HAEMATOLOGICAL STUDIES OF Plasmodium berghei (ANKA STRAIN) INDUCED

ALBINO MICE MODEL TREATED WITH RUZU BITTERS

BY

JIMOH, ABIDEMI ABISOLA.

MATRIC NO: SCI/16/17/0559

A PROJECT RESEARCH SUBMITTED TO THE DEPARTMENT OF ZOOLOGY AND

ENVIRONMENTAL BIOLOGY, FACULTY OF SCIENCE, OLABISI ONABANJO
UNIVERSITY, AGO-IWOYE, OGUN STATE

IN FULFILMENT OF THE ACADEMIC REQUIREMENT FOR THE AWARD IN

BACHELOR IN SCIENCE (B.Sc.) HONS. DEGREE IN ZOOLOGY

MARCH, 2021.

1
 CERTIFICATION

I certify that this research was carried out by JIMOH, ABIDEMI ABISOLA with Matriculation

Number SCI/16/17/0559 in the Department of Zoology and Environmental Biology, Faculty of

Science, Olabisi Onabanjo University, Ago-iwoye, Ogun State under my able supervision.

___________________________ __________________

Dr. (Mrs) Titilola Salisu Date

B.Sc., M.Sc., Ph.D

Supervisor

DEDICATION
2
This work is dedicated to almighty God for his guidance over me, who keeps me in his love and

made this work a success.

ACKNOWLEDGMENTS

3
It is my earnest intention to express my profound gratitude to Almighty God for His enabling

grace and to all that have contributed or help in one way or the other to the successful completion

of my project.

I wish to express my gratitude to my Project Supervisor Dr. (Mrs) Titilola Salisu who not only

supervised in spite of her other pressing duties, was able to read the whole scripts thoroughly and

carefully made useful suggestions and correction of my errors.

I wish to express my joy to Mr. and Mrs. Jimoh for their moral and financial support in making

my studies sweet. I remain indebted to my parents for their continual support throughout the

period of carrying out this research work and my academic pursuit. And to my colleagues I say a

very big thank you for their constant support and availability.

Finally, I thank my part adviser Mrs. Adeleke for being very good to me, and Dr. Adekunle, May

God bless and reward you all for your understanding and patience. Also to my Lecturers, Dr.

Owagboriaye and Dr. Aina and my Head of Department professor Fafioye Oyebamiji , I say

thank you for impacting great knowledge into me.

TABLE OF CONTENTS

CONTENTS Pages

TITLE PAGE I

4
CERTIFICATION II

DEDICATION III

ACKNOWLEDGEMENTS IV

TABLE OF CONTENTS V-VIII

LIST OF TABLES IX

LIST OF FIGURES X

LIST OF PLATES XI

ABSTRACT XII

CHAPTER ONE

1.1 INTRODUCTION 1
1.2 AIMS OF THE STUDY 1

1.3 OBJECTIVE OF THE STUDY 2

CHAPTER TWO: LITERATURE REVIEW

2.0 MALARIA INFECTION AND EPIDEMIOLOGY OF MALARIA 3

2.1 LIFE CYCLE OF Plasmodium spp 4

2.1.1 Diagram Showing the Life Cycle of Malaria (Plasmodium Falciparum) 6

2.2 HOST PARASITE INTERACTION AND PATHOPYSIOLOGY OF

MALARIA 7

2.3 DIAGNOSIS OF MALARIA 8

2.3.1 CLASSIFICATION OF MALARIA 9

5
2.4 PREVENTION OF MALARIA 10

2.4.1 MOSQUITO CONTROL 11

2.5 MALARIA TREATMENT 11

2.6 THE USE OF RUZU HERBAL BITTER IN MALARIA STUDIES 12

CHAPTER THREE: MATERIALS AND METHODS

3.1 EXPERIMENTAL DESIGN 14

3.2 CAGING SYSTEM 14

3.3 FEED AND NUTRITION 14

3.4 WATER SYSTEM 15

3.5 CAGE IDENTIFICATION AND LABELLING 15

3.6 BEDDING 15

3.7 ENRICHMENT 17

3.8 SANITATION AND CLEANING 17

3.9 FEED QUANTIFICATION 17

3.10 WATER QUANTIFICATION 18

3.11. MEASUREMENT OF ANIMALS’ WEIGHTS 18

3.12 TEMPERATURE MONITORING OR MEASUREMENTS 18

3.13 INOCULATION 19

3.14 STAINING OF THE SLIDES 20

3.15 DRUG ADMINISTRATION 21

3.16 SACRIFICING 21
6
CHAPTER FOUR

4.0 RESULTS 23

CHAPTER FIVE

5.0 DISCUSSION, RECOMMENDATIONS AND CONCLUSIONS 32

5.1 CONCLUSION 33

5.2 RECOMENDATIONS 33

REFERENCES 34

7
8
CHAPTER ONE

INTRODUCTION

Malaria is a parasitic disease transmitted by the anopheles mosquito carrying the

plasmodium parasites. The plasmodium species include plasmodium falciparum, plasmodium

vivax, plasmodium ovale, plasmodium malariae and plasmodium knowlesi. However, virtually

all deaths from malaria are caused by plasmodium falciparum (Smyth, 1994). Some of the

symptoms in malaria infections are anemia, thrombocytopenia, leucocytosis, antioxidant

reduction, increased lipid peroxidation, lactic acidosis, coma and death (Olayemi et al., 2012).

Patients with malaria often develop haematological complications and alterations in

biochemical parameters (Khan et al., 2002). Plasmodium berghei has been used in studying the

activity of potential antimalarials in mice because it produces diseases similar to those of human

plasmodium infection (Kumar et al., 2006: Peter, 1998). Plasmodium berghei is one of the four

plasmodium species that has been described in Africa murine rodents, the others being:

plasmodium chabaudi, plasmodium Vinckei and plasmodium Yoelii. Laboratory mouse strains is

frequently used in research as a model for human malaria. In the laboratory, the natural hosts

have been placed by a number of commercially available laboratory mouse strains. The use of

conventional drugs in the treatment of malaria has been exasperated by the resistance of the

plasmodium falciparum to most of the recognized antimalarial drugs (Borst and Oullette, 1995).

