Brieuc-COSSIC-Dissertation-As time flies, adapting trypanosomiasis control methods through a longitudinal study of cattle management in an area of low Tsetse challenge South of Gabon

As time flies by, adapting trypanosomiasis control methods through a
longitudinal study of cattle management in an area of low Tsetse
challenge South of Gabon
By
Brieuc Cossic
May 2015
A dissertation submitted in partial fulfilment for the award of the Degree of
Master of Science in International Animal Health at the University of
Edinburgh
Word count: 15988 words
Ranch
Nyanga,
Gabon

Abstract
A longitudinal study was conducted in a cattle ranch, South of Gabon, to determine the
Diminazen-Aceturate Index (DAI) or Berenil Index among three different breeds, N’Damas,
Zebus and Ndapol, raised under identical management conditions. The objective was to
develop a tool to define more adapted trypanosomiasis control methods under the ranch’s
livestock conditions. Eighty-five cattle have been monitored for 22 weeks during the dry-season,
55 N’Damas, 20 Zebus and 10 Ndapol. A total of 2023 blood samples have been collected on a
weekly basis and were subjected to parasitological and haematological analysis. Moreover,
cattle were weighed on a monthly basis. Samples were examined using the buffy coat method
and the packed cell volume (PCV) value of each animal was also measured. Parasitemia was
evaluated with a microscopic counting method. Infected animals were treated with a single
intramuscular injection of Diminazen-Aceturate (8 mg/kg). 78 single infectious events have been
observed (3,8% CI 95% 3,1 to 4,8%), and a DAI of 1,45 for Zebus, 0,21 for adults N’Damas,
0,23 for calves N’Damas and 1,7 for Ndapol have been calculated. 42 animals remained clear
of infection, mostly N’Damas (32). Two trypanosome species were identified: Trypanosoma
congolense (96,2%) and T. vivax (3,8%). Zebus were significantly more often infected than
adults N’Damas (Chi-square = 69,1, P<0,001). Ndapol were significantly more often infected
than N’Damas calves (Chi-square = 17,49, P<0,001). The mean PCV value of the infected
animals was lower (26,6 for Zebus, 34,2 for adults N’Damas, 32,2 for calves N’Damas and 27,3
for Ndapol) compared to non-infected animals (32,0 for Zebus, 37,7 for adults N’Damas, 34,7
for calves N’Damas and 33,5 for Ndapol). In conclusion, this study shows that
chemoprophylaxis should be adapted to each breed. DAI may be a useful tool in order to
assess trypanosomiasis risk, to adapt control methods to each area and to each breed.
However it is a time consuming method that may be improved by using randomly selected
sentinels animals in each herd.

Dissertation Statement
I, Brieuc Cossic (s1267853)
hereby declare that this dissertation is my own work and that I have
not plagiarized work from other sources. I confirm that I have cited all the sources, including
books, journals, conference proceedings and websites from which I obtained information for
completing this work. The work in this dissertation has not been submitted to any other
University for the award of any degree.
Signature: Date: 5
th
June 2015
Key words
African Animal Trypanosomiasis, cattle, Ndama, Zebus, Diminazen-Aceturate, Berenil index,
Tsetse, Gabon.

Acknowledgments
I would like to thank my supervisor Dr. Kim Picozzi and my program director Dr. Ewan MacLeod
from the University of Edinburgh, for their support and advice.
I am very grateful to SIAT Gabon for allowing the experiment to take place. A particular thanks
goes to Pierre-Antoine Couvreur for his help in realizing this project.
I would like to thank Pr. Jean-Paul Dehoux from the Université Catholique de Louvain for
making me discover the Berenil Index.
I would like to thank the University of Liège and more particularly Pr. Pascal Leroy, for allowing
the addition of this protocol to the Genetic Selection Program that was under his supervision.
I would like to thank Dr. Brice Adjahoutonon for his support, his advice and help during the
entire study. Our conversations were always very useful to me.
Etienne Hambursin, the ranch’s cartographer among a lot of others abilities was a great friend
and helped me a lot by creating well-adapted parks for the purpose of our studies.
Maïga Mamadou Ousseyni and Cheikna Sakho who were in charge of the herd assisted me in
the fieldwork. By their excellent work, they made the study possible and I learnt a great deal
about herd management with them.
I am very grateful to Pierre Gloagen for his great help in the results statistical analyses and to
Céline Joie for her help in reviewing this manuscript.
During the last two weeks, I have been assisted in the field and the laboratory work by Gui Lov
Dibanganga, a final year undergraduate at the INSAB, an Agronomic engineer school in Gabon
and I am very grateful for his help.
My family and friends have been very supportive throughout the three years of this MSc, I owe
them a big thank you for this, and particularly to my wife, Charlène.

Abbreviations
AAT: African Animal Trypanosomiasis
ABT: African Bovine Trypanosomiasis
BCT: Buffy Coat Technique
DAI: Diminazen-Aceturate Index
DDT: Dichlorodiphenyltrichloroethane
EDTA: Ethylenediaminetetraacetic acid
ELISA: Enzyme-Linked Immunosorbent Assay
FAO: Food and Agriculture Organisation
IFAT: Indirect Fluorescent Antibody Test
MCT: Microhaematocrit Centrifuge Technique
OGAPROV: The Office Gabonais d'Amélioration et de Production de Viande
OIE: Office International des Epizooties
PCR: Polymerase Chain Reaction
PCV: Packed Cell Volume
TTT: Tsetse Transmitted Trypanosomiasis
VSG: Variable Surface Glycoproteins

Table of Contents
1.
INTRODUCTION
1

1.1 AFRICAN ANIMAL TRYPANOSOMIASIS
1

1.1.1 GLOSSINA AND TRYPANOSOMIASIS
2

1.1.2 IMPACT OF TRYPANOSOMIASIS ON ANIMAL PRODUCTION
7

1.1.3 DIAGNOSIS - LABORATORY METHODS
8

1.1.4 TREATMENTS AND CONTROL
10

1.2
STUDY AREA DESCRIPTION AND TRYPANOSOMIASIS
12

1.2.1 GEOGRAPHICAL SITUATION
13

1.2.2 TRYPANOSOMIASIS IN GABON AND WITHIN THE STUDY SITE
15

1.2.3 BREEDS
17

1.3 THE DIMINAZEN ACETURATE INDEX
21

1.4 AIM OF THE STUDY
21

2. MATERIALS AND METHODS
23

2.1 STUDY AREA DESCRIPTION
23

2.2 ANIMALS
25

2.2.1 STUDY COHORT IDENTIFICATION AND COMPOSITION.
26

2.2.2 WEEKLY ANIMAL COLLECTIONS
26

2.2.3 ANIMAL HEALTH MANAGEMENT
27

2.3 SAMPLING AND LABORATORY WORK
27

2.3.1 SAMPLES COLLECTION AND PRESERVATION
27

2.3.2 TREATMENTS
30

2.3.3 WEIGHING
30

2.3.4 LABORATORY METHODS
31

2.4 DATA MANAGEMENT AND STATISTICAL ANALYSIS
37

3.
RESULTS
38

3.1
OVERALL
TRYPANOSOMIASIS
SITUATION
38

3.2
RESULTS
AMONG
ZEBUS
41

3.3
RESULTS
AMONG
NDAMA
44

3.3.1
RESULTS
AMONG
ADULTS
44

3.3.2
RESULTS
AMONG
CALVES
46

3.4
RESULTS
AMONG
NDAPOL
47

3.5
PARASITEMIA
AND
TRYPANOSOMA
SPECIES
50

4. DISCUSSION
52

4.1 DISCUSSION OF THE RESULTS
52

4.1.1 THE DAI AND INFECTIONS
52

4.1.2 ANALYSIS OF WEIGHTING RESULTS
54

4.1.3 ANALYSIS OF PCV VALUE RESULTS
55

4.1.4 THE DETERMINATION OF A CUT-OFF VALUE FOR PCV
56

4.1.5 TRYPANOSOMES SPECIES
56

4.1.6 FALSE NEGATIVE RESULTS
56

4.4 CRITICISM OF METHODOLOGY
57

Table
of
Contents

4.4.1 SAMPLING AND TREATMENT
57

4.4.2 TIMELINE
57

4.4.3 LABORATORY ANALYSIS
58

5.
CONCLUSIONS
59

6.
REFERENCES
I

List of Tables and Figures
Tables

Table
1
Test
methods
for
the
diagnosis
of
TTT
and
their
purpose
(OIE,
2013)
___________________________
9

Table
2
Trypanocidal
for
domestic
animals
(Dia
and
Desquesnes,
2007;
Hunter
et
al.,
2006)
_________
10

Table
3
Mean,
standard
deviation
and
confidence
interval
for
PCV
values
for
N'Damas
(adapted
from

Host
et
al.,
1983)
___________________________________________________________________________________________
19

Table
4
Distribution
frequency
of
infected
animals
during
the
entire
period
___________________________
39

Table
5
Distribution
frequency
of
infected
animals
during
the
pre-‐treatment
period
for
Zebus
and

N’Damas
____________________________________________________________________________________________________
39

Table
6
Distribution
frequency
of
infected
animals
during
the
pre-‐treatment
period
for
Ndapol
_____
39

Table
7
Distribution
frequency
of
infected
animals
during
the
post-‐treatment
period
for
all
the

animals
_____________________________________________________________________________________________________
40

Table
8
Distribution
of
animals
infected
at
least
once,
positive
samples
and
false
negative
___________
40

Table
9
Weight
(kg)
among
Zebus infected at least once and non-infected Zebus
________________
42

Table
10
Weight
(kg)
among
infected
and
non-‐infected
adults
N’Damas
_______________________________
44

Table
11
Weight
(kg)
among
infected
and
non-‐infected
calves
Ndamas
________________________________
46

Table
12
Weight
(kg)
among
infected
and
non-‐infected
Ndapol
________________________________________
48

Table
13
Parasitemia
levels
for
the
four
different
groups
(scale
ranging
from
5,4
log
to
9,0
log
;
based

on
Herbert
and
Lumsden
(1976))
_________________________________________________________________________
51

Figures

Figure
1
Blood
stream
forms
of
Trypanosoma
congolense
(a),
T.
vivax
(b)
and
T.
brucei
(c)
(FAO,
1998)

________________________________________________________________________________________________________________
3

Figure
2
Trypanosoma
spp.
simplified
life
cycle
(Lee
et
al.,
2007).
________________________________________
4

Figure
3
Maps
representing
the
predicted
areas
of
suitability
for
the
three
Tsetse
flies
subgenus.
a)

Morsitans
b)
Palpalis
c)
Fusca
(fao.org,
February
2014,

http://www.fao.org/ag/againfo/programmes/en/paat/maps.html)
____________________________________
5

