Equine Trypanosomiasis: Molecular Detection, Hematological, and Oxidative Stress Profiling

Surra caused by Trypanosoma evansi (T. evansi) is widely distributed and has significant impact on equine sector and international trades. However, there are no available data about the genetic characterization of this parasite in horses in Egypt. So, the goal of this study was to study the molecular characterization of T. evansi in horses and determine the changes in hematological parameters and oxidative stress associated with T. evansi infection. A total of 12 horses were examined using PCR targeting RoTat 1.2 VSG gene, and we evaluated the changes in hematological and oxidative stress between infected and healthy animals. The results revealed a notable reduction in red blood cell (RBC), hematocrit (HCT), and hemoglobin (Hb) levels in the infected horses, as compared to the control healthy group. In contrast, the infected group showed a substantial increase in mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV). In addition, the infected group exhibited monocytopenia, eosinopenia, and notable lymphocytopenia. Regarding oxidative stress profile, the infected horses showed decreased levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and antioxidant capacity (TAC) compared to the control group. Moreover, the PCR assay targeting RoTat 1.2 VSG gene revealed positive specific band (205 bp) in all examined samples for T. evansi and the phylogenetic analysis demonstrated that strain of this study is closely related to T. evansi isolate in horses from India (MT501210) while showed difference from sequences of other species. The results emphasize the changes in blood composition and the body's response to oxidative stress caused by T. evansi infection in horses.


Introduction
Many diseases of major importance in equine go undiagnosed and unreported [1][2][3][4][5].Trypanosomiases, caused by unicellular fagellate protozoa from the genus Trypanosoma, are among these diseases.T. evansi and T. vivax, often found in Africa, the Middle East, Asia, and Latin America, are the most clinically signifcant species that can infect horses [6].T. evansi is mostly transmitted mechanically by hematophagous insects from the Tabanidae and Stomoxidae families; however, ticks have also been transmitting the disease [7].
Clinical signs can vary depending on the animal's immune system and disease stage.Tey can include progressive weight loss despite an insatiable appetite, intermittent fever, weakness, anemia, pale mucous membranes, limb and ventral edema, urticaria plaques, keratitis and conjunctivitis, and paralysis of hindquarters and lips.If animal left untreated, these symptoms can lead to death [8,9].Moreover, the infection with T. evansi is commonly associated with reductions in hemoglobin (Hb), packed cell volume (PCV), and red blood cell count (RBC) [10,11].
T. evansi has been reported in many geographical locations in Egypt, afecting bufalo, cattle, donkeys, camels, sheep, and goats [12,13].Unfortunately, there is a lack of data regarding the occurrence of T. evansi in horses in Egypt [14].
Trypanosome infection produces large amounts of reactive oxygen species (ROS) and free radicals, which function as cytotoxic agent [15].Accumulation of ROS within cells has the ability to damage biomolecules, with lipids being particularly vulnerable unless regulated by an efcient antioxidant scavenging mechanism [16].
Antioxidant enzymes have a variety of defence mechanisms that successfully regulate reactive oxygen species (ROS) levels to ensure they remain in sufcient quantities.Two antioxidant enzymes, SOD and CAT, work along with nonenzymatic antioxidants such as GSH to remove free radicals and reduce oxidative damage to cells.Even minor diferences in the amounts of these antioxidant enzymes can have a major impact on the ability of cellular lipids, proteins, and DNA to resist oxidative damage [17].In experimental horses infected with T. evansi, blood levels of catalase, superoxide dismutase, and glutathione showed signifcant decreases [18].
Samples of blood, lymph, milk, cerebrospinal fuid, and preputial or vaginal discharges are used for detection of T. evansi [7].Numerous tests can be employed, such as the inoculation of laboratory animals as mice, blood smears, molecular detection using polymerase chain reaction (PCR), and serological analysis like the complement fxation test, indirect immunofuorescence antibody test (IFAT), enzymelinked immunosorbent assay (ELISA), and card agglutination test for trypanosomiasis (CATT) [19].
Molecular approaches are increasingly becoming more important as the preferred technique to detect pathogens [20].DNA-based technologies, such as polymerase chain reaction (PCR), have been widely employed for diagnosing trypanosomiasis infection in a variety of animal species, including camels, horses, cattle, and pets.Tis approach is extremely sensitive and specifc, capable of detecting all phases of parasite infection [21].Several target sequences, including internal transcribed spacer region (ITS), ribosomal DNA, VSG genes, and kinetoplast DNA, have been identifed as dependable targets for detecting T. evansi [21].
Trypanosomes have the unique capacity to switch the class of Variant Surface Glycoproteins (VSGs), which allows the parasite to survive by dodging the host's immune response.Among these, RoTat 1.2 VSG emerges as the primary variable antigen type (VAT), expressed during the early, middle, and late stages of T. evansi infection [22].Consequently, RoTat 1.2 VSG serves as a validated molecule for both serological [23] and molecular detection [24] of T. evansi infection.
Tis study aimed to evaluate the alteration in hematobiochemical parameters and oxidative stress profle associated with T. evansi infection.In addition, genetic identifcation and phylogenetic analysis were performed for T. evansi isolate from horses based on RoTat 1.2 VSG gene.

