Research paperComparison of serological and molecular tests for detection of Trypanosoma evansi in domestic animals from Ghardaïa district, South Algeria
Introduction
Surra is a trypanosomosis due to Trypanosoma (T.) evansi, the first ever pathogenic trypanosome described in horses and dromedaries from India (Hoare, 1972). Trypanosoma evansi is a flagellated protozoan parasite transmitted mechanically by different fly species like Tabanus sp. and Stomoxys sp. (Luckins, 1988; Brun et al., 1998) and in South America also by vampire bats (Hoare, 1972). Trypanosoma evansi affects a huge range of domestic and wild mammals around the world and especially camels and horses in Africa (Dia et al., 1997; Njiru et al., 2004; Gari et al., 2010; Birhanu et al., 2015; Fikru et al., 2015); camels, horses, water buffalo and cattle in Asia (Abo-Shehada et al., 1999; Verloo et al., 2000; Hasan et al., 2006; Elshafie et al., 2013; Tehseen et al., 2015; Alanazi et al., 2018; Yadav et al., 2019) and horses, cattle and dogs in South America (Herrera et al., 2004; Jaimes-Dueñez et al., 2017; Ramírez-Iglesias et al., 2017). Sporadically, the parasite has caused outbreaks in Europe by importation of infected animals from endemic countries as was recently the case in Spain and France (Gutierrez et al., 2006; Desquesnes et al., 2009; Tamarit et al., 2010).
Clinical signs differ from one host species to another and vary from unapparent to lethal. In camels, surra causes intermittent fever, weakness, abortion and oedema. It is sometimes fatal within a few months but more chronic evolution lasting 2 to 3 years is demonstrated (Singh and Momin, 2008). Infection in horses is characterised by anaemia, icterus, weight loss and neurological signs with a mortality rate of about 50 % (Rodrigues et al., 2009). In cattle and water buffaloes, surra is usually a chronic disease but clinical signs occur like abortion, weight loss and neurological disorders (Luckins, 1988). Sheep and goats are mainly asymptomatic (Desquesnes et al., 2013). In dogs, T. evansi causes a serious disease, often accompanied with blindness, and frequent fatal outcome (Echeverria et al., 2019).
Clinical signs are not pathognomonic and diagnosis can only be confirmed by laboratory tests. In routine practice, parasitological examination is usually limited to microscopic observation of a Giemsa stained thin blood smear or thick drop which are poorly sensitive in chronic infections with low numbers of parasites circulating in peripheral blood. Serological antibody detection tests on the other hand, have been proven to be very useful, especially for epidemiological surveys on surra (Atarhouch et al., 2003). Recommended serological techniques listed in the Terrestrial Manual of the World Organisation for Animal Health (OIE) are: ELISA with native T. evansi variant surface glycoprotein (VSG) RoTat 1.2 as antigen (ELISA/RoTat1.2), ELISA with whole T. evansi cell lysate as antigen (ELISA/WCL), the Card Agglutination Test for T. evansi (CATT/T.evansi), and an antibody-mediated complement lysis test (immune trypanolysis or TL) with T. evansi variable antigen type (VAT) RoTat 1.2 (OIE, 2018). Major disadvantages of serological tests are cross-reactivity with non-specific antibodies caused by other infections and the persistence of specific antibodies for weeks or months after successful treatment. As surrogate for parasitological diagnosis, molecular tests detect parasite-specific DNA or RNA and therefore are very specific. Moreover, DNA and RNA disappear within days after successful treatment and RNA presence indicates active infection. Molecular tests for the diagnosis of surra are either specific for the subgenus Trypanozoon, to which T. evansi belongs together with T. brucei and T. equiperdum or specific for a certain T. evansi subtype (Claes et al., 2004; Njiru et al., 2006; Carnes et al., 2015). The OIE Terrestrial Manual recommends TBR PCR which targets a 177 bp repetitive microsatellite sequence present in Trypanozoon (Masiga et al., 1992; OIE, 2008). The high copy number of the 177 bp repeats renders the TBR PCR very sensitive but makes is also very prone to contamination during sampling, especially in large scale surveys (unpublished observations). Alternative tests for Trypanozoon detection, such as 18S PCR targeting a ribosomal gene and ITS1 PCR targeting the internal transcribed spacer 1 within the ribosomal locus have been described (Desquesnes et al., 2001). The former is Trypanozoon specific while the latter has the advantage that it detects the ITS1 in the genome of T. congolense, T. vivax, and Trypanozoon subgenus (including T. brucei, T. evansi, T. equiperdum). To replace the conventional 18S PCR, Deborggraeve and co-workers (2011) developed a quantitative real time PCR targeting the 18S gene (18S qPCR). Recently, a touch-down variant of the ITS1 PCR (ITS1 TD PCR) was developed to reduce non-specific reactions often observed with specimens from cattle (Tran et al., 2014). It detects the ITS1 in the genome of T. congolense, T. vivax, and Trypanozoon subgenus (including T. brucei, T. evansi, T. equiperdum).
