The first molecular detection of equine piroplasmosis in Vietnam

The first molecular detection of equine piroplasmosis in Vietnam | 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 The first molecular detection of equine piroplasmosis in Vietnam Thanh Dao Thi Ha, Tamás Szűts, Ngoc Duong Nhu, Duong Truong Thi Quoy, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7848604/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2026 Read the published version in Parasitology Research → Version 1 posted 7 You are reading this latest preprint version Abstract Equine piroplasmosis, which is caused by Theileria equi and Babesia caballi , has a significant impact on the veterinary and economic aspects of the global horse industry. However, many countries, including Vietnam, have not yet conducted epidemiological surveys to determine the prevalence of these haemoparasites. The aim of this study was therefore to detect T. equi and/or B. caballi infections in horses and to identify their genotypes. Blood samples were collected from 154 seemingly healthy horses in 8 districts of Hanoi, Thai Nguyen, and Son La provinces located in the northern part of the country. Twenty-four horses (15.58%, 95% CI: 10.70–22.14%) were infected with T. equi in six districts. Babesia caballi was not detected. No significant association was found between gender, host age, and the number of infected horses. Phylogenetic analysis of the 18S rRNA sequences from the positive DNA samples revealed genotypes A, C, and E. The results of this study confirm the presence of equine theileriosis in northern Vietnam and highlight the need for nationwide studies of equine piroplasmosis involving a large sample size. Theileria equi Horses Molecular identification 18S rRNA gene Vietnam Figures Figure 1 Figure 2 1. Introduction Equine piroplasmosis is a tick-borne infectious disease caused by two main causative agents, Theileria equi (formerly Babesia equi ) and Babesia caballi (Apicomplexa: Piroplasmida) (Rothschild 2013 ; Wise et al. 2013 ). Recently, a new species, Theileria haneyi , closely related to T. equi , has been discovered (Knowles et al. 2018 ). Preliminary studies of the small subunit ribosomal RNA gene (18S rDNA) revealed different genotypes for T. equi and B. caballi (Bhoora et al. 2009 ; Qablan et al. 2012 ; Onyiche et al. 2019 ). The disease caused by T. equi and/or B. caballi has also been detected in donkeys, mules, zebras (Tirosh-Levy et al. 2020a ), and in non-equids, including camels (Qablan et al. 2012 ), waterbucks (Githaka et al. 2014 ), tapirs (Da Silveira et al. 2017 ), dogs and cattle (Salim et al. 2019 ). Horses may clear B. caballi within a few years, while T. equi persists for life unless treated (Wise et al. 2013 ; Scoles & Ueti 2015 ). Under natural and experimental conditions more than 30 tick species of many genera such as Hyalomma , Rhipicephalu s and Dermacentor can transmit trans-stadially T. equi and B. caballi (Tirosh-Levy et al. 2020a ). Other genera such as Ixodes , Haemaphysalis , and Amblyomma are suspected of being capable of transmitting these hemoparasites, but they have not been confirmed (Scoles and Ueti 2015 ). The transovarian transmission has only been mentioned for B. caballi which means that infected ticks without re-infection can also transmit this species over several generations (Scoles and Ueti 2015 ). Trans-placental transmission has been reported for T. equi which may lead to abortion or the birth of unapparent carrier foal (Allsopp et al. 2007 ; Chhabra et al. 2012 ). There are many reports of iatrogenic transmission of both T. equi and B. caballi by surgical needles, equipment, and blood transfusions (Rothschild 2013 ; Wise et al. 2013 ). After infection the incubation period ranges between 12 and 19 days for T. equi and between 10 and 30 days for B. caballi (Knowles et al., 2018 ). Traditionally, equine piroplasmosis is classified in peracute, acute, and chronic forms. Nevertheless, many infected horses remain asymptomatic or exhibit no specific clinical signs, such as high fever, tachycardia, anorexia, anaemia, icterus. The peracute form may be life-threatening (Rothschild 2013 ; Wise et al. 2013 ; Tirosh-Levy et al 2020a ). The disease caused by T. equi and/or B. caballi is endemic in several countries of Africa, Asia, America, and Europe (Onyiche et al. 2020; Tirosh-Levy et al. 2020a ) because the seemingly healthy infected horses are carriers of these pathogens for an extended period between endemic and non-endemic regions (Chauvin et al. 2009 ). Equine piroplasmosis has a considerable veterinary and economic impact on the horse industry worldwide (Tamzali 2013 ; Tirosh-Levy et al. 2020). Therefore, equine piroplasmosis is a reportable disease worldwide according to the World Organisation for Animal Health (WOAH) ( https://www.woah.org ). Currently, only a few countries, including Japan, New Zealand, Iceland and Ireland are officially considered to be free from this parasitosis (Mendoza et al. 2024 ). Although equine piroplasmosis is known to have a cosmopolitan distribution of (Tirosh-Levy et al. 2020a ), including many Asian countries (Kamyingkird et al. 2014 ; Nugrahaa et al. 2018; Wang et al. 2019 ; Kumar et al. 2020 ; Khaing et al. 2025 ), this veterinary and economically important equine parasitosis has not been reported from Vietnam until now. The aim of this study was therefore to investigate the occurrence of piroplasmosis in horses in the country using a PCR-based method, which is much more sensitive and specific than traditional smear examinations or serology (Mendoza et al. 2024 ). 2. Materials and methods 2.1 Study areas and sample collection A total of 154 horses older than 5 months of age were sampled in 8 districts of Hanoi, Thai Nguyen, and Son La provinces located in the northern part of Vietnam. These animals were born in Vietnam and used by their owners for carrying wood or working in rice fields. They were kept outdoors almost year-round in communal pasture lands grazing together with cattle, water buffaloes, and small ruminants where hard tick species occur making them frequently exposed to tick bites. Blood samples, 5 ml from each horse, were randomly collected in 2022 and 2023 from the jugular vein into EDTA-coated vacutainer tubes and transferred to the laboratory in iceboxes. The protocol used in this study was followed the QCVN 01–83:2011/BNN&PTNT ( https://cucthuy.gov.vn/en/tieu-chuan-quy-chuan/-/standards/detail/77582 ) and approved by the Proposal Committee of Ministry of Science and Technology, Vietnam (No. 02/2022/HD-NDT). All samples were taken from apparently healthy horses. Furthermore, a questionnaire has been used to gain information about main characteristics of each farm and sampled horse. 