Accepted Manuscript

Title: Trypanosome infection in dromedary camels in EasternEthiopia: prevalence, relative performance of diagnostic toolsand host related risk factors

Author: Regassa Fikru Yimer Andualem Terefe GetachewJoris Menten Epco Hasker Bekana Merga Bruno MariaGoddeeris Philippe Buscher

PII: S0304-4017(15)00194-6DOI: http://dx.doi.org/doi:10.1016/j.vetpar.2015.04.008Reference: VETPAR 7600

To appear in: Veterinary Parasitology

Received date: 29-3-2014Revised date: 27-3-2015Accepted date: 2-4-2015

Please cite this article as: Fikru, R., Andualem, Y., Getachew, T., Menten,J., Hasker, E., Merga, B., Goddeeris, B.M., Buscher, P.,Trypanosome infectionin dromedary camels in Eastern Ethiopia: prevalence, relative performance ofdiagnostic tools and host related risk factors, Veterinary Parasitology (2015),http://dx.doi.org/10.1016/j.vetpar.2015.04.008

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative 1

performance of diagnostic tools and host related risk factors2

3

Regassa Fikrua,b,c*, Yimer Andualemd, Terefe Getachewa, Joris Mentene, Epco Haskerf, Bekana 4

Mergaa, Bruno Maria Goddeerisc, Philippe Büscherb5

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aCollege of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Debre7

Zeit, Ethiopia.8

bInstitute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-9

2000 Antwerp, Belgium.10

cKU Leuven, Faculty of Bioscience Engineering, Department Biosystems, Kasteelpark Arenberg 11

30, B-3001 Leuven, Belgium.12

dSchool of Veterinary Medicine, Wollo University, P.O. Box 1145, Dessie, Ethiopia13

eInstitute of Tropical Medicine, Department of Clinical Sciences, Nationalestraat 155, B-2000 14

Antwerp, Belgium.15

fInstitute of Tropical Medicine, Department of Public , Nationalestraat 155, B-2000 Antwerp, 16

Belgium.17

* Corresponding author. Tel: +25191190705618

E-mail address: fikruregassa@yahoo.com19

20

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Abstract21

A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern 22

Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk 23

factors of trypanosome infection in camel. An overall prevalence of 2 %, 24.1 %, 21.3 %, 9.5 %24

and 7.8 % was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi, 25

RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection 26

was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting 27

camels in the study area. No single animal was positive when tested with T. evansi type B 28

specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular 29

tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate 30

compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate 31

observed in each gender of camels (p > 0.05). The positivity rate was significantly higher in 32

camels with poor body condition and in older animals when tested using the CATT/T.evansi or 33

RoTat 1.2 PCR (p > 0.05). Camels that tested positive with all tests had significantly lower 34

PCV’s (p < 0.05). This study provides further evidence that T. evansi is endemic in the Afar 35

region of Ethiopia.. The latent class analysis indicated an estimate overall prevalence of 19% 36

(95% CI: 13 - 28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and 37

the PCR tests (39%-53%) but higher specificity of the PCR tests (86% -99%) and low specificity 38

of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the 39

tests would help diagnosis of trypanosomosis. Further studies are required to determine the 40

prevalence of clinical disease and losses due to trypanosomosis.41

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Keywords: Dromedary camel, Ethiopia, prevalence, risk factor, trypanosomosis, Trypanosoma43

evansi, Trypanosoma vivax44

Introduction45

Trypanosomosis is one of the major health problems with high morbidity and mortality in camels46

in Ethiopia (Demeke, 1998; Tekle and Abebe, 2001). The disease is caused by infection with 47

hemoflagellated parasites belonging to the genus Trypanosoma. Trypanosoma evansi belongs to 48

Trypanozoon subgenus and is the most commonly reported cause of camel trypanosomosis called 49

surra (Röttcher et al., 1987). T. evansi is mechanically transmitted by blood sucking flies, such as 50

Tabanidae and Stomoxys sp (Hoare, 1972). A number of researchers have investigated the 51

epidemiology of camel trypanosome infection in different parts of Ethiopia. These studies were 52

mainly based on parasitological and serological tests (Richard, 1979; Elias, 2003; Hailemariam 53

et al., 2008; Hagos et al., 2009; Kassa et al., 2011; Tadesse et al., 2012). Parasitological 54

examination suffers from limited sensitivity even when using the haematocrit centrifugation 55

technique. Serological tests are unable to distinguish past and current infections as the antibodies 56

persist in the circulation (Büscher, 2014). The serological tests also are not fully specific due to57

the possibility of cross reactions with antibodies produced against other infections. For example 58

