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Trypanosome infection in dromedary camels in Eastern Ethiopia: Prevalence, relative performance of diagnostic tools and host related risk factors
Accepted Manuscript Title: Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative performance of diagnostic tools and host related risk factors Author: Regassa Fikru Yimer Andualem Terefe Getachew Joris Menten Epco Hasker Bekana Merga Bruno Maria Goddeeris Philippe B¨uscher PII: DOI: Reference:
S0304-4017(15)00194-6 http://dx.doi.org/doi:10.1016/j.vetpar.2015.04.008 VETPAR 7600
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
29-3-2014 27-3-2015 2-4-2015
Please cite this article as: Fikru, R., Andualem, Y., Getachew, T., Menten, J., Hasker, E., Merga, B., Goddeeris, B.M., B¨uscher, P.,Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative performance of diagnostic 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 proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative
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performance of diagnostic tools and host related risk factors
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Regassa Fikrua,b,c*, Yimer Andualemd, Terefe Getachewa, Joris Mentene, Epco Haskerf, Bekana
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Mergaa, Bruno Maria Goddeerisc, Philippe Büscherb
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Zeit, Ethiopia.
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b
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College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Debre
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Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-
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2000 Antwerp, Belgium.
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30, B-3001 Leuven, Belgium.
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d
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Antwerp, Belgium.
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f
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Belgium.
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* Corresponding author. Tel: +251911907056
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E-mail address: [email protected]
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KU Leuven, Faculty of Bioscience Engineering, Department Biosystems, Kasteelpark Arenberg
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School of Veterinary Medicine, Wollo University, P.O. Box 1145, Dessie, Ethiopia
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Institute of Tropical Medicine, Department of Clinical Sciences, Nationalestraat 155, B-2000
Institute of Tropical Medicine, Department of Public , Nationalestraat 155, B-2000 Antwerp,
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Abstract
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A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern
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Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk
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factors of trypanosome infection in camel. An overall prevalence of 2 %, 24.1 %, 21.3 %, 9.5 %
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and 7.8 % was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi,
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RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection
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was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting
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camels in the study area. No single animal was positive when tested with T. evansi type B
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specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular
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tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate
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compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate
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observed in each gender of camels (p > 0.05). The positivity rate was significantly higher in
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camels with poor body condition and in older animals when tested using the CATT/T.evansi or
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RoTat 1.2 PCR (p > 0.05). Camels that tested positive with all tests had significantly lower
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PCV’s (p < 0.05). This study provides further evidence that T. evansi is endemic in the Afar
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region of Ethiopia.. The latent class analysis indicated an estimate overall prevalence of 19%
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(95% CI: 13 - 28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and
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the PCR tests (39%-53%) but higher specificity of the PCR tests (86% -99%) and low specificity
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of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the
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tests would help diagnosis of trypanosomosis. Further studies are required to determine the
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prevalence of clinical disease and losses due to trypanosomosis.
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Keywords: Dromedary camel, Ethiopia, prevalence, risk factor, trypanosomosis, Trypanosoma
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evansi, Trypanosoma vivax
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Introduction
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Trypanosomosis is one of the major health problems with high morbidity and mortality in camels
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in Ethiopia (Demeke, 1998; Tekle and Abebe, 2001). The disease is caused by infection with
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hemoflagellated parasites belonging to the genus Trypanosoma. Trypanosoma evansi belongs to
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Trypanozoon subgenus and is the most commonly reported cause of camel trypanosomosis called
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surra (Röttcher et al., 1987). T. evansi is mechanically transmitted by blood sucking flies, such as
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Tabanidae and Stomoxys sp (Hoare, 1972). A number of researchers have investigated the
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epidemiology of camel trypanosome infection in different parts of Ethiopia. These studies were
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mainly based on parasitological and serological tests (Richard, 1979; Elias, 2003; Hailemariam
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et al., 2008; Hagos et al., 2009; Kassa et al., 2011; Tadesse et al., 2012). Parasitological
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examination suffers from limited sensitivity even when using the haematocrit centrifugation
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technique. Serological tests are unable to distinguish past and current infections as the antibodies
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persist in the circulation (Büscher, 2014). The serological tests also are not fully specific due to
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the possibility of cross reactions with antibodies produced against other infections. For example
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Desquesnes et al. described cross reactivity of T. evansi antigen with anti T. cruzi antibodies
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(Desquesnes et al., 2007). Control of camel trypanosomosis depends mainly on the use of
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curative and prophylactic drugs and their application ideally should take into account actual
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information of the disease. However to properly understand the epidemiology of a disease, up-to-
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date and high-quality data are required through application of accurate diagnostic tools (Ali et
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al., 2009). In Ethiopia, a number of studies indicated that T. evansi is the sole pathogenic
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trypanosome infecting camels (Hailemariam et al., 2008; Kassa et al., 2011; Tadesse et al.,
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2012). These findings need also to be verified with more accurate diagnostic techniques like
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PCR to assess the possibility of infection with other mechanically transmitted trypanosomes like
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T. vivax (Birhanu et al., 2013). Therefore, this study was undertaken to assess the prevalence of
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camel trypanosome infection in the Afar region using different diagnostic techniques, to assess
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the relative performance of different diagnostic PCRs and to identify host related risk factors
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associated with trypanosome infection.
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Materials and methods
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Study area
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The present study was conducted in Chifra and Dewe districts of Afar Region, located in the
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North-Eastern part of Ethiopia between 39°34' and 42°28'E longitude and 8°49' and 14°30'N
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latitude in the rift valley (Figure 1). The districts have similar arid and semi-arid agro-ecology,
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where livestock production is the main occupation of the community. The average elevation of
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the districts is 802-825 meters above sea level with annual temperature ranging from 25ºC to
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33ºC. The rainfall is bimodal with erratic distribution, the long rainy season (kerma) taking place
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between Mid-June to Mid-September and the short rainy season (sugum) occurring between
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March and April. The average annual rainfall is between 400 and 600 mm.
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Study animals and design
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Dromedary camels ≥ 6 months old were considered for blood sampling. Age, gender, and
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previous treatment history were recorded. Body condition was assessed according to Faye et al.
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(Faye et al., 2001). Animal age groups were defined as follows: 6 months to 1 year, >1 to 5
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years, >5 to 10 years and >10 years.
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A cross-sectional study was conducted on 399 camels (199 camels from Chifra and 200 camels
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from Dewe) from November 2011 to April 2012. A combination of convenience, purposive and
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multistage stratified sampling methods was applied according to Toma et al. (Toma et al., 1999).
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First, the two study districts were selected based on their camel population and reported cases of
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camel trypanosomosis. A complete list (sampling frame) of the pastoral villages was obtained
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from which five pastoral associations (PAs) within each district were selected based on their
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accessibility. From these PAs, three to five herds were selected from volunteer herders.
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Pastoralists keep their camel herd in groups based on age, gender and reproductive status
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(lactating) in separate stables which were considered as stratum. However, some dominant
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lactating females could not be caught. An average of 10 camels (8–12) were randomly selected
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from each herd depending on the number of camels available from each stratum.. The samples
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size required to estimate the prevalence of infection in the camel population was 384 based on
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the formula given by Thrusfield with a design prevalence of 50%, a precision of 5% and a 95%
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confidence interval (Thrusfield, 2005). The sample size was increased to 399 to provide an
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allowance for sample loss..
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Specimen collection and diagnostic test
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Blood samples were collected from the jugular vein into 10 ml heparinised vacutainer tubes.
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From the collected blood, thin smears were prepared, air dried, fixed with methanol and stained
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with Giemsa for parasitological examination. Packed cell volume (PCV), expressed in
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percentage, was estimated after 5 minutes centrifugation at 12000 rpm of blood in two capillary
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tubes.
