Introduction

Trypanosoma evansi is a single-celled eukaryotic parasite which is closely related to T. brucei, the causative agent of sleeping sickness and nagana disease in human and animals, respectively [1]. T. evansi is the etiological agent of “Surra”, or mal de cadeiras, characterized by anemia, immunosuppression and damage to the central nervous system causing ocular and reproductive disorders [2]. It affects a wide range of domestic and wild mammalian species [2], and it is one of the most pathogenic trypanosomes transmitted mechanically by different blood-sucking fly species such as Tabanus spp. and Stomoxys spp. [3] as well as by vampire bats Desmodus rotundus [4]. Due to the high mortality and morbidity rates affecting livestock productivity, this disease is responsible for disastrous economic losses estimated at $404,630 USD per year [5, 6]. Although some authors have suggested that T. evansi originated and mainly affected camels in Africa [7], all domestic mammals studied to date are susceptible [8]. The disease results in a high rate of mortality in horses, camels, and dogs [2, 8]. Outbreaks of animal surra have been reported in North Africa, the Middle East, South America, Asia, and recently Europe [9]. T. evansi infection in humans was not only reported initially in India [10] but also confirmed recently in Vietnam [11]. Other probable cases in humans have been reported worldwide, but molecular parasite detection was not performed [2].

Many diagnostic methods (with varying degrees of sensitivity and specificity) are available to detect T. evansi infections including parasitological, serological, and molecular assays [12, 13]. Some methods detect trypanosome infection by microscopical examination of fresh or stained bloodsmears [14], whereas others identify different T. evansi strains, being classified as Type A or Type B, according to their Kinetoplast DNA minicircle sequences [15] and the presence of the RoTat1.2 variant surface glycoprotein (VSG) gene [16, 17] or antigen [18]. Other molecular-based methods target sequences within ribosomal genes of Trypanosoma spp., such as the small sub-unit ribosomal gene (18S) [19] or the internal transcribed spacers (ITS) [20, 21], and have been used for species identification.

In Algeria, a huge number of dromedary camels (Camelus dromedarius) is concentrated in the dry desert of the Sahara which occupies 87% of the total area of Algeria (2,381,741 km2). In this region, camels constitute the major source of animal protein and transport for nomads; however, there is a paucity of information regarding camel diseases—especially those concerning hemoparasites [22]. A variety of diagnostic tests has been used to assist in the detection of T. evansi infections in Algeria, and these tests have demonstrated species differences in infection susceptibility among camels, dogs, horses, and donkeys [23,24,25,26]. However, those previous studies did not extensively study the genetic structure and/or the genetic diversity of T. evansi, particularity in camels from Algeria. Therefore, the present study was devoted to the molecular characterization of T. evansi derived from naturally infected camels in eastern Algeria and comparison between isolates from different parts of the world in order to describe distinct T. evansi haplotypes.

Materials and Methods

Study Area

This study was conducted in four provinces (Ouargla, El Oued, Biskra and Ghardaia) located at 2°04′–7°35′ E and 28°32′–34°56′ N (Fig. 2). These areas are known for extreme aridity and extreme heat; they are among the hottest places on Earth during the height of summer. These regions comprise one of the most significant camel rearing areas in Algeria and play a key role in food security and transportation of Saharan and steppe community people [22].

Camel Blood Sampling

Between July 2018 and June 2019, a total of 12 camel herds ranging in size from 10 to 70 head were randomly selected for this study. Blood samples (n = 148) were collected from a random set of animals which included both sexes (20 males and 128 females) and animals in various age groups:  < 1 year (n = 7), 1–3 years (n = 33), 4–9 years (n = 53), 10–15 years (n = 45), and  > 15 years (n = 10) based on dental wear and owner information. Whole blood was collected from each camel via jugular venipuncture into a 5-mL Vacutainer®tube containing the anticoagulant ethylene diamine tetra acetic acid (EDTA). Samples were stored (in the blood collection tubes) at  − 20 °C until DNA extraction was performed.