Ruzu herbal bitters is a poly herbal mixture widely used for treatment of many diseases such as

diabetes, fever, obesity, hypertension, arthritis and infertility among others. Ruzu herbal bitters is

made up of 40% Curculigo pilosa root, 30% Uvaria chamae stem and 40% Citrullus colocynthis

bark.
9
1.2 STATEMENT OF THE PROBLEM

The world is faced with a global epidemic called Malaria. It is one of the major threats to

the public and economic sector in Nigeria. Malaria is caused by a parasite called Plasmodium. It

is transmitted from one person to another through the biting of certain species of mosquitos.

Those who have the greatest risk of severe forms of the disease, and death, are children who are

under the age of 5 years, and pregnant women.

An estimate of 50 million pregnancies that occur each year globally, approximately 25 million is

thought to occur in developing countries (African countries etc.). Pregnant women and children

are thought to be the most vulnerable to malaria. Several initiatives has been implemented over

the years by World Health Organization (WHO) to control malaria all of over the world, there

has been slightly improved progress. Without any intervention, malaria would cause 10,000 of

these women and 200,000 of their infants’ death as a result of malaria infection and severe

malarial anaemia. Several Services offered all over the world, which includes clinical care,

prevention of disease and health promotion activities. The health system is also supported by

about several pharmacy and chemical shops. The health care system of the urban areas is quite

encouraging because the cities are endowed with public health nurses, doctors, antenatal, health

education, disease control and commonly health nurses who have involved in outreach services

(KMHD Annual Report, 2007). Traditional medicines has been used to treat malaria for

thousands of years and they are the source of the modern antimalarial drugs. Traditional

medicines are important and sustainable source of treatment in poor areas whereby effective

antimalarial drugs can not be afforded(Merlin and Gerard, 2004).

10
1.3 AIM OF THE STUDY

 To evaluate the blood indices of Plasmodium berghei induced albino rat treated with

Ruzu bitters.

1.4 OBJECTIVES OF THE STUDY

 To compare the blood indices of Plasmodium berghei induced albino rat treated with

ruzu bitters.

 To determine the efficiency of ruzu bitters in Plasmodium berghei induced albino rat.

11
 CHAPTER TWO

LITERATURE REVIEW

2.0 MALARIA INFECTION AND EPIDEMIOLOGY OF MALARIA

Malaria is a mosquito borne infectious disease that affects humans and other animals

(WHO, 2014). Malaria causes symptoms that typically include fever, tiredness, vomiting and

headaches (Caraballo, 2014). In severe cases, it can cause yellow skin, seizures, coma or death.

Symptoms usually begin ten to fifteen days after being bitten by an infected mosquito. Malaria is

caused by single-celled micro-organisms of the plasmodium group commonly spread by an

infected female anopheles’ mosquito. The mosquito bite introduces the parasites from the

mosquito saliva into a person's blood. The parasite travels to the liver where they mature and

reproduce. Five plasmodium species causes malaria in human which includes; plasmodium

vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum and Plasmodium

Knowlesi (Brooker et al., 2007).

However, most deadly species are Plasmodium vivax and Plasmodium falciparum, with

Plasmodium falciparum being the deadliest species in sub-saharan Africa (Melo et al., 2010).

The wide distribution of these pathogens acrossthe globe is responsible for the morbidity and

mortality especially in tropical countries (Salazar castanon et al., 2014). Malaria is a major

health problem in Africa, Asia, central America, Oceania and south America. About 40% of the

world's population lives in areas where malaria is common (WHO, 2007). According to recent

studies on malaria incidence, malaria has been estimated to be a threat to close to 3.3 billion

people and death record of 1.2 million (Melo et al., 2010).

12
 Most of the mortality is noticeable in the African continent, particularly in sub-saharan

Africa among children below the age of 5 years and pregnant women (onkoba et al., 2015). The

sub-saharan region therefore serves as the core of parasite transmission. Even though recent

studies on malaria have documented a marked decline in mortality up to about 54% in the

African continent (Benelli, 2016). The disease still remains a serious public health threat that

needs attention.

2.1 LIFE CYCLE OF Plasmodium spps.

The life cycle of plasmodium Involves several distinct stages in insect and vertebrate

host. Parasites are generally Introduced into a vertebrate host by the bite of an Insect host

generally a mosquito with exception of some plasmodium species of reptiles (vernick et al.,

2005). The transmission of malaria to the host (vertebrates such as humans), is through the

anopheles female mosquitoes (Crawley and Nahien, 2004). Most life cycles of plasmodium spp.

Occur during Infected female anopheles mosquito bites during blood meal, which releases

infective sporozoites through the dermis into the peripheral blood circulation of the host.

Sporozoites then migrate to the hepatocytes, where they duplicate through asexual schizogony to

produce schizonts containing merozoites.

Then, the schizont ruptures and releases several merozoites which enter the blood stream. In the

cases of Plasmodium vivax and Plasmodium ovale the hypnozoites are formed which remains

dormant in the liver until future phase. However, this does not occur in P. falciparum (Trampuz

et al., 2003). The process occurring in the liver is referred to as exoerythrocytic cycle which is

usually asymptomatic. The intra-erythrocytic stage commences when the merozoites leaves the

liver and enters red blood cells (RBCs), developing into a ring

13
form which later matures into trophozoites. During the ring stage, the parasite lies dormant for a

while and becomes metabolically active after about 15 hours post invasion. Therefore, there is an

increase in the rate of metabolism and biosynthesis trophozoites (Kirk 2004; Kirk, 2001). It then

converts hemoglobin of the RBCs to amino acid and it is also dependent on other nutrient

sources for survival (Ramasamy, 1998; Milner et.al., 2012).