The aims of this study were: i) to determine the prevalence of T. evansi in dromedary camels, ruminants and dogs, with particular interest in detection of subclinical infections and putative reservoir hosts, ii) to compare the accuracy and to assess the concordance between six diagnostic methods used for diagnosis of T. evansi: TL, ELISA/RoTat 1.2, ELISA/WCL, CATT/T.evansi, 18S quantitative PCR and ITS1 touchdown PCR.
Section snippets
Study area
The study was carried out from May 2017 to February 2018 in the wilaya of Ghardaïa (Fig. 1). Ghardaïa is located in the north of the Algerian Sahara, about 600 km from the capital Algiers, between 33° and 31°15′ N, 2°30′ and 5 °E. The climate is arid with very low rainfall (160 mm/year), very high summer temperatures (20 °C–45 °C) and low winter temperatures. The wilaya of Ghardaïa covers a total area of 86,560 km2 (4 % of the Algerian territory). The pastoral area of Ghardaia covers an area of
Prevalence
The number of positive samples, according to the various diagnostic methods and host species, are shown in Table 2. Regarding the serological tests, the highest proportion of positive animals was recorded with ELISA/RoTat 1.2, ranging from 0.8 % in goat to 9.9 % in camels and overall positivity rate of 4.9 %. With CATT/T.evansi, the overall positivity rate was 3.1 % ranging from 0.6 % in sheep to 9.3 % in camels. With ELISA/WCL, the overall positivity rate was 3.4 % ranging from 0% in horses to
Discussion
Our study confirms that T. evansi is present in Algeria particularly in dromedary camels, as observed in other studies carried out elsewhere in this country (Bennoune et al., 2013; Boushaki et al., 2019). Depending on the diagnostic test, prevalences ranged from 6.2 %–13 % which is much lower than what is reported from some other countries. For example, observed prevalences of T. evansi in dromedaries in Egypt were 31 % and 71 % in respectively RoTat 1.2 PCR and TBR PCR (Elhaig and Sallam, 2018
CRediT authorship contribution statement
Karima Benfodil: Conceptualization, Methodology, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision. Philippe Büscher: Conceptualization, Methodology, Resources, Writing - original draft, Writing - review & editing, Visualization, Funding acquisition. Amine Abdelli: Writing - review & editing, Formal analysis. Nick Van Reet: Writing - review & editing, Validation. Abdellah Mohamed-herif: Methodology, Validation. Samir Ansel: Methodology, Validation. Said
Declaration of Competing Interest
None.
Acknowledgements
We thank the veterinarians of the agriculture service of the wilaya of Ghardaia for their help in collecting samples. We thank all owners for field assistance.
We thank the OIE Reference Laboratory for Surra for allowing us to carry out the diagnostic tests at the Institute of Tropical Medicine in Antwerp and the High National Veterinary School of Algiers, Algeria for funding the stay of Karima Benfodil in Belgium.
We also thank Dr. Meryam Krit of the Institute of Tropical Medicine Antwerp for
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