The first molecular detection of benzimidazole resistance in Haemonchus contortus from Sheep in some areas of Southern Xinjiang

The first molecular detection of benzimidazole resistance in Haemonchus contortus from Sheep in some areas of Southern Xinjiang | 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 benzimidazole resistance in Haemonchus contortus from Sheep in some areas of Southern Xinjiang Reyilanmu Tuerhong, Lianxi Xin, Ying Zhang, Abudusaimaiti Tuoheti, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4545411/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Aug, 2024 Read the published version in Parasitology Research → Version 1 posted 3 You are reading this latest preprint version Abstract To understand the benzimidazole (BZ) resistance of Haemonchus contortus in Southern Xinjiang, three single nucleotide polymorphisms (SNPs, designated F167Y, E198A and F200Y) in the isotype-Ⅰβ-tubulin gene which are associated with BZ resistance were investigated for H. contortus populations from sheep in some areas of Southern Xinjiang༎In brief, a total of 190 H. contortus adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang, and species identity of each adult worm was confirmed by PCR amplification of ITS-2 using H. contortus -specific primers targeting the ITS-2. The samples were then investigated by PCR-sequencing of the isotypeⅠβ-tubulin gene for analyzing BZ-related SNPs at locus 167, 198 and 200༎The results showed that only E198A and F200Y mutations were detected in the investigated H. contortus populations. E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing county, respectively) was predominant compared with F200Y (homozygous and heterozygote resistant: found in 14% and 13.33% of sequenced samples from Minfeng and Hejing county, respectively). The results indicate a high prevalence of BZ resistance in H. contortus populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of H༎contortus in areas with similar condition༎ Haemonchus contortus benzimidazole resistance isotypeⅠβ-tubulin gene single nucleotide polymorphism Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Xinjiang is abundant in natural and social resources. Over the past few years, the region's livestock industry has experienced significant growth alongside its economic development (Xiaoxian 2013 ). Currently, the livestock industry is a crucial component of the agricultural economy in Xinjiang, with its output value consistently increasing. In Southern Xinjiang, historically known as a key pastoral region, livestock farming becomes one of the most important income sources for pastoralists (Reyaniguli 2023 ). In this region, small ruminants such as sheep (includes about 25 million sheep) are very popular and occupying a prominent position in the livestock herd structure, as they are in demand and are easy to manage (Yanfeng 2016 ). However, the spread and prevalence of gastrointestinal nematodes (GINs) are steadily increasing, posing a significant threat to the development of sheep breeding in southern Xinjiang. In particular, the harm caused by Haemonchus contortus infection is particularly severe (Gaojie 2020 ). Haemonchus contortus is a highly significant parasite that commonly infects small ruminants on a global scale (Naeem et al. 2020 ). In most of the world, large burdens of larvae can rapidly develop when environmental conditions favor the free-living stages, and lead to sudden animal deaths with minimal warning (Besier et al. 2016 ). The blood-sucking H. contortus causes anemia, weight loss, and potentially fatal outcomes in severely affected animals. In chronic cases of haemonchosis, which lead to decreased animal productivity and eventual fatalities, long-lasting infections are more common, particularly in cases of prolonged inadequate nutrition (Mohamed et al. 2024 ). In addition, the high biotic potential of this parasite, such as high egg production, strong survival ability of L3, and a lengthy parasitism period of one year within the host body (Jacquiet et al. 1995 ), contributes to its global prevalence (Besier et al. 2016 ). In China, H. contortus is widespread throughout the country (includes Xinjiang), with varying prevalence rates observed among different provinces (Wang et al. 2017 ). The control of GINs in small ruminants primarily relies on the use of anthelmintics. The rapid succession of anthelmintics over the past sixty years indeed provided many options to successfully control a wide range of GIN infections (Pavičić et al. 2023 ). Anthelmintics, especially benzimidazoles based on albendazole, have long played a miraculous role in the control of parasitic diseases. When benzimidazoles were first introduced in the 1960s, they were hailed as wonder drugs. However, the continuous and frequent medication usage of BZs have caused BZs resistance to develop, resulting in failure in controlling the spread of small ruminant GIN, especially H. contortus (Rufener et al. 2009 ). At present, the BZs resistance in H. contortus has reached alarming levels worldwide and even multi-resistance in small ruminant is redefining how anthelmintics are used in GIN control programs (dos Santos et al. 2014 ; Redman et al. 2015 ; Zhang et al. 2016 ). Recently, in parts of Southern Xinjiang, some pastoralists have been complaining a decline in the efficacy of BZs, especially in small ruminants. Although studies on resistance to benzimidazoles have been conducted in China (detecting the frequency of resistance mutation in the β-tubulin isotype 1 gene in H. contortus ), there is a lack of data on the presence of known resistance mutations (F200Y, F167Y, and E198A) in H. contortus isolated from native sheep in the Southern Xinjiang. The aim of this study was to investigate BZs resistance in H. contortus in native sheep from two regions of Southern Xinjiang by analyzing the frequency of the three SNP in the β-tubulin isotype 1 gene. The findings of this research are crucial for assessing the extent of BZs resistance and could inform strategies for farm management and anthelmintic treatment programs, ultimately helping to mitigate economic losses due to anthelmintic resistance in Xinjiang. 2. Materials and Methods 2.1. Parasite material In this work, adult worms were collected from two different geographical regions (Hejing county in Bazhou and Minfeng county in Hotan) of Southern Xinjiang. First of all, the origin information of the sheep was verified through the daily records of the abattoir's office, which records the provenance of the sheep from various regions and was confirmed with the owners who transport the sheep to the abattoir. Then, a total of 190 H. contortus adults (100 adult worms were collected from Hejing county and 90 adult worms were collected from Minfeng county) were collected from 52 (Hejing:23, Minfeng:29) out of 70 (Hejing:30, Minfeng:40) slaughtered sheep (native naturally infected sheep) in county abattoirs in two different regions of Southern Xinjiang. However, the history of anthelmintic use was unclear in all cases. After the worms were washed in physiological saline, they were sent to the laboratory for identification. 