Title Identification of a New Metacyclic/Blood Stage

Title Identification of a New Metacyclic/Blood Stage Specific Proteinand its Application for the Control of Nagana( 本文(Fulltext) )

Author(s) MOCHABO, Kennedy Miyoro

Report No.(DoctoralDegree) 博士(獣医学) 甲第404号

Issue Date 2014-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/49027

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

I

Identification of a New Metacyclic/Blood Stage Specific Protein and its Application for

the Control of Nagana

( /)

2013

The United Graduate School of Veterinary Sciences, Gifu University

(Obihiro University of Agriculture and Veterinary Medicine)

MOCHABO, Kennedy Miyoro

II

Table of contents

Table of contents .............................................................................................................. II

Abbreviations ................................................................................................................. VI

Chapter 1 .......................................................................................................................... 1

General introduction ......................................................................................................... 1

1.1 Life cycle and general biology of trypanosomes ............................................... 1

1.2 Economic importance ........................................................................................ 4

1.3 Diagnosis ........................................................................................................... 5

1.3.1 Direct examination techniques ................................................................... 6

1.3.2 Indirect examination techniques ................................................................. 8

1.4 Treatment and prevention ................................................................................ 10

1.5 Vaccine development against African trypanosomosis ................................... 12

1.6 Aims of the present study ................................................................................ 14

Chapter 1 ........................................................................................................................ 16

Expression, immunolocalization and serodiagnostic value of Tc38630 protein from

Trypanosoma congolense ............................................................................................... 16

1.1 Introduction ...................................................................................................... 16

1.2 Materials and methods ..................................................................................... 18

1.2.1 Parasites .................................................................................................... 18

III

1.2.2 DNA extraction ........................................................................................ 18

1.2.3 DNA amplification and gel extraction...................................................... 19

1.2.4 Cloning and sequencing ........................................................................... 19

1.2.5 Recombinant protein expression .............................................................. 19

1.2.6 Polyclonal antibody production ................................................................ 20

1.2.7 Southern blot analysis ............................................................................... 20

1.2.8 Western blot analysis ................................................................................ 21

1.2.9 Indirect fluorescent antibody test (IFAT) ................................................. 21

1.2.11 ELISA ....................................................................................................... 22

1.2.12 Data management and analysis ................................................................ 23

1.3 Results and discussion ..................................................................................... 24

1.4 Summary .......................................................................................................... 26

Chapter 2 ........................................................................................................................ 33

Establishment and evaluation of the potential use of recombinant Tc38630 protein of

Trypanosoma congolense in enzyme-linked immunosorbent assay and

immunochromatographic test ......................................................................................... 33

2.1 Introduction ...................................................................................................... 33


2.2.1 Parasites and animals ................................................................................ 36

2.2.2 Recombinant protein production .............................................................. 36

2.2.3 Trypanosome lysate antigens.................................................................... 36

IV

2.2.4 Immunization and production of anti-rTc38630 sera ............................... 37

2.2.5 ELISA ....................................................................................................... 37

2.2.6 Preparation of gold colloid-conjugated antigens and ICT strip ................ 37

2.2.7 Sera ........................................................................................................... 39



2.4 Summary .......................................................................................................... 43

Chapter 3 ........................................................................................................................ 49

Evaluation of the recombinant Tc38630 protein as a potential vaccine for nagana ....... 49

3.1 Introduction ...................................................................................................... 49


3.2.1 Parasites and animals ................................................................................ 51

3.2.2 Construction and expression of recombinant 38630 ................................ 51

3.2.3 Homogenate preparation........................................................................... 51

3.2.4 Immunization of mice ............................................................................... 52

3.2.5 Trypanosome challenge and parasitaemia monitoring ............................. 53



3.4 Summary .......................................................................................................... 57

General discussion .......................................................................................................... 63

Conclusion ...................................................................................................................... 65

V

Acknowledgements ........................................................................................................ 66

References ...................................................................................................................... 68

VI

Abbreviations

A AAT: animal African trypanosomosis

B BCA: bicinchoninic acid assay

BCT: buffy coat technique

bp: base pair

BSF: bloodstream form

Bst: Bacillus stearothermophilus.

C CATT: card agglutination test for trypanosomosis

CFT: compliment fixation test

D DAB: Diaminobenzidine

DDW: double distilled water

DEAE: diethyl amino-ethyl

DNA: deoxyribonucleic acid

dNTP: deoxynucleotide triphosphate

DPI: days post infection

E EDTA: ethylenediaminetetraacetic acid

ELISA: enzyme-linked immunosorbent assay

VII

F FAO: Food and Agriculture Organization of the United Nations

G GST: Glutathione S-transferase

H HAT: human African trypanosomosis

HCl: hydrochloric acid

HCT: haematocrit centrifugation technique

HMI-9: Hirumi’s modified Iscoves’s medium 9

I ICT: immunochromatographic test

i.p.: intraperitoneal

IFAT: indirect fluorescent antibody test

Ig: immunoglobulin

ILRI: International Livestock Research Institute

ISG: invariant surface glycoprotein

K kbp: Kilo base pair

kDNA: kinetoplast DNA

KIVI: kit for in vitro isolation

L LAMP: loop mediated isothermal amplification

LAT: latex agglutination test

M m-AECT: miniature anion-exchange column technique

VIII

MCF: metacyclic form

MEXT: Ministry of Education, Culture, Sports, Science and Technology

N NPV: negative predictive value

NRCPD: National Research Center for Protozoan Diseases

O OIE: Office International Des Epizooties

P PAGE: polyacrylamide gel electrophoresis

PBS: phosphate buffered saline

PCF: procyclic form

PCI: phenol-chloroform-isoamyl alcohol

PCR: polymerase chain reaction

PCV: packed red cell volume

PPV: positive predictive value

R RIA: radioimmunoassays

RNA: ribonucleic acid

S SC: subcutaneous

SDS: sodium dodecyl sulfate

T Taq: Thermus aquaticus

T. b. brucei: Trypanosoma brucei brucei

IX

T. b. gambiense: Trypanosoma brucei gambiense

T. b. rhodesiense: Trypanosoma brucei rhodesiense

T. congolense: Trypanosoma congolense

T. evansi: Trypanosoma evansi

T. theileri: Trypanosoma theileri

T. vivax: Trypanosoma vivax

TBV: transmission blocking vaccine

TMB: tetramethylbenzidine

TVM: Trypanosoma vivax medium

V VSG: variant surface glycoprotein

W WHO: World Health Organization

Unit abbreviations

D оC: degree celcius

E EU: endotoxin unit

H h: hour

K kDa: kilo Dalton

M μg: microgram

mg: milligram

X

min: minute

ml: milliliter

mM: milliMole

V v/v: volume/volume

W w/v: water/volume

μl: microliter

1

Chapter 1

General introduction

1.1 Life cycle and general biology of trypanosomes

Parasitic protozoa affect plants, invertebrates and most vertebrates including

humans (41). Trypanosomes are some of the protozoan parasites that have been studied

for a long time and cause disease in both humans and livestock commonly known as

sleeping sickness and nagana, respectively (36, 149). Efforts to control the disease they

cause date as far back as 1800s but the diagnosis of human pathogenic from the non-

pathogenic ones was in 1950s and 1960s. However, the cyclical transmission was

implied as early as 1909 in tsetse flies.

Animal African trypanosomosis or nagana disease is caused by T. congolense, T.

vivax, T. brucei spp and T. simiae. The four species are members of salivarian group

transmitted via the mouth parts of tsetse (129). Nagana comes from a Zulu word of the

disease “N’gana” which means “useless” or “to be depressed” and all domestic animals

can be infected with signs of fever, listlessness, emaciation, hair loss, lacrimation,

oedema, anaemia and paralysis. Animal African trypanosomosis (AAT), caused majorly

by T. congolense is widespread in the whole of sub-Saharan Africa and cause a

considerable loss to livestock production thus compromising food security (71, 170).

Trypanosomes are single-celled organisms, like all parasitic protozoa, they

display extreme adaptation to their environment that constitutively exhibit complex life

cycles (95). They undergo both biochemical and morphological changes during their life

2

cycle (76, 96). Following a bite from tsetse fly (Glossina spp), non-dividing forms of

metacyclic trypomastigote (MCFs) are injected into a mammalian host (95, 168). The

trypanosomes multiply locally at the site of the bite for a few days before entering the

lymphatic and vascular systems. They differentiate into dividing bloodstream forms

(BSFs) that survive free in the bloodstream and evade immune responses through

antigenic variation. These forms are able to be taken up tsetse in the next feeding where

the BSFs differentiate into procyclic forms (PCFs). The dividing PCFs first establish in

the tsetse midgut before a few of them migrate to proboscis (T. congolense) or salivary

gland (T. brucie spp) to mature as differentiated dividing epimastigote forms (EMFs).

The EMFs continuously divide while adhering to the epithelial cells throughout the life

span of tsetse. They undergo a process called metacyclogenesis (51, 164) to form MCFs

ready to be propagated back to the mammalian host to complete the life cycle. A

complete cycle takes 5-13 days (44, 55).

African trypanosomes have two genomes, in the nucleus and in the

mitochondrion (kinetoplast) (100). Trypanosomes are eukaryotic cells with typical

cytoplasmic structures and organelles (Fig.1). They possess a nucleus, mitochondrion,

Golgi apparatus, endoplasmic reticulum and lysosomes. In addition, they have other

organelles like the kinetoplast, glycosomes and flagellar pocket (95).

The mitochondrion spreads along the entire body as a single organelle that

contains the kinetoplast (100, 167). It is indeed only active in the PCFs where there is

less glucose in the midgut of tsetse. These organelles are positioned within the

cytoskeletal corset between the posterior end and the centre of the cell. The most

posterior structure is the flagellar pocket and this is the only point where endocytosis

and exocytosis processes occur (120), thus protecting themselves from adverse host

3

environment. It is at this pocket where VSGs are recycled (35, 174). The motility of

trypanosome is dependent on the single flagellum (11).

In the morphological changes, there is repositioning of the kinetoplast relative to

the posterior end and the nucleus that is at the centre. However, these changes and

mechanisms in the life cycle are not clearly understood but is speculated that they assist

in motility of BSFs and PCFs and for the attachment of EMFs (95). The kinetoplast is

composed of circular DNA referred to as maxicircles and minicircles (95, 146). The

maxicircles contain genes that encode mitochondrial proteins while the minicircles

encode short guide RNAs.

