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DECLARATION

I, ORUNI AMBROSE, hereby do declare that to the best of my knowledge, this is my

original research report and has not been submitted to any University or institution for the

award of any degree or certificate in the same or related field.

SignedDate.

This research report has been submitted for examination with the approval of my

supervisors:

Ass. Prof. Enock Matovu

Principal Investigator, Molecular Biology Laboratory II

Department of Veterinary Parasitology and Microbiology,

College of Veterinary Medicine, Animal resource development and Biosecurity,

Makerere University,

P.O. Box 7062,

Kampala, Uganda.

Signed..Date

Ms. Monica Namayanja (MSc)

Senior Researcher, Molecular Biology Laboratory I,

Department of Veterinary Parasitology and Microbiology,

College of Veterinary Medicine, Animal resource development and Biosecurity,

Makerere University,

P.O. Box 7062,

Kampala, Uganda.

Signed..Date


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DEDICATION

I dedicate this report to my parents; Mr. and Mrs. Okee and to my brothers and sisters;

Ojok, Acellam, Kolo, Ajok, Adong, Aryemo for always supporting me in all that I do.


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ACKNOWLEDGEMENT

I would like to send my sincere gratitude to Ms. Monica Namayanja for the effort she put

in from the time I began my research to the finish, no words can estimate how thankful I

am. I would also like to greatly thank Ass. Prof. Enock Matovu for supervising and

funding my research project, without which I wouldnt have done any work. A special

thank you also goes to my parents, brothers and sisters for supporting me throughout my

research period. And last but not least Prof. G. W. Lubega, members of Molecular

Biology Laboratory I and II, my fellow students; Wachiuri Kelvin, Cuu Gloria and my

dear friends; Allan, Henry, Steven, Rebecca, Austin and Wilson, who all contributed in

one way or the other for the success of this project.


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TABLE OF CONTENTS

DECLARATION.............................................................................................................................

DEDICATION.................................................................................................................................

ACKNOWLEDGEMENT...............................................................................................................TABLE OF CONTENTS ...............................................................................................................

LIST OF FIGURES AND TABLES..............................................................................................

ABBREVIATIONS AND ACRONYMS.......................................................................................

ABSTRACT.....................................................................................................................................

CHAPTER ONE ..............................................................................................................................

1.0INTRODUCTION.....................................................................................................................

1.1Background ..................................................................................................................................

1.2 Statement of the problem ............................................................................................................

1.3 Objectives ....................................................................................................................................

1.3.1 General objective .....................................................................................................................

1.3.2 Specific objective .....................................................................................................................

1.4 Justification and significance ......................................................................................................

1.5Research Question .......................................................................................................................

CHAPTER TWO .............................................................................................................................

2.0 LITERATURE REVIEW ........................................................................................................

2.1 African Trypanosomiasis ............................................................................................................

2.2 Trypanosoma brucei ...................................................................................................................

2.2.1 Life cycle ofTrypanosoma brucei ...........................................................................................

2.3 Management of Human African Trypanosomiasis .....................................................................

2.3.1 Diagnosis of Human African Trypanosomiasis .......................................................................

2.3.1.1 Serological techniques ..........................................................................................................

2.3.1.1.1 The Card Agglutination Test for Trypanosomiasis (CATT) .............................................

2.3.1.1.2 Antibody detection .............................................................................................................

2.3.1.1.3 The LATEX agglutination test forT. b. gambiense...........................................................

2.3.1.1.4 Immunofluorescence Assays .............................................................................................


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2.3.1.2 Parasitological techniques .....................................................................................................

2.3.1.2.1 Chancre aspirate .................................................................................................................

2.3.1.2.2 Lymph node aspirate ..........................................................................................................

2.3.1.2.3 Wet and thick blood films ..................................................................................................

2.3.1.2.4 Microhematocrit centrifugation technique .........................................................................

2.3.1.2.5 Quantitative buffy coat .......................................................................................................

2.3.1.2.6 Mini-anion-exchange centrifugation technique .................................................................

2.3.1.3 Molecular diagnosis ..............................................................................................................

2.3.1.3.1 Polymerase Chain Reaction (PCR) ....................................................................................

2.3.1.3.2 Loop-mediated Isothermal Amplification (LAMP) ...........................................................

2.3.1.4 Diagnosis to Stage Human African Trypanosomiasis ..........................................................

2.3.1.4.1 White blood cell count .......................................................................................................

2.3.1.4.2 Protein concentration .........................................................................................................

2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF...................................................

2.3.1.4.4 Trypanosomes detection in the CSF ..................................................................................

2.3.1.5 Treatment of the disease .......................................................................................................

2.4 Pyroglutamyl peptidase 1 (PGP 1) ..............................................................................................

2.4.1 Clan CF of Pyroglutamyl peptidase 1 ......................................................................................

2.4.2 Family C15 of Pyroglutamyl peptidase 1 ................................................................................

2.4.3 Trypanosoma brucei PGP 1 .....................................................................................................

2.4.4 Sequence ofTrypanosoma PGP 1............................................................................................

2.5 Immunogenicity ..........................................................................................................................

2.5.1The Nature of the Protein ..........................................................................................................

2.5.1.1 Degree of foreignness ...........................................................................................................

2.5.1.2 Molecular size .......................................................................................................................

2.5.1.3 Structure of the protein .........................................................................................................

2.5.1.4 Ability to be processed...........................................................................................................

2.5.2 Route of administration............................................................................................................

2.5.3 Genetic makeup of the organism .............................................................................................


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2.5.4 Adjuvant ...................................................................................................................................

2.5.5 Dose of protein given ...............................................................................................................

2.5.6 Formulation and purity of the protein ......................................................................................

2.6 Overview of the techniques to be used .......................................................................................

2.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........................

2.6.2 Western Blotting ......................................................................................................................

2.6.3 Enzyme-Linked Immunosorbent Assay (ELISA) ....................................................................

2.7 pET-28a (+) vector ......................................................................................................................

CHAPTER THREE ........................................................................................................................

3.0 MATERIALS AND METHODS .............................................................................................

3.1.0 Study design .............................................................................................................................

3.2.0 Materials: .................................................................................................................................

3.2.1 Transformed BL21DE3 cells ...................................................................................................

3.3.0 Methods: ..................................................................................................................................

3.3.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells

provided ............................................................................................................................................

3.3.1.1 Growth of glycerol stocks containing pET28a (+) ...............................................................

3.3.1.2 Plasmid extraction .................................................................................................................

3.3.1.3 Agarose Gel Electrophoresis .................................................................................................

3.3.1.4. Restriction enzyme digestion ...............................................................................................

3.3.2 Expression of the recombinant Trypanosoma PGP 1 ..............................................................

3.3.2.1 Small scale expression ..........................................................................................................

3.3.2.1.1 Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................

3.3.2.1.2 Western Blotting ................................................................................................................

3.3.2.2 Large scale expression ..........................................................................................................

3.3.3 Extraction, Purification and Quantification Trypanosoma PGP 1 and bacterial

protein ...............................................................................................................................................

3.3.3.1 Extraction ..............................................................................................................................

3.3.3.1.1 Extraction ofTrypanosoma PGP 1 ....................................................................................


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3.3.3.1.2 Extraction of Bacterial protein ...........................................................................................

3.3.3.1.2.1 Growth of competent BL21 cells from the glycerol stock ..............................................

3.3.3.1.2.2 Extraction of bacterial protein ........................................................................................

3.3.3.2 Purification of the Trypanosoma PGP 1 and bacterial protein from inclusion

bodies ................................................................................................................................................

3.3.3.2.1 Purification of inclusion bodies .........................................................................................

3.3.3.2.2 Purification of the recombinant Trypanosoma PGP 1 and bacterial protein .....................

3.3.3.3 Quantification of the Trypanosoma PGP 1 and bacterial protein ........................................

3.3.4 Immunisation of mice of the Trypanosoma PGP 1 and bacterial protein ................................

3.3.5 Analysis of immune sera ..........................................................................................................

3.3.5.1 Western blot analysis ............................................................................................................

CHAPTER FOUR............................................................................................................................

4.0 RESULTS ...................................................................................................................................

4.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells .............................

4.2 Expression of recombinant Trypanosoma PGP 1 .......................................................................

4.3 Extraction and Purification ofTrypanosoma PGP 1 and bacterial protein .................................

4.3.2 Purification ...............................................................................................................................

4.3.2.1 Purification of recombinant Trypanosoma PGP 1 ................................................................

4.3.2.2 Purification of Bacterial protein............................................................................................

