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: 5th 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 10th 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 km2 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 km2 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 15th parallel North (southern parts of Mali and Niger), and a line drawn between the

13th parallel South (Angola’s Atlantic coast) and the 27th 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°E30

°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 groupPrediction 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 tsetsegroupMorsitans

´0 1,500 3,000750

Kilometers

0° 30°E

30°S

30°S

0° 0°

30°N

30°N






Tsetse: Palpalis groupPrediction of suitability

10% - 40%40% - 70%70% - 95%> 95%

Lakes



Predicted areas of suitability for riverine tsetsegroupPalpalis

´0 1,500 3,000750

Kilometers

0° 30°E

30°S

30°S

0° 0°

30°N

30°N






Tsetse: Fusca groupPrediction of suitability

10% - 40%40% - 70%70% - 95%> 95%

Lakes



Predicted areas of suitability for forest tsetsegroupFusca

´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$/km2, 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. vivax, T. congolense, T. brucei

Homidium bromide Ruminants and horses T. vivax, T. congolense, 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. vivax, T. congolense, 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).


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 18th and October 3rd 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.


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


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


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


29

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

A

B

C


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’.


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


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.


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


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


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


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"fields4"5$tryps 6.6$log 2"3$tryps 6.0$log 2"3$tryps 5.7$log2"3$tryps 6.3$log 1$tryps 5.4$log

0$tryps <$5.4$log


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 18th for N’Damas and Zebus and they were all treated with

Diminazen-Aceturate on April 22nd. Sampling started on May 2nd for Ndapol and they were all

treated on May 8th. 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 1st 2013 and an Isometamidium treatment three weeks later on November 28th 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 5Ndamas,adults 32 9 1 0 0 0Ndamas,calves 8 5 0 0 0 0Zebus 1 4 9 5 0 1Ndapol 1 2 5 1 0 1

43 20 15 6 0 278 20 30 18 0 10

Number,of,infections

Breed

Total,of,infected,animalsTotal,of,infections


17 17 0 0 0 017 17 0 0 0 0



Breed


3 3 0 0 0 03 3 0 0 0 0



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.


38 23 12 1 2 058 23 24 3 8 0



Breed

Number'of'infected'animals Number'of'positive'samples Number'of'false'negativeNdamas'adults 10 15 43Ndamas'calves 5 8 12Zebus 19 65 14Ndapol 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 MaxZebus+(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 426non8infected+(n=1) 347 347 347 376 376 376 367,6%(SD=13,1) 347 380

Mean Min Max Mean Min Max Mean Min MaxZebus+(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 43non8infected+(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

Results

43

At the start of the program, on the prophylactic treatment day, average PCV level of the

Zebus was 23,5 (SD=6,6); Min = 8; Max = 33. One week later, it was 28,6 (SD=5,1). At the end

of the experiment in October, it was 27,8 (SD=4,5); Min = 14; Max = 34.

PCV values for non-infected animals have a mean at 32,0 (SD=4,9), Min = 14, Max =

43. When PCV values are considered at the moment of the infection for infected animals, lower

values are found with a mean PCV value at 25,6 (SD=5,9), Min = 8 and Max = 37. On Figure

31, variations within the herd are clearly represented and a large variation of PCV values within

the herd can be seen.

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.

During the laboratory analysis, false negative for trypanosomes presence have been

detected. False negative are considered when an animal is positive for trypanosomiasis one

week but negative the next week before receiving the Diminazen-Aceturate. Because blood is

collected before the treatment, these animals are supposed to be infected; however analysis

sometimes showed a negative result, classified as a false negative. In the case of Zebus, 14

false negative situations have been noticed. It represents 17,7 % of the 79 analyses performed

on animals supposed to be infected.

Results

44

3.3 RESULTS AMONG NDAMA

3.3.1 RESULTS AMONG ADULTS

Forty-two adults N’Damas have been monitored, 25 heifers of 3,5 years old and 17

cows of an average of seven years old. Two heifers had to be removed during the program

because of brucellosis, based on a Rose Bengal Test. Cows had an average weight of 272,7 kg

(SD=27,4) and a range of 236 to 342 kg. Heifers had an average weight of 266,6 kg (SD=17,8)

and a range of 236 to 322 kg; there was no significant changes in body weight across the

duration of the study (table 10).

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

Ten N’Damas out of 42 have been positive to trypanosomiasis at least once during the

experiment, which represents 23,8% of the group and a total of 11 different infections have

been detected. Over the pre-treatment period, two infections have been detected on two

different adults N’Damas. Over the post-treatment period, nine infections among nine different

adults N’Damas have been detected.

