Prevalence and molecular characterization of Theileria sp. from endangered Arabian gazelles in Saudi Arabia

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Omer, Esam Al-Shaebi, Abdulaziz N. Alagaili, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8348152/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Wildlife conservation in Saudi Arabia is gaining much attention from the authorities. Habitat destruction, climate change, and human encroachment, as well as disease outbreaks, negatively impact conservation efforts. Hemoparasites among endangered Saudi wildlife have not been studied before. The aim of this study is to investigate the prevalence of hemoparasites among two endangered gazelle species { Gazella marica (Reem) and Gazella arabica (Idmi)} raised at King Khalid Wildlife Research Center (KKWRC) for breeding and reintroduction purposes. A total of 233 blood samples were collected from the jugular veins of gazelles and examined microscopically for the detection of piroplasm or any relevant blood parasites. Ticks were collected from some gazelles at KKWRC to identify them and see their potential role in the transmission of hemoparasites. DNA was extracted from the collected blood and ticks for the detection of hemoparasites, and amplified targeting the partial 18S rRNA gene. The prevalence of intraerythrocytic bodies suggestive of Theileria sp. was detected in 41 (17.9%) gazelles of both species, whereas PCR products of Theileria sp. DNA were detected in 65 (27.9%) gazelles. The sensitivity of the molecular technique compared to the direct microscopy was 76.74%, whereas the specificity was 83.16%. Ticks were morphologically identified as Hyalomma impeltatum and confirmed with molecular techniques. Sequences obtained from Theileria sp. from both Reem and Idmi gazelles were identical with only one mutation (transition) at position 140, where it was a T in Reem sequences while it was a C consistently in all sequences obtained from Idmi gazelles. Phylogenetic studies indicated that sequences obtained from gazelles in the present study group were related to Theileria sp. which was isolated from the Chinkara gazelle from Pakistan and related to isolates from the dama gazelle. Theileria sp. DNA was amplified from two ticks collected from Reem gazelles, and they were found to be identical to the Theileria sp. sequences obtained from Reem gazelles. This finding suggested that H. impeltatum can potentially be a vector for Theileria sp. in Reem gazelles. No Theileria sp. was amplified from ticks collected from Idmi gazelles. The prevalence of Theileria infection in gazelles at KKWRC in Saudi Arabia was reported for the first time. The possible vector responsible of the transmission was also deciphered. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Zoology Gazella marica Gazella arabica Theileria sp. Molecular characterization Saudi Arabia Figures Figure 1 Figure 2 Introduction Theileria parasites are obligate intracellular hemoprotozoan parasites and transmitted by ixodid ticks, they belong to the order Piroplasmida [ 1 ]. Theileria species are known to cause theileriosis in a range of domestic and wild ungulates [ 2 ]. According to Sivakumar and colleagues [ 3 ], Theileria spp. are designated into two major forms or groups; schizont “transforming” species, and “non-transforming” species. Members of the first group are regarded as virulent and pathogenic species, whereas those of the other group are regarded as benign, nonpathogenic or of low pathogenicity [ 3 ]. Members of the first such as T. parva and T. annulata in cattle and T. lestoquardi in sheep and goats, induced proliferation of schizonts, which results in clinical disease [ 4 , 5 ]. While the second group of non-transforming Theileria spp., included T. taurotragi, T. orientalis , and T. buffeli . Some of these species, however, may cause schizont-associated pathology and may also results into lymphoid hyperplasia similar to that found in pathogenic group of Theileria spp. [ 4 , 6 , 7 ]. The disease caused by pathogenic Theileria spp. is characterized by a marked fever, where the body temperature may reach 40–41.5°C, depression, lacrimation, nasal discharge, and enlargement of regional superficial lymph nodes and marked anemia [ 8 ]. Studies dealt with hemoparasites in Saudi Arabia were restricted to studying the prevalence of the parasites in domestic livestock [ 9 – 11 ]. Recently molecular evidences of such parasites were shown from different regions in Saudi Arabia [ 12 – 15 ]. There are only two reports on blood parasites in non-domestic animals; where Theileria spp. were detected from the hedgehog ( Paraechinus aethiopicus ) in one study and from Gerbillus nanus and Rattus rattus in another study [ 16 , 17 ]. Wildlife conservation in Saudi Arabia is gaining much attention from the authorities. Reintroduction programs for the endangered Saudi wildlife has been established in various protected areas [ 18 , 19 ]. Endangered species of gazelles including the sand gazelle ( Gazella marica ) and the mountain gazelles ( Gazella arabica ) are raised at King Khalid Wildlife Research Centre (KKWRC) for breeding, conservation and reintroduction purposes into their natural habitats from which they vanished. Some of the protozoan parasites particularly enteric as well as cysts-forming parasites have been reported from theses earlier [ 20 – 24 ]. No hemoparasites have been reported from gazelles in Saudi Arabia. Therefore, the present study aims to investigate the prevalence of hemiparasites in some endangered Arabian gazelles kept at KKWRC, particularly; the sand gazelle ( Gazella marica ) and the mountain gazelle ( Gazella arabica ) using microscopic and molecular methods. Furthermore, the risk factors associated with the prevalence of hemoparasites in gazelles were also evaluated. An attempt was made to investigate and characterize the possible vector involved in the transmission of the Theileria sp. detected in gazelles at KKWRC. Results Microscopic and molecular prevalence Microscopic examination of Giemsa-stained blood smears from gazelles revealed that intraerythrocytic stages suggestive of Theileria spp. were detectable from 42 out of 233 (18.0%) Reem and Idmi gazelles investigated (Fig. 1 ). The prevalence of Theileria infection using both microscopic and PCR methods, together with the prevalence in males and females as well as in juveniles and adult gazelles, is given in Table (1). Using microscopy, intraerythrocytic bodies suggestive of Theileria sp. were detected in 18.0% of both Reem (22 out of 122) and Idmi (20 out of 111) gazelles investigated in the present study. PCR amplification of a fragment ~ 450bp suggestive of Theileria sp. DNA was detected in 27.9% (65 out of 233) of Reem and Idmi gazelles screened. The prevalence was 29.5% (36 out of 122) in Reem gazelles while it was 26.1% (29 out of 111) in Idmi gazelles. The difference in the prevalence of Theileria sp. infection between the two gazelles’ species, using both methods, was not significant (p > 0.05). The difference in prevalence of Theileria sp. infection between the two species of gazelles, using molecular method, was not significant (p > 0.05). There was a significant difference (p 0.05) in the prevalence between males and females using both methods. Whereas, there was a significant difference (p < 0.05) in the prevalence between adults and juveniles using molecular method, with prevalence in juveniles being higher (Table 1 ). Table 1 Results of the prevalence of Theileria sp. in Reem ( Gazella marica ) and Idmi ( Gazella arabica ) gazelles at King Khalid Wildlife Research Center, Saudi Arabia, using direct microscopy and Polymerase Chain Reaction. Variables Number examined Microscopic Results (%) P value PCR Results (%) P value Reem Idmi Reem Idmi Reem Idmi Sex Male Female > 0.05 > 0.05 54 58 12 (22.2) 10 (17.2) 17 (31.5) 13 (22.4) 68 53 9 (14.7) 10 (18.9) 19 (27.9) 16 (30.2) Age Adult Juvenile > 0.05 < 0.05 70 82 7 (10.0) 16 (19.5) 6 (8.6) 20 (34.4) 52 29 15 (28.8) 4 (13.8) 30 (57.7) 9 (31.0) Table 2 Taxa and their 18S rRNA sequences GenBank accession numbers, their hosts, and their origin were used in the present analyses. Accession number Taxon Host Country PX457129 Theileria sp. Gazella arabica Saudi Arabia PX457130 Theileria sp. Gazella arabica Saudi Arabia PX457131 Theileria sp. Gazella marica Saudi Arabia PX457132 Theileria sp. Gazella marica Saudi Arabia PQ050069.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050070.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050067.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050065.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050064.