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About 250 µl of blood were preserved in an equal volume of AS-1 buffer (QIAgen) and stored at
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ambient temperature for DNA extraction. The remaining blood was centrifuged and plasma was
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collected and preserved at -20°C until the antibody detection was performed with the Card
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Agglutination Test for Trypanosomiasis (CATT/T. evansi). CATT/T. evansi was performed as
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per the manufacturer‘s instructions (Institute of Tropical Medicine, Antwerp, Belgium). Briefly,
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25μl of camel plasma, diluted 1:4 in CATT-buffer, were dispensed onto a reaction zone of a
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plastic test card. After adding one drop (about 45 μl) of CATT reagent, the reaction mixture was
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spread by a stirring rod and allowed to react on a CATT rotator for 5 min at 70 rpm. A specimen
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was considered positive when blue agglutinates were visible (Bajyana Songa and Hamers, 1988;
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Verloo et al., 2000).
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Molecular analysis
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For the molecular analysis, DNA was extracted with the QIAamp mini blood kit (Qiagen) from
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200 µL blood/AS1 buffer into 200 µL elution buffer according to the manufacturer's instructions
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as described elsewhere (Fikru et al., 2012). Extracted DNA was stored at -20°C until tested by
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the respective PCRs. The PCRs with their target sequence, primer sequences and amplicon
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lengths are represented in Table 1. Interpretation of the results after electrophoresis in 2%
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agarose gels and staining with ethidium bromide (EtBr), is based on the characteristic amplicon
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lengths. The different PCRs were conducted on all the samples to assess their performance to
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detect and identify the different trypanosome species affecting camels.
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Furthermore, comparison of the analytical sensitivity of the RoTat 1.2 PCR, 18S PCR and ITS-1
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PCR was carried out on purified DNA of two T. evansi isolates, from Kazakhstan and Morocco.
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The DNA was tested as fivefold dilution series in water, ranging from 1 ng/μl down to 0.064
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pg/μl.
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Data analysis
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Data were analysed using SPSS (Statistical Package for Social Sciences, version 20) and STATA
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(Stata Statistical Software: Release 12. StataCorp LP). McNemar chi-square (χ2) test and odds
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ratios (OR) were calculated to compare the prevalence of trypanosome infection in the two study
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areas. The level of agreement between diagnostic tests was determined using Cohen's kappa
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coefficient interpreted following Landis and Koch (Cohen, 1960; Landis and Koch, 1977).
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ANOVA was used to assess the difference in mean PCV of parasitemic and aparasitemic camels.
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95% binomial confidence interval and p < 0.05 was set to decide on statistical significance.
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For an exploratory assessment of the diagnostic accuracy of the different tests, allowing for the
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imperfect nature of all diagnostic methods used, we performed latent class analysis (LCA). This
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model-based approach allows an unbiased estimation of the sensitivity and specificity of
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diagnostic methods when a gold standard test that is 100% sensitive and 100% specific is
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unavailable. We performed LCA using WinBUGS (Stata Statistical Software: Release 12.
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StataCorp LP) and R, assessing several models allowing for dependence between test results
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within infected and non-infected subjects, as well as the standard conditional independence
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model (Menten et al., 2008; R Core Team, 2014). The best fitting models presumed 100%
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specificity of the thin blood smear Giemsa staining technique and allowed false negative test
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results on the 3 PCR tests to be correlated. This model showed a good fit to the data (Bayesian
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lack-of-fit p-value = 0.238).
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Results
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The observed prevalences of trypanosome infection in camel in both districts, estimated using
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the diagnostic tests are given in table 2.
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The overall prevalence of camel trypanosome infection was significantly (p < 0.05) lower in
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Giemsa stained thin smear (Table 2) than in the other tests. The serological test, CATT/T. evansi
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and the molecular test, RoTat1.2 PCR (T. evansi type A-specific), revealed a significantly higher
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prevalence than ITS-1 and 18S PCRs (p < 0.05). A number of specimens were shown to contain 7 Page 7 of 33
T. vivax based on the results of the ITS-1 PCR. The parasites observed by thin blood smear
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examination had morphology characteristic of T. evansi with a small subterminal kinetoplast at
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the posterior end and a flagellum (Hoare, 1972). With the T. vivax proline racemase PCR, only
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one T. vivax infection could be confirmed. No single T. evansi type B infection was detected
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with EVAB PCR. No statistically significant difference in prevalence of trypanosome infection
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was recorded between the study districts by any of the diagnostic tools.
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Out of 96 CATT/T. evansi positives samples, respectively 26, 13 and 10 samples were also
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positive in RoTat 1.2 PCR, 18S PCR and ITS-1 PCR (Table 3 ). On the other hand, out of 303
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CATT/T. evansi negative samples, respectively 59, 25 and 21 samples were positive for
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RoTat1.2 PCR, 18S PCR and ITS-1 PCR. There was slight agreement between CATT/T. evansi
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and 18S PCR (K = 0.07), CATT/T. evansi and RoTat1.2 PCR (K = 0.08), and CATT/T. evansi
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and ITS-1 PCR (K = 0.05). Out of the 85 RoTat1.2 PCR positives, 52 were negative in 18S PCR
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and 58 were negative in ITS1 PCR. 18S PCR and ITS-1 PCR picked respectively 5 and 4
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positive specimens out of 314 RoTat 1.2 PCR negative specimens (Table 3 ). There was slight
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agreement between RoTat1.2 PCR and Giemsa stained thin blood smear (K = 0.03) but there was
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a moderate agreement between RoTat 1.2 PCR and 18s PCR (K = 0.47) and a fair agreement
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between RoTat1.2 PCR and ITS-1 PCR (K = 0.39). There was a substantial agreement between
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18S PCR and ITS-1 PCR (K = 0.79) though out of 38 positives for 18S PCR, 10 specimens were
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negative for ITS-1 PCR and out of 31 ITS-1 PCR positives, 3 were negative for 18S PCR (Table
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3). There was slight agreement between Giemsa stained thin blood smear and 18S PCR (K =
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0.05).
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The frequency distribution of tests results (N) and probability of being infected for each outcome
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pattern as estimated with LCA is presented in Table 3. Further results from LCA are summarised
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in Table 4. The estimated prevalence from the model was 19% (95% CI: 13 - 28). The model
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indicated for CATT/T. evansi a low sensitivity of 43% with relatively low specificity of 80%.
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The PCR tests showed higher specificity (86% for RoTat 1.2, >99% for 18S and ITS-1) with
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sensitivity similar to CATT/T. evansi in the 40 to 50% range (53% for RoTat 1.2 PCR, 48% for
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18S PCR, and 39% for ITS-1 PCR). The LCA results confirmed the very low sensitivity (11%)
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of Giemsa stained thin blood smear in this setting.
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The latent class model classified all those with Giemsa stained thin blood smear positive as
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infected, as well as all those with at least 2 PCRs positive, or CATT/T. evansi and at least one of
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18S PCR or ITS-1 PCR positive. Those with only CATT/ T. evansi or at most a single PCR
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positive were classified as not infected, as were those with only CATT/T. evansi and RoTat 1.2
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PCR positive.
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When tested on purified DNA of a Kazakhstan and a Moroccan T. evansi strain, the analytical
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sensitivity of the ITS-1 PCR was 0.064 pg/µl with both T. evansi strains and lower than the
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RoTat1.2 PCR (0.32 pg/µl) and 18S PCR (0.32 pg/µl). However, at the lower DNA
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concentrations, aspecific amplicons start to appear besides the Trypanozoon-specific 450 bp in
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the ITS-1PCR (Figure 2).
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None of the PCR tests showed a significant (p > 0.05) difference in positivity rate between
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gender groups and between animals with poor and moderate body condition (Table 5). However
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a significantly higher prevalence in female animals (χ2 = 5.4, p < 0.05; OR = 0.6, CI 0.3-0.9 by
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CATT/T. evansi) and in camels with poor body condition (χ2 = 20.3, p < 0.05; OR = 26.8, CI
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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
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PCR) was recorded. No significant difference in positivity rate was recorded between age groups
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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 =
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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
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PCR (χ2 = 5.6, p < 0.05;, OR = 2.7, CI 1.2-6.2) there were significantly more positives in older
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camels. History of trypanocidal treatment had no significant (p > 0.05) impact on positivity rate
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by all the diagnostic tests.