There is no significant difference between heifers and cows. Only one N’Damas has

been infected twice (Animal number 5037) after 15 weeks, therefore, it is difficult to have

information on re-infection time.


by the number of animals providing an index of 0,21 for adults N’Damas.

Mean Min Max Mean Min Max Mean Min MaxNdamas+adults+(n=42) 269,1&(SD=22,1) 236 342 282,0&(SD=25,9) 242 370 276,5&(SD=24,7) 233 370

+infected+(n=10) 275,9&(SD=33,0) 244 342 282,7&(SD=39,4) 242 370 275,2&(SD=20,2) 233 318non9infected+(n=32) 266,9&(SD=17,6) 236 307 281,7&(SD=20,5) 244 317 280,6&(SD=35,1) 242 370

Mean Min Max Mean Min Max Mean Min MaxNdamas+adults+(n=42) 34,8&(SD=3,1) 29 43 38,2&(SD=4,2) 28 48 37,5&(SD=4,7) 20 53

+infected+(n=10) 34,3&(SD=2,8) 31 40 38,9&(SD=5,4) 28 45 37,7&(SD=4,5) 20 53non9infected+(n=32) 34,9&(SD=3,2) 29 43 37,9&(SD=3,8) 30 48 37,0&(SD=5,2) 21 48



Results

45

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

On the fourth week, on May 15th, a significant drop is observed in PCV values among all

adults N’Damas (figure 32). However, no particular event is registered at this time.


53. When PCV values are considered at the moment of the infections for infected animals,

lower values are found with a mean PCV value at 34,2 (SD=6,2), Min = 21 and Max = 44.


adults N’Damas was 34,8 (SD=3,0); Min = 29; Max = 43. One week later, it was 35,75

(SD=4,3). At the end of the experiment in October, it was 38,2 (SD=4,2); Min = 28; Max = 48

and large variation of PCV values within the herd can be seen (figure 6).

On Figure 32, variations within the herd are clearly represented. A large variation of

PCV values within the herd can be seen.

Unlike Zebus that were treated one week after being diagnosed as infected, N’Damas

were treated 5 weeks later for the purpose of the genetic research program. Therefore,

additional analyses were performed on infected animals. Forty-three false negative results have

been detected. Those represent 74,1 % of the 58 analyses performed on N’Damas supposed to

be infected.

Results

46

3.3.2 RESULTS AMONG CALVES

Thirteen N’Damas calves were monitored, seven females and six males, born between

October 2014 and January 2015.

Five calves out of 13 have been positive to trypanosomiasis at least once during the

experiment, which represents 50% of the group and a total of five different infections have been

detected meaning that no re-infections occurred. There is no significant difference between

male and female.

Over the pre-treatment period, two infections have been detected on two different

calves N’Damas. Over the post-treatment period, three infections among three different calves

N’Damas have been detected.


by the number of animals providing an index of 0,23 for calves N’Damas.

At the beginning of the post treatment period, their weight ranged from 51 to 133

kilograms with a mean weight at 99,3 kg (SD = 23,8). At the end of the study, their weight

ranged from 81 to 168 kg with a mean weight of 138,7 kg (SD = 24,6) (table 11).

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

The growth rate is interesting when calves are considered in studies, as it is the only

way to compare different weights and ages. With 127 days between the first and last weighing,

the average growth rate is of 0.31 kg/day for N’Damas calves. The growth rates for infected and

non-infected calves are respectively of 0,33 kg/day and 0,30 kg/day.

Age difference between infected and non-infected is not significant, with an average of

169 day (SD=23,7) for infected calves and 164 day (SD=38,7) at the beginning of the

experiment on April 18th.





calves N’Damas was 31,8 (SD=5,7); Min = 24; Max = 43. One week later, it was 32,1 (SD=4,6).

At the end of the experiment in October, it was 35,1 (SD=5,5); Min = 26; Max = 49 and a large

variation of PCV values within the herd can be seen (figure 33).