Molecular Characterization of T. evansi

DNA Extraction from Camel Blood

Total genomic DNA was extracted from 200 µL of EDTA-preserved whole blood of each of the 148 camel blood samples using the DNeasy Blood and Tissue Kit (Qiagen®, Hilden, Germany), according to the manufacturer's instructions. DNA was eluted in a final volume of 200 μL and stored at  − 20 °C until used for molecular characterization.

DNA Amplification and Sequencing of ITS Ribosomal Spacer

Genomic DNA samples were subjected to a standard conventional PCR assay to amplify DNA. In order to obtain the ITS1 complete sequence, it was necessary to incorporate the partial 18S and 5.8S sequences so that they could act as anchors for the ITS1 sequence using the two previously described primers ITS1 CF (5′CCG-GAA-GTT-CAC-CGA-TAT-TG3′) and ITS1 BR (5′TTG-CTG-CGT-TCT-TCA-ACG-AA3′) amplifying African Trypanosomal species [21]. Standard PCR reaction was performed in a final volume of 25 μL containing 3.5 mM of MgCl2, 0.2 mM of each deoxyribonucleoside triphosphate (dNTPs) mixture, 1 × PCR buffer, 1 U of Taq DNA Polymerase (Roche, Germany), 0.2 μM of each primer (Eurogentec, Belgium), and 5 μL of genomic DNA. DNA-free distilled water was used as negative control. The PCR reactions were performed in an automated DNA thermal cycler (Biometra TRIO Thermoblock Heat Cycler, Germany) using the following reaction conditions: 95 °C for 10 min for initial denaturation, 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The PCR products were subject to electrophoresis in a 1.5% agarose gel stained with ethidium bromide and then visualized using UV light. Six positive PCR product samples were subsequently purified using QIAquick PCR Purification Kit (QIAGEN, Germany) according to manufacturer’s recommendation. Purified PCR products were sequenced in both directions with the same primers used in the PCR amplification. Sequencing reactions were performed by an ABI 3100 Genetic Analyzer automated sequencer (Applied Biosystems, USA) using an ABI PRISM BigDye™ terminator cycle sequencing kits (Applied Biosystems, Foster City, USA).

Phylogenetic and Phylogeographic Analyses

The ChromasPro software (Technelysium PTY, Australia) was used to analyze, assembl and edit the DNA sequences obtained in this study. A nucleotide BLAST was made to compare the identity of the sequences with the NCBI database. Multiple sequence alignment was performed based on ITS1 complete sequences after ligation of the amplicons products using ClustalW. In order to determine the potential novelty of haplotypes identified in our experimental animals, an exhaustive search on GenBank was performed to obtain all sequences of T. evansi for which the ITS1 region was complete. Altogether 96 available sequences were included in the analysis: 69 from Asia, 21 from Africa, and six from South America. A ClustalW alignment was performed on sequences belonging to the same country, and representative haplotypes from different countries, including haplotypes of the present study, were subsequently aligned. According to previous studies on gene diversity of different parasites, each sequence containing an original nucleotide signature is considered a new haplotype [27,28,29].

The phylogeny reconstruction was performed with the DNA sequences of our 6 T. evansi isolates and 39 other representative haplotypes of T. evansi (previously identified in Africa, Asia and South America and deposited in GenBank). The tree was inferred with complete deletion using the maximum likelihood (ML) method, with 500 bootstrap replicates, using Molecular and Evolution Genetic Analysis (MEGA v5 software) [30]. For the latter, the best model was the Tamura 3-parameter with five rate categories and assuming that a certain fraction of sites are evolutionarily invariable (+ I). Genetic distances [Kimura 3-parameter distance] were estimated with MEGA v5 software [30]. All codon positions were used, and all positions containing gaps or missing data were eliminated. Haplotype diversity  < Hd >  and nucleotide diversity (π) were calculated using DNA Sequence Polymorphism software (DNASP 5.10.01) [31].

