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However, the prevalence of <jats:italic>Trichomonas<\/jats:italic> spp., a group of parasites known to induce diarrhea in various hosts, presents significant challenges in breeding facilities. These parasites pose a substantial threat to the pig breeding industry. Furthermore, despite its prevalence, diagnosing <jats:italic>Trichomonas<\/jats:italic> spp. is often challenging, primarily owing to the presence of mixed infections involving different species within clinical samples. To address this concern, we developed a novel isolation method that combines a single-cell isolation culture technique with an antimicrobial drug susceptibility test.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>Trichomonas was isolated and cultured by using the established single-worm separation technology combined with antibacterial drug screening method, and it was identified as <jats:italic>Pentatrichomonas hominis<\/jats:italic> by molecular biological identification and morphological identification. The in vitro culture conditions of the isolate were optimized to establish a stable in vitro culture system.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The method developed in this study was effective in successfully isolating a pure species of trichomonad from fecal samples obtained from weaned piglets in Guangdong Province. By optimizing important variables such as the culture medium, serum type, and inoculum quantity, we established a stable in vitro culture system utilizing a modified Diamond medium supplemented with 10% Procell fetal bovine serum without the use of antibiotics. Subsequent analysis of the isolate\u2019s 18S rRNA gene, ITS1-5.8S rRNA-ITS2 gene, and EF-\u03b1 gene, through polymerase chain reaction, DNA sequencing, and phylogenetic analysis, revealed its close association to <jats:italic>Pentatrichomonas hominis<\/jats:italic>. Light microscopy and scanning electron microscopy demonstrated the presence of various distinct cellular structures, including four anterior flagella, recurrent flagellum, undulating membrane, pelta and axostyle. Additionally, transmission electron microscopy revealed the existence of organelles such as the Golgi complex, rough endoplasmic reticulum, food vacuoles, and hydrogenosomes. This study represents the first successful isolation of monoclonal cells of <jats:italic>P. hominis<\/jats:italic> to our knowledge and serves as a valuable baseline for future research focused on the isolation and purification of various other parasites. Additionally, it offers practical guidance for the diagnosis and management of <jats:italic>Trichomonas<\/jats:italic> spp. infections in pigs.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>In summary, our findings underscore the efficacy of our novel isolation technique as a valuable tool for the diagnosis and management of <jats:italic>Trichomonas<\/jats:italic> spp. infections, which can help mitigate the significant economic losses encountered in the pig breeding industry.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Graphical Abstract<\/jats:title>\n          <\/jats:sec>","DOI":"10.1186\/s13071-025-06752-9","type":"journal-article","created":{"date-parts":[[2025,4,5]],"date-time":"2025-04-05T07:55:28Z","timestamp":1743839728000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development of a single-cell cloning technique for isolation of Pentatrichomonas hominis: a promising tool for diagnosing Trichomonas spp. infections in the pig breeding industry"],"prefix":"10.1186","volume":"18","author":[{"given":"Yibin","family":"Zhu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiming","family":"Cai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shenquan","family":"Liao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyun","family":"Fang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanqin","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dingai","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuanqiang","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minna","family":"Lv","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuhui","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junjing","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongle","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangjie","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijun","family":"Yin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianfei","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nanshan","family":"Qi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingfei","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,5]]},"reference":[{"key":"6752_CR1","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1186\/s12917-021-02904-y","volume":"17","author":"A Mahittikorn","year":"2021","unstructured":"Mahittikorn A, Udonsom R, Koompapong K, Chiabchalard R, Sutthikornchai C, Sreepian PM, et al. 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