Background
Surra is an animal disease occurring in Africa, Asia and Latin America, caused by Trypanosoma evansi. T. evansi belongs to the subgenus Trypanozoon, together with T. equiperdum and T. brucei. The parasite can infect different host species and is mechanically transmitted by different biting flies such as Tabanidae and Stomoxys as well as by vampire bats such as Desmodus rotondus 1. Camels and horses are very susceptible to the infection and death can occur within weeks or months. Moreover, T. evansi infections of cattle and buffaloes usually lead to a pronounced immunosuppression resulting in an increased susceptibility to other opportunistic diseases such as Pasteurella and anthrax 2.
Diagnosis of a T. evansi infection usually starts with clinical symptoms or the detection of antibodies to T. evansi. Conclusive evidence of T. evansi infection, however, relies on detection of the parasite in the blood or tissue fluids of infected animals. Unfortunately, parasitological techniques cannot always detect ongoing infections as the level of parasitaemia is often low and fluctuating, particularly during the chronic stage of the disease 3.
As an alternative to parasitological tests, DNA detection based on PCR has been proposed. Trypanozoon specific primers have been designed previously: TBR primers which target a 177 bp repeat 4, pMUTEC primers targeting a retrotransposon 5 and ORPHON primers which target the spliced leader sequence 6. Most of them have been tested on cattle 78, water buffaloes 9 or goats 10. PCR tests for diagnosis of T. congolense and T. vivax infections exist as well 11. The development of a PCR test that would be able to differentiate between the different members of the Trypanozoon subgenus still remains a challenging issue. For T. evansi infections, the only specific test available so far is based on the detection of a kinetoplast DNA sequence 1213. However, the existence of dyskinetoplastic trypanosomes such as T. evansi RoTat 5.1 14 and E152 12 casts doubt about the diagnostic potential of such tests to detect all infections caused by T. evansi parasites. Recently, Ventura et al. 15 developed a PCR (PCR-Te664) for the detection of T. evansi based on a Random Amplified Polymorphic DNA (RAPD) fragment. The taxon specificity of this PCR remains uncertain since it was only tested on nine T. evansi strains, one T. equiperdum, two T. b. gambiense and one T. b. rhodesiense. Following evidence that the variable epitope of RoTat 1.2 VSG is expressed by all T. evansi strains tested so far 16, and that the gene encoding RoTat 1.2 VSG is present in all T. evansi but not in T. brucei isolates 17, we designed primers derived from the sequence of this VSG cDNA. In this article we will present and discuss the results obtained with these primers and compare them to the results we obtained using the PCR-Te664.
Results
PCR RoTat 1.2 : taxon specificity
The 39 different trypanosome stocks used in this study are listed in Table 1 [see Additional file 1]. They were derived from a wide range of hosts and from distinct geographical locations. In all PCR runs, RoTat 1.2 DNA was used as a positive control. As shown in Figure 1, the RoTat 1.2 PCR yielded a 205 bp amplicon in the positive control (lane 1) as well as in all other T. evansi populations (lanes 3–8). Moreover, the same fragment was found in seven out of the nine T. equiperdum populations tested. The T. equiperdum BoTat 1.1 was PCR negative (lane 10), while the T. equiperdum OVI strain yielded a PCR product shorter than 205 bp (lane 11) probably due to mispriming. All other tested trypanosome populations, including six T. b. brucei, eight T. b. gambiense, five T. b. rhodesiense, two T. congolense, one T. vivax and one T. theileri, were negative. (lanes 18–40). As a negative control, a PCR-mix without template DNA was included (lane 2). Sequencing of the positive samples revealed that all amplicon were identical (data not shown). The weak band in OVI did not yield sufficient material to enable sequencing.
