Molecular identification of new Trypanosoma evansi type non-A/B isolates from buffaloes and cattle in Indonesia

Abstract Trypanosoma evansi is reportedly divided into two genotypes: types A and B. The type B is uncommon and reportedly limited to Africa: Kenya Sudan, and Ethiopia. In contrast, type A has been widely reported in Africa, South America, and Asia. However, Trypanosoma evansi type non-A/B has never been reported. Therefore, this study aims to determine the species and genotype of the Trypanozoon subgenus using a robust identification algorithm. Forty-three trypanosoma isolates from Indonesia were identified as Trypanosoma evansi using a molecular identification algorithm. Further identification showed that 39 isolates were type A and 4 isolates were possibly non-A/B types. The PML, AMN-SB1, and STENT3 isolates were likely non-A/B type Trypanosoma evansi isolated from buffalo, while the PDE isolates were isolated from cattle. Cladistic analysis revealed that Indonesian Trypanosoma evansi was divided into seven clusters based on the gRNA-kDNA minicircle gene. Clusters 6 and 7 are each divided into two sub-clusters. The areas with the highest genetic diversity are the provinces of Banten, Central Java (included Yogyakarta), and East Nusa Tenggara. The Central Java (including Yogyakarta) and East Nusa Tenggara provinces, each have four sub-clusters, while Banten has three.


Introduction
Species identification in the Trypanozoon subgenus based on morphology and molecular markers still causes disputes among researchers.It is difficult to morphologically identify the three Trypanozoon subgenus species because of their morphological similarities (Li et al., 2006;Sánchez et al., 2016;Wen et al., 2016;Gizaw et al., 2017).Their molecular identification is similarly challenging, with some commonly used primer pairs such as ITS1 and ITS2 or TBR known to detect pan-trypanosomes (WOAH, 2021).The three primer pairs can detect a broad range of species, such as Trypanosoma congolense, Trypanosoma simiae, Trypanosoma vivax, Trypanosoma theileri, Trypanozoon subgenus, and several other species (Salim et al., 2014;Isaac et al., 2016;Alanazi et al., 2018;Gaithuma et al., 2019;Marsela et al., 2020).Therefore, it is more suitable to screen based on their DNA sequence.
However, some primer pairs can be used to distinguish Trypanosoma brucei from T. evansi by targeting the kinetoplast DNA (kDNA) minicircle gene (Artama et al., 1992).T. evansi can also be distinguished from T. brucei and Trypanosoma equiperdum by targeting the kDNA maxicircle gene (Li et al., 2007).It has recently been reported to use several primer pairs successively for species identification (Subekti et al., 2023).Therefore, the appropriate algorithm design will greatly increase the accuracy of molecular identification of Trypanozoon species.Genetically, T. evansi has also been reported to be divided into two genotypes: types A and B (Birhanu et al., 2016;Boushaki et al., 2019;Li et al., 2020).Molecular identification for genotype classification relies on two primer pairs: ILO7957/8091 targeting the VSG RoTat 1.2 gene and EVAB targeting the kDNA minicircle type B (Njiru et al., 2006;Birhanu et al., 2016;Boushaki et al., 2019).
T. evansi type B is uncommon and reportedly limited to Africa: Kenya, Sudan, Chad, and Ethiopia (Birhanu et al., 2016;Boushaki et al., 2019).In contrast, T. evansi type A has been widely reported in Africa, South America, and Asia (Njiru et al., 2006;Birhanu et al., 2016).This study aims to identify T. evansi from Indonesian isolates with molecular identification algorithms while establishing genotypes and their genetic diversity.

Trypanosome and DNA extraction
Forty-three trypanosome isolates from several regions of Indonesia were grown in Deutschland, Denken, and Yoken (DDY) mice.When their parasitemia was high, the mice were euthanized, and blood was collected by heart puncture.Next, Trypanosoma-containing blood was purified using the Toyopearl 650M DEAE-methacrylate polymer (Tosoh Bioscience, Philadelphia, PA, USA; Subekti et al., 2023).DNA was extracted from pure trypanosomes using DNAzol (Molecular Research Center Inc., Cincinnati, OH, USA) according to the manufacturer's instructions.All extracted DNA was stored in the freezer (−20°C) until needed.

PCR primer and program
The primers used in the study and their amplification program are briefly described in Table 1.Polymerase chain reaction (PCR) was performed using a GTC96S, 96-well Thermal Cycler (Cleaver Scientific, Rugby, Warwickshire, UK).The 50 μL reaction mixture contained 1 µL (100 ng/µL) DNA, 1 µL (20 µM) of each primer (forward and reverse), 25 µL of MyTaq™ HS Red Mix 2x (Meridian Life Science Inc., Memphis, TN, USA), and 22 μL of nuclease-free water (Promega, Madison, WI, USA).The PCR product (amplicon) was electrophoresed in a 1.5% agarose gel with 1 st Base FloroSafe DNA stain (Axil Scientific Pte Ltd., Singapore) using the RunVIEW real-time gel visualization system (Cleaver Scientific) and visualized using a Clear View UV Transilluminator (Cleaver Scientific).