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050063.1 Theileria sp. Marsh Deer + Brown Brocket Brazil OR135739.1 Theileria sp. Marsh Deer + Brown Brocket Brazil OR135738.1 Theileria sp. Marsh Deer + Brown Brocket Brazil OR135737.1 Theileria sp. Marsh Deer + Brown Brocket Brazil OR135736.1 Theileria sp. Marsh Deer + Brown Brocket Brazil PQ050071.1 Theileria sp. Marsh Deer + Brown Brocket Brazil MW008519.1 Theileria cervi Marsh Deer + Brown Brocket Texas MW008528.1 Theileria cervi Marsh Deer + Brown Brocket Texas KM186958.1 Uncultured Theileria sp. Roe deer + Tick China KM186960.1 Uncultured Theileria sp. Roe deer + Tick China MG041373.1 Theileria cervi Roe deer + Tick Russia MH085203.1 Theileria cervi Roe deer + Tick China PP024227.1 Theileria sp. Goat + Sheep + Dog India MW307318.1 Theileria luwenshuni Goat + Sheep + Dog Thailand LC602484.1 Theileria luwenshuni Goat + Sheep + Dog Japan MW307320.1 Theileria luwenshuni Goat + Sheep + Dog Thailand MZ734314.1 Theileria luwenshuni Goat + Sheep + Dog Thailand AB650504.1 Theileria sp. giraffe East Africa AY748464.1 Theileria sp. Malelane roan antelope South Africa AY748462.1 Theileria sp. Malelane sable antelope South Africa HQ179766.1 Theileria sp. Damaliscus lunatus South Africa MT814741.1 Theileria sp. Alcelaphus buselaphus Zambia FJ155996.1 Theileria sp. Rhipicephalus evertsi evertsi South Africa MT814752.1 Theileria sp. Antelopes Zambia GU733378.1 Theileria sp. sable South Africa MT814729.1 Theileria sp Alcelaphus buselaphus Zambia MT814730.1 Theileria sp. Alcelaphus buselaphus Zambia AY260175.1 Theileria separata Sheep + Rhipicephalus evertsi South Africa OP763595.1 Theileria sp. Dama gazelle UAE OP763603.1 Theileria sp. Dama gazelle UAE AY735115.1 Theileria sp. Dama gazelle UAE AY735116.1 Theileria sp. Dama gazelle UAE MN209937.1 Theileria sp. Chinkara gazelle Pakistan MN209938.1 Theileria sp. Chinkara gazelle Pakistan MK131256.1 Theileria sp. waterbuck South Africa KF597064.1 Theileria sp. waterbuck Kenya KF597067.1 Theileria sp. waterbuck Kenya KF597069.1 Theileria sp. waterbuck Kenya L24381.1 Toxoplasma gondii Tachyzoites ( T. gondii ) Australia Sensitivity and specificity The sensitivity of the PCR test, designating the true positives, was found to be 76.74% (95% CI: 61.37% to 88.24%) whereas the specificity, designating the true negatives, was found to be 83.16% (95% CI: 77.06% to 88.19%). The positive predictive value (PPV) of the PCR method was found to be 63.81% (95% CI: 55.25% to 71.57%) whereas the negative predictive value (NPV) was found to be 90.24% (95% CI: 84.25% to 94.11%). The sensitivity of the direct microscopy was found to be 49.23% (95% CI: 36.60% to 61.93%) while the specificity was found to be 94.05% (95% CI: 89.33% to 97.11%). The positive predictive value (PPV) of the direct microscopy method was found to be 76.19% (95% CI: 62.56% to 85.97%) whereas the negative predictive value (NPV) was found to be 82.72% (95% CI: 78.98% to 85.92%). Sequence data and phylogenetic analysis DNA sequencing resulted in acquisition of 33 sequences from Reem and 25 sequences from Idmi gazelles. Some of the PCR products did not yield good sequences and they were eliminated from the analysis. Representative DNA sequences from Theileria sp. from Idmi gazelles were given the accession numbers PX457129 and PX457130, whereas those from Reem gazelles were given the numbers PX457131 and PX457132. Theileria sp. sequences from both gazelles’ species were identical with only one mutation which was consistently found in each gazelle species. At position 140 of the alignment a transition from T in Reem to C in Idmi gazelles was noticed. Furthermore, one sequence (PX457130) from Idmi gazelles showed a mutation at position 143 having an A while it was T in all other sequences including sequences from Reem gazelles as well. Phylogenetic analysis revealed that Theileria sp. sequences obtained from gazelles at KKWRC grouped with two sequences from Theileria sp. dama gazelle isolates T1-1 and T4-1 (MN209937 and MN209938) from Chinkara gazelles in Pakistan. Both sequences from Idmi and Reem gazelles showed 100% and 99.71% identities to sequences from the Chinakra (MN209937 and MN209938) respectively. They shared common ancestor with the other group of sequences of Theileria sp. obtained from dama gazelles of the United Arab Emirates and USA (OP763595, OP763603, AY735115, AY735116). Sequences AY735115, AY735116 were from Theileria sp. dama gazelle isolates with a note that the source of blood was cervine blood without any indication of the host. Whereas sequences OP763595, OP763603 were labeled Theileria sp. from dama gazelle ( Nanger dama ), without any indication regarding the source of blood. Theileria spp. reported from other antelopes or related taxa were placed in different clades as shown in Fig. 2 . Morphological identification of ticks collected from gazelles revealed their identity as Hyalomma impeltatum . Partial DNA sequences from the COXI gene from ticks were related to H. impeltatum as well confirming the morphological identification. Interestingly, Theileria sp. from Reem gazelles were also isolated from 2 of the H. impeltatum ticks which were collected from Reem gazelle (Deposited in GenBank and were given the accession numbers PX457133 and PX457134). Six sequences from H. impeltatum , three from each from Reem and Idmi gazelles were deposited in GenBank under accession numbers PX457490, PX457493 and PX457495 for Reem and PX457491, PX457492 and PX457494for Idmi gazelles. H. impleltatum sequences obtained in the present study were 99% identical to DNA sequences from H. impeltatum reported from Madina (ON138788.1) and Tabuk (ON138785.1) in Saudi Arabia. Discussion Hemoparasites belonging to the Genus Theileria has been detected in both Reem and Idmi gazelles at KKWRC. This finding represents the first microscopic as well as molecular-based report of Theileria sp. in these species of gazelles. Intraerythrocytic bodies suggestive of Theileria sp. have been detected in both species of gazelles investigated. Using microscopy, the overall prevalence of Theileria sp. in gazelles investigated was 18.0%, while it was found to be higher (27.9%) using molecular techniques. This report is considered the first microscopic and molecular-based report of hemoparasites in members of the Genus Gazella in Saudi Arabia. The sensitivity and specificity of the PCR method of detection of Theileria sp. in both species of gazelles was found to be 76.74% and 83.16% respectively. Whereas the sensitivity and specificity of the direct microscopy method was found to be 49.23% and 94.05% respectively. It appeared that the PCR method is more sensitive for the detection of Theileria sp. in gazelles. Similar records have been reported recently from sheep, goats and cattle [ 25 – 27 ]. The sensitivity and specificity for the detection of Theileria spp. in sheep and goats’ samples were 26.98% and 98.31% for the blood smear method, and 73.01% and 100% for the PCR test, respectively [ 27 ]. The higher prevalence of Theileria sp. detected through molecular techniques compared to microscopic examination, in several studies, as well as in this study, is likely due to the increased specificity of the PCR method, which can detect low levels of parasitemia that may not be visible microscopically. Whereas, the microscopic method depends on only small volume of the blood samples from which the smear was prepared, furthermore, if the level of parasitemia is low then lower chance of demonstrating the piroplasms in the blood smear. Direct microscopy is a valid diagnostic tool in Theileria infection and was found to be valid in several studies [ 25 , 28 ]. However, the presence of artifacts, hemolysis of the erythrocytes, poor staining, microscopic expertise and low parasitemia may indirectly interfere with the detection of piroplasms within the erythrocytes and render diagnosis more difficult. There was no significant difference in the prevalence between males and females in gazelles at KKWRC. There is contradicting results as for the prevalence of Theileria spp. in males and females. A recent study on the prevalence of Theileria ovis in sheep in Saudi Arabia showed that the prevalence in males in greater than in females [ 15 ]. In some studies, however, the prevalence was found to be more in females compared to males [ 29 , 30 ]. The difference in the prevalence between males and females in farm animals may be understandable as keeping females for longer periods, hence, greater chances of exposure to ticks whereas males are managed differently and are not kept for long time in the farms. In wildlife the situation may be different and animals are bred for reintroduction purposes and the idea of keeping females for longer does not apply to wildlife. Unlike what is generally believed, the prevalence of Theileria sp. in juvenile gazelles was found to be significantly higher than in adult gazelles. Similar results were obtained from some studies in domestic animals [ 31 , 32 ]. Underdeveloped immune system of juvenile animals in addition to vaccination and weaning may be contributory factors to high prevalence of Theileria sp. in juveniles compared to adult animals. The general belief was that adult animals are more prone to infection with Theileria sp. which has been reported earlier in several investigations [13,33,34]. It was attributed to; that ticks are associated with adult animals for longer periods compared to young animals hence higher chances of infection. Previously, two DNA sequences in GenBank (MN209937 and MN209938) from the Chinkara ( Gazella bennettii ) from Pakistan, Punjab were found to show high identity with the sequences from both species of gazelles in the present study. Both sequences from the chinkara were 100% identical to sequences from G. marica , whereas, it was 99% identical to those from G. arabica . It is worth mentioning that the sequences obtained from the chinkara were labelled dama gazelle, and they were 97% identical to sequences from dama gazelle ( Nanger dama ). Four sequences were available in GenBank from dama gazelles ( N. dama ); OP763595, OP763603, AY735115, and AY735116 were designated as Theileria sp. from dama gazelle. These sequences were 96.9% to 97.7% identical to sequences obtained from gazelles in the present study and those obtained from the Chinkara. Sequences of Theileria sp. from Reem, Idmi and Chinkara gazelles i.e. which belong to members of the genus Gazella ; formed one clade which shared a common ancestor to Theileria sp. from the dama gazelle which belong to the genus Nanger . Both clades grouped with Theileria spp. reported from antelopes with significant designation of