malala JB, Anjili CO, Orwa
J, Mibei EK, Ndeti CM, Muchiri FW, Sifuna
PO, Oginga FO, Otundo DO, and Karanja
SM. Annonaceae fruits growing in coast
region of Kenya as an alternative source of
dietary carotenoids. International Journal
of Food Science and Biotechnology. 2017;
2(5):114-120.
15. Rupprecht JK, Hui YH, McLaughlin JL.
Annonaceous acetogenins: A review. J.
Nat. Prod. 1990;53:237-278.
16. Pinto AC, de Q, Cordeiro de Andrade
SRM, Ferreira FR, Filgueiras HA, de C,
Alves RE, Kinpara DI. Annona species.
International centre for underutilised crops,
University of Southampton, Southampton,
UK; 2005.
ISBN: 0854327851
17. Maundu PM, Ngugi GW, Kabuye CHS.
Traditional food plants of Kenya. KENRIK,
National museums of Kenya, Nairobi,
Kenya; 1999.
18. AOAC. Official methods of analysis.
Association of Official Analytical Chemists,
Washington DC; 2000.
19. Carvalho LJ, Ferreira-da-Cruz M, Daniel-
Ribeiro FM, Pelajo-Machado CT, Henrique
LL. Plasmodium berghei ANKA infection
induces thymocyte apoptosis and
thymocyte depletion in CBA mice.
Memórias do Instituto Oswaldo Cruz.
2006;101:5-9.
20. Schnur LE, Zuckerman A, Montillio B.
Dissemination of leishmaniasis to the
organs of Syrian hamsters following
intrasplenic inoculation of promastigotes.
Exp. Parasitol. 1973;34:432-447.
21. Nolan TJ, Farrell JP. Experimental
infections of the multimmate rat (Mastomys
natalensis) with L. donovani and L. major.
American Journal of Tropical Medicine and
Hygiene. 1987;36(2):264-269.
22. Kumar V, Akinleye AO, Makkar HPS,
Angulo-Escalante MA, Becker K. Growth
performance and metabolic efficiency in
Nile tilapia (Oreochromis niloticus L.) fed
on a diet containing Jatropha platyphylla
kernel meal as a protein source. J Anim
Physiol Anim Nutr. 2012;96(1):37-46.
23. Bradley DJ, Kirkley J. Regulations of
Leishmania population within the host. The
variable course of Leishmania donovani
infection in mice. Clinical Experimental
Immunology. 1977;30:119-129.
24. Payne RW, Murray DA, Harding SA, Baird
DB, Soutar DM. An introduction to gen stat
for windows, 14th Ed. VSN international,
Hemel Hempstead. UK; 2011.
25. Makwali JA, Wanjala FME, Kaburi JC,
Ingonga J, Byrum WW, Anjili CO.
Combination and monotherapy of
Leishmania major infection in BALB/c mice
using plant extracts and herbicides. Vector
Borne Dis. 2012;49:123–130.
26. Kaur S, Kaur G, Sachdeva H, Kaur J. In
vivo evaluation of the antileishmanial
activity of two immunomodulatory plants,
Emblica officinalis And Azadirachta indica
In Balb/C Mice. International Journal of
Ayurvedic and Herbal Medicine. 2013;3(1):
1066-1079.
27. Salgado FN, Ferreira TM, Costa JM.
Involvement of the renal function in
patients with visceral leishmaniasis (kala-
azar). Rev Soc Bras Med Trop. 2003;
36(2):217-21.
28. NAS. Nutritional requirements of laboratory
animals. 4
th
Revised edition. National
academy press. Washington DC; 1995.

Chimbevo et al.; IJTDH, 30(2): 1-14, 2018; Article no.IJTDH.39784
14
29. Santos EW, Oliveira DC, Hastreiter A,
Silva GB, Beltran JSO, Tsujita M, Crisma
AR, Neves SMP, Fock RA, Borelli P.
Hematological and biochemical reference
values for C57BL/6, Swiss Webster and
BALB/c mice. Braz. J. Vet. Res. Anim. Sci.
2016;53(2):138-145.
30. Sackow MA, Danneman P, Brayton C. The
laboratory Mouse. 2005.
31. Achliya GS, Wadodkar SG, Dorle AK.
Evaluation of hepatoprotective effect of
Amalkadi Ghrita against carbon
tetrachloride-induced hepatic damage in
rats. J Ethnopharmacol 2004;90(2-3):229-
232.
32. Mathur P, Samantaray JC, Samanta P.
High prevalence of functional liver
derangement in visceral leishmaniasis at
an Indian tertiary care centre. Clin
Gastroenterol Hepatol. 2008;6(10):70-
1172.
33. Jarallah HM. Pathological Effects of
Leishmania donovani promastigotes on
liver and spleen of experimentally infected
Balb/c mice. Medical Journal of Babylon.
2016;13(1):134–140.
34. Beaver PC, June RC, Cupp EW. Clinical
parasitology. 9th Ed. Lea and Febiger;
Philadelphia. 1984;70-77.
35. Fernandes LR, Ribeir, ACC, Segatt, M,
Santos LFFF, Amaral J, Portugal LR, Leite
JIA. Leishmania major Self-Limited
Infection Increases Blood Cholesterol and
Promotes Atherosclerosis Development.
Cholesterol; 2013.
(Article ID 754580)
36. Murray LJ, Bamford KB, Reilly DPJO,
McCrum EE, Evans AE. Helicobacter pylori
infection: Relation with cardio- vascular
risk factors, ischaemic heart disease, and
social class. British Heart Journal. 1995;
74 (5):497–501.
37. Doenhoff MJ, Stanley RG, Griffiths K,
Jackson CL. An anti-atherogenic effect of
Schistosoma mansoni infections in mice
associated with a parasite-induced
lowering of blood total cholesterol.
Parasitology. 2002;125(5):415–421.
38. Burnett MS, Durrani S, Stabile E, et al.
Murine cytomega-lovirus infection
increases aortic expression of
proatherosclerotic genes. Circulation.
2004;109(7):893–897.
39. Portugal LR, Fernandes LR, Cesaretal GC.
Infectionwith Toxoplasma gondii increases
atherosclerotic lesion in Apo E-deficient
mice. Infection and Immunity. 2004;72:
3571–3576.
40. Mussa FF, Chai H, Wang X, Yao Q,
Lumsden AB, Chen C. Chlamydia
pneumoniae and vascular disease: “An
update”. Journal of Vascular Surgery.
2006;43:1301–1307.
41. Meurman H, Sanz M, Janket SJ.
Oral health, athero- sclerosis and
cardiovas-cular disease. Critical Reviews
in Oral Biology and Medicine. 2004;
15:403–413.
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