2.2. Genomic DNA isolation Upon arrival, the individual adult worms were identified based on the morphological features (Naeem et al. 2020 ; Lichtenfels et al. 1994 ). Then, total genomic DNA was extracted from individual adults using a QIAamp DNA kit (QIAamp, Hilden, Germany) following the manufacturer’s directions and then stored at − 20°C. 2.3. Species identification The species identification of each adult worm as H. contortus was confirmed by molecular biological method. With reference to the primers used in previous research, the primers targeting the ribosome second internal transcribed spacer (ITS-2) of H. contortus were synthesized by Shanghai Sheng Gong Bioengineering Technology Service Co., LTD. A 265 bp region of the ITS-2 was specifically amplified by conventional PCR from each adult worm genomic DNA using the primer pair ITS-2 F: 5’-CAAATGGCATTTGTCTTTTAG-3’ ITS-2 R: 5’-TTAGTTTCTTTTCCTCCGCT-3’ (Bott et al.2009). In brief, PCR was performed in 25 µl containing DNA 2.0 µL, upstream primers (ITS-2-F; 10 mmol/L) 1.0 µL, downstream primer (ITS-2-R; 10 mmol/L) 1.0 µL, 2×TransStart FastPfu Fly PCR SuperMix (TransGen Biotech) 12.5 µL, DEPC H2O 8.5 µL in thermal cycler using the following protocol: 94°C/5 min, followed by 35 cycles of 94°C/30 s, 55°C/30 s and 72°C/30 s, with a final extension at 72°C/5 min. A negative control (without template DNA) sample was included in each PCR run. All PCR amplicons were examined on 1% agarose gels, and detected using ultraviolet transillumination to confirm a single band of the appropriate size. 2.4. PCR amplification and direct sequencing of the isotype-1 β-tubulin gene fragment of H. contortus A 385 bp region of the isotype-1 β-tubulin gene was amplified from individual adult worm genomic DNA samples using forward and reverse primers pair (von Samson-Himmelstjerna et al. 2009 ). β-tubulin -F-5′-GACGCATTCACTTGGAGGAG-3′ β-tubulin -R-5′-CATAGGTTGGATTTGTGAGTT-3′ The PCR was performed in 25 µl as described in Subsection 2.3 using the following cycling protocol: 94 ℃/5 min, followed by 35 cycles at 94 ℃/30 s, 54 ℃/30 s and 72 ℃/30 s, with a final extension at 72 ℃/5 min. PCR amplicons were examined by using agarose gel (1%) electrophoresis. The PCR product was purified using the PCR purification kit (Jiangsu Cowin Biotech Co., Ltd, China) and direct sequencing (sent to Wuhan Qingke Bioengineering Technology Service Co., Ltd) with forward and reverse primers (in both directions). 2.5. Determination of the frequencies of BZ resistance-associated SNPs in H. contortus The sequencing results were examined by using SnapGene 6.0.2 software to obtain the original sequence peak map, and the mutations of three SNP sites (F167Y, E198A and F200Y.) were analyzed. As described in a previous study (Kotze et al. 2012 ), in single sequences, if a single peak occurs at any of the sites (F167Y, E198A, and F200Y), it was recorded as homozygous sensitive or homozygous resistant allele. When double peaks appeared, and the sequence with a ‘height’ of the secondary peak equal to or higher than at least 50% of the dominant peak at that position was considered heterozygous. Finally, the genotypic and allelic frequencies of H. contortus from two different regions were calculated. 3. Results 3.1. Morphological identification of H. contortus Nematodes were discovered attached to the wall of the abomasum during dissection, and morphological identification was performed on the collected adult worms. The results indicated that the adult worms collected exhibited distinct morphological characteristics that allowed for easy identification under a microscope. The female worm, measuring approximately 2–3 cm in length, displayed a characteristic 'twist' feature (Fig. 1 A). In contrast, the male worm was smaller than the female and exhibited a 'Y'-shaped spicules at the tail end (Fig. 1 B & C). Through morphological characteristics of the adult worms (Wang 2009), H. contortus worms were identified. 3.2. Molecular identification of H. contortus To confirm the identity of the collected adult worms as H. contortus , DNA samples extracted from the 190 adult worms were subjected to PCR amplification using specific primers targeting the ITS-2 gene of H. contortus , producing a single band of approximately 265 bp in length in each of 190 samples (selected results shown in Fig. 2 ). In addition, 20 purified PCR products were sequenced and the sequencing results were consistent with those of H. contortus sequence. This confirmed that the adult worm samples selected were indeed H. contortus and suitable for further experimentation. 3.3. Detection of SNPs in the isotype-1 β-tubulin gene associated with resistance to BZs in H. contortus The isotype-1 β-tubulin gene of H. contortus was successfully amplified from each of 190 samples using gene-specific primers, producing a single band of 385 bp in length for each sample (representative results shown in Fig. 3 ). PCR products were then purified and subjected to sequencing. The resulting sequence peaks were then analyzed for SNP, as illustrated in Fig. 4 . The peak chart data indicates that the 167 position in samples from the two regional populations is homozygous sensitive, while positions 198 and 200 in these samples exhibit varying degrees of heterozygous benzimidazoles-resistant and homozygous benzimidazoles-resistant types. 3.4. The SNP frequency and genotypes in the isotype-1 β-tubulin gene in two H. contortus populations For all 190 individual adult worms from two H. contortus populations, three SNPs positions including F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) were examined (Table 1 ). The results revealed that for SNP F167Y, all H. contortus samples were homozygous sensitive. For SNP E198A, in the Bazhou and Hotan populations, 70% and 60% of the H. contortus samples were homozygous sensitive, respectively; 18% and 7.78% were heterozygous resistant, respectively and 12% and 32.2% were homozygous resistant, respectively. Moreover, for SNP F200Y, 86% of H. contortus samples (from Bazhou population) and 86.7% (from Hotan population) were homozygous sensitive, respectively; 7% (Bazhou) and 2.22% (Hotan) were heterozygous resistant, respectively; 32.22% (Bazhou) and 11.11% (Hotan) were homozygous resistant, respectively. Table 1 Frequencies of three SNPs in the isotype-1 ß tubulin gene that associated with benzimidazole resistance of Haemonchus contortus from two populations in Southern Xinjiang Population Allele No of homozygous sensitive (%) No of heterozygous resistant (%) No of homozygous resistant (%) Bazhou 167 100(100) 