Various organisms, including bacteria, protozoa, fungi and humans have been

found to have glycosylphosphatidylinositol (GPI)-anchored membranes (42). The GPI-

anchors consist of carbohydrates, lipids, phosphates and amines. They have been well

documented due to VSGs that are abundant in African trypanosomes whereby they are

responsible for antigenic variation (32, 123, 165). The GPI anchor in general (61, 108),

serves at least three possible functions: 1) to enhance mobility of protein in the plasma

membrane which is important in facilitating rapid responses to appropriate stimuli; 2) to

connect with signal transduction pathways; 3) important in targeting certain proteins to

apical domains of plasma membrane of some epithelial cells.

It has been found out that at each stage, the African trypanosomes

predominantly express stage-specific surface molecules. In both Trypanozoon and

Nannomonas groups, the BSFs and MCFs express variant surface glycoprotein which

has been extensively studied (10, 140, 162). The T. brucei PCFs express EP procyclin

and GPEET procyclin that are glycoproteins resistant to tsetse proteases (1) while T.

4

congolense PCFs, express glutamic acid/alanine-rich protein (GARP) and a congolense

procyclin (22, 160). In addition, EMFs of T. congolense express GARP and congolense-

epimastigote specific protein (CESP) while brucei alanine-rich protein (BARP) is

surface coat for T. brucei (142, 159). All these coats are GPI-anchored glycoproteins.

There are various ways that the parasites escape from immune defences of the

host (145). These include antigenic masking, blocking, intracellular location,

immunosuppression and antigenic variation. The latter is especially important for

African trypanosomes and is well documented. Other parasites with similar

phenomenon, although less characterized are Plasmodium, Babesia and Giardia.

1.2 Economic importance

With a burgeoning of human population in developing world, there is need to

ensure food security which implies that crops and livestock production must be

intensified (98). Livestock plays an important role in the agricultural expansion and

intensification through provision of traction, manure, nutrient recycling and acting as a

means of enhancing wealth and distributing income. The supply and value of animal

products and contribution of livestock to crop production is severely compromised in

tsetse-infested areas of Africa through the effects of especially bovine trypanosomosis.

It has been known that trypanosomosis reduces cattle density by up to 70% and the sale

of meat and milk by 50%. Calving rates and calf mortality are both reduced by 20%

(106). From the domestic livestock perspective, trypanosomosis has been described as

the ‘scourge of Africa’ whereby livestock consume resources without being productive

as compared to other diseases that kill quickly thereby fodder and water are not wasted

(105).

5

Trypanosomosis has been linked to both direct impacts (mortality, fertility, milk

yield, manure and draught animals) while indirect impacts include loss of potential for

production (i.e., the production that could be achieved if trypanosomosis did not occur)

(39, 124, 147, 152). Estimated loss to African agriculture is of US$4.5 billion per year

by nagana with over 50 million of cattle under risk. Tsetse flies inhabit about 10 million

km2 of sub-Saharan Africa (45, 71, 106, 119, 129, 161) covering about 40 countries thus

making nagana as the most important livestock disease in the continent. If there was

absence of the disease, three or four times more livestock would be kept. Adding to the

risk to human infections, it leads to disruption of socio-economic and agricultural

development in rural areas. Generally, there are three control strategies employed for

trypanosomosis so far that include tsetse control, chemotherapy and chemoprophylaxis,

and trypanotolerant animals (99). The fourth, a vaccine which is yet to be developed,

would have the greatest impact. The choice of the breeds as well as herd size and

migration patterns of pastoralists, to rear livestock in tsetse infested areas is influenced

on economic impacts of trypanosomosis (147).

For either curative or prophylaxis, it was estimated that trypanocide use in cattle

is 1.9 to 2 doses per head per annum in tsetse infested areas in Africa (73). For farmer’s

willingness to participate in control of tsetse and trypanosomosis, the issues of public

versus private goods would have to be considered.

1.3 Diagnosis

In the epidemiology and control of any disease, precise diagnosis and definitive

identification of the causative agent is extremely important. The diagnosis of

trypanosomosis is basically divided into direct and indirect detection techniques of the

6

parasite. More often, improved diagnostic tests are needed to support treatment and for

research purposes, especially in epidemiological surveys (9, 34). The clinical signs of

any infectious disease are always a manifestation of interaction of the host and the

microorganism (173) but for clinical diagnosis of nagana, generally, there are no

pathognomonic signs to be relied on (129). Therefore, the methods used to diagnose the

parasite need to be sensitive and specific.

1.3.1 Direct examination techniques

1.3.1.1 Blood films

This involves examination under light microscope of wet, thick or thin films of

fresh blood, usually obtained from the ear vein, jugular vein or the tail. So far, it is very

simple and gives immediate results (118). Stained thin and thick blood smears may be

used though blood films are less sensitive. Lymph and chancre fluid may also be

examined by above methods. Giemsa-stained blood smears for microscopic

examination were introduced in 1904 by Gustav Giemsa and have become the “gold

standard” diagnostic test for many protozoan parasites (43).

1.3.1.2 Concentration techniques

The microhaematocrit centrifugation technique (HCT), or the Woo method (179),

has been used and this can further be improved to buffy coat technique (BCT) by

cutting the capillary tube and expressing the buffy coat onto slide and examining under

the microscope (107). These two methods improve sensitivity (500 parasites per ml) and

at the same time the degree of anaemia can be assessed by reading packed red cell

volume (PCV).

7

1.3.1.3 Anion exchange column technique

The miniature anion-exchange column technique (m-AECT) has been used for

the diagnosis especially for human sleeping sickness (85). Blood is passed through a

diethyl amino-ethyl (DEAE)-cellulose column to separate trypanosomes from red blood

cells. However, it is not widely used in animals under field conditions as the technique is

very expensive and time consuming (118).

1.3.1.4 Sub-inoculation methods

These methods involve transmitting a suspect case infection to another

vertebrate host, to an invertebrate host or to an in vitro culture system (34, 118). In

animal sub-inoculation, rodent is commonly used and is more sensitive than the

concentration techniques and sometimes PCR as well, thus it is particularly useful in

revealing subpatent infections. The inoculation is expensive, results are not immediate

and some species of trypanosomes cannot grow in rodents. Use of invertebrate host also

referred as xenodiagnosis, is the feeding of clean susceptible vector on a suspect case.

After feeding, it is either dissected and examined for presence of infection or allowed to

feed on a clean animal which is itself examined for the transmitted infection. This

method is sensitive but requires the maintenance of clean colony of tsetse flies and is

time consuming. The first report on isolating trypanosomes using in vitro culture

method was on T. b. brucei and T. evansi from animals with low parasitaemias which

would not be detected via blood film or HCT (185). A kit for in vitro isolation of

trypanosomes (KIVI) was developed which is especially important for T. b. gambiense.

The kit has been applied in domestic animals though it is expensive and not practical for

routine use.

8

1.3.2 Indirect examination techniques

1.3.2.1 Serodiagnosis

Serological techniques have been employed in the diagnosis of African

trypanosome infections (118). However, the disadvantage of serology as a diagnostic

tool is that there is usually a lag between the onset of infection and the development of

antibodies to the infecting microorganism and often do not differentiate between the

current and past infections (173).

Antibody detection techniques include complement fixation test (CFT) that has

been used in the diagnosis of T. equiperdum in equines (50). Indirect fluorescent

antibody test (IFAT) has been used in herd diagnosis of trypanosomes (29). IFAT has

been shown to be sensitive and specific, although there is cross-reactivity between the

trypanosome species, in addition to it being expensive. Card agglutination test for

trypanosomosis (CATT), the simplest for T. evansi, has also been used (83, 110). When

antibodies are detected, however, they do not distinguish between current and past

infections, and also cross-reactions may occur between trypanosome species (84).

Enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA)

have also been used (50). ELISA was developed in early 1970s and now widely applied

in biomedical science (34). ELISA has particularly been used for epidemiological

surveys to detect trypanosome antibodies. However, in the detection techniques, involve

use of either whole parasite or crude parasite lysate as the antigen which are not often

standardized. It can be varied to use blood spotted filter papers thus obviating the use of

cold chain facilities (56).

9

Enzyme immunoassays have been developed for the detection of antigens rather

than antibodies in the diagnosis of diseases (110, 112). These assays detect the

circulating antigens of T. congolense, T. vivax and T. brucei in blood of infected

animals. Latex agglutination test (LAT) is such that has been used specifically for T.

evansi (111). The demonstration of trypanosome antigens is equivalent to

parasitological diagnosis and thus an indicator of active infection if an animal has not

been recently treated for the disease (112, 171).

The ELISA technique may give false negative results even in parasitologically

proven cases. This occurs in sera from acute or early phase of infection and has been

observed in T. congolense, T. vivax and T. brucei infections in cattle and goats (92, 112).

The monoclonal antibody used in antigen ELISA is directed at an internal or somatic

unsecreted antigen that is only released after trypanosome lysis. Thus, in early infection,

before the first parasitaemic peak, the test can give negative results due to absence or

low levels of antigens in blood (92, 112). It is, therefore, important to combine antigen

detection ELISA with the parasitological techniques for effective diagnosis of

trypanosomosis (92, 110).

1.3.2.2 Molecular techniques

Molecular detection techniques have been developed for diagnosis of infections

with African trypanosomes in humans, animals and tsetse flies (118). First practical

polymerase chain reaction (PCR) technique was performed in 1983 (28) and now

various primer sets are available that can amplify different trypanosome subgenus,

species and types (31, 94). In addition, species-specific probes are now available to

identify the known trypanosome species in both host and vector (89). Masake et al. (93)

10

reported that PCR can detect infection as early as 5 days following an infective tsetse

bite. Using the quantitative PCR rather than the conventional PCR confers an additional

advantage of identification as well as establishing the parasite burden (180).

Although these DNA techniques are extremely sensitive and better diagnostic

tools, their adoption in developing countries in Africa is still low and so far, they are

used in the confines of well-established laboratories (34, 118). Now that most genomes

are getting completed, the methods will be improved.

A relatively new molecular technique known as Loop mediated isothermal

amplification (LAMP) has been developed. In this technique, the target sequence is

amplified at a constant temperature of 60 - 65 °C using either two or three sets of

primers and a Bst DNA polymerase with high strand displacement activity in addition to

a replication activity (116, 155). LAMP is a simple (using water bath / heating block),

rapid (1h amplification), highly sensitive and specific in addition, cost effective

molecular technique. It is now increasingly being explored in the detection of various

infectious diseases such as viral (125) tuberculosis (46, 63) malaria, (127) and human

African trypanosomosis (114) and has the potential to replace conventional gene

amplification methods once it is validated (155, 156).

1.4 Treatment and prevention

There are a few drugs in the market that were discovered long time ago for

treatment and prevention of animal African trypanosomosis with some degree of

toxicity and approximately a million doses are administered annually in Africa (40).