Mean Min Max Mean Min Max Mean Min MaxNdamas+calves+(n=13) 99,30%(SD=23,8) 51 133 138,7%(SD=24,6) 81 168 120,5%(SD=28,0) 51 168

+infected+(n=5) 108,4%(SD=24,7) 69 133 150,4%(SD=20,9) 115 168 130,9%(SD=27,1) 69 168non8infected+(n=8) 93,60%(SD=23,0) 51 123 131,4%(SD=25,0) 81 166 114,0%(SD=26,9) 51 166

Mean Min Max Mean Min Max Mean Min MaxNdamas+calves+(n=13) 32,1%(SD=4,6) 23 38 35,1%(SD=5,5) 26 49 34,6%(SD=4,2) 23 49

+infected+(n=5) 31,0%(SD=4,9) 23 35 32,8%(SD=6,1) 26 40 33,6%(SD=3,8) 23 42non8infected+(n=8) 32,8%(SD=4,6) 24 38 37,3%(SD=5,0) 34 49 35,2%(SD=4,3) 24 49



Results

47

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

Four false negative results have been detected, representing 33,3 % of the 12 analyses

performed on animals supposed to be infected.

3.4 RESULTS AMONG NDAPOL

Ten Ndapol calves were monitored, five females and five males. Eight of them born in

September 2014 and two in March 2015.

Nine Ndapol out of 10 have been positive to trypanosomiasis at least once during the

experiment, which represents 90% of the group and a total of 20 different infections have been

detected. There is no significant difference between male and female. Over the pre-treatment

period, three infections have been detected on three different Ndapol. Over the post-treatment

period, 17 infections among eight different Ndapol have been detected.


by the number of animals providing an index of 1,7 for Ndapol.

At the beginning of the study, during the post treatment period, their weight ranged from

99 to 164 kilograms with a mean weight at 128,6 kg (SD = 22,2). At the end of the study, their

weight ranged from 112 to 208 kg with a mean weight of 165,6 kg (SD = 31,8) (table 12).

Results

48

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

With 127 days between the first and last weighting, the average growth rate is of 0,29

kg/day for Ndapol calves. The growth rates for infected and non-infected calves respectively of

0,29 kg/day and 0,35kg/day. However, there is only one non-infected individual.

Seven animals have been re-infected for a total of re-infections 11 observed and one of

the animals has even been infected up to five times during the protocol (Animal number 417).

It is worth noticing that for 10 of these 11 re-infections (91%) the re-infection time was

between four to eight weeks (figure 34).

Figure 34 Number of weeks between two infections for Ndapol





adults Ndapol was 28,0 (SD=6,1); Min = 18; Max = 36. One week later, it was 31,3 (SD=2,9). At

the end of the experiment in October, it was 30,8 (SD=4,6); Min = 21; Max = 37 and a large

variation of PCV values within the herd can be seen (figure 35).

Mean Min Max Mean Min Max Mean Min MaxNdapol,(n=10) 128,6&(SD=22,2) 99 164 165,6&(SD=31,8) 112 208 154,4&(SD=28,9) 99 208,infected,(n=9) 128,0&(SD=23,5) 99 164 164,2&(SD=33,4) 112 208 153,7&(SD=30,0) 99 208

non6infected,(n=1) 134 134 134 178 178 178 159,6&(SD=18,7 134 178

Mean Min Max Mean Min Max Mean Min MaxNdapol,(n=10) 28,0&(SD=6,0) 18 36 30,8&(SD=4,6) 21 37 32,4&(SD=4,7) 16 46,infected,(n=9) 29,7&(SD=4,5) 23 36 30,1&(SD=4,3) 21 35 32,2&(SD=4,7) 16 46

non6infected,(n=1) 18 18 18 37 37 37 33,8&(SD=4,0) 18 38

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

Results

49

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

PCV values are of interest and a pattern is seen for animals with multiple infections.

PCV value significantly drops at the moment of the infection before returning to normal after

treatment (figure 36).

Results

50

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

Eight false negative results have been detected. It represents 21,6 % of the 37 analyses

performed on animals supposed to be infected.

3.5 PARASITEMIA AND TRYPANOSOMA SPECIES

Based on direct observation, two species of Trypanosoma have been observed, T.

congolense and T. vivax. T. congolense was more often present, with 61 certified identification,

however data on the matter are not sufficient to be significant. T. vivax has been identified

twice. As the species could not be positively determined in every sample, there are less

identified samples than positive ones. No mixed infections have been observed.

Trypanosoma congolense was small and slow and often adhered to RBCs in small

groups, whereas T. vivax was large and extremely active under the microscope, quickly

crossing the field in straight direction (OIE, 2013).

Moreover, parasitemia levels have been collected when possible thanks to the Herbert

and Lumsden’s counting method (1976) (table 13). Nevertheless the method’s accuracy seems

insufficient to have a good insight in our case.

Results

51

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))

It is worth noticing that parasitemia levels are different according to the breed. Ndapol

have the higher parasitemia level, followed by Zebus, N’Damas calves and finally N’Damas

adults. However, this information should be handled with care and considered more like a

tendency.