0(0) 0(0) 198 70(70) 18(18) 12(12) 200 86(86) 7(7) 7(7) Hotan 167 90(100) 0(0) 0(0) 198 54(60) 7(7.78) 29(32.22) 200 78(86.67) 2(2.22) 10(11.11) In addition, based on manual comparison and analysis of mutations at the E198A and F200Y sites, a total of 6 genotypes were detected, namely Hs-198, Hs-200; Hs-198, Het-200; Hs-198, HR-200; Het-198, Hs-200; Het-198, Het-200; and HR-198, Hs-200 (Table 2 ). The genotypes (Hs-198, Hs-200) that are homozygous sensitive at both 198 and 200 sites have the highest frequency in the two populations, with the frequencies of 56% and 45.6%, respectively. The frequencies of HR-198 (HR-198, Hs-200) were 12% and 32.22%, respectively. The frequencies of HR-200 (Hs-198, HR-200) were 7% and 11.11%, respectively. The frequency of HR-198 genotype was higher than that of HR-200. Heterozygous genotype at two positions (Het-198, Het-200) was detected in Hotan populations with 1.11% of frequency. Table 2 The genotype frequencies (%) of isotype-I β-tubulin gene in Xinjiang H. contortus populations Population genotype(frequencies %) Hs-198, Hs-200 Het-198, Hs-200 Hs-198, Het-200 Het-198, Het-200 Hs-198, HR-200 HR-198, Hs-200 Bazhou 56(56) 18(18) 7(7) 0(0) 7(7) 12(12) Hotan 41(45.6) 7(7.78) 2(2.22) 1(1.11) 10(11.11) 29(32.22) Note: Hs: pure sensitized HR: pure resistant Het: heterozygous resistant 4. Discussion BZs are commonly used anthelmintics to treat infections with GIN in both humans and animals. The extensive use of BZs, such as albendazole, in control programs, coupled with limited alternative anthelmintic options and high rates of reinfection in endemic regions globally, has led to the development of BZs resistance in GIN populations infecting animals (Mohammedsalih et al. 2019 ; Pitaksakulrat et al. 2021 ). The majority of previous studies focus on the species belonging to the three most important genera of small ruminant nematodes, Haemonchus , Trichostrongylus and Teladorsagia , and highlights the serious issue of resistance to BZs. For instance, for Haemonchus nematodes, the presence of BZs resistance in South America, Oceania, Africa, Europe, and Asia is well documented, especially in Brazil, Australia, Sudan, France, Thailand, as well as in China (Kotze et al. 2016). Furthermore, H. contortus is widely recognized as the primary BZ-resistant nematode or even multi-resistant species in many countries, but there is no relevant research on this issue in southern Xinjiang. This is the first initial documentation of BZs resistance from H. contortus in Southern Xinjiang by screening the frequency of the three SNPs polymorphism in the β-tubulin isotype 1 gene. In the two populations, the mutations of resistance SNP E198A and F200Y were detected, and the SNP E198A was most frequently encountered, whereas F167Y was not detected. The results indicate that the SNP E198A was the most common BZ resistance-associated SNP in the two populations, in accordance with previous reports that E198A is more prevalent than other two SNPs in H. contortus populations in China (Zhang et al. 2016 ; Shen et al. 2019 ). It has been proposed that a population is considered BZ resistant if ≥ 10% of individuals carry a resistant genotype (Barrere et al. 2013). In this study, both E198A and F200Y were detected in two H. contortus populations, with frequencies ranging from 30–40% and 13.33–14%, respectively (Table 1 ). In addition, in the two populations, homozygous resistant genotypes (Hs-198, HR-200 and HR-198, Hs-200) ranged from 7–32.22% (Table 2 ). These findings indicate that there is a high frequency of BZs resistance in both regions, which is similar to the previous studies in China (Zhang et al. 2016 ; Yang et al., 2017; Xin et al. 2018 ). This suggests a potential overuse of BZ drugs in these regions over time, as evidenced by the presence of heterozygosity at positions E198A and F200Y in both populations. Furthermore, in this study, no H. contortus genotypes were identified as 'homozygous resistant' at both codon 198 and 200, or 'homozygous resistant' at one codon (198 or 200) and heterozygous at another codon (200 or 198) within both populations in Southern Xinjiang. This observation could be attributed to a 'mutual exclusion' phenomenon between the two homozygous alterations, as both mutations would lead to worm lethality (Kotze et al. 2012 ; Ghisi et al. 2007 ). In Xinjiang, the treatment of GIN disease mainly relys on the usage of two groups of anthelmintics, including benzimidazole and ivermectin. The veterinarians and farmers commonly utilize BZ drugs like albendazole over an extended period. The prolonged administration of albendazole may lead to alterations in the genes associated with BZs resistance in H. contortus . Small ruminant agriculture, particularly sheep farming, is predominantly concentrated in the southern Xinjiang region as they are in demand for local people. Sheep have been a staple livestock for generations, resulting in prolonged exposure to anthelmintics and ultimately leading to the development of resistance. These results confirm the rapid selection and emergence of allele resistance in South Xinjiang. Conclusions In conclusion, this is the first analysis of all three BZ resistance-associated SNPs (F167Y, E198A and F200Y) in the isotype-1 β-tubulin gene of H. contortus populations from sheep in Southern Xinjiang. Different frequencies of E198A and F200Y were detected in two populations studied. E198A occurred more frequently than did F200Y in field populations, whereas F167Y was not detected. Finally, in our opinion, there is an urgent need for detailed studies on BZ resistance in H. contortus of livestock animals in Southern Xinjiang. Declarations Acknowledgments The authors would like to thank the members of the College of Veterinary Medicine, Xinjiang Agricultural University for their scientific input. Thanks to Professor Hu Min from Huazhong Agricultural University for his revision and guidance Funding The work was funded by the “Tianchi Talents” Introduction Program of the Autonomous Region Talent Development Fund. Availability of data and materials Not applicable. Ethics approval Not applicable. Consent to participate and consent to publish Not applicable. Competing interests The authors declare no competing interests. 