2.2. DNA extraction, polymerase chain reaction (PCR), and sequence analysis Genomic DNA was extracted in the Department of Parasitology, National Institute of Veterinary Research, Hanoi, from 200 µl of each blood sample according to the manufacturer’s instructions (QIAGEN DNA Blood Mini-Kit, Germany). The DNA samples were stored at − 20 ◦ C until use. A conventional PCR was used with the primers BJ1 5’- GTC TTG TAA TTG GAA TGA TGG– 3’ forward and BN2 5’ –TAG TTT ATG GTT AGG ACT ACG − 3’reverse to amplify a ~ 500 bp long fragment of the 18S rRNA gene. Five µl of extracted DNA were added to 20 µl of reaction mixture containing 1.0 U HotStar Taq Plus DNA Polymerase (5U/µl) (QIAGEN, Hilden, Germany), 0.5 µl dNTP Mix (10mM), 0.5 µl of each primer (50µM), 2.5 µl of 10x Coral Load PCR buffer (15mM MgCl 2 included), and 15.8 µl DW. An initial denaturation step at 95°C for 10 min was followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s and extension at 72°C for 40 s. Final extension was performed at 72°C for 5 min. then kept at 4°C. DNA of Babesia sp. served as positive control. PCR products were electrophoresed in 1.5% agarose gel (100V, 50 min), stained with ethidium-bromide and visualized under ultra-violet light. Ten representative T. equi 18S rRNA sequences from this study were deposited in GenBank under the accession numbers PV688145–PV688154. Purification and sequencing of the positive PCR products were performed by Eurofins Biomi Ltd. (Gödöllő, Hungary). Ten sequences were aligned to references with NCBI BLAST, National Institutes of Health, USA ( http://www.ncbi.nlm.nih.gov ). 2.3. Phylogenetic analysis To test the placement of the Vietnamese 18S rRNA samples the sequence list and clade assortment (Supplement table) from Tirosh-Levy et al. (2020) was used. To provide a robust phylogenetic background to our ~ 500-bp-long diagnostic sequences, we have selected among the longer, 1500 + bp long sequences. We aimed to select 10–10 sequences from the five genotypes reported in T. equi (Tirosh-Levy et al. 2020a ). However, for genotype B we were able to get only two over 1500-bp-long long sequences (AB515310, EU642507) from horses, thus we supplemented our selection with four sequences (KF597073, KF597077, KF597078, KF597081) from samples of waterbucks thus raising the number of sequences in genotype B to six. Besides the T. equi samples, five Theileria parva sequences were used as outgroups, thus we used 51 terminals from the literature. In the analysis 75 terminals were analysed. The full list of the sequences’ GenBank accession numbers, distribution data together with their assigned clade could be found in Supplement Table. The sequences have been aligned with MAFFT (Katoh and Toh, 2008 ), alignment was curated in BGME (Criscuolo and Gribaldo, 2010 ), the substitution model were selected via SMS (Lefort et al. 2017 ), then analysed with maximum likelihood in PhyML (Guindon et al. 2010 ) on the NGPhylogeny server (Lemoine et al. 2019 ). Boostrap values were calculated after Lemoine et al. ( 2018 ), and the tree has been visualized and edited in iTol (Letunic and Bork 2024 ). The final alignment was analysed by the MrBayes (Huelsenbeck and Ronquist 2001) v3.2.7 as well on the NGPhylogeny server. The alignment, the SMS model selection process, the chosen Model, the ML tree, the MrBayes command line, the MrBayes tree with the posterior probabilities in newick format could be found in the Supplement information. 2.4. Statistical analysis The Fisher exact was applied as independence test within R-environment (R Core Team 2025). 3. Results 3.1. The occurrence of horse piroplasmosis Targeting the 18S rDNA gene with specific PCR of 154 examined horses, 24 were found to be positive in 6 districts of the three provinces (Fig. 1 ). The overall prevalence was 15.58% (95%CI: 10.70-22.14) (Table 1 ). Based on the sequence analysis, all of the positive samples were identified as Theileria equi . This species was detected in 6 districts of the three provinces (Fig. 1 ). The prevalence of infection was very similar in Hanoi and Son La provinces. Neither Babesia caballi was not detected. There were no significant differences in the frequency of PCR-positive animals among the provinces (p = 0.2444). There was no significant association between gender and the number of horses infected with T. equi (p = 0.1822), nor between age, converted to three categories (0.6–3, > 3–6, and over six years old), and the number of horses infected with T. equi (p = 0.4684). Table 1 Prevalence of Theileria equi infection of horses detected with molecular method in the three provinces of Vietnam Province Number examined Number infected Prevalence (%) 95%CI Ha Noi 29 6 20.69 9.85–38.39 Son La 42 9 21.43 11.71–35.94 Thai Nguyen 83 9 10.84 5.81–19.34 Overall 154 24 15.58 10.70-22.14 3.2. Sequences analysis of T. equi Based on the NCBI BLAST results, newly obtained T. equi 18S rRNA sequences (PV688145–PV688154, PX369340–PX369353) shared 99.09–100% identity to each other and to T. equi sequences in GenBank. The results of the Maximum likelihood analysis are shown in Fig. 2 . Our result was consistent with the published tree by Tirosh-Levy et al. (2020), although the placement of genotype A with B + E did not receive any support. Twelve samples (PV688149–PV688151, PV688153, PX369342, PX369343, PX369345, PX369347–PX369351) nested in two separate groups within the genotype A, three samples (PX369340, PX369341, PX369344) in genotype C, whereas the other nine samples (PV688144–PV688148, PV688152, PV688154, PX369353, PX369352, PX369346) were placed in genotype E (Fig. 2 ). 4. Discussion Since the beginning of the 20th century when Laveran ( 1901 ) gave the name Piroplasma equi to the intraerythrocytic parasite found in the blood of horses, equine piroplasmosis has been reported from many European, American and African countries (Onyiche et al. 2019 ; Tirosh-Levy et al. 2020a ). Although it was speculated more than one hundred years ago that piroplasmosis occurred in a flock of mules in French Indochina (Schein 1917 ), no information about the occurrence of this economically important parasitosis in Asia was available for many decades. Since the 2010s, however, serological and/or molecular studies have revealed the presence of equine piroplasmosis in various Asian countries where one or both haemoparasite species were detected (Munkhjargal et al. 2013 ; Kamyingkird et al. 2014 ; Nugraha et al. 2018 ; Ybañez et al. 2018 ; Wang et al. 2019 ; Zhao et al. 2020; Kumar et al. 2020 ; Khaing et al. 2025 ). To the best of our knowledge, no serological or molecular examinations of this tick-borne disease had been carried out in Vietnam prior to this survey. The results revealed that 24 out of 154 blood samples collected in Vietnam contained the DNA of T. equi , which is the predominant species in some Asian countries (Tirosh-Levy et al. 2020a ; Kumar et al. 2020 ). On the contrary, a higher prevalence of B. caballi than T. equi was reported in Mongolia (Munkhjargal et al. 2013 ). Neither B. caballi nor dual infections with the two protozoan species were found. There are two possible explanations for the absence of B. caballi . Either this species does not occur among horses in the studied provinces, or it was present but efficiently eliminated by the host immune system. This is in contrast to the lifelong persistence of T. equi in untreated horses (Brüning 1996 ). When we compared our result (15.58%) with the findings of other Asian studies, we found that a higher prevalence was reported from China (Wang et al. 2019 ; Zhao et al. 2020), Iran (Kalantari et al. 2022 ) and the Philippines (Ybañez et al. 2018 ), while a lower prevalence was found in India (Kumar et al. 2020 ), and Myanmar (Khaing et al. 2025 ). The difference in the prevalence of equine theileriosis among countries may be due to variations in the occurrence and abundance of competent vectors, the environmental factors of ticks and the effectiveness of tick control measures (Onyiche et al. 2019 ). No significant correlation was found between the PCR results and the age of the horses. Ruegg et al. ( 2007 ) reported that a positive correlation was found