Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections

Background Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.


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 susceptibil-ity 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 [7,8], 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 [12,13]. 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.

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.

PCR RoTat 1.2 : analytical sensitivity
A tenfold dilution series (10 5 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 PCR specificity results for the different Trypanosoma (T.) spe-cies and subspecies in this study

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 [18][19][20]. 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 [22,23]. 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. 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 Multiplexendonuclease 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.

Conclusions
PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.

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 (  [26][27][28][29][30].
Twenty µl of trypanosome sediment (approximately 2.10 7 cells) were resuspended in 200 µl of Phosphate Buffered Saline (PBS) (8.1 mM Na 2 HPO 4 .2H 2 0, 1.4 mM NaHPO 4, 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, T ann. 59°C and RoTat 1.2 Reverse ATT AGT GCT GCG TGT GTT CG, T ann. 59°C.
All amplifications were carried out in a Biometra ® Trioblock 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 elec-trophoresis 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).