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The overall mean PCV in the studied camels was low (23.6%) indicating that the animals were
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generally in poor condition. It is noteworthy that in our study, no camels were observed with
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good body condition. In all diagnostic tests PCV was significantly (p < 0.05) lower in test
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positive animals compared to test negative animals (table 6).
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Discussion
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In this study, Giemsa stained thin blood smear examination, antibody detection by CATT/T.
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evansi and molecular tests by RoTat 1.2, 18S and ITS1-PCRs were used to assess prevalence and
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host related risk factors of trypanosome infection in camels in Afar region, Eastern Ethiopia.
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Only 2% of the examined camels were found infected by T. evansi by microscopy. This
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corresponds with previous findings in camels in different parts of Ethiopia, including two other
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districts in Afar Region, Gowani and Awash Fentale (Getahun, 1998; Hailemariam et al., 2008;
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Kassa et al., 2011; Tadesse et al., 2012) and in other African countries, for instance 2.3% in
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Kenya (Ngaira et al., 2004) and 2.5% in Nigeria (Egbe-Nwiyi and Chaudry, 1994). However,
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some other studies conducted in Ethiopia reported higher prevalences 6.5 %-12.1% (Richard,
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1979; Abebe, 1991; Hailu, 2000; Hagos et al., 2009). This could be due to differences in the
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management and husbandry regimens of the camels under study, or to seasonal effects as well as
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to study design. Previous studies have shown that the prevalence of surra in camels is higher
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during the rainy season and could be explained by higher densities of Tabanid and Stomoxys flies
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during that season (Löhr et al., 1985; Luckins, 1988; Hailemariam et al., 2008).
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In the absence of a golden standard test, the LCA revealed an overall estimate prevalence of
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19.4%, with low sensitivity of CATT/T. evansi and the PCR tests (43% - 53%) but better than
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the sensitivity of Giemsa stained thin blood smear examination (11%). When considering
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microscopy, one should keep in mind that the observed prevalence reflects only a fraction of the
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real prevalence since most microscopic techniques are poorly sensitive (OIE, 2012). In case of
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Giemsa stained thin smear examination, the lower detection limit is greater than 500,000
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trypanosomes/ml of blood (OIE, 2012). Therefore, indirect diagnosis, e.g. by detection of T.
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evansi specific antibodies, may give a better estimation of the infection burden as has been
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shown in previous studies (Diall et al., 1994; Gutierrez et al., 2000). An overall seroprevalence
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of 24.1% was recorded with CATT/T. evansi and corresponds with the CATT seroprevalence
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observed in Bale zone (24.9%) (Hagos et al., 2009).
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The observed seroprevalences may be overestimates of the actual prevalence since antibody
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detection tests have the inherent shortcoming that past infections cannot be distinguished from
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current infections (Luckins, 1988; Ngaira et al., 2003; Zeyed et al., 2010). Molecular diagnostics
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targeting parasite DNA are considered good surrogates for parasite detection (Büscher, 2014).
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An overall positivity rate of 21.1%, 9.5% and 7.8% was observed respectively by RoTat 1.2
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PCR, 18S PCR and ITS-1 PCR, which is significantly higher than recorded by thin smear
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examination (2%). CATT/T evansi and RoTat1.2 PCR yielded comparable positivity rates that
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were significantly higher than in 18S and ITS-1 PCRs. Similar observations were made in
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Uganda where very low molecular prevalence of Trypanozoon was recorded with the ITS-1 PCR
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as compared to a PCR for the single copy gene phospholipase C (GPI-PLC) (Ahmed et al.,
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2013). The higher positivity rate recorded with RoTat1.2 PCR needs special attention but could
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partly be explained by the low RoTat1.2 PCR specificity estimate by LCA. Our data show that
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the analytical sensitivity of RoTat 1.2 PCR based on the single copy gene is lower than the ITS-1
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and 18S PCR based on multi copy sequences. Yet, RoTat 1.2 PCR unexpectedly yielded a higher
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positivity rate than ITS-1 and 18S PCR. The lowest positivity rate obtained by ITS-1 PCR
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remains unexplained but it could be related to limited specificity of the ITS-1 primers. We
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observed that when the concentration of target DNA decreases in the presence of host DNA, the
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primers become less specific and tend to bind to the host DNA. On the other hand, we cannot
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rule out that the RoTat1.2 PCR is less specific in the presence of host DNA as it is supported by
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the LCA where RoTat1.2 PCR is less specific than ITS-1 PCR and 18S PCR. Noteworthy, only
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one out of 8 thin blood smear positive animals was negative for CATT/T. evansi whereas out of
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303 CATT/T. evansi negative samples 19.5%, 8.3% and 6.9% respectively, were positive for
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RoTat1.2, 18S and ITS-1 PCRs. It is possible but unlikely that all these seronegative animals
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carried early infections and had not yet formed detectable antibody levels. The number of RoTat
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1.2 PCR positives that could not be detected by CATT/T. evansi is significantly higher than with
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18S and ITS-1 PCRs. This could be explained by the presence but not yet the expression of the
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RoTat1.2 gene in the circulating trypanosomes (Verloo et al., 2001). On the other hand there are
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a number of RoTat1.2 PCR and CATT/T. evansi negatives that are positive in 18S and ITS-1
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PCR that could be explained by the absence of the RoTat1.2 gene like it has been described for
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the T. evansi type B strains isolated in Kenya and suspected to circulate also in Ethiopia but not
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confirmed in our study (Elsaid et al., 1998; Davison et al., 1999; Ngaira et al., 2003; Hagos et al.,
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2009; Zeyed et al., 2010; Salim et al., 2011).
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Even though 18S PCR and ITS-1 PCR cannot differentiate between the taxa of the Trypanozoon
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group, the positive results reported in this study are most likely due to T. evansi, since most of
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them are positive in the T. evansi specific RoTat1.2 PCR and there are no records of tsetse flies
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in the Afar region excluding the presence of T. brucei but not of non-RoTat 1.2 T. evansi (Njiru
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et al., 2006). In Ethiopia, tsetse flies and tsetse-transmitted trypanosomes prevail in the
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Southwestern and Northwestern parts of the country following major river basins between
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longitude 33o and 38o E and latitude 5o and 12o N while the rest of the country, the North,
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Southeast and East are considered tsetse free (National Tsetse and Trypanosomosis Investigation
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and Control Center (NTTICC), 2004; Sinshaw et al., 2006). Our study revealed, for the first time
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in Ethiopia, a T. vivax infection in camels. That there are no previous reports of camels infected
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with T. vivax in Ethiopia is probably due to the fact that the commonly used serological and
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parasitological tests fail to detect T. vivax or to distinguish it from T. evansi. Mixed infections of
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T. congolense, T. vivax and Trypanozoon have been reported in camels in Nigeria but were based
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on microscopic examination while in Sudan, using the ITS-1 PCR, only T. evansi (Trypanozoon)
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has been detected in camel (Mbaya et al., 2010; Salim et al., 2011). Pathogenicity of T. vivax to
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camels is not well described but infected animals could act as reservoir and spread the parasite to
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the more susceptible livestock species (Enwezor and Sackey, 2005).
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Whatever diagnostic technique used, the overall observed prevalence of surra was similar in both
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study sites which can be explained by comparable camel husbandry practices and ecological
289
biotopes in both districts. On the other hand, a significantly higher prevalence was recorded in
290
female animals by CATT/T. evansi and Giemsa stained thin smear. Similar findings were
291
reported in Kenya and in Israel (Singh et al., 2004; Berlin et al., 2010). The fact that
292
seropositivity in CATT increases with age is in agreement with previous studies and probably
293
reflects time of exposure to biting flies and persistence of circulating antibodies, even after
294
successful treatment (Dia et al., 1997; Gutierrez et al., 2000; Atarhouch et al., 2003). Camels
295
with poor body conditions were more likely to be positive in Giemsa stained thin smear and
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CATT/T. evansi, compared to those with moderate body condition. Nutritional stress or other
297
diseases, whether infectious or not, could render camels more susceptible to infection with T.