Figure
4
Young
N’Damas
showing
emaciation,
a
chronic
Trypanosoma
infection
sign
__________________
7

Figure
5
Injection
of
trypanocidal
drugs
to
Zebus
________________________________________________________
11

Figure
6
Map
demonstrating
the
location
of
the
Gabonese
Republic
in
Africa
(Wikipedia,
January

2014)
________________________________________________________________________________________________________
13

Figure
7
Map
demonstrating
the
location
of
the
Nyanga
province
and
of
the
Ranch
de
la
Nyanga
(red

rectangle)
(mapsof.net,
January
2014)
____________________________________________________________________
14

Figure
8
The
Ranch
de
la
Nyanga,
divided
in
three
administrative
blocks
(Green,
Yellow,
red)

(Hambursin,
2014)
_________________________________________________________________________________________
14

Figure
9
A
view
of
the
ranch's
park
in
Mukelengui
_______________________________________________________
15

Figure
10
A
Zebus
jumping
into
the
dipping
tank.
Flumethrin
dip
is
used
in
order
to
protect
against

ticks
and
Tsetse
flies
________________________________________________________________________________________
16

Figure
11
A
Zebus
cow
_____________________________________________________________________________________
17

Figure
12
A
dehorned
N’Damas
heifer.
Iron
branding
marks
can
be
seen
on
its
thigh
_________________
18

Figure
13
A
dehorned
male
Ndapol
calf,
iron
branding
marks
can
be
seen
on
its
thigh
________________
20

Figure
14
The
park
number
2
of
the
Mukelengui
Section.
The
health
centre
is
also
located
on
the

picture
(yellow
circle)
______________________________________________________________________________________
23

Figure
15
Maïga
conducting
the
herd
into
the
park
after
weekly
cares
_________________________________
24

Figure
16
The
Mukelengui
health
centre,
where
manipulations
on
cattle
are
done
____________________
24

Figure
17
Animals
of
the
program
gathered
at
the
health
center
_______________________________________
26

Figure
18
Jumping
(A)
and
swimming
(B)
into
the
flumethrin
dip
______________________________________
27

Figure
19
Maïga
Mamadou
Ousseyni
(right)
and
Cheikna
Sakho
(left)
performing
blood
collection
_
28

Figure
20
Animals
randomly
entering
the
crowding
alley
(A,
B),
checking
for
injuries
(C)
____________
29

Figure
21
Diminazen-‐aceturate,
curative
trypanocid
(VERIBEN®,
CEVA
Africa)
(ceva-‐africa.com)
__
30

Figure
22
The
weighing
dispositive
(A),
a
Zebus
being
weighed
in
the
"squeeze
chute"
(B)
___________
31

Figure
23
Picture
representing
a
blood
collection
tube
(a),
capillary
tubes
(b),
play
dough
(c)
and

capillary
tubes
after
blood
centrifugation
(d)
____________________________________________________________
33

Figure
24
Rotor
of
the
centrifuge,
after
centrifugation
of
24
samples
__________________________________
33

Figure
25
Different
layers
at
the
end
of
the
centrifugation.
The
Buffy
Coat,
containing
trypanosomes

are
in
the
middle
(adapted
from
Wikipedia,
January
2014)
_____________________________________________
34

Figure
26
Device
to
directly
measure
PCV
on
a
centrifuged
capillary
tube.
The
capillary
tube,
is
placed

in
a
central
rail,
the
buffy
coat
is
on
a
line
(orange).
The
grey
disc
is
moved
until
both
side
of
grey

angle
represented
on
it
correspond
to
their
marks.
One
at
each
end
of
the
liquid
in
the
tube
(yellow

and
red).
Here
PCV
is
41%
_________________________________________________________________________________
34

Figure
27
Materials
used
to
prepare
slides.
Centrifuged
capillary
tube
(a),
identified
slide
and
coverslip

(b),
diamond
pointed
pencil
(c)
and
plastic
pasteur's
pipette
___________________________________________
35

Figure
28
«
Chart
and
table
for
estimating
trypanosome
parasitaemia.
The
circles
are
used
for

matching
when
more
than
one
organism
per
microscope
field
is
present,
the
tables
for
lower

concentrations.
The
values
in
the
boxes
in
the
charts
and
in
the
tables
indicate
the
logarithm
of
the

number
of
trypanosomes
per
millilitre
as
computed
for
Trypanosoma
brucei
infections
in
mouse
blood

inspected
under
x400
magnification.
For
viewing
at
25
cm,
the
circles
are
drawn
with
a
diameter
of

6.5
cm.
They
contain
representations
of
trypanosomes
(6
mm)
that
decrease
in
number
by
twofold

steps
»
(A),
representation
of
the
tables
(B)
(Herbert
and
Lumsden,
1976)
_____________________________
36

Figure
29
Number
of
treatments
per
week.
The
prophylactic
treatment
for
N’Damas
and
Zebus
was
on

April
22nd;
for
Ndapol
it
was
on
May
8th.
__________________________________________________________________
41

Figure
30
Number
of
weeks
between
two
infections
for
Zebus
__________________________________________
42

Figure
31
PCV
values
for
Zebus.
The
median
of
the
herd
is
represented
in
red.
The
mean
PCV
value
for

non-‐infected
animal
is
represented
in
green
and
the
mean
PCV
value
at
the
moment
of
the
infection
is

represented
in
orange.
_____________________________________________________________________________________
43

Figure
32
PCV
values
for
adults
N’Damas.
The
median
of
the
herd
is
represented
in
red.
The
mean
PCV

value
for
non-‐infected
animal
is
represented
in
green
and
the
mean
PCV
value
at
the
moment
of
the

infection
is
represented
in
orange
_________________________________________________________________________
45

Figure
33
PCV
values
for
calves
N’Damas.
The
median
of
the
herd
is
represented
in
red.
The
mean
PCV

value
for
non-‐infected
animal
is
represented
in
green
and
the
mean
PCV
value
at
the
moment
of
the

infection
is
represented
in
orange
_________________________________________________________________________
47

Figure
34
Number
of
weeks
between
two
infections
for
Ndapol
_________________________________________
48

Figure
35
PCV
values
for
Ndapol.

The
median
of
the
herd
is
represented
in
red.
The
mean
PCV
value

for
non-‐infected
animal
is
represented
in
green
and
the
mean
PCV
value
at
the
moment
of
the
infection

is
represented
in
orange
___________________________________________________________________________________
49

Figure
36
PCV
values
for
three
Ndapol.
Infections
are
represented
by
black
triangles
_________________
50

1

1. INTRODUCTION
1.1 AFRICAN ANIMAL TRYPANOSOMIASIS
African trypanosomiasis, both human and animal, are vector borne diseases of
antiquity; some historians even refer to these conditions from the 10
th
century in relation with
Moors’ invasions of sub-Saharan Africa. In those records they were mostly described because
of their role in stopping invaders by infecting soldiers and their horses while crossing humid
areas with a high Glossina pressure (Laveissière and Penchenier, 2005; N’Diaye, 2001).
Nowadays, according to the Programme Against African Trypanosomosis (2008) the
disease “lies at the heart of Africa’s struggle against poverty” and is one of the most important
factors inhibiting the development of the area and achieving the first Millennium Development
Goal of the United Nations, to eradicate extreme poverty and hunger, with 37 countries affected
by the disease and 21 of them among the world’s 25 poorest.
African Animal Trypanosomiasis (AAT) are endemic to a large part of sub-Saharan
Africa and remain a considerable economic burden for the area. Being a major obstacle to the
development of animal breeding, they decrease the access to proteins of animal origin in
countries where they are essential and where a large part of the population relies on livestock
(de La Rocque et al., 2001).
This pathology, also called by the Zulu word “nagana” meaning “to be depressed”, has
the same area of distribution as the Glossina or Tsetse flies; or even “tsêtsê” meaning in
Tswana (Bantu) “Fly that kills cattle”. These are blood-eating dipterous which is the main vector
for the trypanosome parasites (Krafsur, 2009). Almost a third of Africa is infested, accounting for
10 millions km
2
of humid and semi-humid land (Samdi et al., 2010).
However, these areas also offer a great potential for livestock breeding and may be
exploited for that purpose under certain conditions. AAT control therefore constitutes a major
challenge, being considered that this disease is the most constraining factor among the seven
more feared vector-born diseases for cattle in that part of the world, namely trypanosomiasis,
theileriosis, cowdriosis, anaplasmosis, babesiosis, dermatophilosis and African swine fever
(Winrock Institute for Agricultural Development, 1992; Hursey and Slingerberg, 1995).
Nevertheless, disease and vector control remain a considerable challenge and finding
appropriate ways of dealing with these infestations and the infections that they carry is
important for the continent’s development. Areas are very extensive, often their accessibility is
restrained, control methods are expensive and offer great differences in terms of costs-benefits
depending on the situation. Therefore, an approach to assessing the potential benefits from
improving control has to be implemented (Shaw, 2009).
The first step of this assessment is to have a clear view of the trypanosomiasis situation
in each area. A good way to start is to gather data on the prevalence of the disease and the
burden that it represents toward animals. Diminazen-Aceturate Index (DAI), also known as the
Berenil Index, represents a good indicator to have a quick overview of the situation by giving the
number of treatments per animal over a certain period in an area.

Introduction

2

1.1.1 GLOSSINA AND TRYPANOSOMIASIS
1.1.1.1 Aetiology and Life Cycle
AAT are caused by the parasite Trypanosoma spp., a flagellated protozoan belonging
to the order Trypanosomatidae, genus Trypanosoma. They are mostly located in the
extracellular compartment of vertebrate’s blood plasma, lymph and various tissues (OIE, 2013).
African bovine trypanosomiasis (ABT) are mainly caused by Trypanosoma congolense, T. vivax
and to a lesser extent T. brucei (Blood et al., 2007) as represented on figure 1.
Trypanosomes require two hosts, one is said intermediate and welcomes an asexual
multiplication cycle by binary division, the other one is said final and is where asexual and
sexual multiplication occur to prepare infective forms (Peacock et al., 2014). Parasites are
ingested by hematophagous invertebrate (the final host) during their vertebrate’s blood meal
(the intermediate host), therefore becoming the vector (Coetzer and Tutsin, 2004). As shown in
figure 2, where the best-studied stages are represented, colonization of Tsetse flies and
mammalian hosts occurs through the multiplication by division of trypanosomes. Once
colonization is achieved, parasites may eventually transform into resting (non-dividing) forms,
waiting for a change in their environment, i.e. a host change (Lee et al., 2007).
African trypanosomes belong to the Salivaria group because infective metacyclic form is
located in the salivary glands of the vector. It differs from the Stercoraria group characterized by
the parasite’s development terminating in the rear part of the digestive tract of the vector as with
T. cruzi in triatomine bugs in South America. Transmission of AAT is therefore inoculative by
the injection of infective metacyclic forms during vector’s blood meal. Once they are into the
bloodstream, parasites undergo a multiplication in the form of trypomastigote. The vector is
most of the time Tsetse flies (Glossina spp.) (Stuart et al., 2008).
Trypomastigote forms are motile cells with a fusiform and undulating membrane along
the body continuing with a free flagellum that originates near their large single mitochondrion.
Kinetoplast, a characteristic structure of the genus containing DNA, is located at the rear end
(figure 1) (Coetzer and Tutsin, 2004).