Mean Min Max Number,of,dataNdapol 6,3$log$(SD=0,8) 5,4$log 8,1$log 22

N'damas,adults 6,0$log$(SD=0,2) 5,4$log 7$log 13N'damas,calves 6,1$log$(SD=0,9) 5,4$log 7,2$log 5

Zebus 6,2$log$(SD=0,7) 5,4$log 7,4$log 45

52

4. DISCUSSION

ABT represents an economic burden in Africa. At the ranch, in a context of low

trypanosomiasis pressure, control methods appear efficient but have a great cost in term of

money, manpower and time. In order to use those methods more adequately, it is essential to

benefit from a clear insight of the trypanosomiasis situation in the ranch, depending on the

breed, the category and even the location. The focus of this study was to test the DAI

determination method as a manner to undergo this assessment, among three different breeds,

Zebus, N’Damas and Ndapol, in one park of the ranch. Zebus are well known trypanosensitive

animals and they received a particular care at the ranch with three prophylactic trypanocidal

treatments a year against two for N’Damas and Ndapol. In this study, differences between the

three breeds will be evaluated and discussed in order to adapt control methods more

adequately.

In the present study, it was hypothesised in the introduction that the number of

treatments for the Zebus would be significantly higher than for the Ndama, with the Ndapol at an

average level as the result of the different levels of trypanotolerance.

4.1 DISCUSSION OF THE RESULTS

4.1.1 THE DAI AND INFECTIONS

First infections occurred three weeks after the post-prophylactic treatment for Ndapol,

five for Zebus, six for calves N’Damas and eight for adults N’Damas. This may illustrates

different levels of resistance to trypanosomiasis, i.e. different levels of infections and of

parasitemia.

There is no marked influence of the time on weekly incidence expect for the higher rate

on the last week if we refer to the figure 29. The fact that animals are rapidly treated lowered the

risk of the appearance of a reservoir effect into the herd may help in keeping a stabilised

incidence rate.

Ordner et al., (1988) found that in Gabon, observed that the Nguni cows (a cross breed

between Bos taurus indicus and Bos taurus) had 3,2 infections/cow/year, crossbreds between

N’Damas and Nguni 2,1 and N’Damas only one. Therefore N’Damas are either less infected or

have a better control of parasitemia. They also explained that: “when exposed to a medium

Tsetse challenge at the management level and conditions of OGAPROV, N’Damas performed

well, but Nguni could not survive (i.e. would die) without chemotherapy and have average

performance without chemoprophylaxis. The Nguni x N’Damas crosses were intermediate

between the two parent breeds for the indications of trypanotolerance.“ (Ordner et al., 1988).

At the ranch, average prevalence of 7,5%, 10,1% and 5,4% have been reported for

N’Damas between 1985 and 1991. Prevalence percentages of 25,9% for Nguni and 16,6% for a

Discussion

53

crossbred between Nguni and N’Damas have also been described (see .1.2.2). These results

may be compared to the pre-treatment period where a prevalence of 65% for Zebus, 4,8% for

adults N’Damas, 15,4% for calves N’Damas and 30% for Ndapol was found. This is what was

expected for N’Damas. Higher prevalence for Zebus may be explained by the length of time

since the last treatment and the susceptibility of the breed to the disease.

Over the entire protocol, only one Zebu out of 20 and one Ndapol out of 10 remained

clear of infections. However, 32 adults N’Damas out of 42 and eight calves N’Damas out of 13

remained clear of infections. This highlights the difference of trypanotolerance between breeds

and the possibilities for individual selection for resistance base on infectious events as

developed in the conclusion.

The DAI can be used to establish the challenge in an area and represents the average

number of infections each animal is likely to contract over a period. Most of the time, DAI index

are determined over an annual period, which is considered as a minimum in order to have a

representative result. It is performed in the field, under normal cattle management conditions.

The DAI is nevertheless often underestimated because of the relatively short but inevitable

persistence of the Diminazen-Aceturate, because of the different level of trypanotolerance, i.e.

ability to control and keep parasitemia under the detection threshold and of by the sensitivity of

the laboratory tests detecting parasitemia. In this study, the period of time between the first

positive diagnosis and the treatment is also a source for underestimation of the DAI: during this

period, animal cannot be infected again and is therefore unavailable for the protocol.

