title identification of a new metacyclic/blood stage
TRANSCRIPT
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 Materials and methods ..................................................................................... 36
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
2.2.8 Data management and analysis ................................................................ 39
2.3 Results and discussion ..................................................................................... 40
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 Materials and methods ..................................................................................... 51
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
3.2.6 Data management and analysis ................................................................ 53
3.3 Results and discussion ..................................................................................... 54
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
Figu
re 5
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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 Materials and methods
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.
2.2.8 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. 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
2.3 Results and discussion
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 Materials and methods
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.
3.2.6 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. 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
3.3 Results and discussion
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.
rTc38630 = recombinant protein; rGST = recombinant glutathione S-transferases; MCF =
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.
rTc38630 = recombinant protein; rGST = recombinant glutathione S-transferases; MCF =
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
References
1) Acosta-Serrano A, Vassella E, Liniger M, Kunz Renggli C, Brun R, Roditi I,
Englund PT (2001). The surface coat of procyclic Trypanosoma brucei:
programmed expression and proteolytic cleavage of procyclin in the tsetse fly. Proc
Natl Acad Sci USA 98(4): 1513-1518.
2) Alarcon JB, Waine GW, McManus DP (1999). DNA vaccines: technology and
application as anti-parasite and anti-microbial agents. Adv Parasitol. 42: 343-410.
3) Alsford S, Eckert S, Baker N, Glover L, Sanchez-Flores A, Leung KF, Turner DJ,
Field MC, Berriman M, Horn D (2012). High-throughput decoding of
antitrypanosomal drug efficacy and resistance. Nature 482(7384): 232-236.
4) Anene BM, Onah DN, Nawa Y (2001). Drug resistance in pathogenic African
trypanosomes: what hopes for the future? Vet Parasitol. 96: 83-100.
5) Antoine-Moussiaux N, Buscher P, Desmecht D (2009). Host-parasite interactions in
trypanosomiasis: on the way to an antidisease strategy. Infect Immun. 77(4): 1276-
1284.
6) Authié E, Boulangé A, Muteti D, Lalmanach G, Gauthier F, Musoke AJ (2001).
Immunisation of cattle with cysteine proteinases of Trypanosoma congolense:
targetting the disease rather than the parasite. Int J Parasitol. 31(13): 1429-1433.
69
7) Balaban N, Waithaka HK, Njogu AR, Goldman R (1995). Intracellular antigens
(microtubule-associated protein copurified with glycosomal enzymes)--possible
vaccines against trypanosomiasis. J Infect Dis. 172(3): 845-850.
8) Bannai H, Sakurai T, Inoue N, Sugimoto C, Igarashi I (2003). Cloning and
sxpression of mitochondrial heat shock protein 70 of Trypanosoma congolense and
potential use as a diagnostic antigen. Clin Diagn Lab Immunol. 10(5): 926-933.
9) Barrett MP, Croft SL (2012). Management of trypanosomiasis and leishmaniasis. Br
Med Bull. 104:175-196.
10) Barry JD (1997). The relative significance of mechanisms of antigenic variation in
African trypanosomes. Parasitol Today 13(6): 212-218.
11) Bastin P, Sherwin T, Gull K (1998). Paraflagellar rod is vital for trypanosome
motility. Nature 391(6667): 548.
12) Behbehani AM (1983). The smallpox story: life and death of an old disease.
Microbiol Rev. 47(4): 455.
13) Blake AD; Junyi M, Jia-Qiang H (2009). "Identifying Cardiotoxic Compounds".
Genetic Engineering & Biotechnology News. Technical Note 29 (9) (Mary Ann
Liebert). pp. 34–35.
14) Bosompem KM, Ayi I, Anyan WK, Arishima T, Nkrumah FK, Kojima S (1997). A
monoclonal antibody-based dipstick assay for diagnosis of urinary schistosomiasis.
Trans R Soc Trop Med Hyg. 91(5): 554-556.
70
15) Bossard G, Boulange A, Holzmuller P, Thévenon S, Patrel D, Authié E (2010).
Serodiagnosis of bovine trypanosomosis based on HSP70/BiP inhibition ELISA.
Vet Parasitol. 173 (1–2): 39-47.
16) Boulangé A, Katende J, Authié E (2002). Trypanosoma congolense: expression of a
heat shock protein 70 and initial evaluation as a diagnostic antigen for bovine
trypanosomosis. Exp Parasitol. 100(1): 6-11.
17) Boulangé A, Serveau C, Brillard M, Minet C, Gauthier F, Diallo A, Lalmanach G,
Authié E (2001). Functional expression of the catalytic domains of two cysteine
proteinases from Trypanosoma congolense. Int J Parasitol. 31(13): 1435-1440.
18) Boyer TD (1989). "The glutathione S-transferases: an update". Hepatology. 9(3):
486–496.
19) Boyt WP (1980). “A field guide for the diagnosis, treatment and prevention of
African animal trypanosomiasis”, FAO, pp. 88-119.
20) Brito LA, Singh M (2011). Acceptable levels of endotoxin in vaccine formulations
during preclinical research. J Pharm Sci. 100(1): 34-37.