between host age and T. equi infection in the surveyed provinces. As in a previous study (Steinman et al. 2012 ), gender was not found to be a risk factor for T. equi infection in this study. However, other authors (Ruegg et al. 2007 ; Moretti et al. 2010 ) did find a correlation between gender and infection. No tick infestations of horses were observed during the sampling period. Therefore, we could not obtain any relevant data on tick species that could be the vector of T. equi . However, most of the suspected tick genera ( Hyalomma , Dermacentor , and Haemaphysalis ), that may act as vectors (de Waal 1992 ; Scoles and Ueti 2015 ), are present in Vietnam (Hornok et al. 2024 ; Ngoc et al. 2025 ) and stable flies, one of the most important mechanical vectors of Theileria spp. including T. equi (Hornok et al. 2020 ) are commonly found on the pasture. Furthermore, iatrogenic transmission cannot be ruled out, because contaminated needles and other equipment have been reported by many authors (Rothschild 2013 ; Wise et al. 2013 ) as a source of such infections. Preliminary studies have revealed five 18S rRNA genotypes of T. equi (A, B, C, D and E) (Bhoora et al. 2009 ; Qablan et al. 2012 ; Onyiche et al. 2019 ). The genetic diversity of T. equi may be important for the transmission of this pathogen (Manna et al. 2018 ; Tirosh-Levy et al. 2020b ); however, more molecular epidemiological data are needed to confirm this. This study provides the first data on the genetic diversity of T. equi in Vietnam. Three genotypes (A, C and E) were identified through sequence analysis of T. equi taken from 24 horses. The most prevalent genotype was A, occurring in half of the infected horses living in three provinces. This genotype has been isolated in many countries and is more commonly associated with clinical theileriosis than other genotypes (Tirosh-Levy et al. 2021 ). The second most prevalent genotype was E, which was found in nine of the animals in this study. It has mainly been reported in China (Chen et al. 2022 ) and Mongolia (Otgonsuren et al. 2024 ). Genotype C was found in two horses in the province Hanoi and one horse in the province Thai Nguyen. The time and place of arrival of T. equi , the agent of theileriosis in horses, in Vietnam is unknown. However, according to reports by Laveran ( 1901 ) and Schein ( 1917 ), it can be stated that this haemoparasite species was already present in the north of the country at the beginning of the 20th century, causing clinical symptoms and death in mules living in the Haiphong area. In the following decades, mainly during the Vietnam War, it can be assumed that three genotypes of T. equi were introduced to the northern part of the country from China, where the three genotypes have been found in infected equines (Wang et al. 2019 ; Zhao et al. 2020). From there, they spread via tick vectors and contaminated tools. 5. Conclusion In conclusion, the results of the first molecular survey suggest that Theileria equi , the agent that causes equine piroplasmosis, is present in northern Vietnam, albeit in a subclinical form. Further studies are needed to assess the risk posed by T. equi and Babesia caballi in other parts of the country. This should be accompanied by tick vector surveillance and educational programmes for veterinarians about this notifiable disease. Declarations Data availability The sequences obtained during this study are deposited in GenBank under the following accession numbers: 16S rRNA gene PV688145.1-PV197254.1. All other relevant data are included in the manuscript and the supplementary material or are available upon request by the corresponding author. Acknowledgement We thank the veterinarians who participated in this study for their kind cooperation. In addition, we would like to thank to local owners of horses in the three provinces for their excellent collaboration during samplings. Funding The study was performed and funded in the frame of the project 2019-2.1.12-TÉTVN-2020-00012 supported by National Research, Development and Innovation Office (Hungary) and project no. NDT/HU/22/02 (Decision No. 2822/QD-BKHCN, 9 November 2021) supported by the Ministry of Science and Technology (Vietnam). T.Sz. was supported by the strategic research fund of the University of Veterinary Medicine Budapest (Grant No. SRF-002)” Authors information Department of Parasitology, National Institute of Veterinary Research, Hanoi, Vietnam Thanh Dao Thi Ha, Ngoc Duong Nhu, Duong Truong Thi Quy Department of Zoology, University of Veterinary Medicine, Budapest, Hungary Tamás Szűts Centre for Bioinformatics, University of Veterinary Medicine, Budapest, Hungary Norbert Solymosi Department of Parasitology and Zoology, University of Veterinary Medicine, Budapest, Hungary Nóra Takács, Sándor Hornok, Róbert Farkas HUN-REN-UVMB Climate Change: New Blood-sucking Parasites and Vector-borne Pathogens Research Group, Hungary Nóra Takács, Sándor Hornok Contributions TDTH: conceptualization, study design, manuscript writing, review. TSz: data curation, data analysis, searched for relevant literature. NDN: coordination of sample collection, data analysis. DTTQ: study design, searched for relevant literature. NS: data curation, data analysis, searched for relevant literature. NT: PCR tests, sequencing, data analysis. SH: conceptualization, manuscript writing and review. RF: conceptualization, study design, manuscript writing, review. All authors reviewed the manuscript. Ethical declaration The protocol used in this study was followed the QCVN 01-83:2011/BNN&PTNT (https://cucthuy.gov.vn/en/tieu-chuan-quy-chuan/-/standards/detail/77582) and approved by the Proposal Committee of Ministry of Science and Technology, Vietnam (No. 02/2022/HD-NDT). Competing interests The authors declare no competing interests. Supplementary information Supplement table: GenBank accession numbers, locality and assigned clades of the examined sequences. 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Parasitol 134:939–947.https://doi.org/10.1017/S0031182007002405 Salim B, Alanazi AD, Omori R et al (2019) Potential role of dogs as sentinels and reservoirs for piroplasms infecting equine and cattle in Riyadh City, Saudi Arabia Acta Trop 193:7883. DOI: 10.1016/j.actatropica.2019.02.029 Schein H (1917) Equine piroplasmosis in South Annam, French Indo-China. Bull Soc Exot Pathol 10:871–873. Scoles GA, Ueti MW (2015) Vector ecology of equine piroplasmosis. Annu Rev Entomol 60: 561-580. https://doi.org/10.1146/annurev-ento-010814-021110 Steinman A, Zimmerman T, Klement E et al (2012) Demographic and environmental risk factors for infection by Theileria equi in 590 horses in Israel. Vet Parasitol 187:558–562. https://doi.org/10.1016/j.vetpar.2012.01.018 Tamzali Y (2013) Equine piroplasmosis: an updated review. Equine Vet Educ 25:590–598. https://doi.org/10.1111/eve.12070 Tirosh-Levy S, Gottlieb Y, Fry LM et al (2020a) Twenty years of equine piroplasmosis research: global distribution, molecular diagnosis, and phylogeny. Pathogens 9(11):926. https://doi.org/10.3390/pathogens9110926 Tirosh-Levy S, Steinman A, Levy H et al (2020b) Parasite load and genotype are associated with clinical outcome of piroplasm-infected equines in Israel. Parasit Vectors 13:267. https://doi.org/10.1186/s13071-020-04133-y Tirosh-Levy S, Mazuz ML, Savitsky I (2021) A serological and molecular prevalence of Babesia caballi in apparently healthy horses in Israel. Pathogens 10:445. https://doi.org/10.3390/pathogens10040445 Wang J, Liua J, Yanga J et al (2019) The first molecular detection and genetic diversity of Babesia caballi and Theileria equi in horses of Gansu province, China. Ticks and Tick Born Dis 10:528–532. https://doi.org/10.1016/j.ttbdis.2019.01.003 Wise LN, Kappmeyer LS, Mealey RH, Knowles DP (2013) Review of equine piroplasmosis. J Vet Intern Med 27:1334–1346. https://doi.org/10.1111/jvim.12168 Ybañez AP, Ybañez RHD, Talle MG et al (2018) Serological and molecular detection of Theileria equi and Babesia caballi in Philippine horses. Ticks and Tick Born Dis 9:1125–1128. https://doi.org/10.1016/j.ttbdis.2018.04.010 Zhao S, Wang H, Zhang S (2200) First report of genetic diversity and risk factor analysis of equine piroplasm infection in equids in Jilin, China. Parasit Vector 13:459. https://doi.org/10.1186/s13071-020-04338-1 Additional Declarations No competing interests reported. 