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Yanfeng X (2016) Comparative study of the animal husbandry specialized cooperative business modes in Xinjiang. Xinjiang Agricultural University. Zhang Z, Gasser RB, Yang X, Yin F, Zhao G, Bao M, Pan B, Huang W, Wang C, Zou F, Zhou Y, Zhao J, Fang R, Hu M (2016) Two benzimidazole resistance-associated SNPs in the isotype-1 β-tubulin gene predominate in Haemonchus contortus populations from eight regions in China. Int J Parasitol Drugs Drug Resist 6, 199-206. https://doi.org/10.1016/j.ijpddr.2016.10.001 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 14 Aug, 2024 Read the published version in Parasitology Research → Version 1 posted Editor assigned by journal 10 Jun, 2024 Submission checks completed at journal 10 Jun, 2024 First submitted to journal 07 Jun, 2024 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-4545411","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315947971,"identity":"476a96c9-90d7-4c48-82dc-eddec014ff5d","order_by":0,"name":"Reyilanmu Tuerhong","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Reyilanmu","middleName":"","lastName":"Tuerhong","suffix":""},{"id":315947972,"identity":"5e29c632-29fc-4d3d-a453-660725a8ed00","order_by":1,"name":"Lianxi Xin","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lianxi","middleName":"","lastName":"Xin","suffix":""},{"id":315947973,"identity":"14e74013-cc0e-4167-b4c3-beb2dc367884","order_by":2,"name":"Ying Zhang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":315947975,"identity":"57727407-62f3-4dc8-b9b9-0bf82d95b340","order_by":3,"name":"Abudusaimaiti Tuoheti","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Abudusaimaiti","middleName":"","lastName":"Tuoheti","suffix":""},{"id":315947976,"identity":"5532396e-d180-4e0a-9858-0fd45daae780","order_by":4,"name":"Yi Zhang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":315947979,"identity":"0bc35ea3-72ee-4b96-9c7e-d9fda0c8b9d4","order_by":5,"name":"Saifuding Abula","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Saifuding","middleName":"","lastName":"Abula","suffix":""},{"id":315947980,"identity":"abd5e36c-2705-4a5b-b9b2-f196dbb13eca","order_by":6,"name":"Adelijiang Wusiman","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Adelijiang","middleName":"","lastName":"Wusiman","suffix":""},{"id":315947982,"identity":"aa8f7d9d-523b-435a-a00c-f21d544578ff","order_by":7,"name":"kalibixiati aimulajiang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"kalibixiati","middleName":"","lastName":"aimulajiang","suffix":""},{"id":315947984,"identity":"b2249899-09fe-4f1f-96bd-28ed51684ed4","order_by":8,"name":"Waresi Tuersong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie3RMQrCMBTG8RcCcQm6Pqm0JxACBaFLvUpjwKnudSsITh5AsbcQxDEgOOlecbAidOqgu6CrTo2bYP77D97HA7DZfjDmFbJoP55h/7zVZqQJ0VUEKVWQs8iMuCBLHKeUpDkXhofBdojHDaNkObvnFYRuN60jZKqCxZ43aeewCjJQfk/XEcrlCRlSBqO1w0HLdS1hrZvzYILMIC4NCYcBtqcRmWPMDAmCErjXSvCdH2TCYIs3B1lgokPRmFzyKgndWvIRmr7mnXwrbDab7S96AWKIPwyK2dt9AAAAAElFTkSuQmCC","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Waresi","middleName":"","lastName":"Tuersong","suffix":""}],"badges":[],"createdAt":"2024-06-07 10:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4545411/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4545411/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00436-024-08314-x","type":"published","date":"2024-08-14T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58919067,"identity":"7c262786-23fa-4111-91f5-b947a814c900","added_by":"auto","created_at":"2024-06-24 06:34:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2712538,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological features of adult female and male worms of \u003cem\u003eHaemonchus contortus\u003c/em\u003e. a \u0026amp; b: Tail end (Spicules and copulatory bursa) of a male worm; c: Intestine and gonad of a female worm; d: Vulval flap of an adult female (the arrow is pointing).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4545411/v1/2b688cf34cb84bca15626227.png"},{"id":58918667,"identity":"8e3d114f-9381-4074-ae07-820777b7d7c0","added_by":"auto","created_at":"2024-06-24 06:26:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203322,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis of PCR products of ITS-2 amplified from \u003cem\u003eHaemonchus contortus\u003c/em\u003e individual worms. Lane M DNA ladder (100–2000 bp); 1–7 DNA from individual adult worm; Lane 9, 10 negative controls.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4545411/v1/5f8fd0d65c291923e583455b.png"},{"id":58919068,"identity":"11a09d97-a0ea-4df1-8b43-ac78a90b9b95","added_by":"auto","created_at":"2024-06-24 06:34:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120206,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis of PCR products of I-β tubulin gene amplified from Haemonchus contortus individual worms. Lane M DNA ladder (100–2000 bp); 1–7 DNA from individual adult worm; Lane 9, 10 negative controls.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4545411/v1/03e2801b42a5fe60046ad62d.png"},{"id":58918670,"identity":"cea9445f-ef6c-411f-8590-49c95c950f9a","added_by":"auto","created_at":"2024-06-24 06:26:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2568346,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms of homozygous resistant and heterozygote allele sequences of I-β tubulin gene. a: The homozygous resistant (RR) genotype at codons 198 (GAA to GCA, E198A); b: The homozygous resistant (RR) genotype at codons 200 (TTC to TAC; F200Y); c \u0026amp; d: The heterozygous (SR) genotype at codons 198 and 200.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4545411/v1/9385071cf58e10ef63aceff3.png"},{"id":63071239,"identity":"77223d40-09d9-42b5-b1eb-71da1aeb39ab","added_by":"auto","created_at":"2024-08-22 20:05:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11524522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4545411/v1/c5172713-bff9-4ba0-9ee6-48973cdc5478.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The first molecular detection of benzimidazole resistance in Haemonchus contortus from Sheep in some areas of Southern Xinjiang","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eXinjiang is abundant in natural and social resources. Over the past few years, the region's livestock industry has experienced significant growth alongside its economic development (Xiaoxian \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Currently, the livestock industry is a crucial component of the agricultural economy in Xinjiang, with its output value consistently increasing. In Southern Xinjiang, historically known as a key pastoral region, livestock farming becomes one of the most important income sources for pastoralists (Reyaniguli \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this region, small ruminants such as sheep (includes about 25\u0026nbsp;million sheep) are very popular and occupying a prominent position in the livestock herd structure, as they are in demand and are easy to manage (Yanfeng \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the spread and prevalence of gastrointestinal nematodes (GINs) are steadily increasing, posing a significant threat to the development of sheep breeding in southern Xinjiang. In particular, the harm caused by \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection is particularly severe (Gaojie \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e is a highly