Molecular Based Evidence, Genetic Profile and Risk Factors of Theileria spp. Infection in Caprine in Sulaymaniyah Province /Iraq

Molecular Based Evidence, Genetic Profile and Risk Factors of Theileria spp. Infection in Caprine in Sulaymaniyah Province /Iraq | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Based Evidence, Genetic Profile and Risk Factors of Theileria spp. Infection in Caprine in Sulaymaniyah Province /Iraq Shadan Hassan Abdullah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7055150/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Theileriosis is a significant protozoal infection affecting livestock health status and productivity worldwide. Study aimed to detect Theileria infection in native goats from Sulaymaniyah province, Northern region of Iraq. For current study random blood samples were taken from clinically healthy goats belonging to mixed small ruminant flocks from four regions of Sulaymaniyah province. PCR based on the gene coding for 18S rRNA was applied for detecting Theileria parasite from goat blood samples, followed by sequencing of two isolates and defining the genetic diversity. Effects of various risk factors on theileriosis prevalence also evaluated. The overall infection rate of caprine theileriosis was 55.83%. Higher infection rate of 61.67% was reported from Sharazoor in compare to the low infection rate of 51.67% from Sitak. Risk factors including age, flock size and the tick burden represented significant determinants (p < 0.005), although herd composition no significantly effects on Theileria infection. Blast analysis of study sequences established the detection of T. ovis , and the constructed phylogenetic tree defined that current isolates PV575340.1 and PV575340.1 were clustered to gather with previously detected T. ovis isolates. The study provides an insight to the existence of Theileria species among goat population with higher infection rates in spite of observing of no obvious clinical signs, therefore attention directed to word the animal status should be highlighted as they are associated with financial and health impacts, along with the role of epidemiological risk factors on the incidence rate should not be neglected. Theileria goat theileriosis Phylogeny Sulaymaniyah Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Theileriosis is a widespread tick-borne hemoprotozoan disease that affects many wild and domestic animals worldwide (Islam et al. 2021). Theileria species is an obligate intracellular protozoan parasite of the phylum Apicomplexa(Sitotaw et al. 2014). In small ruminant theileriosis caused by several Theileria species including T. lestoquardi, T. luwenshuni, T. uilenbergi, T. ovis, T. recondita, T. separata, Theileria sp. MK and Theileria sp. OT3 . Among these, T. ovis causes sheep and goats to contract benign theileriosis, a subclinical infection(Udonsom et al. 2022). Fever, lymphadenopathy, icterus, hemorrhage, and diarrhea are possible symptoms of clinical ovine theileriosis, but intermittent fever, wasting, anemia, and appetite loss are associated with long-lasting infection (Stuen 2020). Respiratory signs of cough, nasal discharge, shortness of breath have been also reported (Hassen and Meerkhan 2020). In addition to significant financial losses, theileriosis associated with high mortality rates, decreased production, costs associated with the treatment of diseased animals and the necessity for effective vector control strategies (Al-Hamidhi et al. 2021). Current studies have demonstrated the disease's ongoing prevalence and effects on domestic ruminant output, particularly when contrasted with cattle(Riaz et al. 2023). It is crucial to identify the risk factors that lead to theileriosis in order to control the disease globally (Islam et al. 2021). Where the vector is present, the carrier state significantly increases the chance of disease transmission to adjacent areas free of theileriosis. chronic asymptomatic carriage of Theileria spp. (Gharbi et al. 2011). Goats are the important species of food animals that possess a special ability to adjust to difficult situations with multiple economically relevant traits such as short reproduction cycles, higher rates of reproduction and high consumer acceptability worldwide (Tawfeeq and Albakri 2024). Molecular methods emerge as a valuable tool for detecting apicomplexan parasites, their high sensitivity and specificity allowed to identify parasitic species in tick vectors and animals, even under difficult conditions like mixed infections or low parasite burdens (Rahmani-Varmale et al. 2019). Often, the 18S small subunit ribosomal RNA (18S rRNA) gene is used to identify Theileria spp. (Cao et al. 2013). Wide- range studies have been carried out in various parts of Iraq regarding Theileriosis in sheep and cattle, while paucity of information about haemoparasitic infection in caprine and their impacts on health and productivity necessitate to design such study, in order to ascertain the occurrence of the Theileria infection in indigenous goats, via applying of PCR based molecular approach, in relation to risk factors evaluation, as well as assessing of their genetic diversity based on the amplification of 18S rRNA. Materials and Methods Study design and sampling condition The study population was goats, a total of 240 indigenous goats were selected randomly from 10 selected small ruminant farms, whose owner show agreement to participate in this study. The semi-extensive technique used to keep the animals was typified by their free grazing on pastures. No specific health problems were observed in selected animals, goats more than 6 months till 3 years of ages were involved irrespective of sex and breed. During the period from April to October 2023 sampling was performed and the enrolled goats were belonging to four districts of Sulaymaniyah province, Northern region of Iraq. Data on the risk factors was collected via a questionnaire including age, flock size, herd composition and tick burden. The selected animals were categorized into two age groups and they