Genotyping of Trypanosoma brucei evansi in Egyptian camels: detection of a different non-RoTat 1.2 Trypanosoma brucei evansi in Egyptian camels

Trypanosoma brucei evansi (T. b. evansi) is an enzootic organism found in Egyptian camels, which genetically classified into types A and B. To detect the parasite genotype circulating in Egyptian camels, we collected 94 blood samples from three distant districts and subjected them to different PCR assays; T. brucei repeat (TBR), internal transcribed spacer-1 (ITS-1), and variable surface glycoproteins (VSG) (RoTat 1. 2, JN 2118Hu) and EVAB PCRs. The highest prevalence was obtained with TBR (80/91; 87.9%), followed by ITS-1 (52/91; 57.1%), VSG JN 2118Hu (42/91; 46.2%), and VSG RoTat 1. 2 (34/91; 37.4%). We reported a different non-RoTat 1. 2 T. b. evansi for the first time in Egyptian camels. Results showed that 47 (58.7%) out of 80 samples were classified as T. b. evansi. Of these, 14 (29.8%) were RoTat 1. 2 type, 13 (27.6%) were non-RoTat 1. 2 type, and 20 (42.6%) samples were from mixed infection with both types. All samples were tested negative with EVAB PCR. RoTat 1. 2 T. b. evansi was the most prevalent in Giza and El Nubariyah, whereas, in Aswan, the only type detected was non-RoTat 1. 2 T. b. evansi. The nucleotide sequences of the VSG RoTat 1.2 and JN 2118Hu PCR products were submitted to DNA Data Bank of Japan (DDBJ) and GenBank under the accession numbers LC738852, and (OP800400-OP800403). Further research is required to increase the sample size and verify the new sequences to corroborate the prevalence of a new variant of non-RoTat 1.2 T. b. evansi in Egypt.


Introduction
Trypanosoma brucei evansi, belonging to the family Trypanosomatidae, is a unicellular protozoan parasite distributed in the blood of wild and domestic animals but rarely in humans (Joshi et al. 2005;Desquesnes et al. 2013). Previously, T. evansi and T. equiperdum were classified as separate species but recent evolutionary studies proposed that T. evansi and T. equiperdum originate from genetically diverse T. brucei strains and hence, are T. brucei subspecies (Lai et al. 2008;Carnes et al. 2015). It causes a wasting disease called surra that imposes significant economic losses due to reduced fertility, productivity, and mortality of untreated animals (Desquesnes et al. 2013).
Trypanosomes can be distinguished by the presence of kinetoplasts containing DNA (kDNA) that matches their mitochondrial DNA. The T. brucei kDNA is characterized by 20 to 50 maxicircles (approximately 23 kb) together with thousands of extremely varied minicircles (approximately 1 kb). Due to a partial loss of kDNA, T. b. equiperdum and T. b. evansi are termed dyskinetoplastic. T. b. evansi exhibits homogeneous minicircles. Although T. b. equiperdum retains its maxicircles, occasionally with significant deletions, it has lost the diversity of its minicircles (Claes et al. 2005;Lai et al. 2008;Schnaufer 2010).
According to the restriction digestion pattern of the minicircles, T. b. evansi is distinguished into types A and B (Njiru et al. 2006), with the former being more prevalent.
Distributed in Africa, Asia, and South America, it is defined by the gene for the variable surface glycoprotein (VSG) RoTat 1.2. Its antibodies can be detected in T. b. evansi type A-infected animals as VSG is expressed during the early infection stages . T. b. evansi type B is relatively less frequent and was initially detected in Africa in camels from Kenya (Borst et al. 1987;Ngaira et al. 2005). Lately, it has been reported in Ethiopia (Birhanu et al. 2016) then, more recent work by Oldrieve et al. (2021) using genomic data from various Trypanozoon strains, reclassifies the IVM-t1 strain from Mongolia as the first Asian T. brucei evansi type B strain.
As the RoTat 1.2 gene sequence is missing in T. b. evansi type B, serological and molecular assays targeting RoTat 1.2 VSG, including the CATT/ RoTat 1.2 and RoTat 1.2 PCR cannot detect type B infections (Claes et al. 2004). So far, three molecular assays have been established for identifying T. b. evansi type B: (1) EVAB PCR: based on a minicircle DNA sequence specific to type B, (2) a PCR, and (3) a LAMP, both based on VSG JN 2118Hu type B-specific sequence (Ngaira et al. 2005;Njiru et al. 2010). In Egypt, T. b. evansi is endemic in camels and horses, while water buffaloes are considered reservoirs (Elhaig and Sallam 2018). Previous molecular studies reported that RoTat1.2 VSG PCR could not detect T. b. evansi in some infected animals from different species (Elhaig and Sallam 2018;Behour et al. 2019). Therefore, in this study, our objective was to detect and identify the trypanosome type, specifically for T. b. evansi, found in Egyptian camels, using type-specific molecular tools.