Desquesnes et al. described cross reactivity of T. evansi antigen with anti T. cruzi antibodies59

(Desquesnes et al., 2007). Control of camel trypanosomosis depends mainly on the use of 60

curative and prophylactic drugs and their application ideally should take into account actual 61

information of the disease. However to properly understand the epidemiology of a disease, up-to-62

date and high-quality data are required through application of accurate diagnostic tools (Ali et 63

al., 2009). In Ethiopia, a number of studies indicated that T. evansi is the sole pathogenic 64

trypanosome infecting camels (Hailemariam et al., 2008; Kassa et al., 2011; Tadesse et al., 65

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2012). These findings need also to be verified with more accurate diagnostic techniques like 66

PCR to assess the possibility of infection with other mechanically transmitted trypanosomes like67

T. vivax (Birhanu et al., 2013). Therefore, this study was undertaken to assess the prevalence of 68

camel trypanosome infection in the Afar region using different diagnostic techniques, to assess 69

the relative performance of different diagnostic PCRs and to identify host related risk factors 70

associated with trypanosome infection.71

Materials and methods 72

Study area73

The present study was conducted in Chifra and Dewe districts of Afar Region, located in the 74

North-Eastern part of Ethiopia between 39°34' and 42°28'E longitude and 8°49' and 14°30'N 75

latitude in the rift valley (Figure 1). The districts have similar arid and semi-arid agro-ecology, 76

where livestock production is the main occupation of the community. The average elevation of 77

the districts is 802-825 meters above sea level with annual temperature ranging from 25ºC to 78

33ºC. The rainfall is bimodal with erratic distribution, the long rainy season (kerma) taking place 79

between Mid-June to Mid-September and the short rainy season (sugum) occurring between 80

March and April. The average annual rainfall is between 400 and 600 mm.81

Study animals and design82

Dromedary camels ≥ 6 months old were considered for blood sampling. Age, gender, and 83

previous treatment history were recorded. Body condition was assessed according to Faye et al. 84

(Faye et al., 2001). Animal age groups were defined as follows: 6 months to 1 year, >1 to 5 85

years, >5 to 10 years and >10 years.86

A cross-sectional study was conducted on 399 camels (199 camels from Chifra and 200 camels 87

from Dewe) from November 2011 to April 2012. A combination of convenience, purposive and 88

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multistage stratified sampling methods was applied according to Toma et al. (Toma et al., 1999). 89

First, the two study districts were selected based on their camel population and reported cases of 90

camel trypanosomosis. A complete list (sampling frame) of the pastoral villages was obtained 91

from which five pastoral associations (PAs) within each district were selected based on their 92

accessibility. From these PAs, three to five herds were selected from volunteer herders.93

Pastoralists keep their camel herd in groups based on age, gender and reproductive status 94

(lactating) in separate stables which were considered as stratum. However, some dominant 95

lactating females could not be caught. An average of 10 camels (8–12) were randomly selected 96

from each herd depending on the number of camels available from each stratum.. The samples 97

size required to estimate the prevalence of infection in the camel population was 384 based on 98

the formula given by Thrusfield with a design prevalence of 50%, a precision of 5% and a 95% 99

confidence interval (Thrusfield, 2005). The sample size was increased to 399 to provide an 100

allowance for sample loss..101

Specimen collection and diagnostic test102

Blood samples were collected from the jugular vein into 10 ml heparinised vacutainer tubes. 103

From the collected blood, thin smears were prepared, air dried, fixed with methanol and stained 104

with Giemsa for parasitological examination. Packed cell volume (PCV), expressed in 105

percentage, was estimated after 5 minutes centrifugation at 12000 rpm of blood in two capillary 106

tubes.107

About 250 µl of blood were preserved in an equal volume of AS-1 buffer (QIAgen) and stored at 108

ambient temperature for DNA extraction. The remaining blood was centrifuged and plasma was 109

collected and preserved at -20°C until the antibody detection was performed with the Card 110

Agglutination Test for Trypanosomiasis (CATT/T. evansi). CATT/T. evansi was performed as 111

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per the manufacturer‘s instructions (Institute of Tropical Medicine, Antwerp, Belgium). Briefly, 112