were examined for ticks throughout their entire bodies. Blood sample collection From the jugular vein of a selected animals, about 4ml of blood were drawn, collected in tubes with EDTA using disposable syringes and transported to the Vetyerinary Medicine College laboratory at University of Sulaimani. Blood samples were stored at – 80 until DNA extraction, subsequent to the preparing of thin blood smears from each herd for microscopic examination. After being fixed with methanol for five minutes, the dried blood smears were stained using the Giemsa stain procedure. Examination under oil-immersion microscopic fields have been performed for the presence of Theileria spp. piroplasmic stages (Altay et al., 2005). DNA extraction and PCR amplification Genomic DNA was extracted from 200µ using DNA extraction kit (GeNet Bio, South Korea) following the manufacture's instruction, the extracted DNA was kept at – 20°C for PCR. PCR analysis was carried out on 250 extracted DNA aliquots using primer sets targeting 18SrRNA for Thieleria spp. by amplifying 1700 bp with previously designed primer sets of Forward primer 5’-AACCTGGTTGATCCTGCCAG-3’ and Reverse primer5’-AAACCTTGTTACGACTTCTC-3’ (Heidarpour Bami et al. 2009), with modification. The amplification was performed in automated Prime Thermal cycler in a total reaction volume of 20µ with thermoprofiles that started with initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 30 s with a final extension step of 72°C for 5 min. All amplified products were subjected to 1% agarose gel and visualized under UV illuminator. Sequencing and Phylogenetic To verify Theileria spp. two positive samples were selected and sequenced following the standard procedure of Sanger sequencing at Macrogen, South Korea. for confirming the recovered Theielria species, the identified partial sequences were checked using BLASTn homology through NCBI (The National Center for Biotechnology Information: http://blast.ncbi.nlm.nih.gov/Blast.cgi website. Confirmed partial gene of current sequences were submitted in GenBank and received the accession numbers of PV575340.1 and PV575341.1. The multiple alignment of nucleotides and phylogenetic analysis were achieved by Mega 10 software using Neighbor joining method (Saitou and Nei 1987), with the tree stability estimation by a boot strap analysis for 1000 replications (Felsenstien 1985). Data analysis The Chi square test of significance has been used for finding the association between the variables, using the SPSS statistical program, P-value < 0.05 were considered statistically significant. Results The data represented existence of Theileria pathogens with higher prevalence rates of 55.83% Fig. 1 . Theileria spp. was detected from all examined flocks Fig. 2, and the higher infection rate was reported among examined animals belong to Sharazoor region 61.67% followed by Qaradagh 56.92%, then Tanjaro 52.73% and 51.67% was the lowest infection rate reported from Sitak, with no significant association between areas regarding the infection prevalence rate as it was illustrated in Table 1 . Table 1 Frequency of Theileria infection among goats’ population in Sulaymaniyah Province. Locations Total examined no. No. of positive goats Frequency of infection Chi - square value [P- value] Qaradagh 65 37 56.92 1.49 [0.6] Sharazoor 60 37 61.67 Sitak 60 31 51.67 Tanjaro 55 29 52.73 Total 240 134 55.83 from the goat’s blood samples. A fragment of 1700 bp Theileria spp. (arrows) in blood (18S ribosomal RNA gene) was detected in Lane 1, 2, smear of infected goat. Giemsa stain (100X). 4, 5 and 6 representing positive samples, Lane M: DNA marker, and Lane 3 represent the negative sample. Risk factor evaluation including the herd size, tick burden being with the age of infected host represented significantly (p ≤ 0.05) association with the occurrence of theileriosis in goats, however herd composition and coexistence of sheep with goats has no significant effect on incidence rate of Theileria infection in rearing goats in the study areas showed in Table 2 . Table 2 Frequency of Theileria infection in relation to epidemiological risk factors in goats from Sulaymaniyah province. Determinants Variables No. of examined animals No. of positive animals Frequency of positive Chi - square value [P- value] Age ≤ 1 year 98 46 34.33 5.313 [0.02*] > 1 year 142 88 65.67 Herd size ≤ 50 47 20 14.93 4.179 [0.04*] > 50 193 114 85.07 Co-existence with sheep Yes 184 98 73.13 2.116 [0.14] No 56 36 27.48 Tick burden present 173 120 89.55 46.00 [0.00*] absent 67 14 10.45 Total 240 134 55.83 To conduct phylogenetic analysis, the neighbor joining method was used. Phylogenetic analyses revealed that the detected Theileria spp. is genetically related to T. ovis . Through BLASTn analysis of the partial 18S rRNA gene sequence of T. ovis , high identity of 97–99% was observed to be shared between the study sequence isolates with the available isolates in GenBank database from different countries. Through constructed phylogeny topology of T. ovis tree was defined and revealed close relationship among isolates PV575340.1 and PV575341.1 of current study with other T. ovis isolates previously reported in Iraq (OR854547.1), Turkey (AY508454.1, AY508457.1 and MN493111.1), India (MW440586.1), China (PV202473.1) and France (EU622911.1) as they were clustered together Fig. 3 . The nucleotide sequence identity value of T. ovis of current study isolated sequences PV575340.1 and PV575341.1 was 100%, and 99% with previously published sequence isolates (AY508457.1, EU622911.1, PV202473.1, MN544931.1, JQ737135.1, MG738321.1 and MW440584.1) Fig. 4 Discussion Goats are one of the important livestock animals as providing source human foods. In current study by applying of PCR procedure Theileriosis in caprine was detected in higher frequency rate of 55.83% which is aligns with infection rate of 52.60% from another region of Iraq (Tawfeeq and Albakri 2024) .The recognized current infection rate of Thielria pathogen 55.83% in goats was exceeded what were reported from Saudi Arabia 57.8% (Metwally