The malaria parasite relies on the host stored energy because it has just a single

mitochondrion and lacks a functional citric acid cycle. Trophozoites undergo schizogony to form

merozoites. The parasitized red blood cells (PRBCs) are leased to release abundant merozoites,

which enter blood circulation to restart a new cycle of schizogony within the RBCs. Some of the

merozoites in the blood stream develop into the male and female gametocytes. These male and

female gametes are then ingested by the mosquito during subsequent blood meal and undergo

gametogenesis in the mosquito midgut. During the process, the gametocytes undergo fertilization

to produce motile ookinete. The ookinete then crosses the epithelial cell of the midgut into the

haemocoele and develops into oocyst, the oocyst then produces sporozoites. The sporozoites

formed in the mosquito moves to the salivary glands ready for re-infection of the vertebrae host

in another blood meal and the cycle resumes. The life cycle of malaria is similar in all species of

plasmodium.

14
DIAGRAM SHOWING THE LIFE CYCLE OF MALARIA (Plasmodium falciparum)

PARASITE IN FEMALE ANOPHELES MOSQUITO AND IN HUMAN.

15
2.2 HOST PARASITE INTERACTION AND PATHOPHYSIOLOGY OF MALARIA

The pathogenesis and clinical outcome induced by P. falciparum is a multi-complex

interaction (Buffett et al., 2011). Complication arises from an interplay between parasite and the

host and might lead to multiple responses within the host. These responses could either be

mechanical, humoral or immunological, ready to eliminate parasite but end up affecting the host

(Elsheikha and Shaeshaa, 2007; Mishra and Das, 2008). Once the host is infected, parasite-

induced red blood cell, alteration and microcirculatory abnormalities which lead to local and

systemic reaction occur (Buffett et al., 2011). Malaria infection is known to be associated with

severe anemia, cerebral malaria and metabolic acidosis, hypoglycemia, hypovolemia (mock

enhaupt et al., 2004).

Hypovolemia, which has been identified as significant feature might be exacerbated by

the severe anemia caused by red blood cells destruction, metabolic acidosis and microvascular

impediment by sequestrated parasites resulting in the decrease delivery of oxygen to tissue

(mocken haupt et al., 2004). Microcirculation perturbations might lead to decrease in metabolites

exchange and hypoxia, may trigger the release of pro-inflammatory and anti-inflammatory

cytokines mediators (Clark and cowden, 2003; murambiwa et al., 2013). Hypoxia has also been

attributed to the disproportionate release of pro-inflammatory and anti-inflammatory cytokine.

Therefore, it has been associated to be the main factor in the pathogenesis of malaria (Clark and

cowden, 2003). Understanding the host immune response is a vital contribution that will help

unveil protective immunological strategies. Another main feature of the life cycle of P.

falciparum is cytoadherence, whereby the mature parasitized red blood cells (trophozoites and

schizonts) are capable of displaying significant change by adhering to the endothelial cells

(Rosenthal, 2008: Buffett et al., 2011; cobbold et al., 2016). This could favor the parasite
16
because the process can possibly obscure the passage of abnormal erythrocyte to the spleen

(Rosenthal, 2008). On the other hand, the adherence of mature Parasitized red blood cells on

small blood vessels obstructs the peripheral blood circulation contributing to the pathogenesis of

malaria. However, the early stages (ring) of the infection present slight modification of adhesion

or deformability, which can be seen within the blood circulation.

2.3 DIAGNOSIS OF MALARIA

Diagnosis of malaria in non-endenmic areas requires a high degree of suspicion which

might be elicited by any of the following; recent travel history, enlarged spleen, fever, low

number of platelets in the blood and levels of bilirubin in the blood higher than normal combined

with a normal level of white blood cells (Nadjm et al., 2012). Reports in 2016 and 2017 from

countries where malaria is common suggest high levels of over diagnosis due to insufficient or

inaccurate laboratory testing (Manguins et al., 2017). Malaria is usually confirmed by the

microscopic examination of blood films or by antigen -based rapid diagnostic tests (RDT)

(Katten et al., 2011).

In some areas, rapid diagnostic tests must be able to distinguish whether the malaria

symptoms are caused by plasmodium falciparum or by other species of parasites since treatment

strategies could differ for non-p.falciparum infections (Abba et al., 2014). Microscopy is the

most commonly used method to detect the malaria parasite. About 165 million blood films were

examined for malaria in 2010 (Wilson, 2012). Despite its wide spread usage, diagnosis by

microscopy suffers from two main drawbacks. Many settings especially the rural are not

equipped to perform the test, and the accuracy of the results depends on both the skill of the

person examining the blood film and the levels of the parasite in the blood. The sensitivity of the

17
blood films ranges from 75 - 90%. In optimum conditions to as low as 50%. Commercially

available rapid diagnostic tests are often more accurate than blood films at predicting the

presence of malaria parasites, but they are widely variable in diagnostic sensitivity and

specificity depending on manufacturer and are unable to tell how many parasites are present.

In regions where laboratory tests are readily available, malaria should be suspected and

tested for in any unwell person who has been in an area where malaria is endemic. In areas

where laboratory diagnostic tests cannot be afforded use only a history of fever as the indication

to treat malaria. Thus, the common teaching "fever" equals malaria unless proven otherwise. A

drawback of this practice is over diagnosis of malaria and mismanagement of non-malaria fever

which wastes limited resources, erodes confidence in the health care system and contributes to

drug resistance (Perkins et al., 2008).

Although polymerase chain reaction base tests have been developed, they are not widely

used in areas where malaria is common as of 2012, due to their complexity (Nadjm et al., 2012).