DAI for each breed is in accordance with other results tendencies with an index of 1,45

for Zebus, 0,21 for adults N’Damas, 0,23 for calves N’Damas, and 1,7 for Ndapol. Theoretically,

DAI reflects the trypanosomiasis challenge, the higher it is, the higher the risk. Some cases may

have not been detected due to a too low parasitemia, in particular within trypanotolerant cattle

N’Damas, which is highlighted by the relatively high number of false negative tests (see 4.1.6).

However, these results show a clear difference between breeds. Zebus and Ndapol are more

infected than adults and calves N’Damas respectively. This is consistent with different levels of

trypanotolerance for Zebus. It is interesting to see that Ndapol do not have an intermediate level

of trypanotolerance but on the contrary an enhanced sensitivity to the disease. It is important to

keep in mind that this study has only been conducted over a 22 weeks period, during the dry-

season, were trypanosomiasis challenge is supposed to be less important than during the rainy-

season.

According to Uilenberg G. (1998), in mixed herds DAI is significantly higher in Zebus

than in N’Damas. He also classifies DAI determined over a one-year period as follows:

- A DAI of three for Zebus is relatively low and requires only curative treatment of

infected animals detected on a monthly or two-monthly basis.

- A DAI of four to six indicates a medium to high challenge and requires monthly curative

treatments or a prophylactic treatment.

Discussion

54

- A higher DAI indicates a high to very high challenge and requires prophylactic

treatment. However the use of sensitive breed have to be review

It is difficult to extend these results to situation treated in this study since only the dry-

season is concerned. Such quantification is arbitrary and it would be more adequate to pursue

the DAI determination under ranch’s conditions for each breed and area and to determine levels

adapted to each situation. It is nevertheless clear with these results that each breed should be

handled independently.

Re-infection times are interesting and consistent with the incubation period of one to

two weeks, the length of the protection offered by the Diminazen-Aceturate, the sensitivity of the

analysis and the cyclical activity of the parasite. There are four to eight weeks between two

consecutive infections for 52% of the 23 re-infections for Zebus, and 91% of the 11 re-infections

for Ndapol. In both breed, an animal has shown five different infectious events. It shows that

during the year these breeds will need several treatments. Some animals seem to be more

sensitive than others; this may be interesting in genetic selection schemes. Only one adult

N’Damas has been re-infected after 15 weeks while no calf was infected twice. Even if the

protocol was different with five weeks between the first positive diagnosis and the treatment, it

shows that N’Damas are more resistant and/or have lower parasitemia.

4.1.2 ANALYSIS OF WEIGHTING RESULTS

Weigh analysis should be handled with care as there is a lot of factors that may

interfere with the results. Besides, as animals were weighed on a monthly basis, findings are

less precise than expected. It is difficult to consider the weigh gain or loss as an indicator of

health. For instance, a lot a of animals are still growing (calves and heifers), some of them are

pregnant and Zebus have undergone an important weigh loss just before the experiment

because of lactation.

However, it is worth noticing that there is a difference between animals infected at least

once and non-infected animals. For Zebus, the non-infected animal gained 29 kg over the entire

period while infected animals gained an average of 14,8 kg. Non-infected N’Damas adults

gained 14,3 kg while infected animals gained 6,8 kg. Non-infected N’Damas calves gained 37,8

kg while infected animals gained 42 kg. Non-infected Ndapol gained 44 kg while infected

animals gained 36,2 kg.

Concerning growth rates among calves, different results are observed among N’Damas

and Ndapol. For N’Damas and Ndapol, there is a growth rate of 0,33 kg/day and 0,29 kg/day for

infected calves and 0,30 kg/day and 0,35 kg/day for non-infected calves respectively. Therefore,

the influence of infectious events is different according to the breed. Maybe the effect of the

infection is more important in Ndapol than in trypanotolerant calves, N’Damas are able to better

handle trypanosomiasis when infected.

Discussion

55

It is also important to keep in mind that more sensitive laboratory tests for trypanosome

detection might allow a more accurate classification of animals into infected/non infected

categories. Results for N’Damas may be underestimated more importantly than other breeds

because they have most of the time a lower parasitemia; i.e. under the detection threshold.

4.1.3 ANALYSIS OF PCV VALUE RESULTS

Normal PCV values expected are of 37,6 CI 30-45 for adults N’Damas, 43,2 CI 35-51

for N’Damas calves of 6 months old, 23,7 CI 25-34 for N’Damas between 12-20 months (Host et

al., 1983) and 34,9 for adults Zebus (Merlin P., 1986). The four PCV figures 31, 32, 33 and 35

also show a great variability among individuals in each breed.