et al. 2021), Iran 38% (Habibi et al. 2024), Pakistan 30% (Arif et al. 2023), Thailand 10.30% (Udonsom et al. 2022), and in Bangladesh 8.50% (Islam et al. 2021), even though higher infection rates of 85.3% and 86.8%, from Iran one of the neighboring region to Sulaymaniyah province was reported by (Rahmani-Varmale et al. 2019; Rahrvani et al. 2023). Geographical diversity and climate of different study areas and variation in potential mechanical and biological vectors are remarkable factors associated with differences in reported prevalence rates (Rahman et al. 2022). Despite detecting the high infection rate of theileriosis, no obvious clinical signs were not observed from the infected goats. This can be a sign of endemicity of theileriosis in the study area. Moreover, in regions where the disease is thought to be endemic, persistent infections, a characteristic of natural infections (Stuen 2020). However, it is important to consider the impact of subclinical infections because different species and genotypes have different levels of pathogenicity depending on the host. (Sivakumar et al. 2014). Theileria ovis is the primary and prevalent causes of theileriosis small ruminates, result in substantial financial losses for sheep and goats in tropical and sub-tropical areas (Naz et al. 2012). Despite not being as dangerous as T. lestoquardi , T. ovis infection can cause fever, increasing weight loss, decreased productivity, and even death (Tumwebaze et al. 2020). Various factors associated with variation in occurrence rate tick borne pathogens including Theileria spp. for instance the host, age, climate, systems for management, density of tick infestation, immunity of the host, size of the sample, sampling time and the detection methods (Altay et al. 2024). The reported higher infection rate in current study might related to the proper sampling period, during April to October which is defined as tick activation period. In general, tick activation peaks between spring and fall (Dumanli et al. 2012). The wide-ranging system of rearing animals used for pasture grazing has a direct impact on diseases carried by ticks because it increases the likelihood of exposure to natural vectors (Rahman et al. 2022). In addition, trans-boundary movement of animals might be another factor between different neighboring regions were Theileria is endemic, related to that (Tawfeeq and Albakri 2024) reported higher infection rate of theileriosis among imported goats. Among the risk factors, age of infected host, flock size and tick burden were appeared to be significantly associated (p ≤ 0.05), although herd composition, co-existence of goats with sheep represented non-significantly associated (p > 0.05) with occurrence of caprine theileriosis. Current study defines significant increase in Theileria infection rate with the age of infected animals (p < 0.02), the higher infection rate of 65.67% was reported from aged goats in compared to younger animals, this finding consistence with the previous results by (Shi et al. 2019; Islam et al. 2021; Metwally et al. 2021 Zhou et al. 2023) were they observed an increase in theileriosis occurrence rate with the age, which represent an association with the tick contact period extension. However, the obtained data by (Niaz et al. 2021; Riaz et al. 2024) disagreed with current finding in which high infection rate was reported from younger animals. Data also represented that goats belong to large sized flocks are significantly prone to Theileria infection 85.07% (p < 0.04) in compare to animals reared in small sized herds 14.93%, similarly (Islam et al. 2021) reported lower infection rate in small sized than medium sized small ruminant flocks. Tick infestation reported as a significant risk factor in current study that associated with significant higher infection rate of 89.55% (p < 0.00) compared to those with no ticks, in an agreement to present finding similar data was stated by (Niaz et al. 2021;Nassiba et al. 2024). The incidence of theileriosis in small ruminants might be influenced by the distribution and abundance of tick species in various geographical areas (Kumar et al. 2022; Prajapati et al. 2023). Study data represented non-significant higher infection rates among goats that are co-existent with sheep 73.13%, which is align with obtained results by (Ashraf et al. 2024; Nassiba et al. 2024) in which prevalence Theileria was not associated with herd composition. Riaz et al. (2024) reported higher infection rate of Theileriosis in mixed species herd in compare to goat -only herds. Studies performed on both sheep and goats find out higher infection rate among goats which indicated that infected sheep might be an existence source for tick vector among herds, (Arif et al., 2023) reported higher infection rate of theileriosis among sheep than goats. The constructed phylogenetic tree based on 18SrRNA gene by the Neighbor Joining test using the Mega 10 software, revealed that current Theileria isolates PV575340.1 and PV575340.1 were represented higher degree of similarity to and are clusters together with T. ovis previously deposited in GenBank data base. Topology of T. ovis tree represented close relationship between study isolates and the isolates from Iraq (OR854547.1), Turkey (AY508454.1, AY508457.1 and MN493111.1), India (MW440586.1), China (PV202473.1) and France (EU622911.1) as they are clustered to gather in one clade. Conclusion The study results illustrate notable infection by Thielrias pp. from clinically healthy goats in Sulaymaniyah province, which reveal the endemicity of available pathogens. Significant increasing in the number of importing small ruminants from neighboring regions might increase the chance for spreading the blood pathogens. T. ovis was the known species through sequence analysis which is T. ovis emerges as the main causes of caprine theileriosis in the area of study. Risk factors evaluation for disease expression revealed significant association for some of them. Large-scale studies must be conducted to define other circulation Theileria spp.as well as to investigate the tick vectors transmitting T. ovis . Further studies to assess risk variables for illness manifestation linked to tick-borne pathogen incidence is an essential step for improving the production. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose Ethics approval The study design was reviewed and approved by Ethical Committee of Veterinary Medicine College, University of Sulaimani. Informed consent was obtained from the owners for the participation of their animals in this study. Funding The author declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions The author contributed to the study conception and design. Material preparation, data collection and analysis, and manuscript writing by Shadan Hassan Abdullah also read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this published article. Nucleotide sequences were submitted to the GenBank database under the accession numbers PV575340.1 and PV575341.1 References Al-Hamidhi S, Elshafie EI Yaghfoori S et al (2021). 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BMC Veterinary Research 19(1). https://doi.org/10.1186/s12917-023-03766-2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7055150","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483255626,"identity":"3c7fda4d-76ca-44f8-a153-66329a50fa44","order_by":0,"name":"Shadan Hassan Abdullah","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8625-2193","institution":"University of Sulaimani","correspondingAuthor":true,"prefix":"","firstName":"Shadan","middleName":"Hassan","lastName":"Abdullah","suffix":""}],"badges":[],"createdAt":"2025-07-05 23:30:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7055150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7055150/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86636941,"identity":"6b3ae9d5-51fb-4a47-97c6-6281d8b7a3a9","added_by":"auto","created_at":"2025-07-14 07:25:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165364,"visible":true,"origin":"","legend":"\u003cp\u003ePCR amplification of \u003cem\u003eTheileria\u003c/em\u003e spp. obtained\u003c/p\u003e\n\u003cp\u003efrom the goat’s blood samples. A fragment of 1700 bp\u003c/p\u003e\n\u003cp\u003e(18S ribosomal RNA gene) was detected in Lane 1, 2,\u003c/p\u003e\n\u003cp\u003e4, 5 and 6 representing positive samples, Lane M:\u003c/p\u003e\n\u003cp\u003eDNA marker, and Lane 3 represent the negative sample.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7055150/v1/bf0ee1a00ac1c1dc075bc23e.png"},{"id":86636946,"identity":"9c10ef54-0bbb-46f8-a457-577087577f5f","added_by":"auto","created_at":"2025-07-14 07:25:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326455,"visible":true,"origin":"","legend":"\u003cp\u003eIntra -erythrocytic stages of \u003cem\u003eTheileria\u003c/em\u003e spp. (arrows) in blood smear of infected goat. Giemsa stain (100X).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7055150/v1/d55fba320ca6bf1455e80083.png"},{"id":86637235,"identity":"856c8b9b-305d-4970-bb43-712452aa8006","added_by":"auto","created_at":"2025-07-14 07:33:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167581,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenic analysis of \u003cem\u003eTheileria ovis\u003c/em\u003eisolated from current study with previously documented isolated from database based on 18SrRNA gene partial sequence. the tree was constructed by maximum likelihood with the Kimura-2 method and 1000 bootstrap replications.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7055150/v1/2a005e1ca4a851482dbd38aa.png"},{"id":86636945,"identity":"971b3211-6dc6-4dbf-8424-787324832f32","added_by":"auto","created_at":"2025-07-14 07:25:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":824394,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide sequence alignment results of \u003cem\u003eTheileria ovis\u003c/em\u003e 18S rRNA gene isolate from goat in current study (PV575340.1, PV575341.1) and reported sequences from the database.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7055150/v1/e8820f90a4c2886fdf9215c0.png"},{"id":94598973,"identity":"3169224c-55c2-4324-a3ba-2361b56887bd","added_by":"auto","created_at":"2025-10-28 18:59:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2281053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7055150/v1/98ff8c33-aeee-411d-a339-df0fe6b40115.pdf"}],"financialInterests":"","formattedTitle":"Molecular Based Evidence, Genetic Profile and Risk Factors of Theileria spp. Infection in Caprine in Sulaymaniyah Province /Iraq","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTheileriosis is a widespread tick-borne hemoprotozoan disease that affects many wild and domestic animals worldwide (Islam et al. 2021). \u003cem\u003eTheileria\u003c/em\u003e species is an obligate intracellular protozoan parasite of the phylum Apicomplexa(Sitotaw et al. 2014).\u003c/p\u003e\u003cp\u003eIn small ruminant theileriosis caused by several \u003cem\u003eTheileria\u003c/em\u003e species including \u003cem\u003eT. lestoquardi, T. luwenshuni, T. uilenbergi, T. ovis, T. recondita, T. separata, Theileria sp. MK\u003c/em\u003e and \u003cem\u003eTheileria\u003c/em\u003e sp. \u003cem\u003eOT3\u003c/em\u003e. Among these, \u003cem\u003eT. ovis\u003c/em\u003e causes sheep and goats to contract benign theileriosis, a subclinical infection(Udonsom et al. 2022). Fever, lymphadenopathy, icterus, hemorrhage, and diarrhea are possible symptoms of clinical ovine theileriosis, but intermittent fever, wasting, anemia, and appetite loss are associated with long-lasting infection (Stuen 2020). Respiratory signs of cough, nasal discharge, shortness of breath have been also reported (Hassen and Meerkhan 2020).\u003c/p\u003e\u003cp\u003eIn addition to significant financial losses, theileriosis associated with high mortality rates, decreased production, costs associated with the treatment of diseased animals and the necessity for effective vector control strategies (Al-Hamidhi et al. 2021).