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Budapest","correspondingAuthor":false,"prefix":"","firstName":"Tamás","middleName":"","lastName":"Szűts","suffix":""},{"id":537673782,"identity":"4ff60ca4-41ef-4057-aa65-91c538bc7df9","order_by":2,"name":"Ngoc Duong Nhu","email":"","orcid":"","institution":"National Institute of Veterinary Research","correspondingAuthor":false,"prefix":"","firstName":"Ngoc","middleName":"Duong","lastName":"Nhu","suffix":""},{"id":537673787,"identity":"701b0325-72a3-4947-adb7-43db0854a3a0","order_by":3,"name":"Duong Truong Thi Quoy","email":"","orcid":"","institution":"National Institute of Veterinary Research","correspondingAuthor":false,"prefix":"","firstName":"Duong","middleName":"Truong Thi","lastName":"Quoy","suffix":""},{"id":537673789,"identity":"54fb957e-12e8-4c0f-b630-85b1f45e6e6d","order_by":4,"name":"Norbert Solymosi","email":"","orcid":"","institution":"University of Veterinary 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14:36:18","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110689,"visible":true,"origin":"","legend":"","description":"","filename":"0cf50506667f4ae6ada356b96a4801951structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/4550fcdf425baeb504b77202.xml"},{"id":94869564,"identity":"ed2b08d0-bbb8-403a-b83f-3f6e810db00c","added_by":"auto","created_at":"2025-10-31 14:36:18","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123079,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/1fc1c0b58808ac15404dbacb.html"},{"id":94869547,"identity":"4160d48d-7db5-482f-8b6e-165f97acfbce","added_by":"auto","created_at":"2025-10-31 14:36:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106454,"visible":true,"origin":"","legend":"\u003cp\u003eThe three provinces with the number of samples are illustrated on the map of Vietnam. The black dots indicate those districts where \u003cem\u003eTheileria equi\u003c/em\u003einfected horses were detected.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/41b5ea0c739b456721a3fc54.png"},{"id":94869548,"identity":"08836ddc-3233-408d-b45b-03b9fcc3b9df","added_by":"auto","created_at":"2025-10-31 14:36:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":266320,"visible":true,"origin":"","legend":"\u003cp\u003eThe inferred Maximum likelihood tree of \u003cem\u003eT. equi 18S \u003c/em\u003erRNA sequences. The tree included 75 sequences and 1548 positions. GTR +G+I substitution models were used, bootstrap values below 70 are not shown. Five \u003cem\u003eTheileria parva\u003c/em\u003e sequences were used as outgroups.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/629fa1fbe2cd7b7f36994282.png"},{"id":102233997,"identity":"9eacabd3-52fa-465d-b5b9-038c09e6e6d9","added_by":"auto","created_at":"2026-02-09 16:02:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":980204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/43e0e278-81b7-455f-8938-5000181053fa.pdf"},{"id":94987017,"identity":"b9977edf-a894-406c-81eb-5ae255c43fe7","added_by":"auto","created_at":"2025-11-03 07:01:06","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14572,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/4dd8500fb1cbadcc0177eaca.xlsx"},{"id":94986383,"identity":"6b8b60c9-61ba-4f14-945f-021ba447a65a","added_by":"auto","created_at":"2025-11-03 07:00:14","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":181095,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinfo.txt","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/49791fc1ebc419f8f83dc93b.txt"},{"id":94869555,"identity":"33c0d335-d614-49e3-822e-84c517ed0b11","added_by":"auto","created_at":"2025-10-31 14:36:18","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":107582,"visible":true,"origin":"","legend":"","description":"","filename":"ThephotoofthefulluncroppedGelsandBlotsimages.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7848604/v1/1448bde03bf0013976ab7968.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"The first molecular detection of equine piroplasmosis in Vietnam","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEquine piroplasmosis is a tick-borne infectious disease caused by two main causative agents, \u003cem\u003eTheileria equi\u003c/em\u003e (formerly \u003cem\u003eBabesia equi\u003c/em\u003e) and \u003cem\u003eBabesia caballi\u003c/em\u003e (Apicomplexa: Piroplasmida) (Rothschild \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wise et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Recently, a new species, \u003cem\u003eTheileria haneyi\u003c/em\u003e, closely related to \u003cem\u003eT. equi\u003c/em\u003e, has been discovered (Knowles et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Preliminary studies of the small subunit ribosomal RNA gene (18S rDNA) revealed different genotypes for \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e (Bhoora et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Qablan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Onyiche et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The disease caused by \u003cem\u003eT. equi\u003c/em\u003e and/or \u003cem\u003eB. caballi\u003c/em\u003e has also been detected in donkeys, mules, zebras (Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e), and in non-equids, including camels (Qablan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), waterbucks (Githaka et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), tapirs (Da Silveira et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), dogs and cattle (Salim et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Horses may clear \u003cem\u003eB. caballi\u003c/em\u003e within a few years, while \u003cem\u003eT. equi\u003c/em\u003e persists for life unless treated (Wise et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Scoles \u0026amp; Ueti \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Under natural and experimental conditions more than 30 tick species of many genera such as \u003cem\u003eHyalomma\u003c/em\u003e, \u003cem\u003eRhipicephalu\u003c/em\u003es and \u003cem\u003eDermacentor\u003c/em\u003e can transmit trans-stadially \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e (Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Other genera such as \u003cem\u003eIxodes\u003c/em\u003e, \u003cem\u003eHaemaphysalis\u003c/em\u003e, and \u003cem\u003eAmblyomma\u003c/em\u003e are suspected of being capable of transmitting these hemoparasites, but they have not been confirmed (Scoles and Ueti \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The transovarian transmission has only been