However, incorporating RDTs into the diagnosis of malaria can reduce antimalarial prescription.

Although RDTs does not improve the health outcome of those infected with malaria, it also does

not lead to worse outcomes when compared to presumptive antimalarial treatment.

2.3.1 CLASSIFICATION OF MALARIA

Malaria is classified into either severe or uncomplicated by the World Health

Organization (WHO). It is said to be severe when any of the criteria below occurs:

 Decreased consciousness

 Significant weakness such that the person is unable to walk.

 Inability to feed
18
  Two or more convulsions

 Low blood pressure (less than 70mm Hg in adults and 50mm Hg in children)

 Breathing problems

 Circulator shock

 Kidney failure or haemoglobin in urine

 Bleeding problems or hemoglobin less than 50g/L (5g/dl).

 Pulmonary oedema

 Blood glucose less than 2.2m mol/L(40mg/dl)

 Acidosis or lactate levels of greater than 5mmol/L

 A parasite level in the blood of greater than 100,000 per micro liter in low intensity

transmission areas, or 250,000 per micro liter in high intensity transmission areas.

2.4 PREVENTION OF MALARIA

Methods used to prevent malaria include medications, mosquito elimination and

prevention of bites. As of 2020, there is one vaccine for malaria know as RTS which is licensed

for use. The presence of malaria in an area requires a combination of high human population

density, high anopheles mosquito population density and high rates of transmission from humans

to mosquito and from mosquitoes to humans. Prevention of malaria may be more cost-effective

than treatment of the disease in the long run, but the initial costs required are out of reach of

many of the world's poorest people. There is a wide difference in the cost of control (i.e

maintenance of low endemicity) and elimination programs between countries. For example, in

China whose government in 2010 announced a strategy to pursue malaria elimination in the

Chinese province, the required investments are a small proportion of public expenditure on

19
health. In contrast, a similar programme in Tanzania would cost an estimated one-fifth of the

public health budget.

2.4.1 MOSQUITO CONTROL

Vector control refers to methods used to decrease malaria by reducing the levels of

transmission by mosquitoes. For individual protection, the most effective insect repellents are

based on meet or picaridin (kajfasz, 2009). However, there is insufficient evidence that mosquito

repellent can prevent malaria infection (Mia et al., 2018). Insecticide treated mosquito nets

(ITNs) and indoor residual spraying (IRS) are effective and have been commonly used to prevent

malaria and their use has contributed significantly to the decrease in malaria in the 21st century

(Furnival et al,.2020). ITNs and IRS may not be sufficient to completely eliminate the disease as

these interventions depend on how many people use nets, how many gaps in insecticide there

are, if people are not protected when outside of the home, and an increase in mosquitoes that are

resistant to insecticides.

2.5 MALARIA TREATMENT

Malaria is treated with antimalarial medications; the ones used depends on the type and

severity of the disease. While medications against fever are commonly used, their effect on

outcomes are not clear. Providing free antimalarial drugs to households may reduce childhood

deaths when used appropriately. Programmes which presumptively treat all causes of fever with

antimalarial drugs may lead to overuse of anti-malarials and undertreat other causes of fever.

Nevertheless, the use of malaria rapid diagnostic kits can help to reduce over usage of

antimalarials.

2.6 THE USE OF RUZU HERBAL BITTER IN MALARIA STUDIES

20
Ruzu herbal bitters (RHB) is a poly herbal mixture prepared in Nigeria and it is widely used as

an anti-obesity concoction. Ruzu herbal bitters is claimed for the management and treatment of

obesity, diabetes, hypertension, arthritis and infertility among others. Ruzu herbal bitters is an

aqueous preparation of 40% curculigo pilosa root, 30% uvaria chamae stem and 40% citrullus

colocynthis bark. The genus curculigo belongs to the family Hypoxidaceace and consists of

approximately 20 species of exclusively tropical origin (palazzino; Gale; ederici; Monache

Cometa; palmery, 2000). The members of the family are small to medium herbs, with grass-like

leaves and an Invisible stem, modified into a corm or a rhizomes. The rhizomes of curligo pilosa

(cp) shum and thorn, was the first African species to be described of the Curculigo genus (RJ

Hamid; D Amir; D Farnoush; V Ghasem; G Hasan; M Sadrollah; RG Mohammed; F Mehrdad,

Emergency Medical Journal Article). The uvaria chamae is a Nigerian medicinal plant that

belongs to the family Annonaceae . It is commonly called by the igala people of kogi state as

Ayiloko, kaskaifi by the Hausa and Oko oja by the Yorubas in Nigeria, as well as Akotompo by

the fula-fainte of Ghana. It is a medicinal plant used in the treatment of fever and injuries (P

Kumar; M Clark. Diabetes Mellitus and other disorders of metabolism. Kumar and Clark’s

Clinical medicine 6th edition). There are other claims that the plant can currently some oral

sickness such as Abdominal pain, treatment for piles, wounds, sore throat and diarrhea. Citrullus

colocynthis Schrad, belongs to the family of cucurbitaceae and it is popularly named bitter apple

or bitter cucumber in English and called Hendevaneh Abujahl (Abujahl watermelon) or Kadu

Hanzal (bitter ground). In Persian, it well known medicinal plant used alone or in compounds for

many medical purposes (Hassan; Abdel; Mohammeda. 2000). Citrullus colocynthis has been

widely applied in the management of diabetes, leprosy, common cold, cough, asthma, bronchitis,

jaundice, joint pain, cancer, toothache, wound, mastitis and gastrointestinal disorders Marchesini,

21
petta and R.D. Grave, 2016. Diet, weight loss and liver health in non- alcoholic fatty liver

disease: pathophysiology, evidence and practice. Hepathology, 63: 2032-2043).