different species detected in different wildlife. From the phylogenetic tree; it appears that species of Theileria infecting members of the genus Gazella and those infecting members of the genus Nanger are distinct and they shared common ancestor. Theileria sp. detected in gazelles is among the benign non-transforming Theileria parasites such as T. sergenti/T. buffeli/T. orientalis group [35]. Ticks which were collected from gazelles raised at KKWRC were identified as H. impeltatum and the identification was confirmed molecularly. This finding indicated that the predominant type of ticks infesting gazelles is related to H. impeltatum . Furthermore, finding DNA related to Theileria sp. from Reem gazelles clearly demonstrated that this tick species is a potential vector for Theileria sp. in Reem gazelles. The inability to detect Theileria sp. from the DNA of H. impeltatum ticks doesn’t mean that this tick is not a suitable vector for transmitting Theileria infection in Idmi gazelles, however, some more work is required to prove the assumption or reject it. Furthermore, some of the ticks collected in the present study were dried up and the preservation of some of them was inadequate and were collected over a long period of time and kept at KKWRC. This may limit the recovery of good quality DNA from some of the ticks, therefore, affected recovery of the parasite DNA. Previously, Almahallawi and colleagues [ 15 ] reported that both H. impeltatum and H. dromedarii as potential vectors for T. ovis from sheep in Northwestern Saudi Arabia. Omer and colleagues [ 14 ] were also able to detect T. annulata DNA from both H. anatolicum and H. dromedarii ticks from the eastern province in Saudi Arabia. Finding the possible vector incriminated in transmitting Theileria sp. in gazelles raises a question of controlling such vector, furthermore it is recommended that all gazelles at KKWRC which are kept for breeding and reintroduction purposes must be screened regularly against Theileria infections and animals proved positive for this parasite must be excluded from reintroduction programs and must be treated before release. At KKWRC, one of the routine practices is administering a dose of ivermectin to all handled gazelles annually, which may help in minimizing tick infestation in gazelles hence; negatively influencing the transmission of Theileria infection among gazelles. Methods This study was conducted at the Department of Zoology, College of Science, under the permit issued by King Saud University Animal Ethics Committee (KSU-SE.23–104). Gazelle species and blood collection Herds of gazelles are kept at King Khalid Wildlife Research Center (KKWRC) for breeding and reintroduction purposes. KKWRC is located 80 km north east the capital of Saudi Arabia; of Riyadh. Species of gazelles kept at KKWRC included the Arabian sand gazelle, locally know as Reem ( Gazella marica ) and the Arabian mountain gazelle, locally known as Idmi ( Gazella arabica ). The gazelles are kept in breeding pens which are 50x100 meters wide. Food and water are provided for animals in daily basis. Gazelles are subjected to routine capturing for veterinary intervention including vaccination, deworming and translocation. At the end of each breeding pen there is a triangular shape boma to facilitate the process of capturing and easy sampling. The animal record keeping system of the animals at the center are maintained using ZIMS software, where all the movements, interventions with gazelles are recorded since birth. Gazelles were clinically healthy when blood samples were collected. The collection of blood samples was performed during the routine capturing process for veterinary interventions. The sample size (n) was determined using the statistical formula by Thrusfield [ 36 ]: $$\:n=\frac{{Z}^{2}\:P(1-P)}{{d}^{2}}$$ where n is the desired sample size; Z is 95% confidence interval (1.96), P is the estimated prevalence of infections and d is the desired precision level (5%). Since the prevalence of Theileria infections in the study area was unknown and this study was undertaken to determine the prevalence of Theileria spp. infections in gazelles species at KKWRC, 13% prevalence was assumed [ 36 ]. During capturing gazelles were bled by jugular venipuncture. A volume of 5 ml of blood was collected in vacutainer tubes with EDTA as anticoagulant. Blood samples were collected from a total of 233 gazelles. Samples included 111 from Idmi gazelles and 122 from Reem gazelles. Gazelles which are less than one-year old are designated as juveniles whereas those more than one-year old are regarded as adult. Of Idmi gazelle samples; 47 were from male while 64 were female, furthermore, 82 samples were collected from adult whereas 29 were from juvenile gazelles. With regard to samples from Reem gazelles, 50 samples were from males while 72 were from females; 70 samples were from adult while 52 samples were from juveniles. Blood smears were prepared from all samples collected from gazelles, air-dried, fixed with methanol and Giemsa-stained following the standard procedures [ 15 ]. DNA was extracted from blood using DNeasy blood and tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA was stored at − 80°C until use. A fragment of ~ 450-base pair (bp) of the 18S small subunit ribosomal RNA (rRNA) gene was intended to amplify using the primers RLB-F 5′- GACACAGGGAGGTAGTGACAAG-3′ as a forward primer and RLB-R 5′- CTAAGAATTTCACCTCTGACAGT-3′ as a reverse primer [ 37 ]. The region intended for amplification includes the hypervariable region (V4) of the 18S rRNA gene. The polymerase chain reaction (PCR) was set up using 2 µl of the DNA extracted from blood with 10 µM of each of the forward and reverse primers, 5 µl of 5× PCR buffer, and 0.2 µl (1 unit) of Taq DNA polymerase (Bioline, London, UK). Double distilled water was used as the negative control to detect contamination. The PCR was performed on a thermocycler (Multigene, Labnet, Maryland, USA) and the conditions for the PCR were as follows: initial denaturation cycle at 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec, and a final extension at 72°C for 5 min. A total of 41 ticks were collected from 24 gazelles (9 Reem and 15 Idmi) at KKWRC. Ticks were not noticed on animals during the blood collection. They were collected at different times. Ticks were preserved in 70% ethanol and identified to the species level according to the keys given previously [38,39,40]. DNA was extracted from ticks using Qiagen extraction kit (Qiagen, Hilden, Germany) after being washed in water, same as the protocol followed for blood extraction. Extracted DNA was subjected to PCR amplification of cytochrome oxidase subunit I gene using primers, COXI-F 5′-GGAACAATATATTTAATTTTTGG-3′ as a forward primers and COXI-R 5′-ATCTATCCCTACTGTAAATATATG-3′ as a reverse primer which amplify ~ 800 bp of the COXI [ 41 ]. The PCR products from the amplification of 18S rRNA region from Theileria spp. or COXI region from ticks were evaluated using 1.5% agarose gel (with ethidium bromide) electrophoresis and visualized with ultraviolet light using a transilluminator. Digital images were taken of the amplicons. The PCR products were sequenced following the standard procedure of Sanger sequencing at Macrogen, South Korea. All sequences were aligned analyzed and edited using MEGA 11 software [42]. Further, the phylogenetic trees were based on the comparison with sequences of Theileria spp. detected in the present study and those available in GenBank. Trees were constructed using the maximum likelihood (ML) and neighbor-joining (NJ) methods of the MEGA 11 program and bootstrap analysis with 1000 replications was used to estimate the confidence of the branching patterns of the trees [42]. Calculation of sensitivity and specificity The sensitivity and specificity of the polymerase chain reaction test compared to the direct microscopy were calculated according to the following formula[ 36 ]. \(\:\:\) $$\:\text{S}\text{e}\text{n}\text{s}\text{i}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\text{%}=\frac{\text{P}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{s}\:\text{o}\text{n}\:\text{d}\text{i}\text{r}\text{c}\text{t}\:\text{m}\text{i}\text{c}\text{r}\text{o}\text{s}\text{c}\text{o}\text{p}\text{y}\:\text{a}\text{n}\text{d}\:\text{P}\text{C}\text{R}\:\:}{\text{P}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{s}\:\text{o}\text{n}\:\text{P}\text{C}\text{R}}\text{X}\:100$$ $$\:\text{S}\text{p}\text{e}\text{c}\text{i}\text{f}\text{i}\text{c}\text{i}\text{t}\text{y}\:\text{%}=\frac{\text{N}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{s}\:\text{o}\text{n}\:\text{d}\text{i}\text{r}\text{e}\text{c}\text{t}\:\text{m}\text{i}\text{c}\text{r}\text{o}\text{s}\text{c}\text{o}\text{p}\text{y}\:\text{a}\text{n}\text{d}\:\text{P}\text{C}\text{R}\:\:}{\text{N}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{s}\:\text{o}\text{n}\:\text{P}\text{C}\text{R}}\text{X}\:100$$ Statistical Analysis Differences in the prevalence rates of Theileria specie in different gazelle species as well as from males, females, and age groups were tested using χ2 -test, which was performed in SPSS 22.0. The significance was set at 95%. Declarations Competing interests The authors declare no competing interests. Funding The authors wish to thank KKWRC staff for their cooperation and help with sample collection and professional handling of gazelles. This work was funded by the Ongoing Research Funding program (ORF-2025-94), King Saud University, Riyadh, Saudi Arabia. Author Contribution S.A., A.N.A., and O.B.M. conceived the study, S.A., S.A.O. and E.A. conducted laboratory work, A.A., A.N.A., S.A., and O.B.M. performed data analysis. S.A.O., A.A. E.A., and O.B.M. drafted the manuscript. All authors reviewed and commented on the manuscript. Data Availability Data used in this study are available on request. References - Norval, R. A. I., Perry, B. D. & Young, A. S. The epidemiology of theileriosis in Africa. ILRI (1992). (aka ILCA and ILRAD). - Mans, B. J., Pienaar, R. & Latif, A. A. A review of Theileria diagnostics and epidemiology. Int. J. 