25μl of camel plasma, diluted 1:4 in CATT-buffer, were dispensed onto a reaction zone of a 113

plastic test card. After adding one drop (about 45 μl) of CATT reagent, the reaction mixture was 114

spread by a stirring rod and allowed to react on a CATT rotator for 5 min at 70 rpm. A specimen115

was considered positive when blue agglutinates were visible (Bajyana Songa and Hamers, 1988; 116

Verloo et al., 2000). 117

Molecular analysis118

For the molecular analysis, DNA was extracted with the QIAamp mini blood kit (Qiagen) from 119

200 µL blood/AS1 buffer into 200 µL elution buffer according to the manufacturer's instructions120

as described elsewhere (Fikru et al., 2012). Extracted DNA was stored at -20°C until tested by 121

the respective PCRs. The PCRs with their target sequence, primer sequences and amplicon 122

lengths are represented in Table 1. Interpretation of the results after electrophoresis in 2% 123

agarose gels and staining with ethidium bromide (EtBr), is based on the characteristic amplicon 124

lengths. The different PCRs were conducted on all the samples to assess their performance to 125

detect and identify the different trypanosome species affecting camels. 126

Furthermore, comparison of the analytical sensitivity of the RoTat 1.2 PCR, 18S PCR and ITS-1 127

PCR was carried out on purified DNA of two T. evansi isolates, from Kazakhstan and Morocco. 128

The DNA was tested as fivefold dilution series in water, ranging from 1 ng/μl down to 0.064 129

pg/μl.130

Data analysis131

Data were analysed using SPSS (Statistical Package for Social Sciences, version 20) and STATA 132

(Stata Statistical Software: Release 12. StataCorp LP). McNemar chi-square (χ2) test and odds 133

ratios (OR) were calculated to compare the prevalence of trypanosome infection in the two study 134

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areas. The level of agreement between diagnostic tests was determined using Cohen's kappa 135

coefficient interpreted following Landis and Koch (Cohen, 1960; Landis and Koch, 1977).136

ANOVA was used to assess the difference in mean PCV of parasitemic and aparasitemic camels. 137

95% binomial confidence interval and p < 0.05 was set to decide on statistical significance.138

For an exploratory assessment of the diagnostic accuracy of the different tests, allowing for the 139

imperfect nature of all diagnostic methods used, we performed latent class analysis (LCA). This 140

model-based approach allows an unbiased estimation of the sensitivity and specificity of 141

diagnostic methods when a gold standard test that is 100% sensitive and 100% specific is 142

unavailable. We performed LCA using WinBUGS (Stata Statistical Software: Release 12. 143

StataCorp LP) and R, assessing several models allowing for dependence between test results 144

within infected and non-infected subjects, as well as the standard conditional independence 145

model (Menten et al., 2008; R Core Team, 2014). The best fitting models presumed 100% 146

specificity of the thin blood smear Giemsa staining technique and allowed false negative test 147

results on the 3 PCR tests to be correlated. This model showed a good fit to the data (Bayesian 148

lack-of-fit p-value = 0.238). 149

150

Results151

The observed prevalences of trypanosome infection in camel in both districts, estimated using 152

the diagnostic tests are given in table 2.153

The overall prevalence of camel trypanosome infection was significantly (p < 0.05) lower in 154

Giemsa stained thin smear (Table 2) than in the other tests. The serological test, CATT/T. evansi155

and the molecular test, RoTat1.2 PCR (T. evansi type A-specific), revealed a significantly higher 156

prevalence than ITS-1 and 18S PCRs (p < 0.05). A number of specimens were shown to contain 157

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T. vivax based on the results of the ITS-1 PCR. The parasites observed by thin blood smear 158

examination had morphology characteristic of T. evansi with a small subterminal kinetoplast at 159

the posterior end and a flagellum (Hoare, 1972). With the T. vivax proline racemase PCR, only 160

one T. vivax infection could be confirmed. No single T. evansi type B infection was detected 161

with EVAB PCR. No statistically significant difference in prevalence of trypanosome infection 162

was recorded between the study districts by any of the diagnostic tools.163

Out of 96 CATT/T. evansi positives samples, respectively 26, 13 and 10 samples were also 164

positive in RoTat 1.2 PCR, 18S PCR and ITS-1 PCR (Table 3 ). On the other hand, out of 303 165

CATT/T. evansi negative samples, respectively 59, 25 and 21 samples were positive for 166