21) Broadhead R, Dawe HR, Farr H, Griffiths S, Hart SR, Portman N, Shaw MK,
Ginger ML, Gaskell SJ, McKean PG, Gull K (2006). Flagellar motility is required
for the viability of the bloodstream trypanosome. Nature 440(7081): 224-227.
22) Bütikofer P, Vassella E, Boschung M, Renggli CK, Brun R, Pearson TW, Roditi I
(2002). Glycosylphosphatidylinositol-anchored surface molecules of Trypanosoma
71
congolense insect forms are developmentally regulated in the tsetse fly. Mol
Biochem Parasitol. 119(1): 7-16.
23) Carvalho J, Monteiro GA, Atouguia J, Prazeres DMF, Rodgers J (2008).
Developing a vaccine for African trypanosomiasis: only wishful thinking or a
definite possibility? BMC Proc. 2(1): P9.
24) Chandler J, Gurmin T, Robinson N (2000). The place of gold in rapid tests. IVD
Technol., 6: 37–49.
25) Chappuis F, Loutan L, Simarro P, Lejon V, Büscher P (2005). Options for field
diagnosis of human African trypanosomiasis. Clin Microbiol Rev. 18(1): 133-146.
26) Chappuis F, Rijal S, Soto A, Menten J, Boelaert M (2006). A meta-analysis of the
diagnostic performance of the direct agglutination test and rK39 dipstick for
visceral leishmaniasis. BMJ. 333(7571): 723.
27) Cheng Z, Goto Y, Nguyen T-T, Sakurai T, Zang J, Kawazu S, Inoue N (2012).
Preliminary studies for development of an immunochromatographic test (ICT) for
detection of antibody against salivarian trypanosomes by using recombinant
ribosomal P0 protein. Protozool. Res. 22: 10-18.
28) Comfort N (1999). Essay reviews. [Review of: Rabinow P. Making PCR: a story of
biotechnology. University of Chicago Press, and Fujimura J. Crafting science: a
socio-history of the quest for the genetics of cancer. Harvard University Press]. Oral
Hist Rev. 26(2):181-186.
72
29) Connor, R. J. (1994). African Animal Trypanosomiasis. In: Coetzer, W. A. J.
Thomson, G. R. and Tustin, R. C. (eds.), Infectious Diseases of Livestock with a
Special Reference to Southern Africa. Oxford University Press, South Africa, pp.
167-212.
30) Coustou V, Plazolles N, Guegan F, Baltz T (2012). Sialidases play a key role in
infection and anaemia in Trypanosoma congolense animal trypanosomiasis. Cell
Microbiol. 14(3): 431-445.
31) Desquesnes M, Dávila AM (2002). Applications of PCR-based tools for detection
and identification of animal trypanosomes: a review and perspectives. Vet Parasitol.
109(3-4): 213-231.
32) Donelson JE (2003) Antigenic variation and the African trypanosome genome. Acta
Trop. 85(3): 391-404.
33) Donelson JE, Hill KL, El-Sayed NM (1998). Multiple mechanisms of immune
evasion by African trypanosomes. Mol Biochem Parasitol. 91(1): 51-66.
34) Eisler MC, Dwinger RH, Majiwa PAO, Picozzi K (2004). Diagnosis and
epidemiology of African animal trypanosomiasis. In: The trypanosomiasis. Eds I
Maudlin, P H Holmes and M A Miles. Wallingford, UK: CABI International, pp
253-267.
35) Engstler M, Boshart M (2004). Cold shock and regulation of surface protein
trafficking convey sensitization to inducers of stage differentiation in Trypanosoma
brucei. Genes Dev. 18(22): 2798-2811.
73
36) Enyaru JC, Ouma JO, Malele II, Matovu E, Masiga DK (2010). Landmarks in the
evolution of technologies for identifying trypanosomes in tsetse flies. Trends
Parasitol. 26(8): 388-394.
37) Esser KM, Schoenbechler MJ, Gingrich JB (1982). Trypanosoma rhodesiense blood
forms express all antigen specificities relevant to protection against metacyclic
(insect form) challenge. J Immunol. 129(4): 1715-1718.
38) Eyford BA, Sakurai T, Smith D, Loveless B, Hertz-Fowler C, Donelson JE, Inoue N,
Pearson TW (2011). Differential protein expression throughout the life cycle of
Trypanosoma congolense, a major parasite of cattle in Africa. Mol Biochem
Parasitol. 177(2): 116-125.
39) FAO (2000a). Impacts of trypanosomosis on African agriculture, by B.M. Swallow.
PAAT Technical and Scientific Series No. 2. Rome.
40) FAO (2000b). A field guide for the diagnosis, treatment and prevention of African
animal trypanosomosis, 2nd edition. FAO, Rome, Italy.
41) Ferella M (2008). Detection and characterization of novel proteins in Trypanosoma
cruzi. PhD dissertation, Karolinska Institute, Stockholm, Sweden.
42) Ferguson MA (1999). The structure, biosynthesis and functions of
glycosylphosphatidylinositol anchors, and the contributions of trypanosome
research. J Cell Sci. 112 (17): 2799-2809.
43) Fleischer B (2004). Editorial: 100 years ago: Giemsa's solution for staining of
plasmodia. Trop Med Int Health 9(7): 755-756.