<p>Additional File 1</p>
Table 1. Data on the different Trypanosoma (T.) populations used in this study
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<p>Figure 1</p>
PCR specificity results for the different Trypanosoma (T.) species and subspecies in this study
PCR specificity results for the different Trypanosoma (T.) species and subspecies in this study. Lane 1 pos. control RoTat 1.2, Lane 2 neg. control, Lanes 3–8 (T. evansi) are, respectively, AnTat 3.1, STIB 816, Zagora I.17, Colombia, Merzouga 56, CAN 86 K; Lanes 9–17 (T. equiperdum) are, respectively, AnTat 4.1, BoTat 1.1, OVI, STIB 818, Alfort, Hamburg, SVP, Am. Strain, Can. Strain ; Lanes 18–23 (T.b.brucei) are, AnTat 1.8, AnTat 2.2, AnTat 5.5, KETRI 2494, TSW 196, STIB 348; Lanes 24–31 (T.b.gambiense) are, respectively, AnTat 9.1, AnTat 11.6, AnTat 22.1, NABE, SEKA, ABBA, LIGO, LiTat 1.6; Lanes 32–36 (T.b. rhodesiense) are STIB 884, STIB 850, AnTat 25.1/S, Etat 1.2/S, AnTat 12.1/S ; Lanes 37–38 (T. congolense) are IL1180, TRT 17; Lane 39 (T. vivax) is ILRAD 700 and Lane 40 (T. theileri) is MELSELE ; Lanes M 100 bp molecular marker (MBI Fermentas, Germany).
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PCR RoTat 1.2 : analytical sensitivity
A tenfold dilution series (105 trypanosomes down to 1 trypanosome per 200 μl sample) of RoTat 1.2 trypanosomes in mouse blood was prepared to determine the analytical sensitivity of the PCR. As shown in figure 2, the PCR was able to detect as few as 10 trypanosomes per PCR reaction, which corresponds with a lower detection limit of 50 trypanosomes per ml. In principle, this limit can still be lowered if a blood sample of 200 μl extracted with the QIAamp DNA mini kit is eluted in less than 200 μl.
<p>Figure 2</p>
Analytical sensitivity of the RoTat 1.2 PCR
Analytical sensitivity of the RoTat 1.2 PCR. Lanes M 100 bp molecular marker (MBI Fermentas, Germany); lane 1: 105 trypanosomes, lane 2: 104 trypanosomes, lane 3: 103 trpyanosomes, lane 4: 102 trypanosomes, lane 5: 10 trypanosomes, lane 6: 1 trypanosome, lane 7: 0.1 trypanosome, lane 8: negative control.
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PCR-Te664 : taxon specificity
To evaluate the RoTat 1.2 diagnostic system alongside other published methods, we compared our method to the PCR-Te664 method as published by Ventura et al. 15 using the same trypanosome stocks. The PCR-Te664 method yielded the expected amplicon in all seven T. evansi strains and in seven out of nine T. equiperdum. As with the RoTat 1.2 PCR only T. equiperdum strains OVI and BoTat 1.1 remained negative. Unexpectedly, four out of six T. b. brucei (AnTat 2.2, AnTat 5.2, TSW 196 and KETRI 2494) and two T. b. gambiense type II strains (ABBA and LIGO) tested positive in this PCR (data not shown).
Discussion
This study was initiated to develop a specific PCR test that would be able to distinguish T. evansi from the other members of the Trypanozoon subgenus. The study is an extension of the initial observation that the RoTat 1.2 VSG gene only is found in T. evansi and not in T. brucei strains 17. This study mainly focused on the presence and expression of the RoTat 1.2 VSG gene in T. evansi rather than the use of this VSG in diagnosis of Salivarian trypanosomes.