Introduction

3

Figure
1
Blood
stream
forms
of
Trypanosoma
congolense
(a),
T.
vivax
(b)
and
T.
brucei
(c)
(FAO,
1998)

a

b

c

Introduction

4

Figure
2
Trypanosoma
spp.
simplified
life
cycle
(Lee
et
al.,
2007).

1.1.1.2 Different mode of transmission and the predominant role of Glossina spp.
AAT are mainly transmitted by blood-sucking insect vector belonging to the Diptera
order, cyclically by the genus Glossina but also for a small amount, mechanically by biting flies
such as Tabanidae, Stomoxys and Hippoboscidae (Desquesnes, 2004; OIE, 2013).
Transmission is said mechanical when pathogens are in mouthparts without multiplying or
suffering any modifications while they are carried. Transmission is said cyclical and specific
when multiplication and biological modifications occur which is the case in salivary glands of
Glossina (Krafsur, 2009).
Glossina have a vast distribution area of almost 10 millions km
2
in sub-Saharan Africa
representing a third of the continent (figure 3), and many species are inventoried with different
requirements in terms of humidity, temperature and ecology, resulting in different areas of
distribution (Samdi et al., 2010). Shrubs savannahs and gallery forests are their main habitat
since Tsetse flies need the protection offered by vegetation against solar radiations and wind
(Taïgue, 1994). According to Morlais (1996) distribution is therefore confined to the area
between the 15
th
parallel North (southern parts of Mali and Niger), and a line drawn between the
13
th
parallel South (Angola’s Atlantic coast) and the 27
th
parallel South (at the border between
South Africa and Mozambique) as shown on figure 3. Distribution North of this area is limited by

Introduction

5

low rainfalls (less than 600 mm per year) and South of this area, annual average temperature
lower than 20 °C also prevents the expansion of Glossina species.

Figure
3
Maps
representing
the
predicted
areas
of
suitability
for
the
three
Tsetse
flies
subgenus.
a)
Morsitans
b)

Palpalis
c)
Fusca
(fao.org,
February
2014,
http://www.fao.org/ag/againfo/programmes/en/paat/maps.html)

0° 30°E
30°S
30°S
0°
0°
30°N
30°N
This map shows the predicted areas of suitability for tsetse flies.
It was produced for FAO - Animal Health and Production Division
and DFID - Animal Health Programme by Environmental Research Group
Oxford (ERGO Ltd) in collaboration with the Trypanosomosis and Land Use in
Africa (TALA) research group at the Department of Zoology, University of Oxford
in November 1999. The modelling process relies on logistic regression of fly
presence against a wide range of predictors. The predictor variables include
remotely sensed (satellite image) surrogates of climate: vegetation, temperature,
moisture. Demographic, topographic and agroecological predictors are also used.
The prediction was created at 5 kilometers resolution for the whole sub-Saharan Africa.
Tsetse: Morsitans group
Prediction of suitability
10% - 40%
40% - 70%
70% - 95%
> 95%
Lakes
Areas cleared of tsetse since 1967
sub-Saharan African Countries
Predicted areas of suitability for savanna tsetse
groupMorsitans
´0 1,500 3,000750
Kilometers
0° 30°E
30°S
30°S
0°
0°
30°N
30°N
Tsetse: Palpalis group
10% - 40%
40% - 70%
70% - 95%
> 95%
Lakes
Predicted areas of suitability for riverine tsetse
groupPalpalis
´0 1,500 3,000750
Kilometers
0° 30°E
30°S
30°S
0°
0°
30°N
30°N
Tsetse: Fusca group
10% - 40%
40% - 70%
70% - 95%
> 95%
Lakes
Predicted areas of suitability for forest tsetse
groupFusca
´0 1,500 3,000750
Kilometers
a

c

b

Introduction

6

1.1.1.3 Antigenic Variation
Variable Surface Glycoproteins (VSG) covering of trypanosomes represent the main
targets for the host’s immune system. During the each wave of parasitaemia, due to the
parasite clonal expansion, the VSG are identical within the population; the host’s immune
system reacts toward them by producing appropriate antibodies. This leads to the specific
activation of complement and the lysis of the infectious agents (Coetzer and Tutsin, 2004).
However, VSG facilitate immune evasion of the parasite by randomly changing their
sequences enabling persistence of trypanosomes that will evade the immune system; with
successive waves of parasitemia, the infection becomes chronic. The switch occurs by
changing the expression of different versions of the VSG genes, which are estimated to several
hundreds. A switch in the expression of the gene randomly occurs at a rate of 2 X 10
-3
switches
per division of the parasite for T. brucei, leading to a new population by clonal expansion after
the previous population has been destroyed by the immune system (Turner, 1997).
The changes in the sequence of the VSG and therefore the absence of a stable
antigenic target to aim at partly explain the inability to develop a reliable vaccine against the
disease.
1.1.1.4 Clinical signs and species affected
First signs of infection appearing after an incubation period of one to two weeks
following the first infective bite, these are often unnoticed and are followed by a chronic
evolution with intermittent crises related to differential parasitaemia (Hunter et al., 2006). There
are no pathognomonic signs and ABT mostly cause anaemia and body condition loss (figure 4).
Intermittent fever attacks; oedema, abortion, emaciation and a decreased fertility are observed
(OIE, 2013). Lymphadenopathy is also described (Hunter, 2006). Milk production and ability to
work decrease (Murray et al., 1991), however their impact on the economy depends on the
animal use. The infection eventually ends up with the death of the animal by exhaustion after
three to four months in chronic cases. Still, the disease’s evolution seems to be strongly
influenced by individual susceptibility and may greatly differ depending on breed, age or even
individuals. In acute cases, death can occur within one week (Tabel et al., 2000; Toure, 1977).
A lot of mammals can be infected by at least one of the three main Trypanosoma
species involved in ABT. These animals are of importance because they act like reservoirs and
play a substantial role in ABT epidemiology.

Introduction

7

Figure
4
Young
N’Damas
showing
emaciation,
a
chronic
Trypanosoma
infection
sign

1.1.2 IMPACT OF TRYPANOSOMIASIS ON ANIMAL PRODUCTION
In Africa, economic losses caused by AAT are important and Delespaux et al.,
estimated in 2008 that an average 60 millions of cattle were infected on the continent. Samdi et
al., (2010) estimated that costs linked to AAT in Africa represent five billion dollars.
According to Kristjanson et al., (1999), 46 million cattle are bred in Tsetse infested
areas at an annual cost of $1340 million, and it may cost even more if all additional costs are
considered. Costs estimation are difficult to handle because there are a lot of parameters to
take into account. Sometimes, only direct costs are considered such as veterinary costs or
mortalities. However, effects on population, on governments etc. have also to be considered but
are more difficult to evaluate.

Introduction

8

Costs may be direct and linked to livestock’s health such as mortality and morbidity
associated to smaller growth rates, weight losses and infertility (Trail et al., 1985). ABT reduce
the production of meat and milk by at least 50% as a result of emaciation and anaemia of
infected animals (Swallow, 1999). Direct costs also include veterinary expenses, vector’s control
campaign and trypanocidal drugs (Samdi et al., 2010)
Indirect effects on land use occur where the presence of Glossina spp. affects livestock
production by reducing the access to some grazing areas, avoiding settling of nomadic
population and the use of less productive but more resistant breeds such as N’Damas. The
ability to work, and in particular the draught power that is very important in fieldwork, is also
decreased and affects population’s production (Samdi et al., 2010; Shaw, 2009).
Kristjanson et al., (1999) also explain that the potential benefits of AAT control in terms
of meat and milk production could represent $700 million per year in Africa. 17 million of them
are treated with trypanocids and assuming that animals are treated twice a year at a price of
approximately one dollar per treatment, curative and preventive treatments would represent an
estimated $35 million annual cost for African livestock producer (Kristjanson et al., 1999).
More recently, Shaw (2009) presented a cost-benefits analysis to address the potential
benefits of AAT control, the output indicated gains in US$/km
2
, these ranged from under $500 to
over $7000 over 20 years depending on the cattle and work oxen distribution.
1.1.3 DIAGNOSIS - LABORATORY METHODS
In the absence of pathognomonic sign for ABT, diagnosis relies on laboratory methods
to confirm the presence of the parasite. Those methods can be either direct like microscopic
visualisation or indirect such as serological tests (Enzyme-Linked Immunosorbent Assay or
ELISA for instance) or molecular analysis utilising the Polymerase Chain Reaction (PCR).
Serological diagnosis such as the ELISA and the Indirect Fluorescent Antibody Test
(IFAT) has a good sensitivity and a good specificity for Trypanosoma (Desquesnes, 2004),
which is also the case with PCR (table 1). However, they are expensive and require
sophisticated equipment. Moreover, serological methods detect immune responses to current
and past infections and therefore active infections are only presumptive. According to
Desquesnes (2004), antibodies may stay an average of 3-4 months after curing while for Van
den Bossche et al., (2000) it can go up to 13 months.

Introduction

9

Table
1
Test
methods
for
the
diagnosis
of
TTT
and
their
purpose
(OIE,
2013)
As shown in table 1, the Haematocrit Centrifuge Technique or Woo’s Method and the
Buffy Coat Technique or Murray’s Method, are well adapted to a situation corresponding to
active infection, where confirmation of clinical cases and Pack Cell Volume (PCV) are needed.
Those methods rest on centrifugation to concentrate parasites to improve the sensitivity and on
microscopic observation directly into the microtube or expressed on a slide. They also allow a
direct observation and identification of pathogens. For all these reasons, the laboratory protocol
will be based on the Woo’s MCT Method (Woo, 1970) and on the Murray’s BCT Method
(Murray, 1977).