\u003c/p\u003e\u003cp\u003eCurrent studies have demonstrated the disease's ongoing prevalence and effects on domestic ruminant output, particularly when contrasted with cattle(Riaz et al. 2023). It is crucial to identify the risk factors that lead to theileriosis in order to control the disease globally (Islam et al. 2021).\u003c/p\u003e\u003cp\u003eWhere the vector is present, the carrier state significantly increases the chance of disease transmission to adjacent areas free of theileriosis. chronic asymptomatic carriage of \u003cem\u003eTheileria\u003c/em\u003e spp. (Gharbi et al. 2011).\u003c/p\u003e\u003cp\u003eGoats are the important species of food animals that possess a special ability to adjust to difficult situations with multiple economically relevant traits such as short reproduction cycles, higher rates of reproduction and high consumer acceptability worldwide (Tawfeeq and Albakri 2024).\u003c/p\u003e\u003cp\u003eMolecular methods emerge as a valuable tool for detecting apicomplexan parasites, their high sensitivity and specificity allowed to identify parasitic species in tick vectors and animals, even under difficult conditions like mixed infections or low parasite burdens (Rahmani-Varmale et al. 2019). Often, the 18S small subunit ribosomal RNA (18S rRNA) gene is used to identify \u003cem\u003eTheileria spp.\u003c/em\u003e (Cao et al. 2013).\u003c/p\u003e\u003cp\u003eWide- range studies have been carried out in various parts of Iraq regarding Theileriosis in sheep and cattle, while paucity of information about haemoparasitic infection in caprine and their impacts on health and productivity necessitate to design such study, in order to ascertain the occurrence of the \u003cem\u003eTheileria\u003c/em\u003e infection in indigenous goats, via applying of PCR based molecular approach, in relation to risk factors evaluation, as well as assessing of their genetic diversity based on the amplification of 18S rRNA.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eStudy design and sampling condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study population was goats, a total of 240 indigenous goats were selected randomly from 10 selected small ruminant farms, whose owner show agreement to participate in this study. The semi-extensive technique used to keep the animals was typified by their free grazing on pastures. No specific health problems were observed in selected animals, goats more than 6 months till 3 years of ages were involved irrespective of sex and breed. During the period from April to October 2023 sampling was performed and the enrolled goats were belonging to four districts of Sulaymaniyah province, Northern region of Iraq. Data on the risk factors was collected via a questionnaire including age, flock size, herd composition and tick burden. The selected animals were categorized into two age groups and they were examined for ticks throughout their entire bodies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBlood sample collection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom the jugular vein of a selected animals, about 4ml of blood were drawn, collected in tubes with EDTA using disposable syringes and transported to the Vetyerinary Medicine College laboratory at University of Sulaimani. Blood samples were stored at \u0026ndash; 80 until DNA extraction, subsequent to the preparing of thin blood smears from each herd for microscopic examination. After being fixed with methanol for five minutes, the dried blood smears were stained using the Giemsa stain procedure. Examination under oil-immersion microscopic fields have been performed for the presence of \u003cem\u003eTheileria\u003c/em\u003e spp. piroplasmic stages (Altay et al., 2005).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and PCR amplification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from 200\u0026micro; using DNA extraction kit (GeNet Bio, South Korea) following the manufacture's instruction, the extracted DNA was kept at \u0026ndash; 20\u0026deg;C for PCR. PCR analysis was carried out on 250 extracted DNA aliquots using primer sets targeting 18SrRNA for \u003cem\u003eThieleria\u003c/em\u003e spp. by amplifying 1700 bp with previously designed primer sets of Forward primer 5\u0026rsquo;-AACCTGGTTGATCCTGCCAG-3\u0026rsquo; and Reverse primer5\u0026rsquo;-AAACCTTGTTACGACTTCTC-3\u0026rsquo; (Heidarpour Bami et al. 2009), with modification. The amplification was performed in automated Prime Thermal cycler in a total reaction volume of 20\u0026micro; with thermoprofiles that started with initial denaturation at 94\u0026deg;C for 5 min, followed by 30 cycles at 94\u0026deg;C for 30 s, 52\u0026deg;C for 30 s and 72\u0026deg;C for 30 s with a final extension step of 72\u0026deg;C for 5 min. All amplified products were subjected to 1% agarose gel and visualized under UV illuminator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequencing and Phylogenetic\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify \u003cem\u003eTheileria\u003c/em\u003e spp. two positive samples were selected and sequenced following the standard procedure of Sanger sequencing at Macrogen, South Korea. for confirming the recovered \u003cem\u003eTheielria\u003c/em\u003e species, the identified partial sequences were checked using BLASTn homology through NCBI (The National Center for Biotechnology Information: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e website. Confirmed partial gene of current sequences were submitted in GenBank and received the accession numbers of PV575340.1 and PV575341.1.\u003c/p\u003e\u003cp\u003eThe multiple alignment of nucleotides and phylogenetic analysis were achieved by Mega 10 software using Neighbor joining method (Saitou and Nei 1987), with the tree stability estimation by a boot strap analysis for 1000 replications (Felsenstien 1985).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe Chi square test of significance has been used for finding the association between the variables, using the SPSS statistical program, P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe data represented existence of \u003cem\u003eTheileria\u003c/em\u003e pathogens with higher prevalence rates of 55.83% Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eTheileria\u003c/em\u003e spp. was detected from all examined flocks Fig.