Previously, other research groups have used VSG genes as target sequences for PCR detection of T.b. gambiense infections (sleeping sickness). In these studies, five different primers derived from VSG genes, AnTat 11.17, LiTat 1.3, 117, 2 K and U2 were used in PCR screening of different trypanosome populations, originating from distinct geographical locations 181920. AnTat 11.17 based PCR tests were capable of distinguishing T.b. gambiense from T.b. brucei parasites from most foci of sleeping sickness in countries such as Nigeria, Cameroon, Côte d'Ivoire, R. P. Congo/Brazza. and Sudan. However, populations originating from the Moyo focus in North-west Uganda and from Cameroon were shown to be negative in AnTat 11.17 and in LiTat 1.3 (2 K) PCRs respectively. According to Bromidge et al. 18, this might be due to antigenic variation and genetic evolution of the VSG genes. On the other hand, the presence of 117 and U2 genes was shown to be a common feature among all T. brucei populations tested. In T. evansi, a similar phenomenon may occur in certain Kenyan isolates. A recent study by Ngaira et al. 21 pointed out that some T. evansi stocks in the Isiolo district in Kenya seem to lack the Rotat 1.2 VSG gene. It is believed that these stabilates belong to the T. evansi type B group. So far, this type of T. evansi has only been observed in this specific region in Kenya 2223. To our knowledge, all other T. evansi isolated elsewhere, are from the classical T. evansi type A group. Thus, we assume that, except for these few Kenyan strains belonging to the type B group, our PCR is specific for T. evansi.
Compared to the PCR-Te664 presented by Ventura et al. 15, the PCR RoTat 1.2 seems to have a higher taxon specificity, since no reaction with T. b. brucei, nor with T. b. gambiense type II was observed. However, regarding T. equiperdum, both PCR test positive for the same seven T. equiperdum strains and are both negative for the BoTat 1.1 and OVI strains. Since the RAPD fragment (AF397194) shares no homology with the Rotat 1.2 VSG gene (AF317914) and is not found within the expression site of trypanosomes, both sequences can be considered as independent molecular markers. Based on the observations with both markers, it appears that on the genomic level the Botat 1.1 and the OVI strains are different from the other T. equiperdum and T. evansi strains. The observed analytical sensitivity with the RoTat 1.2 PCR is comparable to what was reported for the Te664 PCR (25 cells per reaction) 15.
The presence of a RoTat 1.2 specific DNA sequence in some T. equiperdum strains corresponds with the serological evidence that rabbits experimentally infected with these strains develop RoTat 1.2 specific lytic antibodies within 30 days post infection 24. In contrast, rabbits infected with the BoTat 1.1 clone and the OVI strain, which are negative in the present PCR, did not produce specific antibodies to the RoTat 1.2 clone when tested in immune trypanolysis. This might be explained by the loss of the RoTat 1.2 gene in the OVI and the BoTat 1.1 strain. It is also possible that there has been a sequence drift at the sites where these primers could bind. However, we hypothesize that RoTat 1.2 VSG truly is T. evansi specific and that RoTat 1.2 PCR positive T. equiperdum strains are actually T. evansi and not T. equiperdum. Indeed, in a previous molecular characterization study using Random Amplified Polymorphic DNA (RAPD) and the Multiplex-endonuclease Genotyping Approach (MEGA) it appeared that the T. equiperdum collection is not as homogenous as previously believed and that the generally followed concept that T. equiperdum is very closely related to T. evansi and more distant from T. b. brucei, seems incorrect. From the cluster analysis on the available strains, it appeared that only two clusters can be identified: a homogeneous T. evansi/T. equiperdum cluster and a more heterogeneous T. b. brucei/T. equiperdum cluster 25. Interestingly, all strains of that homogeneous T. evansi/T. equiperdum cluster are all PCR RoTat 1.2 VSG positive while the strains found in the more heterogeneous T. b. brucei/T. equiperdum cluster, in casu BoTat 1.1 and OVI are PCR RoTat 1.2 VSG negative.
Methods
Trypanosome populations
A total of 39 different trypanosome populations were used in this study. They belong to 39 stocks and six species, isolated from a variety of host species at distinct geographical locations (Table 1 [see Additional file 1]). Only three T. equiperdum strains, BoTat 1.1, OVI and STIB 818 are well documented, i.e. known origin and host. The other six are putative T. equiperdum, based on publications or on their use as reference strains in different national dourine reference laboratories 2627282930.