298
evansi (Eyob and Matios, 2013). On the other hand, camel trypanosomosis by itself is one of the
299
major causes resulting in poor body condition (Röttcher et al., 1987). Significantly lower PCV
300
values were recorded in seropositive and PCR positive camels which corresponds with other
301
studies using serological, parasitological and molecular diagnostic tests (Diall et al., 1994;
302
Atarhouch et al., 2003; Tadesse et al., 2012).
303
In this study, we used LCA to estimate the accuracy of the different diagnostic tests in the
304
absence of a gold standard. LCA revealed lower sensitivity for CATT/T. evansi (43%) than
305
expected from previous studies (80%-92%) while the specificity (80.4%) falls within the
306
expected range (69%-100%)(Diall et al., 1994; Diall et al., 1997; Verloo et al., 1998; Ngaira et
307
al., 2003). Unfortunately, for reasons of specimen import regulations, we were not able to
308
perform immune trypanolysis with T. evansi RoTat 1.2 as reference test for T. evansi type A
309
specific antibodies (Verloo et al., 2001). The low sensitivity of the CATT/T. evansi in this study
310
may be due to unusual low amounts of specific antibodies circulating in the infected camels, that
311
might have been detected in immune trypanolysis, or to infections with T. evansi type B that
312
remained under the lower detection limit of the EVAB PCR.
313
In conclusion, this study confirms that trypanosome infection is a potential threat affecting the
314
health and productivity of camel, in particular adult female animals, in Afar, Ethiopia. T. evansi
315
is the prominent trypanosome species affecting camels since only one case among 399 camels
316
was confirmed infected with T. vivax by TvPRAC PCR. There are significant discrepancies in
317
sensitivity of different diagnostic tests used in this epidemiological study necessitating
318
improvement of the available diagnostic tests to provide high quality epidemiological data.
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14 Page 14 of 33
Acknowledgements
320
The PhD fellowship of Fikru Regassa was financed by the Belgian Directorate General for Development
321
Cooperation.
322
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458 459
21 Page 21 of 33
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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
Target group
Target
M an
from Eastern Ethiopia. Primers
Primer sequence
Amplicon (bp)
Reference
18S
18s F
5′-CGCCAAGCTAATACATGAACCAA-3′
110
(Deborggraeve
rRNA
18S R
5′-TAATTTCATTCATTCGCTGGACG-3′
ITS-1
ITS-1 F
5’-TGTAGGTGAACCTGCAGCTGGATC-3’
rRNA
ITS-1 R
5-CCAAGTCATCCATCGCGACACGTT-3’
RoTat1.2
RoTat1.2F
5′-GCGGGGTGTTTAAAGCAATA-3′
T. evansi type A
T. evansi type B
T. vivax
minicircle
Proline
ce pt
Trypanozoon
RoTat1.2R
5′-ATTAGTGCTGCGTGTGTTCG-3′
EVAB-1
5’-ACAGTCCGAGAGATAGAG-3'
EVAB-2
5'-CTGTACTCTACATCTACCTC-3’
TvPRAC F
5’-CGCAAGTGGACCGTTCGCCT-3’
TvPRAC-R
5’-ACGCGGGGCGAACAGAAGTG-3’
Ac
Trypanosomatidae
racemase
ed
sequence
et
al., 2006) 450
(Desquesnes et al., 2001)
205
(Claes et al., 2004)
436
(Njiru et al., 2006)
239
(Fikru et al., 2014)
22 Page 22 of 33
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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.
District
Thin blood
CATT/T. evansi
ed
smear
RoTat1.2 PCR
18S PCR
ITS-1 PCR
Total
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
Ac
ce pt
Chifra
23 Page 23 of 33
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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.
PCR RoTat 1.2
18S
ITS1
+
+
+
+
+
+
+
+
+
-
+
-
-
+
-
-
Thin blood smear
N
Probability infected
+
2
100
ed
CATT/T. evansi
M an
evansi infection performed on 399 camels from Eastern Ethiopia, as estimated with latent class analysis.
-
6
100
-
+
1
100
ce pt
+
-
+
4
100
+
+
-
18
100
-
-
+
1
100
-
+
+
-
1
96.8
+
+
-
-
3
94.1
-
-
+
+
-
1
92.2
-
+
+
-
-
4
85.3
+ +
Ac
-
24 Page 24 of 33
+
-
+
-
+
-
-
-
-
+
-
-
-
-
-
+
-
+
+
-
-
+
-
-
-
-
+
-
-
-
-
-
-
1
ip t
-
84.7
cr
+
1
us
+
60.5 38.2
2
19.6
-
13
18.4
-
64
17.8
-
37
7.2
-
238
6.7
Ac
ce pt
ed
M an
2
25 Page 25 of 33
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Table 4: An exploratory assessment of the diagnostic accuracy of the different diagnostic tests performed on 399 camels from Eastern
M an
Ethiopia, estimated by latent class analysis with a prevalence of 19.4 % (95% CI: 12.8 – 28.0) Sensitivity (95% CI)
CATT
42.5 (32.4-52.9)
PCR RoTat1.2
52.9 (39.3-67.9)
PCR 18S
47.7 (33.0-64.2)
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)
Specificity (95% CI)
80.4 (77.8-84.6) 85.9 (84.1-88.1) 99.2 (98.0-100)
Ac
ce pt
ed
Tests
26 Page 26 of 33
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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.
Number Positive
Positive
Positive
18S PCR
ITS-1 PCR
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)
Male
148
0.7
17.6 (11.5-23.7)
10.8 (5.8-
Moderate
condition
Poor
score No
history
Yes
Age category
≤ 1yr
9.5 (4.8-14.2)
15.8) 17.7 (13.5-21.9)
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)
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)
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)
50
0
20 (8.9-31.1)
22.0 (10.5-33.5)
12.0 (13.0-
8.0 (0.5-15.5)
311
Ac
Treatment
18.2 (12.0-24.4)
ed
Body
0.3
ce pt
Gender
M an
Thin blood smear CATT/ T. evansi RoTat1.2 PCR
22.5 (17.9-27.1)
10 (6.7-13.3)
7.4 (4.5-10.3)
31.0)
27 Page 27 of 33
15.3 (7.0-23.6)
ip t
0
25.0 (15.0-35.0)
cr
72
us
1 < x ≤ 5 yr
11.1 (3.8-
20.1)
18.4)
2.4 (0.5-4.3)
25.8 (20.4-31.2)
> 10 yr
8 (0.0-18.3)
40.0* (20.8-59.2) 40.0* (20.8-59.2) 12.0 (0.0-
M an
5 < x ≤ 10 yr 252 25
7.9 (4.6-11.2) 6.3 (3.3-9.3)
24.7)
12.0 (0.0-247)
Ac
ce pt
ed
* significant difference
18.3 (13.5-23.1)
12.5 (4.9-
28 Page 28 of 33
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Table 6: Mean PCV (%) with standard deviation (SD) and 95% confidence interval of 399 camels from Eastern Ethiopia tested using
Test
PCV
CATT/T. evansi
2.7
23.4-24.0
2,1
19.0-22.5
2.6
24.0-24.6
21.5*
1.8
21.1-21.9
23.8
2.7
23.5-24.1
23.7
Positive
20.7*
Negative
24.3
ce pt
Negative Positive
23.0*
2.7
22.4-23.5
Negative
23.7
2.7
23.5-24.0
Positive
22.6*
2.7
21.8-23.5
Negative
23.7
2.7
23.4-24.0
Positive
22.6*
2.7
21.7-23.6
Ac
18S PCR
ITS-1 PCR
95% confidence interval
Negative
Positive RoTat1.2 PCR
SD
ed
Thin blood smear
M an
different tests to detect T. evansi infection.