significant parasite that commonly infects small ruminants on a global scale (Naeem et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In most of the world, large burdens of larvae can rapidly develop when environmental conditions favor the free-living stages, and lead to sudden animal deaths with minimal warning (Besier et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The blood-sucking \u003cem\u003eH. contortus\u003c/em\u003e causes anemia, weight loss, and potentially fatal outcomes in severely affected animals. In chronic cases of haemonchosis, which lead to decreased animal productivity and eventual fatalities, long-lasting infections are more common, particularly in cases of prolonged inadequate nutrition (Mohamed et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, the high biotic potential of this parasite, such as high egg production, strong survival ability of L3, and a lengthy parasitism period of one year within the host body (Jacquiet et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), contributes to its global prevalence (Besier et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In China, \u003cem\u003eH. contortus\u003c/em\u003e is widespread throughout the country (includes Xinjiang), with varying prevalence rates observed among different provinces (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe control of GINs in small ruminants primarily relies on the use of anthelmintics. The rapid succession of anthelmintics over the past sixty years indeed provided many options to successfully control a wide range of GIN infections (Pavičić et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Anthelmintics, especially benzimidazoles based on albendazole, have long played a miraculous role in the control of parasitic diseases. When benzimidazoles were first introduced in the 1960s, they were hailed as wonder drugs. However, the continuous and frequent medication usage of BZs have caused BZs resistance to develop, resulting in failure in controlling the spread of small ruminant GIN, especially \u003cem\u003eH. contortus\u003c/em\u003e (Rufener et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). At present, the BZs resistance in \u003cem\u003eH. contortus\u003c/em\u003e has reached alarming levels worldwide and even multi-resistance in small ruminant is redefining how anthelmintics are used in GIN control programs (dos Santos et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Redman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, in parts of Southern Xinjiang, some pastoralists have been complaining a decline in the efficacy of BZs, especially in small ruminants. Although studies on resistance to benzimidazoles have been conducted in China (detecting the frequency of resistance mutation in the β-tubulin isotype 1 gene in \u003cem\u003eH. contortus\u003c/em\u003e), there is a lack of data on the presence of known resistance mutations (F200Y, F167Y, and E198A) in \u003cem\u003eH. contortus\u003c/em\u003e isolated from native sheep in the Southern Xinjiang. The aim of this study was to investigate BZs resistance in \u003cem\u003eH. contortus\u003c/em\u003e in native sheep from two regions of Southern Xinjiang by analyzing the frequency of the three SNP in the β-tubulin isotype 1 gene. The findings of this research are crucial for assessing the extent of BZs resistance and could inform strategies for farm management and anthelmintic treatment programs, ultimately helping to mitigate economic losses due to anthelmintic resistance in Xinjiang.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Parasite material\u003c/h2\u003e \u003cp\u003eIn this work, adult worms were collected from two different geographical regions (Hejing county in Bazhou and Minfeng county in Hotan) of Southern Xinjiang. First of all, the origin information of the sheep was verified through the daily records of the abattoir's office, which records the provenance of the sheep from various regions and was confirmed with the owners who transport the sheep to the abattoir. Then, a total of 190 \u003cem\u003eH. contortus\u003c/em\u003e adults (100 adult worms were collected from Hejing county and 90 adult worms were collected from Minfeng county) were collected from 52 (Hejing:23, Minfeng:29) out of 70 (Hejing:30, Minfeng:40) slaughtered sheep (native naturally infected sheep) in county abattoirs in two different regions of Southern Xinjiang. However, the history of anthelmintic use was unclear in all cases. After the worms were washed in physiological saline, they were sent to the laboratory for identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Genomic DNA isolation\u003c/h2\u003e \u003cp\u003eUpon arrival, the individual adult worms were identified based on the morphological features (Naeem et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lichtenfels et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Then, total genomic DNA was extracted from individual adults using a QIAamp DNA kit (QIAamp, Hilden, Germany) following the manufacturer\u0026rsquo;s directions and then stored at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Species identification\u003c/h2\u003e \u003cp\u003eThe species identification of each adult worm as \u003cem\u003eH. contortus\u003c/em\u003e was confirmed by molecular biological method. With reference to the primers used in previous research, the primers targeting the ribosome second internal transcribed spacer (ITS-2) of \u003cem\u003eH. contortus\u003c/em\u003e were synthesized by Shanghai Sheng Gong Bioengineering Technology Service Co., LTD. A 265 bp region of the ITS-2 was specifically amplified by conventional PCR from each adult worm genomic DNA using the primer pair\u003c/p\u003e \u003cp\u003eITS-2 F: 5\u0026rsquo;-CAAATGGCATTTGTCTTTTAG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eITS-2 R: 5\u0026rsquo;-TTAGTTTCTTTTCCTCCGCT-3\u0026rsquo; (Bott et al.2009). In brief, PCR was performed in 25 \u0026micro;l containing DNA 2.0 \u0026micro;L, upstream primers (ITS-2-F; 10 mmol/L) 1.0 \u0026micro;L, downstream primer (ITS-2-R; 10 mmol/L) 1.0 \u0026micro;L, 2\u0026times;TransStart FastPfu Fly PCR SuperMix (TransGen Biotech) 12.5 \u0026micro;L, DEPC H2O 8.5 \u0026micro;L in thermal cycler using the following protocol: 94\u0026deg;C/5 min, followed by 35 cycles of 94\u0026deg;C/30 s, 55\u0026deg;C/30 s and 72\u0026deg;C/30 s, with a final extension at 72\u0026deg;C/5 min. A negative control (without template DNA) sample was included in each PCR run. All PCR amplicons were examined on 1% agarose gels, and detected using ultraviolet transillumination to confirm a single band of the appropriate size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. PCR amplification and direct sequencing of the isotype-1 β-tubulin gene fragment of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA 385 bp region of the isotype-1 β-tubulin gene was amplified from individual adult worm genomic DNA samples using forward and reverse primers pair (von Samson-Himmelstjerna et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eβ-tubulin -F-5\u0026prime;-GACGCATTCACTTGGAGGAG-3\u0026prime;\u003c/p\u003e \u003cp\u003eβ-tubulin -R-5\u0026prime;-CATAGGTTGGATTTGTGAGTT-3\u0026prime;\u003c/p\u003e \u003cp\u003eThe PCR was performed in 25 \u0026micro;l as described in Subsection \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e using the following cycling protocol: 94 ℃/5 min, followed by 35 cycles at 94 ℃/30 s, 54 ℃/30 s and 72 ℃/30 s, with a final extension at 72 ℃/5 min. PCR amplicons were examined by using agarose gel (1%) electrophoresis. The PCR product was purified using the PCR purification kit (Jiangsu Cowin Biotech Co., Ltd, China) and direct sequencing (sent to Wuhan Qingke Bioengineering Technology Service Co., Ltd) with forward and reverse primers (in both directions).