Introduction

10

1.1.4 TREATMENTS AND CONTROL
Control methods mostly rely on two aspects, on one hand the control of the infection
once animals have been infected and on the other hand the control of the vector population to
reduce the challenge of infection and the risk of transmission.
1.1.3.1 Control of the trypanosome
Treatments rely on chemotherapy (figure 5) to address the trypanosomal infection, in
order to limit losses due to morbidity and mortality and to decrease the reservoir effect in a herd.
Two different approaches are described and must be combined in order to get the best
efficiency, curative treatments to eliminate parasites once the animal is infected and preventive
treatments to protect animals against infection during a long-term period. Table 2 gathers some
of the molecules that are used as trypanocidal in Africa (Dia and Desquesnes, 2007).
The level of risk of infection, the seasonality of ABT as well as the trypanotolerance
degree of animals must define the trypanocids use strategy. Dia and Desquesnes described
different situations in a manual written in 2007 to help for a rational use of drugs. If the risk is
low over the whole year, a targeted curative treatment for infected animals only is
recommended. If there is a high risk during some seasons, preventive prophylaxis is advised
during the period at risk. Finally if the risk is high during the entire year, trypanotolerant cattle
should be preferred and a program offering a permanent protection has to be selected. Every
situation is different and it reflects the importance of having a good assessment of risks in each
area to adapt control methods.
Drugs Domestic species Trypanosomes
Curative trypanocidal
Diminazen Aceturate Ruminants
T. vivax, T. congolense,
T. brucei
Homidium chloride Ruminants and horses
T. brucei
Homidium bromide Ruminants and horses
T. brucei
Suramin Camels, horses, ruminants and dogs T. brucei, T. evansi
Quinapyramin
Camels, horses, ruminants, pigs and
dogs
T. spp.
Preventive trypanocidal
Isometamidium chloride Cattle, horses
T. brucei
Suramin Camels, horses and ruminants T. brucei, T. evansi
Quinapyramin
Camels, horses, ruminants, pigs
T. spp.
And dogs
Table
2
Trypanocidal
for
domestic
animals
(Dia
and
Desquesnes,
2007;
Hunter
et
al.,
2006)

Introduction

11

However, trypanocidal drugs face a major difficulty, which is the appearance of the
drug-resistant Trypanosoma. For instance, overreliance on trypanocids in villages in South-East
Mali to deal with AAT led to the development of a multi-drugs resistant Trypanosoma
congolense sub-population resisting to both Diminazen-Aceturate and Isometamidium chloride
because of widespread use and more importantly misuse of trypanocidal drugs. (Mungube et
al., 2012)
Figure
5
Injection
of
trypanocidal
drugs
to
Zebus

Chemo resistance appears when dose and time of contact are not sufficient. Most
frequently it is due to an underestimation of body weight, a too diluted product, a too large
period of time between two treatments, the use of fraudulent products with active molecule in
small amount or even absent, or drugs being stored too long after reconstitution (Coetzer and
Tutsin, 2004; Dia and Desquesnes, 2007). Problems of dilution may also appear when 2,36 g
VERIBEN® packs are used instead of 23,6 g (Personal experience, 2014). An alternation in
molecules used is also highly recommended to lower the risk of drug-resistance appearance
and to increase product diversity (Dia and Desquesnes, 2007).
Moreover, drug use is expensive and is dependent on supply chains and animals
restrain capacity of livestock holders. Vectors’ control is therefore also very important to fight
AAT in Africa.
1.1.3.2 Control of the vector
Indirect methods such as actions on the habitats consisting in bush removal and the
use of sterile males are used (Hunter et al., 2006; Kgori et al., 2006; Shaw, 2009).
Direct methods such as the use of insecticides on a large scale in the environment or
associated with traps or insecticide treated targets baited with synthetic attracting products
(Vreysen et al., 2013; Black and Seed, 2002). Cattle are also used as natural baits and

Introduction

12

insecticide spraying on cattle’s legs and belly (Bourn et al., 2005) by pour-on (Shaw, 2009) or
by dipping (Personal, 2013) is also efficient.
Spraying directly in Tsetse fly habitat using aerial and ground aspersion, especially
where they rest and where they emerge from the soil (Shaw, 2009) can also be achieved. The
aerial spraying of pyrethroid such as deltamethrin offers good results, as observed in the
Okavango Delta (Kgori et al., 2006). Such spraying may have a lower environmental impact
than what have been observed with organo-chlorine such as the
Dichlorodiphenyltrichloroethane (DDT) in the past (Kurugundla et al., 2010)
However, these methods remain insufficient to control ABT. Infected areas are indeed
too large to be systematically treated and there is often a lack of sustainable transboundary
programs to reduce the prevalence of trypanosomiasis on a long-term basis.
1.1.3.2 Trypanotolerant cattle
Another way to control the effect of ABT is to use trypanotolerant cattle breeds such as
N’Damas or Baoule that are coming from a co-evolution together with the parasite since their
arrival in Africa 6000 years BC (Jousse, 2004).
N’Damas cattle have the genetic ability (Murray et al., 1982) to control their
parasitaemia (intensity and frequency of crisis) (Paling et al., 1991) and this ability leads to a
lower number of Trypanosoma spp. in the bloodstream and a less important decrease of PCV.
Numbers are particularly low during the chronic phase of the infection (Mattioli and Faye, 1996).
Therefore, some infections, with a parasitaemia below the detection threshold may not be
detected.
1.2 STUDY AREA DESCRIPTION AND TRYPANOSOMIASIS
The study took place in the Gabonese Republic, a country located on the Atlantic coast
of Central Africa (figure 6).
The Gabonese economy mostly relies on oil, wood, and mineral extraction such as
manganese for instance. The country imports 60% of its food and its meat production is almost
non-existent despite of the very good agronomic conditions in rural areas. However, the sector
of animal production has to cope with low prices rivalry for imported products, relative high
prices for labour and animals aliments, difficult access to credits, the absence of basic training
and the scarcity and dilapidation of the roads (NEPAD, 2005).
Data about the agricultural sector are generally scarce in Gabon and official reports or
papers about animal health are difficult to find due to a low level of reporting. Agriculture is very
poorly developed in the country and represented less than 5% of Gross Domestic Product in
2010 (Faostat, 2015).

Introduction

13

In 2008, there were 4115 cattle in the whole country with 3000 heads at the ranch de la
Nyanga alone and the 1115 others divided in 15 places. In 2009, 7500 cattle were inventoried
for the whole country. Information is missing for more recent years (WAHID, 2015).
Although trypanosomiasis is not the major problem for the livestock production in the
country yet, it has to be taken in account from the beginning to manage the burden.
Unfortunately AAT in Gabon are not well documented. In 2011, trypanosomiasis was officially
present in the country according to the Office International des Epizooties (OIE) (WAHID, 2015)
no information since and no notification in Promed (Promed, 2015).
1.2.1 GEOGRAPHICAL SITUATION
The study area is located in the administrative region of Nyanga, the southernmost
province of Gabon, near Congo’s border (figure 7). This is the least developed and least
populated region of the country with 50,297 people including 19,204 in the province’s capital,
Tchibanga (2,4 pers/km2) (Direction Générale de la Statistique et des Etudes Economiques,
2004). Population is mostly rural and live in small villages of about 50 inhabitants. Animal
husbandry is generally poorly developed and consists in small groups of small ruminant and
poultry kept in the vicinity of the household.
Figure
6
Map
demonstrating
the
location
of
the
Gabonese
Republic
in
Africa
(Wikipedia,
January
2014)

Introduction

14

Figure
7
Map
demonstrating
the
location
of
the
Nyanga
province
and
of
the
Ranch
de
la
Nyanga
(red
rectangle)

(mapsof.net,
January
2014)

The study was conducted in a private concession, the Ranch de La Nyanga, a cattle
ranch belonging to the Belgian agro-industrial group “Société d’Investissement pour l’Agriculture
Tropicale” whose role in to develop livestock in Gabon (figure 7 and 8).

Figure
8
The
Ranch
de
la
Nyanga,
divided
in
three
administrative
blocks
(Green,
Yellow,
red)
(Hambursin,
2014)
The ranch represents a rectangle of 100.000 ha, located in a valley oriented according
to a North-West/South-East axis and between 3°10’45.S; 11°10’45E and 3°29’07S; 11°44’47E
along the national road L116 going from Tchibanga to the Congo border. The northern limit
being the Nyanga River and the Southern limit the mountains chain of the Mayombe. The mean
altitude is at 150 m high and the area is relatively hilly. The shale and limestone plain is mostly

Introduction

15

covered with herbaceous vegetation type and dotted with shrubs (figure 9). Larger trees are
observable along streams and form a gallery forest around them. The savannahs are covered
with grassland predominantly Brachiaria, Hyparrhenia, Panicum, Andropogon and Digitaria
species. Forest galleries are present along the gullies and rivers.
Climate is equatorial with two dry seasons (May-September and December-January)
and two wet seasons February-May and September-December). Average annual precipitation is
2000 mm but it varies greatly during the year. Average annual temperature is around 28°C
during the day and 22°C at night.

Figure
9
A
view
of
the
ranch's
park
in
Mukelengui
1.2.2 TRYPANOSOMIASIS IN GABON AND WITHIN THE STUDY SITE
In 1982, high mortality rates were recorded in Gabonese livestock and mostly
attributed to the rift valley Fever and trypanosomiasis (Hoste et al., 1992). In 1991, Trail et al.,
(1991a) reported an average prevalence of 25% in 1987, 31% in 1988 and 9% in 1989. They
observed T. congolense and T. vivax.
Over a three-years period, between 1985 and 1988, Ordner et al., (1988) studied
trypanosomiasis prevalence among two strains of N’Damas cattle, Nguni cattle, a cross breed
between Bos taurus indicus and Bos taurus, and a cross breed between N’Damas and Nguni
cattle. The study was conducted into three ranches in Gabon, including Nyanga’s ranch.
Average prevalence of 7,5%; 10,1%; 25,9% and 16,5 % respectively was found.
In 1991, Leak et al., reported a 5,4% trypanosomiasis prevalence in N’Damas cattle at
the ranch de la Nyanga, lately the Office Gabonais d'Amélioration et de Production de Viande
(OGAPROV).
It is clear that prevalence varies widely and this may be attributed to very different
conditions in terms of animal husbandry, research area, diagnosis technique, methods and
seasons. It confirms that there is a great need in a wide up-to-date trypanosomiasis challenge
evaluation in the country.