RoTat1.2 PCR, 18S PCR and ITS-1 PCR. There was slight agreement between CATT/T. evansi167

and 18S PCR (K = 0.07), CATT/T. evansi and RoTat1.2 PCR (K = 0.08), and CATT/T. evansi168

and ITS-1 PCR (K = 0.05). Out of the 85 RoTat1.2 PCR positives, 52 were negative in 18S PCR 169

and 58 were negative in ITS1 PCR. 18S PCR and ITS-1 PCR picked respectively 5 and 4 170

positive specimens out of 314 RoTat 1.2 PCR negative specimens (Table 3 ). There was slight171

agreement between RoTat1.2 PCR and Giemsa stained thin blood smear (K = 0.03) but there was 172

a moderate agreement between RoTat 1.2 PCR and 18s PCR (K = 0.47) and a fair agreement 173

between RoTat1.2 PCR and ITS-1 PCR (K = 0.39). There was a substantial agreement between 174

18S PCR and ITS-1 PCR (K = 0.79) though out of 38 positives for 18S PCR, 10 specimens were 175

negative for ITS-1 PCR and out of 31 ITS-1 PCR positives, 3 were negative for 18S PCR (Table 176

3). There was slight agreement between Giemsa stained thin blood smear and 18S PCR (K = 177

0.05).178

The frequency distribution of tests results (N) and probability of being infected for each outcome 179

pattern as estimated with LCA is presented in Table 3. Further results from LCA are summarised 180

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in Table 4. The estimated prevalence from the model was 19% (95% CI: 13 - 28). The model 181

indicated for CATT/T. evansi a low sensitivity of 43% with relatively low specificity of 80%. 182

The PCR tests showed higher specificity (86% for RoTat 1.2, >99% for 18S and ITS-1) with 183

sensitivity similar to CATT/T. evansi in the 40 to 50% range (53% for RoTat 1.2 PCR, 48% for 184

18S PCR, and 39% for ITS-1 PCR). The LCA results confirmed the very low sensitivity (11%) 185

of Giemsa stained thin blood smear in this setting.186

The latent class model classified all those with Giemsa stained thin blood smear positive as 187

infected, as well as all those with at least 2 PCRs positive, or CATT/T. evansi and at least one of 188

18S PCR or ITS-1 PCR positive. Those with only CATT/ T. evansi or at most a single PCR 189

positive were classified as not infected, as were those with only CATT/T. evansi and RoTat 1.2 190

PCR positive.191

When tested on purified DNA of a Kazakhstan and a Moroccan T. evansi strain, the analytical 192

sensitivity of the ITS-1 PCR was 0.064 pg/µl with both T. evansi strains and lower than the 193

RoTat1.2 PCR (0.32 pg/µl) and 18S PCR (0.32 pg/µl). However, at the lower DNA 194

concentrations, aspecific amplicons start to appear besides the Trypanozoon-specific 450 bp in 195

the ITS-1PCR (Figure 2). 196

None of the PCR tests showed a significant (p > 0.05) difference in positivity rate between 197

gender groups and between animals with poor and moderate body condition (Table 5). However 198

a significantly higher prevalence in female animals (χ2 = 5.4, p < 0.05; OR = 0.6, CI 0.3-0.9 by 199

CATT/T. evansi) and in camels with poor body condition (χ2 = 20.3, p < 0.05; OR = 26.8, CI 200

3.3-220.9 by thin smear examination, χ2 = 31.4, p < 0.05; OR = 4.1, CI 2.4-6.8 by RoTat1.2 201

PCR) was recorded. No significant difference in positivity rate was recorded between age groups 202

with ITS-1 PCR (χ2 = 0.7, p = 0.3; OR = 1.7, CI 0.5-6.0) and 18S PCR (χ2 = 0.2, p = 0.4; OR = 203

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1.3, CI 0.4-4.6) but in CATT/T. evansi (χ2 = 3.8, p < 0.05;, OR = 2.3, CI 1.0-5.2) and RoTat1.2 204

PCR (χ2 = 5.6, p < 0.05;, OR = 2.7, CI 1.2-6.2) there were significantly more positives in older205

camels. History of trypanocidal treatment had no significant (p > 0.05) impact on positivity rate206

by all the diagnostic tests. 207

The overall mean PCV in the studied camels was low (23.6%) indicating that the animals were 208

generally in poor condition. It is noteworthy that in our study, no camels were observed with 209

good body condition. In all diagnostic tests PCV was significantly (p < 0.05) lower in test 210

positive animals compared to test negative animals (table 6). 211

Discussion212

In this study, Giemsa stained thin blood smear examination, antibody detection by CATT/T.213

evansi and molecular tests by RoTat 1.2, 18S and ITS1-PCRs were used to assess prevalence and 214

host related risk factors of trypanosome infection in camels in Afar region, Eastern Ethiopia. 215