Ethical statement
The animal manipulation and sample collection methods used in this study were approved by The Ethical Research Committee of the Animal Reproduction Research Institute (ARRI), Agricultural Research Center (Code No.1 14 1 4 2 9).

Samples collection
We collected 94 blood samples from adult apparent healthy camels in the districts of Aswan (26 samples from quarantined animals), Giza (43 samples from slaughterhouses), and El Nubariyah (20 samples from small holders) as shown in Fig. 1. The blood samples (3 ml/camel) were collected in EDTA by jugular vein puncture. Then, the samples were transferred on ice to the laboratory for molecular analysis.

DNA extraction
Genomic DNA was extracted from the whole blood samples using GeneDirex genomic DNA Isolation Kit (Gen-eDirex, Inc., Taiwan) following the manufacturer's instructions. DNA quantity and purity were measured using a spectrophotometer (BioPhotometer plus, Eppendorf, Hamburg, Germany) and stored at − 20 °C until further use. The positive control DNA of T. b. evansi RoTat1.2 was previously isolated from naturally infected camels and was stored at − 20 °C (Behour et al. 2019). Also DNA of DEAE purified T. b. evansi type B was kindly provided by Institute of Tropical Medicine, Antwerp, Belgium.

PCR assays
The DNA isolated from each sample was subjected to different PCR assays for detecting and typing the trypanosomes. Table 1 indicates each PCR assay's target, primers sequence, amplicon size, annealing temperature, and reference. T. brucei repeat (TBR) PCR assay is the gold standard for detecting Trypanozoon in addition to Internal Fig. 1 Map of Egypt shows the locations from which camels blood samples were collected. Samples were collected from small holders in El Nubariyah (20 samples), slaughterhouses in Giza (43), quarantined animals in Aswan (26 samples). The generated map was done using ARcGris online from Esri, Map data © OpenStreetMap contributors, Microsoft, Facebook, and its affilates, Esri community Map contributors, map layer by Esri transcribed spacer-1 (ITS-1) PCR. VSG RoTat 1.2 and JN 2118Hu PCRs ( for RoTat 1.2 and non-RoTat 1.2 T. b. evansi, respectively) were applied to detect T. b. evansi VSG genes, and EVAB PCR targeting class B minicilcles for T. b. evansi type B. Moreover, all the VSG JN 2118Hu-positive samples were tested with maxicircle gene NADH-dehydrogenase subunit (ND5) PCR to exclude other T. brucei spp. The success of extraction method was tested with DRB-Exon2 PCR for camel DNA on the TBRnegative samples. The same, a single copy gene namely a phospholipase C (GPI-PLC) PCR was performed for samples that are RoTat1.2 negative to demonstrate that enough parasite material was available for amplification. For each assay, the amplification mixture (25 µl) consisted of Dream Taq TM Green PCR Master Mix (2x) (Thermo Scientific, Lithuania), 25 pmol of each assay-specific primer, and 100 ng of DNA template. Positive and negative controls were also included in each PCR assay. Amplification was performed using the SimpliAmp thermocycler (Applied Biosystems, Thermo Fisher Scientific, Singapore) using the following conditions: initial denaturation for 5 min at 95 °C, followed by 35 cycles of denaturation at 94 °C for 45 s, the specified annealing for 45 s, extension at 72 °C for 60 s, and finally, an extension cycle at 72 °C for 10 min. Each amplicon (7 µl) was detected using electrophoresis on 1.5% agarose gel and then visualized under ultraviolet light after staining with ethidium bromide.