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B., Lindsay, N., Rietkerk, F. E. & Williamson, D. T. The reintroduction of mountain gazelle Gazella gazella in Saudi Arabia. Int. Zoo Yearbook . 32 (1), 107–116 (1993). - Wacher, T. et al. The conservation status of the threatened gazelles of Ar Rub’al-Khālī, Saudi Arabia. J. Arid Environ. 229 , 105396 (2025). - Hussein, H. S. & Mohammed, O. B. Eimeria rheemi sp-n (Apicomplexa, Eimeriidae) from the Arabian sand gazelle, Gazella subgutturosa marica (Artiodactyla, Bovidae) in Saudi Arabia. J. Helminthological Soc. Wash. 59 (2), 190–194 (1992). - Mohammed, O. B. & Hussein, H. S. Eimeria idmii sp. n. (Apicomplexa: Eimeriidae) from the Arabian mountain gazelle, Gazella gazella , in Saudi Arabia. J. Helminthological Soc. Wash. 59 (1), 120–124 (1992). - Mohammed, O. B. & Hussein, H. S. Antibody prevalence of toxoplasmosis in Arabian gazelles and oryx in Saudi Arabia. J. Wildl. Dis. 30 (4), 560–562 (1994). - Mohammed, O. B. & Flamand, J. R. 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Ticks (Acari: Ixodidae) parasitizing indigenous livestock in northern and eastern Saudi Arabia. Arab. Gulf J. Sci. Res. Agric. Biol. Sci. B-5 , 273–286 (1987). - Walker, A. R. Ticks of domestic animals in Africa: a guide to identification of species Vol. 74 (Bioscience Reports, 2003). - Chitimia, L. et al. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox 1 and nad 5 genes. Exp. Appl. Acarol. 52 (3), 305–311 (2010). - Tamura, K., Stecher, G. & Kumar, S. MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38 (7), 3022–3027 (2021). Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":137859,"visible":true,"origin":"","legend":"\u003cp\u003eGiemsa-stained blood film prepared from an Idmi gazelle showing intraerythrocytic bodies (arrows) suggestive of \u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8348152/v1/6c3373cf9b3da69588269cce.jpg"},{"id":98825901,"identity":"2ef1811b-5566-47b2-a9a5-aa63575f2da1","added_by":"auto","created_at":"2025-12-22 18:42:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85783,"visible":true,"origin":"","legend":"\u003cp\u003eConsensus phylogenetic tree constructed with maximum-likelihood (ML) and neighbor-joining (NJ) methods, showing phylogenetic relationships between \u003cem\u003eTheileria\u003c/em\u003e sp. detected in Reem (Blue triangles) and Idmi (Solid red circles) from King Khalid Wildlife Research Centre (KKWRC) and 44 related taxa obtained from NCBI GenBank with \u003cem\u003eToxoplasma gondii\u003c/em\u003e as an out-group. Numbers indicated at branch nodes are bootstrap values (NJ/ML). Only bootstraps \u0026gt;50% are shown.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8348152/v1/4fa2339b9cd9967f3aa51d04.png"},{"id":103251019,"identity":"8abb1ad4-bdc5-4713-b59f-1d8057a24ab2","added_by":"auto","created_at":"2026-02-23 16:01:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1075751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8348152/v1/324493e5-963d-4e58-a653-49cf2d693f89.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence and molecular characterization of Theileria sp. from endangered Arabian gazelles in Saudi Arabia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eTheileria\u003c/em\u003e parasites are obligate intracellular hemoprotozoan parasites and transmitted by ixodid ticks, they belong to the order Piroplasmida [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eTheileria\u003c/em\u003e species are known to cause theileriosis in a range of domestic and wild ungulates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to Sivakumar and colleagues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], \u003cem\u003eTheileria\u003c/em\u003e spp. are designated into two major forms or groups; schizont \u0026ldquo;transforming\u0026rdquo; species, and \u0026ldquo;non-transforming\u0026rdquo; species. Members of the first group are regarded as virulent and pathogenic species, whereas those of the other group are regarded as benign, nonpathogenic or of low pathogenicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Members of the first such as \u003cem\u003eT. parva\u003c/em\u003e and \u003cem\u003eT. annulata\u003c/em\u003e in cattle and \u003cem\u003eT. lestoquardi\u003c/em\u003e in sheep and goats, induced proliferation of schizonts, which results in clinical disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While the second group of non-transforming \u003cem\u003eTheileria\u003c/em\u003e spp., included \u003cem\u003eT. taurotragi, T. orientalis\u003c/em\u003e, and \u003cem\u003eT. buffeli\u003c/em\u003e. Some of these species, however, may cause schizont-associated pathology and may also results into lymphoid hyperplasia similar to that found in pathogenic group of \u003cem\u003eTheileria\u003c/em\u003e spp. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The disease caused by pathogenic \u003cem\u003eTheileria\u003c/em\u003e spp. is characterized by a marked fever, where the body temperature may reach 40\u0026ndash;41.5\u0026deg;C, depression, lacrimation, nasal discharge, and enlargement of regional superficial lymph nodes and marked anemia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies dealt with hemoparasites in Saudi Arabia were restricted to studying the prevalence of the parasites in domestic livestock [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recently molecular evidences of such parasites were shown from different regions in Saudi Arabia [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There are only two reports on blood parasites in non-domestic animals; where \u003cem\u003eTheileria\u003c/em\u003e spp. were detected from the hedgehog (\u003cem\u003eParaechinus aethiopicus\u003c/em\u003e) in one study and from \u003cem\u003eGerbillus nanus\u003c/em\u003e and \u003cem\u003eRattus rattus\u003c/em\u003e in another study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWildlife conservation in Saudi Arabia is gaining much attention from the authorities. Reintroduction programs for the endangered Saudi wildlife has been established in various protected areas [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Endangered species of gazelles including the sand gazelle (\u003cem\u003eGazella marica\u003c/em\u003e) and the mountain gazelles (\u003cem\u003eGazella arabica\u003c/em\u003e) are raised at King Khalid Wildlife Research Centre (KKWRC) for breeding, conservation and reintroduction purposes into their natural habitats from which they vanished. Some of the protozoan parasites particularly enteric as well as cysts-forming parasites have been reported from theses earlier [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. No hemoparasites have been reported from gazelles in Saudi Arabia. Therefore, the present study aims to investigate the prevalence of hemiparasites in some endangered Arabian gazelles kept at KKWRC, particularly; the sand gazelle (\u003cem\u003eGazella marica\u003c/em\u003e) and the mountain gazelle (\u003cem\u003eGazella arabica\u003c/em\u003e) using microscopic and molecular methods. Furthermore, the risk factors associated with the prevalence of hemoparasites in gazelles were also evaluated. An attempt was made to investigate and characterize the possible vector involved in the transmission of the \u003cem\u003eTheileria\u003c/em\u003e sp. detected in gazelles at KKWRC.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic and molecular prevalence\u003c/h2\u003e \u003cp\u003eMicroscopic examination of Giemsa-stained blood smears from gazelles revealed that intraerythrocytic stages suggestive of \u003cem\u003eTheileria\u003c/em\u003e spp. were detectable from 42 out of 233 (18.0%) Reem and Idmi gazelles investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The prevalence of \u003cem\u003eTheileria\u003c/em\u003e infection using both microscopic and PCR methods, together with the prevalence in males and females as well as in juveniles and adult gazelles, is given in Table\u0026nbsp;(1). Using microscopy, intraerythrocytic bodies suggestive of \u003cem\u003eTheileria\u003c/em\u003e sp. were detected in 18.0% of both Reem (22 out of 122) and Idmi (20 out of 111) gazelles investigated in the present study. PCR amplification of a fragment\u0026thinsp;~\u0026thinsp;450bp suggestive of \u003cem\u003eTheileria\u003c/em\u003e sp. DNA was detected in 27.9% (65 out of 233) of Reem and Idmi gazelles screened. The prevalence was 29.5% (36 out of 122) in Reem gazelles while it was 26.1% (29 out of 111) in Idmi gazelles. The difference in the prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. infection between the two gazelles\u0026rsquo; species, using both methods, was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The difference in prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. infection between the two species of gazelles, using molecular method, was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the prevalence in \u003cem\u003eTheileria\u003c/em\u003e sp. in gazelles at KKWRC using microscopic and molecular methods, having more prevalence by molecular method. There was no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the prevalence between males and females using both methods. Whereas, there was a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the prevalence between adults and juveniles using molecular method, with prevalence in juveniles being higher (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of the prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. in Reem (\u003cem\u003eGazella marica\u003c/em\u003e) and Idmi (\u003cem\u003eGazella arabica\u003c/em\u003e) gazelles at King Khalid Wildlife Research Center, Saudi Arabia, using direct microscopy and Polymerase Chain Reaction.