Introduction

16

1.2.2.1 Trypanosomiasis control methods at the ranch
Trypanosomiasis is a well-known problem within the ranch and several control methods
are already implemented. However there is no or very few differences depending on the breed,
the category or the area.
Chemoprophylaxis is mostly based on systematic trypanocidal drug treatments with a
curative dose of Diminazen-Aceturate, followed by a preventive drug, Isometamidium chloride
two weeks later. This treatment is applied twice a year for N’Damas, when seasons change,
and three times a year for Zebus.
It represents an average cost of 2,4€ (£1,75)/year/N’Damas and 4€ (£2,91)/year/Zebus
for the drugs alone. At the end of the meat production process, with a price fixed at 3000 francs
cfa/kg (4,58 euros) and a dressing percentage of 40% and 45% respectively, it represents 5,2%
of the meat of a 10 years old N’Damas and 5,5% of a 10 years old Zebus.
Using cattle as natural baits carries out control of Tsetse flies. The cows are dipped into
flumethrin, a pyrethroid every two weeks (figure 10). This process is part of the tick-control plan
but also plays a role into the Trypanosoma vector control, as the flies get intoxicated when they
come for their blood meal on pyrethroid-treated cattle.
Trials have also been conducted on environment modifications in order to limit bush
expansions in some areas and therefore limit Tsetse-resting places where cattle are present.
2,4-D, a dicotyledonous selective systemic herbicidal has been sprayed in some areas with
good results.

Figure
10
A
Zebus
jumping
into
the
dipping
tank.
Flumethrin
dip
is
used
in
order
to
protect
against
ticks
and
Tsetse

flies

Introduction

17

1.2.3 BREEDS
There are two predominant breeds in the ranch Zebus (figure 11), N’Damas (figure 12)
a third one is currently developed, Ndapol (figure 13). They have different characteristics and
react differently toward trypanosomiasis.
1.2.3.1 Zebus

Figure
11
A
Zebus
cow
After being considered as species for a long time, Zebus is now considered as sub-
species of Bos Taurus, Bos taurus indicus. Three different theories explain their first arrival in
Africa. The first one claims an arrival through Mesopotamia and Egypt three to four thousands
years ago and then spread into the continent following pastoral communities. Humped cattle
represented on Egyptian tomb paintings appearing at the second millennium BC suggest that
role (Marshall, 2000; Payne and Wilson, 1999; Epstein, 1971). The second one argues that
there has been a separate domestication of wild cattle in the region, based on archaeological
findings in the Sahara (Muzzolini, 2000).
Finally, Hanotte et al., conducted a molecular genetic research in 2002 where fifty
populations from 23 African countries were studied, both B. taurus and B. taurus indicus. This
research suggested that Zebus cattle spread from the East to the West by genetic introgression
with Bos taurus already present in the area rather than by replacement.
Another major arrival is documented in 1887 when Italian missionaries brought animals
from Aden or Bombay to Massowah (Eritrea) to improve productivity, introducing Rinderpest in
the area at the same time. This is the first incursion of the disease into sub-Saharan Africa and
results were disastrous with eighty to ninety per cent of cattle but also wildlife such as buffalos,
wildebeest, giraffe and antelopes that died. To cope with considerable damage produced by the
disease in livestock, a lot of Zebus were imported from India (Taylor et al., 2005; Edington,
1899).

Introduction

18

At the ranch, Zebus are supposed to come from crossbreeding between Bororo, Fulani,
Adamawa Gudali and mostly Ngaundere Zebus, all belonging to West African Zebus (DAGRIS,
2007). They come from livestock located in North Nigeria and North Cameroon, in the
Adamawa mountains, where Ngaundere is the main city.
Zebus are considered as trypanosensitive and therefore their breeding in Tsetse-
infested areas faces a lot of difficulties and is often restricted to area above 1,200 m elevation or
with less than 800 mm yearly rainfall. Tropical sub-humid lowlands are generally avoided
(Houérou, 2008; Hanotte et al., 2003; Black and Seed, 2002). However, these animals are very
effective in withstanding drought conditions and can be very productive under the right
conditions (DAGRIS, 2007).
A study conducted in 1986 by Merlin P., on 330 Zebus Gudali revealed a mean PCV
value of 34,9.
1.2.3.2 N’Damas

Figure
12
A
dehorned
N’Damas
heifer.
Iron
branding
marks
can
be
seen
on
its
thigh

According to Jousse (2004) N’Damas arrived in Africa 6000 years BC from Egypt and
descending from the first domesticated cattle in the “Fertile Crescent” 9000 BP. However,
recent genetic research and archaeological findings also indicated that there might have been a
different centre of domestication in Africa in the Sahara in the mean time (Gifford-Gonzalez and
Hanotte, 2011; Hanotte et al., 2002; Bradley and Loftus, 2000).
They are Bos taurus belonging to the Humpless Longhorns group are “considered to
be a pure descendant of the original Hamitic Longhorns of north-east Africa” (DAGRIS, 2007).
However, recent genetic investigations also showed that a slow genetic introgression by the
Zebus has later influenced them as well as a minor genetic influence from European cattle (Bos
taurus) (Hanotte et al., 2002).
The breed is known for its trypanotolerance and its resistance to tick-borne diseases
(Mattioli et al., 1995; Ngamuna, 1988). They are also adapted to stressful humid and dry tropical

Introduction

19

climates. The selective pressure associated with their long history under African conditions may
explain these abilities (Black and Seed, 2002; Jousse, 2004).
N’Damas are part of a traditional husbandry management in villages located in Tsetse-
infested areas. Livestock breeders own a few cattle as draught animals, partial milking even if
milk production is low, meat production and as a form of capitalization (Itty, 1990).
N’Damas is a compact medium sized breed with a beef conformation, an average 115
cm high at the shoulders. The average adult weighs range from 320 to 360 kg and 250 to 285
kg for females (Payne and Wilson, 1999; Coulomb, 1976). They have a short and broad head
with average 60 cm long lyre-shaped horns. The typical coat is shorthaired and the colour is
fawn or wheat coloured with darker extremities and a lighter belly and underside. Sexual
dimorphism is well marked and bulls are stocky with large and strong heads (Coulomb, 1976).
The skin is thin and forms a small dew-lap in the inferior part of the chest (Hoste et al., 1988)
A study conducted in between July 1980 and august 1981 on 600 head of cattle, with
6000 samples in order to determine normal PCV value of N’Damas revealed that it mostly
varies with the age and sex. It is also at the individual level a characteristic highly repeatable
also linked to the first month of growth. Therefore, it is an important criterion for genetic
selection. Expected values are represented in the table 3 (Hoste et al., 1983).
Age

Female
Male

Mean
SD
CI
Mean
SD
CI

3
months
45,0
5,2
35-‐55
44,7
4,8
35-‐54

6
months
43,2
4,0
35-‐51
42
3,6
35-‐49

12-‐20
months
29,7
2,4
25-‐34
28,8
2,5
24-‐34

Adult
37,6
3,9
30-‐45
34,3
3,9
27-‐42

Table
3
Mean,
standard
deviation
and
confidence
interval
for
PCV
values
for
N'Damas
(adapted
from
Host
et
al.,

1983)
N’Damas at the ranch come from a large herd kept for beef under ranching condition in
Democratic Republic of the Congo.

Introduction

20

1.2.3.3 Ndapol

Figure
13
A
dehorned
male
Ndapol
calf,
iron
branding
marks
can
be
seen
on
its
thigh
The third breed present at Nyanga is a crossbreed between Senepol, a Brazilian Bos
taurus and N’Damas (Senepol x N’Damas) obtained by artificial insemination, in order to
conduct studies to evaluate its productivity under ranch’s conditions. They are called Ndapol on
the ranch.
Senepol are Bos taurus cattle developed in the 1800’s in the Caribbean’s Islands. It
offers a gentle disposition, no horns and an easy calving, which simplifies their handling.
Moreover they have a high heat tolerance, tick-borne diseases resistance and a good
production of meat. This breed fits particularly well into the ranch’s husbandry practices.
Producers say that this breed has been developed by a crossbred between Red Poll
from Europe and N’Damas cattle from Senegal. However, a recent study genotyped 152
Senepol individuals on 47,365 Single Nucleotide Polymorphism and compared it with results
available for 18 other populations representative of Senepol, N’Damas and Zebus. Results
showed that Senepol is a crossbreed between Red Poll (89%) and Zebus (10,4%) and that only
0,6% of ancestry comes from N’Damas. If there is any N’Damas ancestry, its genes have been
counter-selected in the beginning, probably because they did not fit in breeding objectives of
meat production and hornless phenotype (Flori et al., 2012). More importantly, Zebus and Red
Poll are known to be trypanosensitive. Therefore Senepol might not be trypanotolerant as
expected and promoted by some breeding societies, mostly because Caribbean Islands are
Trypanosoma and Tsetse free. So even if they are more productive than other cattle under
Tsetse free tropical conditions, their importation in Tsetse-infested areas should be conducted
carefully. A rigorous assessment of trypanotolerance in Senepol has not been done yet and is
required to make the appropriate decisions for the importation of Senepol in West and Central
Africa (Flori et al., 2012).

Introduction

21

1.3 THE DIMINAZEN ACETURATE INDEX
Control methods are numerous and all have pros and cons. Therefore an integrated
approach combining proven trypanosomiasis control approaches is most desirous and depends
on risk and conditions in each area. DAI determination helps in assessing the trypanosomiasis
challenge thus allowing a better adaptation to each specific case.
DAI, also known as the Berenil index has first been developed by Whiteside (1962),
when he observed that when trypanosomiasis challenge increases, the protection offered by
trypanocidal drugs decreases.
Uilenberg, in a field guide written on behalf of the FAO in 1988, explains that this
method is realistic and practical, but it is just an estimation that might be underestimated
depending on the sensitivity of the diagnosis test. It also varies along with the trypanotolerance
of the breed. For him, DAI must be calculated after weekly sampling at least 10 animals over a
year, to represent the average number of infections each animal contracts over a year.
In his book, Tsetse Biology and Ecology: Their Role in the Epidemiology and Control of
Trypanosomosis, Leak (1999) gives this definition of the DAI: “The Berenil Index (i.e. DAI) is a
relatively simple way of measuring trypanosomiasis risk by measuring the frequency of
infections in susceptible Zebus cattle when each infection, as soon as it is detected, is treated
with the trypanocidal drug, Diminazen-Aceturate (Berenil®)”. According to him, this index
proposes a less precise but quicker appraisal of disease risk than other methods such as
Tsetse counting and their infection rate, thus being of immediate beneficial for livestock
producers. However, he points out that the drug resistance may lead to an overestimation of the
risk.
According to Takken et al., (1988), DAI is a useful indicator of trypanosome risk and
helps in defining treatments frequencies. DAI also provides an alternative and complementary
method of assessing trypanosomiasis challenge than those commonly used. It has the same
accuracy than collection of Tsetse data and of prevalence rates of infection, particularly where
trypanotolerant are bred (Claxton et al., 1991).
1.4 AIM OF THE STUDY
This study is designed to determine the DAI of an area south of Gabon during the dry
season in order to have a better understanding of the infection process and the trypanosomiasis
challenge. It may help in adapting treatments and animal husbandry in the area. Effective
methods for control, breeds to select and grazing areas will be easier to determine. For now,
there are few differences in trypanosomiasis management among breeds and areas into the
ranch. It would be interesting to avoid chemoprophylaxis when possible, because of the risk of
resistance and also because it represents an important cost at the ranch’s scale.