Only 2% of the examined camels were found infected by T. evansi by microscopy. This216

corresponds with previous findings in camels in different parts of Ethiopia, including two other 217

districts in Afar Region, Gowani and Awash Fentale (Getahun, 1998; Hailemariam et al., 2008; 218

Kassa et al., 2011; Tadesse et al., 2012) and in other African countries, for instance 2.3% in 219

Kenya (Ngaira et al., 2004) and 2.5% in Nigeria (Egbe-Nwiyi and Chaudry, 1994). However, 220

some other studies conducted in Ethiopia reported higher prevalences 6.5 %-12.1% (Richard, 221

1979; Abebe, 1991; Hailu, 2000; Hagos et al., 2009). This could be due to differences in the 222

management and husbandry regimens of the camels under study, or to seasonal effects as well as 223

to study design. Previous studies have shown that the prevalence of surra in camels is higher 224

during the rainy season and could be explained by higher densities of Tabanid and Stomoxys flies 225

during that season (Löhr et al., 1985; Luckins, 1988; Hailemariam et al., 2008).226

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In the absence of a golden standard test, the LCA revealed an overall estimate prevalence of 227

19.4%, with low sensitivity of CATT/T. evansi and the PCR tests (43% - 53%) but better than 228

the sensitivity of Giemsa stained thin blood smear examination (11%). When considering 229

microscopy, one should keep in mind that the observed prevalence reflects only a fraction of the 230

real prevalence since most microscopic techniques are poorly sensitive (OIE, 2012). In case of 231

Giemsa stained thin smear examination, the lower detection limit is greater than 500,000 232

trypanosomes/ml of blood (OIE, 2012). Therefore, indirect diagnosis, e.g. by detection of T. 233

evansi specific antibodies, may give a better estimation of the infection burden as has been 234

shown in previous studies (Diall et al., 1994; Gutierrez et al., 2000). An overall seroprevalence 235

of 24.1% was recorded with CATT/T. evansi and corresponds with the CATT seroprevalence 236

observed in Bale zone (24.9%) (Hagos et al., 2009).237

The observed seroprevalences may be overestimates of the actual prevalence since antibody 238

detection tests have the inherent shortcoming that past infections cannot be distinguished from 239

current infections (Luckins, 1988; Ngaira et al., 2003; Zeyed et al., 2010). Molecular diagnostics 240

targeting parasite DNA are considered good surrogates for parasite detection (Büscher, 2014). 241

An overall positivity rate of 21.1%, 9.5% and 7.8% was observed respectively by RoTat 1.2 242

PCR, 18S PCR and ITS-1 PCR, which is significantly higher than recorded by thin smear 243

examination (2%). CATT/T evansi and RoTat1.2 PCR yielded comparable positivity rates that 244

were significantly higher than in 18S and ITS-1 PCRs. Similar observations were made in 245

Uganda where very low molecular prevalence of Trypanozoon was recorded with the ITS-1 PCR 246

as compared to a PCR for the single copy gene phospholipase C (GPI-PLC) (Ahmed et al., 247

2013). The higher positivity rate recorded with RoTat1.2 PCR needs special attention but could 248

partly be explained by the low RoTat1.2 PCR specificity estimate by LCA. Our data show that 249

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the analytical sensitivity of RoTat 1.2 PCR based on the single copy gene is lower than the ITS-1 250

and 18S PCR based on multi copy sequences. Yet, RoTat 1.2 PCR unexpectedly yielded a higher 251

positivity rate than ITS-1 and 18S PCR. The lowest positivity rate obtained by ITS-1 PCR252

remains unexplained but it could be related to limited specificity of the ITS-1 primers. We 253

observed that when the concentration of target DNA decreases in the presence of host DNA, the 254

primers become less specific and tend to bind to the host DNA. On the other hand, we cannot 255

rule out that the RoTat1.2 PCR is less specific in the presence of host DNA as it is supported by 256

the LCA where RoTat1.2 PCR is less specific than ITS-1 PCR and 18S PCR. Noteworthy, only 257

one out of 8 thin blood smear positive animals was negative for CATT/T. evansi whereas out of 258

303 CATT/T. evansi negative samples 19.5%, 8.3% and 6.9% respectively, were positive for 259

RoTat1.2, 18S and ITS-1 PCRs. It is possible but unlikely that all these seronegative animals 260

carried early infections and had not yet formed detectable antibody levels. The number of RoTat 261