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNumber examined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMicroscopic Results (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003ePCR Results (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIdmi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIdmi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIdmi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale\u003c/p\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12 (22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (17.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17 (31.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13 (22.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (14.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (18.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19 (27.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16 (30.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAdult\u003c/p\u003e \u003cp\u003eJuvenile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;0.05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (10.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (19.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6 (8.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20 (34.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (28.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (13.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30 (57.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9 (31.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTaxa and their 18S rRNA sequences GenBank accession numbers, their hosts, and their origin were used in the present analyses.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaxon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCountry\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePX457129\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGazella arabica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSaudi Arabia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePX457130\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGazella arabica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSaudi Arabia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePX457131\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGazella marica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSaudi Arabia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePX457132\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGazella marica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSaudi Arabia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050069.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050070.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050067.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050065.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050064.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050063.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOR135739.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOR135738.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOR135737.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOR135736.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePQ050071.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMW008519.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria cervi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTexas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMW008528.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria cervi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMarsh Deer\u0026thinsp;+\u0026thinsp;Brown Brocket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTexas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKM186958.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUncultured \u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoe deer\u0026thinsp;+\u0026thinsp;Tick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKM186960.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUncultured \u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoe deer\u0026thinsp;+\u0026thinsp;Tick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG041373.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria cervi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoe deer\u0026thinsp;+\u0026thinsp;Tick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRussia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMH085203.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria cervi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoe deer\u0026thinsp;+\u0026thinsp;Tick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePP024227.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGoat\u0026thinsp;+\u0026thinsp;Sheep\u0026thinsp;+\u0026thinsp;Dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIndia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMW307318.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria luwenshuni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGoat\u0026thinsp;+\u0026thinsp;Sheep\u0026thinsp;+\u0026thinsp;Dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThailand\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLC602484.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria luwenshuni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGoat\u0026thinsp;+\u0026thinsp;Sheep\u0026thinsp;+\u0026thinsp;Dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJapan\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMW307320.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria luwenshuni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGoat\u0026thinsp;+\u0026thinsp;Sheep\u0026thinsp;+\u0026thinsp;Dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThailand\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMZ734314.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria luwenshuni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGoat\u0026thinsp;+\u0026thinsp;Sheep\u0026thinsp;+\u0026thinsp;Dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThailand\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAB650504.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003egiraffe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAY748464.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMalelane roan antelope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAY748462.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMalelane sable antelope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHQ179766.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDamaliscus lunatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMT814741.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAlcelaphus buselaphus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZambia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFJ155996.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRhipicephalus evertsi evertsi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMT814752.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntelopes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZambia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGU733378.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMT814729.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAlcelaphus buselaphus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZambia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMT814730.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAlcelaphus buselaphus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZambia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAY260175.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria separata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSheep\u0026thinsp;+\u0026thinsp;\u003cem\u003eRhipicephalus evertsi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOP763595.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDama gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUAE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOP763603.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDama gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUAE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAY735115.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDama gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUAE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAY735116.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDama gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUAE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMN209937.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChinkara gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePakistan\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMN209938.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChinkara gazelle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePakistan\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMK131256.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewaterbuck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouth Africa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKF597064.