Introduction

22

A group of selected animals will be sampled on a weekly basis for 24 weeks during the
dry season. Active infections will be confirmed by microscopic observation and infected animal
will be treated with Diminazen-Aceturate. In the mean time, PCV values and weighs will be
measured to see if there are of any significance.
DAI determination for Ndapol into the ranch may provide further information on the
subject and be of great interest to know if whether or not this cross breed is a good lead in this
area and if Senepol benefits from the N’Damas trypanotolerance.

23

2. MATERIALS AND METHODS
This longitudinal study looked at the trypanosome infectivity status of 85 animals,
residing within the Nyanga ranch in Gabon, over a period of six months (April to October 2014).
2.1 STUDY AREA DESCRIPTION
The study area is located in the park number two (figure 14) of the Moukelengui section
of the ranch, identified on the ranch’s map by a blue circle (figure 8). A 1,5 m high fence with
five levels of barbed wire maintains the boundary of the park. The fence’s integrity is checked
every day to inspect for damage caused by elephants, buffalos and warthogs, present in large
number in the area.

Figure
14
The
park
number
2
of
the
Mukelengui
Section.
The
health
centre
is
also
located
on
the
picture
(yellow

circle)

Materials
and
Methods

24

This park has a surface area of 948 hectares, which is divided in five blocks; these
isolations are grazed in rotation during the year with pasture management (using fire) practiced
to provide food in sufficiency. The herd stay under the watch of two herdsmen during the week
(figure 15).
Figure
15
Maïga
conducting
the
herd
into
the
park
after
weekly
cares
The park also has a veterinary health centre, where cattle are easily manipulated (figure
16).

Figure
16
The
Mukelengui
health
centre,
where
manipulations
on
cattle
are
done
Water is available at all times within small ponds and a lake located in MUK2A; those
humid areas are surrounded by vegetation and gallery forest. For the study area, precipitations
are quite low, because of the dry season: April 47,3 mm; May 100,5 mm, June five mm, no
rainfall was recorded in the later months of this study.
Among the animals living onto the ranch, goats, sheep, dogs and horses are of interest
but also wild animals such as buffalos (Syncerus caffer nanus) and waterbuck (Kobus
ellipsiprymnus) that represent a reservoir for the disease (Hunter et al., 2006; OIE, 2013).

Materials
and
Methods

25

Animals are free to go everywhere within Moukelengui two, however they spend most of
their time within dedicated rotation block boundaries as fresh grass is present due to the on-
going pasture management.
The area was known for being a Tsetse habitat in the 1990s (Leak et al., 1991), more
recently (2014) Tsetse were trapped using the windows-opened approach as a car was slowly
driving (10 km/h) within the section as described by Pollock (1982). At the ranch in the 1990’s,
Glossina tabaniformis was the main species while G. palpalis and G. nashi were also present.
The Tsetse challenge was considered as medium with a low fly density (Leak et al., 1991).
The program lasted for 24 weeks between April 18
th
and October 3
rd
2014.
2.2 ANIMALS
Animals of the program were available in collaboration with another research program
on animal genetic improvement by selection and crossbreeding. The genetic program already
led the research team to select animals according to different criteria, in order to create in fine a
group of genetically superior reproducers. The program aims at improving animals’
characteristics on growth rate, dressing percentage, final weights; number of weaned calves,
docility and for N’Damas and Ndapol, trypanotolerance. They were selected on weight, colours,
conformation, reproduction, ability to raise their calf for cows and character.
Zebus were selected after the weaning of their first calf. They are all cows of an
average of six years old. Animals with the highest body weights among Zebus cattle were
selected. The weights range from 301 to 393 kilograms with an average weight at 352,5 kg (SD
= 22,7). Brownish colours were preferred for no particular reason besides esthetical reasons.
Animal with a bad temper were not selected to facilitate manipulations.
N’Damas adults were selected according to their colours, that needed to meet breed
criteria (see 1.2.3.2). Among cows and heifers, animals with the right colour and the highest
body weights were kept. 25 heifers of an average of 3,5 years old and 17 cows of an average of
seven years old were selected. Among cows, only individual that had raised at least three
calves were kept, because it was considered as a good sign for their reproduction ability. At the
ranch, a cow is considered a good reproducer not only if it can produce one calf every year but
also if it is able to wean it properly at the age of eight months. Animals with a gentle character
were preferred. Cows had an average weight of 272,7 (SD=27,4) and a range of 236 to 342 kg.
Heifers had an average weight of 266,6 (SD=17,8) and a range of 236 to 322 kg.
N’Damas calves were selected according to their mother. If a selected cow has a calf,
then the calf is also selected. It is also a part of the progeny test for the genetic program. Calves
ages ranged from four to seven months.

Materials
and
Methods

26

Ndapol come from artificial insemination trials. Their mothers are kept into the herd but
not included in the program. Five males and five females of eight months old for eight of them
and of two months old for the last two calves.
When possible, i.e. when animal were born at the ranch, they were dehorned in their
young age in order to limit risks for them and for herdsmen.
2.2.1 STUDY COHORT IDENTIFICATION AND COMPOSITION.
Individual ear tags, each with a unique identification number were used to identify the
animals included within this research project.
In the program, 85 animals are monitored (figure 17):
- 10 Ndapol calves: (five females and five males),
- 20 Zebus cows
- 55 N’Damas, (25 heifers, 17 cows and 13 calves [seven females and six males]).

Figure
17
Animals
of
the
program
gathered
at
the
health
center
2.2.2 WEEKLY ANIMAL COLLECTIONS
Animals were gathered once a week at the veterinary health centre, where they could
be easily handled (figure 16). They were gathered by the livestock keepers on the evening
before the weekly examination, and spent the night in the corral. On occasion, some individuals
were not present at a particular sampling event, instead they remained on the pasture; in that
case, animals are noted “Absent” within the weekly report. These omissions were generally
caused by a lack of manpower. These happened especially in the earliest months of the trial, as
the cohort was larger (n=345). In addition during the dry season, cattle are much more
scattered across the pasture in search of forage.
Within this study, animals were in the block MUK2A in April and May. In April, MUK2B
was burnt, allowing animals to go into that block in June, July and August as enough new grass
had grown. In September and October, animals stayed in MUK2E, burnt in July. Animals stayed
longer in MUK2B because the herd was composed of 345 animals until mid-June. Animals that

Materials
and
Methods

27

were not part of the program were removed at this date following the identification of cases of
brucellosis in the herd.
The affected animals were mostly pregnant heifers coming from a brucellosis non-free
area in Africa, put with the program’s herd because of a lack of information and a lack of
available pastures at this time on the ranch.
2.2.3 ANIMAL HEALTH MANAGEMENT
Prophylactic treatments, such as Pasteurellosis and Contagious Bovine
Pleuropneumonae vaccination, deworming (ivermectin) were provided as necessary. Every two
weeks, animals were dipped into a flumethrin bath (BAYTICOL®, Bayer) to repel tick
attachment (figure 18 A, B). Flumethrin also have a repulsive effect on Tsetse flies. These
processes aimed at systematically control possible causes of anaemia, others than
trypanosomiasis.

Figure
18
Jumping
(A)
and
swimming
(B)
into
the
flumethrin
dip

At the beginning of this project, on the April 22nd, every animal of the program was
treated with a high dose (8 mg/kg) of Diminazen-Aceturate, a curative trypanocidal, in order to
treat them for trypanosomiasis.
2.3 SAMPLING AND LABORATORY WORK
2.3.1 SAMPLES COLLECTION AND PRESERVATION
A B

Materials
and
Methods

28

Each animal of the program was sampled on a weekly basis, every Thursday between
10 a.m. and 4 p.m.. Adults are separated from calves in order to avoid injuries by squashing
during the sampling procedure.
The sampling was conducted using a wooden crowding alley. Animals were managed
in groups of 15, with systematic sampling along the apparatus (figure 19, 20). At this time,
animals were also checked for injuries and treatments were given as required (see Section
2.2.3).
Blood samples were collected from the coccygeal vein for adults (figure 19); this access
is preferred to jugular or ear veins due to the ease of access and avoidance of issues with the
restraint of these animals. On the other hand, blood was collected from the jugular vein for
calves, as they are easier to handle, and to avoid damaging the coccygeal vein that is too small
at this age. Three milliliters of blood were collected from each animal using a 21G needle (BD
Vacutainer Precision Glide Multiple Sample Needle 21G x 1’ (0,8 x 25 mm) combined with 5 or
10 ml EDTA blood collection tubes (BD Vacutainer). Needles and tubes were used only once in
order to prevent any cross-contamination of samples and to avoid cross-infection by blood-
transmitted diseases; such as brucellosis that was circulating in the area at the time of this
study.
Labelling with the unique animal eartag number identified each sample. Following
collection the vacutainer was slowly turned upside down three times in order to ensure a good
mixing of EDTA and blood.
Before releasing the cattle from the alley, samples were checked in order to be sure
that each animal was sampled and that the blood collected could be identified. Samples were
kept during the operation in a cool box (Pelicase Elite 35) with icepacks, and transported to the
laboratory where they are stored in a refrigerator at 4 °C to be processed the day after.
The man in charge of the herd, Maïga Mamadou Ousseyni on figure 15 and 19, was
also trained to perform blood samples in order streamline the collection process and release
animals in pastures earlier than with only one operator performing sampling.

Figure
19
Maïga
Mamadou
Ousseyni
(right)
and
Cheikna
Sakho
(left)
performing
blood
collection

Materials
and
Methods

29

Figure
20
Animals
randomly
entering
the
crowding
alley
(A,
B),
checking
for
injuries
(C)
A

B

C

Materials
and
Methods

30

2.3.2 TREATMENTS
If necessary, treatments were given directly after the blood sampling, before releasing
animals from the crowding alley. Prophylactic operations such as vaccination (CBPP,
Pasteurellosis) and deworming were done if necessary. Otitis, pneumonia, abscesses and
myiasis and other diseases were also treated if needed, such cases are recorded in a notebook.
At this time, animals that appeared positive for trypanosomiasis from the previous
weeks laboratory tests (depending on the breed), were treated with a curative trypanocidal,
namely Diminazen-Aceturate (VERIBEN®, Ceva Africa, figure 21), directed against infections
with Trypanosoma brucei, T. vivax and T. congolense. Treatment consists in a single deep
intra-muscular injection in the neck.