The drugs can be grouped as curative, prophylactic or sanative (19, 137). The curative

drugs are suramin (since 1916/1921), diminazene aceturate (since 1944), melarsenoxide

11

cysteamine (since 1949), quinapyramine sulphate (since 1950s), homidium (since 1963),

and isometamidium chloride (since 1963). The prophylactic drugs are quinapyramine

sulphate and isometamidium chloride. The latter drug becomes prophylactic only if

used at a high dose (19).

A sanative drug is one that has not been in use for sometime but when used will

eliminate trypanosomes that are resistant to the drugs used previously. It should provide

moderate prophylaxis and avoid development of resistance to the prime drug, but this

has not been well implemented, leading to a multiple resistance to curative, prophylactic

and sanative drugs (40).

In the first stage management of sleeping sickness (69, 176), Pentamidine,

discovered in 1941, is used for T. b. gambiense sleeping sickness and has less adverse

effects, therefore, well tolerated by patients. While for T. b. rhodesiense, Suramin,

discovered in 1921, is used but has adverse effects on the urinary tract and allergic

reactions. In the second stage management of the disease in humans, Melarsoprol,

discovered in 1949, it is used in both forms of infection. It is derived from arsenic and

has many toxic effects whereby 3-10% patients die of complications of encephalopathic

syndrome. Another drug used, Eflornithine (since 1990), which is less toxic than

melarsoprol, but only effective against T. b. gambiense. A combination treatment of

nifurtimox and eflornithine has been introduced since 2009 but still not effective for T.

b. rhodesiense. Nifurtimox (since 1960s) is registered for the treatment of American

trypanosomiasis but not for human African trypanosomiasis. Pafuramidine (DB289) an

oral drug for second stage HAT, is still under evaluation (157).

12

The present strategy of chemotherapy and chemoprophylaxis is faced with the

following technical drawbacks: a limited number of drugs for use; the emergent drug

resistance; cross-resistance to the present drugs; and, toxicity of the drugs. However, the

research and development of more effective drugs which takes an average of 8-12 years,

has not been pursued as is not profitable for pharmaceutical companies (13).

Other trypanosomosis control methods include the use of trypanotolerant breeds

for livestock farming and vector control in endemic countries to complement chemo-

therapy/phylaxis in a three-approach strategy (4).

1.5 Vaccine development against African trypanosomosis

Vaccination comes from the Latin word vacca which means ‘cow’. This was

when it was noted that an infection with cowpox virus conferred protection from

smallpox virus (12, 104) and since it was first used by Jenner in 1796, a vaccine has

been defined as a biological preparation that improves immunity to a particular disease.

A search for a vaccine against trypanosomosis started quite early in an infection and

treatment strategy (76) which is currently applied for Theileria parva. However, this

was not successful for trypanosomosis and was attributed to antigenic variation

phenomenon in African trypanosomes. Researchers have changed strategy in the

development of vaccines targeting invariant molecules (101). Use of recombinant

protein has already been deployed (172). Understanding host-pathogen interactions

would be essential in vaccine development (163). Owing to limited control options for

AAT, ways to develop a vaccine will go a long way in enhancing agricultural expansion

(30). However, with the advent of research focused on the identification of invariant

components to be used as therapeutic targets, possible immunogens (136) and the

13

evolution of DNA vaccine technology, the possibility is beckoning in the near future

(23). Indeed, DNA vaccines have been termed as third generation of vaccines (2, 104)

compared to the first generation that used whole organism vaccines – either live and

attenuated, or killed forms. The second generation vaccines were developed to reduce

risks associated with the first ones. These vaccines consist of some protein antigens

(e.g., tetanus or diphtheria toxoid) or recombinant protein components (e.g., hepatitis B

surface antigen). Since description genomic organization (183) of a gene family for the

invariant surface glycoproteins (ISG) from T. b. brucei parasites, attempts have been

made use them as a DNA vaccine by Lança et al., (74) in experimental model. There are

three approaches in vaccination strategies; anti-parasite, transmission blocking (TBVs)

and anti-disease vaccine (87). The first one is the traditional one while the latter two are

relatively new. In TBVs, the aim is to disrupt the lifecycle of the parasite in the vector

however, it does not protect the vaccinated individuals but will result in reduced vectors

and transmission. The aim of anti-disease is to protect the host from disease associated

complications (6) which is borrowed from observations on trypanotolerant animals. The

strategy leads to better control of parasitaemia and anaemia thus, improved survival

rates. The common denominator for all these approaches has been lack of strong

stimulation of B cell memory, and if they do, trypanosomes actively get rid of it (87,

130). Future prospects for anti-trypanosome vaccination should aim to stimulate the

IgM memory response.

14

1.6 Aims of the present study

Therefore, given this general background, the overall objective of this study

design was to apply molecular biological tools for the diagnosis and control of nagana

with specific objectives: 1) To identify and characterize T. congolense metacyclic

(MCF) stage specific surface protein for application in the serodiagnosis (chapter 1); 2)

To establish and evaluate the potential use of recombinant Tc38630 protein from T.

congolense in enzyme-linked immunosorbent assay and immunochromatographic test

(chapter 2); and, 3) To evaluate the rTc38630 protein as a potential vaccine candidate in

the anti-disease strategy (chapter 3).

15

Figure 1: Schematic drawing of a trypanosome (62).

16

Chapter 1

Expression, immunolocalization and serodiagnostic value of Tc38630

protein from Trypanosoma congolense

1.1 Introduction

Animal African trypanosomosis (AAT), caused majorly by T. congolense, is

widespread in the whole of sub-Saharan Africa and causes a considerable loss to

livestock production thus compromising on food security. Estimated loss to African

agriculture is US$1.6-5 billion per year by nagana with over 46 million of cattle and

millions of small ruminants under risk (45, 71, 119). In the absence of vaccine, there is

also a limitation in the treatment and diagnosis of the disease (88). So far, various

serological techniques have been employed in the diagnosis of AAT (118). ELISA has

particularly been used for epidemiological surveys to detect antibodies against

trypanosome antigens. However, the detection techniques use parasite cell lysate as the

antigen which are not often standardized. Molecular techniques with a detection level of

as low as 0.1 parasite/ml, especially LAMP have been established (114, 155), though

realistic field conditions require simpler approaches (88). Currently, there is a promising

use of recombinant antigens to improve on the available trypanosome cell lysate to

detect antibodies (48, 113). With the completion of sequencing of T. congolense whole

genome, in silico cloning of novel diagnostic antigens will be expected (64). For control

of AAT in Africa, there is need to acquire accurate epidemiological information on the

prevalence in different livestock species and to achieve this, sensitive and specific

diagnostic tests are required. From time immemorial, diagnosis of a disease usually is

based on the clinical signs and symptoms, by demonstration of the causative organism

17

or by reactions to diagnostic tests. For AAT, though often reliable, the sensitivity of

microscopy is limited due to low parasitaemias of infected animals (91, 126). Therefore,

there is an urgent need for simple, rapid diagnostic techniques to replace microscopy

and currently available serological tests that are of variable sensitivity and specificity.

Recently, ELISA based on recombinant invariant surface glycoprotein (ISG) 75 antigen

has been applied for diagnosis of T. evansi infection in camels (158). The aim of this

study was to clone ISG orthologue from T. congolense, and to establish an ELISA using

recombinant T. congolense ISG for the diagnosis of T. congolense infections.

18

1.2 Materials and methods

1.2.1 Parasites

T. congolense IL3000 savannah strain isolated near Kenya/Tanzania border was

used. PCF and EMF were propagated at 27 oC using Trypanosoma vivax medium

(TVM)-1 medium composed of Eagle’s minimum essential medium (EMEM, M4655,

Sigma–Aldrich, St. Louis, MO, USA) supplemented with 20% heat-inactivated foetal

bovine serum (FBS), 2 mM L-glutamine and 10 mM L-proline. PCFs were routinely

maintained by diluting 3ml of log-phase parasite suspension with 7ml of fresh medium

every 2 days while the plastic-adherent EMF cultures were maintained by replacing the

entire culture supernatant with fresh medium every 2 days. BSF was maintained at 33

oC using HMI-9 medium modified from Iscove's Dulbecco's MEM (IMDM, I-3390,

Sigma–Aldrich, St. Louis, MO, USA). The modification was done by supplementing

the medium with 0.05 mM bathocuproine sulphonate, 1.5 mM L-cysteine, 0.12 mM 2-

mercaptoethanol, 1 mM sodium pyruvate, supplemented with 20% heat-inactivated

foetal bovine serum (FBS) (53, 142). The BSFs were maintained daily by splashing and

replacing the entire supernatant with fresh medium at log-phase.

1.2.2 DNA extraction

Total genomic DNA was extracted from Trypanosoma congolense axenically

maintained in the laboratory. Phenol-chloroform-isoamyl alcohol (25:24:1) method was

used for DNA extraction (143). The parasite DNA in the aqueous phase was

precipitated with 2 volumes of cold 99.5% ethanol, and then centrifuged at 10,000 x g

for 10 minutes, dried, and dissolved in sterile double distilled water (DDW).

19

1.2.3 DNA amplification and gel extraction

Primers were designed using Genetyx software (GENETYX Co., Japan) from

identified genes that are relatively highly expressed in MCF of T. congolense (38). The

PCR reactions were conducted in a total volume of 50 μl that contained 5 μl 10× buffer,

1.5 μl 50 mM MgCl2, 4 μl 2.5 mM dNTPs, 0.5 μl (5 units) Taq DNA Polymerase

(Invitrogen, USA), 5 μl primer mix (10 pmol/μl each, F: 5’-ATG CCG CGC CTG ATG

ACA CA-3’; R: 5’-GCC GTC AGG GTT GTA CGG AT-3’), 29 μl DDW, and 5 μl

DNA sample. The reaction mixture was incubated in a thermal cycler (VERITI™

Thermal Cycler, Applied Biosystems, Foster City, CA, USA) under conditions as

follows: 94 oC for 10 min (denaturation step) and subjected to 40 cycles at 94 oC for 45

sec, 1 min at 55 oC, and 1 min at 72 oC with a final extension at 72 oC for 7 min. The

PCR products were electrophoresed in 1% agarose gel, stained with ethidium bromide

and visualised under UV light. The target band was cut from the gel and purified to get

DNA. The concentration of the purified DNA was determined using NANODROP®

(Thermo Fisher Scientific Inc., Delaware, USA).