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewaterbuck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKenya\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKF597067.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewaterbuck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKenya\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKF597069.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTheileria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewaterbuck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKenya\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL24381.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eToxoplasma gondii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTachyzoites (\u003cem\u003eT. gondii\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAustralia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSensitivity and specificity\u003c/h3\u003e\n\u003cp\u003eThe sensitivity of the PCR test, designating the true positives, was found to be 76.74% (95% CI: 61.37% to 88.24%) whereas the specificity, designating the true negatives, was found to be 83.16% (95% CI: 77.06% to 88.19%). The positive predictive value (PPV) of the PCR method was found to be 63.81% (95% CI: 55.25% to 71.57%) whereas the negative predictive value (NPV) was found to be 90.24% (95% CI: 84.25% to 94.11%). The sensitivity of the direct microscopy was found to be 49.23% (95% CI: 36.60% to 61.93%) while the specificity was found to be 94.05% (95% CI: 89.33% to 97.11%). The positive predictive value (PPV) of the direct microscopy method was found to be 76.19% (95% CI: 62.56% to 85.97%) whereas the negative predictive value (NPV) was found to be 82.72% (95% CI: 78.98% to 85.92%).\u003c/p\u003e\n\u003ch3\u003eSequence data and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eDNA sequencing resulted in acquisition of 33 sequences from Reem and 25 sequences from Idmi gazelles. Some of the PCR products did not yield good sequences and they were eliminated from the analysis. Representative DNA sequences from \u003cem\u003eTheileria\u003c/em\u003e sp. from Idmi gazelles were given the accession numbers PX457129 and PX457130, whereas those from Reem gazelles were given the numbers PX457131 and PX457132. \u003cem\u003eTheileria\u003c/em\u003e sp. sequences from both gazelles\u0026rsquo; species were identical with only one mutation which was consistently found in each gazelle species. At position 140 of the alignment a transition from T in Reem to C in Idmi gazelles was noticed. Furthermore, one sequence (PX457130) from Idmi gazelles showed a mutation at position 143 having an A while it was T in all other sequences including sequences from Reem gazelles as well.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis revealed that \u003cem\u003eTheileria\u003c/em\u003e sp. sequences obtained from gazelles at KKWRC grouped with two sequences from \u003cem\u003eTheileria\u003c/em\u003e sp. dama gazelle isolates T1-1 and T4-1 (MN209937 and MN209938) from Chinkara gazelles in Pakistan. Both sequences from Idmi and Reem gazelles showed 100% and 99.71% identities to sequences from the Chinakra (MN209937 and MN209938) respectively. They shared common ancestor with the other group of sequences of \u003cem\u003eTheileria\u003c/em\u003e sp. obtained from dama gazelles of the United Arab Emirates and USA (OP763595, OP763603, AY735115, AY735116). Sequences AY735115, AY735116 were from \u003cem\u003eTheileria\u003c/em\u003e sp. dama gazelle isolates with a note that the source of blood was cervine blood without any indication of the host. Whereas sequences OP763595, OP763603 were labeled \u003cem\u003eTheileria\u003c/em\u003e sp. from dama gazelle (\u003cem\u003eNanger dama\u003c/em\u003e), without any indication regarding the source of blood. \u003cem\u003eTheileria\u003c/em\u003e spp. reported from other antelopes or related taxa were placed in different clades as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMorphological identification of ticks collected from gazelles revealed their identity as \u003cem\u003eHyalomma impeltatum\u003c/em\u003e. Partial DNA sequences from the COXI gene from ticks were related to \u003cem\u003eH. impeltatum\u003c/em\u003e as well confirming the morphological identification. Interestingly, \u003cem\u003eTheileria\u003c/em\u003e sp. from Reem gazelles were also isolated from 2 of the \u003cem\u003eH. impeltatum\u003c/em\u003e ticks which were collected from Reem gazelle (Deposited in GenBank and were given the accession numbers PX457133 and PX457134). Six sequences from \u003cem\u003eH. impeltatum\u003c/em\u003e, three from each from Reem and Idmi gazelles were deposited in GenBank under accession numbers PX457490, PX457493 and PX457495 for Reem and PX457491, PX457492 and PX457494for Idmi gazelles. \u003cem\u003eH. impleltatum\u003c/em\u003e sequences obtained in the present study were 99% identical to DNA sequences from \u003cem\u003eH. impeltatum\u003c/em\u003e reported from Madina (ON138788.1) and Tabuk (ON138785.1) in Saudi Arabia.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHemoparasites belonging to the Genus \u003cem\u003eTheileria\u003c/em\u003e has been detected in both Reem and Idmi gazelles at KKWRC. This finding represents the first microscopic as well as molecular-based report of \u003cem\u003eTheileria\u003c/em\u003e sp. in these species of gazelles. Intraerythrocytic bodies suggestive of \u003cem\u003eTheileria\u003c/em\u003e sp. have been detected in both species of gazelles investigated. Using microscopy, the overall prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. in gazelles investigated was 18.0%, while it was found to be higher (27.9%) using molecular techniques. This report is considered the first microscopic and molecular-based report of hemoparasites in members of the Genus \u003cem\u003eGazella\u003c/em\u003e in Saudi Arabia. The sensitivity and specificity of the PCR method of detection of \u003cem\u003eTheileria\u003c/em\u003e sp. in both species of gazelles was found to be 76.74% and 83.16% respectively. Whereas the sensitivity and specificity of the direct microscopy method was found to be 49.23% and 94.05% respectively. It appeared that the PCR method is more sensitive for the detection of \u003cem\u003eTheileria\u003c/em\u003e sp. in gazelles. Similar records have been reported recently from sheep, goats and cattle [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The sensitivity and specificity for the detection of \u003cem\u003eTheileria\u003c/em\u003e spp. in sheep and goats’ samples were 26.98% and 98.31% for the blood smear method, and 73.01% and 100% for the PCR test, respectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The higher prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. detected through molecular techniques compared to microscopic examination, in several studies, as well as in this study, is likely due to the increased specificity of the PCR method, which can detect low levels of parasitemia that may not be visible microscopically. Whereas, the microscopic method depends on only small volume of the blood samples from which the smear was prepared, furthermore, if the level of parasitemia is low then lower chance of demonstrating the piroplasms in the blood smear. Direct microscopy is a valid diagnostic tool in \u003cem\u003eTheileria\u003c/em\u003e infection and was found to be valid in several studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the presence of artifacts, hemolysis of the erythrocytes, poor staining, microscopic expertise and low parasitemia may indirectly interfere with the detection of piroplasms within the erythrocytes and render diagnosis more difficult.\u003c/p\u003e \u003cp\u003eThere was no significant difference in the prevalence between males and females in gazelles at KKWRC. There is contradicting results as for the prevalence of \u003cem\u003eTheileria\u003c/em\u003e spp. in males and females. A recent study on the prevalence of \u003cem\u003eTheileria ovis\u003c/em\u003e in sheep in Saudi Arabia showed that the prevalence in males in greater than in females [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In some studies, however, the prevalence was found to be more in females compared to males [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The difference in the prevalence between males and females in farm animals may be understandable as keeping females for longer periods, hence, greater chances of exposure to ticks whereas males are managed differently and are not kept for long time in the farms. In wildlife the situation may be different and animals are bred for reintroduction purposes and the idea of keeping females for longer does not apply to wildlife. Unlike what is generally believed, the prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. in juvenile gazelles was found to be significantly higher than in adult gazelles. Similar results were obtained from some studies in domestic animals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Underdeveloped immune system of juvenile animals in addition to vaccination and weaning may be contributory factors to high prevalence of \u003cem\u003eTheileria\u003c/em\u003e sp. in juveniles compared to adult animals. The general belief was that adult animals are more prone to infection with \u003cem\u003eTheileria\u003c/em\u003e sp. which has been reported earlier in several investigations [13,33,34]. It was attributed to; that ticks are associated with adult animals for longer periods compared to young animals hence higher chances of infection.