Figure
21
Diminazen-‐aceturate,
curative
trypanocid
(VERIBEN®,
CEVA
Africa)
(ceva-‐africa.com)
Diminazen is a curative drug expected to treat the animal and suppresses
trypanosomiasis, but without preventive effect. Diminazen-Aceturate is presented as powder
and the solution must be reconstituted with sterile water. A fresh solution was prepared each
week to avoid storage and ensure that the same product was available each week without
degradation.
A strong dosage was used in order to ensure that the administration was sufficient, and
to avoid the appearance of drug resistance. Therefore a dose of 8 mg of Diminazen acetate per
kg of body weight was administered by injection; this ration is at the top end of the
recommended dosing regimen. Body weigh is based on the last weigh recorded for each animal
(see below).
2.3.3 WEIGHING
Animals were weighed on a monthly basis, one-by-one using Avery-Weigh Tronix Chute
Weigh 1.75 and a 640 XL indicator, plugged directly on the car’s battery. As represented on
figure 22 A, B where a Zebus cow is being weighed, animals were blocked onto a wooden
board that rests upon the weighing bars, this apparatus is placed within the crowding alley.
Their ear tag number identifies them and weight was registered within a notebook, and they
were released through the sliding door in front of the weighing ‘pen’.

Materials
and
Methods

31

Animals were weighed as they present themselves in the alley and in the same
conditions each week, with a night having an empty stomach and between 10 a.m. and 4 p.m..
Results of the day were entered into a Microsoft Excel spread sheet later in the evening.

Figure
22
The
weighing
dispositive
(A),
a
Zebus
being
weighed
in
the
"squeeze
chute"
(B)
2.3.4 LABORATORY METHODS
To suit to the materials available at the time in the laboratory, in geographic isolation
conditions and at low cost, parasite concentration technique associated to direct microscopic
observation have been selected. Therefore, Microhaematocrit Centrifuge Technique (MCT) and
the Buffy Coat Technique (BCT) are preferred. Besides, according to Toro et al., (1981),
microhaematocrit centrifuge technique also gives better results for the diagnosis of bovine
trypanosomiasis than Thick Stained Blood and Wet Blood Film techniques.
The Woo Method (Woo, 1970) allows a parasite concentration, based on the separation
of blood components’ depending on their specific gravity. Samples are then processed
according to the BCT first described by Murray in 1977 allowing a direct visualisation of
Trypanosoma and the exploration of 70 µl of blood, the microtube volume.
Sensitivity of the method depends on the level of parasitemia as well as on the species
of Trypanosoma. A detection of parasites of almost 100 % can be achieved when at least 700
trypanosomes per ml of blood are present. It decreases to 80%-46% of detection between 700
and 60 parasites/ml and almost 0% below 60 tryps/ml for T. vivax with the Woo method
(Desquesnes, 2004). Therefore, it may vary accordingly to cyclical parasitemia peaks.
With this method, the PCV can be assessed at the same time (OIE, 2013), which
reflects anaemic conditions. Anaemia can be caused by AAT and is therefore an important
indicator with 94% specificity and 89% sensitivity when a cut-off value of 26 is observed if
combined to parasitological diagnosis (Marcotty et al., 2008).
B

A

Materials
and
Methods

32

Marcotty et al., (2008) showed that a combination of parasitological diagnosis and PCV
determination improved the accuracy of the diagnostic outcome; the determination of a cut-off
value for the PCV that is geographically appropriate may further improve the process’s
effectiveness.
2.3.4.1.Sample
preparation

Samples examination was conducted every Friday, a period of no more that 24 hours
maximum after sampling. Samples were taken out of the refrigerator, 24 units at a time, and
kept at room temperature (24°C). Other samples are kept in the refrigerator at 4°C until the first
batch processing was over. Samples are slowly put upside down three times in order to have
homogenous blood. A 75 mm/ 75 microliters heparinised haematocrit capillary tube
(Hirschmann Laborgerate) was dipped into sample’s tube in order to collect materials via
capillary action.
The heparinised capillary tubes are sealed with sealing Crystaseal (Wax Seal Plate
Capillary -Hirschmann Laborgerate) and placed with the sealed ends pointing towards outside
in a GriCel micro-hematocrito MOD.61 microtube centrifuge (figure 23). They were spun at the
maximum rotation for four minutes, 24 samples at a time as represented on figure 24. Blood
elements separated into layers according to their density as represented in figure 25.

Materials
and
Methods

33

Figure
23
Picture
representing
a
blood
collection
tube
(a),
capillary
tubes
(b),
play
dough
(c)
and
capillary
tubes

after
blood
centrifugation
(d)

Figure
24
Rotor
of
the
centrifuge,
after
centrifugation
of
24
samples
a

b

c

d

Materials
and
Methods

34

2.3.4.2.
Packed
Cell
Volume
measurement

The Packed Cell Volume (PCV) is the volume percentage (%) of red blood cells in
blood. PCV is easily determined by dividing the length of the packed red blood cells by the total
length of the blood sample in the microtube (figure 25).

Figure
25
Different
layers
at
the
end
of
the
centrifugation.
The
Buffy
Coat,
containing
trypanosomes
are
in
the

middle
(adapted
from
Wikipedia,
January
2014)
For capillary tubes, the PCV is directly measured thanks to a manual device
represented in figure 26 (GriCel).

Figure
26
Device
to
directly
measure
PCV
on
a
centrifuged
capillary
tube.
The
capillary
tube,
is
placed
in
a
central

rail,
the
buffy
coat
is
on
a
line
(orange).
The
grey
disc
is
moved
until
both
side
of
grey
angle
represented
on
it

correspond
to
their
marks.
One
at
each
end
of
the
liquid
in
the
tube
(yellow
and
red).
Here
PCV
is
41%
Trypanosomes

Materials
and
Methods

35

2.3.4.3
Parasitemia
evaluation

Following blood centrifugation, trypanosomes are mainly concentrated in the buffy coat
zone (figure 25). Thus the following observations are directed toward this part of the
microhaematocrit capillary tube.
The capillary tube was cut with a diamond pointed pencil 1 mm below the buffy coat to
include the uppermost layer of red blood cells. Then using a plastic Pasteur’s pipette, whose
extremity has been heated, to fit around micro-haematocrit tube, the contents of the capillary
tube are expressed onto a 76 mm x 26 mm microscope slide. The next step consists of
overlaying the content with a coverslip by slowly making contact on one side of the drop and
then carefully lowering the coverslip down to avoid air bubbles (figure 27). Each slide is
identified with the ear tag number of the corresponding animal.

Figure
27
Materials
used
to
prepare
slides.
Centrifuged
capillary
tube
(a),
identified
slide
and
coverslip
(b),

diamond
pointed
pencil
(c)
and
plastic
pasteur's
pipette

Slides were examined using a Leica DM1000 microscope. The first examination
consisted of a rapid review of the slide surface, at x 10 eyepieces and x 10 objective to assess
for trypanosome movements. This scan take about 30 seconds. The second examination is
done with the x 40 objective. The entire coverslip area was then examined using a systematic
scan from the upper-left corner to the lower-right corner. This examination takes about 4-5
minutes. If trypanosomes were observed during this part, then the counting method is applied.
The Herbert and Lumsden’s charts and tables (1976) (figure 28) were used to provide
an estimate of the trypanosome concentrations. However, results can’t be used in order to
provide a true number of trypanosomes per millilitre as the Lumsden charts were developed for
estimating parasites counts of wet blood films whereas in our cases, centrifugation concentrated
a

b

c

d

Materials
and
Methods

36

them. However, the estimate can be used as an indication of concentration and offers the
possibility to obtain results of relative values allowing comparing animals.
If observation revealed trypanosomes’ presence, the use of the Lumsden charts or
tables was decided based on this observation (figure 28). When large numbers are present,
charts are preferred. If there is one organism per field or fewer, tables were used. The first
count was made of five fields. If two or more trypanosomes appear, then the result is read in the
corresponding table. If there are fewer parasites then 10 fields are counted using the same
principle and if it’s not enough, it goes to 20 fields.
If no trypanosomes are seen, parasitemia is recorded as inferior to antilog 5.4. It is not
possible to declare the animal negative for trypanosomiasis because concentrations may be too
low for being detected with this method.

Figure
28
«
Chart
and
table
for
estimating
trypanosome
parasitaemia.
The
circles
are
used
for
matching
when
more

than
one
organism
per
microscope
field
is
present,
the
tables
for
lower
concentrations.
The
values
in
the
boxes
in

the
charts
and
in
the
tables
indicate
the
logarithm
of
the
number
of
trypanosomes
per
millilitre
as
computed
for

Trypanosoma
brucei
infections
in
mouse
blood
inspected
under
x400
magnification.
For
viewing
at
25
cm,
the

circles
are
drawn
with
a
diameter
of
6.5
cm.
They
contain
representations
of
trypanosomes
(6
mm)
that
decrease
in

number
by
twofold
steps
»
(A),
representation
of
the
tables
(B)
(Herbert
and
Lumsden,
1976)
5"fields 10"fields 20"fields
4"5$tryps 6.6$log 2"3$tryps 6.0$log 2"3$tryps 5.7$log
2"3$tryps 6.3$log 1$tryps 5.4$log
0$tryps <$5.4$log

Materials
and
Methods

37

2.3.4.4
Determination
of
the
Diminazen-‐treated
animals
for
the
next
week

Zebus and Ndapol positive for trypanosomiasis were put on the list of animals to be
treated with Diminazen-Aceturate at the next period of sampling.
N’Damas that are positive for the first time were treated five weeks later in order to
respect another research program on genetic selection and trypanotolerance. It is necessary to
see how each individuals reacts to the infestation.
2.4 DATA MANAGEMENT AND STATISTICAL ANALYSIS
Data was entered into a Microsoft Excel spread sheet on a weekly basis. A pivot-table
has been designed in order to easily extract information from the data.
The Diminazen-Aceturate Index (DAI) was calculated for the dry season (April until
October 2014). This method allows us to determine trypanosomiasis challenge in the area
(Uilenberg G., 1998). Diminazen is used because its lack of persistent effect with an elimination
half-life of 107.5±8.50 h in calves (Kaur et al., 2000).
Blood samples of cattle are examined at weekly intervals and infested animals are
treated with Diminazen-Aceturate. The DAI is calculated with this formula:
DAI = number of infection recorded over the 6 months / number of animals
The DAI for this period is easily determined by dividing the number of cases of infection
by the number of animals that is the average number of infections per animal. In our case, we
want to have a global six months - DAI for the area and one for each breed (N’Damas, Zebus,
Ndapol) and age class (calves, adults) separately.
Statistical analysis was conducted using the free software “R”. This software was also
used to draw most of the figures. Chi-square tests have been performed on by-hand.