22
 CHAPTER THREE

METHODOLOGY

3.1 EXPERIMENTAL DESIGN

Study animals Mus musculus was grouped into three, five animal per group. Group are;

Experimental group (Group 1), Positive control group (Group 2) and Negative control group

(Group 3). Group 1 which is the experimental group contain animals which are inoculated with

plasmodium berghei (parasite) which are later treated with Ruzu bitter herb. Group 2 or negative

control group contains animals with parasite without treatment, while Group 3 which is the

positive contains animal that are not inoculated with parasite rather no treatment.

Experiment was conducted with in the space of 3 weeks excluding acclimatization week which

was two weeks. Due to induced stress and effect of transportation rats are left for two weeks

before the commencement of the study in view of acclimatization to their new environment.

3.2 CAGING SYSTEM

Improvised Individual ventilation caging system (IVC) was used in housing the rats in a well

conducive environment. The cages are opaque (Weiss and Taylor, 1985), consisting of

Aluminium base container of 30cm(L)×27cm(W)×15cm(H), and a metallic lid cover which

provides space for pelletized compounded feed and as well as automated water system each,

where rats feeds via ad-libitum.

23
3.3 FEED AND NUTRITION

Mice were fed with compounded pelletized ad-libitum over the period of 21 days or 3 weeks,

feed was quantified daily by weighting with electronic weighing scale (Castelhano-Carlos et al.,

2014) and counting of pellets. The feed was compounded and pelletized into 6mm pellet at

Fasolab Animal and Aquaculture Feeds depot. Ijebu Igbo, Ogun State, Nigeria. Table 1. below

shows the composition feed.

3.4 WATER SYSTEM

Water was provided ad-libitium via locally made improvised water bottle which permits mice to

drink by sucking. Water intakes per day, per group are measured by using 500ml measuring

cylinder daily after consumption (Castelhano-Carlos et al., 2014).

3.5 CAGE IDENTIFICATION AND LABELLING

Each cage was labelled with the aid of masking tape and marker while rats are labelled at

different part of the skin with picric solution which was prepared by mixing picric acid with 70%

methanol. Animals are stained (labelled) at the following part of the body as follow; Head (H),

right hand (RH), left hand (LH), right leg (RL), left leg (LL) and tail (T) so as to avoid mix up as

well as proper management of data for each rat both male and female. Animal are also relabelled

when label fade away.

3.6 Bedding

Wood shavings were gotten from furniture workshop at Ijebu-Igbo, these shavings were used as

bedding material in each cage (Blom et al., 1996). Beddings or wood shavings were changed as

well as routine cleaning of cages which was observed twice a week at three days interval in

24
accordance with (Burn and Mason, 2008.) in order to stabilize the ammonia concentration from

urine in cages also to reduction cannibalism risk.

Table1: Showing Percentages of Constituents in Compounded Feed for the Rats.

25
S/N Constituent Percentage (%)
01 Wheat offal 9.76

02 Bone meal 3.25

03 Soya beans meal 0.27

04 Palm kernel 14.1

05 Groundnut 11.9

06 Maize 43.3

07 Salt 0.27

08 Lysine 1.08

09 Methionine 1.08

10 Oyster 14.9

26
3.7 ENRICHMENT

Enrichment material (Plastic bottles) were improvised to serve as plastic tunnel by cutting both

end and leaving the middle that part serves as plastic enrichment which was used as enrichment

for rats over the period of study (Rattazzi et al., 2016). The enrichment materials are cleaned

during routine cleaning and are properly replaced if consumed by mice which had no adverse

effect according to (Townsend, 1997).

3.8 SANITATION AND CLEANING

The animal house was been clean daily to ensure proper environmental status and cages where

routinely cleaned twice a week within three days of interval as stated by (Burn and Mason,

2008.) in order to stabilize the ammonia concentration in cages also to reduction cannibalism

risk. There is total displacement of animal in another cage containing wood shaving in order to

buffer the effect of change in environment. Note, gloves and accurate personal protective

equipment are used during sanitation.

3.9 FEED QUANTIFICATION

Routinely feed was quantified daily with two methods namely by weighting and pellet counting

(Castelhano-Carlos et al., 2014). Weighing of feed was done with the aid of high precision

weighing scale (Camry ISO 9001, Model EK5055,) and weighing bowl where feed is weighted

in grammas. Pellet counting was done manually by hand count after weighing of the feed, the

number of pellets was counted are record accordingly in the record book following cage labels

and this was done daily over the period of study.

27
3.10 WATER QUANTIFICATION

Throughout the study water was given to the rat’s ad-litium via improvised water bottles, two

bottles per each cage. Water bottles were washed daily to ensure proper sanitation and water was

change and measured with the aid of 500ml glass measuring cylinder in order to quantify what

was consumed daily (Castelhano-Carlos et al., 2014). Collected data were recorded properly in

the record book according to cage label.

3.11. MEASUREMENT OF ANIMALS’ WEIGHTS

Mice body weight was monitored and recorded in order to account for body weight loss or gain;

this was done daily throughout the study (Castelhano-Carlos et al., 2014). Mice were weighted at

weekly and this was done with the aid of weighing bowl and weighting scale. Wood shavings are

placed in weighing bowl in order to compensate for the change in animals ’ environment since

rats are totally displaced when weighing. Weighing balance was properly set for use after which

weighing bowl was placed and tarred, rat was gently picked by soft handling at tail end and

displaced into the weighing bowl and reading was taken and recorded appropriately into the

record book. This was done weekly throughout the study following same process.

3.12 TEMPERATURE MONITORING OR MEASUREMENTS

Temperature data was measured and recorded routinely every day at exactly 12:00 pm. Data

were collected with the aid of two different thermometers both mercury in glass and clinical was

implored, the major reason for this is to test the sensitivity and ensure minimum error as both are

properly correlated and record in record book. Thermometer was affixed or anchored to the cage

shelve throughout the study period in order to be at close range and for accurate collection of

data.