1.2 PCR positives that could not be detected by CATT/T. evansi is significantly higher than with262

18S and ITS-1 PCRs. This could be explained by the presence but not yet the expression of the 263

RoTat1.2 gene in the circulating trypanosomes (Verloo et al., 2001). On the other hand there are 264

a number of RoTat1.2 PCR and CATT/T. evansi negatives that are positive in 18S and ITS-1 265

PCR that could be explained by the absence of the RoTat1.2 gene like it has been described for 266

the T. evansi type B strains isolated in Kenya and suspected to circulate also in Ethiopia but not 267

confirmed in our study (Elsaid et al., 1998; Davison et al., 1999; Ngaira et al., 2003; Hagos et al., 268

2009; Zeyed et al., 2010; Salim et al., 2011).269

Even though 18S PCR and ITS-1 PCR cannot differentiate between the taxa of the Trypanozoon270

group, the positive results reported in this study are most likely due to T. evansi, since most of 271

them are positive in the T. evansi specific RoTat1.2 PCR and there are no records of tsetse flies272

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in the Afar region excluding the presence of T. brucei but not of non-RoTat 1.2 T. evansi (Njiru 273

et al., 2006). In Ethiopia, tsetse flies and tsetse-transmitted trypanosomes prevail in the 274

Southwestern and Northwestern parts of the country following major river basins between 275

longitude 33o and 38o E and latitude 5o and 12o N while the rest of the country, the North, 276

Southeast and East are considered tsetse free (National Tsetse and Trypanosomosis Investigation 277

and Control Center (NTTICC), 2004; Sinshaw et al., 2006). Our study revealed, for the first time 278

in Ethiopia, a T. vivax infection in camels. That there are no previous reports of camels infected 279

with T. vivax in Ethiopia is probably due to the fact that the commonly used serological and 280

parasitological tests fail to detect T. vivax or to distinguish it from T. evansi. Mixed infections of 281

T. congolense, T. vivax and Trypanozoon have been reported in camels in Nigeria but were based 282

on microscopic examination while in Sudan, using the ITS-1 PCR, only T. evansi (Trypanozoon) 283

has been detected in camel (Mbaya et al., 2010; Salim et al., 2011). Pathogenicity of T. vivax to284

camels is not well described but infected animals could act as reservoir and spread the parasite to 285

the more susceptible livestock species (Enwezor and Sackey, 2005).286

Whatever diagnostic technique used, the overall observed prevalence of surra was similar in both 287

study sites which can be explained by comparable camel husbandry practices and ecological 288

biotopes in both districts. On the other hand, a significantly higher prevalence was recorded in 289

female animals by CATT/T. evansi and Giemsa stained thin smear. Similar findings were 290

reported in Kenya and in Israel (Singh et al., 2004; Berlin et al., 2010). The fact that 291

seropositivity in CATT increases with age is in agreement with previous studies and probably 292

reflects time of exposure to biting flies and persistence of circulating antibodies, even after 293

successful treatment (Dia et al., 1997; Gutierrez et al., 2000; Atarhouch et al., 2003). Camels 294

with poor body conditions were more likely to be positive in Giemsa stained thin smear and 295

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CATT/T. evansi, compared to those with moderate body condition. Nutritional stress or other 296

diseases, whether infectious or not, could render camels more susceptible to infection with T. 297

evansi (Eyob and Matios, 2013). On the other hand, camel trypanosomosis by itself is one of the 298

major causes resulting in poor body condition (Röttcher et al., 1987). Significantly lower PCV 299

values were recorded in seropositive and PCR positive camels which corresponds with other 300

studies using serological, parasitological and molecular diagnostic tests (Diall et al., 1994; 301

Atarhouch et al., 2003; Tadesse et al., 2012).302

In this study, we used LCA to estimate the accuracy of the different diagnostic tests in the 303

absence of a gold standard. LCA revealed lower sensitivity for CATT/T. evansi (43%) than 304

expected from previous studies (80%-92%) while the specificity (80.4%) falls within the 305

expected range (69%-100%)(Diall et al., 1994; Diall et al., 1997; Verloo et al., 1998; Ngaira et 306

al., 2003). Unfortunately, for reasons of specimen import regulations, we were not able to 307

perform immune trypanolysis with T. evansi RoTat 1.2 as reference test for T. evansi type A 308