1.2.4 Cloning and sequencing

Zero blunt TOPO® vector (Invitrogen, Carlsbad, CA) was used to clone PCR

products. Sequencer reaction was carried out by using M13 primers and big dye

terminator (Applied Biosystems). DNA sequence was determined by the ABI Prism

3100 Genetic Analyzer (Applied Biosystems).

1.2.5 Recombinant protein expression

Recombinant protein was expressed as a glutathione S-transferase (GST)-fusion

protein using pGEX4T-1 expression plasmid vector (GE Healthcare Bio-Sciences Corp.,

20

UK) in Escherichia coli (E. coli) DH5 . The expression of the GST-fusion recombinant

protein was induced in E. coli DH5 by adding 1 mM isopropyl- -thiogalactoside at 25

oC. Recombinant proteins were purified using glutathione sepharose 4B beads (GE

Healthcare Bio-Sciences Corp.) according to the manufacturer’s instructions. The

protein concentrations were determined using a protein quantification kit (BCA Protein

Assay Kit, PIERCE Chemical Company, Rockford, IL, USA.).

1.2.6 Polyclonal antibody production

Three female BALB/c mice (eight-week-old) purchased from Clea, Japan, were

immunized with a recombinant protein using 100μl (100μg) emulsified in equal volume

of TITERMAX® Gold (TiterMax USA Inc., Norcross, GA, USA). The immunizations

were done intraperitoneally (i.p.) for primary and two boosters at a two-week interval.

Two other mice were immunized with GST while one mouse was used as a negative

control. One week after last booster injection, blood was collected by cardiac puncture

at terminal anaesthesia. Sera were prepared by centrifugation at 17,000 x g for 10 min at

4 oC and stored at -30 oC until use. This experiment was conducted in accordance with

the Standards Relating to the Care and Management of Experimental Animals of

Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

(No. 24-135).

1.2.7 Southern blot analysis

T. congolense IL3000 EMF genomic DNA was digested with the following

restriction enzymes (Roche Diagnostics K.K., Japan): Eco RI, Xho I and Cla I, Bam HI,

Xma I, Nar I and Xcm I. Five g of the restriction enzyme treated genomic DNA

samples were loaded and separated in 1% agarose gel. The DNA sizes were estimated

21

according to migration of 1 kbp DNA ladder (Takara Bio Inc., Japan). The separated

DNA fragments were transferred onto nylon membrane (Hybond-N+, Amersham

Biosciences, UK) as previously described (143). Hybridization and labelling of the

probe were performed using AlkPhos Direct Labelling and Detection Systems

(Amersham Biosciences). Imaging was done by X-ray film (Eastern Kodak Company,

USA).

1.2.8 Western blot analysis

Parasites were sonicated in a sample buffer (2% sodium dodecyl sulfate (SDS)

62.5mM Tris HCl pH 6.8, 5% (v/v) 2-β mercaptoethanol, 10% (v/v) glycerol, 0.05%

(w/v) bromophenol blue) and were heated at 100 oC for 10 min. Then the proteins were

separated in a 10% SDS-polyacrylamide gel electrophoresis (PAGE). Afterwards, the

proteins were transferred onto a nitrocellulose membrane for 1 hour at 110 mA and

blocked in 5% PBS skim-milk overnight at 4 oC. The blots were incubated with

polyclonal antibody (1:200) for 1 hr at room temperature followed by incubation with

anti-mouse IgG (1:2,500) for 1 hr at room temperature. The blots were incubated in 3,3'-

Diaminobenzidine (DAB) tetrahydrochloride hydrate D5637-10G, Sigma–Aldrich, St.

Louis, MO, USA) to visualize results.

1.2.9 Indirect fluorescent antibody test (IFAT)

Parasites were obtained from in vitro cultures and cell suspensions spread over

glass slides (Well-printed Diagnostic Slides, Erie Scientific Company, IL, USA.),

individually as PCFs, EMFs, MCFs and BSFs. The slides were air-dried and fixed with

methanol for 10 min at room temperature. The slides were incubated with anti-serum

(1:200) at 37 oC for two hours. After washing with phosphate buffered saline (PBS), the

22

slides were incubated with anti-mouse IgG for 2 hrs at 37 oC. The nucleus and

kinetoplast were stained with Hoechst 33342 (Dojindo Co. Ltd.) (1:200) for 30 min at

37 oC. Confocal laser scanning microscope (TCS-NT, Leica Microsystems GmbH,

Wetzlar, Germany) was used to analyse the prepared specimens.

1.2.10 Mice infections

After collection of blood to obtain pre-infection sera, four female ICR mice

(eight-week-old) purchased from Clea, Japan were inoculated i.p. with in vitro prepared

BSF with 104 parasites/mouse. Sampling and parasitaemias were conducted and

checked respectively every other day for the first month and thereafter done weekly for

two more months. The levels of parasitaemias were estimated according to modified of

matching method (52). This experiment was conducted in accordance with the

Standards Relating to the Care and Management of Experimental Animals of Obihiro

University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan (No. 24-

45).

1.2.11 ELISA

ELISA was performed according to the OIE Manual of Diagnostic Tests and

Vaccines for Terrestrial Animals (118) by using either PCF cell lysate or a recombinant

antigen. Each well of microplates (Nunc Maxisorp, Thermo Fisher Scientific Inc.) was

coated with 200 ng of antigen and incubated for overnight at 4 oC. Antigen-coated

plates were washed five times with PBS containing 0.05% Tween 20 (PBST), once with

PBS and incubated with blocking solution (PBST containing 1% bovine serum albumin

(BSA)). Serum samples were diluted 200 times with PBST containing 0.1% BSA, and

100 μl/well were incubated at 37°C for 1 hr. According to the manufacturer’s

23

instructions, horseradish peroxidase-conjugated protein IgG (1:2,000) (Invitrogen) and

tetramethylbenzidine were utilized for detection of antigen-antibody reaction. Finally,

100 μl of stop solution (1M phosphoric acid) was added into the wells and the

absorbance was read at 450 nm. All samples were analysed in triplicates.

ELISA was performed to check whether the recombinant antigen would react

with Trypanosoma theileri, which is a widely distributed non-pathogenic bovine

trypanosome, using archived infected field bovine serum samples from Japan. The

infection with T. theileri was confirmed through microscopy prior to preparation of

serum samples.

1.2.12 Data management and analysis

The data were entered and analysed in both MS Excel 2007 and Graph Pad

Prism Version 5.04 using descriptive statistics at 95% confidence interval and t-test.

Differences were considered statistically significant at P < 0.05.

24

1.3 Results and discussion

The purpose of characterizing the hypothetical protein was to identify novel

pathogenic factors of T. congolense that may be used as a candidate in the disease

control strategy. These may include either diagnostic or drug targets, and potential

vaccine candidates (49). So far the vaccine has been elusive because of the

immunodominant variant surface glycoprotein (VSG) in a phenomenon known as

antigenic variation (32, 33). The three control strategies employed for trypanosomosis

so far include tsetse control, chemotherapy and trypanotolerant animals (99). Therefore

development of accurate, sensitive and rapid diagnostic tests is of importance as a

realistic control measure for African trypanosomosis. The invariant surface

glycoproteins (ISG65 and ISG75) have been characterized in T. brucei (181, 182).

Recently it was reported that the ISG75 has been associated with suramin metabolism

(3). The ISGs are polypeptides consisting of an N-terminal signal sequence, a

hydrophilic extracellular domain, single trans-membrane alpha-helix and a short

cytoplasmic domain (158, 181). They are expressed in the BSF but not in the PCFs of T.

brucei, and are distributed over the entire surface of the parasite. Because of its

invariable nature, ISG75 was successfully utilized as a diagnostic antigen for surra in

camel (158). However, T. congolense ISGs have not been identified yet. In order to

identify T. congolense ISGs, differential protein expression data of all the life cycle

stages of T. congolense were utilized (38). Among others, one gene, Tc38630 (Gene ID:

TcIL3000.0.38630, 1,236 bp), is expressed more in MCF and BSF than procyclic form

(PCF) and epimastigote form (EMF) by 8.5 times. The Tc38630 gene encodes 411

amino acids, which consists of the predicted N-terminal signal peptide, single trans-

membrane alpha-helix, and N-glycosylation sites (Fig. 2). According to these domain

25

structures, Tc38630 was assumed as a T. congolense orthologue of the T. brucei ISG.

Although the T. brucei ISG genes are present as multiple copies in the parasite genome

(183), Southern blot analysis demonstrated that Tc38630 was a single copy gene with

some allelic polymorphism at one restriction enzyme site (Fig. 3). At this Nar I (a

single-cutter enzyme) restriction site, multiple bands were demonstrated than expected

as compared to two other single cutter enzymes. This result was surprising because in

trypanosomes many genes are present in multiple copies (183). The polyclonal

antibodies against Tc38630 were used to immunolocalize the protein whereby normal

and GST immunized sera were used as controls. The Tc38630 appeared to be expressed

in both cytosol and cell surface of MCF and BSF (Fig. 5). This clearly indicates that

Tc38630 protein (a putative T. congolense ISG) starts expressing from MCF stage. Like

T. brucei ISGs, Tc38630 also had the discrepancy between the apparent and the

predicted molecular mass. This probably would be due the protein running as a diffuse

band in SDS-PAGE even after deglycosylation suggesting some further covalent

modifications (181). In addition, Tc38630 protein was highly negatively charged with

an isoelectric point of 4.86 making it bind little to SDS-PAGE gel leading to a slower

mobility relative to the molecular protein standards. Although the predicted molecular

mass of the Tc38630 was 44.7 kDa, native Tc38630 was expressed as approximately 70

kDa in both BSF and MCF (Fig. 4) (181). A series of rTc38630-based ELISA

experiments were conducted to investigate whether the rTc38630 was usable for

serodiagnosis of T. congolense infection or not. As a standard test, the OIE

recommended ELISA using the PCF cell lysate antigen was utilized (Fig. 6). The cut-

off value was 0.013 for both ELISAs (calculated as a mean + 3 standard deviation for

day 0 sera). As a result, use of rTc38630 as an antigen in ELISA and PCF cell lysate

26

ELISA was not statistically different (p>0.05). Alongside, parasitaemias were

determined by wet blood smears to confirm the mice were infected (Fig. 6C). The

rTc38630 ELISA showed high titre consistently from 7 days post infection (DPI), which

is 2 days earlier than PCF cell lysate ELISA. On further analysis, from day 7, the

rTc38630 was better in diagnosis of infection (p<0.05). Worth to note, the recombinant

antigen did not react with Trypanosoma theileri, which is a widely distributed non-

pathogenic bovine trypanosome, using archived infected serum samples (data not

shown). In conclusion, in order to confirm the specificity of ELISA, additional

experiments on cross-reaction to sera infected with T. brucei, T. vivax, etc, are needed.

Similarly, to confirm the sensitivity, additional experiments on dose-dependent of the

antigen are also needed. Although further evaluation is required prior to field

application, the Tc38630-based ELISA is a promising diagnostic antigen for nagana.