4.4 Quantification .............................................................................................................................

4.5 Analysis of sera for the different groups of mice .......................................................................

4.5.1. Western blot analysis of pre-immune sera ..............................................................................

4.5.2 Western blot analysis of immune sera .....................................................................................

4.5.3 ELISA analysis of sera.............................................................................................................

4.5.2.1 Group one (Test group) .........................................................................................................

4.6.2.2 Group two (Adjuvant group) .................................................................................................

4.6.2.3 Group three (PBS group) ......................................................................................................

4.6.2.4 Group Four (Bacterial protein group) ...................................................................................


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CHAPTER FIVE .............................................................................................................................

5.0 DISCUSSION .............................................................................................................................

CHAPTER SIX ................................................................................................................................

6.0 CONLUSION AND RECOMMENDTAION .........................................................................

6.1 CONCLUSION ..........................................................................................................................

6.2 RECOMMENDATION ............................................................................................................

REFERENCES.................................................................................................................................

APPENDIX I ....................................................................................................................................

APPENDIX II...................................................................................................................................


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LIST OF FIGURES AND TABLES

Figure 1: TheLife cycle ofTrypanosoma brucei in the human and the tsetse fly...6

Figure 2: Vector map of pET28a (+)....23

Figure 3: A 1% agarose gel showing confirmation of insert digested from plasmid

extract...34

Figure 4: A 15% SDS-PAGE gel and western blot showing successful expression of

Trypanosoma PGP

1... .34

Figure 5: A 15% SDS-PAGE gel showing cell lysis BL21DE3 and BL21 cells....35

Figure 6: A 15% SDS-PAGE gel showing purification ofTrypanosoma PGP 1...........35

Figure 7: A 15% SDS-PAGE gel showing purification of bacterial protein..36

Figure 8: Graph for quantification .....37

Figure 9: Western blots showing analysis of pre-immune sera .....37

Figure 10: Western blots showing analysis of immune sera..37

Figure 11: ELISA fortest group....38

Figure 12: ELISA for Adjuvant group..38

Figure 13: ELISA for PBS group......39

Figure 14: ELISA for bacterial protein group...39

Figure 15: Immunogenecity curve40

Table 1: Calculated concentration of the purified portions ofTrypanosoma PGP 1....36


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ABBREVIATIONS AND ACRONYMS

BBB Blood Brain Barrier

Bst Bacillus staeorothermophilus

bp base pairs

CAT Card Agglutination Test

CATT Card Agglutination Test for Trypanosomes

CDC Center for Disease Control

CNS Central Nervous System

CSF Cerebral Spinal Fluid

DAB 3, 3-diaminobenzidine

E. coli Escherichia coli

ECL Enzyme Chemiluminescence

ELISA Enzyme Linked Immuno-sorbent Assay

EDTA Ethylene diamine tetra acetic acid

HAT Human African Trypanosomiasis

HRP Horse Radish Peroxidase

IPTG Isopropyl-beta-D-thiogalactopyranoside

Kda Kilodaltons

LH-RH Luteinising Releasing Hormone


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mHAET Mini-anion-exchange centrifugation technique

mHCT Microhematocrit Centrifugation Technique

Ni-NTA Nickel-Nitrilotriacetic acid

O.D Optical Density

OPD o-phenylenediamine dihydrochloride

PARP Procyclic acidic repetitive protein

PCR Polymerase Chain Reaction

PGP 1 Pyroglutamyl Peptidase

rpm rotations per minute

SDS Sodium Dodecyl Sulfate

SDS-PAGE SDS-Poly Acrylamide Gel Electrophoresis

spp. Species

T. b. Trypanosoma brucei

T. b. gambiense Trypanosoma brucei gambiense

T. b. rhodesiense Trypanosoma brucei rhodesiense

TRH Thyrotropin-Releasing Hormone

W H O World Health Organisation


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ABSTRACT

Diagnosis of Human African Trypanosomiasis (HAT) still remains a challenge despite a

number of diagnostic techniques available. There are no simple and reliable screening

tests for the disease especially forT. b. rhodesiense. This therefore puts a hindrance in the

control of the disease in Africa. This study was carried out to determine the immunogenic

potential of Trypanosoma Pyroglutamyl peptidase type 1 which is released during

intravascular destruction of Trypanosomes in blood during the infection; this would

determine whether Trypanosoma Pyroglutamyl peptidase type 1can be used as a

diagnostic antigen for screening HAT in endemic areas. The gene for Trypanosoma

Pyroglutamyl peptidase type 1 was previously cloned in pET28a and transformed in E.

coli (BL21DE3) cells. In this study, recombinant Trypanosoma Pyroglutamyl peptidasetype 1 was successfully expressed in the E. coli (BL21DE3) cells. The protein was

extracted by cell lysis from theE. coli cells and purified using Ni-NTA agarose column.

The purified recombinant Trypanosoma Pyroglutamyl peptidase type 1 was used to

immunise 7 to 8 weeks old male Swiss albino mice using 40g/ml as the initial dose

(prime dose) of the protein. The first and second boosts were done using 20g/ml of the

purified protein. Production of specific antibodies was determined using western blotting

and ELISA. The western blots showed strong signal detection by the protein for sera after

second boost at a dilution of 1:2000. However, at the same dilution of 1:2000, very weak

signals were also detected on the lane ofTrypanosoma Pyroglutamyl peptidase type 1 on

the membrane. The ELISA results showed that the Trypanosoma Pyroglutamyl peptidase

type 1 gave an antibody IgG titer of 1:486,000 for the first and second boost sera and this

was depicted in the immunogenicity curve that showed no change in titers after first

boosting and second boosting post imunisation. The results obtained from this study

therefore show that, Trypanosoma Pyroglutamyl peptidase type 1 was capable of eliciting

specific and quantifiable antibodies in mice. This means that the Trypanosoma

Pyroglutamyl peptidase type 1 is immunogenic and could be a good candidate for a

diagnostic antigen for screening for HAT, however, wider studies on the protein like full

study of the structure, antigenecity study, evaluation to see if the protein can pick up

some cases of HAT, among others, should be conducted.


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CHAPTER ONE

1.0INTRODUCTION1.1BackgroundAfrican trypanosomes are parasitic hemo-flagellated protozoans of the genus

Trypanosoma, transmitted to the host bloodstream by the tsetse fly (Glossina spp.) and

cause African Trypanosomiasis in humans and domestic animals. The different species of

trypanosomes in Africa include; Trypanosoma congolense, Trypanosoma evansi,

Trypanosoma vivax and Trypanosoma brucei with the sub species ofT. b .brucei, T. b.

gambiense, T .b .rhodesiense. T. b. brucei is one of the causative agents of Animal

African trypanosomiasis, it is not human infective due to its susceptibility to lysis by

human apolipoprotein L1(Vanhamme et al., 2003), T. b. gambiense causes chronic

Human African Trypanosomiasis (HAT) most common in central and western Africa,

while T. b. rhodesiense causes acute HAT most common in southern and eastern Africa

(Barrett et al., 2003).

African Trypanosomiasis is endemic in some regions of sub-Saharan Africa, covering

about 37 countries and 60 million people. In 2010, it was estimated that 50,000 to 70,000

people were infected, the number showed a decline smaller compared to earlier years

(WHO, 2010). The disease has devastating effects on both humans and livestock

populations, contributing to poverty in some of these affected regions of Africa. Control

of African Trypanosomiasis mainly depends on proper diagnosis and treatment, however

HAT diagnosis is still unsatisfactory (Njiru et al., 2007); much as the current

parasitological tests are cheap and simple, they are tedious, time consuming, and are of

low sensitivities because of characteristic low and fluctuating parasitemia of infected

individuals. The CATT detection technique is sensitive and works well in the diagnosis

of HAT due T .b. gambiense, but it is not always reliable in the diagnosis of T. b.

rhodesiense (Lejon et al., 2002) hence some cases may be missed out. There is therefore

need to develop tests similar to CATT that can accurately diagnose the disease due to

both sub species. The molecular techniques are relatively sensitive and specific but
http://en.wikipedia.org/wiki/Naganahttp://en.wikipedia.org/wiki/Naganahttp://en.wikipedia.org/wiki/APOL1http://en.wikipedia.org/wiki/Endemic_%28epidemiology%29http://en.wikipedia.org/wiki/Sub-Saharan_Africahttp://en.wikipedia.org/wiki/Sub-Saharan_Africahttp://en.wikipedia.org/wiki/Endemic_%28epidemiology%29http://en.wikipedia.org/wiki/APOL1http://en.wikipedia.org/wiki/Naganahttp://en.wikipedia.org/wiki/Nagana


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involve sophisticated and expensive equipments that would need simplification, not to

mention that most of the molecular techniques are still experimental and hence

impracticable for field diagnosis.