Ethical Statement.
Te study design received approval from the Research Ethical Committee of Faculty of Veterinary Medicine, Benha University, Egypt (ethical number: BUFVTM39-09-23).All procedures of the study were carried out in accordance with regulation of ethical committee of Faculty of Veterinary Medicine, Benha University.

Animals and Sampling.
A total of 12 horses were subjected for examination, and seven of them (infected group) showed clinical signs of trypanosomiasis like emaciation, pale mucous membrane, and difculty in walking as shown in Figure 1, while the rest of the fve horses were apparently healthy.Blood samples (5 mL) were collected aseptically from jugular vein using vacutainer tubes have EDTA which used for hematological and molecular examination.In addition, another samples was collected in clean tube without anticoagulant to separate the serum for biochemical analysis.

Oxidative Stress Evaluation.
Te levels of MAD, SOD, CAT, TAC, and GSH were measured in serum samples using commercial kits (Biodiagnostic, Giza, Egypt) according to the guidelines of the manufacturer.

DNA Extraction and PCR Assay.
Te DNA was extracted from the prepared blood sample according to the manufacturer's instructions using the QIAamp DNA Kit (Qiagen, Hilden, Germany).Te extracted DNA was kept at −20 °until use.Amplifcation of RoTat 1.2 VSG gene for detection of T. evansi was performed using the specifc pair of primers RoTat-F:5′-GCGGGGTGTTTAAAGCAATA-3′ and RoTat-R:5-ATTAGTGCTGCGTGTGTTCG-3′ for detection of 205 bp product size [25].Te PCR assay was performed in 25 μl volumes containing 5 µl DNA template, 1 μl of each primer (20 pmol/μl), 12.5 μl of emerald Amp GT PCR Master mix (2xprimer), and 5.5 μl PCR grade water under the following condition: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 52 °C for 30 sec, and extension at 72 °C for 40 sec.After the last the cycle, the mixture was incubated at 72 °C for 10 min.Te positive control for T. evansi was provided from Animal health Research Center, Giza, Cairo.Te amplifcation products were analyzed by electrophoresis on 1.5% agarose gel.

Sequencing and Phylogenetic Analysis.
Te purifed PCR products of a highly concentrated DNA samples was used for sequencing using the sample primers of PCR assay.Te automated DNA sequencer Applied Biosystems 3130 was used to sequence the DNA (ABI, 3130, USA), employing the BigDye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA).Tis was followed by an initial BLAST ® analysis to determine sequence identity with GenBank database [26].

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Veterinary Medicine International Te sequences were assembled and edited using the BioEdit program before being entered into GenBank with an accession number (LC789026).Te partial RoTat 1.2 VSG gene sequences were aligned with previously reported gene sequences in the GenBank database (https://www.clustalw.genome.jp)using CLUSTAL W [27]. Te neighbor-joining tree approach was performed with 500 bootstrap replicates to generate a phylogenetic tree based on the Kimura 2parameter model for nucleotide sequences using MEGA7 [28].