Preparation of trypanosome DNA
Procyclic trypanosome populations were grown in vitro in Cunningham's medium 31 and in the Kit for In Vitro Isolation (KIVI) 32. Pure procyclic trypanosomes were obtained by repeated centrifugation (20 min., 2000 g) and sediment washes with Phosphate Glucose Sacharose buffer (PGS) (38 mM Na2HPO4.2H20, 2 mM NaHPO4, 80 mM glucose, 100 mM sacharose, pH 8.0). Bloodstream form trypanosomes were expanded in mice and rats and were purified from the blood by di-ethyl-amino-ethyl (DEAE) chromatography 33, followed by repeated centrifugation (20 min., 2000 g) and sediment washes with Phosphate Buffered Saline Glucose (PSG) (38 mM Na2HPO4.2H20, 2 mM NaHPO4, 80 mM glucose, 29 mM NaCl, pH 8.0). Trypanosome sediments were subsequently stored at -80°C.
Twenty μl of trypanosome sediment (approximately 2.107 cells) were resuspended in 200 μl of Phosphate Buffered Saline (PBS) (8.1 mM Na2HPO4.2H20, 1.4 mM NaHPO4, 140 mM NaCl, pH 7.4) and the trypanosome DNA was extracted using the commercially available QIAamp DNA mini kit (Westburg, Leusden, The Netherlands), resulting in pure DNA in 200 μl of TE buffer. The typical yield of DNA extracted from a 20 μl pellet was 150 ng/μl or 30 μg total DNA. The extracts obtained were diluted 200 times in water and divided into aliquots of 2 ml in microcentrifuge tubes for storage at -20°C.
For trypanosome dilution series, 180 μl of each heparinized blood sample were mixed with an equal volume of the Qiagen AS-1 storage buffer and subsequently extracted using the QIAamp DNA blood mini kit (Westburg, Leusden, The Netherlands) resulting in 200 μl of extracted DNA in Millipore water. Manipulation was performed according to the manufacturer's instructions.
PCR RoTat 1.2
Primers were derived from the RoTat 1.2 VSG sequence (AF317914), recently cloned and sequenced by Urakawa et al. 17. Using DNA sequence homology search programs to interrogate databases at TIGR (The Institute for Genomic Research) and GenBank, primer sequences were identified within the region (608–812 bp) lacking homology with any other known VSG sequence present in the databases. Primer sequences (in 5'-3' direction) and annealing temperatures were as follows: RoTat 1.2 Forward GCG GGG TGT TTA AAG CAA TA, Tann. 59°C and RoTat 1.2 Reverse ATT AGT GCT GCG TGT GTT CG, Tann. 59°C.
Twenty μl of extracted DNA were mixed with 30 μl of a PCR-mix containing: 1 U Taq DNA recombinant polymerase (Promega, UK), PCR buffer (Promega, UK), 2.5 mM MgCl2 (Promega, UK), 200 μM of each of the four dNTPs (Roche, Mannheim, Germany) and 0.8 μM of each primer (Gibco BRL, UK).
All amplifications were carried out in a Biometra® Trio-block thermocycler. Cycling conditions were as follows: denaturation for 4 min. at 94°C, followed by 40 amplification cycles of 1 min. denaturation at 94°C, 1 min. primer-template annealing at 59°C and 1 min. polymerization at 72°C. A final elongation step was carried out for 5 min. at 72°C.
Twenty μl of the PCR product and ten μl of a 100 bp size marker (MBI Fermentas, Germany) were subjected to electrophoresis in a 2 % agarose gel (25 min. at 100 V). Gels were stained with ethidium bromide (0.5 μg/ml) (Sigma, USA) and analyzed on an Imagemaster Video Detection System (Pharmacia, UK).
PCR Te-664
PCR on purified DNA samples was performed using primers and PCR conditions according to Ventura et al. 15. Only the Taq DNA polymerase was purchased from another distributor, i.e. Promega (UK) instead of Gibco BRL (UK).