* significant difference
29 Page 29 of 33
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Figure legends
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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:
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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
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30 Page 30 of 33
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Latent class analysis estimated an overall prevalence of 19%
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We conducted a cross-sectional study on camel trypanosome infection in Eastern Ethiopia
All infections were due to Trypanosoma evansi except one with Trypanosoma vivax
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Risk factors for infection were poor body condition and age
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S0304-4017(15)00194-6 http://dx.doi.org/doi:10.1016/j.vetpar.2015.04.008 VETPAR 7600
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
29-3-2014 27-3-2015 2-4-2015
Please cite this article as: Fikru, R., Andualem, Y., Getachew, T., Menten, J., Hasker, E., Merga, B., Goddeeris, B.M., B¨uscher, P.,Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative performance of diagnostic 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 proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Trypanosome infection in dromedary camels in Eastern Ethiopia: prevalence, relative
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performance of diagnostic tools and host related risk factors
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Regassa Fikrua,b,c*, Yimer Andualemd, Terefe Getachewa, Joris Mentene, Epco Haskerf, Bekana
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Mergaa, Bruno Maria Goddeerisc, Philippe Büscherb
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a
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Zeit, Ethiopia.
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b
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College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Debre
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Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, B-
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2000 Antwerp, Belgium.
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c
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30, B-3001 Leuven, Belgium.
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d
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Antwerp, Belgium.
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f
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Belgium.
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* Corresponding author. Tel: +251911907056
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E-mail address: [email protected]
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KU Leuven, Faculty of Bioscience Engineering, Department Biosystems, Kasteelpark Arenberg
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School of Veterinary Medicine, Wollo University, P.O. Box 1145, Dessie, Ethiopia
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Institute of Tropical Medicine, Department of Clinical Sciences, Nationalestraat 155, B-2000
Institute of Tropical Medicine, Department of Public , Nationalestraat 155, B-2000 Antwerp,
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Abstract
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A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern
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Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk
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factors of trypanosome infection in camel. An overall prevalence of 2 %, 24.1 %, 21.3 %, 9.5 %
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and 7.8 % was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi,
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RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection
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was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting
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camels in the study area. No single animal was positive when tested with T. evansi type B
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specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular
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tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate
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compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate
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observed in each gender of camels (p > 0.05). The positivity rate was significantly higher in
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camels with poor body condition and in older animals when tested using the CATT/T.evansi or
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RoTat 1.2 PCR (p > 0.05). Camels that tested positive with all tests had significantly lower
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PCV’s (p < 0.05). This study provides further evidence that T. evansi is endemic in the Afar
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region of Ethiopia.. The latent class analysis indicated an estimate overall prevalence of 19%
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(95% CI: 13 - 28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and
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the PCR tests (39%-53%) but higher specificity of the PCR tests (86% -99%) and low specificity
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of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the
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tests would help diagnosis of trypanosomosis. Further studies are required to determine the
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prevalence of clinical disease and losses due to trypanosomosis.
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Keywords: Dromedary camel, Ethiopia, prevalence, risk factor, trypanosomosis, Trypanosoma
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evansi, Trypanosoma vivax
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Introduction
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Trypanosomosis is one of the major health problems with high morbidity and mortality in camels
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in Ethiopia (Demeke, 1998; Tekle and Abebe, 2001). The disease is caused by infection with
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hemoflagellated parasites belonging to the genus Trypanosoma. Trypanosoma evansi belongs to
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Trypanozoon subgenus and is the most commonly reported cause of camel trypanosomosis called
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surra (Röttcher et al., 1987). T. evansi is mechanically transmitted by blood sucking flies, such as
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Tabanidae and Stomoxys sp (Hoare, 1972). A number of researchers have investigated the
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epidemiology of camel trypanosome infection in different parts of Ethiopia. These studies were
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mainly based on parasitological and serological tests (Richard, 1979; Elias, 2003; Hailemariam
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et al., 2008; Hagos et al., 2009; Kassa et al., 2011; Tadesse et al., 2012). Parasitological
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examination suffers from limited sensitivity even when using the haematocrit centrifugation
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technique. Serological tests are unable to distinguish past and current infections as the antibodies
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persist in the circulation (Büscher, 2014). The serological tests also are not fully specific due to
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the possibility of cross reactions with antibodies produced against other infections. For example
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Desquesnes et al. described cross reactivity of T. evansi antigen with anti T. cruzi antibodies
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(Desquesnes et al., 2007). Control of camel trypanosomosis depends mainly on the use of
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curative and prophylactic drugs and their application ideally should take into account actual
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information of the disease. However to properly understand the epidemiology of a disease, up-to-
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date and high-quality data are required through application of accurate diagnostic tools (Ali et
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al., 2009). In Ethiopia, a number of studies indicated that T. evansi is the sole pathogenic
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trypanosome infecting camels (Hailemariam et al., 2008; Kassa et al., 2011; Tadesse et al.,
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2012). These findings need also to be verified with more accurate diagnostic techniques like
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PCR to assess the possibility of infection with other mechanically transmitted trypanosomes like
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T. vivax (Birhanu et al., 2013). Therefore, this study was undertaken to assess the prevalence of
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camel trypanosome infection in the Afar region using different diagnostic techniques, to assess
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the relative performance of different diagnostic PCRs and to identify host related risk factors
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associated with trypanosome infection.
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Materials and methods
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Study area
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The present study was conducted in Chifra and Dewe districts of Afar Region, located in the
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North-Eastern part of Ethiopia between 39°34' and 42°28'E longitude and 8°49' and 14°30'N
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latitude in the rift valley (Figure 1). The districts have similar arid and semi-arid agro-ecology,
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where livestock production is the main occupation of the community. The average elevation of
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the districts is 802-825 meters above sea level with annual temperature ranging from 25ºC to
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33ºC. The rainfall is bimodal with erratic distribution, the long rainy season (kerma) taking place
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between Mid-June to Mid-September and the short rainy season (sugum) occurring between
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March and April. The average annual rainfall is between 400 and 600 mm.
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Study animals and design
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Dromedary camels ≥ 6 months old were considered for blood sampling. Age, gender, and
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previous treatment history were recorded. Body condition was assessed according to Faye et al.
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(Faye et al., 2001). Animal age groups were defined as follows: 6 months to 1 year, >1 to 5
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years, >5 to 10 years and >10 years.
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A cross-sectional study was conducted on 399 camels (199 camels from Chifra and 200 camels
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from Dewe) from November 2011 to April 2012. A combination of convenience, purposive and
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multistage stratified sampling methods was applied according to Toma et al. (Toma et al., 1999).
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First, the two study districts were selected based on their camel population and reported cases of
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camel trypanosomosis. A complete list (sampling frame) of the pastoral villages was obtained
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from which five pastoral associations (PAs) within each district were selected based on their
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accessibility. From these PAs, three to five herds were selected from volunteer herders.
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Pastoralists keep their camel herd in groups based on age, gender and reproductive status
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(lactating) in separate stables which were considered as stratum. However, some dominant
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lactating females could not be caught. An average of 10 camels (8–12) were randomly selected
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from each herd depending on the number of camels available from each stratum.. The samples
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size required to estimate the prevalence of infection in the camel population was 384 based on
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the formula given by Thrusfield with a design prevalence of 50%, a precision of 5% and a 95%
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confidence interval (Thrusfield, 2005). The sample size was increased to 399 to provide an
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allowance for sample loss..
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Specimen collection and diagnostic test
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Blood samples were collected from the jugular vein into 10 ml heparinised vacutainer tubes.
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From the collected blood, thin smears were prepared, air dried, fixed with methanol and stained
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with Giemsa for parasitological examination. Packed cell volume (PCV), expressed in
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percentage, was estimated after 5 minutes centrifugation at 12000 rpm of blood in two capillary
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tubes.
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About 250 µl of blood were preserved in an equal volume of AS-1 buffer (QIAgen) and stored at
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ambient temperature for DNA extraction. The remaining blood was centrifuged and plasma was
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collected and preserved at -20°C until the antibody detection was performed with the Card
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Agglutination Test for Trypanosomiasis (CATT/T. evansi). CATT/T. evansi was performed as
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per the manufacturer‘s instructions (Institute of Tropical Medicine, Antwerp, Belgium). Briefly,
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25μl of camel plasma, diluted 1:4 in CATT-buffer, were dispensed onto a reaction zone of a
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plastic test card. After adding one drop (about 45 μl) of CATT reagent, the reaction mixture was
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spread by a stirring rod and allowed to react on a CATT rotator for 5 min at 70 rpm. A specimen
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was considered positive when blue agglutinates were visible (Bajyana Songa and Hamers, 1988;
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Verloo et al., 2000).