\u0026nbsp;2, and the higher infection rate was reported among examined animals belong to Sharazoor region 61.67% followed by Qaradagh 56.92%, then Tanjaro 52.73% and 51.67% was the lowest infection rate reported from Sitak, with no significant association between areas regarding the infection prevalence rate as it was illustrated in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eFrequency of \u003cem\u003eTheileria\u003c/em\u003e infection among goats\u0026rsquo; population in Sulaymaniyah Province.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocations\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal examined no.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo. of positive goats\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFrequency of infection\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eChi - square value\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e[P- value]\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQaradagh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e56.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003cp\u003e[0.6]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSharazoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e61.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSitak\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTanjaro\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e240\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e134\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e55.83\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003efrom the goat\u0026rsquo;s blood samples. A fragment of 1700 bp \u003cem\u003eTheileria\u003c/em\u003e spp. (arrows) in blood\u003c/p\u003e\u003cp\u003e(18S ribosomal RNA gene) was detected in Lane 1, 2, smear of infected goat. Giemsa stain (100X).\u003c/p\u003e\u003cp\u003e4, 5 and 6 representing positive samples, Lane M:\u003c/p\u003e\u003cp\u003eDNA marker, and Lane 3 represent the negative sample.\u003c/p\u003e\u003cp\u003eRisk factor evaluation including the herd size, tick burden being with the age of infected host represented significantly (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) association with the occurrence of theileriosis in goats, however herd composition and coexistence of sheep with goats has no significant effect on incidence rate of \u003cem\u003eTheileria\u003c/em\u003e infection in rearing goats in the study areas showed in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eFrequency of \u003cem\u003eTheileria\u003c/em\u003e infection in relation to epidemiological risk factors in goats from Sulaymaniyah province.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDeterminants\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo. of examined\u003c/p\u003e\u003cp\u003eanimals\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo. of positive animals\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFrequency of positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eChi - square value\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e[P- value]\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;1 year\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e34.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e5.313\u003c/p\u003e\u003cp\u003e[0.02*]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1 year\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHerd size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e4.179\u003c/p\u003e\u003cp\u003e[0.04*]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e193\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCo-existence with sheep\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e73.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.116\u003c/p\u003e\u003cp\u003e[0.14]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e27.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTick burden\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e89.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e46.00\u003c/p\u003e\u003cp\u003e[0.00*]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eabsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e240\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e134\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e55.83\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo conduct phylogenetic analysis, the neighbor joining method was used. Phylogenetic analyses revealed that the detected \u003cem\u003eTheileria\u003c/em\u003e spp. is genetically related to \u003cem\u003eT. ovis\u003c/em\u003e. Through BLASTn analysis of the partial 18S rRNA gene sequence of \u003cem\u003eT. ovis\u003c/em\u003e, high identity of 97\u0026ndash;99% was observed to be shared between the study sequence isolates with the available isolates in GenBank database from different countries. Through constructed phylogeny topology of \u003cem\u003eT. ovis\u003c/em\u003e tree was defined and revealed close relationship among isolates PV575340.1 and PV575341.1 of current study with other \u003cem\u003eT. ovis\u003c/em\u003e isolates previously reported in Iraq (OR854547.1), Turkey (AY508454.1, AY508457.1 and MN493111.1), India (MW440586.1), China (PV202473.1) and France (EU622911.1) as they were clustered together Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe nucleotide sequence identity value of \u003cem\u003eT. ovis\u003c/em\u003e of current study isolated sequences PV575340.1 and PV575341.1 was 100%, and 99% with previously published sequence isolates (AY508457.1, EU622911.1, PV202473.1, MN544931.1, JQ737135.1, MG738321.1 and MW440584.1) Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGoats are one of the important livestock animals as providing source human foods. In current study by applying of PCR procedure Theileriosis in caprine was detected in higher frequency rate of 55.83% which is aligns with infection rate of 52.60% from another region of Iraq (Tawfeeq and Albakri 2024) .The recognized current infection rate of \u003cem\u003eThielria\u003c/em\u003e pathogen 55.83% in goats was exceeded what were reported from Saudi Arabia 57.8% (Metwally et al. 2021), Iran 38% (Habibi et al. 2024), Pakistan 30% (Arif et al. 2023), Thailand 10.30% (Udonsom et al. 2022), and in Bangladesh 8.50% (Islam et al. 2021), even though higher infection rates of 85.3% and 86.8%, from Iran one of the neighboring region to Sulaymaniyah province was reported by (Rahmani-Varmale et al. 2019; Rahrvani et al. 2023).