74
44) Gardiner PR (1989). Recent studies of biology of Trypanosoma vivax. Adv
Parasitol. 28: 229-317.
45) Geerts S, Holmes PH, Eisler MC, Diall O (2001). African bovine trypanosomiasis:
the problem of drug resistance. Trends Parasitol. 17(1): 25-28.
46) Geojith G, Dhanasekaran S, Chandran SP, Kenneth J (2011). Efficacy of Loop
Mediated Isothermal Amplification (LAMP) assay for the laboratory identification
of Mycobacterium tuberculosis isolates in a resource limited setting. J Microbiol
Methods 84(1): 71-73.
47) Goo YK, Lee N, Terkawi MA, Luo Y, Aboge GO, Nishikawa Y, Suzuki H, Kim S,
Xuan X (2012). Development of a rapid immunochromatographic test using a
recombinant thrombospondin-related adhesive protein of Babesia gibsoni. Vet
Parasitol. 190(3-4): 595-598.
48) Goto Y, Duthie MS, Kawazu S, Inoue N, Carter D (2011). Biased cellular locations
of tandem repeat antigens in African trypanosomes. Biochem Biophys Res Commun.
405(3): 434-438.
49) Grebaut P, Chuchana P, Brizard J-P, Demettre E, Seveno M, Bossard G, Jouin P,
Vincendeau P, Bengaly Z, Boulange A, Cuny G, Holmuller P (2009). Identification
of total and differentially expressed excreted-secreted proteins from Trypanosoma
congolense strains exhibiting different virulence and pathogenicity. Int J Parasitol.
39(10): 1137-1150.
75
50) Hagebock JM (1992). Dourine. Foreign animal disease report, United States
Department of Agriculture, 19-4: 9-13.
51) Hendry KA, Vickerman K (1988). The requirement for epimastigote attachment
during division and metacyclogenesis in Trypanosoma congolense. Parasitol Res.
74(5): 403-408.
52) Herbert WJ, Lumsden WHR (1976). Trypanosoma brucei: A rapid matching
method for estimating the hosts parasitemia. Exp Parasitol. 40(3): 427-431.
53) Hirumi H, Hirumi K (1991) In vitro cultivation of Trypanosoma congolense
bloodstream forms in the absence of feeder cell layers. Parasitology 102 Pt 2: 225-
236.
54) Hirumi H, Hirumi K, Doyle JJ, Cross GA (1980). In vitro cloning of animal-
infective bloodstream forms of Trypanosoma brucei. Parasitology 80(2): 371-382.
55) Hoare CA (1972). Trypanosomes of mammals: a zoological monograph. Oxford
and Edinburgh: Blackwell Scientific Publications.
56) Hopkins JS, Chitambo H, Machila N, Luckins AG, Rae PF, van den Bossche P
Eisler MC (1998). Adaptation and validation of antibody-ELISA using dried blood
spots on filter paper for epidemiological surveys of tsetse-transmitted
trypanosomosis in cattle. Prev Vet Med. 37: 91-99.
57) Huang X, Xuan X, Hirata H, Yokoyama N, Xu L, Suzuki N, Igarashi I (2004).
Rapid immunochromatographic test using recombinant SAG2 for detection of
antibodies against Toxoplasma gondii in cats. J Clin Microbiol. 42(1): 351-353.
76
58) Huang X, Xuan X, Verdida RA, Zhang S, Yokoyama N, Xu L, Igarashi I (2006).
Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and
B. equi infections in horses. Clin Vaccine Immunol. 13(5): 553-555.
59) Huang X, Xuan X, Xu L, Zhang S, Yokoyama N, Suzuki N, Igarashi I (2004).
Development of an immunochromatographic test with recombinant EMA-2 for the
rapid detection of antibodies against Babesia equi in horses. J Clin Microbiol.
42(1): 359-361.
60) Hutchinson OC, Webb H, Picozzi K, Welburn S Carrington M (2004). Candidate
protein selection for diagnostic markers of African trypanosomiasis. Trends
Parasitol. 20 (11): 519-523.
61) Ikezawa H (2002). Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol
Pharm Bull. 25(4): 409-417.
62) ILRAD (1989). Annual report of the International Laboratory for Research on
Animal Diseases: ilri.org.
63) Iwamoto T, Sonobe T, Hayashi K (2003). Loop-mediated isothermal amplification
for direct detection of Mycobacterium tuberculosis complex, M. avium, and M.
intracellulare in sputum samples. J Clin Microbiol. 41(6): 2616-2622.
64) Jackson AP, et al. (2012) Antigenic diversity is generated by distinct evolutionary
mechanisms in African trypanosome species. Proc Natl Acad Sci USA 109(9):
3416-3421.
77
65) Jackson DG, Windle HJ, Voorheis HP (1993). The identification, purification, and
characterization of two invariant surface glycoproteins located beneath the surface
coat barrier of bloodstream forms of Trypanosoma brucei. J Biol Chem. 268(11):
8085-8095.
66) Jamonneau V, Ravel S, Garcia A, Koffi M, Truc P, Laveissière C, Herder S,
Grébaut P, Cuny G, Solano P (2004). Characterization of Trypanosoma brucei s.l.
infecting asymptomatic sleeping-sickness patients in Côte d'Ivoire: a new genetic
group? Ann Trop Med Parasitol. 98(4): 329-337.