1.4 Summary

Animal African trypanosomosis is a serious constraint to livestock sector

development in sub-Saharan Africa. The disease, mainly caused by T. congolense, has a

limitation in its diagnosis and treatment. There is urgent need for a simple, rapid

detection technique to replace the few available serological tests that are of variable

sensitivity and specificity. Currently, there is a promising use of recombinant proteins to

improve on the trypanosome lysate to detect antibodies. In this respect, a stage-specific

gene that is relatively highly expressed in metacyclic and blood trypomastigotes of T.

congolense was identified. According to previously obtained differential protein

expression data, the gene TcIL3000.0.38630 (1,236 bp) is by 8.5 times more expressed

more in metacyclic and blood trypomastigotes than in procyclic trypomastigotes and

epimastigotes. The same stage specific expression pattern was shown in Western blot

27

analysis. In addition, in confocal laser scanning microscopy the Tc38630 protein was

present in the cytosol and on the cell surface of metacyclic and blood trypomastigotes.

Through bioinformatics, the Tc38630 had N-terminal signal sequence, hydrophilic

extracellular domain, single transmembrane alpha-helix and short cytoplasmic domain,

which is characteristic of the T. brucei invariant surface glycoprotein. However, unlike

T. brucei invariant surface glycoprotein, the Tc38630 existed as a single copy gene with

a probable allelic polymorphism at the Nar I restriction site. The recombinant Tc38630-

based ELISA detected antibodies against Tc38630 as early as 7 days post infection in

experimentally infected mouse model. Taken together, these results suggest that the

Tc38630 is a novel potential diagnostic antigen of animal African trypanosomosis.

28

Figure 2: Deduced amino acid sequence of Tc38630. Possible N-glycosylation sites are

indicated in red. An underline indicates a predicted N-terminal signal peptide. Predicted

single trans-membrane alpha-helix domain is highlighted in bold.

29

Figure 3: Southern blot analysis of Tc38630 gene. Total DNA from Trypanosoma

congolense was treated by the following restriction enzymes: Eco RI (Lane 1), Xho I

(Lane 2), Cla I (Lane 3), Bam HI (Lane 4), Xma I (Lane 5) and Nar I (Lane 6). PCR

amplified full length Tc38630 gene was utilized as a probe. The enzymes used for lanes

1-3 do not cut Tc38630 gene, while for lanes 4-6 are single cutter. Bars on the left side

indicate 1 kbp DNA ladder.

30

Figure 4: Differential expression of Tc38630 was analysed by Western blot. B, M, E

and P indicate blood stream form, metacyclic form, epimastigote form, and procyclic

form, respectively.

31

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32

Figure 6: Detection of IgG responses against rTc38630 (A) and PCF cell lysate (B)

antigens in infected mouse sera by ELISA. Course of parasitaemia in infected mice is

shown in panel C.

33

Chapter 2

Establishment and evaluation of the potential use of recombinant

Tc38630 protein of Trypanosoma congolense in enzyme-linked

immunosorbent assay and immunochromatographic test

2.1 Introduction

African trypanosomosis has been known to be a serious constraint to livestock

sector development in sub-Saharan Africa. The animal form also known as nagana is

caused by tsetse-transmitted protozoan parasites, T. brucei, T. congolense, T. vivax among

others (118). The parasites may also be transmitted mechanically by biting flies. Despite

the disease’s economic losses and having been studied for a long time, there are still

challenges in diagnosis and treatment (88). Therefore, the aim of this study was to find a

new ELISA-based diagnostic test using recombinant proteins for the diagnosis of T.

congolense infections and also apply it in immunochromatographic test (ICT). According to

amino acid domain structures, Tc38630 was assumed as a T. congolense orthologue of T.

brucei ISG and further found it to be a novel diagnostic antigen in experimental mouse

model (102). The use of non-variable surface proteins such as ISG75 is promising

alternative in the improvement of diagnostic tests (60).

Several diagnostic tests including PCR, IFAT, ELISA and microscopy are used for

trypanosome detection and vary in their sensitivity and specificity. In addition, these tests

vary in their cost and ease of their application (118). It is advisable that proper diagnosis

34

may be achieved by combining appropriate diagnostic tests. Microscopy detection, still

considered as a gold standard, has low sensitivity whereby if trypanosomes are 100

parasites per ml or less cannot be detected (25) and cannot be deployed for large scale

screening. In serodiagnosis, there is detection of either circulating antigens or antibodies

and if an assay is able to detect the former, then it would demonstrate an active infection

(60). Enzyme-linked immunosorbent assay (ELISA), first used in early 1970s utilizes

antibodies or antigens and colour change to identify a substance, is now frequently applied

as a diagnostic tool in medicine and various fields (34, 77). Moreover, compared to other

detection methods, ELISA technique is suitable for mass screening. The ELISA technique

may give false negative results even in parasitologically proven cases. This often occurs for

sera from acute or early phase of infection and has been observed in T. congolense, T. vivax

and T. brucei infections in cattle and goats (92, 112). Despite its drawbacks by further not

distinguishing between current and past infection, ELISA has particularly been useful for

epidemiological surveys to detect trypanosome antibodies (118). However, in the ELISA

detection techniques, involve use of either whole parasite or crude parasite lysate as the

antigen which are not often standardized.

With the success of ELISA experiment, though labour-intensive and time-

consuming, requires equipment and trained personnel to perform, there is need for a simple

and rapid test which could be used as preliminary screening of the disease by the

veterinarians or pen-side detection of the disease in livestock (59, 70). Thus, a convenient,

rapid, and sensitive diagnostic test, such as an immunochromatographic test (ICT) which

detects antibody and does not require any instrument, is desired (24, 138). An ICT is a

35

nitrocellulose membrane (NC)-based immunoassay. An attempt has been made to utilize

ICT on AAT using a recombinant ribosomal P0 protein (27).

Immunochromatographic test (ICT), which utilizes the model of chromatography,

was first introduced in late 70s and became popular in late 80s, (67, 121, 154). It was first

commercially produced for a home-based test for pregnancy (97). Due to its simplicity and

quick results, it has been extensively applied for different purposes such as diagnosis of

human and animal diseases and detection of target compounds, agricultural and

environmental applications (24, 128). Specifically, the ICT has been developed for tropical

diseases including malaria (178), Kinetoplastids (leishmaniasis and Chagas disease) (26,

139, 151), schistosomiasis (14), babesiosis (47, 59, 70, 86), toxoplasmosis (57) and

neosporosis (80). The test involves a combination of various techniques such as assembly

process, selection of ICT materials, antigen-antibody affinity, and selection of reagents and

buffers at particular pH levels.

The serological assays need improvements to make therapeutic decisions at a herd

or individual level and thus should be highly sensitive and specific. Therefore, the objective

of this work was to establish and evaluate the potential use of recombinant protein from T.

congolense in ELISA and ICT and compared with the reference test lysate antigen-based

ELISA as recommended by OIE.

36


2.2.1 Parasites and animals

T. congolense IL3000 savannah strain isolated near Kenya/Tanzania border was

used. PCF and EMF were propagated at 27 oC using Trypanosoma vivax medium (TVM)-1

while, the BSF was at 33 oC using Hirumi’s modified Iscoves’s 9/10% (HMI-9) medium

(53). Eight-week-old female BALB/c (Clea, Japan) mice were used for sera production and

infections. Maintenance of the parasites was as described in section 1.2.1.

This experiment was conducted in accordance with the Standards Relating to the

Care and Management of Experimental Animals of Obihiro University of Agriculture and

Veterinary Medicine, Obihiro, Hokkaido, Japan (No. 24-135).

2.2.2 Recombinant protein production

Expression and purification of rTc38630 protein in Escherichia coli as a fusion

protein with glutathione S-transferase was conducted as described in section 1.2.5.

2.2.3 Trypanosome lysate antigens

All four stages of T. congolense (IL3000) were propagated by in vitro culture (54),

and utilized as the sources of trypanosome lysate antigens. Preparation of lysate antigens

was done as described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial

Animals, 2013 (118).

37

2.2.4 Immunization and production of anti-rTc38630 sera

Anti-rTc38630 sera were produced as described elsewhere (section 1.2.6). The

recombinant immunized serum was purified for IgG polyclonal antibodies by Protein G

affinity chromatography Kit (Bio-Rad Laboratories, Hercules, CA, USA) and dialyzed in

PBS. The concentration of the polyclonal antibody was determined by BCA Protein Assay

Kit (PIERCE Chemical Company, Rockford, IL, USA). Final purified polyclonal

antibodies were stored at -30 oC until use. This, plus the recombinant protein were used in

the assembly of ICT.

2.2.5 ELISA

Purified rTc38630 protein fused with GST was diluted in a coating buffer (0.5M

carbonate-bicarbonate pH 9.6 to final concentration of 200 ng/ml). Each well of the 96-well

microtitre plates (Nunc Maxisorp, Thermo Fisher Scientific Inc) was coated with 100 μl of

the protein overnight at 4 oC. The subsequent protocol was followed as described in section

2.2.11. The cut-off value was defined as the mean value plus 3 standard deviations of the

mean optical density (OD) obtained from 29 known negative cattle serum samples.

2.2.6 Preparation of gold colloid-conjugated antigens and ICT strip

All of the materials used for ICT (glass fibre, absorbance and nitrocellulose

membranes) were purchased from EMD Millipore Corporation (Billerica, MA, USA).

Purified rTc38630 antigen fused with GST was diluted to an optimal concentration of 500

μg/ml with 5 mM phosphate buffer (pH 7.0) and conjugated with gold colloid particles

(British BioCell International, UK) as described previously (58) with some minor

38

modifications. Briefly, rTc38630 (500 μg/ml) was conjugated with a gold colloid (British

BioCell International, Cardiff, UK) at pH 6.5 by gentle mixing (1:10, vol/vol) and

incubation at room temperature for 10 min. Polyethylene glycol 20,000 (PEG) at 0.05% and

bovine serum albumin (BSA) at 1% were then added to stabilize and block the conjugate

particles. After centrifugation at 18,000 × g for 20 min, the supernatant was discarded and

the pellet was resuspended, sonicated and washed with phosphate-buffered saline

containing 0.5% BSA and 0.05% PEG. After the second centrifugation, the pellet was

resuspended in phosphate-buffered saline with 0.5% BSA and 0.05% PEG. The conjugate

was diluted in 10 mM Tris-HCl (pH 8.2) with 5% sucrose, sprayed onto glass fibre

(Schleicher & Schuell, Inc., Keene, NH, USA), and dried in a vacuum overnight. rTc38630

immunized serum was purified for IgG polyclonal antibodies using protein G affinity

chromatography, dialyzed in PBS and used as control line in ICT. rTc38630 (500 μg/ml),

rGST (200 μg/ml) and IgG (1,500 μg/ml) were linearly jetted onto a nitrocellulose (NC)

membrane (Schleicher & Schuell, NH, USA) as the test, GST, and control lines,

respectively, using a BioDot Biojet 3050 quanti-dispenser (BioDot, Inc., CA, USA) (57,

80). Then the membrane was dried at 50 oC for 30 min and blocked by using 0.5% casein in

a 50 mM boric acid buffer (pH 8.5) for 30 min. After a washing with 50 mM Tris-HCl (pH

7.4) containing 0.5% sucrose and 0.05% sodium cholate, the membrane was dried in air

overnight. The NC membrane, absorbent pad, conjugate pad, and sample pad were

assembled sequentially on an adhesive card (Schleicher & Schuell, NH, USA) in a manner

to effect capillary action and cut into 3-mm-wide strips using a BioDot cutter (BioDot, Inc.,

CA, USA). A typical ICT strip is shown in Fig. 7. Detection was performed by pipetting 10

μl of the diluted serum (1:5 in PBS) on the sample. The result was judged 15-20 min after

39

the application of serum samples. The presence of a control band alone indicated a negative

result, whereas the presence of two bands (control and test bands) indicated a positive result.