Nucleotide Sequence Accession Numbers

The ITS 1 complete sequences generated from this study were deposited in GenBank through accession numbers MT539996 to MT540001.

Results

Descriptive Epidemiology

Of 148 camel blood samples, 19 (12.84%) were PCR-positive for Trypanosoma species. All ITS1 PCR products were approximately 480 bp [specific size of all members of the subgenus Trypanozoon [21]]. The result revealed a non-significant difference between localities (χ2 = 5.83, df = 3, P = 0.12); the prevalence rate was highest in Ghardaia (40.0%), followed by 17.1% in El Oued, 10.0% in Biskra, and 0% in Ouargla (Table 1). Similarly, there was no association of sex (χ2 = 0.45, df = 1, P = 0.50) or age (χ2 = 8.33, df = 4, P = 0.08) with recorded prevalence rate. Prevalence of trypanosome detection was higher in females (13.3%) than in males (10.0%) and higher in camels between 4 and 9 years of age (18.9%) than in other age groups (Table 1).

Table 1 Molecular prevalence rates of Trypanosoma spp. among dromedary camels in eastern Algeria stratified by locality, animal sex and age class
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Molecular Characterization and Phylogenetic Analysis

Sequencing of the rDNA complete internal transcribed spacer (ITS 1) region, including the partial sequences of 18S and 5.8S, generated six sequences corresponding to Trypanosoma evansi with BLAST. Multiple sequence alignment revealed limited heterogeneity among the six sequences (nucleotide diversity π = 0.00550), in the form of five polymorphic or segregating sites and two alignment gaps or missing data, leading to the exhibition of four different haplotypes with a rate of gene diversity Hd corresponding to 0.800. This heterogeneity was localized at the 3′ region of the fragment corresponding to ITS1 (336 bp). Overall, there is one Singleton variable site at position 207 and four Parsimony informative sites at positions 212, 232, 233 and 341(Fig. 1).

Fig. 1
figure 1

Alignment of the four Trypanosoma evansi ITS1 sequence haplotypes. The single nucleotides polymorphisms are marked with colors in the frame.

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The first haplotype is represented by isolate 23 from Biskra. The second haplotype is represented by isolate 74 from El Oued. Three identical sequences corresponding to isolates 81, 99 and 119 from El Oued represent the third haplotype, and isolate 139 from Ghardaia represents the fourth haplotype (Fig. 1).

To determine the phylogeographic relationships of T. evansi isolates in camels from Algeria, ITS1 complete sequences of different haplotypes previously reported in Africa, Asia and South America were used. The resulting alignment of the complete ITS1 region exhibited at least 41 different haplotypes confirmed by DNASP (Table 2), with a high rate of gene diversity Hd corresponding to 0.993. The first 30 haplotypes (H1-H30) were found in Asia, two haplotypes (H32 and H33) were found in South America and the remainder of the haplotypes (H34 to H41) originated in Africa. Some haplotypes are shared across continents; for example, H3 was found in China and Kenya, H8 was found in China, Thailand and Egypt, and H10 which has a worldwide distribution. No host specificity was observed.

Table 2 Species and geographical source of Trypanosoma evansi ITS1 sequences characterized in the phylogeographic analysis
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The new Algerian sequences of T. evansi from camel blood samples are grouped into four different haplotypes: H28 is identical to the isolate found in Saudi Arabia (Genbank N°MN625864; unknown source); H10 is identical to a haplotype found in a wide range of domestic and wild mammalian species and having a worldwide distribution; H40 and H41 are unique and do not match any known haplotypes. Pairwise genetic distances (computed using the Tamura 3-parameter model) between our new haplotypes and other representative haplotypes of the dataset showed a genetic divergence between 0.3 and 14% for H40 and between 0.3 and 15% for H41 (Table 3). Phylogenic analyses were performed with all different representative haplotypes, including our Algerian sequences. The unrooted tree topology shows that each haplotype clustered separately, including the two new haplotypes from Algeria (Fig. 2).