67) Jia H, Liao M, Lee E, Nishikawa Y, Inokuma H, Ikadai H, Matsuu A, Igarashi I,
Xuan X (2007). Development of an immunochromatographic test with recombinant
BgSA1 for the diagnosis of Babesia gibsoni infection in dogs. Parasitol Res.
100(6): 1381-1384.
68) Kateregga J, Lubega GW, Lindblad EB, Authié E, Coetzer TH, Boulangé AF
(2012). Effect of adjuvants on the humoral immune response to congopain in mice
and cattle. BMC Vet Res. 8: 63.
69) Kennedy PG (2008). Diagnosing central nervous system trypanosomiasis: two stage
or not to stage? Trans R Soc Trop Med Hyg. 102: 306-307.
70) Kim CM, Blanco LB, Alhassan A, Iseki H, Yokoyama N, Xuan X, Igarashi I
(2008). Development of a rapid immunochromatographic test for simultaneous
serodiagnosis of bovine babesioses caused by Babesia bovis and Babesia bigemina.
Am J Trop Med Hyg. 78(1): 117-121.
78
71) Kristjanson PM, Swallow BM, Rowlands GJ, Kruska RL, de Leeuw PN (1999).
Measuring the costs of African animal trypanosomosis, the potential benefits of
control and returns to research. Agr Syst. 59(1): 79-98.
72) La Greca F, Magez S (2011). Vaccination against trypanosomiasis: can it be done or
is the trypanosome truly the ultimate immune destroyer and escape artist? Hum
Vaccin. 7(11): 1225-1233.
73) Laker CD (1998). Assessment of the economic impact of bovine trypanosomosis
and its control in dairy cattle in Mukono county, Uganda. Ph.D Thesis, Makerere
University, Kampala, Uganda.
74) Lança AS, de Sousa KP, Atouguia J, Prazeres DM, Monteiro GA, Silva MS (2011).
Trypanosoma brucei: immunisation with plasmid DNA encoding invariant surface
glycoprotein gene is able to induce partial protection in experimental African
trypanosomiasis. Exp Parasitol. 127(1): 18-24.
75) Lanham SM and Godfrey DG (1970). Isolation of salivarian trypanosomes from
man and other mammals using DEAE-cellulose. Exp Parasitol., 28: 521-534.
76) Leak SGA (1999). Tsetse biology and ecology: their role in the epidemiology and
control of trypanosomiasis. Wallingford, UK: CABI Publishing.
77) Lequin R M (2005). Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent
Assay (ELISA). Clin Chem. 51 (12): 2415-2418.
78) Li SQ, Fung MC, Reid SA, Inoue N, Lun ZR (2007). Immunization with
recombinant beta-tubulin from Trypanosoma evansi induced protection against T.
79
evansi, T. equiperdum and T. b. brucei infection in mice. Parasite Immunol. 29(4):
191-199.
79) Li SQ, Yang WB, Ma LJ, Xi SM, Chen QL, Song XW, Kang J, Yang LZ (2009).
Immunization with recombinant actin from Trypanosoma evansi induces protective
immunity against T. evansi, T. equiperdum and T. b. brucei infection. Parasitol Res.
104(2): 429-435.
80) Liao M, Zhang S, Xuan X, Zhang G, Huang X, Igarashi I, Fujisaki K (2005).
Development of rapid immunochromatographic test with recombinant NcSAG1 for
detection of antibodies to Neospora caninum in cattle. Clin Diagn Lab Immunol.
12(7): 885-887.
81) Lubega GW, Byarugaba DK, Prichard RK (2002a). Immunization with a tubulin-
rich preparation from Trypanosoma brucei confers broad protection against African
trypanosomosis. Exp Parasitol. 102(1): 9-22.
82) Lubega GW, Ochola DO, Prichard RK (2002b). Trypanosoma brucei: anti-tubulin
antibodies specifically inhibit trypanosome growth in culture. Exp Parasitol. 102(3-
4): 134-142.
83) Luckins AG (1992). Diagnostic methods in trypanosomiasis of livestock. World
Anim Rev. 70/71: 15-20.
80
84) Luckins, A. G. (1977). Detection of antibodies in trypanosome-infected cattle by
means of a microplate enzyme-linked immunosorbent assay. Trop Anim Health
Prod. 9: 53-62.
85) Lumsden WH, Kimber CD, Evans DA, Doig SJ (1979). Trypanosoma brucei:
Miniature anion-exchange centrifugation technique for detection of low
parasitaemias: Adaptation for field use. Trans R Soc Trop Med Hyg. 73(3): 312-317.
86) Luo Y, Jia H, Terkawi MA, Goo YK, Kawano S, Ooka H, Li Y, Yu L, Cao S,
Yamagishi J, Fujisaki K, Nishikawa Y, Saito-Ito A, Igarashi I, Xuan X (2011).
Identification and characterization of a novel secreted antigen 1 of Babesia microti
and evaluation of its potential use in enzyme-linked immunosorbent assay and
immunochromatographic test. Parasitol Int. 60(2): 119-125.