If a GST line appeared, the test was declared nonspecific (57, 80) and if no band was

visible after 20 min, the result was considered invalid. The strips were stably stored with

dehumidification in foil pouches at ambient temperature until use.

2.2.7 Sera

Sera produced as described elsewhere (section 2.2.10) were used as primary

antibody in ELISA for mouse model. Archived cattle field sera samples from Uganda and

Tanzania (408 samples) were used in ELISA and ICT experiments. Twenty-nine archived

cattle field sera samples from a trypanosome non-endemic area in Tanzania were used to

calculate cut-off values. The 29 samples were further subjected to microscopy and PCR and

found to be negative for trypanosomes.


The data were entered and analysed in both MS Excel 2007 and Graph Pad Prism

Version 5.04 using descriptive statistics at 95% confidence interval. Differences were

considered statistically significant at P < 0.05. The strength of agreement among the tests,

ICT and ELISA and reference test was estimated by a kappa statistic. Kappa statistic values

>0.75, 0.40-0.75, and <0.40 represent excellent agreement, fair to good agreement, and

poor agreement, respectively (169).

40


In any disease control strategies, a sensitive and reliable diagnostic test is crucial. In

the present study, the result of rTc38630-based ICT was compared with rTc38630-based

ELISA. In this respect, an attempt was made to find a diagnostic antigen to be used in

ELISA and in ICT in the diagnosis of African trypanosomosis. The T. congolense

orthologue of the T. brucei ISG was found to be a potential diagnostic antigen. Various

ISGs have been identified and characterised that include ISG 64 (65), ISG 65/70 (182), ISG

75 (181) and ISG 100 (115). In particular, ISG 75 has been used for molecular diagnosis

(141). In serodiagnostics, an ideal antigen should be immunogenic (90) and all antigens of

trypanosomes are potentially immunogenic including the cytosolic ones because they are

released as result immune-mediated cell lyses. The use of recombinant antigens in the

diagnosis will eventually replace the native ones as they can easily be standardized.

The potential of the recombinant protein as a diagnostic antigen was evaluated by

ELISA using archived field cattle serum samples. The cut-off value of rTc38630-ELISA

was 0.5 OD using known negative sera while for crude lysate ELISA was 0.3. Since the

protein was fused with GST, rGST-ELISA was conducted as control that was subsequently

subtracted from the mean OD values.

ELISA was performed in order to determine the immunological reactivity of newly

expressed antigens (Fig. 6A) using serially infected four mice sera. To evaluate the

performance of rTc38630 protein as a diagnostic antigen using a more rapid and simple

method, ICT was performed to detect specific antibodies. As shown in Fig. 8, sera from the

41

experimentally infected mouse model were used to check the potential use in ICT. The

result of ICT was similar to that of ELISA in the experimental mouse model (Fig. 6A) and

it was able to detect antibodies against T. congolense as from day 7.

Recombinant ELISA application on archived field cattle serum samples showed

poor agreement (Kappa = 0.11, Sensitivity = 11%, Specificity = 100%, PPV = 95%, NPV =

60%) compared with the reference test (Table 1). This poor agreement would attributed to

the sera producing higher OD values for GST which when taken into consideration reduced

the overall mean OD values. In addition the cut-off value was relatively high. However,

Boulangé et al., (16) showed also in their use of recombinant heat shock protein 70

homologue to have a limited sensitivity in the detection of trypanosome antibodies in cattle.

In addition, the reference test showed high prevalence rates probably due to persistence of

antibodies in the body system and the instability of lysate antigens. By and large, in the

validation of any new diagnostic test, often there is a drawback as there is no gold standard

or reference test and is difficult in endemic areas to have animals with no known infection

status (15).

ELISA requires equipment and expertise to conduct, on the other hand rapid

diagnostic tests (RDTs) are rapid (10-20 Min), require no capital investment, and are

simple to perform and easy to interpret (154, 178). Among the diseases caused by

kinetoplastid protozoa, ICT has been used to diagnose leishmaniasis (151) and Chagas’

disease (139). Indeed, Huang et al., (58) notes that the ICT is an immunoassay in which

nitrocellulose (NC), migration membrane that relies on capillary mechanism in its

assemblage. The antibodies are captured on the immobile test line of the antigens whereby

42

antigen-antibody reaction develops as a coloured line. The performance of the test is simple

as strip is dipped into a sample fluid, and the result can be determined in a few minutes

with the naked eye. In addition, no instrument or testing skills are required. The ICT has

apparent advantages over routine diagnostic tests as no special expertise or equipment is

required. The ICT strip is quite stable during long storage under ordinary conditions and is

rapid taking less than 20 min to complete. Therefore, this test is more practical to use in the

field than any other test. Pen-side immunoassays would be an advantage in making

therapeutic decisions and therefore ICT will go a long way to solve this. Taking together

the above advantages, an ICT was established using mouse model first and then it was

applied on archived field cattle samples. Field cattle serum samples demonstrated a

relatively strong antibody responses to GST control line thereby making the result non-

specific on the few samples tested (Fig. 9). Or, it would be speculated that the Uganda

samples would have come from Schistosoma endemic areas as the recombinant GST is

from Schistosoma japonicum, however, GST is abundant in mammals (18). The sensitivity

was defined as the proportion of animals positive for trypanosome antibodies according to

the reference test that were correctly identified as positive for recombinant ELISA / ICT.

On the other hand specificity was defined as the proportion of animals found without

antibodies for trypanosomes according to the reference test that were correctly identified as

negative by recombinant ELISA (Table 1). These data on field cattle samples otherwise, are

not supported for both ELISA and ICT. ICT was done on a few samples and the result was

inconsistent with the ICT of the experimental mouse model (Fig. 9) thus making the result

inconclusive. Therefore, it can be concluded that ELISA and ICT application for use in

field samples were not successful at this stage. As further work, there is need to express the

43

protein again and cleave the GST tag and apply in ELISA and ICT for field samples or

alternatively use a different low molecule tag. Important to note, an assay that detects the

antibodies are easier to develop as they require less complicated reagents (60). A notable

further development on ICT to detect acute or early infection will need use of IgM rather

than IgG.

2.4 Summary

Trypanosomes are hemo-flagellate protozoan parasites that cause disease in humans

and livestock called sleeping sickness and nagana, respectively. Recombinant protein

Tc38630 (rTc38630) from T. congolense was successfully expressed, characterized and

found to be antigenic using ELISA in experimentally infected mouse model. According to

amino acid domains structure, Tc38630 was assumed as a T. congolense orthologue of the

T. brucei invariant surface glycoprotein (ISG) therefore, a potential diagnostic antigen.

ELISA experiments though labor-intensive and time-consuming, require equipment and

trained personnel to perform. However, they are suitable for epidemiological surveys.

Among other serological tests, immunochromatographic test (ICT) has an advantage as a

one-step rapid analysis, thus making it a convenient and sensitive diagnostic test. BALB/c

mice were immunized with rTc38630 proteins. rTc38630 immunized serum was purified

for IgG polyclonal antibodies using protein G affinity chromatography, dialyzed in PBS

and used as control line in ICT. The ICT was optimized and assembled whereby the

antigen-antibody reaction was detected by colloidal gold conjugated rTc38630 protein at

test line. Indirect ELISA was performed according to the OIE Manual of Diagnostic Tests

and Vaccines for Terrestrial Animals by using either PCF cell lysate or a recombinant

44

antigen. The ICT result was found to be consistent with rTc38630 protein ELISA in the

experimentally infected mice sera. However, ELISA and ICT application in field samples

were inconclusive as they showed low sensitivity. However, in future plan, there is need to

express the protein again and cleave the GST and apply it in ELISA and ICT for field

samples.

45

Figure 7: Schematic diagram of a typical immunochromatographic test strip (24). The result

was judged 15-20 min after the application of serum samples. The presence of a control

band alone indicated a negative result, whereas the presence of two bands (control and test

bands) indicated a positive result. Absence of bands, indicated invalid result.

46

Figure 8: Detection of IgG responses against r38630 antigens on immunochromatographic

test showing consistency with Fig. 6A on infected mouse experimental sera. 0-13 = Days

post infection; Control line = Blue arrowed; Test line = Red arrowed.

47

Figure 9: A representative immunochromatographic test on Uganda cattle field samples.

Control line = Blue arrowed; GST line = Yellow arrowed; Test line = Red arrowed; +,

positive for T. congolense with only two lines; -, negative for T. congolense with only one

line; Lanes 41, 42, 47, 48, 49 show GST line making the result unspecific.

48

Table 1: Two-by-two table showing a reference test, procyclic form (PCF) lysate ELISA

and rTc38630 ELISA tests.

Poor agreement between reference test and rTc38630, Kappa = 0.11, Sensitivity = 11%,

Specificity = 100%, Positive Predictive Value (PPV) = 95%, Negative Predictive Value

(NPV) = 60%.

49

Chapter 3

Evaluation of the recombinant Tc38630 protein as a potential vaccine for

nagana

3.1 Introduction

Currently, there are few drug regimens with some reportedly toxic while others are

becoming alarmingly resistant. Therefore, eventual vaccine development would be

tremendously beneficial (88). So far, this has not been possible due to the trypanosome’s

surface coat ability to avoid the immune responses known as antigenic variation (32, 33).

However, Wei et al., (175) differ and offer the opinion of immunosuppression induced by

trypanosomal infections. Nonetheless, with the advent of research focused on the

identification of invariant components to be used as therapeutic targets and the evolution of

DNA vaccine technology, the possibility is beckoning in the near future (23, 68). Indeed,

DNA vaccines have been termed as third generation of vaccines (2) compared to the first

generation that used whole organism vaccines – either live/attenuated, or killed forms. The

second generation vaccines were developed to reduce risks associated with the first ones.

