Abstract
Purpose
Surra is an economically important livestock disease in many low- and middle-income countries, including those of Northern Africa. The disease is caused by the biting fly-transmitted subspecies Trypanosoma brucei evansi, which is very closely related to the tsetse-transmitted subspecies T. b. brucei and the sexually transmitted subspecies T. b. equiperdum. At least two phylogenetically distinct groups of T. b. evansi can be distinguished, called type A and type B. These evolved from T. b. brucei independently. The close relationships between the T. brucei subspecies and the multiple evolutionary origins of T. b. evansi pose diagnostic challenges.
Methods
Here we use previously established and newly developed PCR assays based on nuclear and mitochondrial genetic markers to type the causative agent of recent trypanosome infections of camels in Southern Algeria.
Results/conclusion
We confirm that these infections have been caused by T. b. evansi type A. We also report a newly designed PCR assay specific for T. b. evansi type A that we expect will be of diagnostic use for the community.
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Introduction
The single cellular parasite Trypanosoma brucei evansi belongs to the subgenus Trypanozoon that also comprises T. b. brucei and T. b. equiperdum [1, 2] (the taxonomical status of T. b. evansi is controversial [1, 3, 4]; for this study, we will be referring to it as a subspecies of T. brucei). Trypanosoma b. evansi is the most widely distributed of the pathogenic animal trypanosomes, affecting a large number of wild and domesticated animal species in Asia, Africa and Latin America [5, 6]. In Europe, it is present in the Canary Islands, from where recent sporadic incursions into the French and Spanish mainland have occurred [5, 7, 8]. Trypanosoma b. evansi causes a trypanosomosis called “surra” in many countries [8,9,10]. It is an acute, chronic or subclinical disease that is very often fatal in camels, horses and dogs, but can also seriously affect cattle and buffaloes. Other animals, including wildlife, are also susceptible.
In affected countries, surra is an economically important disease, which causes high mortality, reduced milk and meat production, poor carcass quality, reduced reproductive performance, and decreased draft power and manure production [9]. Haematophagous flies of the genera Tabanus and Stomoxys are particularly relevant for transmitting the infection from host to host, acting as mechanical vectors without parasite development in the insect [9]. This is a key difference to T. b. brucei, where transmission is dependent on cyclic development in the tsetse fly [11]. Indeed, it is this mechanical transmission that has allowed the parasite to move beyond the tsetse fly region and out of Africa [12]. In South and Central America, T. b. evansi can also be transmitted by vampire bats (Desmodus rotundus), which act as both vectors and reservoirs [9].
Another key difference to T. b. brucei is that T. b. evansi strains are either ‘dyskinetoplastic’ or ‘akinetoplastic’, i.e., they have either dysfunctional kinetoplast DNA (the mitochondrial DNA network in these organisms) or lack it entirely. Where kDNA is present, T. b. evansi strains typically lack maxicircles—the equivalent of mitochondrial DNA in other eukaryotes—and are characterized by minicircle sequence homogeneity [13, 14]. By contrast, T. b. brucei contains hundreds of different minicircle classes [15]. Trypanosoma b. evansi is therefore incapable of mitochondrial gene expression, and a compensatory mutation in the nuclearly encoded subunit γ of the F1FO ATP synthase is necessary to enable viability [16]. Based on the minicircle class that dominates the kDNA networks, T. b. evansi can be divided into types A and B [1, 13, 17]. Indeed, this difference can be exploited for polymerase chain reaction (PCR)-based diagnostics and molecular characterization of the parasite. PCR-based assays that target Trypanozoon-specific satellite DNA or ribosomal DNA are regarded as the most sensitive for diagnosis or characterization of surra infections [18,19,20], while for genotyping T. b. evansi and/or to distinguish between T. b. evansi types A and B, PCR assays targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, microsatellite markers and the F1-ATP synthase γ subunit gene are being used [4, 17, 21,22,23].