38

3.
RESULTS

A study was conducted over a 24 weeks period in a cattle ranch in Gabon. It aimed at
estimating the DAI for three different cattle breeds raised under identical management
conditions. Each week, 10 Ndapol, 55 N’Damas and 20 Zebus were sampled. N’Damas are
separated in two distinct groups, calves and adults. Three animals had to be removed from the
protocol because of brucellosis.
Positive results were considered when at least one trypanosome was observed under
microscopic observation. Negative results were considered when no parasite was observed.
Nevertheless, it is important to underline the fact that it does not mean that the animal was not
infected, simply that the outcome of this analysis is based upon the visualisation by microscopy;
sub-clinical infections may fall below this level of diagnostic sensitivity (see 2.3.4).
Animals were considered infected from the first point of observation of a trypanosome to
the point of treatment that may be the next week or five weeks later depending on the breed. It
is important to underline the fact that it was considered as one single infection.
False negative results were registered among the four categories of animals. They are
identified when an animal was not seen to be concurrently infected between the positive sample
and the treatment.
Sampling started on April 18
th
for N’Damas and Zebus and they were all treated with
Diminazen-Aceturate on April 22
nd
. Sampling started on May 2
nd
for Ndapol and they were all
treated on May 8
th
. Therefore, the sampling period is divided into two parts the first two
sampling before treatments (the first one and the one of the prophylactic treatment day) and the
22 weeks after the treatment for Zebus and N’Damas and the 20 weeks for Ndapol. DAI will be
calculated on infectious events after the herd treatment, for a period of 22 weeks and 20 weeks
depending on the breed.
It is interesting to know that N’Damas received a Diminazen-Aceturate treatment on
November 1
st
2013 and an Isometamidium treatment three weeks later on November 28
th
2013.
Zebus received the same treatment in December 2013.
In total, over the 24 weeks period, 2023 samples were collected. Over this period, some
animals were occasionally absent from the sampling. This was recorded to have happened
twice for Zebus (0,4% of Zebus’ samples), 22 times for N’Damas adults (2,1%), twice for
N’Damas calves (0,6%) and three times for Ndapol (1,5%).
3.1
OVERALL
TRYPANOSOMIASIS
SITUATION

Of the 2023 samples collected, 117 were seen to be positive. However, when it is
related to animal health, some of them may be due to the same infection of an animal sampled
before the treatment. Therefore, 78 were considered to be single infectious events (3,8% CI

Results

39

95% 3,1 to 4,8%). Across the observation period 42/85 animals remained clear of infection.
Forty-three animals (50,6% CI 95% 40,0 to 61,2%) were infected with trypanosomes at least
once during the course of the experiment. Ten of the 42 N’Damas adults and five of the 13
Ndama calves, 19 of the 20 Zebus and nine of the 10 Ndapol. The distribution of frequency of
infections is shown in table 4, based on Leperre and Claxton (1994).
Table
4
Distribution
frequency
of
infected
animals
during
the
entire
period

When only the pre-treatment period for Zebus and N’Damas is considered, of the 151
samples collected, 31 samples were seen to be positive leading to 17 single infections (11,3%
CI 95% 6,2 to 16,3%). Two adults N’Damas, two calves N’Damas and 13 Zebus considered as
infected. Across the observation period 58/75 animals (Ndapol were not sampled yet) remained
clear of infection. Seventeen animals (22,7%) were infected with trypanosomes at least once
during this period. Two of the 42 N’Damas adults and two of the 13 N’Damas calves, and 13 of
the 20 Zebus (table 5).

Table
5
Distribution
frequency
of
infected
animals
during
the
pre-‐treatment
period
for
Zebus
and
N’Damas

Of the 15 samples collected for Ndapol during their pre-treatment period, five were seen
to be positive and three single infections (20% CI 95% 0 to 40,2%) are considered on three/10
different animals (30%) (table 6).

Table
6
Distribution
frequency
of
infected
animals
during
the
pre-‐treatment
period
for
Ndapol

Therefore, for the post-treatment period for the three breeds, of the 1857 samples
collected, 81 samples were seen to be positive, and 58 single infections (3,1% CI 95% 2,3 to
3,9%) were considered with nine cases among adults N’Damas, three among calves N’Damas,
29 among Zebus and 17 among Ndapol. Across the observation period 46/85 animals remained
clear of infection. Thirty-eight animals (44,7%) were infected with trypanosomes at least once
0 1 2 3 4 5
Ndamas,adults 32 9 1 0 0 0
Ndamas,calves 8 5 0 0 0 0
Zebus 1 4 9 5 0 1
Ndapol 1 2 5 1 0 1
43 20 15 6 0 2
78 20 30 18 0 10
Number,of,infections
Breed
Total,of,infected,animals
Total,of,infections
0 1 2 3 4 5
Zebus 7 13 0 0 0 0
Ndapol 0 0 0 0 0 0
17 17 0 0 0 0
17 17 0 0 0 0
Total,of,infections
Breed
0 1 2 3 4 5
Zebus 0 0 0 0 0 0
Ndapol 7 3 0 0 0 0
3 3 0 0 0 0
3 3 0 0 0 0
Total,of,infections
Breed

Results

40

during the course of this period. Nine of the 42 N’Damas adults and three of the 13 Ndama
calves, 18 of the 20 Zebus and eight of the 10 Ndapol (table 7).

Table
7
Distribution
frequency
of
infected
animals
during
the
post-‐treatment
period
for
all
the
animals
Over the entire period, 22 samples were classified as false negative for the protocol
with one week between a positive sample and the treatment (Zebus and Ndapol). Fifty-five
samples are considered as false negative for the five weeks protocol (N’Damas). A total of 77
samples are considered as false negative, i.e. 39,7% CI 95% 32,8 to 46,6% of the samples
expected to be positive (194 = 117 + 77) (table 8).

Table
8
Distribution
of
animals
infected
at
least
once,
positive
samples
and
false
negative
Zebus are significantly more often infected than adults N’Damas (Chi-square = 69,1,
P<0,001). Ndapol are significantly more often infected than N’Damas calves (Chi-square =
17,49, P<0,001). Therefore each breed is going to be considered independently.
0 1 2 3 4 5
Zebus 2 10 6 1 1 0
Ndapol 2 1 6 0 1 0
38 23 12 1 2 0
58 23 24 3 8 0
Total,of,infections
Breed
Number'of'infected'animals Number'of'positive'samples Number'of'false'negative
Ndamas'adults 10 15 43
Ndamas'calves 5 8 12
Zebus 19 65 14
Ndapol 9 29 8
43 117 77
Breed
Total

Results

41

Figure
29
Number
of
treatments
per
week.
The
prophylactic
treatment
for
N’Damas
and
Zebus
was
on
April
22nd;

for
Ndapol
it
was
on
May
8th.

Figure 29 represents the number of single infections during the experiment. During the
second week for Zebus and N’Damas and the fourth week for Ndapol, the high numbers are
due to infections that may have occurred before the beginning of the protocol because animals
are not supposed to self-cure and therefore entered the protocol already infected. It is
interesting to see that there is a period of three weeks between the prophylactic treatment and
the first post treatment infection for Ndapol, five weeks for Zebus, six weeks for N’Damas calves
and eight weeks for N’Damas calves. After the first infection post-treatment, weekly incidence is
almost the same during the protocol with a higher infection rate at the end of the protocol on the
last week.
3.2
RESULTS
AMONG
ZEBUS

Twenty Zebus cows were monitored in the study. Age has been estimated to six years
old based on cows’ history and information available at the ranch. Their calves had just been
weaned before the beginning of the experiment and weigh loss due to lactation may have
impacted on the mean weight of the group. One of the Zebus had to be removed because it
appeared positive to brucellosis, based on a Rose Bengal Test.
At the beginning of the study, during the post treatment period, their weight ranged from
301 to 393 kilograms with a mean weight at 352,5 kg (SD = 22,7). At the end of the study, their
weight ranged from 277 to 400 kg with a mean weight of 368 kg (SD = 26,67) (table 9).
Numbers of infections have no significant effect on final weights.

Results

42

Table
9
Weight
(kg)
among
Zebus infected at least once and non-infected Zebus
However, comparison between infected and non-infected Zebus should be handled
carefully as there is only one non-infected animal and the analysis is unlikely to be statistically
significant.
Nineteen Zebus out of 20 have been positive to trypanosomiasis at least once during
the experiment, which represents 95% of the group and a total of 42 different infectious events
have been detected. Over the pre-treatment period, 13 infections have been detected on 13
different Zebus. Over the post-treatment period, 29 infections among 18 different Zebus have
been detected.
DAI is calculated by dividing the number of infections during the post-treatment period
by the number of animals, providing an index of 1,45 for Zebus.
Re-infections among Zebus are considered for animals with at least two different
infections and measuring time between these infections determines the re-infection time, as
represented on figure 30. Twenty-three re-infections have been observed and one of the
animals was repeatedly infected five times during the protocol (Animal number 6013). It is worth
noticing that for 12 of these 23 re-infections (52%), the re-infection time was between four to
eight weeks, which is interesting considering incubation period.

Figure
30
Number
of
weeks
between
two
infections
for
Zebus
Mean Min Max Mean Min Max Mean Min Max
Zebus+(n=20) 352,5%(SD=22,7) 301 393 368%(SD=26,7) 277 400 368,8%(SD=23,6) 277 426
+infected+(n=19) 352,8%(SD=23,3) 301 393 367,6%(SD=27,4) 277 400 368,8%(SD=24,0) 277 426
non8infected+(n=1) 347 347 347 376 376 376 367,6%(SD=13,1) 347 380
Mean Min Max Mean Min Max Mean Min Max
Zebus+(n=20) 23,5%(SD=6,6) 8 33 27,8%(SD=4,6) 14 34 31,0%(SD=5,5) 8 43
+infected+(n=19) 23,5%(SD=6,8) 8 33 27,7%(SD=4,7) 14 34 31,1%(SD=5,6) 8 43
non8infected+(n=1) 23 23 23 30 30 30 29,1%(SD=2,7) 23 35
Weight+at+the+beginning+(in+kg) Weight+at+the+end+(in+kg) Weight+during+the+entire+period+(in+kg)
PCV+at+the+beginning PCV+at+the+end PCV+during+the+entire+period

Brieuc-COSSIC-Dissertation-As time flies, adapting trypanosomiasis control methods through a longitudinal study of cattle management in an area of low Tsetse challenge South of Gabon

Brieuc-COSSIC-Dissertation-As time flies, adapting trypanosomiasis control methods through a longitudinal study of cattle management in an area of low Tsetse challenge South of Gabon

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