These vaccines consist of some protein antigens (e.g., tetanus or diphtheria toxoid) or

recombinant protein components (e.g., hepatitis B surface antigen). Since description

genomic organization (183) of a gene family for the invariant surface glycoproteins (ISG)

from Trypanosoma brucei brucei parasites, attempts have been made to use them as a DNA

vaccine by Lança et al., (74) in experimental model. A different approach in the search for

vaccine to combat this menace based on ‘anti-disease’ rather than an anti-parasite strategy

50

has been adopted following observations of trypanotolerance phenomenon in a non-sterile

condition (17). Recombinant proteins have been used in experimental setting in cattle

against T. congolense infections whereby trypanotolerance was exhibited in the otherwise

trypanosusceptible boran cattle. Even though, the trypanolerance breaks when levels of

parasites exceed 107 per ml of blood. Since the immunodominant VSG has failed as a

vaccine, focus has shifted to characterizing invariant glycoproteins as alternative antigens

for potential vaccines. These ISGs are embedded under the VSG that include ISG64 (65),

ISG65 (182), ISG75 (181), ISG100 (115). Other non-variable antigens are cytoskelon

proteins, microtubulins and flagella pocket proteins that have been used as vaccine

candidates experimentally with mixed results (21, 79). In general, so far the research on

vaccines can be summarized as those that elicited partial protection (6, 74, 78, 79, 81, 101,

131, 148, 150) while others elicited no protection (131, 133). An ideal vaccine candidate

has to activate a strong, protective and long lasting immune response (68, 132).

The aim of this study was to produce a vaccine through evaluation of the

recombinant protein (Tc38630) as a potential vaccine candidate. However, it is becoming

increasingly unforeseeable for anti-infection vaccine and thus the focus has shifted to anti-

disease strategy as opposed to anti-parasite (5, 17, 144) whereby pathogenic factors

expressed during infection are counteracted by drugs and/or vaccine but not target the

parasite.

51


3.2.1 Parasites and animals

Trypanosoma congolense IL3000 savannah strain isolated near Kenya/Tanzania

border was used. PCF and EMF were propagated at 27 oC using Trypanosoma vivax

medium (TVM)-1 while, the BSF was at 33 oC using Hirumi’s modified Iscoves’s 9/10%

(HMI-9) medium (53). Maintenance of the parasites was done as described elsewhere

(section 1.2.1). BALB/c (Clea, Japan) mice at eight-week-old were used for vaccine

experiment.

This experiment was conducted in accordance with the Standards Relating to the

Care and Management of Experimental Animals of Obihiro University of Agriculture and

Veterinary Medicine, Obihiro, Hokkaido, Japan (No. 24-135)

3.2.2 Construction and expression of recombinant 38630

Expression and purification of rTc38630 protein in Escherichia coli as a fusion

protein with glutathione S-transferase was conducted as described in section 2.2.5.

3.2.3 Homogenate preparation

Metacyclic form parasites (MCFs) were purified from EMF stage in vitro cultures

by passage through DEAE cellulose (DE-52; Whatman) column chromatography and

elution with PSG (PBS, pH 8.0+1.5g glucose/L) (75). BSF stage in vitro cultures were

harvested and washed three times in PBS as a pellet by centrifugation (1500 x g, 10 min, 4

oC). The isolated pellets were freeze-thawed three times and homogenized in PBS. Protein

52

concentrations for MCF and BSF homogenates were determined by BCA Protein Assay

Reagent (PIERCE Chemical Company, Rockford, IL, USA).

3.2.4 Immunization of mice

Five groups of seven – eight weeks old female BALB/c (Clea, Japan) mice, five per group,

were used in the experiment. Five groups of BALB/c mice (highly susceptible to T.

congolense infections), five per group, were randomized and assigned as recombinant

protein (rTc38630), recombinant GST, MCF homogenate, BSF homogenate and PBS. They

were immunized respectively using 10μg in 100μl PBS emulsified in equal volume of

TITERMAX® Gold (TiterMax USA Inc., Norcross, GA, USA) adjuvant. The

immunizations were done subcutaneously (sc) for primary and after one week, with first

booster. Second booster was done at a two-week interval after the first boost. Responses to

immunization were assessed by ELISA (6). Briefly, the ELISA plates (Nunc Marxisop®)

were coated appropriately with rTc38630 antigen, rGST, BSF homogenate and MCF

homogenate in 50 mM carbonate-bicarbonate buffer, pH 9.6 and incubated at 4 oC. The

plates were blocked with 1% BSA in PBS containing 0.1% Tween-20 (blocking buffer)

before incubation with serum dilutions in blocking buffer for one hour. The second

antibody was anti-mouse IgG conjugated to horseradish peroxidase (Sigma) while

tetramethylbenzidine (TMB) was used as substrate. All washing steps were done with PBS

containing 0.5 Tween 20. Titres to the antibody to the respective antigens were measured

up to dilution of 6,400 times.

53

Endotoxin also known as lipopolysaccharide (LPS) was removed from the

recombinant protein using Pierce® High-Capacity Endotoxin Removal Resin Kit (Thermo-

Scientific, Rockford, IL, USA) and the level was measured using Endospecy® ES-50M Kit

(Seikagaku Corporation, Japan). A 0.57 EU/ml level of LPS in protein was achieved

against the recommended level of 0.25-0.5 EU/ml equivalent to 0.25-0.5 ng endotoxin / ml

(20).

3.2.5 Trypanosome challenge and parasitaemia monitoring

Trypanosomes for experimental challenge were first expanded in BALB/c mice

which were later sacrificed. The concentration was determined using a Naubauer

hemocytometer and trypanosomes were estimated for challenge of five groups of BALB/c

mice. About 5,000 parasites were used i.p. per mouse in the second week after the last

boost. After challenge, the mice were monitored daily to determine the pre-patent period.

Parasitaemias were monitored daily via tail blood by wet blood smears and when

count was over 50 trypanosomes per field, haemocytometer was used to estimate

trypanosomes.


The data were entered and analysed in both MS Excel 2007 and Graph Pad Prism

Version 5.04 using descriptive statistics at 95% confidence interval. Survival analysis was

done using log-rank tests to compare differences in survival curves. Differences were

considered statistically significant at P < 0.05. Period of survival was defined as the number

of days after challenge, the infected animals remained alive.

54


Five groups of BALB/c mice (highly susceptible to T. congolense infections), five

per group, were randomized and assigned as recombinant Tc38630, recombinant GST,

MCF homogenate, BSF homogenate and PBS. They were immunized respectively, using

TITERMAX® as adjuvant. They were challenged with a lethal dose of expanded

bloodstream form of T. congolense intraperitoneally (i.p.). This is the commonly used route

to induce experimental infections, whereas the SC route and use of MCFs would be ideal in

mimicking the natural infection (8). A trial to infect MCFs through i.p. route was made, but

BALB/c mice were resistant as they cleared the parasitaemia. The titre of specific

antibodies (≥1:6400) was achieved before challenge. Endotoxin, a liposaccharide (LPS)

which is known to provoke non-specific immunity (87) was removed and the levels

measured.

In order to evaluate the protective efficacy of rTc38630 protein (having found it to

be immunodominant), BALB/c mice were immunized with rTc38630, followed by two

boosters prior to challenge infection with IL3000 T. congolense. The results showed that

rTc38630-GST immunized mice showed long pre-patent period, higher survival rates

compared with control groups, but no significant difference was observed between the

groups (p value = 0.09) (Fig. 10). One mouse in the r Tc38630 immunized group however,

survived longer than the rest for about 17 days and the median survival time (10 days) (Fig.

11) was one day longer than in control groups. Parasitaemias were monitored daily after

challenge of the mice and one mouse in rTc38630 survived one day longer as shown by a

drop of parasitaemia at day 10 compared to control groups (Fig. 12A). This meant that the

55

mouse that had the drop, it failed to clear the infection once it was established as it

eventually succumbed. This might be attributed to immunosuppression (166). The median

survival time was not any different between the groups (p>0.05). The mean pre-patent

period for all the groups was 3 days with no significance differences between groups (p =

0.11) (Fig. 13). Without any treatment, when infected i.p. with 103 T. congolense, BALB/c

mice survive for 8.5 ± 0.5 days (153). Owing to the fact that developing a conventional

anti-parasite vaccine has been elusive due to antigenic variation (32), and more recently

immunosuppression (175), an anti-disease approach strategy was proposed (6). This idea

was borrowed following observations of trypanotolerance in cattle (109) and in humans

(66). In serodiagnostics, an ideal antigen should be immunogenic (90) and all antigens of

trypanosomes are potentially immunogenic including the cytosolic ones as they are released

as result immune-mediated cell lyses. Tolerant animals normally maintain trypanosome

levels below 103, but tolerance breaks when they reach about 107 parasites/ml of blood

(117).

An attempt has been made to utilize metacyclic stage proteins as a vaccine (37) and

they found out to have a limited protection. Several invariant molecules have been used as

vaccine with mixed results which include tubulin, actin, microtubule-associated proteins

(MAPs), flagellar pocket (FP) and recombinant ISG75 (7, 78, 79, 81, 82, 101). In an ideal

experimental design, there is need to challenge the mice three to six months after the last

immunization and check whether protection is elicited (87). A single bite of tsetse can

inject about 104 metacyclic trypomastigotes; therefore in the estimation 5 x 103 parasites

were adequate for the challenge through i.p. This regime has been used experimentally for

56

ISG75 but the mice were not protected when challenged (87). Other workers, Ramey et al,

(133), used an estimation of 106 parasite load with no protection reported. Again, blood

samples (≈100 μl) were collected from the mice’s tail that ensured anaemia was not due to

sampling rather infection. The target of vaccination often, is to induce B cell memory (131).

The orthologue ISG Tc38630 protein potential as a vaccine candidate was investigated

while the ISG from T. brucei has been used as a DNA vaccine whereby it produced a

partial protection in experimental model (74). Although rTc38630 protein is not

significantly protective, further work need to be done that may include the combination of

different antigens. In addition to combinations, the choice of adjuvants needs to be

considered as they can improve on immunogenicity of the antigens, speed and duration of

immune responses, among other advantages (177).

In vaccinology, all vaccines may be classified into living and non-living vaccines

(135). In the past many vaccines were in the former class but more recently most vaccines

are non-living whereby a whole killed pathogen or components of them (subunit vaccines)

are used to induce protective immunity. In using conventional approaches to vaccinology as

concluded by Mora et al., (103), some proteins may be immunogenic in vivo but not

necessarily protective therefore, the reverse vaccinology would address this concern. Other

workers have classified the vaccines into three, live, killed and subunit (177). Recombinant

DNA and proteins fall in the latter class and largely, they do have fewer side effects thus

making them safe. However, the recombinant antigens, especially those purified from

bacterial system contain some lipopolysaccharide (LPS) which is known to have

adjuvanticity. Vaccines work by priming the antigen-specific T and B cells which will

57

undergo apoptosis, but a small number will convert to the memory cells that will control

subsequent infections by the invader targeted by the vaccine. The choice of adjuvants is

important for ideal immune responses. The Adjuvants are substances that enhance

immunogenicity of antigens by mimicking on how infections activate the innate immunity.