mentioned for \u003cem\u003eB. caballi\u003c/em\u003e which means that infected ticks without re-infection can also transmit this species over several generations (Scoles and Ueti \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Trans-placental transmission has been reported for \u003cem\u003eT. equi\u003c/em\u003e which may lead to abortion or the birth of unapparent carrier foal (Allsopp et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chhabra et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). There are many reports of iatrogenic transmission of both \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e by surgical needles, equipment, and blood transfusions (Rothschild \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wise et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After infection the incubation period ranges between 12 and 19 days for \u003cem\u003eT. equi\u003c/em\u003e and between 10 and 30 days for \u003cem\u003eB. caballi\u003c/em\u003e (Knowles et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Traditionally, equine piroplasmosis is classified in peracute, acute, and chronic forms. Nevertheless, many infected horses remain asymptomatic or exhibit no specific clinical signs, such as high fever, tachycardia, anorexia, anaemia, icterus. The peracute form may be life-threatening (Rothschild \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wise et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tirosh-Levy et al \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). The disease caused by \u003cem\u003eT. equi\u003c/em\u003e and/or \u003cem\u003eB. caballi\u003c/em\u003e is endemic in several countries of Africa, Asia, America, and Europe (Onyiche et al. 2020; Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e) because the seemingly healthy infected horses are carriers of these pathogens for an extended period between endemic and non-endemic regions (Chauvin et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Equine piroplasmosis has a considerable veterinary and economic impact on the horse industry worldwide (Tamzali \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tirosh-Levy et al. 2020). Therefore, equine piroplasmosis is a reportable disease worldwide according to the World Organisation for Animal Health (WOAH) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.woah.org\u003c/span\u003e\u003cspan address=\"https://www.woah.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Currently, only a few countries, including Japan, New Zealand, Iceland and Ireland are officially considered to be free from this parasitosis (Mendoza et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough equine piroplasmosis is known to have a cosmopolitan distribution of (Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e), including many Asian countries (Kamyingkird et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nugrahaa et al. 2018; Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khaing et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), this veterinary and economically important equine parasitosis has not been reported from Vietnam until now. The aim of this study was therefore to investigate the occurrence of piroplasmosis in horses in the country using a PCR-based method, which is much more sensitive and specific than traditional smear examinations or serology (Mendoza et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study areas and sample collection\u003c/h2\u003e\u003cp\u003eA total of 154 horses older than 5 months of age were sampled in 8 districts of Hanoi, Thai Nguyen, and Son La provinces located in the northern part of Vietnam. These animals were born in Vietnam and used by their owners for carrying wood or working in rice fields. They were kept outdoors almost year-round in communal pasture lands grazing together with cattle, water buffaloes, and small ruminants where hard tick species occur making them frequently exposed to tick bites.\u003c/p\u003e\u003cp\u003eBlood samples, 5 ml from each horse, were randomly collected in 2022 and 2023 from the jugular vein into EDTA-coated vacutainer tubes and transferred to the laboratory in iceboxes. The protocol used in this study was followed the QCVN 01\u0026ndash;83:2011/BNN\u0026amp;PTNT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cucthuy.gov.vn/en/tieu-chuan-quy-chuan/-/standards/detail/77582\u003c/span\u003e\u003cspan address=\"https://cucthuy.gov.vn/en/tieu-chuan-quy-chuan/-/standards/detail/77582\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and approved by the Proposal Committee of Ministry of Science and Technology, Vietnam (No. 02/2022/HD-NDT). All samples were taken from apparently healthy horses. Furthermore, a questionnaire has been used to gain information about main characteristics of each farm and sampled horse.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. DNA extraction, polymerase chain reaction (PCR), and sequence analysis\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted in the Department of Parasitology, National Institute of Veterinary Research, Hanoi, from 200 \u0026micro;l of each blood sample according to the manufacturer\u0026rsquo;s instructions (QIAGEN DNA Blood Mini-Kit, Germany). The DNA samples were stored at \u0026minus;\u0026thinsp;20\u003csup\u003e◦\u003c/sup\u003eC until use.\u003c/p\u003e\u003cp\u003eA conventional PCR was used with the primers BJ1 5\u0026rsquo;- GTC TTG TAA TTG GAA TGA TGG\u0026ndash; 3\u0026rsquo; forward and BN2 5\u0026rsquo; \u0026ndash;TAG TTT ATG GTT AGG ACT ACG \u0026minus;\u0026thinsp;3\u0026rsquo;reverse to amplify a\u0026thinsp;~\u0026thinsp;500 bp long fragment of the 18S rRNA gene. Five \u0026micro;l of extracted DNA were added to 20 \u0026micro;l of reaction mixture containing 1.0 U HotStar Taq Plus DNA Polymerase (5U/\u0026micro;l) (QIAGEN, Hilden, Germany), 0.5 \u0026micro;l dNTP Mix (10mM), 0.5 \u0026micro;l of each primer (50\u0026micro;M), 2.5 \u0026micro;l of 10x Coral Load PCR buffer (15mM MgCl\u003csub\u003e2\u003c/sub\u003e included), and 15.8 \u0026micro;l DW. An initial denaturation step at 95\u0026deg;C for 10 min was followed by 40 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 54\u0026deg;C for 30 s and extension at 72\u0026deg;C for 40 s. Final extension was performed at 72\u0026deg;C for 5 min. then kept at 4\u0026deg;C. DNA of \u003cem\u003eBabesia\u003c/em\u003e sp. served as positive control. PCR products were electrophoresed in 1.5% agarose gel (100V, 50 min), stained with ethidium-bromide and visualized under ultra-violet light. Ten representative \u003cem\u003eT. equi\u003c/em\u003e 18S rRNA sequences from this study were deposited in GenBank under the accession numbers PV688145\u0026ndash;PV688154.