Results for non-infected animals are equivalent to those described in the literature with

32,0 (SD=4,9) for Zebus, 37,7 (SD=4,5) for adults N’Damas. However, calves N’damas, which

were an average of 6 months old at the beginning of the program and therefore almost 12

months old at the end, have a PCV value at 34,7 (SD=4,2), which was expected. Ndapol have a

value at 33,5 (SD=3,9), consistent with other observations on the breed.

PCV values animals actually infected are lower than values for non-infected animals as

expected with 25,6 (SD=5,9) for Zebus, 34,2 (SD=6,2) for adults N’Damas, 32,2 (SD=4,1) for

calves N’Damas and 27,3 (SD=4,5) for Ndapol. Drop is more important for Zebus and Ndapol

than N’Damas.

On prophylactic treatment day, the average PCV was initially of 23,5 (SD=6,6) for

Zebus, 34,8 (SD=3,0) for adults N’Damas, 31,8 (SD=5,7) for calves N’Damas and 28,0

(SD=6,1). This was after a period of challenge. After only one week, the value raised to 28,6

(SD=5,1) for Zebus, 35,8 (SD=4,3) for adults N’Damas, 32,1 (SD=4,6) for calves N’Damas and

31,3 (SD=2,9) for Ndapol. It shows that Diminazen-Aceturate is efficient and PCV goes back

quickly to its normal level after the treatment. However, it also shows that the effect of

trypanosomiasis on PCV is much more important for Zebus and Ndapol than for N’Damas.

The difference between PCV values at the beginning and at the end of the program

shows that there is an improvement for each breed. It could be explained by better veterinary

support along the program and rapidly treated when diagnose positive to trypanosomiasis.

For re-infected Ndapol, there is a correlation between levels of PCV and infections as

represented on figure 36. The pattern is illustrated for some re-infected animals. A decrease in

PCV value is almost clearly observable on each infection. However, some infections are not

associated with anaemia. The pattern was also not very pronounced for the other breeds. It is in

accordance with what is developed at the point 4.1.4.

The PCV drop observed on May 15th for adults N’Damas is difficult to explain and may

be due to a problem during samples analysis.

Discussion

56

4.1.4 THE DETERMINATION OF A CUT-OFF VALUE FOR PCV

The determination of a cut-off value for PCV value would be very interesting for the

diagnosis of trypanosomiasis. However, results show that it is complicated and not as reliable

as expected. 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). However, minimum and maximum levels do not allow the establishment of a cut-off

value adapted to each breed and each category (age, sex). This is because minimum PCV

values for non-infected animals are lower than maximum values for infected animals with

respectively 8 and 34 for Zebus, 29 and 48 for adults N’Damas, 23 and 39 for calves N’Damas

and 18 and 37 for Ndapol. The determination of a cut-off value should be handled with care and

based on further observation. Moreover, it should only be considered as an indication. This is

also what Marcotty et al., (2008) explained when showing that this is the combination of

parasitological diagnosis and PCV determination that may improved the accuracy of the

diagnostic outcome.

4.1.5 TRYPANOSOMES SPECIES

T. congolense is the most represented species with 96,2% of the identification, followed

by T. vivax (3,8%). This predominance is consistent with previous work in the area in 1991 by

Trail et al. (1991). They observed that T. congolense was responsible of 65%, 74% and 64% of

the identified infections in 1987, 1988 and 1989. Other part of the infections was attributed to T.

vivax. The methodology that combines the diagnosis with a Diminazen-Aceturate treatment of

all animals found positive does not allow us to appreciate the debilitating power of different

trypanosome species by simple comparison of the average PCV in infected and non-infected

animals. However, T. congolense, the dominant species in this ranch, is deemed more

pathogenic for cattle (Trail et al., 1991a).

Ndapol have the higher parasitemia level (6,3 log), followed by Zebus (6,2 log),

N’Damas calves (6,1 log) and finally N’Damas adults (6,0 log). It is consistent with results

observed with DAI. It seems that the more infections there are according to the breed, the more

the parasitemia observed is high. It shows that resistance to trypanosomiasis may be breed

dependant. These results have no absolute meaning but they may provide relative information.

Unfortunately, our results do not allow us to clearly see if there is a difference in the T.

congolense and T. vivax ratio between breeds and category.

4.1.6 FALSE NEGATIVE RESULTS

The amount of false negative results confirm that the BCT method was not sensitive

enough to detect low parasitemia. Depending on the breed, 17,7 % false negative were

observed for Zebus, 21,6 % for Ndapol, 33,3 % for calves N’Damas and 74,1 % for adults

Discussion

57

N’Damas. This is consistent with the other results of parasitemia levels and infections rates.