Identification of proteins that are released during the pathogenesis of HAT could help in

identification of diagnostic antigens that can be used in diagnosis of HAT. This would for

help simplify the diagnosis of HAT since the above techniques are not reliable for the

diagnosis of HAT especially due to T. b. rhodesiense. Among such proteins are Cysteine

peptidases like Oligopeptidase A and B as well as Pyroglutamyl peptidase 1 (PGP 1).

Pyroglutamyl peptidase type 1 (PGP 1) belongs to a group of peptidases called Cysteine

peptidases. Cysteine peptidases are divided into clans and further into families (Barret

and Rawlings 2001); PGP 1 belongs to Clan CF and Family C15. The enzyme isintracellular and soluble. In mammals, PGP 1 has been shown to release Pyroglutamate

from Thyrotropin-Releasing Hormone (TRHI), Luteinising Hormone Releasing Hormone

(LH-RH), neurotensin, bombesin and leukopyrokinin (Dando et al., 2003). In

Trypanosoma brucei, PGP 1 is a 25.1 Kda soluble cystosolic cysteine peptidase that is

released into the host blood stream during intravascular destruction of trypanosomes in

the host blood stream during the infection and is expressed in all life cycle stages of

Trypanosoma brucei as well as four other African Trypanosomes(Morty et al., 2006). It

is a factor involved in the pathogenesis of HAT and is known to degrade peptides

including Thyrotrophin Releasing Hormone (TRH) and Gonadotropin Releasing

Hormone (GnRH) (Morty et al., 2006) by removing the N-terminal Pyroglutamyl residue

of these peptides that protects them from proteolysis. A study conducted on Trypanosoma

PGP 1 showed that the protein can be recognised by infected human sera (Anywar,

2009), however, the study was not conclusive on the immunogenicity of Trypanosoma

PGP 1.

1.2 Statement of the problem

Lack of a field applicable screening test forT .b .rhodesiense is recruiting investigation of

proteins predictably capable of eliciting immune response that could be exploited to

develop new diagnostic tests. A previous study had already been conducted on


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Trypanosoma PGP 1 to determine its recognition by infected human sera using field

samples (Anywar, 2009). The results obtained form that study demonstrated that

Trypanosoma PGP 1 was indeed recognised by patient sera by western blotting.

However, the immunogenic potential of the Trypanosoma PGP 1 was not done. This

study therefore aimed to carry out an independent well controlled research in mice to

show whether immunisation with Trypanosoma PGP 1 leads to generation of specific and

quantifiable antibodies.

1.3 Objectives

1.3.1 General objective

Determine the immunogenic potential ofTrypanosoma PGP 1

1.3.2 Specific objective

Determine whether recombinant Trypanosoma PGP 1 elicits specific immune response in

mice.

1.4 Justification and significance

Trypanosoma PGP 1 has been postulated in the pathogenesis of HAT and is released due

to intravascular destruction of trypanosomes in the blood stream of the host (Morty et al.,

2006). This therefore means that Trypanosoma PGP 1 could be a good candidate for a

diagnostic antigen for HAT. A previous study also showed that Trypanosoma PGP 1 has

a diagnostic potential (Anywar, 2009), however, its immunogenicity is not conclusively

known. Conducting this study on Trypanosoma PGP 1 will demonstrate if the protein can

elicit specific and quantifiable antibodies. Such a confirmation would make Trypanosoma

PGP 1 a suitable candidate for further exploration as diagnostic antigen for HAT andcould lead to development of a new diagnostic test for HAT which would help save time

and lives that could have been lost due to poor and unreliable diagnostic techniques.


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1.5Research QuestionDoes Trypanosoma PGP 1 elicit specific immune response in mice?

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 African Trypanosomiasis

Human African Trypanosomiasis (HAT) (Sleeping sickness) and Animal African

Trypanosomiasis (Nagana), is a parasitic disease that affects humans and animals

respectively. The disease is caused by African trypanosomes of genus Trypanosoma and

these include; Trypanosoma congolense, evansi, vivax and brucei that included the sub

species ofT. b. brucei, T. b. gambiense, T. b. rhodesiense.

Sleeping sickness has been reported in 37 countries in sub-Saharan Africa. Many of the

affected populations live in remote areas with limited access to adequate health services,

which hampers the surveillance and therefore the diagnosis and treatment of cases. In

addition, displacement of populations, war and poverty are important factors leading to

increased transmission and this alters the distribution of the disease due to weakened or

non-existent health systems (WHO Fact sheet N259, 2010). Recent estimates indicate

that over 60 million people living in some 250 locations are at risk of contracting the

disease. There were under 10,000 cases reported in 2009 according to WHO figures


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which represents a huge decrease from the estimated 300,000 new cases in 1998 (WHO,

1998).

There are two forms of the disease depending on the parasite involved; Trypanosoma

brucei gambiense is found in west and central Africa. This form currently accounts for

over 95% of reported cases of sleeping sickness and causes a chronic infection. A person

can be infected for months or even years without major signs or symptoms of the disease.

When symptoms emerge, the patient is often already in an advanced disease stage where

the central nervous system is affected; Trypanosoma brucei rhodesiense is found in

eastern and southern Africa. Nowadays, this form represents fewer than 5% of reported

cases and causes an acute infection. First signs and symptoms are observed a few months

or weeks after infection. The disease develops rapidly and invades the central nervoussystem. Other parasite species and sub-species of the Trypanosoma genus are pathogenic

to animals and cause Animal Trypanosomiasis Nagana in cattle. Animals can host the

human pathogen parasites, especially T. b. rhodesiense; thus domestic and wild animals

are an important parasite reservoir. Animals can also be infected with T. b. gambiense

and act as a reservoir. However the precise epidemiological role of this reservoir is not

yet well known.

The disease in domestic animals, particularly cattle, is a major obstacle to the economic

development of affected rural areas due to its devastating effects on both humans and

livestock populations hence contributing to poverty in some of these endemic areas of

Africa.

2.2 Tr ypanosoma brucei

Trypanosoma brucei species is one of the causative agents ofAfrican Trypanosomiasis

(or sleeping sickness). There are 3 sub-species ofT. brucei: T. b. brucei, T. b. gambiense

and T. b. rhodesiense.

T. brucei gambiense causes chronic Trypanosomiasis in humans most common in central

and western Africa, where humans are thought to be the primary reservoirs (Barrett et al.,

2003).
http://en.wikipedia.org/wiki/African_trypanosomiasishttp://en.wikipedia.org/wiki/African_trypanosomiasis


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T. brucei rhodesiense causes acute Trypanosomiasis in humans most common in southern

and eastern Africa, where game animals and livestock are thought to be the primary

reservoir (Barrett et al., 2003).

Uganda is the only country where both forms of the disease are present; most likely, apotential geographical overlap of the two endemic areas (Picozzi et al., 2005). This may

therefore hinder the field identification of the correct infective sub-species of

Trypanosoma brucei hence hindering treatment as well.

T. brucei brucei causes Animal African Trypanosomiasis along with several other species

ofTrypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by

human apolipoprotein L1 (Vanhamme et al., 2003).

2.2.1 Life cycle ofTrypanosoma brucei


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Figure 1: Life cycle ofTr ypanosoma bruceiin the human and the tsetse fly. Image credit: Alexander J.

da Silva and Melanie Moser, Centers for Disease Control Public Health Image Library .

The life cycle of a trypanosome involves various developmental stages involving a series

of differentiation in both the vector (tsetse fly) and the mammalian host. During the

different stages of its life cycle, the parasites changes in morphology and cell structure.

The infection in the host begins when the metacyclic trypomastigotes form of the parasite

is injected into the host through the skin by the infected tsetse fly (Figure 1:-1). At the

site of infection, the metacyclic trypomastigotes multiply locally for a few days after theyenter into the lymphatic system and pass into the blood stream. Once in the hosts blood,

the metacyclic trypomastigotes go through development then transform into long slender

bloodstream forms covered by the variant surface glycoprotein (VSG), (Biebingeret al.,

1996) (Figure 1:-2). These bloodstream trypomastigotes are carried to other sites

throughout the body and also reach other body fluids (e.g. lymph, spinal fluid) and


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continue replication by binary fission as well as differentiate into intermediate forms and

stumpy forms which have the VSG (Figure 1:-3-4). The entire life cycle of the African

trypanosomes is represented by extracellular stages. A tsetse fly becomes infected with

bloodstream trypomastigotes when taking a blood meal from an infected mammalian host

and it is the stumpy forms are then taken up by the vector (Figure 1:-5). Once in the

midgut of the fly, they transform into procyclic trypomastigotes, the VSG coat is then

shed within a few hours replaced by a coat of procyclic acidic repetitive protein (PARP)

also known as procyclin (Biebinger et al., 1996) (Figure 1:-6). The procyclic

trypomastigotes then multiply by binary fission, leave the mid gut and migrate to into the

ectoperitrophic space then into the foregut (mouth parts) through the proventriculus.