\u003c/p\u003e\u003cp\u003eGeographical diversity and climate of different study areas and variation in potential mechanical and biological vectors are remarkable factors associated with differences in reported prevalence rates (Rahman et al. 2022).\u003c/p\u003e\u003cp\u003eDespite detecting the high infection rate of theileriosis, no obvious clinical signs were not observed from the infected goats. This can be a sign of endemicity of theileriosis in the study area. Moreover, in regions where the disease is thought to be endemic, persistent infections, a characteristic of natural infections (Stuen 2020). However, it is important to consider the impact of subclinical infections because different species and genotypes have different levels of pathogenicity depending on the host. (Sivakumar et al. 2014).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTheileria ovis\u003c/em\u003e is the primary and prevalent causes of theileriosis small ruminates, result in substantial financial losses for sheep and goats in tropical and sub-tropical areas (Naz et al. 2012). Despite not being as dangerous as \u003cem\u003eT. lestoquardi\u003c/em\u003e, \u003cem\u003eT. ovis\u003c/em\u003e infection can cause fever, increasing weight loss, decreased productivity, and even death (Tumwebaze et al. 2020).\u003c/p\u003e\u003cp\u003eVarious factors associated with variation in occurrence rate tick borne pathogens including \u003cem\u003eTheileria\u003c/em\u003e spp. for instance the host, age, climate, systems for management, density of tick infestation, immunity of the host, size of the sample, sampling time and the detection methods (Altay et al. 2024).\u003c/p\u003e\u003cp\u003eThe reported higher infection rate in current study might related to the proper sampling period, during April to October which is defined as tick activation period. In general, tick activation peaks between spring and fall (Dumanli et al. 2012).\u003c/p\u003e\u003cp\u003eThe wide-ranging system of rearing animals used for pasture grazing has a direct impact on diseases carried by ticks because it increases the likelihood of exposure to natural vectors (Rahman et al. 2022).\u003c/p\u003e\u003cp\u003eIn addition, trans-boundary movement of animals might be another factor between different neighboring regions were \u003cem\u003eTheileria\u003c/em\u003e is endemic, related to that (Tawfeeq and Albakri 2024) reported higher infection rate of theileriosis among imported goats.\u003c/p\u003e\u003cp\u003eAmong the risk factors, age of infected host, flock size and tick burden were appeared to be significantly associated (p\u0026thinsp;\u0026le;\u0026thinsp;0.05), although herd composition, co-existence of goats with sheep represented non-significantly associated (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) with occurrence of caprine theileriosis.\u003c/p\u003e\u003cp\u003eCurrent study defines significant increase in \u003cem\u003eTheileria\u003c/em\u003e infection rate with the age of infected animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.02), the higher infection rate of 65.67% was reported from aged goats in compared to younger animals, this finding consistence with the previous results by (Shi et al. 2019; Islam et al. 2021; Metwally et al. 2021 Zhou et al. 2023) were they observed an increase in theileriosis occurrence rate with the age, which represent an association with the tick contact period extension. However, the obtained data by (Niaz et al. 2021; Riaz et al. 2024) disagreed with current finding in which high infection rate was reported from younger animals.\u003c/p\u003e\u003cp\u003eData also represented that goats belong to large sized flocks are significantly prone to \u003cem\u003eTheileria\u003c/em\u003e infection 85.07% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.04) in compare to animals reared in small sized herds 14.93%, similarly (Islam et al. 2021) reported lower infection rate in small sized than medium sized small ruminant flocks.\u003c/p\u003e\u003cp\u003eTick infestation reported as a significant risk factor in current study that associated with significant higher infection rate of 89.55% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00) compared to those with no ticks, in an agreement to present finding similar data was stated by (Niaz et al. 2021;Nassiba et al. 2024). The incidence of theileriosis in small ruminants might be influenced by the distribution and abundance of tick species in various geographical areas (Kumar et al. 2022; Prajapati et al. 2023).\u003c/p\u003e\u003cp\u003eStudy data represented non-significant higher infection rates among goats that are co-existent with sheep 73.13%, which is align with obtained results by (Ashraf et al. 2024; Nassiba et al. 2024) in which prevalence \u003cem\u003eTheileria\u003c/em\u003e was not associated with herd composition. Riaz et al. (2024) reported higher infection rate of Theileriosis in mixed species herd in compare to goat -only herds. Studies performed on both sheep and goats find out higher infection rate among goats which indicated that infected sheep might be an existence source for tick vector among herds, (Arif et al., 2023) reported higher infection rate of theileriosis among sheep than goats.