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Molecular analysis
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For the molecular analysis, DNA was extracted with the QIAamp mini blood kit (Qiagen) from
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200 µL blood/AS1 buffer into 200 µL elution buffer according to the manufacturer's instructions
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as described elsewhere (Fikru et al., 2012). Extracted DNA was stored at -20°C until tested by
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the respective PCRs. The PCRs with their target sequence, primer sequences and amplicon
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lengths are represented in Table 1. Interpretation of the results after electrophoresis in 2%
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agarose gels and staining with ethidium bromide (EtBr), is based on the characteristic amplicon
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lengths. The different PCRs were conducted on all the samples to assess their performance to
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detect and identify the different trypanosome species affecting camels.
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Furthermore, comparison of the analytical sensitivity of the RoTat 1.2 PCR, 18S PCR and ITS-1
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PCR was carried out on purified DNA of two T. evansi isolates, from Kazakhstan and Morocco.
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The DNA was tested as fivefold dilution series in water, ranging from 1 ng/μl down to 0.064
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pg/μl.
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Data analysis
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Data were analysed using SPSS (Statistical Package for Social Sciences, version 20) and STATA
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(Stata Statistical Software: Release 12. StataCorp LP). McNemar chi-square (χ2) test and odds
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ratios (OR) were calculated to compare the prevalence of trypanosome infection in the two study
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areas. The level of agreement between diagnostic tests was determined using Cohen's kappa
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coefficient interpreted following Landis and Koch (Cohen, 1960; Landis and Koch, 1977).
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ANOVA was used to assess the difference in mean PCV of parasitemic and aparasitemic camels.
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95% binomial confidence interval and p < 0.05 was set to decide on statistical significance.
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For an exploratory assessment of the diagnostic accuracy of the different tests, allowing for the
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imperfect nature of all diagnostic methods used, we performed latent class analysis (LCA). This
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model-based approach allows an unbiased estimation of the sensitivity and specificity of
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diagnostic methods when a gold standard test that is 100% sensitive and 100% specific is
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unavailable. We performed LCA using WinBUGS (Stata Statistical Software: Release 12.
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StataCorp LP) and R, assessing several models allowing for dependence between test results
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within infected and non-infected subjects, as well as the standard conditional independence
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model (Menten et al., 2008; R Core Team, 2014). The best fitting models presumed 100%
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specificity of the thin blood smear Giemsa staining technique and allowed false negative test
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results on the 3 PCR tests to be correlated. This model showed a good fit to the data (Bayesian
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lack-of-fit p-value = 0.238).
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Results
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The observed prevalences of trypanosome infection in camel in both districts, estimated using
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the diagnostic tests are given in table 2.
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The overall prevalence of camel trypanosome infection was significantly (p < 0.05) lower in
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Giemsa stained thin smear (Table 2) than in the other tests. The serological test, CATT/T. evansi
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and the molecular test, RoTat1.2 PCR (T. evansi type A-specific), revealed a significantly higher
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prevalence than ITS-1 and 18S PCRs (p < 0.05). A number of specimens were shown to contain 7 Page 7 of 33
T. vivax based on the results of the ITS-1 PCR. The parasites observed by thin blood smear
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examination had morphology characteristic of T. evansi with a small subterminal kinetoplast at
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the posterior end and a flagellum (Hoare, 1972). With the T. vivax proline racemase PCR, only
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one T. vivax infection could be confirmed. No single T. evansi type B infection was detected
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with EVAB PCR. No statistically significant difference in prevalence of trypanosome infection
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was recorded between the study districts by any of the diagnostic tools.
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Out of 96 CATT/T. evansi positives samples, respectively 26, 13 and 10 samples were also
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positive in RoTat 1.2 PCR, 18S PCR and ITS-1 PCR (Table 3 ). On the other hand, out of 303
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CATT/T. evansi negative samples, respectively 59, 25 and 21 samples were positive for
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RoTat1.2 PCR, 18S PCR and ITS-1 PCR. There was slight agreement between CATT/T. evansi
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and 18S PCR (K = 0.07), CATT/T. evansi and RoTat1.2 PCR (K = 0.08), and CATT/T. evansi
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and ITS-1 PCR (K = 0.05). Out of the 85 RoTat1.2 PCR positives, 52 were negative in 18S PCR
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and 58 were negative in ITS1 PCR. 18S PCR and ITS-1 PCR picked respectively 5 and 4
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positive specimens out of 314 RoTat 1.2 PCR negative specimens (Table 3 ). There was slight
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agreement between RoTat1.2 PCR and Giemsa stained thin blood smear (K = 0.03) but there was
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a moderate agreement between RoTat 1.2 PCR and 18s PCR (K = 0.47) and a fair agreement
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between RoTat1.2 PCR and ITS-1 PCR (K = 0.39). There was a substantial agreement between
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18S PCR and ITS-1 PCR (K = 0.79) though out of 38 positives for 18S PCR, 10 specimens were
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negative for ITS-1 PCR and out of 31 ITS-1 PCR positives, 3 were negative for 18S PCR (Table
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3). There was slight agreement between Giemsa stained thin blood smear and 18S PCR (K =
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0.05).
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The frequency distribution of tests results (N) and probability of being infected for each outcome
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pattern as estimated with LCA is presented in Table 3. Further results from LCA are summarised
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in Table 4. The estimated prevalence from the model was 19% (95% CI: 13 - 28). The model
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indicated for CATT/T. evansi a low sensitivity of 43% with relatively low specificity of 80%.
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The PCR tests showed higher specificity (86% for RoTat 1.2, >99% for 18S and ITS-1) with
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sensitivity similar to CATT/T. evansi in the 40 to 50% range (53% for RoTat 1.2 PCR, 48% for
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18S PCR, and 39% for ITS-1 PCR). The LCA results confirmed the very low sensitivity (11%)
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of Giemsa stained thin blood smear in this setting.
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The latent class model classified all those with Giemsa stained thin blood smear positive as
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infected, as well as all those with at least 2 PCRs positive, or CATT/T. evansi and at least one of
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18S PCR or ITS-1 PCR positive. Those with only CATT/ T. evansi or at most a single PCR
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positive were classified as not infected, as were those with only CATT/T. evansi and RoTat 1.2
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PCR positive.
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When tested on purified DNA of a Kazakhstan and a Moroccan T. evansi strain, the analytical
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sensitivity of the ITS-1 PCR was 0.064 pg/µl with both T. evansi strains and lower than the
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RoTat1.2 PCR (0.32 pg/µl) and 18S PCR (0.32 pg/µl). However, at the lower DNA
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concentrations, aspecific amplicons start to appear besides the Trypanozoon-specific 450 bp in
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the ITS-1PCR (Figure 2).
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None of the PCR tests showed a significant (p > 0.05) difference in positivity rate between
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gender groups and between animals with poor and moderate body condition (Table 5). However
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a significantly higher prevalence in female animals (χ2 = 5.4, p < 0.05; OR = 0.6, CI 0.3-0.9 by
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CATT/T. evansi) and in camels with poor body condition (χ2 = 20.3, p < 0.05; OR = 26.8, CI
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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
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PCR) was recorded. No significant difference in positivity rate was recorded between age groups
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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 =
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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
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PCR (χ2 = 5.6, p < 0.05;, OR = 2.7, CI 1.2-6.2) there were significantly more positives in older
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camels. History of trypanocidal treatment had no significant (p > 0.05) impact on positivity rate
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by all the diagnostic tests.
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The overall mean PCV in the studied camels was low (23.6%) indicating that the animals were
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generally in poor condition. It is noteworthy that in our study, no camels were observed with
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good body condition. In all diagnostic tests PCV was significantly (p < 0.05) lower in test
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positive animals compared to test negative animals (table 6).