Once in the foregut, the procyclic trypomastigotes then change into elongated and

asymmetrically dividing epimastigotes (Hill, 2003) (Figure 1:-7) which then multiply

actively in the proboscis and move to the salivary glands for final development. Once in

the salivary glands, the epimastigotes continue to multiply by binary fission to generating

short epimastigotes which attach themselves to the salivary gland epithelium (Figure 1:-

8). The attached epimastigotes differentiate into VSG-coated metacyclic trypomastigotes

that suited for mammalian bloodstream environment (Figure 1:-1). The cycle then

continues. The cycle takes about 3 weeks in the fly, the tsetse fly then remains infective

for the rest of its life (Chappuis et al., 2005).

2.3 Management of Human African Trypanosomiasis

The management of the disease is basically on three steps; Screening for potential

infection; which may involve use of serological techniques which are mostly available for

T. b. gambiense, Diagnosing for the presence of the parasite and Staging to determine the

state of the disease progression after which treatment can be effected.

2.3.1 Diagnosis of Human African Trypanosomiasis

The diagnosis of HAT is based on three techniques; Serological technique,

Parasitological technique and Molecular technique (WHO Fact sheet N259 2010).

Diagnosis must be made as early as possible and before the neurological stage in order to


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avoid complicated, difficult and risky treatment procedures. However, for proper,

effective and accurate treatment, staging of the disease has to first be performed since the

drugs used in the treatment of first stage is different from that used to treat second stage.

Drugs used to treat first stage are normally effective and of low toxicity compared to

drugs used to treat second stage therefore staging has to be accurate.

Laboratory diagnosis range from simple procedures like microscopy to detect

trypanosomes in body fluids; lymph node aspirates, chancres fluid, blood and cerebro-

spinal fluid (CDC 2006) to complicated procedures like most of the molecular techniques

such as Polymerase chain reaction (PCR).

2.3.1.1 Serological techniques

This involves using serological tests (mostly available for T. b .gambiense). Due to

fluctuating parasitemia in T. b .gambiense, serological tests are important in screening for

infection.

2.3.1.1.1 The Card Agglutination Test for Trypanosomiasis (CATT)

Developed in the late 1970s,the CATT is a fast and simple agglutination assay for

detectionof trypanosome antigens however, CATT is only effective forT. b. gambiense,

it is not effective and reliable forT. b. rhodesiense. It is mostly used forT .b. gambiense

specific antibodies in the blood, plasma,or serum of HAT patients (Magnus et al., 1978).

The antigen consists of lyophilised bloodstream forms ofT. b. gambiense variable

antigen type LiTat1.3. The trypanosomes

are fixed, stained with Coomassie blue, and

freeze-dried.One drop the CATT reagent is mixed with one drop of blood and shaken for

5 minon the rotator, and the result is visible to the naked eye. The reported sensitivity

of

the CATT on undiluted whole blood (CATT-wb) varies from 87to 98%, and the negative

predictive value is excellent duringmass population screening (Noireau et al., 1987; Truc

et al., 2002). Nevertheless,false-negative CATT results can occur (Penchenier et al.,

1991), as suspected inpatients infected with strains of trypanosomes that lack or

do not

express the LiTat 1.3 gene (Dukes et al., 1992; Enyaru et al., 1998). Furthermore, when

the CATT is performedon undiluted blood or serum with a low dilution of less than 1:4,


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theagglutination can be inhibited, a phenomenon called prozone which can be overcome

by adding EDTA tothe dilution buffer (Pansaerts et al., 1988), this increases the

sensitivity (Magnus et al., 2002). The test has a reported specificity of around 95% but

the positive predictivevalue is limited because the test is used

for mass screening in

populations where the prevalence of HATis usually below 5% (Robays et al., 2004).

False-positive results can occur in patients with malaria and other parasiticdiseases such

as transient infection by nonhuman trypanosomes(Magnus et al., 1978).

2.3.1.1.2 Antibody detection

Indirect evidence for trypanosome infection can be obtainedby demonstrating specific

antibodies in serum of infected hosts. Trypanosomes have a complex antigenicstructure

evoke production of a large spectrum of antibodies.T. b. gambiense specific IgG and IgM

antibodies are presentin high concentrations and are directed mainly against the Immuno-

dominant surface glycoprotein antigens of the parasite. The sensitivity and specificity of

the test to be used to detect these antibodies greatly depends on the antigen(s). The

available current serological used to detect antibodies include Enzyme-linked

immunosorbent assay (ELISA) that can antibodies after 3 to4 weeks of infection

(Vanhamme et al., 2001) with strict standardisation and quantification (Lejon et al.,

1998) but sero-positivity must be interpretedwith caution in previously treated patients

since antibodiescan persist for up to 3 years after cure (Paquet et al., 1992). ). ELISA can

also detect specific antibodiesin the saliva HAT

(Lejon et al., 2003). However, ELISA

requires time and sophiscated equipments like ELISA reader and does some of the other

antibody detection techniques hence limiting their use in field diagnosis and

referencelaboratories for remote testing of samples collected in the

field during surveys.

2.3.1.1.3 The LATEX agglutination test for T. b. gambiense

The test has been developed as a field alternativeto the CATT (Bscheret al., 1999). The

test is based on the combination of threepurified variable surface antigens, LiTat 1.3, 1.5,

and 1.6,coupled with suspended latex particles. The test procedure is

similar to the

CATT, including the use of a similar rotator.Compared to the CATT, the LATEX shows
http://g/diagnosis%20of%20HAT%201.htm%23R74http://g/diagnosis%20of%20HAT%201.htm%23R126http://g/diagnosis%20of%20HAT%201.htm%23R126http://g/diagnosis%20of%20HAT%201.htm%23R74


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a higherspecificity (96 to 99%) but a lower or similar sensitivity (71

to 100 (Penchenier

et al., 2003; Magnus et al., 2002). However, the LATEX agglutination test is only

available T. b. gambiense and not available forT. b. rhodesiense.

2.3.1.1.4 Immunofluorescence Assays

Immunofluorescence assays have been used with success for HATcontrol in Equatorial

Guinea, Gabon, and the Republic of Congo,where they were shown to be highly sensitive

and specific (Noireau et al., 1988).The availability of standardised antigen commercially

in the market at low cost has greatlyimproved the reliability of the test (Magnus et al.,

1978). It can be used withserum but the test sensitivity has been reported

to be as low as

75% when used with impregnated filter papers (Simarro et al., 1999). However this

technique requires sophisticated equipment like an immunofluorescent microscope and

this limits its use in remote areas.

2.3.1.2 Parasitological techniques

The diagnosis of the presence of parasites has been achieved greatly through a number of

parasitological tests. Parasitological diagnosis is made by microscopic examinationof

lymph node aspirate, blood, or CSF and this provides direct evidencefor trypanosome

infection thus allowing definite diagnosis. Unfortunately, it is estimated that 20% to 30%

of patients are missed by the standard parasitological techniques (Robays et al., 2004).

There is also always fluctuation in parasite numbers in T. b. gambiense infection10,000

trypanosomes per ml, being easilydetectable and less than 100 trypanosomes per ml,

being below thedetection limit of the most sensitive methods in use. This implies, failure

todemonstrate parasites does not necessarily exclude

infection. Parasite detection can be

rather labor-intensive. Some of the available parasitological detection methods that are

currently field use are mentioned below (WHO Trypanosomiasis Control

Manual 1983).

2.3.1.2.1 Chancre aspirate

Trypanosomes can be detected in the chancre a few days earlierthan in the blood. The

chancre is punctured, and the fluid obtainedis microscopically examined as a fresh or


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fixed and Giemsa-stainedpreparation. This method is very seldom applied in the

fieldbecause most infections are detected much later, when the chancre

has already

disappeared.