Hematological Parameter Findings.
In the infected horses, there was a notable decrease in the mean values of RBCs and HCT, whereas Hb exhibited a statistically signifcant reduction (p � 0.021) compared to the healthy group.Conversely, a signifcant increase (p < 0.05) in the mean values of MCHC, MCH, and MCV was observed in the infected animals relative to the control healthy group.Additionally, the infected animals exhibited monocytopenia, eosinopenia, and signifcant lymphocytopenia (p � 0.022) when compared to the control healthy group as shown in Table 1.

Oxidative Stress Parameters.
In the infected horses, a reduction in the levels of GSH, SOD, CAT, and TAC was observed compared to the control group.Conversely, there was a signifcant (p < 0.0001) increase in the level of MDA in the infected animals relative to the control healthy group as shown in Table 2.

PCR Amplifcation and Phylogenetic Analysis.
Te considered positive samples for T. evansi showed single specifc band of 205 bp size upon agarose gel electrophoresis, Figure 2. Te T. evansi sequence of this study was deposited in the NCBI database, receiving the accession number LC789026.Te phylogenetic analysis revealed that the sequence of T. evansi of this study was seen to be located in the same clade with the T. evansi isolate from horses from India (MT501210) while it is genetically distinct from other T. evansi sequences of other species as MK867833 from camels from Kenya, OL310520 from dogs from India, KF726106 from cattle from Egypt, and other strains from horses from other countries like India and Israel as shown in Figure 3.