\u003c/p\u003e \u003cp\u003ePreviously, two DNA sequences in GenBank (MN209937 and MN209938) from the Chinkara (\u003cem\u003eGazella bennettii\u003c/em\u003e) from Pakistan, Punjab were found to show high identity with the sequences from both species of gazelles in the present study. Both sequences from the chinkara were 100% identical to sequences from \u003cem\u003eG. marica\u003c/em\u003e, whereas, it was 99% identical to those from \u003cem\u003eG. arabica\u003c/em\u003e. It is worth mentioning that the sequences obtained from the chinkara were labelled dama gazelle, and they were 97% identical to sequences from dama gazelle (\u003cem\u003eNanger dama\u003c/em\u003e). Four sequences were available in GenBank from dama gazelles (\u003cem\u003eN. dama\u003c/em\u003e); OP763595, OP763603, AY735115, and AY735116 were designated as \u003cem\u003eTheileria\u003c/em\u003e sp. from dama gazelle. These sequences were 96.9% to 97.7% identical to sequences obtained from gazelles in the present study and those obtained from the Chinkara. Sequences of \u003cem\u003eTheileria\u003c/em\u003e sp. from Reem, Idmi and Chinkara gazelles i.e. which belong to members of the genus \u003cem\u003eGazella\u003c/em\u003e; formed one clade which shared a common ancestor to \u003cem\u003eTheileria\u003c/em\u003e sp. from the dama gazelle which belong to the genus \u003cem\u003eNanger\u003c/em\u003e. Both clades grouped with \u003cem\u003eTheileria\u003c/em\u003e spp. reported from antelopes with significant designation of different species detected in different wildlife. From the phylogenetic tree; it appears that species of \u003cem\u003eTheileria\u003c/em\u003e infecting members of the genus \u003cem\u003eGazella\u003c/em\u003e and those infecting members of the genus \u003cem\u003eNanger\u003c/em\u003e are distinct and they shared common ancestor. \u003cem\u003eTheileria\u003c/em\u003e sp. detected in gazelles is among the benign non-transforming \u003cem\u003eTheileria\u003c/em\u003e parasites such as \u003cem\u003eT. sergenti/T. buffeli/T. orientalis\u003c/em\u003e group [35].\u003c/p\u003e \u003cp\u003eTicks which were collected from gazelles raised at KKWRC were identified as \u003cem\u003eH. impeltatum\u003c/em\u003e and the identification was confirmed molecularly. This finding indicated that the predominant type of ticks infesting gazelles is related to \u003cem\u003eH. impeltatum\u003c/em\u003e. Furthermore, finding DNA related to \u003cem\u003eTheileria\u003c/em\u003e sp. from Reem gazelles clearly demonstrated that this tick species is a potential vector for \u003cem\u003eTheileria\u003c/em\u003e sp. in Reem gazelles. The inability to detect \u003cem\u003eTheileria\u003c/em\u003e sp. from the DNA of \u003cem\u003eH. impeltatum\u003c/em\u003e ticks doesn’t mean that this tick is not a suitable vector for transmitting \u003cem\u003eTheileria\u003c/em\u003e infection in Idmi gazelles, however, some more work is required to prove the assumption or reject it. Furthermore, some of the ticks collected in the present study were dried up and the preservation of some of them was inadequate and were collected over a long period of time and kept at KKWRC. This may limit the recovery of good quality DNA from some of the ticks, therefore, affected recovery of the parasite DNA. Previously, Almahallawi and colleagues [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] reported that both \u003cem\u003eH. impeltatum\u003c/em\u003e and \u003cem\u003eH. dromedarii\u003c/em\u003e as potential vectors for \u003cem\u003eT. ovis\u003c/em\u003e from sheep in Northwestern Saudi Arabia. Omer and colleagues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] were also able to detect \u003cem\u003eT. annulata\u003c/em\u003e DNA from both \u003cem\u003eH. anatolicum\u003c/em\u003e and \u003cem\u003eH. dromedarii\u003c/em\u003e ticks from the eastern province in Saudi Arabia. Finding the possible vector incriminated in transmitting \u003cem\u003eTheileria\u003c/em\u003e sp. in gazelles raises a question of controlling such vector, furthermore it is recommended that all gazelles at KKWRC which are kept for breeding and reintroduction purposes must be screened regularly against \u003cem\u003eTheileria\u003c/em\u003e infections and animals proved positive for this parasite must be excluded from reintroduction programs and must be treated before release. At KKWRC, one of the routine practices is administering a dose of ivermectin to all handled gazelles annually, which may help in minimizing tick infestation in gazelles hence; negatively influencing the transmission of \u003cem\u003eTheileria\u003c/em\u003e infection among gazelles.\u003c/p\u003e \n\n "},{"header":"Methods","content":"\u003cp\u003e This study was conducted at the Department of Zoology, College of Science, under the permit issued by King Saud University Animal Ethics Committee (KSU-SE.23–104).\u003c/p\u003e\u003ch3\u003eGazelle species and blood collection\u003c/h3\u003e\u003cp\u003eHerds of gazelles are kept at King Khalid Wildlife Research Center (KKWRC) for breeding and reintroduction purposes. KKWRC is located 80 km north east the capital of Saudi Arabia; of Riyadh. Species of gazelles kept at KKWRC included the Arabian sand gazelle, locally know as Reem (\u003cem\u003eGazella marica\u003c/em\u003e) and the Arabian mountain gazelle, locally known as Idmi (\u003cem\u003eGazella arabica\u003c/em\u003e). The gazelles are kept in breeding pens which are 50x100 meters wide. Food and water are provided for animals in daily basis. Gazelles are subjected to routine capturing for veterinary intervention including vaccination, deworming and translocation. At the end of each breeding pen there is a triangular shape boma to facilitate the process of capturing and easy sampling. The animal record keeping system of the animals at the center are maintained using ZIMS software, where all the movements, interventions with gazelles are recorded since birth. Gazelles were clinically healthy when blood samples were collected. The collection of blood samples was performed during the routine capturing process for veterinary interventions.\u003c/p\u003e\u003cp\u003eThe sample size (n) was determined using the statistical formula by Thrusfield [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e]:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:n=\\frac{{Z}^{2}\\:P(1-P)}{{d}^{2}}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003en\u003c/em\u003e is the desired sample size; \u003cem\u003eZ\u003c/em\u003e is 95% confidence interval (1.96), \u003cem\u003eP\u003c/em\u003e is the estimated prevalence of infections and \u003cem\u003ed\u003c/em\u003e is the desired precision level (5%). Since the prevalence of \u003cem\u003eTheileria\u003c/em\u003e infections in the study area was unknown and this study was undertaken to determine the prevalence of \u003cem\u003eTheileria\u003c/em\u003e spp. infections in gazelles species at KKWRC, 13% prevalence was assumed [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDuring capturing gazelles were bled by jugular venipuncture. A volume of 5 ml of blood was collected in vacutainer tubes with EDTA as anticoagulant. Blood samples were collected from a total of 233 gazelles. Samples included 111 from Idmi gazelles and 122 from Reem gazelles. Gazelles which are less than one-year old are designated as juveniles whereas those more than one-year old are regarded as adult. Of Idmi gazelle samples; 47 were from male while 64 were female, furthermore, 82 samples were collected from adult whereas 29 were from juvenile gazelles. With regard to samples from Reem gazelles, 50 samples were from males while 72 were from females; 70 samples were from adult while 52 samples were from juveniles. Blood smears were prepared from all samples collected from gazelles, air-dried, fixed with methanol and Giemsa-stained following the standard procedures [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDNA was extracted from blood using DNeasy blood and tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA was stored at − 80°C until use. A fragment of ~ 450-base pair (bp) of the 18S small subunit ribosomal RNA (rRNA) gene was intended to amplify using the primers RLB-F 5′- GACACAGGGAGGTAGTGACAAG-3′ as a forward primer and RLB-R 5′- CTAAGAATTTCACCTCTGACAGT-3′ as a reverse primer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The region intended for amplification includes the hypervariable region (V4) of the 18S rRNA gene. The polymerase chain reaction (PCR) was set up using 2 µl of the DNA extracted from blood with 10 µM of each of the forward and reverse primers, 5 µl of 5× PCR buffer, and 0.2 µl (1 unit) of Taq DNA polymerase (Bioline, London, UK). Double distilled water was used as the negative control to detect contamination. The PCR was performed on a thermocycler (Multigene, Labnet, Maryland, USA) and the conditions for the PCR were as follows: initial denaturation cycle at 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec, and a final extension at 72°C for 5 min.\u003c/p\u003e\u003cp\u003eA total of 41 ticks were collected from 24 gazelles (9 Reem and 15 Idmi) at KKWRC. Ticks were not noticed on animals during the blood collection. They were collected at different times. Ticks were preserved in 70% ethanol and identified to the species level according to the keys given previously [38,39,40]. DNA was extracted from ticks using Qiagen extraction kit (Qiagen, Hilden, Germany) after being washed in water, same as the protocol followed for blood extraction. Extracted DNA was subjected to PCR amplification of cytochrome oxidase subunit I gene using primers, COXI-F 5′-GGAACAATATATTTAATTTTTGG-3′ as a forward primers and COXI-R 5′-ATCTATCCCTACTGTAAATATATG-3′ as a reverse primer which amplify ~ 800 bp of the COXI [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe PCR products from the amplification of 18S rRNA region from \u003cem\u003eTheileria\u003c/em\u003e spp. or COXI region from ticks were evaluated using 1.5% agarose gel (with ethidium bromide) electrophoresis and visualized with ultraviolet light using a transilluminator. Digital images were taken of the amplicons.