Sequencing of PCR products
The PCR amplicons of T. b. evansi RoTat 1.2 (2 samples) and non-Rotat 1.2 (4 samples) were purified using Gene JET Gel Extraction Kit (Thermo Scientific K0691, Germany) according to the manufacturer's instructions. We performed forward and reverse sequencing in an automated DNA sequencer (ABI 3730XL, Applied Biosystems, USA). The results were compared with available NCBI GenBank sequences and submitted to DNA Data Bank of Japan (DDJB) and Gen-Bank. The sequences were aligned using Clustal W with the BioEdit program v7.2.5 (Hall 1999). The phylogenetic tree was constructed using the Maximum Likelihood method and Jukes-Cantor model (Jukes and Cantor 1969) with 1000 bootstrap replicates conducted using Mega 11 (Tamura et al. 2021).

Results
We performed different PCR assays to detect T. b. evansi in the sample DNA. Using TBR PCR, a specific 164-bp band was obtained in 80 out of 94 samples. Of the 14 negative samples, 3 were tested negative for camel DNA with The detected trypanosomes were typed by comparing the results of the four PCR assays (Table 3)       respectively, from India. This sequence was not previously recorded from camel origin in the GenBank database. Our isolate was significantly related to the Indian and Indonesian isolates according to the phylogenetic tree (Fig. 7), which showed a close relationship among T. b. evansi targeting RoTat 1.2 VSG. The sequencing results of the VSG JN 2118Hu PCR products for non-RoTat 1.2 T. b. evansi were analyzed and submitted to GenBank as Trypanosoma evansi isolate Egy_ARRI1, 2, 3, 4 sequences under the accession numbers: OP800400; OP800401; OP800402; OP800402; OP800403. The nBLAST results for the VSG JN 2118Hu PCR sequences indicated a lack of similarity with all sequences obtained from GenBank (except primers sequence region). However, as the four sequence isolates were identical (Fig. 8), they were submitted as unverified sequences. The NEBcutter computer program exposed three restriction sites of Hae III enzyme in the sequence of non-RoTat1.2 PCR fragment. The restricted fragments were 149 bp (right end), 67 bp (left end), 31 bp, and 21 bp. Fig. 7 The original phylogenetic tree constructed using 14 partial cd sequences of T. b. evansi RoTat 1.2 and one mRNA complete cd sequence using the Maximum Likelihood method and Jukes-Cantor model (Jukes and Cantor 1969) with 1000 bootstrap replicates conducted in Mega 11 (Tamura et al. 2021). The T. b. evansi RoTat 1.2 type sequences with accession numbers, isolate names, host names, and country where it was isolated, are listed. LC738852 is the accession number of the sequence for our isolate Trypanosoma evansi ARRI VSG gene for variable surface glycoprotein, partial cds indicated by the red square Fig. 8 Multiple sequence alignment of the sequences of the four sample isolates (OP800400, OP800401, OP800402, OP800403) amplified by VSG JN 2118Hu PCR assay. The sequences were aligned using Clustal W conducted in BioEdit program v7.2.5 (Hall 1999), which indicated that the isolates were identical and sequences were repetitive Tropical Animal Health and Production (2023) 55:279