\u003c/p\u003e\u003cp\u003ePurification and sequencing of the positive PCR products were performed by Eurofins Biomi Ltd. (G\u0026ouml;d\u0026ouml;llő, Hungary). Ten sequences were aligned to references with NCBI BLAST, National Institutes of Health, USA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Phylogenetic analysis\u003c/h2\u003e\u003cp\u003eTo test the placement of the Vietnamese 18S rRNA samples the sequence list and clade assortment (Supplement table) from Tirosh-Levy et al. (2020) was used. To provide a robust phylogenetic background to our\u0026thinsp;~\u0026thinsp;500-bp-long diagnostic sequences, we have selected among the longer, 1500\u0026thinsp;+\u0026thinsp;bp long sequences. We aimed to select 10\u0026ndash;10 sequences from the five genotypes reported in \u003cem\u003eT. equi\u003c/em\u003e (Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). However, for genotype B we were able to get only two over 1500-bp-long long sequences (AB515310, EU642507) from horses, thus we supplemented our selection with four sequences (KF597073, KF597077, KF597078, KF597081) from samples of waterbucks thus raising the number of sequences in genotype B to six. Besides the \u003cem\u003eT. equi\u003c/em\u003e samples, five \u003cem\u003eTheileria parva\u003c/em\u003e sequences were used as outgroups, thus we used 51 terminals from the literature. In the analysis 75 terminals were analysed. The full list of the sequences\u0026rsquo; GenBank accession numbers, distribution data together with their assigned clade could be found in Supplement Table. The sequences have been aligned with MAFFT (Katoh and Toh, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), alignment was curated in BGME (Criscuolo and Gribaldo, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), the substitution model were selected via SMS (Lefort et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), then analysed with maximum likelihood in PhyML (Guindon et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) on the NGPhylogeny server (Lemoine et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Boostrap values were calculated after Lemoine et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and the tree has been visualized and edited in iTol (Letunic and Bork \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The final alignment was analysed by the MrBayes (Huelsenbeck and Ronquist 2001) v3.2.7 as well on the NGPhylogeny server. The alignment, the SMS model selection process, the chosen Model, the ML tree, the MrBayes command line, the MrBayes tree with the posterior probabilities in newick format could be found in the Supplement information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe Fisher exact was applied as independence test within R-environment (R Core Team\u003c/p\u003e\u003cp\u003e2025).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1. The occurrence of horse piroplasmosis\u003c/h2\u003e\u003cp\u003eTargeting the 18S rDNA gene with specific PCR of 154 examined horses, 24 were found to be positive in 6 districts of the three provinces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe overall prevalence was 15.58% (95%CI: 10.70-22.14) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on the sequence analysis, all of the positive samples were identified as \u003cem\u003eTheileria equi\u003c/em\u003e. This species was detected in 6 districts of the three provinces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The prevalence of infection was very similar in Hanoi and Son La provinces. Neither \u003cem\u003eBabesia caballi\u003c/em\u003e was not detected. There were no significant differences in the frequency of PCR-positive animals among the provinces (p\u0026thinsp;=\u0026thinsp;0.2444). There was no significant association between gender and the number of horses infected with \u003cem\u003eT. equi\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.1822), nor between age, converted to three categories (0.6\u0026ndash;3, \u0026gt;\u0026thinsp;3\u0026ndash;6, and over six years old), and the number of horses infected with \u003cem\u003eT. equi\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.4684).\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\u003ePrevalence of \u003cem\u003eTheileria equi\u003c/em\u003e infection of horses detected with molecular method in the three provinces of Vietnam\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProvince\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber examined\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber infected\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrevalence (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95%CI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHa Noi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.85\u0026ndash;38.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSon La\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.71\u0026ndash;35.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThai Nguyen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.81\u0026ndash;19.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOverall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.70-22.14\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\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Sequences analysis of T. equi\u003c/h2\u003e\u003cp\u003eBased on the NCBI BLAST results, newly obtained \u003cem\u003eT. equi\u003c/em\u003e 18S rRNA sequences (PV688145\u0026ndash;PV688154, PX369340\u0026ndash;PX369353) shared 99.09\u0026ndash;100% identity to each other and to \u003cem\u003eT. equi\u003c/em\u003e sequences in GenBank. The results of the Maximum likelihood analysis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Our result was consistent with the published tree by Tirosh-Levy et al. (2020), although the placement of genotype A with B\u0026thinsp;+\u0026thinsp;E did not receive any support. Twelve samples (PV688149\u0026ndash;PV688151, PV688153, PX369342, PX369343, PX369345, PX369347\u0026ndash;PX369351) nested in two separate groups within the genotype A, three samples (PX369340, PX369341, PX369344) in genotype C, whereas the other nine samples (PV688144\u0026ndash;PV688148, PV688152, PV688154, PX369353, PX369352, PX369346) were placed in genotype E (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSince the beginning of the 20th century when Laveran (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1901\u003c/span\u003e) gave the name \u003cem\u003ePiroplasma equi\u003c/em\u003e to the intraerythrocytic parasite found in the blood of horses, equine piroplasmosis has been reported from many European, American and African countries (Onyiche et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Although it was speculated more than one hundred years ago that piroplasmosis occurred in a flock of mules in French Indochina (Schein \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1917\u003c/span\u003e), no information about the occurrence of this economically important parasitosis in Asia was available for many decades. Since the 2010s, however, serological and/or molecular studies have revealed the presence of equine piroplasmosis in various Asian countries where one or both haemoparasite species were detected (Munkhjargal et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kamyingkird et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nugraha et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yba\u0026ntilde;ez et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al. 2020; Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khaing et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To the best of our knowledge, no serological or molecular examinations of this tick-borne disease had been carried out in Vietnam prior to this survey.