2.3.1.2.2 Lymph node aspirate

Cervical Lymph Node (CLN) palpation is done systematically in conjunction with

CATT, in all patientswith a positive CATT result. Enlarged CLNs are punctured and

fresh aspirate is expelled onto a slide,and a cover slip is applied to spread the sample and

facilitatethe reading. The wet preparation is then immediately examined under X400

magnification for the presence of motiletrypanosomes. The technique is simple and

cheap. The sensitivityvaries between 40 and 80% depending on the parasite strain,

the

stage of the disease (sensitivity is higher during the firststage), and the prevalence of

other diseases causing lymphadenopathy (Simarro et al., 2003; Van Meirvenne, 1999).

2.3.1.2.3 Wet and thick blood films

In wet blood films, 5 to 10 l of finger prick blood isplaced on a slide, cover slipped and

examined microscopically at X400 magnification. Trypanosomes can be seen moving

betweenthe erythrocytes. Although this method has very low sensitivity of about 10,000

trypanosomes per ml, it is still usedin some centers because of its low cost and

simplicity.Examination of 20 l of stained thick blood

film slightly improves sensitivity,

with a detection thresholdof around 5000 trypanosomes per ml. It is the technique of

choicefor blood examination only when no centrifuge is available (Henry et al.,

1981).The technique is quite time consuming and requires expertise to recognize the

parasite, which is frequentlydeformed in this preparation.

2.3.1.2.4 Microhematocrit centrifugation technique

The blood concentration microhematocrit centrifugation technique(mHCT) sometimes

referred to as the capillary tube centrifugationtechnique or as the Woo test, was

developed more than 30 yearsago and is still in use in many HAT control programs

(Woo P. T., 1971, 1970).In brief, capillary tubes containing anticoagulant are filled

three-


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quarters full with finger prick blood. The dry end issealed with plasticine. By high-speed

centrifugation in a haematocritcentrifuge for 6 to 8 min, trypanosomes are concentrated

atthe level of the white blood cells, between the plasma and the

erythrocytes. The

capillary tubes, mounted in a special holder,can be directly examined at low

magnification (x100 or x200)for mobile parasites. The sensitivity of mHCT increases

withthe number of tubes examined, with an estimated detection threshold

of 500

trypanosomes per ml. This technique is moderately time-consuming,and the concomitant

presence of microfilaria in the blood canrender the visualisation of the much smaller

trypanosomes verydifficult. Nevertheless, this relatively simple technique can

be applied

during mass screening by mobile teams.

2.3.1.2.5 Quantitative buffy coat

The quantitative buffy coat (QBC) initiallydeveloped for the rapid assessment of

differential cell counts,has been extended to the diagnosis of hemoparasites

includingtrypanosomes (Levine et al., 1989; Bailey et al., 1992). It has the advantages of

concentratingthe parasites by centrifugation and, by staining the nucleus

and kinetoplast

of trypanosomes with acridine orange, allowing a better discrimination from white blood

cells. After high-speedcentrifugation of the blood in special capillary tubes

containingEDTA, acridine orange, and a small floating cylinder, motile trypanosomes

can be identified by their fluorescent kinetoplastsand nuclei in the expanded buffy coat.

UV light is generatedby a cold light source connected by a glass fiber to a

specialobjective containing the appropriate filter and the procedure is done in a

darkroom. However the relative sophistication and fragilityof the material prevents its

daily use during active screeningsessions. QBC technique is a very sensitive technique

with 95% sensitivity for trypanosome concentrations of 450 per ml.The QBC can detect

more patients with low parasitemia than themHCT when fewer than eight capillary tubes

are used (Ancelle et al., 1997). Itis as sensitive as the mini-anion-exchange

centrifugation technique(mAECT) (Ancelle et al., 1997; Truc et al.,1998).


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2.3.1.2.6 Mini-anion-exchange centrifugation technique

The mAECT was introduced by Lumsden et al, based on atechnique developed by

Lanham and Godfrey (Lanham and Godfrey, 1970). An initial evaluationshowed that the

mAECT was more sensitive than the thick bloodfilm and the mHCT (Lumsden et al.,

1981). An updated version has been describedby Zillmann et al., 1996. The technique

consists of separatingthe trypanosomes, which are less negatively charged than

bloodcells, from venous blood by anion-exchange chromatography and

concentrating

them at the bottom of a sealed glass tube by low-speedcentrifugation. The tip of the glass

tube is then examined ina special holder under the microscope for the presence of

trypanosomes.The large blood volume (300 l) enables the detection

of less than 100

trypanosomes/ml and therefore giving a high sensitivity. However, the manipulations arequite tedious and time consuming.

2.3.1.3 Molecular diagnosis

Molecular diagnosis also offers another alternative in the diagnosis of HAT. The methods

used in molecular diagnosis are known for being sensitive (Bscheret al., 2004). These

methods range from Polymerase Chain Reaction (PCR) for amplifying DNA to Loop

Mediated Isothermal Amplification. However, most of the molecular methods fordiagnosis of HAT are under trial and have not yet been recommended for the diagnosis of

HAT.

2.3.1.3.1 Polymerase Chain Reaction (PCR)

Different assays now exist; however, none of them have beenvalidated for diagnostic

purposes. PCRs targeting repetitive sequences are in theory more sensitive than those

targetinglow-copy or single-copy sequences like the recently developed

tests for

distinguishing T. b. gambiense and T. b. rhodesiense (Jamonneau et al., 2001; Kabiri et

al., 1999; Radwanska et al., 2002; Schares et al., 1996). In principle, PCR can be applied

to any patient sample thatmay contain trypanosome DNA, such as whole blood or buffy

coat,lymph node fluid, or CSF. Samples should be stabilized in special

buffers. However,

the amount of samplethat can be applied on filter paper is small, thus limiting

the chance


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to contain enough DNA for detection. Samples shouldbe protected from sunlight to avoid

DNA degradation. PCR resultsare not always unequivocal. Unexplained false-negative

and false-positiveresults were observed in CATT-sero-positive but parasitologically

non-

confirmed persons and in CATT-negative controls (Garcia et al., 2000; Solano et al.,

2002).Also, the significance of a positive PCR on a CSF sample is

unclear. PCR is 100%

sensitive comparedto double centrifugation of CSF (Truc et al., 1999), but a number of

patients with positive PCR resultswith CSF were successfully treated with pentamidine,

thus showingthem to be in the first stage of the disease (Jamonneau et al., 2003). The

methods needs simplification hence PCR is not an option forfield diagnosis and for the

time being is restricted to researchpurposes.

2.3.1.3.2 Loop-mediated Isothermal Amplification (LAMP)

LAMP is a rapid, simple and highly sensitive technique that is used for gene

amplification (Notomi et al., 2000). The technique bases on autocycling strand

displacement synthesis of DNA by Bacillus sterothermophillus (Bst) DNA polymerase

under isothermal conditions (60-65oC). It uses 6 primers recognized by 8 selections of

target DNA hence increasing specificity, rapidity and efficiency. It amplifies target DNA

three fold every half cycle producing large amounts of product within 30-60 minutes

(Notomi et al., 2000). Visualisation is achieved through addition of a fluorescent dye

SYBR Green I to the DNA formed (Poon et al., 2006). The technique takes a short time

and can be carried out in an incubator.

2.3.1.4 Diagnosis to Stage Human African Trypanosomiasis

Staging of patients with HAT relies on examination of CSF obtainedby lumbar puncture.

This is a vital step in the diagnosis process and determination of treatment for HAT.The

first stage corresponds to presence of parasites in the in the blood and lymph, the second

stage involves presence of parasites in the CNS (Chappuis et al., 2005). Staging of the

disease is critical and must be made as accurate as possible due to different drugs used to

treat first stage and second stage. Treatment success in the second stage depends on a

drug that can cross the blood-brain barrier to reach the parasite such drugs like


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melarsoprol, an arsenicalderivative associated with a 2 to 10% fatality rate (Pepin et al.,

1994) are toxic ,complicated to administer and have side effects such as the brain

disorder and damage like post-treatment reactive encephalopathy (PTRE). Therefore

wrong staging may result not only to administering wrong treatment to the patient but

also trauma and even death of the patient due to wrong drug given. A number of methods

for staging the disease are available.

2.3.1.4.1 White blood cell count from the CSF

The CSF white blood cell count is the most widely used techniquefor stage

determination. After collecting the CSF sample by lumbar puncture, thecell count should

be carried out as soon as possible to preventcell lysis. Due to the small number of cells in

normal CSF, a cell-counting chamber has a volume of at least 1 l,such as the Fuchs-

Rosenthal and the Neubauer devices. It isnot recommended to dilute the CSF with Trck

solution sincethis solution can lyse trypanosomes. Patients

with 6 to 20 white blood cells

per l in the CSF are sometimes referredto as being in the "early second stage" or

"intermediate stage"of the illness.