3.13 INOCULATION
28
Plasmodium berghei, ANKA strain was used as parasite in the experiment and blood was

collected via in-situ from infected mice blood.

Aim: To collect in-situ parasitized blood samples, and culture malaria parasite (P. berghei) in

pure healthy animals.

Material needed: Syringe, Acid citrate dextrose (ACD), Normal saline.

Procedure:

 Parasitized blood sample are collected directly from tail ending (intravenously).

 0.01mls of anticoagulant was drawn into a syringe via needle.

 The parasitized animal was handled with care and the tip of the tail was cut to obtain

blood.

 Then a little pressure was applied to the tail alongside swiping upward, to get blood

which was collected through a sterile syringe.

 After which I mixed the blood properly with anticoagulant (ACD) in syringe to avoid

clotting of blood.

 Then 0.6 ml of normal saline water was added for proper dilution.

 Animal in experimental group and negative control was Injected or inoculated with 0.2ml

of the solution, Using either Intraperitonous method injection method.

29
 Making Blood smear

 After collecting the blood sample, two or three drops of blood were dropped on a cleaned

and well labelled slide.

 Then it was stirred gently with another slide in a circular manner in such a way that it was

not more than 1 cm in diameter, this makes up the thick film.

 A drop of blood is put on the same slide.

 After putting a drop of blood on the slide.

 Then a pure and sterile slide is used as the spreader and is been held at the edge resting

on the slide that contain the blood at 450.

 Push firmly along the slide once.

 Then fix thin films after 5min with methanol so as to avoid dehaemoglobinization. Note,

the thick films are not meant to be fixed.

3.14 STAINING OF THE SLIDES

 10% Giemsa solution was prepared using de-ionized water with value pH 7.2.

 Then slides are placed staining trough.

 Slides are gently stained by pouring the stain onto the slides using dropper

 Slides were left for10 minutes to permit proper staining

 wash slide gently flushing the stain off the slides by adding drops of running water

through the help of wash bottle

 After that, then place the slides downward in a slide rack to drain and dry making sure the

film doesn't touch the rack.

30
Microscopical examination of slides

 Mount the slides on the microscope stage

 Add one or two drops of oil immersion on the slide

 Position the objective lens

 Then view through the ocular or the eye piece and adjust to the area where the parasites

are seen clearly

 In identifying the parasite with in the red blood cell, and count parasitamia.

3.15 DRUG ADMINISTRATION

Ruzzu herb solution which was purchased at local shop at Ijebu-Igbo was is administered to

animals (mice) orally using oral cannula which is affixed to a syringe. Only the experimental

group is involved in treatment with Ruzzu herb.

3.16 SACRIFICING

At the end of the study mice are killed and dissected in order to take blood samples in large

volume using cardiac tap method.

Aim: To kill (euthanatize), dissect mice, collect blood sample from animal humanely.

Apparatus used: Surgical blade, scissors, syringe and needle, forceps, cotton wool, dessicator,

dissecting pin, dissecting board, chloroform, methylated spirit.

Procedure:

 Mice was anesthetized by placing animal in desiccator containing chloroform, causing

few minutes sub-consciousness in animal.

31
 Then mice are pinned dorsa-ventrally on the dissecting board at fore limbs and hind limbs

using dissecting pin.

 Then I dissect posterior-anterior with the aid of forceps, scissors and surgical blade.

 Then I opened the thoracic cavity and blood was collected via cardiac tap using needle

and syringe.

CHAPTER FOUR
32
 RESULTS

Groups/Parameter PCV (%) HB (g/dl) RBC (x106uL) WBC (x106uL)

Experimental 20.0 7.0 3.0 10050.0

Negative 26.0 8.0 2.9 7275.0
Positive 26.6 8.5 2.9 7690.0

Table 2: Table showing average value of important hematological parameter or index.

33
Groups Mortality

Experimental 3
Positive control 1
Negative control 4

Table 3: showing acute toxicity.

34
Figure 2: Showing weight of mice during experiment.

35
Figure 3: Showing feed, number of pellet and water intake for Positive control group.

36
Figure 4. Showing feed, number of pellet and water intake for experimental group.

37
Figure 5: Showing feed, number of pellet and water intake for Negative control group.

38
Plate 1: Micrograph showing blood film of un-infected mice (Positive control group)

39
Plate 2: Micrograph showing blood film of infected mice (negative control group)

40
Plate 3: Micrograph showing blood film of infected mice (experimental group)

41
 CHAPTER FIVE

DISCUSSION, RECOMMENDATIONS AND CONCLUSION

5.1 DISSCUSSION

From table 2, it was observed that the Pack cell volume which is the PCV is high in negative

control, positive control and it low in experimental group this suggest that high level of

parasitemia count had affected red blood cells. White blood cells are numerous in experimental

group this suggest that immunological effect is induced by parasite in mice. Hemoglobin level is

very low in experimental group due to the depletion of red blood cell.

From figure 2 it was observed that experimental group had the lowest weight at the inception of

the experiment and weight increases along the week showing that Ruzza herb had annul the

effect of P berghei on weight, which could have been loos of weight. The negative control group

had a declined weight at week 3, at highest parasiteamia level since no treatment was

administered to this group. The positive control group had a linear increase in weight since they

are parasite free as well as no treatment administered also.

Positive control group from figure 3, appears to deviate from normal expected linear model as

intake increases and reduce during week two environmental factor might have account for this.

Figure 4., display weekly decline of intake maintenance hence reduction in feeding and drinking

which drops drastically along the week this can be as a result of the P. beighei infection,

parasiteamia level is inversely proportional to the level of intake.