specific antibodies (Verloo et al., 2001). The low sensitivity of the CATT/T. evansi in this study 309

may be due to unusual low amounts of specific antibodies circulating in the infected camels, that 310

might have been detected in immune trypanolysis, or to infections with T. evansi type B that 311

remained under the lower detection limit of the EVAB PCR.312

In conclusion, this study confirms that trypanosome infection is a potential threat affecting the 313

health and productivity of camel, in particular adult female animals, in Afar, Ethiopia. T. evansi314

is the prominent trypanosome species affecting camels since only one case among 399 camels 315

was confirmed infected with T. vivax by TvPRAC PCR. There are significant discrepancies in 316

sensitivity of different diagnostic tests used in this epidemiological study necessitating 317

improvement of the available diagnostic tests to provide high quality epidemiological data.318

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Acknowledgements319

The PhD fellowship of Fikru Regassa was financed by the Belgian Directorate General for Development 320

Cooperation.321

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323

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Table 1: Primer sequences, target sequences and expected amplicon size of the different PCRs used to test blood from 399 camels

from Eastern Ethiopia.

Target group Target

sequence

Primers Primer sequence Amplicon (bp) Reference

18s F 5′-CGCCAAGCTAATACATGAACCAA-3′Trypanosomatidae 18S

rRNA 18S R 5′-TAATTTCATTCATTCGCTGGACG-3′

110 (Deborggraeve et

al., 2006)

ITS-1 F 5’-TGTAGGTGAACCTGCAGCTGGATC-3’Trypanozoon ITS-1

rRNA ITS-1 R 5-CCAAGTCATCCATCGCGACACGTT-3’

450 (Desquesnes et al.,

2001)

RoTat1.2F 5′-GCGGGGTGTTTAAAGCAATA-3′T. evansi type A RoTat1.2

RoTat1.2R 5′-ATTAGTGCTGCGTGTGTTCG-3′

205 (Claes et al., 2004)

T. evansi type B minicircle EVAB-1 5’-ACAGTCCGAGAGATAGAG-3' 436 (Njiru et al., 2006)

EVAB-2 5'-CTGTACTCTACATCTACCTC-3’

TvPRAC F 5’-CGCAAGTGGACCGTTCGCCT-3’T. vivax Proline

racemase TvPRAC-R 5’-ACGCGGGGCGAACAGAAGTG-3’

239 (Fikru et al., 2014)

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Table 2: Observed prevalences in % and with 95% confidence interval of T. evansi infection in camels in two districts of Eastern

Ethiopia using different diagnostic tests.

TotalDistrict Thin blood

smear

CATT/T. evansi RoTat1.2 PCR 18S PCR ITS-1 PCR

Chifra 2 (0.06-3.94) 20.6 (15-26.2) 21.1(15.3-26.8) 8 (4.2-11.8) 8 (4.2-11.8) 199

Dewe 2(0.05-3.95) 27.5(21.3-33.7) 21.5 (15.8-27.2) 11 (6.7-15.3) 7.5 (3.9-11.2) 200

Total 2(0.6-3.4) 24.1(19.9-28.3) 21.3 (17.3-25.3) 9.5 (6.6-12.4) 7.8 (5.2-10.4) 399

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Table 3: Frequency distribution (N) and probability of being infected for each outcome pattern observed in five diagnostic tests for T.

evansi infection performed on 399 camels from Eastern Ethiopia, as estimated with latent class analysis.

PCR

CATT/T. evansi RoTat 1.2 18S ITS1 Thin blood smear N Probability infected

+ + + + + 2 100

+ + + + - 6 100

+ + - - + 1 100

+ - - - + 4 100

- + + + - 18 100

- - - - + 1 100

+ - + + - 1 96.8

+ + + - - 3 94.1

- - + + - 1 92.2

- + + - - 4 85.3

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+ + - + - 1 84.7

+ - + - - 1 60.5

- - + - - 2 38.2

- - - + - 2 19.6

+ + - - - 13 18.4

+ - - - - 64 17.8

- + - - - 37 7.2

- - - - - 238 6.7

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Table 4: An exploratory assessment of the diagnostic accuracy of the different diagnostic tests performed on 399 camels from Eastern

Ethiopia, estimated by latent class analysis with a prevalence of 19.4 % (95% CI: 12.8 – 28.0)

Tests Sensitivity (95% CI) Specificity (95% CI)

CATT 42.5 (32.4-52.9) 80.4 (77.8-84.6)

PCR RoTat1.2 52.9 (39.3-67.9) 85.9 (84.1-88.1)

PCR 18S 47.7 (33.0-64.2) 99.2 (98.0-100)

PCR ITS-1 39.1 (26.4-52.8) 99.4 (98.8-100)

Giemsa stained thin smear 10.8 (7.3-14.6) 100 (fixed)

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Table 5: Host related variables and positivity rate obtained with different tests for T. evansi infection diagnosis, performed on 399

camels from Eastern Ethiopia.