2.3.1.4.2 Protein concentration

In normal healthy individuals, proteins in the CSF consist mainlyof albumin (70%) and

IgG (30%), both originating from the serum.Protein concentrations in the CSF are

elevated in HAT patientsand range from 100 to 2,000 mg/liter (Bisseret al., 2002; Lejon

et al., 2003). Protein concentrationscan also be raised in first-stage illness due to the

diffusionof IgG into the CSF, which can be present in very high concentrations

in the

serum. Recent evidence suggests that the protein concentrationthreshold set by WHO

(370 mg/liter) is too low and should beraised to 750 mg/liter to reflect blood-brain barrier

(BBB) impairment,

astrocyte activation, and neuro-degeneration (Lejon et al., 2001). CSF

protein concentration is simple and accurately determines the totalprotein concentration

in CSF. However the technique is rather difficult and the CSF proteinconcentrations

obtained by different methods and different standardsare not comparable. Due to the

sophistication of methods, the absence of standardisation, the instabilityof reagents, and


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the limited added value compared toCSF cell count (Miezan et al., 1998), it is no longer

recommended and has been virtually abandoned infield laboratories.

2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF

CSF of second-stageHAT patients contains high levels of immunoglobulins,

especiallyIgM (Bisseret al., 1997). An increased CSF IgM concentration has

thus been

considered by some as a strong potential marker ofsecond-stage HAT. High IgM levels

in CSF is due to intrathecalsynthesis, the dominance of IgM presence is an early

markerof CNS invasion whereas blood-CSF barrier dysfunction is found in

late CNS

involvement (Lejon et al., 2003). Despite its relevance to stage determination, IgM

detectionin CSF has not been carried out in the field, owing to the lack

of simple and

robust tests. A latex agglutination test for IgMin CSF (LATEX/IgM) has recently been

developed and is designedfor field use (Lejon et al., 2004). This method is however still

not being used due to specific trypanosome antibodies, anti-galactocerebrosides and

trypanosome DNA in the CSF, hence other methods are still undergoing evaluation and

development for staging HAT (Lejon et al., 2004).

2.3.1.4.4 Trypanosomes detection in the CSF

The finding of trypanosomes in CSF allows immediate classificationof a patient as being

in the second stage of illness. It isimportant to examine the CSF immediately after

lumbar puncture,because trypanosomes in CSF start to lyse within 10 min.

Directdetection of trypanosomes (e.g., during cell counting) is a

simple and cheap

technique but suffers from insufficient sensitivity. Increased sensitivity of trypanosome

detection is obtained bycentrifugation of the CSF sample, especially when a double

centrifugation method is used (Cattand et al., 1988). The latter method is relatively time-

consuming

and requires two different types of centrifuges; therefore,

it is not applicable in

every field setting. A modified andsimplified single centrifugation of CSF using a sealed

Pasteurpipette has been proposed as an alternative to double centrifugation

(Miezan et

al., 2000).


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2.3.1.5 Treatment of the disease

Treatment is better done after staging of the disease. The drugs used in the first stage of

the disease are of lower toxicity and easier to administer hence, the earlier the disease is

identified, the better the prospect of a cure. Treatment success in the second stage

depends on a drug that can cross the blood-brain barrier to reach the parasite. Such drugs

are toxic and complicated to administer. Four drugs are registered for the treatment of

sleeping sickness in endemic countries; Pentamidine; discovered in 1941, used for the

treatment of the first stage ofT. b. gambiense sleeping sickness. Despite non-negligible

undesirable effects, it is in general well tolerated by patients. Suramin; discovered in

1921, used for the treatment of the first stage of T. b. rhodesiense though it provokes

certain undesirable effects, in the urinary tract and as well as allergic reactions.Melarsoprol; discovered in 1949, it is used in second stage of both forms of infection. It

is derived from arsenic and has many undesirable side effects. The most dramatic is

reactive encephalopathy (encephalopathic syndrome) which can be fatal (3% to 10%). An

increase in resistance to the drug has been observed in several foci particularly in central

Africa. Eflornithine; is less toxic than melarsoprol and was registered in 1990. It is only

effective against treatment of second stage infection due to T. b. gambiense. A

combination treatment of nifurtimox and eflornithine has been recently introduced in

2009. It simplifies the use of eflornithine in monotherapy, but unfortunately it is not

effective for T.b. rhodesiense. Nifurtimox is registered for the treatment of American

trypanosomiasis but not for human African trypanosomiasis. Nevertheless, after safety

and efficacy data provided by clinical trials, its use in combination with eflornithine has

been accepted and included in the WHO List of Essential Medicine, and it is provided

free of charge for this purpose by WHO (WHO Fact sheet N259, 2010).

2.4 Pyroglutamyl peptidase 1 (PGP 1)

PGP 1 is a putative protein and belongs to a group of peptidase called Cysteine

peptidases. Cysteine peptidases have characteristic molecular topologies in 3 dimensions

and 2 dimensions (MEROPS database). They possess cysteine nucleophile and catalytic

residues in the order Glutamate, Cysteine, Histidine in sequence. Cysteine peptidases are


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divided into clans and further into families (Barret and Rawlings 2001). PGP 1 belongs to

Clan CF and Family C15.

2.4.1 Clan CF of Pyroglutamyl peptidase 1

The clan contains a single family, C15. The structure of PGP 1 qualifies it to clan CF

because its protein fold is unlike that of any other cysteine peptidase. The tertiary

structure for Pyroglutamyl peptidase 1 has been determined (Odagak et al., 1999) and

shows an alpha/beta protein with an alpha/beta/alpha sandwich. PGP 1 enzyme is

intracellular and soluble and in mammals, PGP 1 has been shown to release pGlu from

thyrotropin-releasing hormone, luteinising hormone releasing hormone, neurotensin,

bombesin and leukopyrokinin (Dando et al., 2003), but the physiological significance of

this is unclear.

2.4.2 Family C15 of Pyroglutamyl peptidase 1

Peptidase family C15 contains omega peptidases that release an N-terminal

pyroglutamate residue. There is a catalytic triad which occurs in the order Glu, Cys, His

in the sequence. The only known activity of family C15 is removal of a pyroglutamate

(pGlu) residue from the N-terminus of a peptide and typical synthetic substrates include;

pyroglutamate (pGlu), 7-(4-methyl-) coumarylamide (NHMec) and pyroglutamate 4-

naphthylamine (pGluNHNap). The protein fold presented by PGP 1 is unlike that of any

other cysteine peptidase and thus PGP 1 has a type structure for clan CF.

2.4.3 Trypanosoma bruceiPGP 1

In Trypanosoma brucei, PGP 1 is present as a soluble cystosolic cysteine peptidase. It is

located in chromosome Tb927_04_v4; 707328 - 708134 and is encoded by a single gene

copy of 669 encoding 222 amino acids and protein of 25.1 Kda with a predicted charge of

-4 and an isoelecric point of 5.4. Trypanosoma PGP 1 has no signal transmembrane

domain or GPI anchor (GenDB) and is liberated into the host blood stream during

intravascular destruction of trypanosomes in the host during an infection and therefore it

has been postulated to take part in the pathogenesis of HAT (Morty et al., 2006). It is


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expressed in all life cycle stages of Trypanosoma brucei and four other blood stream

African trypanosomes (Morty et al., 2006). Trypanosoma PGP 1 is optically active and

stable at bloodstream pH and it is insensitive to host plasma cysteine peptidase inhibitors

such as cystatin C, kininogen and alpha-macroglobulin (Morty et al., 2006) and this

makes it a possible factor in the pathogenesis of HAT. During infection, Trypanosoma

PGP 1 is liberated into the blood stream and causes degradation of the peptides; TRH and

GnRH (Morty et al., 2006) by removing the N-terminal Pyroglutamyl residue of these

peptides which protects the peptides from proteolysis (Odakagi et al., 1999) and this

exposes the peptides to proteolysis.