\u003c/p\u003e\u003cp\u003eThe results revealed that 24 out of 154 blood samples collected in Vietnam contained the DNA of \u003cem\u003eT. equi\u003c/em\u003e, which is the predominant species in some Asian countries (Tirosh-Levy et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the contrary, a higher prevalence of \u003cem\u003eB. caballi\u003c/em\u003e than \u003cem\u003eT. equi\u003c/em\u003e was reported in Mongolia (Munkhjargal et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Neither \u003cem\u003eB. caballi\u003c/em\u003e nor dual infections with the two protozoan species were found. There are two possible explanations for the absence of \u003cem\u003eB. caballi\u003c/em\u003e. Either this species does not occur among horses in the studied provinces, or it was present but efficiently eliminated by the host immune system. This is in contrast to the lifelong persistence of \u003cem\u003eT. equi\u003c/em\u003e in untreated horses (Br\u0026uuml;ning \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). When we compared our result (15.58%) with the findings of other Asian studies, we found that a higher prevalence was reported from China (Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al. 2020), Iran (Kalantari et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the Philippines (Yba\u0026ntilde;ez et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while a lower prevalence was found in India (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and Myanmar (Khaing et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The difference in the prevalence of equine theileriosis among countries may be due to variations in the occurrence and abundance of competent vectors, the environmental factors of ticks and the effectiveness of tick control measures (Onyiche et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). No significant correlation was found between the PCR results and the age of the horses. Ruegg et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) reported that a positive correlation was found between host age and \u003cem\u003eT. equi\u003c/em\u003e infection in the surveyed provinces. As in a previous study (Steinman et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), gender was not found to be a risk factor for \u003cem\u003eT. equi\u003c/em\u003e infection in this study. However, other authors (Ruegg et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Moretti et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) did find a correlation between gender and infection. No tick infestations of horses were observed during the sampling period. Therefore, we could not obtain any relevant data on tick species that could be the vector of \u003cem\u003eT. equi\u003c/em\u003e. However, most of the suspected tick genera (\u003cem\u003eHyalomma\u003c/em\u003e, \u003cem\u003eDermacentor\u003c/em\u003e, and \u003cem\u003eHaemaphysalis\u003c/em\u003e), that may act as vectors (de Waal \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Scoles and Ueti \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), are present in Vietnam (Hornok et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ngoc et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and stable flies, one of the most important mechanical vectors of \u003cem\u003eTheileria\u003c/em\u003e spp. including \u003cem\u003eT. equi\u003c/em\u003e (Hornok et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) are commonly found on the pasture. Furthermore, iatrogenic transmission cannot be ruled out, because contaminated needles and other equipment have been reported by many authors (Rothschild \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wise et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) as a source of such infections.\u003c/p\u003e\u003cp\u003ePreliminary studies have revealed five 18S rRNA genotypes of \u003cem\u003eT. equi\u003c/em\u003e (A, B, C, D and E) (Bhoora et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Qablan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Onyiche et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The genetic diversity of \u003cem\u003eT. equi\u003c/em\u003e may be important for the transmission of this pathogen (Manna et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tirosh-Levy et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e); however, more molecular epidemiological data are needed to confirm this. This study provides the first data on the genetic diversity of \u003cem\u003eT. equi\u003c/em\u003e in Vietnam. Three genotypes (A, C and E) were identified through sequence analysis of \u003cem\u003eT. equi\u003c/em\u003e taken from 24 horses. The most prevalent genotype was A, occurring in half of the infected horses living in three provinces. This genotype has been isolated in many countries and is more commonly associated with clinical theileriosis than other genotypes (Tirosh-Levy et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The second most prevalent genotype was E, which was found in nine of the animals in this study. It has mainly been reported in China (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Mongolia (Otgonsuren et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Genotype C was found in two horses in the province Hanoi and one horse in the province Thai Nguyen.\u003c/p\u003e\u003cp\u003eThe time and place of arrival of \u003cem\u003eT. equi\u003c/em\u003e, the agent of theileriosis in horses, in Vietnam is unknown. However, according to reports by Laveran (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1901\u003c/span\u003e) and Schein (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1917\u003c/span\u003e), it can be stated that this haemoparasite species was already present in the north of the country at the beginning of the 20th century, causing clinical symptoms and death in mules living in the Haiphong area. In the following decades, mainly during the Vietnam War, it can be assumed that three genotypes of \u003cem\u003eT. equi\u003c/em\u003e were introduced to the northern part of the country from China, where the three genotypes have been found in infected equines (Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al. 2020). From there, they spread via tick vectors and contaminated tools.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, the results of the first molecular survey suggest that \u003cem\u003eTheileria equi\u003c/em\u003e, the agent that causes equine piroplasmosis, is present in northern Vietnam, albeit in a subclinical form. Further studies are needed to assess the risk posed by \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eBabesia caballi\u003c/em\u003e in other parts of the country. This should be accompanied by tick vector surveillance and educational programmes for veterinarians about this notifiable disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe sequences obtained during this study are deposited in GenBank under the following accession numbers: 16S rRNA gene PV688145.1-PV197254.1. All other relevant data are included in the manuscript and the supplementary material or are available upon request by the corresponding author.\u003c/p\u003e\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eWe thank the veterinarians who participated in this study for their kind cooperation. In addition, we would like to thank to local owners of horses in the three provinces for their excellent collaboration during samplings.