This underlines the fact that some infections among N’Damas may have not been discovered

because of a too low parasitemia.

4.4 CRITICISM OF METHODOLOGY

4.4.1 SAMPLING AND TREATMENT

The sampling was depending on the dimensions of the genetic selection program.

Therefore, animals were already selected according to some criteria exposed in the Materials

and Methods (2.2). In order to have better results and to be able to easily compare different

groups, animals should have been randomly sampled. It would have been interesting to have

groups with animals randomly sampled over the entire population of the ranch and to have

groups of the same number of animals. Some animals were sometimes missing during the

sample collection because of a lack of manpower and the difficulty to gather them during the dry

season in a very large pasture and to bring them to the health centre.

It would have been interesting to sample animals in different areas of the ranch

especially as there are great differences in Tsetse population depending inside the ranch

according to observation made by workers.

Because of the other protocol, it was not possible to apply the same treatment for

Zebus and Ndapol, and for N’Damas. The first two breed were treated one week after the

diagnosis whereas N’Damas were treated five weeks after. Therefore, there is an important

difference between the two groups. Moreover, it would have been more efficient to treat animals

the day of the diagnosis of trypanosomiasis to avoid a reservoir effect during this period and to

have a better accuracy in the DAI determination.

Some animals have been missing during the study (0,4% of Zebus, 2,1% of adults

N’Damas, 0,6% of calves N’Damas and 1,5% of Ndapol). This reflects the character of the

Zebus that are easier to handle while N’Damas are wilder. Because of a lack of manpower

because during the dry-season animals are more scattered in the field, absence are inevitable.

However, it would have been interesting to avoid such breach.

4.4.2 TIMELINE

It would have been more interesting to have an experiment going on during the entire

year in order to have more accurate results and to study the seasonal effect. The study was

planned to last until April 2015, unfortunately it has to be shortened. A year should be a

minimum but a period of two years would be preferable (Uilenberg G., 1998).

Moreover, the experiment should have started at the same time for the three breeds.

Ndapol were added to the protocol two weeks later and it should have been better organized.

Discussion

58

4.4.3 LABORATORY ANALYSIS

Laboratory analyses were performed the day after the sampling. According to Murray

(1977) it should have been performed soon after collection and at least within four to six hours.

The risk is to see a great decrease in the number of detectable parasites, especially with T.

congolense. Unfortunately, because of the number of animals to collect, the distance between

the experimentation park and the laboratory, and the other protocol to deal with it was not

possible. Another possibility would be to make smaller groups and to have one group every day

in order to have results quicker, however it was not compatible with the ranch’s activities.

The microscopic observation seriously underestimates the relapsing rate after treatment

whereas PCR appears to be 3 to 4 times more sensitive and better at species identification for

T. congolense and T. vivax infections (Gall et al., 2004). The low sensitivity of the BCT method

is well known (see 2.3.4) and may be one of the explanations of the high numbers of false

negatives. It is even more important, if the sample is collected between two peaks of

parasitemia (Paris et al., 1982; OIE, 2013). However, it is a method that can be used in a range

of settings and is not reliant on the availability of a clod chain or expensive laboratory

equipment.

Moreover, Ndama cattle have the ability to control their parasitemia (Paling et al. 1991),

leading to lower numbers of parasites in the bloodstream especially during the chronic phase of

the infection (Mattioli and Faye, 1996). Thus some infections, especially when they are chronic,

may not be detected.

Concerning the determination of trypanosomes species, microscopic observation is not

as sensitive as molecular diagnosis and depends a lot on the user experience and ability. It

would have been interesting to use molecular diagnosis to confirm species determination.

Nevertheless, costs of such analyses are still prohibitive and require highly skilled personal.

Another technician has realized the analyses for the last two weeks of the protocol. It

may have influenced the results as they often vary a lot from one operator to another with the

BCT method (OIE, 2013).

59

5. CONCLUSIONS

Livestock is not developed yet in Gabon. Its development is one of the government’s

goals in order to reach the food autonomy and a part of a government’s global plan of

agriculture development called the “Gabon Vert”. Trypanosomiasis is endemic in the country

and as to be taken into account in order to develop livestock in an effective way from the

beginning. Tsetse control methods are in limited use in Gabon. Information on Tsetse

population dynamics is scarce and for now, regular treatment will continue to be an important

measure for ABT control. Even if this method seems efficient, it is important to consider issues

such as drug-resistant trypanosomes emergence as it is already reported in 18 African

countries (Delespaux et al., 2008) and difficulties of supply.