2.4.4 Sequence ofTrypanosomaPGP 1

Source: http://www.genedb.org/featureSeq/Tb927.4.2670

2.4.4.1 Gene sequence

ATGAAGCCTA CAAAACCACT ACTTTACATA ACGGGATACG GACCCTTCTT GGAAGTAACG

GAGAACCCCA

GCGCCACCAT TGCGCAAAGT GTAGCGGAAC AGGTGAGACA AAGTGGCGAA

GCGGATGTCC ATCATGAAAC ACTAGACGTG AACTTAGAGG CCGTTTCCAA ATATTTCAAC

CGCCTCAATG AATCCGTCAC CGCTCATCTG GAAGCCACAC ATCCCGAGAA TCGAGTACTT

CTCGTCAACG TGGGCCTTCA CAGTCGCGAA AAGGAAAAGG TACTGCGGCT GGAAGTGCGC

GCCTTCAATG AACTGGAGGG AAACCCCATC GATGATGAGC TTCCCTTGAG TACATGCAAA

GACAGTGCTT TCGTGAAGGG ATGCAAGCTC GAAACAACAA CAGCCCTCAT AGAGGAACTC

AATGCGATTG AGAGAAATGG TAGCGATCAT CACGAAAAGC CTCGTTGGAT TATTTCTTAC

GACGCGGGGC GATATTACTG CAACTATGCA CTGTACAGAG GCGTGAAGAT GCAGGAAGCT

CTAAACAGCC GCGTGTTTGC CGTGTTTTTG CACATTGTAG CATCCACTGT CGTGTGCATG

GAAGAGCAGG TTGCGCAGGT CCGCATGCTT GTGTCGCACC TCTTGAAACA CATGGAAGCA

GTTGAATGA

2.4.4.2 Amino acid sequence

MKPTKPLLYI TGYGPFLEVT ENPSATIAQS VAEQVRQSGE ADVHHETLDV NLEAVSKY

FN

RLNESVTAHL EATHPENRVL LVNVGLHSRE KEKVLRLEVR AFNELEGNPI DDELPLST

CK


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DSAFVKGCKL ETTTALIEEL NAIERNGSDH HEKPRWIISY DAGRYYCNYA LYRGVKM

QEA

LNSRVFAVFL HIVASTVVCM EEQVAQVRML VSHLLKHMEA VE

2.5 Immunogenicity

Immunogenicity refers to the characteristic that endows a protein with the ability to

provoke an immune response (Singh, 2011), this should not be confused with

Antigenicity which is the ability of a protein to combine specifically with the final

products of the immune response (i.e. secreted antibodies and/or surface receptors on T-

cells) (Kuby Immunology, 2006). Several immunogenicity studies have been conducted

on several proteins both therapeutic and diagnostic. A number of factors affect the

immunogenicity of a protein and these include the following;

2.5.1The Nature of the Protein

Under this a number of factors are examined; Degree of foreignness, Molecular size,

Chemical structure and heterogeneity (structural properties) and Ability to be processed

and presented by an APC

2.5.1.1 Degree of foreignness

Protein capacity to induce the synthesis of specific antibodies is shown to be correlated

with protein evolution rate (Ogievetskaya, 1977), the greater the phylogenetic distance

between the two organisms, the higher the immunogenicity

2.5.1.2 Molecular size

Proteins with high molecular weights are strong immunogens i.e. give higher levels ofimmunogenicity than low molecular weight proteins (Dintzis et al., 1976).Most

immunogens are large, complex molecules with a molecular weight generally greater than

about 100,000 daltons. In general large molecules are better immunogens as compared to

smaller molecules.
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2.5.1.3 Structure of the protein

A number of structural properties affect the immunogenicity of the protein ranging from

sequence variation, glycosylation, complexity in structure, and domain sites.

Glycosylation is believed to interfere with antibody binding and to have an impact on

auto immunity (von Delwig et al., 2006). The structure of a protein ranging from the

amino acid sequence to the tertiary structure and quaternary structure to the presence

domain sites and glycosylation all affect the immunogenicity of a protein.

2.5.1.4 Ability to be processed

Downstream processing of a product can also influence its immunogenicity. Impurities

and contaminants associated with antibody development have also been found in studies

on insulin and growth hormone products (Reeves W. G., 1986).

2.5.2 Route of administration

The route of administration can influence the immunogenicity of the protein

(Schellekens, 2005); however there have been no published cases whereby a change in

administration route completely negated immunogenicity (Schellekens H., 2003). There

are different routes of administration of a protein to an organism and these include,

subcutaneous, intramuscular, intravenous and topical among others. The route of

administration cannot render a protein immunogenic, although it can enhance the

likelihood of an immune reaction to a protein that is already immunogenic.

2.5.3 Genetic makeup of the organism

The genetic background of an organism can sometimes influence immunogenicity. A

well established example is with hemophilia, whereby the genetic defect determines

whether an individual will or will not produce antibodies (Fakharzadeh et al., 2000). In

some studies, there have been conflicting results from studies into the influence of the

major histo-compatibility complex (MHC) on responses to products such as growth

hormone and insulin indicating that MHC has no real effect.


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2.5.4 Adjuvant

Adjutants increase likelihood of immunogenicity by stimulating innate immune response,

boosting the humoral immune responses to enhance antibody responses (O'Hagan et al.,

2004). Many commonly used adjuvants are effective at elevating serum antibody titers,

but do not elicit significant Th1 responses or cytotoxic T lymphocytes (CTLs) (Pashine et

al., 2005).

2.5.5 Dose of protein given

Proteins have been shown to induce immune responses, in particular when administered

as booster doses over prolonged periods (Porteret al., 2001; Ryff et al., 2002). In some

cases, increasing the dose can help increase the efficacy and immunogenicity of a given

protein.

2.5.6 Formulation and purity of the protein

Appropriate formulation of a protein product is highly important. Stabilisation of a

protein is important since inadequacy in this may result into protein to aggregate or

denature, which may affect immunogenic potential (Cleland et al., 1993). Formulation

becomes even more crucial for products that may not be optimally stored or handled

(EMEA data base). Purity of a protein is very important since contaminating agents like

bacterial proteins can relatively increase the immunogenicity of a given protein (Gooding

et al., 1985) hence giving a false positive result, therefore the protein should be as pure as

possible.

2.6 Overview of the techniques to be used

A few of the methods to be used during the study is mentioned below;

2.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE is a technique widely used in Molecular Biology to separate proteins

according to their molecular weight. In this technique, SDS is a strong anionic detergent
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Molecular_biology


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when mixed with proteins; the proteins acquire a negative charge. In combination with

other factors like -Mercaptoethanol and heat, complete denaturation of proteins with

SDS can be achieved forming a SDS-polypeptide complex. When loaded onto a

Polyacrylamide gel matrix which acts as the support medium for electrophoresis then an

electric field applied, the polypeptide complexes migrate to the positive electrode of the

electrophoretic tank and in the process, the Polyacrylamide provides the molecular

sieving effect that separates the proteins basing on their molecular size/ weight. The

proteins on the gels are the visualised by staining and the some of the protein can be

estimated by comparing the running distance against the standard protein of known

molecular weight (Sambrooket al., 1989).

2.6.2 Western Blotting

Western blotting is an analytical technique used to detect specific proteins. The protein

(s) are first separated by SDS-PAGE then transferred to membrane (usually nitrocellulose

orPVDF) using an electric current; the gel that contains the protein is put on the negative

terminal while the membrane is put on the positive terminal, the proteins are then

transferred form the negative to the positive and in the process they are deposited onto

the membrane. To visualise the proteins, probing is done using antibodies specific to the

target protein (primary antibody) (Towbin et al., 1979; Renart et al., 1979). The primary

antibody is then bound to a secondary antibody (anti-immunoglobulin) that is conjugated

to an enzyme (peroxidase). When a substrate specific to the enzyme is bound on the

secondary antibody, a signal inform of a band on the membrane is detected.

2.6.3 Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay is a biochemical technique used to detect the

presence of an antibody or an antigen in a sample. In simple terms, ELISA is technique

that uses the antigen-antibody reaction. In this technique, an antigen is attached to the

surface of the ELISA plate, the antibody specific to the antigen is then bound to the

antigen. Visualisation is achieved by using an enzyme conjugated antibody, the antibody
http://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Antigenhttp://en.wikipedia.org/wiki/Antigenhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Analytical_technique


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binds to the antigen and the enzyme binds to the substrate; the end result is change in

color that can be quantified by measuring Optical Density (O.D).

2.7 pET-28a (+) vector

The pET-28a (+) vector carries an N-terminal His-Tag configuration plus an optional

C-terminal His-Tag sequence. Unique sites are shown on the vector map below. Note that

the sequence is numbered by the pBR322 convention, so the T7 expression region is

reversed on the circular vector map. The vector has a cloning/expression region of the

coding strand transcribed by T7 RNA polymerase. The f1 origin is oriented so that

infection with helper phage will produce virions containing single-stranded DNA that

corresponds to the coding strand. Therefore, single stranded sequencing is performed

using the T7 terminator primer (Novagen).