Table 3 Estimate of evolutionary divergence between Trypanosoma evansi ITS1 sequences
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Fig. 2
figure 2

Phylogenetic (a) and geographic (b) distribution of Trypanosoma evansi haplotypes identified in dromedary camel populations in eastern Algeria in relation to T. evansi haplotypes reported worldwide

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Discussion

In the present study, Trypanosoma spp. DNA was isolated from 12.84% of camel blood samples collected in eastern Algeria. Six sequences based on ITS1 complete sequence amplification were obtained and identified as T. evansi. Two previous studies concerning T. evansi in camels using molecular tools have been reported in Algeria. Overall molecular prevalence of T. evansi in 1056 camels from four provinces of southern Algeria (El Bayadh, Bechar, Ouargla, Tamanrasset) was 11.2% with RoTat 1.2 PCR [26], and rates of 13% were recorded with the 18S qPCR and 6.2% with the ITS1 PCR in 161 camels from Ghardaia [24], However, those PCR positivity rates were lower compared with results from other countries. For example, T. evansi was detected in 42% of camels from Sudan [32], 32% of camels in Egypt, 31% of camels in Pakistan, 26% of camels in Kenya and 25% of camels in Saudi Arabia [8], as well as in 16.8% of camels in Iran [33]. The lower PCR positivity rate in our study may reflect the fact that the majority of our Algerian samples were collected from apparently healthy camels intended for slaughter (and not from animals showing clinical signs or symptoms of disease). There was a huge difference of sampling among males and females and, independent of previous studies, this difference made analysis concerning the influence of sex on T. evansi infection in the present study difficult. Moreover, the lack of sex or age effects on the prevalence of infection is in agreement with previous studies which revealed that sex and age groups are equally exposed to trypanosomiasis in Pakistan [13] and in Algeria [34].

There are ample reports of characterization studies on trypanosomes in general and T. evansi in particular, among which, DNA targets were used to distinguish T. evansi interspecifically and intraspecifically including 18S rDNA, kinetoplast DNA, microsatellite sequences and the expression-site-associated genes (ESAGs) [35, 36]. Amongst those studies, the molecular targets of variable surface glycoprotein (VSG) RoTat 1.2 gene of T. evansi are widely employed [37] because it is an integral part of the parasite’s surface coat and is known to be expressed in early, middle and late stages of infection [38]. The heterogeneity of T. evansi cannot be demonstrated using those targets; however, studies of ITS regions are more efficient for differentiation and may reflect geographical and/or host range effects [5, 35].

To increase knowledge of genetic diversity of T. evansi in Algeria, and to infer phylogenies and relatedness of the Algerian isolates, molecular analysis based on ITS1 complete sequences was performed on naturally infected dromedary camels. The obtained sequences revealed limited nucleotide variability leading to identification of four different haplotypes. However, in a recent study, identical sequences of T. evansi were reported from partial ITS1 belonging to different dogs in northeastern Algeria [39]. The phylogenetic analysis of our isolates, including representative sequences of the ITS1 complete sequences from Africa, Asia and South America, showed that Algerian sequences isolated from camels belong to four different haplotypes: (i) H28, identical to a sequence isolated from Saudi Arabia;(ii) H10, present worldwide in a different host and (iii) two novel sequences representing new haplotypes H40 and H41.

Phylogenetic analyses of reported T. evansi sequences from various species revealed neither host nor geographic specificity. However, due to lack of nucleotide sequences from previous cases of T. evansi in Algeria, history of these haplotypes could not be determined. For this reason, future population and phylogeographic analyses involving a larger number of samples from different Algerian regions and North African countries are needed to evaluate this evolutionary process.

In conclusion, genetic diversity of T. evansi among dromedary camels from Algeria has been demonstrated and two new haplotypes have been identified. These novel findings can serve as the basis for future studies of T.evansi genetic diversity. Nevertheless, additional studies including more samples from different hosts and geographic areas are needed to fully understand the extent of distribution of T.evansi haplotypes in Algeria.