At the ranch, among the association with other methods for Tsetse control, a more

adequate use of trypanocidal drugs may be of interest depending on the area and on the breed.

The DAI determination seems to be an interesting method of trypanosomiasis risk

assessment at the ranch. However, it is a slow and time consuming operation with high costs in

term of manpower. It would probably be of a better use if only a part of each herd was assessed

by using an average of 5 to 10 % of sentinels depending on the size of the group. Moreover, the

determination of a cut-off value for the PCV value according to the breed and the age and sex

could help during laboratory analysis. Only animals with a value under the cut-off value would

have their blood checked under the microscope.

It would be interesting to have a survey of the Tsetse population in the different areas of

the ranch in order to have a better vision of their pressure on cattle, the prevalence of

trypanosomiasis among them and to see if their eradication is conceivable.

Each situation is a case and therefore different control methods should be used in order

to have an integrated disease control using both trypanosomiasis and vector control. In every

part of such a plan, the cost-benefit principle is essential, especially in a private concession like

the studied ranch. A combination of trypanocidal drug use, insecticidal application on cattle as

well as the use of trypanotolerant cattle is therefore interesting.

However, according to Uilenberg (1998), the association of trypanotolerant livestock

and trypanocidal chemoprophylaxis is likely to be uneconomical and it might be more cost-

effective to reserve treatments for trypanotolerant cattle that actually require a cure. Moreover,

according to Mehlhom and Aspöck (2008), the development of immune responses by the host

toward trypanosomes is not essential for keeping cattle in Tsetse areas as long as they are

treated with Isometamidium, a long-acting trypanocidal drug. However, their results also

indicate that it is essential to allow the infection before the treatment to induce immunity

responses. Therefore, as trypanotolerant cattle are able to keep being productive when

infected, it appears important not to treat them on a prophylaxis basis, in order to avoid the

development of naive animals and to limit the risks.

Conclusions

60

The DAI index may also allow the detection of different levels of sensitivity to the

disease among individuals of the same breed. M. Traore (1989) in a cattle ranch in Mali used

the Diminazen-Aceturate index and found three different groups in an experimental Ndamas

herd with sensitive, tolerant and resistant animals. Therefore, it may be an important tool for

genetic selection in order to improve trypanotolerance in a herd by selecting the most

performing animals, i.e. the least treated. However, according to Verhulst and Pandey (1991),

this method lacks sensitivity and feasibility because of slow results. It would be interesting to

have an integrated program of genetic selection based on weigh, growth and reproduction

criteria, but also taking into account the DAI index and the PCV value, in order to have a better

insight of the cattle productivity.

The limited number of infectious events associated with a lower parasitaemia confirms

the traditionally accepted N'Damas resistance to trypanosomiasis. according to some studies,

trypanotolerance would be as much an individual character than a race character (Roelants et

al., 1983). Our results can not show such variability, but it is a factor worth taking into account

for the selection of the most resistant animals.

Chemotherapy is not advised in the case of trypanotolerant cattle because it may

interfere with the establishment of sufficient natural immunity, exception has to be made when

animals are newly introduced in an infected area from a non-infected one. Into the ranch,

N’Damas could be treated individually and each treatment noted. N’Damas selection herds

should integrate these parameters in order to limit the number of treatments per animal and

improve the productivity. Animals with a certain number of treatments per year might be

eliminated in order to gradually select the most resistant ones. This may be an interesting

method to deal with trypanosomiasis and to avoid a decrease in trypanotolerance and the

appearance of chemo-resistance.

Results collected on Zebus and Ndapol show that these breeds need a particular

attention. Prophylaxis with long-acting drugs such as isomitamidium seems interesting.

However, it is also important to operate a selection between animals to keep the more resistant

on the same model than the one used for N’Damas. It is also important to have a real

assessment of their productivity to see whether or not it is more interesting to breed them under

low Tsetse challenge conditions. Prophylaxis are costly in terms of drugs, time, manpower and

may be dangerous as they imply close cattle manipulation. Isomitamidium offers a longer

protection for the animals but Diminazen-Aceturate allows a better understanding of individual

resistance because it requires more treatments and therefore a better statistical approach of the

issue. Obviously, a combination of the two products is also interesting.

Unlike to what was expected, Ndapol do not show an intermediate level of

trypanotolerance. They actually appear quite sensitive and further studies are required to see

whether introducing Senepol into the herd would really be interesting regarding trypanosomiasis

and productivity.

i

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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