From figure inference shows that there is reduction in feed intake as the weight of feed decreases

across the weeks water intake tends to be very low this can be related to the effect the parasite in

42
relation to appetite and body weight since there was no system administer to this group

parasitemia level increase very high.

Acute toxic was also observed due to the virulence of that ANKA strain mortality was recorded

as shown in table.3

5.2 RECOMMENDATION

I recommend that further analysis should be done in order to know more about the toxicity level

of Ruzu herb and also to the affirm effectiveness of this on other diseases.

5.3 CONCLUSIONS

Ruzu herbal mixture had a lot of effect on haematological index or parameter of which important

effects are increase in pack cell volume, white blood cell counts as well as red blood cell count

of a parasitized cell, which can also be described curing malaria, it can be deduced that Ruzu

herb mixture is effective in tackling malaria parasite in suppressive level.

43
 REFERENCES

Abba, K., Deeks, J.J., Olliaro, P., Naing, C.M., Jackson, SM., Takwoingi ,Y., Donegan, S.,
Garner, P (2011). Abba, K. (ed.). "Rapid diagnostic tests for diagnosing uncomplicated P.
falciparum malaria in endemic countries" . Cochrane Database of Systematic Reviews
(7): CD008122.

Abba, K., Kirkham, AJ., Olliaro, P. L., Deeks, J. J., Donegan, S., Garner, P., & Takwoingi, Y
( 2014). "Rapid diagnostic tests for diagnosing uncomplicated non-falciparum or
Plasmodium vivax malaria in endemic countries". The Cochrane Database of Systematic
Reviews. and outcome of renal dysfunction associated with plasmodia infection.
Parasitology research, 101, 1183.

Arts, J.W.M., Kramer, K., Arndt, S.S., & Ohl, F., 2014a. Sex differences in physiological
acclimatization after transfer in Wistar rats. Animals 4, 693e711.

Arts, J.W.M., Oosterhuis, N.Y., Kramer, K., & Ohl, F., 2014b. Effects of transfer from breeding
to research facility on the welfare of rats. Animals 4, 712e728.

Athuman, M., Kabanywanyi, AM., & Rohwer, AC ( 2015). "Intermittent preventive antimalarial
treatment for children with anaemia" The Cochrane Database of Systematic

Buffet, P. A., Safeukui, I., Deplaine, G., Brousse, V., Prendki, V., Thellier, M., Turner, G. D. &
Mercereau-P. O. 2011. The pathogenesis of Plasmodium falciparum malaria in humans:
insights from splenic physiology. Blood, 117,

Caraballo, H (2014). "Emergency department management of mosquito-borne illness: Malaria,

dengue, and west Nile virus".

Castelhano-Carlos, M., Costa, P.S., Russig H., and Sousa, N. (2014). PhenoWorld: a new
paradigm to screen rodent behaviour. Transl Psychiatry (2014) 4: e399;
doi:10.1038/tp.2014.40.

Castelhano-Carols, M.J., and Baumans, V., 2009. The impact of light, noise, cage cleaning and
in house transport on welfare and stress of laboratory
44
Chaguri, L.C.A.G., Souza, N.L., Teixeira, M.A., Mori, C.M.C. Carissimi, A.S., and Merusse,
J.L.B., 2001. Evaluation of reproductive indices in rats (Rattus norvegicus) housed under
an intracage ventilation system. Contemporary Topics in Laboratory Animal Science. 40:
25e30.

Charlotte, C. Burn and Georgia, J. Mason. Effects of cage-cleaning frequency on laboratory rat
reproduction, cannibalism, and welfare.

Chiogu, I.S., UCHENDU, Chukwuka, N., and IHEDIOHA, John Ikechukwu. (2006). Animal
Research International; 3(3): 527 – 530.

CLARK, I. A., AND COWDEN, W. B. 2003. The pathophysiology of falciparum malaria.

Pharmwacology & therapeutics, 99, 221-260

Clough, G., 1982. Environmental effects on animals used in biomedical research. Biological
Reviews of Cambbridge Philosophical Society. 57: 487e523.

COBBOLD, S. A., LLINÁS, M., AND KIRK, K. 2016. Sequestration and metabolism of host
cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum. Cellular
microbiology.

Cowie, A.T., 1984. Lactation. In: Austin, C.R., Short, R.V. (Eds.), Reproduction in Animals,vol.
3. Cambridge University Press, New York, NY, pp. 195e231.

Elsevier , Elsevier. World Health Organization (1958). "Malaria" (PDF). The First Ten Years of
the World Health Organization . World Health Organization. pp. 172–87.

ELSHEIKHA, H. M. & SHEASHAA, H. A. 2007. Epidemiology, pathophysiology, management

Enayati A, Hemingway J (2010). "Malaria management: Past, present, and future".Annual

Review of Entomology . 55: 569–91.

Furnival-Adams J., Olanga EA., Napier ,M., Garner, P (2020-10-15). "House modifications for
preventing malaria". Cochrane Database of Systematic Reviews 1465-1858 .

45
Gamble C, Ekwaru JP, ter Kuile FO, et al. (Cochrane Infectious Diseases Group) (April 2006).
"Insecticide-treated nets for preventing malaria in pregnancy" . The Cochrane Database
of Systematic Reviews (2): CD003755.

Gleave, K., Lissenden, N., Richardson, M., Choi, L., Ranson H, et al. (Cochrane Infectious
Diseases Group) (November 2018). "Piperonyl butoxide (PBO) combined with
pyrethroids in insecticide-treated nets to prevent malaria in Africa" . The Cochrane
Database of Systematic Reviews .

Howitt, P., Darzi ,A., Yang ,GZ., Ashrafian, H., Atun, R., Barlow, J., Blakemore, A., Bull, AM.,
Car, J., Conteh, L., Cooke, GS., Ford, N., Gregson, SA., Kerr, K., King, D., Kulendran,
M., Malkin, RA., Majeed, A., Matlin, S, Merrifield R, Penfold HA, Reid SD, Smith PC,
Stevens, M. M., Templeton, M. R, Vincent, C., Wilson, E., (2012). "Technologies for
global health". The Lancet. 380 (9840): 507–35.