Thin blood smear CATT/ T. evansi RoTat1.2 PCR 18S PCR ITS-1 PCR

Number Positive Positive Positive Positive Positive

Female 251 2.8* (0.8-4.8) 27.9 (22.6-33.5)* 23.1 (17.9-28.3) 8.8 (5.3-12.3) 6.8 (3.7-9.9)Gender

Male 148 0.7 17.6 (11.5-23.7) 18.2 (12.0-24.4) 10.8 (5.8-

15.8)

9.5 (4.8-14.2)

Moderate 311 0.3 17.7 (13.5-21.9) 22.5 (17.9-27.1) 10 (6.7-13.3) 7.4 (4.5-10.3)Body

condition

score

Poor 88 8* (2.3-10.7) 46.6* (36.4-56.8) 17 (9.1-24.9) 7 (1.7-12.3) 9.1 (3.1-15.1)

No 200 2.0 (0.1-3.9) 27.5 (21.3-33.7) 21.5 (15.8-27.2) 11 (6.7-15.3) 7.5 (3.8-11.2)Treatment

history Yes 199 2.0 (0.0-4.0) 20.6 (15.0-26.2) 21.1 (15.4-26.8) 8 (4.2-11.8) 8 (4.2-11.8)

Age category ≤ 1yr 50 0 20 (8.9-31.1) 22.0 (10.5-33.5) 12.0 (13.0-

31.0)

8.0 (0.5-15.5)

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1 < x ≤ 5 yr 72 0 15.3 (7.0-23.6) 25.0 (15.0-35.0) 12.5 (4.9-

20.1)

11.1 (3.8-

18.4)

5 < x ≤ 10 yr 252 2.4 (0.5-4.3) 25.8 (20.4-31.2) 18.3 (13.5-23.1) 7.9 (4.6-11.2) 6.3 (3.3-9.3)

> 10 yr 25 8 (0.0-18.3) 40.0* (20.8-59.2) 40.0* (20.8-59.2) 12.0 (0.0-

24.7)

12.0 (0.0-24-

7)

* significant difference

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Table 6: Mean PCV (%) with standard deviation (SD) and 95% confidence interval of 399 camels from Eastern Ethiopia tested using

different tests to detect T. evansi infection.

Test PCV SD 95% confidence interval

Negative 23.7 2.7 23.4-24.0Thin blood smear

Positive 20.7* 2,1 19.0-22.5

Negative 24.3 2.6 24.0-24.6CATT/T. evansi

Positive 21.5* 1.8 21.1-21.9

Negative 23.8 2.7 23.5-24.1RoTat1.2 PCR

Positive 23.0* 2.7 22.4-23.5

Negative 23.7 2.7 23.5-24.018S PCR

Positive 22.6* 2.7 21.8-23.5

Negative 23.7 2.7 23.4-24.0ITS-1 PCR

Positive 22.6* 2.7 21.7-23.6

* significant difference

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Figure legends

Figure 1: Map of Ethiopia with the two districts (green) Chifra and Dewe in the Afar Region (orange border).

Figure 2: Analytical sensitivity of the three PCRs: Panel A = RoTat1.2 PCR; Panel B = 18S PCR, Panel C = ITS-1PCR. Lanes 1:

Gene Ruler 100 bp DNA ladder (Fermentas). Lanes 2 -8: DNA of the T. evansi isolate from Kazakhstan at decreasing concentrations

(1000, 200, 40, 8, 0.16, 0.032, 0.0064 pg/µl). Lanes 9-15: DNA of the T. evansi isolate from Morocco at decreasing concentrations

(1000, 200, 40, 8, 0.16, 0.032, 0.0064 pg/µl).

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We conducted a cross-sectional study on camel trypanosome infection in Eastern Ethiopia

Depending on the diagnostic test, positivity rates ranged from 2% to 24%

Latent class analysis estimated an overall prevalence of 19%

All infections were due to Trypanosoma evansi except one with Trypanosoma vivax

Risk factors for infection were poor body condition and age

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Figure 1

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Figure 2