In northern Africa, the first cases of trypanosomosis were officially reported from Algeria, Mauritania, Morocco and Tunisia at the beginning of the last century [24,25,26,27]. In-depth epidemiological studies began at the end of the 1980s and showed that camel trypanosomosis could be considered as a dominant disease, with variable prevalence rates depending on the year, the sampling period and the provinces or wilayate (districts) surveyed [28,29,30,31,32,33,34]. A recent epidemiological study in southern Algeria carried out on 1056 dromedary camels revealed overall prevalence rates of 2.4% by Giemsa-stained thin smear (GST), 32.4% by card agglutination test for trypanosomosis (CATT/T. evansi), 23.1% by enzyme-linked immunosorbent assay (ELISA/VSG RoTat 1.2), 21.0% by immune trypanolysis (TL) and 11.2% by PCR (RoTat 1.2 PCR) [35].
Here, we present a genotyping analysis for six of the camels from the previous study [35], based on sequencing of minicircle DNA and of the F1FO ATP synthase subunit γ gene, and confirm the pathogen as T. b. evansi type A. Furthermore, we present a novel PCR assay based on primers with improved specificity for minicircle type A that will be useful for typing of surra infections.
Materials and Methods
All PCR primers are listed in Table 1. All trypanosome isolates or strains are listed in Table 2. Trypanosoma b. evansi and T. b. equiperdum reference strains were kind gifts from Kirsten Gillingwater, Swiss Tropical Institute [36] and from Philippe Büscher and Nick Van Reet, ITM Antwerp.
Growth of T. b. evansi and T. b. equiperdum Reference Strains and DNA Isolation
Trypanosome reference strains were grown in MFI mice and purified from blood using DEAE cellulose as described [40]. DNA extraction was performed using the QIAamp 250 mini blood kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Preparation of FTA Punches
The Harris Uni-Core punch tool (Merck, Darmstadt, Germany) and cutting mat were prepared by soaking in 2% (w/v) sodium hypochlorite solution for 10 min, followed by three washes with ddH2O, soaking in 70% (v/v) ethanol for 5 min and air drying. Punches from FTA cards were washed three times with 200 µl FTA Purification Reagent (GE Healthcare) for 5 min each and twice with 200 µl TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0) for 5 min each. Punches were dried at 50 °C for 15 min and added directly to PCR reaction tubes.
PCR Assays
All PCR assays were performed in 25 µl volumes and used FTA card punches or trypanosome genomic DNA (1–5 ng) as indicated (negative controls included additional H2O instead). Assays for all targets, with exception of the full-length F1FO ATP synthase subunit γ (Tb927.10.180), used the following reagents:
Reagent | Volume |
---|---|
5 × GoTaq PCR buffer (Promega) | 5 µl |
MgCl2 (25 mM) | 2 µl |
dNTPs (10 mM) | 0.5 µl |
GoTaq G2 Hot Start (Promega) | 0.125 µl |
Specific primers, their volumes, and PCR cycling conditions were as follows:
Target | Primers (10 µM) | Volume (µl) | Cycling conditions |
---|---|---|---|
F1FO ATP synthase subunit γ (Tb927.10.180), 511-bp fragment | #1, #2 | 1 | 95 °C 5 min 35x (95 °C 30 s, 55 °C 30 s, 72 °C 1 min) 72 °C 10 min |
Duplex assay minicircle type A (novel)/F1FO ATP synthase subunit γ 511-bp fragment | #3, #4, #5, #6 | 1.25 | 95 °C 5 min 40 × (95 °C 30 s, 51 °C 30 s, 72 °C 1 min) 72 °C 10 min |
Minicircle type A (novel) | #5, #6 | 2.5 | 95 °C 5 min 40 × (95 °C 30 s, 51 °C 30 s, 72 °C 1 min) 72 °C 10 min |
Minicircle type A (ref [17]) | MiniA, MiniB | 2.5 | 95 °C 5 min 40 × (95 °C 30 s, 51 °C 30 s, 72 °C 1 min) 72 °C 10 min |
Maxicircle gene A6 | #7, #8 | 1 | 95 °C 5 min 35 × (95 °C 30 s, 55 °C 30 s, 72 °C 1 min) 72 °C 10 min |
Maxicircle gene ND4 | #9, #10 | 1 | 95 °C 5 min 40 × (95 °C 30 s, 54 °C 30 s, 72 °C 1 min) 72 °C 10 min |
PCR reagents for the full-length F1FO ATP synthase subunit γ (Tb927.10.180) gene, including flanking regions, were as follows (25 µl total):
Reagent | Volume |
---|---|
5 × Phusion PCR buffer (New England Biolabs) | 5 µl |
Primers #3 and #4 (10 µM) | 1.25 µl |
dNTPs (10 mM) | 0.5 µl |
Hot Start Phusion (New England Biolabs) | 0.25 µl |
PCR cycling conditions for the full-length gene were as follows: 98 °C 30 s, 40 cycles (98 °C 10 s, 60 °C 30 s, 72 °C 1 min), 72 °C 10 min.