Discussion
Recently, molecular diagnostic tests have been used instead of parasitological methods for detecting T. b. evansi to expedite diagnosis, lower cost, and improve our epidemiological understanding (Birhanu et al. 2015). TBR PCR was carried out using TBR primers to amplify a 164 bp highly repeated minichromosome satellite DNA sequence (Masiga et al. 1992). This test efficiently detected 80 positive samples out of 94 tested samples (87.9%). The high sensitivity of this PCR has been previously reported (Fernandez et al. 2009;Elhaig and Sallam 2018;Behour et al. 2019). The TBR primers were the first to be described and are primarily used for detecting Trypanozoon DNA. They are regarded as the "gold standard" for identifying T. b. evansi to avoid the issues related to the failure of the primers targeting kDNA. This can be attributed to the fact that TBR primers target highly repetitive satellite sequences (approximately 10,000-20,000), which results in a highly sensitive PCR test that is independent from the presence of parasite kDNA (Ventura et al. 2000;Fernandez et al. 2009). The ITS-1 PCR assay targets ITS-1, positioned between 18S and 5.8S ribosomal DNA sequences. Its size is species-specific, which enables the detection and identification of polyspecific infections (Desquesnes et al. 2001). The ITS-1 internal primers successfully amplified a 90-bp Trypanozoon DNA fragment (Taylor et al. 2008), which helped identify 52/91 (57.1%) positive samples. Since the primers were designed for detecting T. b evansi (Taylor et al. 2008) and as the genome of T. b. evansi contains lower copies (~ 100-200) of the ITS regions (Fernandez et al. 2009) compared with TBR, the sensitivity of this assay was lower than TBR PCR.
In the RoTat 1.2 PCR assay, a primer pair for T. b. evansi type A, targeting the T. b. evansi RoTat 1.2 VSG gene, was used to amplify a 151-bp DNA fragment (Konnai et al. 2009). Although this specific DNA region lacks homology with other known VSG genes in trypanosomes, it is highly conserved among the T. b. evansi strains (Claes et al. 2004). We confirmed that 34/91 (37.1%) were T. b. evansi RoTat 1.2-positive using RoTat 1.2 PCR. Although most strains of T. b. evansi type A carry RoTat 1.2 VSG gene (Urakawa et al. 2001;Ngaira et al. 2005), some strains of T. b. evansi type A and T. b. evansi type B lack it (Ngaira et al. 2004(Ngaira et al. , 2005Kamidi et al. 2017). Furthermore, T. b. evansi strains can completely lose their kinetoplast, which would produce a false-negative result in a diagnostic PCR assay targeting type A minicircles (Lai et al. 2008;Carnes et al. 2015). Therefore, a VSG JN 2118Hu primer set was used in the JN 2118Hu PCR assay for identifying non-RoTat 1.2 T. b. evansi, which amplified a 273 bp DNA segment within T. b. evansi JN 2118Hu coding region that lacked similarity with any other known trypanosome sequence (Ngaira et al. 2005). Notably, 42/91 (46.2%) samples were positive in VSG JN 2118Hu PCR. However, Ngaira et al. (2005) suggested that the VSG JN 2118Hu, which was initially identified in a T. b. evansi strain from Kenya, is a distinctive marker for non-RoTat 1.2 T. b. evansi, Birhanu et al. (2016) reported that the JN 2118Hu VSG PCR was less specific since T. b. brucei and T. b. gambiense yielded positive amplification in this PCR. Hence, we further verified the absence of maxicircle DNA in JN 2118Hu VSG PCR-positive samples with the ND5 PCR (Dean et al. 2013). We have included the detection of a single copy control gene (GPI-PLC PCR) to corroborate that adequate parasite genomic material would have been present within the reaction to give a positive result. This means that the amplification of GPI-PLC demonstrates T. b. evansi when enough parasitic material is present within the sample to effectively amplify the VSG gene if present. A negative PCR result may signify the absence of VSG gene or simply mean that insufficient genomic material is present (Picozzi et al. 2008). Both RoTat 1.2 VSG PCR and JN 2118Hu VSG PCR failed to detect two samples that showed positive amplification by GPI-PLC PCR although Ngaira et al. (2005) recorded 100% sensitivity of the JN 2118Hu VSG PCR. The JN 2118Hu VSG PCR succeeded to detect non-RoTat 1.2 T. b. evansi for the first time in Egyptian camels. Previous studies reported that RoTat VSG PCR could not detect T. b. evansi in some infected animals from different species (Elhaig and Sallam 2018;Behour et al. 2019). This could support presence of non-RoTat 1.2 T. b. evansit in Egypt since it has been detected in some African countries, including Sudan, Ethiopia, Chad, and Kenya (Ngaira et al. 2005;Hagos et al. 2009;Salim et al. 2011;Birhanu et al. 2015;Sánchez et al. 2015). Notably, all tested samples were negative with EVAB PCR that indicates absence of T. b. evansi type B minicircles. An earlier study in Egypt reported that T. b. evansi prevalence in camels usingTBR and RoTat 1.2 PCR were 54.5% and 21.8%, respectively (Elhaig and Sallam 2018). A lower prevalence of Trypanosoma spp in camels was reported in Oman, which was 78/95, 77%; 30/95, 31.6%; 8/95, and 8.4% using TBR, ITS, and RoTat 1.2 PCRs, respectively, with no amplification obtained by EVAB PCR (Al- Kharusi et al. 2022). Moreover, 39.5% and 36.6% molecular prevalence were obtained using ITS-1 PCR in Saudi Arabia and Sudan, respectively (Salim et al. 2011;Metwally et al. 2021). In Algeria, 11.2% molecular prevalence was reported with RoTat 1.2 PCR (Boushaki et al. 2019).