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of the frequencies of BZ resistance-associated SNPs in \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe sequencing results were examined by using SnapGene 6.0.2 software to obtain the original sequence peak map, and the mutations of three SNP sites (F167Y, E198A and F200Y.) were analyzed. As described in a previous study (Kotze et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), in single sequences, if a single peak occurs at any of the sites (F167Y, E198A, and F200Y), it was recorded as homozygous sensitive or homozygous resistant allele. When double peaks appeared, and the sequence with a \u0026lsquo;height\u0026rsquo; of the secondary peak equal to or higher than at least 50% of the dominant peak at that position was considered heterozygous. Finally, the genotypic and allelic frequencies of \u003cem\u003eH. contortus\u003c/em\u003e from two different regions were calculated.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Morphological identification of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eNematodes were discovered attached to the wall of the abomasum during dissection, and morphological identification was performed on the collected adult worms. The results indicated that the adult worms collected exhibited distinct morphological characteristics that allowed for easy identification under a microscope. The female worm, measuring approximately 2\u0026ndash;3 cm in length, displayed a characteristic 'twist' feature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, the male worm was smaller than the female and exhibited a 'Y'-shaped spicules at the tail end (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; C). Through morphological characteristics of the adult worms (Wang 2009), \u003cem\u003eH. contortus\u003c/em\u003e worms were identified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Molecular identification of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo confirm the identity of the collected adult worms as \u003cem\u003eH. contortus\u003c/em\u003e, DNA samples extracted from the 190 adult worms were subjected to PCR amplification using specific primers targeting the ITS-2 gene of \u003cem\u003eH. contortus\u003c/em\u003e, producing a single band of approximately 265 bp in length in each of 190 samples (selected results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, 20 purified PCR products were sequenced and the sequencing results were consistent with those of \u003cem\u003eH. contortus\u003c/em\u003e sequence. This confirmed that the adult worm samples selected were indeed \u003cem\u003eH. contortus\u003c/em\u003e and suitable for further experimentation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Detection of SNPs in the isotype-1 β-tubulin gene associated with resistance to BZs in\u003c/b\u003e \u003cb\u003eH. contortus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe isotype-1 β-tubulin gene of \u003cem\u003eH. contortus\u003c/em\u003e was successfully amplified from each of 190 samples using gene-specific primers, producing a single band of 385 bp in length for each sample (representative results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). PCR products were then purified and subjected to sequencing. The resulting sequence peaks were then analyzed for SNP, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The peak chart data indicates that the 167 position in samples from the two regional populations is homozygous sensitive, while positions 198 and 200 in these samples exhibit varying degrees of heterozygous benzimidazoles-resistant and homozygous benzimidazoles-resistant types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The SNP frequency and genotypes in the isotype-1 β-tubulin gene in two \u003cem\u003eH. contortus\u003c/em\u003e populations\u003c/h2\u003e \u003cp\u003eFor all 190 individual adult worms from two \u003cem\u003eH. contortus\u003c/em\u003e populations, three SNPs positions including F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) were examined (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results revealed that for SNP F167Y, all \u003cem\u003eH. contortus\u003c/em\u003e samples were homozygous sensitive. For SNP E198A, in the Bazhou and Hotan populations, 70% and 60% of the \u003cem\u003eH. contortus\u003c/em\u003e samples were homozygous sensitive, respectively; 18% and 7.78% were heterozygous resistant, respectively and 12% and 32.2% were homozygous resistant, respectively. Moreover, for SNP F200Y, 86% of \u003cem\u003eH. contortus\u003c/em\u003e samples (from Bazhou population) and 86.7% (from Hotan population) were homozygous sensitive, respectively; 7% (Bazhou) and 2.22% (Hotan) were heterozygous resistant, respectively; 32.22% (Bazhou) and 11.11% (Hotan) were homozygous resistant, respectively.\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\u003eFrequencies of three SNPs in the isotype-1 \u0026szlig; tubulin gene that associated with benzimidazole resistance of \u003cem\u003eHaemonchus contortus\u003c/em\u003e from two populations in Southern Xinjiang\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAllele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo of homozygous sensitive (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo of heterozygous resistant (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo of homozygous resistant (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBazhou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70(70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18(18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12(12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86(86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7(7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7(7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHotan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54(60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7(7.