\u003c/p\u003e\u003cp\u003eThe constructed phylogenetic tree based on 18SrRNA gene by the Neighbor Joining test using the Mega 10 software, revealed that current \u003cem\u003eTheileria\u003c/em\u003e isolates PV575340.1 and PV575340.1 were represented higher degree of similarity to and are clusters together with \u003cem\u003eT. ovis\u003c/em\u003e previously deposited in GenBank data base. Topology of \u003cem\u003eT. ovis\u003c/em\u003e tree represented close relationship between study isolates and the isolates from Iraq (OR854547.1), Turkey (AY508454.1, AY508457.1 and MN493111.1), India (MW440586.1), China (PV202473.1) and France (EU622911.1) as they are clustered to gather in one clade.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study results illustrate notable infection by \u003cem\u003eThielrias\u003c/em\u003e pp. from clinically healthy goats in Sulaymaniyah province, which reveal the endemicity of available pathogens. Significant increasing in the number of importing small ruminants from neighboring regions might increase the chance for spreading the blood pathogens. \u003cem\u003eT. ovis\u003c/em\u003e was the known species through sequence analysis which is \u003cem\u003eT. ovis\u003c/em\u003e emerges as the main causes of caprine theileriosis in the area of study. Risk factors evaluation for disease expression revealed significant association for some of them. Large-scale studies must be conducted to define other circulation \u003cem\u003eTheileria\u003c/em\u003e spp.as well as to investigate the tick vectors transmitting \u003cem\u003eT. ovis\u003c/em\u003e. Further studies to assess risk variables for illness manifestation linked to tick-borne pathogen incidence is an essential step for improving the production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003e The study design was reviewed and approved by Ethical Committee of Veterinary Medicine College, University of Sulaimani. Informed consent was obtained from the owners for the participation of their animals in this study.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe author declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eThe author contributed to the study conception and design. Material preparation, data collection and analysis, and manuscript writing by Shadan Hassan Abdullah also read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article. Nucleotide sequences were submitted to the GenBank database under the accession numbers PV575340.1 and PV575341.1\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Hamidhi S, Elshafie EI Yaghfoori S et al (2021). A comparative study of single \u003cem\u003eTheileria lestoquardi\u003c/em\u003e and mixed infections with \u003cem\u003eTheileria ovis\u003c/em\u003e. Parasites \u0026amp; Vectors \u003cem\u003e14\u003c/em\u003e(1). https://doi.org/10.1186/s13071-021-04864-6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAltay K, Dumanli N, Holman PJ et al (2005) Detection of \u003cem\u003eTheileria ovis\u003c/em\u003e in naturally infected sheep by nested PCR. Vet Parasitol \u003cem\u003e127\u003c/em\u003e(2): 99\u0026ndash;104. https://doi.org/10.1016/j.vetpar.2004.09.012\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAltay K, Erol U, Sahin OF (2024) \u003cem\u003eAnaplasma capra\u003c/em\u003e: a new emerging tick-borne zoonotic pathogen. 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BMC Veterinary Research 19(1). https://doi.org/10.1186/s12917-023-03766-2\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Theileria, goat, theileriosis, Phylogeny, Sulaymaniyah","lastPublishedDoi":"10.21203/rs.3.rs-7055150/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7055150/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTheileriosis is a significant protozoal infection affecting livestock health status and productivity worldwide. Study aimed to detect \u003cem\u003eTheileria\u003c/em\u003e infection in native goats from Sulaymaniyah province, Northern region of Iraq. For current study random blood samples were taken from clinically healthy goats belonging to mixed small ruminant flocks from four regions of Sulaymaniyah province. PCR based on the gene coding for 18S rRNA was applied for detecting \u003cem\u003eTheileria\u003c/em\u003e parasite from goat blood samples, followed by sequencing of two isolates and defining the genetic diversity. Effects of various risk factors on theileriosis prevalence also evaluated.\u003c/p\u003e\u003cp\u003eThe overall infection rate of caprine theileriosis was 55.83%. Higher infection rate of 61.67% was reported from Sharazoor in compare to the low infection rate of 51.67% from Sitak. Risk factors including age, flock size and the tick burden represented significant determinants (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), although herd composition no significantly effects on \u003cem\u003eTheileria\u003c/em\u003e infection. Blast analysis of study sequences established the detection of \u003cem\u003eT. ovis\u003c/em\u003e, and the constructed phylogenetic tree defined that current isolates PV575340.1 and PV575340.1 were clustered to gather with previously detected \u003cem\u003eT. ovis\u003c/em\u003e isolates. The study provides an insight to the existence of \u003cem\u003eTheileria\u003c/em\u003e species among goat population with higher infection rates in spite of observing of no obvious clinical signs, therefore attention directed to word the animal status should be highlighted as they are associated with financial and health impacts, along with the role of epidemiological risk factors on the incidence rate should not be neglected.\u003c/p\u003e","manuscriptTitle":"Molecular Based Evidence, Genetic Profile and Risk Factors of Theileria spp. Infection in Caprine in Sulaymaniyah Province /Iraq","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 07:25:09","doi":"10.21203/rs.3.rs-7055150/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0290cab6-788f-425d-9efe-530f40e86a1b","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-28T18:22:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 07:25:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7055150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7055150","identity":"rs-7055150","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}