Sequencing and cladogram construction
The PCR products were sequenced at Bioneer Corp. (Daejeon, Republic of Korea).The obtained nucleotide sequences were assessed for similarity to other trypanosome isolates using the Basic Local Alignment Search Tool (BLAST) from the US National Center for Biotechnology Information (Altschul et al., 1997).The possible identity of the trypanosome isolates was determined based on all nucleotide sequences in the BLAST alignments with the highest percentage sequence similarity and query coverage for each identified species.
The cladogram was constructed using two approaches.The first used the nucleotide sequence of the gRNA-kDNA minicircle gene, while the other used the binary data derived from the PCR results with primer pairs ESAG6/7, gRNA-kDNA minicircle (MINI), RoTat 1.2, and EVAB.The cladogram based on the nucleotide sequence of the gRNA-kDNA minicircle was constructed using CLC Sequence Viewer 8.0 (Qiagen, Copenhagen, Denmark) with the Neighbor-joining method using Jukes-Cantor nucleotide distance measurement and bootstrap analysis with 1000 replicates.The cladogram was visualized using The Interactive Tree Of Life (https://itol.embl.de)(Letunic & Bork, 2021).The cladograms based on the binary data were constructed using hierarchical cluster analysis (HCA) with Minitab (Minitab LLC, State College, PA, USA) with the average linkage method (Stevens & Godfrey, 1992).