87) Magez S, Caljon G, Tran T, Stijlemans B, Radwanska M (2010). Current status of
vaccination against African trypanosomiasis. Parasitology. 137(14): 2017-2027.
88) Magez S, Radwanska M (2009). African trypanosomiasis and antibodies:
implications for vaccination, therapy and diagnosis. Future Microbiol. 4(8): 1075-
1087.
89) Majiwa PAO (1998). Application of the polymerase chain reaction for detection of
trypanosomes. In: “Towards Livestock Disease Diagnosis and Control in the 21st
Century.” Proceedings of a Symposium, Vienna, 7-11 April 1997.
81
90) Manful T, Mulindwa J, Frank FM, Clayton CE, Matovu E (2010). A search for
Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and
targeted cloning. PLoS One. 5(3): e9630.
91) Marcotty T, Simukoko H, Berkvens D, Vercruysse J, Praet N, Van den Bossche P
(2008). Evaluating the use of packed cell volume as an indicator of trypanosomal
infections in cattle in eastern Zambia. Prev Vet Med. 87(3-4): 288-300.
92) Masake RA, Nantulya VM (1991). Sensitivity of an antigen detection enzyme
immunoassay for diagnosis of Trypanosoma congolense infections in goats and
cattle. J Parasitol. 77: 231-236.
93) Masake RA, Njuguna JT, Brown CC, Majiwa PA (2002). The application of PCR-
ELISA to the detection of Trypanosoma brucei and T. vivax infections in livestock.
Vet Parasitol. 105(3): 179-189.
94) Masiga DK, Smyth AJ, Hayes P, Bromidge TJ, Gibson WC (1992). Sensitive
detection of trypanosomes in tsetse flies by DNA amplification. Int J Parasitol.
22(7): 909-918.
95) Matthews KR (2005). The developmental cell biology of Trypanosoma brucei. J
Cell Sci. 118(2): 283-290.
96) Matthews KR, Gull K (1997). Commitment to differentiation and cell cycle re-entry
are coincident but separable events in the transformation of African trypanosomes
from their bloodstream to their insect form. J Cell Sci. 110(20): 2609-2618.
82
97) May K (1991). Home tests to monitor fertility. Am J Obstet Gynecol. 165: 2000–
2002.
98) McCalla AF (1994). Agriculture and Food Needs to 2025: Why We Should Be
Concerned, Consultative Group on International Agricultural Research, Washington,
D.C.
99) McDermott JJ, Coleman PG (2001). Comparing apples and oranges - model-based
assessment of different tsetse-transmitted trypanosomosis control strategies. Int J
Parasitol. 31(5-6): 603-609.
100) Melville SE, Majiwa PAO, Tait A (2004). The African trypanosome genome. In:
The trypanosomiasis. Eds I Maudlin, P H Holmes and M A Miles. Wallingford, UK:
CABI International, pp 39-57.
101) Mkunza F, Olaho WM, Powell CN (1995). Partial protection against natural
trypanosomiasis after vaccination with a flagellar pocket antigen from Trypanosoma
brucei rhodesiense. Vaccine 13(2): 151-154.
102) Mochabo KM, Zhou M, Suganuma K, Kawazu S, Suzuki Y, Inoue N (2013).
Expression, immunolocalization and serodiagnostic value of Tc38630 protein from
Trypanosoma congolense. Parasitol Res. 112(9): 3357-3363.
103) Mora M, Veggi D, Santini L, Pizza M, Rappuoli R (2003). Reverse vaccinology.
Drug Discov Today 8(10): 459-464.
104) Murphy K (2012). Janeway’s Immunobiology. 8th edn. Garland Science, Taylor and
Francis Group, LLC, pp.669-716.
83
105) Murray M, Dexter TM (1988). Anaemia in bovine African trypanosomiasis. Acta
Trop. 45(4): 389-432.
106) Murray M, Gray AR (1984). The current situation on animal trypanosomiasis in
Africa. Prev Vet Med. 2: 23-30.
107) Murray M, Murray PK, McIntyre WI (1977). An improved parasitological
technique for the diagnosis of African trypanosomiasis. Trans R Soc Trop Med Hyg.
71(4): 325-326.
108) Murray RK, Granner DK, Mayes PA, Rodwell VW (2003). Harper’s Illustrated
Biochemistry 26th ed. McGraw-Hill Companies, Inc., USA.
109) Naessens J (2006). Bovine trypanotolerance: A natural ability to prevent severe
anaemia and haemophagocytic syndrome? Int J Parasitol. 36(5):521-528.
110) Nantulya VM (1990). Trypanosomiasis in domestic animals: the problems of
diagnosis. Rev Sci Tech. 9: 357-367.
111) Nantulya VM (1994). Suratex : A simple latex agglutination test for diagnosis of
Trypanosoma evansi infections (Surra). Trop Med Parasitol. 45: 9-12.
112) Nantulya VM, Lindqvist K J (1989). Antigen detection enzyme immunoassays for
the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infections in
cattle. Trop Med Parasitol. 40: 267-272.
84
113) Nguyen T-T, Goto Y, Lun Z-R, Kawazu S-I, Inoue N (2012). Tandem repeat
protein as potential diagnostic antigen for Trypanosoma evansi infection. Parasitol
Res. 110(2): 733-739.
114) Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, Thompson
RC, Ndung'u JM (2008). African trypanosomiasis: sensitive and rapid detection of
the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of
parasite DNA. Int J Parasitol. 38(5): 589-599.
115) Nolan DP, Jackson DG, Windle HJ, Pays A, Geuskens M, Michel A, Voorheis HP,
Pays E (1997). Characterization of a novel, stage-specific, invariant surface protein
in Trypanosoma brucei containing an internal, serine-rich, repetitive motif. J Biol
Chem. 272(46): 29212-29221.
116) Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T
(2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res.
28(12): E63.
117) O'Beirne C, Lowry CM, Voorheis HP (1998). Both IgM and IgG anti-VSG
antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of
Trypanosoma brucei without damage to the parasite. Mol Biochem Parasitol. 91(1):
165-193.
118) OIE (2013). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 7
edn. OIE, Paris.
119) Omamo SW, d'Ieteren GD (2003). Managing animal trypanosomosis in Africa:
issues and options. Rev Sci Tech. 22(3): 989-1002.
85
120) Overath P, Engstler M (2004). Endocytosis, membrane recycling and sorting of
GPI-anchored proteins: Trypanosoma brucei as a model system. Mol Microbiol.
53(3): 735-744.
121) Paek SH, Lee SH, Cho JH, Kim YS (2000). Development of rapid one-step
immunochromatographic assay. Methods 22(1): 53-60.
122) Pan W, Ogunremi O, Wei G, Shi M, Tabel H (2006). CR3 (CD11b/CD18) is the
major macrophage receptor for IgM antibody-mediated phagocytosis of African
trypanosomes: diverse effect on subsequent synthesis of tumor necrosis factor alpha
and nitric oxide. Microbes Infect. 8(5): 1209-1218.
123) Pays E (2006). The variant surface glycoprotein as a tool for adaptation in African
trypanosomes. Microbes Infect. 8(3): 930-937.
124) Perry BD, Randolph TF (1999). Improving the assessment of the economic impact
of parasitic diseases and of their control in production animals. Vet Parasitol. 84(3-
4): 145-168.
125) Pham HM, Nakajima C, Ohashi K, Onuma M (2005). Loop-mediated isothermal
amplification for rapid detection of Newcastle disease virus. J Clin Microbiol.
43(4): 1646-1650.
126) Picozzi K, Tilley A, Fevre EM, Coleman PG, Magona JW, Odiit M, Eisler MC,
Welburn SC (2002). The diagnosis of trypanosome infections: Applications of novel
technology for reducing disease risk. Afr J Biotechnol. 1(2): 39-45.
86
127) Poon LLM, Wong BWY, Ma EHT, Chan KH, Chow LMC, Abeyewickreme W,
Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JSM (2006). Sensitive
and inexpensive molecular test for falciparum malaria: detecting Plasmodium
falciparum DNA directly from heat-treated blood by loop-mediated isothermal
amplification. Clin Chem. 52(2): 303-306.
128) Posthuma-Trumpie GA, Korf J, van Amerongen A (2009). Lateral flow (immuno)
assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal
Bioanal Chem. 393(2): 569-582.
129) Radostitis OM, Gay CC, Hinchcliff KW, Constable PD (2007). Diseases associated
with trypanosomes (trypanosomoses). In: A Textbook of the Diseases of Cattle,
Sheep, Pigs, Goats, and Horses, 10th ed. Elsevier Science Health Science Division,
pp. 1531-1540.
130) Radwanska M, Guirnalda P, De Trez C, Ryffel B, Black S, Magez S (2008).
Trypanosomiasis-induced B cell apoptosis results in loss of protective anti-parasite
antibody responses and abolishment of vaccine-induced memory responses. PLoS
Pathog. 4(5): e1000078.
131) Radwanska M, Magez S, Dumont N, Pays A, Nolan D, Pays E (2000). Antibodies
raised against the flagellar pocket fraction of Trypanosoma brucei preferentially
recognize HSP60 in cDNA expression library. Parasite Immunol. 22(12): 639-650.
132) Rajput ZI, Hu S, Xiao C, Arijo AG (2007). Adjuvant effects of saponins on animal
immune responses. J Zhejiang Univ Sci B. 8(3): 153-161.
87
133) Ramey K, Eko FO, Thompson WE, Armah H, Igietseme JU, Stiles JK (2009).
Immunolocalization and challenge studies using a recombinant Vibrio cholerae
ghost expressing Trypanosoma brucei Ca(2+) ATPase (TBCA2) antigen. Am J Trop
Med Hyg. 81(3): 407-415.
134) Rappuoli R (2000). Reverse vaccinology. Curr Opin Microbiol. 3(5):445-450.
135) Rappuoli R (2001). Reverse vaccinology, a genome-based approach to vaccine
development. Vaccine 19(17-19): 2688-2691.
136) Rasooly R, Balaban N (2004). Trypanosome microtubule-associated protein p15 as
a vaccine for the prevention of African sleeping sickness. Vaccine 22(8): 1007-
1015.