They are thought to act by converting the soluble protein antigen into particulate material

that is readily ingested by antigen-presenting cells such macrophages and microbial

components whereby the immunogenicity is enhanced though not exactly understood fully

(104).

Another approach being pursued, involves transmission blocking vaccines whereby

the parasite is targeted in the vector and will result in reduced numbers of infectious vectors

hence reduced parasite transmission. In future, vaccine development for trypanosomosis

should involve reverse vaccinology (134), now that the T. congolense genome has been

completed (64). As further work, the immune responses involved after parasite challenge

need to be investigated. The recombinant protein need further evaluation with a large

number of mice as well determine the type of immune mechanisms involved.

3.4 Summary

Trypanosomes are flagellated hemo-protozoan parasites vectored by tsetse that

cause disease in humans and livestock called sleeping sickness and nagana, respectively.

Sleeping sickness is caused by T. b. rhodesiense and T. b. gambiense. Whereas the

livestock form, is mainly caused by T. b. brucei, T. congolense, T. vivax. So far, vaccine

has been elusive because of the immunodominant variant surface glycoprotein (VSG) in a

58

phenomenon known as antigenic variation. However, with the advent of research focused

on the identification of invariant components to be used as therapeutic targets, possible

immunogens and the evolution of DNA vaccine technology, the possibility is beckoning in

the near future. Recombinant protein Tc38630 (rTc38630) from T. congolense was

successfully expressed, characterized and found to be antigenic using ELISA in

experimentally infected mouse model. According to amino acid domains structure,

Tc38630 was assumed as a T. congolense orthologue of the T. brucei invariant surface

glycoprotein (ISG). The aim of this study was to produce a vaccine through evaluation of

the recombinant protein (Tc38630) as a potential vaccine candidate. Five groups of female

BALB/c mice, five per group, were randomized and assigned as recombinant protein

(rTc38630), recombinant GST, MCF homogenate, BSF homogenate and PBS. They were

immunized respectively using TITERMAX® as adjuvant. They were challenged with a

lethal dose of expanded bloodstream form of T. congolense. Focusing on anti-disease

strategy as opposed to anti-parasite, one mouse in rTc38630 group survived longer as

compared to control groups though in overall survival analysis, it was not significant

(P=0.09). In future, there is need to recruit more mice numbers and find out the immune

mechanisms involved and possibly combine with MCF homogenate or other antigens for

potency before applying in the field.

59

Figure 10: Survival analysis curves of five groups of immunized BALB/c mice. Overall

there was no significant difference (P=0.09 between rTc38630 and control groups.

rTc38630 = recombinant protein; rGST = recombinant glutathione S-transferases; MCF =

metacyclic form homogenate; BSF = bloodstream form homogenate; PBS = Phosphate

buffered saline.

60

Figure 11: Median survival period for five groups of immunized BALB/c mice. Overall

there was no significant difference (P>0.05 between rTc38630 and control groups.


metacyclic form homogenate; BSF = bloodstream form homogenate; PBS = Phosphate

buffered saline.

61

Figure 12: Parasitaemia trends of immunized BALB/c mice in five groups. One mouse in rTc38630 survived one day longer as shown by a drop of parasitaemia at day 10 compared to control groups. rTc38630 = recombinant protein; rGST = recombinant glutathione S-transferases; MCF = metacyclic form homogenate; BSF = bloodstream form homogenate; PBS = Phosphate buffered saline.

62

Figure 13: Mean pre-patent period for five groups of immunized BALB/c mice. Overall

there was no significant difference (P=0.11 between rTc38630 and control groups.


metacyclic form homogenate; BSF = bloodstream form homogenate; PBS = phosphate

buffered saline.

63

General discussion

Trypanosomosis disease has been a subjected to study for a long time in both

humans and livestock (36, 55, 149) and efforts to control it date as far back as 1800s. This

dissertation highlights three main chapters with results and discussion, excluding the

chapter on general introduction. The current study’s main thrust was on use of recombinant

antigens in diagnostics and vaccinology. Indeed, the current trend in research is focused on

recombinant proteins and DNA technology and their application in drug targets, diagnosis

and vaccines (6, 78, 79, 113, 133, 158, 184).

In the first chapter, the TcIL3000.0.38630 (1,236 bp) gene was selected from many

genes of T. congolense in TriTryp data base (http://tritrypdb.org/tritrypdb/) that is a stage-

specific and relatively highly expressed in metacyclic and blood trypomastigotes (38).

According to previously obtained differential protein expression data, the gene is by 8.5

times expressed more in MCFs and BSFs than in PCFs and EMFs. Unlike its orthologue

ISG of BSF in T. brucei, it was found to be of a single copy gene. A recombinant protein

was expressed, purified and characterised. The protein was immunolocalized on the cell

surface and in the cytoplasm of MCF and BSF stages of trypanosomes. Moreover, the

protein was found to be antigenic in an experimental mouse model.

In the second chapter, an attempt was made to apply the recombinant protein in

serodiagnosis both in ELISA and ICT. It was hypothesized that since the decoded amino

acids domains for Tc38630 were similar to ISG of T. brucei albeit in the BSF, it was

potentially antigenic. Whereas this technology worked well in the mouse model, it was

64

inconclusive when applied on field cattle samples. Indeed, all assays that are developed in

the laboratory they are expected to be translated into field application. The source of failure

was attributed to the GST tag of the protein which had to be taken into consideration not to

be a confounder. Therefore, this opens ground for more research to conclusively test the

hypothesis that was initially set out in the objectives. In addition, if the assay is found

successful it would be prudent to combine with other recombinant antigens to achieve the

detection of all pathogenic trypanosomes.

In the third and last chapter, a recombinant protein was applied in vaccinology since

the rTc38630 protein was an orthologue of ISG of T. brucei. The use of VSG has been tried

before as a vaccine but always failed because of antigenic variation and production of IgM

isotype which is often short lived (72). Most works which report partial protection from

trypanosomosis use low dose challenge, 1,000 parasites therefore the results would be

inconclusive. The vaccination with flagella pocket did not elicit any protection or rather it

was short lived (131). Now the strategy changed to anti-disease vaccine whereby the

pathology rather than the parasite is the target (72). This approach has been applied in the

field whereby cattle were immunized with congopain, a cysteine protease (CP) and the

trypanosusceptible cattle showed higher IgG (6). However, no follow up has been done on

the same. The experimental application of rTc38630 as a vaccine need further trials using

larger number of murine model and determine the immune responses involved, and if

possible, combine with other antigens to potentiate the protective effects.

65

Conclusion

This current study on the use of recombinant technology in diagnostics and vaccine

will serve as a benchmark for more research to improve on sensitivity and specificity of

assays and for effective anti-disease vaccine approach, respectively, in the control of

African trypanosomosis. Moreover, as a way is found out in serodiagnostics to target IgM

to detect for current infections, this class of antibody is also required to be induced to

protect the host from trypanosomes. This is what has been observed in trypanotolerant

animals (122).

For sustainable and successful control of many diseases in the developing world, the

availability of field applicable diagnostics that are cheap, reliable, simple in design and

application, and which provide immediate results, is crucial (60). In the same vein,

vaccination against trypanosomosis should be the focal point in research to fight against

this disease given the trypanotolerance has been reported in both man and livestock (72).

Indeed, this will eventually accentuate the agricultural development in sub-Sarahan Africa.

In the foregoing of this dissertation, a novel recombinant protein from T.

congolense was expressed, characterised and purified. An endeavour was made to apply the

recombinant protein in serodiagnosis and immunization studies. However, further work

need to be done to check on whether it can diagnose and protect against other trypanosomes,

and successfully actualize its application in the field.

66

Acknowledgements

This study was carried out at the National Research Center for Protozoan Diseases

(NRCPD), Obihiro University of Agriculture and Veterinary Medicine (OUAVM). My

utmost respect goes to the current and former directors of NRCPD for offering me a

conducive environment to undertake my studies. The study was financially supported by

the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan and

Japan Society for the Promotion of Science (JSPS). This work was in part supported by

JST/JICA SATREPS.

I sincerely thank my supervisors Prof. Noboru Inoue and Prof. Shin-ichiro Kawazu

for accepting me in their laboratory and their constant mentorship during my PhD studies.

In the same vein, I am grateful to Prof. Hiroshi Suzuki and Prof. Tadashi Itagaki (Iwate

University) for their help and making my studies possible. I also humbly acknowledge Prof.

Kazuaki Takehara and Associate Prof. Yasuhiro Takashima for carefully reviewing this

dissertation. I especially, owe my immense appreciation to Prof. Noboru Inoue for guidance,

motivation and all the relentless support he generously offered me throughout my stay in

Japan. Indeed, I will forever be indebted to him.

My special thanks go to current and former laboratory members of Vaccine and

Mosquito Units just to mention but a few, Dr Oriel Thekisoe, Dr Dusit Laohasinnarong, Dr

Jose Angeles (Joma), Dr Hassan Hakimi, Keisuke Suganuma (Kero), Thuy-Thu Nguyen,

Miho Usui, Hirono Masuda-Suganuma, Shino Yamasaki, Mo Zhou, Ruttayaporn

67

Ngassaman (Thom) and Victor Zulu for their various assistance in one way or another in

the course of my doctoral studies.

To the current and former international students in Japan, I am glad to have enjoyed

their incessant friendship and company throughout my stay in Japan. My great appreciation

to all individuals who I have not mentioned herein but in one way or another contributed in

many ways to my success. To you all, I say, a big thank you.

My earnest thanks go to my director, Kenya Agricultural Research Institute (KARI)

for granting me a paid study leave and Japanese Government for offering me the highly

competitive Monbukagakusho (MEXT) scholarship to pursue knowledge in Japan. Indeed,

I will endeavour to impart the same to my countrymen and women while maintaining

collaborations with Japan.

Finally, I am heartily blessed to have known Obihiro community for their extended

kindness and hospitality for every moment of my stay in Obihiro, Japan.

Last but not least, I pay profound tribute to my wonderful family, Esther and the

children for their everlasting love and bearing with my long absence from home. Their

daily tireless communication and prayers really encouraged me to soldier on. For, I

dedicate this dissertation to them – Esther, Cyril and Yuki.

68

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Title Identification of a New Metacyclic/Blood Stage