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Discussion
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In this study, Giemsa stained thin blood smear examination, antibody detection by CATT/T.
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evansi and molecular tests by RoTat 1.2, 18S and ITS1-PCRs were used to assess prevalence and
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host related risk factors of trypanosome infection in camels in Afar region, Eastern Ethiopia.
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Only 2% of the examined camels were found infected by T. evansi by microscopy. This
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corresponds with previous findings in camels in different parts of Ethiopia, including two other
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districts in Afar Region, Gowani and Awash Fentale (Getahun, 1998; Hailemariam et al., 2008;
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Kassa et al., 2011; Tadesse et al., 2012) and in other African countries, for instance 2.3% in
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Kenya (Ngaira et al., 2004) and 2.5% in Nigeria (Egbe-Nwiyi and Chaudry, 1994). However,
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some other studies conducted in Ethiopia reported higher prevalences 6.5 %-12.1% (Richard,
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1979; Abebe, 1991; Hailu, 2000; Hagos et al., 2009). This could be due to differences in the
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management and husbandry regimens of the camels under study, or to seasonal effects as well as
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to study design. Previous studies have shown that the prevalence of surra in camels is higher
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during the rainy season and could be explained by higher densities of Tabanid and Stomoxys flies
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during that season (Löhr et al., 1985; Luckins, 1988; Hailemariam et al., 2008).
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In the absence of a golden standard test, the LCA revealed an overall estimate prevalence of
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19.4%, with low sensitivity of CATT/T. evansi and the PCR tests (43% - 53%) but better than
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the sensitivity of Giemsa stained thin blood smear examination (11%). When considering
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microscopy, one should keep in mind that the observed prevalence reflects only a fraction of the
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real prevalence since most microscopic techniques are poorly sensitive (OIE, 2012). In case of
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Giemsa stained thin smear examination, the lower detection limit is greater than 500,000
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trypanosomes/ml of blood (OIE, 2012). Therefore, indirect diagnosis, e.g. by detection of T.
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evansi specific antibodies, may give a better estimation of the infection burden as has been
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shown in previous studies (Diall et al., 1994; Gutierrez et al., 2000). An overall seroprevalence
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of 24.1% was recorded with CATT/T. evansi and corresponds with the CATT seroprevalence
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observed in Bale zone (24.9%) (Hagos et al., 2009).
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The observed seroprevalences may be overestimates of the actual prevalence since antibody
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detection tests have the inherent shortcoming that past infections cannot be distinguished from
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current infections (Luckins, 1988; Ngaira et al., 2003; Zeyed et al., 2010). Molecular diagnostics
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targeting parasite DNA are considered good surrogates for parasite detection (Büscher, 2014).
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An overall positivity rate of 21.1%, 9.5% and 7.8% was observed respectively by RoTat 1.2
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PCR, 18S PCR and ITS-1 PCR, which is significantly higher than recorded by thin smear
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examination (2%). CATT/T evansi and RoTat1.2 PCR yielded comparable positivity rates that
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were significantly higher than in 18S and ITS-1 PCRs. Similar observations were made in
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Uganda where very low molecular prevalence of Trypanozoon was recorded with the ITS-1 PCR
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as compared to a PCR for the single copy gene phospholipase C (GPI-PLC) (Ahmed et al.,
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2013). The higher positivity rate recorded with RoTat1.2 PCR needs special attention but could
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partly be explained by the low RoTat1.2 PCR specificity estimate by LCA. Our data show that
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the analytical sensitivity of RoTat 1.2 PCR based on the single copy gene is lower than the ITS-1
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and 18S PCR based on multi copy sequences. Yet, RoTat 1.2 PCR unexpectedly yielded a higher
252
positivity rate than ITS-1 and 18S PCR. The lowest positivity rate obtained by ITS-1 PCR
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remains unexplained but it could be related to limited specificity of the ITS-1 primers. We
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observed that when the concentration of target DNA decreases in the presence of host DNA, the
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primers become less specific and tend to bind to the host DNA. On the other hand, we cannot
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rule out that the RoTat1.2 PCR is less specific in the presence of host DNA as it is supported by
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the LCA where RoTat1.2 PCR is less specific than ITS-1 PCR and 18S PCR. Noteworthy, only
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one out of 8 thin blood smear positive animals was negative for CATT/T. evansi whereas out of
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303 CATT/T. evansi negative samples 19.5%, 8.3% and 6.9% respectively, were positive for
260
RoTat1.2, 18S and ITS-1 PCRs. It is possible but unlikely that all these seronegative animals
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carried early infections and had not yet formed detectable antibody levels. The number of RoTat
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1.2 PCR positives that could not be detected by CATT/T. evansi is significantly higher than with
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18S and ITS-1 PCRs. This could be explained by the presence but not yet the expression of the
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RoTat1.2 gene in the circulating trypanosomes (Verloo et al., 2001). On the other hand there are
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a number of RoTat1.2 PCR and CATT/T. evansi negatives that are positive in 18S and ITS-1
266
PCR that could be explained by the absence of the RoTat1.2 gene like it has been described for
267
the T. evansi type B strains isolated in Kenya and suspected to circulate also in Ethiopia but not
268
confirmed in our study (Elsaid et al., 1998; Davison et al., 1999; Ngaira et al., 2003; Hagos et al.,
269
2009; Zeyed et al., 2010; Salim et al., 2011).
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Even though 18S PCR and ITS-1 PCR cannot differentiate between the taxa of the Trypanozoon
271
group, the positive results reported in this study are most likely due to T. evansi, since most of
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them are positive in the T. evansi specific RoTat1.2 PCR and there are no records of tsetse flies
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in the Afar region excluding the presence of T. brucei but not of non-RoTat 1.2 T. evansi (Njiru
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et al., 2006). In Ethiopia, tsetse flies and tsetse-transmitted trypanosomes prevail in the
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Southwestern and Northwestern parts of the country following major river basins between
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longitude 33o and 38o E and latitude 5o and 12o N while the rest of the country, the North,
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Southeast and East are considered tsetse free (National Tsetse and Trypanosomosis Investigation
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and Control Center (NTTICC), 2004; Sinshaw et al., 2006). Our study revealed, for the first time
279
in Ethiopia, a T. vivax infection in camels. That there are no previous reports of camels infected
280
with T. vivax in Ethiopia is probably due to the fact that the commonly used serological and
281
parasitological tests fail to detect T. vivax or to distinguish it from T. evansi. Mixed infections of
282
T. congolense, T. vivax and Trypanozoon have been reported in camels in Nigeria but were based
283
on microscopic examination while in Sudan, using the ITS-1 PCR, only T. evansi (Trypanozoon)
284
has been detected in camel (Mbaya et al., 2010; Salim et al., 2011). Pathogenicity of T. vivax to
285
camels is not well described but infected animals could act as reservoir and spread the parasite to
286
the more susceptible livestock species (Enwezor and Sackey, 2005).
287
Whatever diagnostic technique used, the overall observed prevalence of surra was similar in both
288
study sites which can be explained by comparable camel husbandry practices and ecological
289
biotopes in both districts. On the other hand, a significantly higher prevalence was recorded in
290
female animals by CATT/T. evansi and Giemsa stained thin smear. Similar findings were
291
reported in Kenya and in Israel (Singh et al., 2004; Berlin et al., 2010). The fact that
292
seropositivity in CATT increases with age is in agreement with previous studies and probably
293
reflects time of exposure to biting flies and persistence of circulating antibodies, even after
294
successful treatment (Dia et al., 1997; Gutierrez et al., 2000; Atarhouch et al., 2003). Camels
295
with poor body conditions were more likely to be positive in Giemsa stained thin smear and
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CATT/T. evansi, compared to those with moderate body condition. Nutritional stress or other
297
diseases, whether infectious or not, could render camels more susceptible to infection with T.