Molecular identification and genotyping
Forty-three trypanosome isolates were identified as T. evansi using the molecular identification algorithm.The alignment of the nucleotide sequences of PCR amplicons with the ESAG6/7 primer pair showed sequence similarity to three species in the Trypanozoon subgenus.Sequence similarity to T. evansi ranged from 89.90% to 98.11%, T. brucei ranged from 91.33% to 98.33%, and T. equiperdum ranged from 84.69% to 97.17% (Table 2).These results are consistent with several reports that concluded that the ESAG6/7 primer pair could identify the Trypanozoon subgenus but not the species (Holland et al., 2001;Isobe et al., 2003;WOAH, 2021).
In the second step, the MINI primers are used to further refine the species identification by eliminating one of the three possible species in the Trypanozoon subgenus.The MINI primer pair has been reported to amplify the gRNA-kDNA minicircle gene in T. evansi but not T. brucei (Artama et al., 1992).The nucleotide sequences of these PCR amplicons showed sequence similarities to T. evansi, ranging from 93.67% to 99.18%, and T. equiperdum, ranging from 90.33% to 99.09%; none showed sequence similarity to T. brucei (Table 2).This result provides additional information not mentioned by Artama et al. (1992), whose study did not include T. equiperdum.At the same time, PCR using the RoTat 1.2-a primer pair (Table 3) showed positive results for all isolates.These results are consistent with Claes et al. (2004), who explained that the RoTat 1.2 primer pair (RoTat 1.2-a in this study) amplified 100% (8/8) of T. evansi and 77.8% (7/9) T. equiperdum isolates but no T. brucei isolates.It can be concluded that the 43 trypanosome isolates were likely T. evansi or T. equiperdum and not T. brucei.
The third and final species identification step used the MAXI primer pair to PCR amplify the kDNA maxicircle gene.The MAXI primer pair has been reported to amplify only the kDNA maxicircle genes in T. equiperdum and T. brucei (Li et al., 2007;Suganuma et al., 2016).Since T. evansi has lost the maxicircle gene, it cannot be amplified by the MAXI primer pair.PCR using the MAXI primer pair was negative for all isolates, supporting the identification of T. evansi and excluding T. equiperdum (Figure 1 and Table 3).
The fourth step is an additional step to determine the T. evansi genotype, which will be identified as type A with a positive result with the ILO7957/8091 primer pair (RoTat 1.2-b in this study) and negative a result with the EVAB primer pair, while T. evansi type B shows the opposite results (Njiru et al., 2006;Birhanu et al., 2016;Boushaki et al., 2019).T. evansi types A and B are differentiated based on minicircle kDNA (Cuypers et al., 2017).The immunodominant RoTat 1.2 variable surface glycoprotein is primarily used to identify T. evansi type A, while EVAB primer is primarily used to identify T. evansi type B isolates based on present or absent of B minicircle kDNA (Birhanu et al., 2016;Cuypers et al., 2017;Boushaki et al., 2019).To date, T. evansi type B has only been reported in Eastern Africa, probably present but not detected in Western and Northern Africa (Cuypers et al., 2017).However, there have been reports that T. evansi type B has only been isolated from camels and found in a limited geographic area, especially Kenya, Ethiopia (both are Eastern Africa), and Sudan which is known to belong to parts of Northern Africa (Njiru et al., 2006;Njiru et al., 2011;Birhanu et al., 2016).In contrast, T. evansi type A has been frequently isolated from various hosts in Africa, South America, and Asia (Birhanu et al., 2016;Behour & Abd El Fattah, 2023).Previous research has shown that the KETRI 2472 isolate was misclassified, and it has been suggested that it should be reviewed.The KETRI 2472 isolate originates from camels in Sudan and is currently believed to be T. evansi type A (Njiru et al., 2006).However, since data from Njiru et al. (2006) showed that this isolate was negative for RoTat 1.2 and EVAB, so it probably deserved to be classified as T. evansi type non-A/B.T. evansi non-A/B (KETRI 3552 and 3557) has also been reported in Kamidi et al. (2017) but there have been several criticisms of the identification approach.The KETRI 3552 and 3557 were both classified as T. evansi non-A/B despite being PCR positive for RoTat 1.2.There are some criticisms, first, they do not prove whether PCR is positive or not for B minicircles which is the key to identifying T. evansi type B. Second, only relied on A-281-del as a genetic marker and did not consider RoTat 1.2 (using ILO7957/ILO8091 primer set) as the key to identifying T. evansi type A, lead doubts and confusion regarding identification and assignment the true status of KETRI 3552 and 3557.Carnes et al. (2015) reported that T. evansi with negative RoTat 1.2 is likely type B, C or something else.This evidence shows that A-281-del as the main key identification for type A is not appropriate, so KETRI 3552 and 3557 should be categorized as T. evansi type A. Third, Kamidi et al. (2017) doubted the RoTat 1.2 primer (ILO7957/ILO8091) because it could not detect all T. evansi.This is actually supporting evidence that RoTat 1.2 (ILO7957/ILO8091) is able to differentiate T. evansi type A and others (type B or something else).This also happened in our study where four out of 43 isolates showed negative with the primers ILO7957/ILO8091 (RoTat 1.2b in this study).This finding is similar with Ngaira et al. (2004) which only detected positive 72.22% of T. evansi tested using same primer sets.In contrast, the use of another RoTat 1.2 primer (RoTat 1.2a in this study) proved successful in detecting all T. evansi that had been tested as reported by Claes et al. (2004) and in this study.
This study identified four out of 43 (9.30%) T. evansi isolates as negative with both the EVAB and ILO7957/8091 primer pairs and could possibly be considered for classification as non-A and non-B types (non-A/B; Table 3).The T. evansi type non-A/B isolates (PML, AMN-SB1, and STENT3) were isolated from buffalo, while the PDE isolate was isolated from cattle.This is the first study to isolate T. evansi type non-A/B strains from bovines outside of Africa.The STENT1 isolate was classified as T. evansi type A because it showed a positive ILO7957/8091 result.Based on the report by Behour & Abd El Fattah (2023), which classified T. evansi type B based on a negative TeRoTat920/1070 result (RoTat 1.2-c in this study) and a positive EVAB result, STENT1 may also be considered T. evansi type non-A/B because TeRoTat920/1070 and EVAB are both negative.However, we consider the classification of STENT1 as non-A/B type to be inappropriate because the sensitivity of TeRoTat920/1070 is below that of ILO7957/8091.Salim et al. (2011) reported that the TeRoTat920/1070 primer pair could amplify the VSG RoTat 1.2 gene belonging to T. evansi in 63.3% (19/30) of isolates, while 36.7%(11/30) were negative.Overall, the difference in detection of three RoTat 1.2 primer sets from this study and other studies seems to require a more in-depth study regarding the identification of T. evansi type A.
A cladogram constructed based on the nucleotide sequence of the gRNA-kDNA minicircle shows that two T. evansi type non-A/B isolates from Indonesia (PML and AMN-SB1) are grouped into Cluster 3 with other T. evansi type A and B isolates and the KETRI 2472 isolate (Figure 2).The other Indonesian T. evansi type non-A/B (STENT3 and PDE) were grouped into Cluster 1 and 2 respectively (Figure 2).This approach was unsuccessful in classifying each T. evansi genotype separately.A suggested alternative approach for cladogram construction was to use HCA based on binary data derived from positive or negative observational data obtained from nucleic acid amplification using the primer pairs ESAG6/7, MINI, TeRoTat920/1070, ILO7957/8091, and EVAB.The cladogram constructed using HCA successfully grouped T. evansi type A, B, and non-A/B isolates into separate clusters (Figure 3).However, one weakness of this approach is that it cannot explore and classify genetic diversity in more detail based on the nucleotide or amino acid sequences of each isolate.
Geographically, the four T. evansi type non-A/B isolates originate from a province that historically did not have trade routes related to livestock movement (Figure 5), especially buffalo and cattle.The four T. evansi types non-A/B isolates were also isolated over a long period.Therefore, the most likely hypothesis was that they emerged independently in each region.While the T. evansi type non-A/B isolates in South Kalimantan and East Nusa Tenggara (ENT) provinces were isolated in adjacent years, these provinces do not have historical and current buffalo trade routes.
The areas with high genetic diversity are the provinces of East Nusa Tenggara, Central Java (including Yogyakarta), and Banten (Figure 5).Central Java (including Yogyakarta) provinces had four sub-clusters (6A, 6B, 7A, and 7B), while Banten province had three sub-clusters (6B, 7A, and 7B).Historically, livestock movement between Banten and Central Java provinces (vice versa), especially cattle and buffalo, has existed for a long time, it is possible that the isolates from the two provinces originated from same ancestor.
The T. evansi isolates isolated from buffaloes in the ENT province showed interesting patterns.They were all grouped into the same cluster (Cluster 1, Figure 2) when compared with T. evansi isolates from outside Indonesia or separated into four cluster when compared with isolates from Indonesia (Figure 4).All isolates from the ENT province were isolated in 2012 from buffaloes that survived the Surra outbreak in 2010-2012.The Surra outbreak in ENT Province in 2010-2012 killed more than 1700 horses and buffaloes (Subekti & Yuniarto, 2020).Based on   New Trypanosoma evansi type non-A/B from Indonesia their clustering (Figure 5), isolates from ENT appear to be related to isolates from East Java, Central Kalimantan and North Sumatra provinces.However, the difference in the year of origin of ENT isolates and isolates from East Java and North Sumatra provinces is greatly different, 2013 versus 1992.Unfortunately, data on the historical spread of trypanosomes at that time are unavailable, making it difficult to predict the association among isolates from those provinces.The genetic relationship between ENT isolates and isolates from Central Kalimantan and East Java provinces also cannot be confirmed conclusively, even though historically livestock movement from ENT to these two provinces has existed for a long time.Further studies are needed to reveal the distribution of T. evansi between islands by comparing data on animal movements between them in the same or adjacent years.