Kajfasz, P., (2009). "Malaria prevention" . International Maritime Health. Archived from the
original on 2017-08-30.

Kattenberg, J. H., Ochodo, E. A., Boer, K., R., Schallig, H. D., Mens, P. F., Leeflang, M. M.
(2011). "Systematic review and meta-analysis: Rapid diagnostic tests versus placental
histology, microscopy and PCR for malaria in pregnant women" . Malaria Journal . 10:
321.

Maia, M. F., Kliner, M., Richardson, M., Lengeler, C., Moore, S. J., et al. (Cochrane Infectious
Diseases Group), 2018. "Mosquito repellents for malaria prevention". The Cochrane
Database of Systematic Reviews. 2 : CD011595.

Manguin, S., Foumane, V., Besnard, P., Fortes, F., Carnevale, P., ( 2017). "Malaria
overdiagnosis and subsequent overconsumption of antimalarial drugs in Angola:
Consequences and effects on human health". Acta Tropica . 171 : 58–63.

Miller, J. M., Korenromp, E. L., Nahlen, B. L. W., Steketee, R., (2007). "Estimating the number
of insecticide-treated nets required by African households to reach continent-wide
malaria coverage targets". Journal of the American Medical Association . 297 (20):
2241–50.
46
Mockenhaupt, F. P., Ehrhardt, S., Burkhardt, J., Bosomtwe, S. Y., Laryea,

Murambiwa, P., Tufts, M., Mukaratirwa, S., Van, H. F. R. & Murabayane, C. T. 2013.
Evaluation of efficacy of transdermal delivery of chloroquine on Plasmodium berghei-
infected male Sprague [ndash] Dawley rats and effects on blood glucose and renal
electrolyte handling.

Nadjm, B., Behrens, R. H. (2012). "Malaria: An update for physicians". Infectious Disease
Clinics of North America.

Noor, A, M., Mutheu, J. J., Tatem, A. J., Hay, S. I., Snow, R. W., (2009). "Insecticide-treated net
coverage in Africa: Mapping progress in 2000–07" . Lancet . 373 (9657): 58–67.

Odaga, J., Sinclair, D., Lokong, J. A., Donegan, S., Hopkins, H., Garner, P., et al. (Cochrane
Infectious Diseases Group) (April 2014).

Orish, V. N., Ansong, J. Y., Onyeabor. O. S., Sanyaolu, A. O., Oyibo, W. A., Iriemenam, N. C.,
(October 2016). "Overdiagnosis and overtreatment of malaria in children in a secondary
healthcare centre in Sekondi-Takoradi, Ghana" . Tropical Doctor. 46 (4): 191–198.

Pates, H., Curtis, C. (2005). "Mosquito behaviour and vector control". Annual Review of
Entomology . 50: 53–70.

Perkins, M. D., Bell, D. R. (2008). "Working without a blindfold: The critical role of diagnostics
in malaria control" . Malaria Journal . 1 (Suppl 1): S5.

Pryce, J., Richardson, M., Lengeler, C. (November 2018). "Insecticide-treated nets for
preventing malaria" . The Cochrane Database of Systematic Reviews . 11: CD000363.

Raghavendra, K., Barik, T. K., Reddy, B. P., Sharma, P., Dash, A. P. (2011). "Malaria vector
control: From past to future". Parasitology Research . 108 (4): 757–79.

Anemana, S. D., Otchwemah, R. N., Cramer, J. P., Dietz, E. & Gellert, 2004. Manifestation and
outcome of severe malaria in children in northern Ghana. The American journal of
tropical medicine and hygiene, 71, 167-172

47
Sabot, O., Cohen, J. M., Hsiang, M. S., Kahn, J. G., Basu, S., Tang, L., Zheng, B. Gao, Q., Zou,
L., Tatarsky, A., Aboobakar, S., Usas, J., Barrett, S., Cohen, J. L., Jamison, D. T.,
Feachem, R. G. (2010). "Costs and financial feasibility of malaria elimination" . Lancet .
376 (9752): 1604–15. doi:10.1016/S0140-

Schlagenhauf-Lawlor, 2008, Instructions for treatment and use of insecticide-treated mosquito

nets (PDF).

Tanser, F. C., Lengeler, C., Sharp, B. L. (2010). Lengeler, C. (ed.).

UNICEF. WHO. September (2015). ISBN 978-92-4-150944-2 . Archived (PDF) from the
original on 5 January 2016. Retrieved 26 December 2015.

Van Den Berg, H. (2009). "Global status of DDT and its alternatives for use in vector control to
prevent disease" .

WHO (2019). World Malaria Report 2019. Switzerland: World Health Organization. pp. xii–xiii,
4–10.

WHO Position Statement (PDF) (Report). World Health Organization. 2006. Archived (PDF)
from the original on 2008-10-02.

WHO. March (2014). Archived from the original on 3 September 2014. Retrieved 28 August
2014.

Wilson, M.L., (2012). "Malaria rapid diagnostic tests" . Clinical Infectious Diseases. 54 (11):
1637–41. doi:10.1093/cid/cis228 . PMID 22550113 .

World Health Organization. (2002). p. 34.Archived (PDF) from the original on 2015-07-06.

Yegorov, S., Galiwango, R.M., Ssemaganda A, Muwanga M, Wesonga I, Miiro G, et al.

(November 2016). "Low prevalence of laboratory-confirmed malaria in clinically
diagnosed adult women from the Wakiso district of Uganda" . Malaria Journal. 15 (1):
555. doi: 10.1186/s12936-016-1604-z .

48