Cloning and Sequencing
All PCR products were cleaned up using the PCR Clean-Up kit from Macherey–Nagel (Dueren, Germany). Sequencing was either direct, using the same primers that had been used for the PCR reaction, or after cloning into pCR-Blunt (Invitrogen; for Phusion PCR products) or into pGEM-T easy (Promega; for GoTaq PCR products), following the manufacturer’s instructions. Cloned products were sequenced using Sanger technology (Edinburgh Genomics or MRC Sequencing Service, Dundee) and standard M13 forward and reverse primers.
Phylogenetic Analysis
A phylogenetic tree was constructed with IQ-TREE [41], using a maximum likelihood model with HKY + G substitution.
Results and Discussion
PCR assays for TbATPase subunit γ confirm infection with T. b. evansi type A
To confirm the diagnosis of a T. b. evansi infection in camels from 5 different Algerian regions (Table 2) [35], we amplified by PCR a 511 bp fragment of subunit γ of the mitochondrial F1FO ATP synthase (systematic TriTrypDB ID Tb927.10.180). In the T. b. evansi types identified so far, this gene contains adaptive mutations that are differentially diagnostic for types A and B [1, 4, 21]. Punches from FTA cards containing DNA purified from blood samples from cases 1 to 6 were washed and placed in reaction tubes, together with PCR reagents and primers #1 and #2 (Table 1). Initial reactions were carried out with (non-proof-reading) Taq polymerase because of its robust performance. Total cellular DNA from a T. b. brucei strain served as positive control. Reactions for all six cases showed a single amplicon of the expected size, suggesting infection with a Trypanozoon (Fig. 1A). To identify the type of T. b. evansi, we next amplified the entire ATP synthase γ gene with primers #3 and #4 and a proof-reading polymerase, followed by cloning and sequencing. Sequence analysis confirmed presence of a heterozygous A281del mutation in the ATP synthase γ protein for all cases (Fig. 1B), providing conclusive evidence for infection with T. b. evansi type A [1]. These results are consistent with the previously reported RoTat1.2-positive PCR results for these isolates [35]. RoTat1.2 is a VSG gene that, when present, is generally considered as being diagnostic for T. b. evansi type A [5]. These results are also consistent with the fact that the only other type of T. b. evansi currently known, type B, has so far only been reported from countries in East Africa, namely Kenya and Ethiopia [5, 17, 21].
Development of a Novel PCR Assay Specific for Minicircle Type A
A defining characteristic of T. b. evansi type A is that (unless the strain is akinetoplastic [1]) its kDNA is dominated by, or consists entirely of, thousands of copies of a particular class of minicircle [13]. A PCR assay for this minicircle class developed by Njiru and colleagues [17] uses primers (‘MiniA’ and ‘MiniB’) derived from a region semi-conserved among all minicircle classes (Supplementary Figure S1) and can therefore result in false positive reactions [21]. We therefore aimed to develop a PCR assay that is highly specific for type A minicircles. Alignment of type A minicircles available in GenBank identified several regions of perfect conservation outside of the universally conserved region. Based on this information, we designed primer sequences that are predicted to amplify a fragment of ~ 570 bp in a PCR assay (Supplementary Figure S1, primers #5 and #6). Alignment of the minicircle type A consensus to the closest match in the recently defined minicircle population of T. b. brucei EATRO 1125 [15], a minicircle that contains the same set of gRNA genes, suggests that primers #5 and #6 should be specific for T. b. evansi minicircle type A (Supplementary Figure S2). Indeed, when tested against a panel of type A and non-type A strains or isolates, the PCR assay was highly specific (Fig. 2A). Sequencing of the ~ 570 bp amplicons confirmed that they corresponded to the expected minicircle type A. The only unexpected result was absence of a ~ 570 bp amplicon for strain T. b. evansi CAN86/Brazil (Fig. 2A, lane 6). As a PCR reaction using the MiniA/MiniB primers also failed to produce an amplicon for this strain (data not shown), we suspect that this strain has spontaneously lost its kDNA. This phenomenon is not unusual in T. b. evansi and T. b. equiperdum [14, 42].