298
evansi (Eyob and Matios, 2013). On the other hand, camel trypanosomosis by itself is one of the
299
major causes resulting in poor body condition (Röttcher et al., 1987). Significantly lower PCV
300
values were recorded in seropositive and PCR positive camels which corresponds with other
301
studies using serological, parasitological and molecular diagnostic tests (Diall et al., 1994;
302
Atarhouch et al., 2003; Tadesse et al., 2012).
303
In this study, we used LCA to estimate the accuracy of the different diagnostic tests in the
304
absence of a gold standard. LCA revealed lower sensitivity for CATT/T. evansi (43%) than
305
expected from previous studies (80%-92%) while the specificity (80.4%) falls within the
306
expected range (69%-100%)(Diall et al., 1994; Diall et al., 1997; Verloo et al., 1998; Ngaira et
307
al., 2003). Unfortunately, for reasons of specimen import regulations, we were not able to
308
perform immune trypanolysis with T. evansi RoTat 1.2 as reference test for T. evansi type A
309
specific antibodies (Verloo et al., 2001). The low sensitivity of the CATT/T. evansi in this study
310
may be due to unusual low amounts of specific antibodies circulating in the infected camels, that
311
might have been detected in immune trypanolysis, or to infections with T. evansi type B that
312
remained under the lower detection limit of the EVAB PCR.
313
In conclusion, this study confirms that trypanosome infection is a potential threat affecting the
314
health and productivity of camel, in particular adult female animals, in Afar, Ethiopia. T. evansi
315
is the prominent trypanosome species affecting camels since only one case among 399 camels
316
was confirmed infected with T. vivax by TvPRAC PCR. There are significant discrepancies in
317
sensitivity of different diagnostic tests used in this epidemiological study necessitating
318
improvement of the available diagnostic tests to provide high quality epidemiological data.
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14 Page 14 of 33
Acknowledgements
320
The PhD fellowship of Fikru Regassa was financed by the Belgian Directorate General for Development
321
Cooperation.
322
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458 459
21 Page 21 of 33
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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
Target group
Target
M an
from Eastern Ethiopia. Primers
Primer sequence
Amplicon (bp)
Reference
18S
18s F
5′-CGCCAAGCTAATACATGAACCAA-3′
110
(Deborggraeve
rRNA
18S R
5′-TAATTTCATTCATTCGCTGGACG-3′
ITS-1
ITS-1 F
5’-TGTAGGTGAACCTGCAGCTGGATC-3’
rRNA
ITS-1 R
5-CCAAGTCATCCATCGCGACACGTT-3’
RoTat1.2
RoTat1.2F
5′-GCGGGGTGTTTAAAGCAATA-3′
T. evansi type A
T. evansi type B
T. vivax
minicircle
Proline
ce pt
Trypanozoon
RoTat1.2R
5′-ATTAGTGCTGCGTGTGTTCG-3′
EVAB-1
5’-ACAGTCCGAGAGATAGAG-3'
EVAB-2
5'-CTGTACTCTACATCTACCTC-3’
TvPRAC F
5’-CGCAAGTGGACCGTTCGCCT-3’
TvPRAC-R
5’-ACGCGGGGCGAACAGAAGTG-3’
Ac
Trypanosomatidae
racemase
ed
sequence
et
al., 2006) 450
(Desquesnes et al., 2001)
205
(Claes et al., 2004)
436
(Njiru et al., 2006)
239
(Fikru et al., 2014)
22 Page 22 of 33
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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.
District
Thin blood
CATT/T. evansi
ed
smear
RoTat1.2 PCR
18S PCR
ITS-1 PCR
Total
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
Ac
ce pt
Chifra
23 Page 23 of 33
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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.
PCR RoTat 1.2
18S
ITS1
+
+
+
+
+
+
+
+
+
-
+
-
-
+
-
-
Thin blood smear
N
Probability infected
+
2
100
ed
CATT/T. evansi
M an
evansi infection performed on 399 camels from Eastern Ethiopia, as estimated with latent class analysis.
-
6
100
-
+
1
100
ce pt
+
-
+
4
100
+
+
-
18
100
-
-
+
1
100
-
+
+
-
1
96.8
+
+
-
-
3
94.1
-
-
+
+
-
1
92.2
-
+
+
-
-
4
85.3
+ +
Ac
-
24 Page 24 of 33
+
-
+
-
+
-
-
-
-
+
-
-
-
-
-
+
-
+
+
-
-
+
-
-
-
-
+
-
-
-
-
-
-
1
ip t
-
84.7
cr
+
1
us
+
60.5 38.2
2
19.6
-
13
18.4
-
64
17.8
-
37
7.2
-
238
6.7
Ac
ce pt
ed
M an
2
25 Page 25 of 33
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Table 4: An exploratory assessment of the diagnostic accuracy of the different diagnostic tests performed on 399 camels from Eastern
M an
Ethiopia, estimated by latent class analysis with a prevalence of 19.4 % (95% CI: 12.8 – 28.0) Sensitivity (95% CI)
CATT
42.5 (32.4-52.9)
PCR RoTat1.2
52.9 (39.3-67.9)
PCR 18S
47.7 (33.0-64.2)
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)
Specificity (95% CI)
80.4 (77.8-84.6) 85.9 (84.1-88.1) 99.2 (98.0-100)
Ac
ce pt
ed
Tests
26 Page 26 of 33
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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.
Number Positive
Positive
Positive
18S PCR
ITS-1 PCR
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)
Male
148
0.7
17.6 (11.5-23.7)
10.8 (5.8-
Moderate
condition
Poor
score No
history
Yes
Age category
≤ 1yr
9.5 (4.8-14.2)
15.8) 17.7 (13.5-21.9)
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)
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)
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)
50
0
20 (8.9-31.1)
22.0 (10.5-33.5)
12.0 (13.0-
8.0 (0.5-15.5)
311
Ac
Treatment
18.2 (12.0-24.4)
ed
Body
0.3
ce pt
Gender
M an
Thin blood smear CATT/ T. evansi RoTat1.2 PCR
22.5 (17.9-27.1)
10 (6.7-13.3)
7.4 (4.5-10.3)
31.0)
27 Page 27 of 33
15.3 (7.0-23.6)
ip t
0
25.0 (15.0-35.0)
cr
72
us
1 < x ≤ 5 yr
11.1 (3.8-
20.1)
18.4)
2.4 (0.5-4.3)
25.8 (20.4-31.2)
> 10 yr
8 (0.0-18.3)
40.0* (20.8-59.2) 40.0* (20.8-59.2) 12.0 (0.0-
M an
5 < x ≤ 10 yr 252 25
7.9 (4.6-11.2) 6.3 (3.3-9.3)
24.7)
12.0 (0.0-247)
Ac
ce pt
ed
* significant difference
18.3 (13.5-23.1)
12.5 (4.9-
28 Page 28 of 33
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Table 6: Mean PCV (%) with standard deviation (SD) and 95% confidence interval of 399 camels from Eastern Ethiopia tested using
Test
PCV
CATT/T. evansi
2.7
23.4-24.0
2,1
19.0-22.5
2.6
24.0-24.6
21.5*
1.8
21.1-21.9
23.8
2.7
23.5-24.1
23.7
Positive
20.7*
Negative
24.3
ce pt
Negative Positive
23.0*
2.7
22.4-23.5
Negative
23.7
2.7
23.5-24.0
Positive
22.6*
2.7
21.8-23.5
Negative
23.7
2.7
23.4-24.0
Positive
22.6*
2.7
21.7-23.6
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18S PCR
ITS-1 PCR
95% confidence interval
Negative
Positive RoTat1.2 PCR
SD
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Thin blood smear
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different tests to detect T. evansi infection.
* significant difference
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Figure legends
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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:
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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
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(1000, 200, 40, 8, 0.16, 0.032, 0.0064 pg/µl).
30 Page 30 of 33
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Latent class analysis estimated an overall prevalence of 19%
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Depending on the diagnostic test, positivity rates ranged from 2% to 24%
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We conducted a cross-sectional study on camel trypanosome infection in Eastern Ethiopia
All infections were due to Trypanosoma evansi except one with Trypanosoma vivax
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Risk factors for infection were poor body condition and age
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Figure 1
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Figure 2
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