137) Raynaud JP, Sones KR, Friedheim EAH (1989). A review of cymelarsan – new
treatment proposed for animal trypanosomiasis due to Trypanosoma evansi and
other trypanosomes of T. brucei group. In: Proceedings of the 20th meeting:
International Scientific Council for the Trypanosomiasis Research and Control
(ISCTRC), Mombasa, Kenya ed. 115, OAU, pp. 334-338.
138) Richardson DC, Ciach M, Zhong KJ, Crandall I, Kain KC (2002). Evaluation of the
Makromed dipstick assay versus PCR for diagnosis of Plasmodium falciparum
malaria in returned travelers. J Clin Microbiol. 40(12): 4528-4530.
139) Roddy P, Goiri J, Flevaud L, Palma PP, Morote S, Lima N, Villa L, Torrico F,
Albajar-Viñas P (2008). Field evaluation of a rapid immunochromatographic assay
88
for detection of Trypanosoma cruzi infection by use of whole blood. J Clin
Microbiol. 46(6): 2022-2027.
140) Ross CA, Cardoso de Almeida ML, Turner MJ (1987). Variant surface
glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are
anchored by a glycolipid tail. Mol Biochem Parasitol. 22(2-3): 153-158.
141) Rudramurthy GR, Sengupta PP, Balamurugan V, Prabhudas K, Rahman H (2013).
PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein
(ISG) 75 gene. Vet Parasitol. 193(1-3): 47-58.
142) Sakurai T, Sugimoto C, Inoue N (2008) Identification and molecular
characterization of a novel stage-specific surface protein of Trypanosoma
congolense epimastigotes. Mol Biochem Parasitol. 161(1): 1-11.
143) Sambrook J, Russell DW (2001). Preparation and analysis of eukaryotic genomic
DNA Molecular Cloning. 3 edn. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, p 6.1-6.64.
144) Schofield L (2007). Rational approaches to developing an anti-disease vaccine
against malaria Microbes Infect. 9: 784–791.
145) Seed JR (1996). In: Baron S, editor. Medical Microbiology, 4th edition. Galveston
(TX): University of Texas Medical Branch at Galveston. Chapter 78.
146) Shapiro TA, Englund PT (1995). The structure and replication of kinetoplast DNA.
Annu Rev Microbiol. 49: 117-143.
89
147) Shaw APM (2004). The economics of African trypanosomiasis. In: The
trypanosomiasis. Eds I Maudlin, P H Holmes and M A Miles. Wallingford, UK:
CABI International, pp 369-402.
148) Silva MS, Prazeres DM, Lança A, Atouguia J, Monteiro GA (2009). Trans-sialidase
from Trypanosoma brucei as a potential target for DNA vaccine development
against African trypanosomiasis. Parasitol Res. 105(5): 1223-1229.
149) Steverding D (2008). The history of African trypanosomiasis. Parasit Vector 1(1):
3.
150) Stijlemans B, Baral TN, Guilliams M, Brys L, Korf J, Drennan M, Van Den
Abbeele J, De Baetselier P, Magez S (2007). A glycosylphosphatidylinositol-based
treatment alleviates trypanosomiasis-associated immunopathology. J Immunol.
179(6): 4003-4014.
151) Sundar S, Reed SG, Singh VP, Kumar PC, Murray HW (1998). Rapid accurate field
diagnosis of Indian visceral leishmaniasis. Lancet 351(9102): 563-565.
152) Swallow BM. (2000). Impact of Trypanosomiasis on African Agriculture. Vol. 2,
PAAT Technical and Scientific Series, FAO. Rome.
153) Tabel H, Wei G, Shi M (2008). T cells and immunopathogenesis of experimental
African trypanosomiasis. Immunol Rev. 225: 128-139.
90
154) Tanaka R, Yuhi T, Nagatani N, Endo T, Kerman K, Takamura Y, Tamiya E (2006).
A novel enhancement assay for immunochromatographic test strips using gold
nanoparticles. Anal Bioanal Chem. 385(8): 1414-1420.
155) Thekisoe OM, Kuboki N, Nambota A, Fujisaki K, Sugimoto C, Igarashi I, Yasuda J,
Inoue N (2007a). Species-specific loop-mediated isothermal amplification (LAMP)
for diagnosis of trypanosomosis. Acta Trop. 102(3): 182-189.
156) Thekisoe OM, Omolo JD, Swai ES, Hayashida K, Zhang J, Sugimoto C, Inoue N
(2007b). Preliminary application and evaluation of loop-mediated isothermal
amplification (LAMP) for detection of bovine theileriosis and trypanosomosis in
Tanzania. Onderstepoort J Vet Res. 74(4): 339-342.
157) Thuita JK, Wang MZ, Kagira JM, Denton CL, Paine F, Mdachi RE, Murilla GA,
Ching S, Boykin DW, Tidwell RR, Hall JE, Brun R (2012). Pharmacology of
DB844, an orally active aza analogue of Pafuramidine, in a monkey model of
second stage human African trypanosomiasis. PLoS Negl Trop Dis. 6(7): e1734.
158) Tran T, Claes F, Verloo D, De Greve H, Büscher P (2009). Towards a new
reference test for surra in camels. Clin Vaccine Immunol. 16: 999-1002.