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe study was performed and funded in the frame of the project 2019-2.1.12-T\u0026Eacute;TVN-2020-00012 supported by National Research, Development and Innovation Office (Hungary) and project no. NDT/HU/22/02 (Decision No. 2822/QD-BKHCN, 9 November 2021) supported by the Ministry of Science and Technology (Vietnam). T.Sz. was supported by the strategic research fund of the University of Veterinary Medicine Budapest (Grant No. SRF-002)\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eAuthors information\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eDepartment of Parasitology, National Institute of Veterinary Research, Hanoi, Vietnam\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThanh Dao Thi Ha, Ngoc Duong Nhu, Duong Truong Thi Quy\u003c/p\u003e\n\u003col start=\"2\"\u003e\n\u003cli\u003eDepartment of Zoology, University of Veterinary Medicine, Budapest, Hungary\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTam\u0026aacute;s Szűts\u003c/p\u003e\n\u003col start=\"3\"\u003e\n\u003cli\u003eCentre for Bioinformatics, University of Veterinary Medicine, Budapest, Hungary\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eNorbert Solymosi\u003c/p\u003e\n\u003col start=\"4\"\u003e\n\u003cli\u003eDepartment of Parasitology and Zoology, University of Veterinary Medicine, Budapest, Hungary\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eN\u0026oacute;ra Tak\u0026aacute;cs, S\u0026aacute;ndor Hornok, R\u0026oacute;bert Farkas\u003c/p\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003eHUN-REN-UVMB Climate Change: New Blood-sucking Parasites and Vector-borne Pathogens Research Group, Hungary\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eN\u0026oacute;ra Tak\u0026aacute;cs, S\u0026aacute;ndor Hornok\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eTDTH: conceptualization, study design, manuscript writing, review. TSz: data curation, data analysis, searched for relevant literature. NDN: coordination of sample collection, data analysis. DTTQ: study design, searched for relevant literature. NS: data curation, data analysis, searched for relevant literature. NT: PCR tests, sequencing, data analysis. SH: conceptualization, manuscript writing and review. RF: conceptualization, study design, manuscript writing, review. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eEthical declaration\u003c/p\u003e\n\u003cp\u003eThe protocol used in this study was followed the QCVN 01-83:2011/BNN\u0026amp;PTNT (https://cucthuy.gov.vn/en/tieu-chuan-quy-chuan/-/standards/detail/77582) and approved by the Proposal Committee of Ministry of Science and Technology, Vietnam (No. 02/2022/HD-NDT).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eSupplementary information\u003c/p\u003e\n\u003cp\u003eSupplement table: \u003c/p\u003e\n\u003cp\u003eGenBank accession numbers, locality and assigned clades of the examined sequences.\u003c/p\u003e\n\u003cp\u003eSupplement information txt:\u003c/p\u003e\n\u003cp\u003eSettings, raw results, final alignment of the performed analyses.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAllsopp MT, Lewis BD, Penzhorn BL (2007) Molecular evidence for transplacental transmission of \u003cem\u003eTheileria equi\u003c/em\u003e from carrier mares to their apparently healthy foals. 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Parasitol 134:939\u0026ndash;947.https://doi.org/10.1017/S0031182007002405\u003c/li\u003e\n \u003cli\u003eSalim B, Alanazi AD, Omori R et al (2019) Potential role of dogs as sentinels and reservoirs for piroplasms infecting equine and cattle in Riyadh City, Saudi Arabia Acta Trop 193:7883. DOI: 10.1016/j.actatropica.2019.02.029\u003c/li\u003e\n \u003cli\u003eSchein H (1917) Equine piroplasmosis in South Annam, French Indo-China. Bull Soc Exot Pathol 10:871\u0026ndash;873.\u003c/li\u003e\n \u003cli\u003eScoles GA, Ueti MW (2015) Vector ecology of equine piroplasmosis. Annu Rev Entomol 60: 561-580. https://doi.org/10.1146/annurev-ento-010814-021110\u003c/li\u003e\n \u003cli\u003eSteinman A, Zimmerman T, Klement E et al (2012) Demographic and environmental risk factors for infection by \u003cem\u003eTheileria equi\u003c/em\u003e in 590 horses in Israel. 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Ticks and Tick Born Dis\u003cem\u003e\u0026nbsp;\u003c/em\u003e9:1125\u0026ndash;1128. https://doi.org/10.1016/j.ttbdis.2018.04.010\u003c/li\u003e\n \u003cli\u003eZhao S, Wang H, Zhang S (2200) First report of genetic diversity and risk factor analysis of equine piroplasm infection in equids in Jilin, China. Parasit Vector 13:459. https://doi.org/10.1186/s13071-020-04338-1\u003c/li\u003e\n\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":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Theileria equi, Horses, Molecular identification, 18S rRNA gene, Vietnam","lastPublishedDoi":"10.21203/rs.3.rs-7848604/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7848604/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEquine piroplasmosis, which is caused by \u003cem\u003eTheileria equi\u003c/em\u003e and \u003cem\u003eBabesia caballi\u003c/em\u003e, has a significant impact on the veterinary and economic aspects of the global horse industry. However, many countries, including Vietnam, have not yet conducted epidemiological surveys to determine the prevalence of these haemoparasites. The aim of this study was therefore to detect \u003cem\u003eT. equi\u003c/em\u003e and/or \u003cem\u003eB. caballi\u003c/em\u003e infections in horses and to identify their genotypes. Blood samples were collected from 154 seemingly healthy horses in 8 districts of Hanoi, Thai Nguyen, and Son La provinces located in the northern part of the country. Twenty-four horses (15.58%, 95% CI: 10.70\u0026ndash;22.14%) were infected with \u003cem\u003eT. equi\u003c/em\u003e in six districts. \u003cem\u003eBabesia caballi\u003c/em\u003e was not detected. No significant association was found between gender, host age, and the number of infected horses. Phylogenetic analysis of the 18S rRNA sequences from the positive DNA samples revealed genotypes A, C, and E.\u003c/p\u003e\u003cp\u003eThe results of this study confirm the presence of equine theileriosis in northern Vietnam and highlight the need for nationwide studies of equine piroplasmosis involving a large sample size.\u003c/p\u003e","manuscriptTitle":"The first molecular detection of equine piroplasmosis in Vietnam","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 14:36:13","doi":"10.21203/rs.3.rs-7848604/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T19:01:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T16:24:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291056040268820075210911085596304264471","date":"2025-10-23T07:11:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T14:58:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-17T02:18:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-16T14:00:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2025-10-13T11:35:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aec2c888-cc7f-4f31-8dc1-8b049f4e940a","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:00:43+00:00","versionOfRecord":{"articleIdentity":"rs-7848604","link":"https://doi.org/10.1007/s00436-026-08630-4","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2026-02-05 15:57:02","publishedOnDateReadable":"February 5th, 2026"},"versionCreatedAt":"2025-10-31 14:36:13","video":"","vorDoi":"10.1007/s00436-026-08630-4","vorDoiUrl":"https://doi.org/10.1007/s00436-026-08630-4","workflowStages":[]},"version":"v1","identity":"rs-7848604","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7848604","identity":"rs-7848604","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}