Conclusions
Forty-three trypanosoma isolates from Indonesia were identified as Trypanosoma evansi using a molecular identification algorithm.Further identification showed that 39 isolates were type A and 4 isolates were possibly non-A/B types.This study reports the first isolation of T. evansi which is suspected to be type non-A/B from bovines.Non-A/B type of T. evansi was found in isolates originating from the provinces of Aceh, Central Java, South Kalimantan and East Nusa Tenggara.This study is also the first to report high genetic diversity in the Banten, Central Java, and East Nusa Tenggara provinces based on the nucleotide sequences of the gRNA-kDNA minicircle.Further research is needed to uncover and more deeply exploration.

New
Trypanosoma evansi type non-A/B from Indonesia

Figure 2 .
Figure 2. Trypanozoon cladogram based on the nucleotide sequence of the gRNA-kDNA minicircle gene constructed with Neighborjoining method using Jukes-Cantor nucleotide distance measurement and bootstrap analysis with 1000 replicates.The asterisk is T. evansi type non-A/B.Tev = Trypanosoma evansi, Tev B = Trypanosoma evansi type B, Tbr = Trypanosoma brucei, and Teq = Trypanosoma equiperdum.

Figure 3 .
Figure 3. Trypanosoma evansi cladogram constructed using the average linkage method with squared Euclidean distance measurement.Binary data conversion of T. evansi type B isolates (MU014 and MU010) generated from Birhanu et al. (2016).

Figure 4 .
Figure 4. Cladogram of Indonesian Trypanosoma evansi based on the nucleotide sequence of the gRNA-kDNA minicircle gene constructed using Neighbor-joining method using Jukes-Cantor nucleotide distance measurement and bootstrap analysis with 1000 replicates.The asterisk indicates T. evansi type non-A/B in this study

Figure 5 .
Figure 5. Distribution map of the Trypanosoma evansi sub-cluster in Indonesia based on the genetic diversity of the minicircle gene (Figure 4).The red asterisk indicates the origin of T. evansi type non-A/B in this study.WNT province = West Nusa Tenggara province, ENT province = East Nusa Tenggara province.

Table 1 .
The nucleotide sequences and PCR programs of the PCR primers used in this study.

Table 2 .
Subekti et al. (2023)f Indonesian trypanosome isolates based on expression site-associated genes region 6 (ESAG6) and gRNA-kDNA minicircle genes.The results of the PCR test for Indonesian trypanosome isolates used different primers for identification and genotyping. is the highest query coverage and sequence similarity only; *isolate number 1-14 generated fromSubekti et al. (2023).