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29(32.22)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78(86.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(2.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10(11.11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition, based on manual comparison and analysis of mutations at the E198A and F200Y sites, a total of 6 genotypes were detected, namely Hs-198, Hs-200; Hs-198, Het-200; Hs-198, HR-200; Het-198, Hs-200; Het-198, Het-200; and HR-198, Hs-200 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The genotypes (Hs-198, Hs-200) that are homozygous sensitive at both 198 and 200 sites have the highest frequency in the two populations, with the frequencies of 56% and 45.6%, respectively. The frequencies of HR-198 (HR-198, Hs-200) were 12% and 32.22%, respectively. The frequencies of HR-200 (Hs-198, HR-200) were 7% and 11.11%, respectively. The frequency of HR-198 genotype was higher than that of HR-200. Heterozygous genotype at two positions (Het-198, Het-200) was detected in Hotan populations with 1.11% of frequency.\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\u003eThe genotype frequencies (%) of isotype-I β-tubulin gene in Xinjiang \u003cem\u003eH. contortus\u003c/em\u003e populations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003egenotype(frequencies %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHs-198,\u003c/p\u003e \u003cp\u003eHs-200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHet-198,\u003c/p\u003e \u003cp\u003eHs-200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHs-198,\u003c/p\u003e \u003cp\u003eHet-200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHet-198,\u003c/p\u003e \u003cp\u003eHet-200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHs-198,\u003c/p\u003e \u003cp\u003eHR-200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHR-198,\u003c/p\u003e \u003cp\u003eHs-200\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBazhou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56(56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18(18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7(7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7(7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12(12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHotan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41(45.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7(7.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(2.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(1.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10(11.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29(32.22)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Hs: pure sensitized HR: pure resistant Het: heterozygous resistant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBZs are commonly used anthelmintics to treat infections with GIN in both humans and animals. The extensive use of BZs, such as albendazole, in control programs, coupled with limited alternative anthelmintic options and high rates of reinfection in endemic regions globally, has led to the development of BZs resistance in GIN populations infecting animals (Mohammedsalih et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pitaksakulrat et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The majority of previous studies focus on the species belonging to the three most important genera of small ruminant nematodes, \u003cem\u003eHaemonchus\u003c/em\u003e, \u003cem\u003eTrichostrongylus\u003c/em\u003e and \u003cem\u003eTeladorsagia\u003c/em\u003e, and highlights the serious issue of resistance to BZs. For instance, for \u003cem\u003eHaemonchus\u003c/em\u003e nematodes, the presence of BZs resistance in South America, Oceania, Africa, Europe, and Asia is well documented, especially in Brazil, Australia, Sudan, France, Thailand, as well as in China (Kotze et al. 2016). Furthermore, \u003cem\u003eH. contortus\u003c/em\u003e is widely recognized as the primary BZ-resistant nematode or even multi-resistant species in many countries, but there is no relevant research on this issue in southern Xinjiang.\u003c/p\u003e \u003cp\u003eThis is the first initial documentation of BZs resistance from \u003cem\u003eH. contortus\u003c/em\u003e in Southern Xinjiang by screening the frequency of the three SNPs polymorphism in the β-tubulin isotype 1 gene. In the two populations, the mutations of resistance SNP E198A and F200Y were detected, and the SNP E198A was most frequently encountered, whereas F167Y was not detected. The results indicate that the SNP E198A was the most common BZ resistance-associated SNP in the two populations, in accordance with previous reports that E198A is more prevalent than other two SNPs in \u003cem\u003eH. contortus\u003c/em\u003e populations in China (Zhang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been proposed that a population is considered BZ resistant if ≥ 10% of individuals carry a resistant genotype (Barrere et al. 2013). In this study, both E198A and F200Y were detected in two \u003cem\u003eH. contortus\u003c/em\u003e populations, with frequencies ranging from 30–40% and 13.33–14%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, in the two populations, homozygous resistant genotypes (Hs-198, HR-200 and HR-198, Hs-200) ranged from 7–32.22% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings indicate that there is a high frequency of BZs resistance in both regions, which is similar to the previous studies in China (Zhang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al., 2017; Xin et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This suggests a potential overuse of BZ drugs in these regions over time, as evidenced by the presence of heterozygosity at positions E198A and F200Y in both populations. Furthermore, in this study, no \u003cem\u003eH. contortus\u003c/em\u003e genotypes were identified as 'homozygous resistant' at both codon 198 and 200, or 'homozygous resistant' at one codon (198 or 200) and heterozygous at another codon (200 or 198) within both populations in Southern Xinjiang. This observation could be attributed to a 'mutual exclusion' phenomenon between the two homozygous alterations, as both mutations would lead to worm lethality (Kotze et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ghisi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Xinjiang, the treatment of GIN disease mainly relys on the usage of two groups of anthelmintics, including benzimidazole and ivermectin. The veterinarians and farmers commonly utilize BZ drugs like albendazole over an extended period. The prolonged administration of albendazole may lead to alterations in the genes associated with BZs resistance in \u003cem\u003eH. contortus\u003c/em\u003e. Small ruminant agriculture, particularly sheep farming, is predominantly concentrated in the southern Xinjiang region as they are in demand for local people. Sheep have been a staple livestock for generations, resulting in prolonged exposure to anthelmintics and ultimately leading to the development of resistance. These results confirm the rapid selection and emergence of allele resistance in South Xinjiang.