The outcomes of the different PCR assays performed for each sample (Table 3) revealed that only 47 out of 80 positive samples could be classified as T. b. evansi. These samples were successfully amplified using TBR, ITS-1, and RoTat 1.2 or JN 2118Hu VSG PCR. The remaining 33 samples could be classified as Trypanozoon. Nineteen out of the 33 samples were only TBR PCR positive which may be due to the high sensitivity of the TBR primers. Five samples were TBR and ITS-1 PCR positive and neither gave positive amplification by RoTat1.2 PCR nor JN 2118Hu PCR. Also, 9 samples were only TBR and JN 2118Hu PCR positive. This might be due to the variations in the sensitivity of the PCR primers. Notably, all RoTat 1.2 PCR-positive samples were also ITS-1 PCR-positive. The nine samples that tested positive with JN 2118Hu VSG PCR were ITS-1 negative. This indicates the consistency between ITS-1 and RoTat 1.2 PCR assays for detecting T. b. evansi. Regarding typing of T. b. evansi in Egypt (Table 4), RoTat 1.2 T. b. evansi is more prevalent than non-RoTat 1.2 T. b. evansi in El Nubariyah and Giza districts. However, only non-RoTat 1.2 T. b. evansi was detected in Aswan district, which might indicate that the origin of the new non-RoTat 1.2 T. b. evansi, is the quarantined animals coming from Sudan. T. b. evansi type A was most prevalent in Ethiopia, with 11.7% molecular prevalence. However, only 0.53% of it was T. b. evansi type B (Birhanu et al. 2015). Recent studies in Oman and Algeria did not find any evidence for T. b. evansi type B (Boushaki et al. 2019;Al-Kharusi et al. 2022).
The phylogenetic tree (Fig. 7) indicated that the T. b. evansi sequence isolates targeting RoTat 1.2 VSG were closely related, representing that this genetic sequence is highly conserved among these isolates. Our isolate is significantly related to the Indian and Indonesian isolates and is a unique sequence isolate of this region from camels. The alignment of VSG JN 2118Hu PCR sequences showed that these isolates were identical and indicated sequence repetition. Although no significant similarity was found between the obtained sequence and that was published by Ngaira et al. (2005), our sequence showed 3 nucleotide deletion compared with the sequence size of Ngaira et al. (2005). The restriction fragments yielded by Hae III enzyme (using NEBcutter program) exposed that one of the restriction fragments (149 bp) has the same size with that obtained by Ngaira et al. (2005) yet different sequence. Absence of similarity of VSG JN 2118Hu PCR sequence with non-Rotat1.2 sequence obtained by Ngaira et al. (2005) and sequence data on GenBank hypothesizes detection of new structure of non RoTat1.2 T. b. evansi. This is expected, since T. b. evansi is considered as petite mutant of T. brucei (Lai et al. 2008). It is clear now that type A T. b. evansi isolates but RoTat1.2 negative are more common than anticipated, which could lead to a high frequency of falsenegative results by current techniques depend on RoTat1.2 VSG. Therefore, combination of RoTat1.2 VSG based PCR and alternatives like VSG JN 2118Hu PCR and A281del PCR could detect the majority of T. b. evansi (Urakawa et al, 2001;Ngaira et al. 2004Ngaira et al. , 2005Kamidi et al. 2017). Noteworthy, Ngaira et al. (2005) raised a question about considering the non-RoTat1.2 T. b. evansi belongs to type B group according to the speculation of Claes et al. (2004). In conclusion, the TBR PCR assay is the most sensitive among the four PCR assays used to detect T. b. evansi in camels. Regarding the typing of T. b. evansi, RoTat 1.2 T. b. evansi is more prevalent in Egypt, while a new non-RoTat 1.2 T. b. evansi was detected for the first time. T. b. evansi RoTat 1.2 ARRI isolate is significantly closer to the Indian and Indonesian isolates and is considered a unique isolate from the camels. However, we could not find any similarity between the VSG JN 2118Hu PCR sequences with the GenBank sequences, which is a limitation of this study. The new genetic structure could be a variant of of non-RoTat 1.2 T. b. evansi so further research on lager sample size is required to verify the new sequences to corroborate the prevalence of non-RoTat1.2 T. b. evansi in Egypt.
Acknowledgements we are grateful to Dr Birhanu hadush Abera (College of Vetrinary Sciences, Mekelle University, Ethiopia) and Dr Nick Van Reet (Institute of Tropical Medicine, Department of Biomedical Sciences, Antwerp, Belgium ) for helping us to obtain T. b. evansi type B DNA as a precious gift from Institute of Tropical Medicine, Antwerp, Belgium.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Data availability All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.