159) Urwyler S, Studer E, Renggli CK, Roditi I (2007). A family of stage-specific
alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei.
Mol Microbiol. 63(1): 218-228.
91
160) Utz S, Roditi I, Kunz Renggli C, Almeida IC, Acosta-Serrano A, Bütikofer P
(2006). Trypanosoma congolense procyclins: unmasking cryptic major surface
glycoproteins in procyclic forms. Eukaryot Cell 5(8): 1430-1440.
161) Van den Bossche P (2001). Some general aspects of the distribution and
epidemiology of bovine trypanosomosis in southern Africa. Int J Parasitol. 31(5-6):
592-598.
162) Van der Ploeg LH (1987). Control of variant surface antigen switching in
trypanosomes. Cell 51(2): 159-161.
163) Vercruysse J, Knox DP, Schetters TP, Willadsen P (2004). Veterinary parasitic
vaccines: pitfalls and future directions. Trends Parasitol. 20(10): 488-492.
164) Vickerman K (1973). The mode of attachment of Trypanosoma vivax in the
proboscis of the tsetse fly Glossina fuscipes: an ultrastructural study of the
epimastigote stage of the trypanosome. J Protozool. 20(3): 394-404.
165) Vickerman K (1978). Antigenic variation in trypanosomes. Nature 263: 613.
166) Vickerman K, Barry JD (1982). African trypanosomiasis. In: Cohen S, Warren KS
(Eds), Immunology of Parasitic Infections. Blackwell Scientific Publications,
Oxford, pp. 204-260.
167) Vickerman K, Coombs GH (1999). Protozoan paradigms for cell biology. J Cell Sci.
112 (17): 2797-2798.
92
168) Vickerman K, Tetley L, Hendry KA, Turner CM (1988). Biology of African
trypanosomes in the tsetse fly. Biol Cell 64(2): 109-119.
169) Viera AJ, Garrett JM (2005). Understanding inter-observer agreement: the kappa
statistic. Fam Med. 37(5): 360-363.
170) Vitouley HS, Mungube EO, Allegye-Cudjoe E, Diall O, Bocoum Z, Diarra B,
Randolph TF, Bauer B, Clausen PH, Geysen D, Sidibe I, Bengaly Z, Van den
Bossche P, Delespaux V (2011). Improved PCR-RFLP for the detection of
diminazene resistance in Trypanosoma congolense under field conditions using
filter papers for sample storage. PLoS Negl Trop Dis. 5(7): e1223.
171) Voller A, De Savigny D (1981). Diagnostic serology of tropical parasitic diseases. J
Immunol Methods 46: 1-29.
172) Vordermeier HM, Lowrie DB, Hewinson RG (2003). Improved immunogenicity of
DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein
boosting. Vet Microbiol. 93(4): 349-359.
173) Washington JA (1996). Principles of Diagnosis: Serodiagnosis. In: Baron S, editor.
Medical Microbiology, 4th edition. Galveston (TX): University of Texas Medical
Branch at Galveston. Chapter 10.
174) Webster P, Russell DG (1993). The flagellar pocket of trypanosomatids. Parasitol
Today 9(6): 201-206.
93
175) Wei G, Bull H, Zhou X, Tabel H (2011). Intradermal infections of mice by low
numbers of african trypanosomes are controlled by innate resistance but enhance
susceptibility to reinfection. J Infect Dis. 203(3): 418-429.
176) WHO (2013). World Health Organization. Human African trypanosomiasis.
177) Wilson-Welder JH, Torres MP, Kipper MJ, Mallapragada SK, Wannemuehler MJ,
Narasimhan B (2009). Vaccine adjuvants: current challenges and future approaches.
J Pharm Sci. 98: 1278-1316.
178) Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH (2007).
A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT).
Am J Trop Med Hyg. 77(6): 119-127.
179) Woo PT (1969). The haematocrit centrifuge for the detection of trypanosomes in
blood. Can J Zool. 47(5): 921-923.
180) Zarlenga DS, Higgins J (2001). PCR as a diagnostic and quantitative technique in
veterinary parasitology. Vet Parasitol. 101(3-4): 215-230.
181) Ziegelbauer K, Multhaup G, Overath P (1992). Molecular characterization of two
invariant surface glycoproteins specific for the bloodstream stage of Trypanosoma
brucei. J Biol Chem. 267(15): 10797-10803.
182) Ziegelbauer K, Overath P (1992). Identification of invariant surface glycoproteins in
the blood-stream stage of Trypanosoma brucei. J Biol Chem. 267(15): 10791-10796.
94
183) Ziegelbauer K, Rudenko G, Kieft R, Overath P (1995). Genomic organization of an
invariant surface glycoprotein gene family of Trypanosoma brucei. Mol Biochem
Parasitol. 69(1): 53-63.
184) Ziegelbauer K, Stahl B, Karas M, Stierhof YD, Overath P (1993). Proteolytic
release of cell surface proteins during differentiation of Trypanosoma brucei.
Biochemistry 32(14): 3737-3742.
185) Zweygarth E, Kaminsky R (1990). Direct in vitro isolation of Trypanosoma brucei
brucei and T. b. evansi from disease hosts with low parasitaemia. Trop Med
Parasitol. 41(1): 56-58.