\u003c/p\u003e\u003cp\u003eThe PCR products were sequenced following the standard procedure of Sanger sequencing at Macrogen, South Korea. All sequences were aligned analyzed and edited using MEGA 11 software [42]. Further, the phylogenetic trees were based on the comparison with sequences of \u003cem\u003eTheileria\u003c/em\u003e spp. detected in the present study and those available in GenBank. Trees were constructed using the maximum likelihood (ML) and neighbor-joining (NJ) methods of the MEGA 11 program and bootstrap analysis with 1000 replications was used to estimate the confidence of the branching patterns of the trees [42].\u003c/p\u003e\u003ch2\u003eCalculation of sensitivity and specificity\u003c/h2\u003e\u003cp\u003eThe sensitivity and specificity of the polymerase chain reaction test compared to the direct microscopy were calculated according to the following formula[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{e}\\text{n}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:\\text{%}=\\frac{\\text{P}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\text{s}\\:\\text{o}\\text{n}\\:\\text{d}\\text{i}\\text{r}\\text{c}\\text{t}\\:\\text{m}\\text{i}\\text{c}\\text{r}\\text{o}\\text{s}\\text{c}\\text{o}\\text{p}\\text{y}\\:\\text{a}\\text{n}\\text{d}\\:\\text{P}\\text{C}\\text{R}\\:\\:}{\\text{P}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\text{s}\\:\\text{o}\\text{n}\\:\\text{P}\\text{C}\\text{R}}\\text{X}\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{p}\\text{e}\\text{c}\\text{i}\\text{f}\\text{i}\\text{c}\\text{i}\\text{t}\\text{y}\\:\\text{%}=\\frac{\\text{N}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\text{s}\\:\\text{o}\\text{n}\\:\\text{d}\\text{i}\\text{r}\\text{e}\\text{c}\\text{t}\\:\\text{m}\\text{i}\\text{c}\\text{r}\\text{o}\\text{s}\\text{c}\\text{o}\\text{p}\\text{y}\\:\\text{a}\\text{n}\\text{d}\\:\\text{P}\\text{C}\\text{R}\\:\\:}{\\text{N}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\text{s}\\:\\text{o}\\text{n}\\:\\text{P}\\text{C}\\text{R}}\\text{X}\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eDifferences in the prevalence rates of \u003cem\u003eTheileria\u003c/em\u003e specie in different gazelle species as well as from males, females, and age groups were tested using χ2 -test, which was performed in SPSS 22.0. The significance was set at 95%.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors wish to thank KKWRC staff for their cooperation and help with sample collection and professional handling of gazelles. This work was funded by the Ongoing Research Funding program (ORF-2025-94), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.A., A.N.A., and O.B.M. conceived the study, S.A., S.A.O. and E.A. conducted laboratory work, A.A., A.N.A., S.A., and O.B.M. performed data analysis. S.A.O., A.A. E.A., and O.B.M. drafted the manuscript. All authors reviewed and commented on the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData used in this study are available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e- Norval, R. A. I., Perry, B. D. \u0026amp; Young, A. S. The epidemiology of theileriosis in Africa. ILRI (1992). (aka ILCA and ILRAD).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e- Mans, B. J., Pienaar, R. \u0026amp; Latif, A. A. A review of \u003cem\u003eTheileria\u003c/em\u003e diagnostics and epidemiology. \u003cem\u003eInt. J. Parasitology: Parasites Wildl.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (1), 104\u0026ndash;118 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e- Sivakumar, T., Hayashida, K., Sugimoto, C. \u0026amp; Yokoyama, N. Evolution and genetic diversity of \u003cem\u003eTheileria\u003c/em\u003e. \u003cem\u003eInfect. Genet. 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Evol.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e (7), 3022\u0026ndash;3027 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gazella marica, Gazella arabica, Theileria sp., Molecular characterization, Saudi Arabia","lastPublishedDoi":"10.21203/rs.3.rs-8348152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8348152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWildlife conservation in Saudi Arabia is gaining much attention from the authorities. Habitat destruction, climate change, and human encroachment, as well as disease outbreaks, negatively impact conservation efforts. Hemoparasites among endangered Saudi wildlife have not been studied before. The aim of this study is to investigate the prevalence of hemoparasites among two endangered gazelle species {\u003cem\u003eGazella marica\u003c/em\u003e (Reem) and \u003cem\u003eGazella arabica\u003c/em\u003e (Idmi)} raised at King Khalid Wildlife Research Center (KKWRC) for breeding and reintroduction purposes. A total of 233 blood samples were collected from the jugular veins of gazelles and examined microscopically for the detection of piroplasm or any relevant blood parasites. Ticks were collected from some gazelles at KKWRC to identify them and see their potential role in the transmission of hemoparasites. DNA was extracted from the collected blood and ticks for the detection of hemoparasites, and amplified targeting the partial 18S rRNA gene. The prevalence of intraerythrocytic bodies suggestive of \u003cem\u003eTheileria\u003c/em\u003e sp. was detected in 41 (17.9%) gazelles of both species, whereas PCR products of \u003cem\u003eTheileria\u003c/em\u003e sp. DNA were detected in 65 (27.9%) gazelles. The sensitivity of the molecular technique compared to the direct microscopy was 76.74%, whereas the specificity was 83.16%. Ticks were morphologically identified as \u003cem\u003eHyalomma impeltatum\u003c/em\u003e and confirmed with molecular techniques. Sequences obtained from \u003cem\u003eTheileria\u003c/em\u003e sp. from both Reem and Idmi gazelles were identical with only one mutation (transition) at position 140, where it was a T in Reem sequences while it was a C consistently in all sequences obtained from Idmi gazelles. Phylogenetic studies indicated that sequences obtained from gazelles in the present study group were related to \u003cem\u003eTheileria\u003c/em\u003e sp. which was isolated from the Chinkara gazelle from Pakistan and related to isolates from the dama gazelle. \u003cem\u003eTheileria\u003c/em\u003e sp. DNA was amplified from two ticks collected from Reem gazelles, and they were found to be identical to the \u003cem\u003eTheileria\u003c/em\u003e sp. sequences obtained from Reem gazelles. This finding suggested that \u003cem\u003eH. impeltatum\u003c/em\u003e can potentially be a vector for \u003cem\u003eTheileria\u003c/em\u003e sp. in Reem gazelles. No \u003cem\u003eTheileria\u003c/em\u003e sp. was amplified from ticks collected from Idmi gazelles. The prevalence of \u003cem\u003eTheileria\u003c/em\u003e infection in gazelles at KKWRC in Saudi Arabia was reported for the first time. The possible vector responsible of the transmission was also deciphered.\u003c/p\u003e","manuscriptTitle":"Prevalence and molecular characterization of Theileria sp. from endangered Arabian gazelles in Saudi Arabia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 18:42:17","doi":"10.21203/rs.3.rs-8348152/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-16T23:51:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T21:00:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T13:07:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T02:40:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334795355165105416347009560012156061937","date":"2025-12-23T10:50:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302049370568618795220901128911094076063","date":"2025-12-22T15:51:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223587237783527215605581554747404812466","date":"2025-12-22T09:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274034176264890564142209024613492128239","date":"2025-12-21T11:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-19T10:40:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-15T11:16:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T11:13:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-12T17:45:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd2fe345-5d22-47d6-9519-b46bc97026de","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":60020797,"name":"Biological sciences/Ecology"},{"id":60020798,"name":"Earth and environmental sciences/Ecology"},{"id":60020799,"name":"Biological sciences/Genetics"},{"id":60020800,"name":"Biological sciences/Microbiology"},{"id":60020801,"name":"Biological sciences/Molecular biology"},{"id":60020802,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-02-23T16:00:16+00:00","versionOfRecord":{"articleIdentity":"rs-8348152","link":"https://doi.org/10.1038/s41598-026-41035-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-22 15:57:10","publishedOnDateReadable":"February 22nd, 2026"},"versionCreatedAt":"2025-12-22 18:42:17","video":"","vorDoi":"10.1038/s41598-026-41035-y","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41035-y","workflowStages":[]},"version":"v1","identity":"rs-8348152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8348152","identity":"rs-8348152","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}