Discussion
Te hematological parameter fndings in the infected animals revealed signifcant alterations in various blood components compared to the control group.Tere was a reduction in RBC, HCT, and Hb levels in infected horses, which indicated the presence of anemia as previously reported in trypanosomiasis-positive equines [10] and camels [29,30].Anemia is thought to be a signifcant and crucial sign of an animal's trypanosomiasis infection.
Te microtubule-reinforced bodies and forceful fagella lashing activity of the million-strong organisms induce mechanical injury to erythrocytes, which in turn causes the complex pathophysiology of anemia in trypanosomiasis [31].MCV, MCH, and MCHC are red cell indices that are used to identify the type of anemia [32].
In our study, the infected horses exhibited macrocytic anemia characterized by elevated MCHC, MCH, and MCV values, and this fnding was in contrast to those reported by Kagira et al. [33] who observed a sharp decrease in MCV and a little reduction in MCH and MCHC which were the hallmarks of the early stages of microcytic hypochromic type anemia.Tese diferences in the results may be attributed to stage of disease progression and underlying pathophysiological mechanisms [34].
Also, the elevated MCV may be the only indicator of conditions like vitamin B12 or folate defciency and anemia is caused by parasitic infections, which interfere with absorbent surfaces, physically obstruct the intestinal lumen, produce proteolytic substances, and consume nutrients intended for the body [35].Tis may be the underlying reason of our observations, as the increasing  anemia exhibited in this case is known as anemia of chronic disease or infammation and is typically found in susceptible animals [36].It is characterized by heightened erythrophagocytosis and poor erythropoiesis due to altered iron hemostasis and persistent secretion of proinfammatory cytokines such as IFN-c, TNF, IL-1, and IL-6 [37] and that could alter red cell indices, leading to increased MCV, MCH, and MCHC values.Te present fndings showed decrease in eosinophils, and these corroborated with a previous study that demonstrated a reduction in horses infected by T. evansi [34].Tis fnding might be explained by the fact that helminth migration through host tissues plays a crucial part in inducing increases in tissue eosinophilic infammation and blood eosinophils, and helminths that stay inside the intestinal lumen may not cause an eosinophil response [38].
Interestingly, this study revealed a signifcant decrease in lymphocytes in infected horses as previously reported by Pal et al. [34]; this could be related to several infectious pathogens that may produce a drop in lymphocyte numbers due to infammation.Tese reductions are probably caused by enhanced lymphocyte migration to lymphoid tissues, increased margination and emigration of lymphocytes to the site of infammation, and decreased lymphocyte efux out of lymphoid tissues [39].
One of the main processes in the pathophysiology of trypanosomiasis is oxidative stress [40]; this is due to the host's decreased RBC capacity for antioxidants [41].Reductions in the levels of GSH, SOD, CAT, and TAC in the infected animals demonstrate a decrease in antioxidant defence mechanisms, while a signifcant elevation in MDA levels in the infected animals indicates enhanced lipid peroxidation and oxidative damage.Tese fndings are consistent with earlier research by Ranjithkumar et al. [11] and Saleh et al. [42].Tis might be explained by the fact that the T. evansi infection releases sialidase and phospholipase, which cause damage to the erythrocyte membrane and the development of insulted red blood cells (RBCs).Tese RBCs then produce reactive oxygen species (ROS), which increase lipid peroxidation in RBCs and cause oxidative stress [43].Tese fndings suggest that trypanosomosis leads to severe protein oxidation, lipid peroxidation, and reduction in the antioxidant enzyme activity [15].
Te RoTat 1.2 gene is expressed by most T. evansi strains.Te RoTat 1.2-based PCR assay is highly sensitive and specifc, making it efective for diagnosing trypanosomosis in diferent animal species [44,45].Tis understanding is essential for refning diagnostic procedures, developing effective control strategies, and treating T. evansi infections.
In the present study, the PCR assay targeting RoTat 1.2 VSG gene was used for detection of T. evansi in horses because the primers targeting this gene are more sensitive and specifc for pathogen and help in early detection, identifcation of positive host, and consequently early effective treatment [46,47].Tese results come in accordance with fndings of previous studies used the similar part of the RoTat 1.2 VSG gene in PCR assays to detect T. evansi [48].
Moreover, this T. evansi isolate clustered within the same clade with previously T. evansi isolate from India (MT501210), while exhibiting divergence from other sequences, including those from Egypt (MG674185), India (LC008133), and Israel (HM209055).Another noteworthy fnding in this study distinct relationship between T. evansi isolate and other T. evansi isolates from other species from diferent countries like Egypt, India, and other countries.T. evansi isolate of this is clear divergence from isolates of Egypt and India of the same host species (Equine).It could be attributed to the diversity of the RoTat 1.2 VSG gene in Egyptian trypanosome isolates from horses associated to long-term parasite persistence due to chronic nature of the disease.
Our fndings align with previous phylogenetic studies on T. evansi using the RoTat 1.2 VSG gene [49], which  Additionally, it may be linked to non-RoTat 1.2 VSG T. evansi, a variant described from Africa [5,[51][52][53][54].Even though this is just a preliminary fnding, it is undeniable that RoTat 1.2 VSG is selectively present in isolates from various hosts [55].Te present study has some limitations as few number of horse were examined, sequencing was performed for one sample and absent of blood smear diagnosis for the examined animals.

Conclusions
Oxidative damage to erythrocytes may contribute to anemia in horses infected with T. evansi.Tis can be used as a marker for both latent and recent infections when correlated with other hematobiochemical indicators.PCR can quickly and reliably detect T. evansi infection in horses, particularly when parasitemia is low.Using PCR in the feld would not only help diagnose and treat individual animals but also eliminate the reservoir of infection, reducing the threat to equine and camel herds where blood sucking mechanical vectors are present.Moreover, T. evansi is circulating in Egyptian horses, thus regular monitoring at is recommended to implement and efective control measure.Further studies are needed to detect other Trypanosoma spp.and to study the alteration of hematological profle in case of coinfection.

Figure 1 :
Figure 1: Te diseases horses showed severe emaciation and weakness.

Table 1 :
Hematological parameters (mean ± SE) of infected and noninfected equine by T. evansi.

Table 2 :
[50]ative stress markers (mean ± SE) in infected and noninfected equine by T. evansi.Te complicated evolutionary history for T. evansi may be attributed to the movement of diseased animals and insect vectors, leading to repeated hybridization occurrences between diferent strains[50].