Figure 2: Vector map of pET28a (+)


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CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1.0 Study design

This was an experimental study. The study involved use of 25 male Swiss albino mice 7-

8 weeks old, obtained from by the Molecular Biology Laboratory, Department of

Parasitology and Microbiology, College of Veterinary Medicine, Animal resources

development and Biosecurity. These mice were divided into four groups; Test group,

Adjuvant group, Negative control group and the bacterial protein group, each group

containing 5 mice.

During the study, the Trypanosoma PGP 1 as recombinant protein was obtained from

previously transformed BL21DE E. coli cells through expression. The protein was then

extracted from theE. coli cells, purified, quantified and immunised in mice. The results

pertaining to antibody production in mice were to be got after analysis of sera collected

from the mice.

3.2.0 Materials:

3.2.1 Transformed BL21DE3 cells

BL21DE3 cells containing pET28a (+) carrying the gene for PGP 1 were provided as

glycerol stocks by Denis Anywar.

3.3.0 Methods:


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3.3.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells

provided

3.3.1.1 Growth of glycerol stocks containing pET28a (+)

Using a sterile loop, the glycerol stock was streaked on LB agar containing 30g/ ml of

kanamycin under sterile conditions and incubated at 37o

C overnight. A plate without

kanamycin and cells and plate with kanamycin but not streaked were also incubated to

determine the level of sterility of the incubated colony. A colony was picked under sterile

conditions from the plate with kanamycin since it was the only one that had cells and the

other plates had no cells meaning that the LB agar was sterile as well as kanamycin. The

colony was cultured in 10ml of sterile terrific broth containing 30g/ ml of kanamycin, at

37oC overnight at 150 rpm shaking.

3.3.1.2 Plasmid extraction

This was done to confirm if the glycerol stocks still contained the plasmid carrying the

insert. From the glycerol stocks, 1.5 ml of the cells from the media were pipetted and

centrifuged in microcentrifuge tubes to obtain a pellet. The plasmid was extracted from

the tubes using the QIAGEN extraction kit (see Appendix II). Ten micro-liters aliquot

from the plasmid extract was analyzed in a 1% Agarose Gel at 100V for 30 minutes.

3.3.1.3 Agarose Gel Electrophoresis

One percent agarose gel was prepared by dissolving 0.3g of agarose powder in 30 ml of

TAE buffer containing 0.005% ethidium bromide. The mixture was warmed in a

microwave until the agarose dissolved. The solution was poured into a casted plate with

comb and left to polymerise. The sample was prepared by mixing 10 l of sample with 1

l of the DNA sample loading dye; ten micro-liters of the prepared sample was loaded

into the polymerised agarose well. The electrode terminals were connected to the

electrophoretic tank and the gel ran at constant voltage of 100V for 30 minutes. The gel

after running was visualised under UV-light illumination.


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3.3.1.4. Restriction enzyme digestion

The plasmid extracted was digested using BamHI and HindIII enzymes in a reaction

mixture containing; 0.5 l of BSA, 4.5 l of PCR water, 3.0 l of 10X buffer, 10.0 l of

extracted plasmid DNA template, 1.0 l of Bam HI and 1.0 l of Hind III. The mix was

incubated for 2 hours and 30 minutes at 37oC in a water bath. Ten micro-liters aliquot

from the digest was analyzed in a 1% Agarose Gel at constant voltage of 100V for 30

minutes.

3.3.2 Expression of the recombinant TrypanosomaPGP 1

3.3.2.1 Small scale expression

A small scale expression in 100 ml terrific broth was done to confirm whether the protein

can be expressed and also to standardize the expression protocol. Glycerol stocks were

grown as in 3.4.1. The cells were transferred under sterile conditions to 100 ml of terrific

broth containing 30 g/ml of kanamycin and grown to an OD > 0.6 at 37oC at 200 rpm

shaking. An aliquot from the pre-induced expression was taken centrifuged mixed with

protein sample loading buffer and stored for analysis. The expression was then induced

with 1mM IPTG for 2 hours and 30 minutes; an aliquot from the expression was taken,

centrifuged and mixed with protein sample loading buffer. From the pre-induced and

induced prepared samples, 10 l was picked and analyzed together in a 15% SDS-PAGE

gel at 200V for 1 hour. Western Blot analysis using anti-His antibody was also done to

confirm the expressed protein.

3.3.2.1.1 Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

A 15% SDS-PAGE gel was prepared; the casting plates were prepared before preparing

the gel. During the preparation of the gel, 15% Separating gel was prepared by adding the

following volumes in a 15 ml tube; 2.5 ml of (30%) monomer, 1.1 ml of distilled water,

1.3 ml of separating buffer, 60 l of (10%) SDS, 30 l of APS and 10 l of TEMED total

volume 5 ml (the last two reagents were added last). The mix was immediately cast into

the plates, leveled with distilled water and left to polymerise for 30 minutes. The distilled


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water used to level was drained off after polymerisation. The 4% stacking gel was then

prepared by adding the following volumes in a 15 ml tube; 680 l of (30%) monomer,

3.0 ml of distilled water, 1.2 ml of stacking buffer, 100 l of (10%) SDS, 60 l of APS

and 5 l of TEMED in a total volume of 5 ml (the last two reagents were added last). The

mix was immediately cast into the plated and the combs inserted immediately and left to

polymerise for 30 minutes. The protein samples were prepared by mixing with sample

loading buffer (see Appendix III) in a ratio of sample loading buffer to sample of 1:4.

The combs were removed, the plates loaded into the electrophoretic tank and running

buffer (see Appendix III) poured into the tank. Ten micro-liters of the prepared samples

were loaded into the wells in two parts. The electrodes were connected to the tank the gel

ran at constant volume of 200V for 1 hour. One part of the gel was stained with

Coomasie brilliant blue, the other part of the gel was transferred to the nitrocellulose

membrane by western blotting.

3.3.2.1.2 Western Blotting

After SDS-PAGE, the gel and the membrane were equilibrated in transfer buffer (see

Appendix III). The gel and the membrane were fixed on the western blot cassette with the

gel on the negative (black) and the membrane on the positive (white). The cassette

containing the gel and membrane and a dummy cassette were then fixed into the transfer

tank; the transfer buffer was then poured into the tank. An ice pack was inserted into the

tank; the electrodes were then connected to the tank. The transfer was done at constant

voltage of 100V for 1 hour. After transfer, the membrane was blocked in 5% skimmed

milk overnight. The membrane was washed three times in PBS-T buffer with shaking for

15 minutes per wash. The wash was poured off and primary antibody added in a dilution

of 1:2000 in PBS-T and incubated for 1 hour with shaking at room temperature. The

membrane was washed three times using PBS-T buffer with shaking for 10 minutes per

wash. The wash was poured off and secondary antibody added in a dilution of 1:10,000

in PBS-T and incubated for 1 hour with shaking at room temperature. The membrane was

then washed three times using PBS-T buffer with shaking for 10 minutes per wash and

the wash poured off. A solution of Diaminobenzidine (DAB) in PBS was then added and

100 l of hydrogen peroxide added to visualize the bands.


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3.3.2.2 Large scale expression

The small scale expression confirmed that the protein could be expressed; however, the

purification from of the protein form the small scale expression gave very low amount

and hence the need for a large scale expression in 1000 ml. Glycerol stocks were grown

as in 3.4.1; the cells were then transferred under sterile conditions to 100 ml terrific broth

containing 30 g/ml of kanamycin and grown at 37oC overnight at 150 rpm shaking. The

cells from the 100 ml were transferred under sterile conditions to 1000 ml of terrific broth

containing same concentration of kanamycin and grown to an OD>0.6 at 37oC at 200 rpm

shaking. An aliquot from the pre-induced sample was taken and treated as in small scale

expression. The expression was then induced using 1 mM of IPTG for 2 hours and 30

minutes. An aliquot from the induced sample was treated as in small scale expression.From the pre-induced and induced prepared samples, 10 l was picked and analyzed

together in a 15% SDS-PAGE gel at constant voltage of 200V for 1 hour.

3.3.3 Extraction, Purification and Quantification TrypanosomaPGP 1 and bacterial

protein

3.3.3.1 Extraction

3.3.3.1.1 Extraction ofTrypanosomaPGP 1

The protein was extracted using cell lysis protocol described in Sambrook et al, 1989.

The cells after the large scale expression were centrifuged at 10,000 rpm for 10 minute

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