Next, we used the new PCR assay to analyze samples from cases 1 to 6. For cases 1, 2, 3 and 6, we obtained a specific band of ~ 570 bp (Fig. 2B, left panel), and direct sequencing confirmed that the amplicons were the type A fragment (Supplementary Figure S3). We did not obtain a product for cases 4 and 5. The same result was obtained with primers MiniA/MiniB: strong amplification products of the expected size for cases 1, 2, 3 and 6, but no products for cases 4 and 5 (Fig. 2B, right panel). We conclude that, in all six cases, camels had been infected with T. b. evansi type A. In cases 4 and 5, the parasites may have become akinetoplastic, and our typing relies exclusively on the presence of the A281del mutation of ATP synthase subunit γ. A phylogenetic tree based on the ~ 570 bp minicircle type A amplicon shows a separation into two main branches that is supported by strong bootstrap values (Supplementary Fig. 4). The Algerian cases branch together with two other isolates from Africa, and also a single isolate from South America, whereas the other isolates, all from non-African countries, form a separate branch. It will be interesting to expand the phylogenetic analysis of type A T. b. evansi based on their minicircle sequence to include other isolates, perhaps using the entire ~ 1-kb sequence to further improve resolution and reliability.
PCR assays for maxicircle-encoded genes A6 and ND4 (primer pairs #7/#8 and #9/#10, respectively; Table 2) were negative (data not shown), consistent with the expected absence of the maxicircle in T. b. evansi [13, 14].
Conclusion
Based on nuclear and mitochondrial genetic markers, we have confirmed that the recently reported trypanosome infections in southern Algerian camels were caused by T. b. evansi type A, adding to an accumulating body of recent reports of surra infections in that country [45,46,47]. We also report a novel PCR assay based on careful sequence analysis of type A minicircles that we expect will be a useful tool for the community to diagnose T. b. evansi type A infections in livestock. Our data reported here suggest good specificity and sensitivity for type A strains and compatibility with samples prepared on FTA cards. Further studies should compare specificity and sensitivity with other assays, such as the recently reported recombinase polymerase amplification lateral flow assay for T. b. evansi [48].
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Acknowledgements
We thank Kirsten Gillingwater (Swiss Tropical Institute), Philippe Büscher and Nick Van Reet (ITM Antwerp) for the kind gifts of T. b. evansi and T. b. equiperdum reference strains. This work was supported by the UK Medical Research Council (Grant MR/L019701/1 to A.S.).
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Authorisation to conduct the original survey [35] had been obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development and Fisheries). At each wilaya, the study had been authorized and supervised by the respective Inspection Vétérinaire de Wilaya (IVW Béchar, El Bayadh, Ouargla, and Tamanrasset), operating under the umbrella of the Direction des Services Vétérinaires. All experiments in Achim Schnaufer’s laboratory are carried out after local ethical approval at the University of Edinburgh by the School of Biological Sciences Ethics Committee (application “aschnauf-0002 Mitochondrial biology of trypanosomes”).
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Boushaki, D., Wallis, J., Van den Broeck, F. et al. Molecular Analysis of Trypanosome Infections in Algerian Camels. Acta Parasit. 67, 1246–1253 (2022). https://doi.org/10.1007/s11686-022-00577-7
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DOI: https://doi.org/10.1007/s11686-022-00577-7