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this is the first analysis of all three BZ resistance-associated SNPs (F167Y, E198A and F200Y) in the isotype-1 β-tubulin gene of \u003cem\u003eH. contortus\u003c/em\u003e populations from sheep in Southern Xinjiang. Different frequencies of E198A and F200Y were detected in two populations studied. E198A occurred more frequently than did F200Y in field populations, whereas F167Y was not detected. Finally, in our opinion, there is an urgent need for detailed studies on BZ resistance in \u003cem\u003eH. contortus\u003c/em\u003e of livestock animals in Southern Xinjiang.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e The authors would like to thank the members of the College of Veterinary Medicine, Xinjiang Agricultural University for their scientific input.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThanks to Professor Hu Min from Huazhong Agricultural University for his revision and guidance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe work was funded by the \u0026ldquo;Tianchi Talents\u0026rdquo; Introduction Program of the Autonomous Region Talent Development Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and consent to publish\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarrere V, Falzon LC, Shakya KP, Menzies PI, Peregrine AS, Prichard RK (2014) Assessment of benzimidazole resistance in \u003cem\u003eHaemonchus contortus \u003c/em\u003ein sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance. Vet Parasitol 198, 159-165. doi:https://doi.org/10.1016/j.vetpar.2013.07.040\u003c/li\u003e\n\u003cli\u003eBesier RB, Kahn LP, Sargison ND, Van Wyk JA (2016) The pathophysiology, ecology and epidemiology of \u003cem\u003eHaemonchus contortus \u003c/em\u003einfection in small ruminants. Adv Parasitol 93, 95-143.doi: https://doi.org/10.1016/bs.apar.2016.02.022\u003c/li\u003e\n\u003cli\u003eBott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, Gasser RB (2009) A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep. Int J Parasitol 39, 1277-87. doi: 10.1016/j.ijpara.2009.03.002\u003c/li\u003e\n\u003cli\u003edos Santos JM, Monteiro JP, Ribeiro WL, Macedo IT, Camur\u0026ccedil;a-Vasconcelos AL, Vieira Lda S, Bevilaqua CM (2014) Identification and quantification of benzimidazole resistance polymorphisms in \u003cem\u003eHaemonchus contortus\u003c/em\u003e isolated in Northeastern Brazil. 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J Parasitol. 81,1013-5.\u003c/li\u003e\n\u003cli\u003eKotze AC, Cowling K, Bagnall NH, Hines BM, Ruffell AP, Hunt PW, Coleman GT (2012) Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of\u003cem\u003e Haemonchus contortus\u003c/em\u003e. Int J Parasitol Drugs Drug Resist 3, 92-7. doi: https://doi.org/10.1016/j.ijpddr.2012.02.003\u003c/li\u003e\n\u003cli\u003eKotze AC, Prichard RK (2016) Anthelmintic resistance in \u003cem\u003eHaemonchus contortus\u003c/em\u003e: history, mechanisms and diagnosis. Adv Parasitol 93:397-428. doi: 10.1016/bs.apar.2016.02.012.\u003c/li\u003e\n\u003cli\u003eLichtenfels JR, Pilitt PA, Hoberg EP (1994) New morphological characters for identifying individual specimens of \u003cem\u003eHaemonchus spp.\u003c/em\u003e (Nematoda: Trichostrongyloidea) and a key to species in ruminants of North America. 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Int J Parasitol Drugs Drug Resist 6, 199-206. https://doi.org/10.1016/j.ijpddr.2016.10.001\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":"Haemonchus contortus, benzimidazole resistance, isotypeⅠβ-tubulin gene, single nucleotide polymorphism","lastPublishedDoi":"10.21203/rs.3.rs-4545411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4545411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo understand the benzimidazole (BZ) resistance of \u003cem\u003eHaemonchus contortus\u003c/em\u003e in Southern Xinjiang, three single nucleotide polymorphisms (SNPs, designated F167Y, E198A and F200Y) in the isotype-Ⅰβ-tubulin gene which are associated with BZ resistance were investigated for \u003cem\u003eH. contortus\u003c/em\u003e populations from sheep in some areas of Southern Xinjiang༎In brief, a total of 190 \u003cem\u003eH. contortus\u003c/em\u003e adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang, and species identity of each adult worm was confirmed by PCR amplification of ITS-2 using \u003cem\u003eH. contortus\u003c/em\u003e-specific primers targeting the ITS-2. The samples were then investigated by PCR-sequencing of the isotypeⅠβ-tubulin gene for analyzing BZ-related SNPs at locus 167, 198 and 200༎The results showed that only E198A and F200Y mutations were detected in the investigated \u003cem\u003eH. contortus\u003c/em\u003e populations. E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing county, respectively) was predominant compared with F200Y (homozygous and heterozygote resistant: found in 14% and 13.33% of sequenced samples from Minfeng and Hejing county, respectively). The results indicate a high prevalence of BZ resistance in \u003cem\u003eH. contortus\u003c/em\u003e populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of \u003cem\u003eH༎contortus\u003c/em\u003e in areas with similar condition༎\u003c/p\u003e","manuscriptTitle":"The first molecular detection of benzimidazole resistance in Haemonchus contortus from Sheep in some areas of Southern Xinjiang","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 06:26:55","doi":"10.21203/rs.3.rs-4545411/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-10T10:15:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T04:48:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2024-06-07T10:12:52+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":"c5d7586b-cbf4-4bfa-a5ad-63bfe49a6bbf","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:33:00+00:00","versionOfRecord":{"articleIdentity":"rs-4545411","link":"https://doi.org/10.1007/s00436-024-08314-x","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2024-08-14 15:57:28","publishedOnDateReadable":"August 14th, 2024"},"versionCreatedAt":"2024-06-24 06:26:55","video":"","vorDoi":"10.1007/s00436-024-08314-x","vorDoiUrl":"https://doi.org/10.1007/s00436-024-08314-x","workflowStages":[]},"version":"v1","identity":"rs-4545411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4545411","identity":"rs-4545411","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}