{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T14:00:08Z","timestamp":1790172008393,"version":"4.1.0"},"reference-count":33,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32273009"],"award-info":[{"award-number":["32273009"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004608","name":"Natural Science Foundation of Jiangsu Province","doi-asserted-by":"publisher","award":["BK20250095"],"award-info":[{"award-number":["BK20250095"]}],"id":[{"id":"10.13039\/501100004608","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Basic Research Program of China","doi-asserted-by":"publisher","award":["2021YFD1800404"],"award-info":[{"award-number":["2021YFD1800404"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["KJJQ2024005"],"award-info":[{"award-number":["KJJQ2024005"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012246","name":"Priority Academic Program Development of Jiangsu Higher Education Institutions","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100012246","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Jiangsu Provincial Agricultural Science and Technology Independent Innovation Fund","award":["CX(23)1041"],"award-info":[{"award-number":["CX(23)1041"]}],"id":[{"id":"https:\/\/ror.org\/0335n2618","id-type":"ROR","asserted-by":"crossref"}]},{"name":"the Young Scholars cultivation program of College of Veterinary Medicine in Nanjing Agricultural University","id":[{"id":"https:\/\/ror.org\/05td3s095","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2026,9,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Advances in microscopy have enabled the widespread application of\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      pathogen tracking approaches to bacterial pathogenesis.\n                      <jats:italic toggle=\"yes\">Pasteurella multocida<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      ) is a major zoonotic pathogen causing severe respiratory diseases in livestock. However, genetic manipulation of clinical swine-derived isolates remains hindered by the lack of stable genetic tools. As a result, no genetically engineered\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      tracking strain limits research in\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      infection dynamics. In this study, we evaluated diverse replication mechanisms and identified that rolling circle replication (RCR)-type replicons exhibit superior transformation robustness in swine isolates. Leveraging the temperature-sensitive pSET4s backbone and an optimized\n                      <jats:italic toggle=\"yes\">mpheS<\/jats:italic>\n                      counter-selection marker, we developed a high-efficiency, markerless genetic manipulation system. The system exhibited high copy numbers and significantly enhanced transformation yields across diverse clinical swine isolates. To validate gene knockout efficiency, we performed markerless deletions of the capsule biosynthesis genes\n                      <jats:italic toggle=\"yes\">hyaD<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">hyaE<\/jats:italic>\n                      . Phenotypic characterization revealed that hyaluronic acid (HA) deficiency led to complete attenuation of virulence in a murine model. Furthermore, we engineered a 15-Pm::GFP reporter strain via site-specific gene knock-in without detectable effects on bacterial growth or virulence. This strain enables direct visualization of bacterial colonization in the lung and liver by fluorescence imaging and allows real-time,\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      monitoring of pathogen and Kupffer cell interactions. Collectively, our results established an effective genetic toolkit for swine-derived\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      and developed fluorescently labeled strains for\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      tracking. This plasmid provides a useful tool for investigating pathogenic mechanisms and\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      infection dynamics of\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      is a critical threat to global animal health, while the difficulty of genetic modification in swine isolates has interrupted the link of the genomic and pathogenic phenotypes. This study provides a highly efficient, RCR-based markerless manipulation system optimized with cross-serogroup applicability for swine isolates. Validated across 15 clinical swine isolates, this plasmid offers a robust tool for genetic analysis and the rational design of next-generation vaccines. By defining the essential roles of\n                      <jats:italic toggle=\"yes\">hyaD<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">hyaE<\/jats:italic>\n                      , we demonstrate that the hyaluronic acid capsule is required for virulence and pulmonary colonization. Moreover, the development of a biologically fluorescent reporter strain enables high-resolution,\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      tracking of infection dynamics, providing a powerful tool for real-time visualization during\n                      <jats:italic toggle=\"yes\">P. multocida<\/jats:italic>\n                      infection.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00787-26","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T13:00:21Z","timestamp":1786539621000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Development of a GFP-labeled\n                    <i>Pasteurella multocida<\/i>\n                    strain for\n                    <i>in vivo<\/i>\n                    organ infection tracing using a stable RCR-based genetic manipulation plasmid"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-1054-9007","authenticated-orcid":false,"given":"Chen","family":"Yuan","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05td3s095","id-type":"ROR","asserted-by":"publisher"}],"name":"Ministry of Agriculture Key Laboratory of Animal Bacteriology, the International Joint Laboratory of Animal Health and Food Safety, and College of Veterinary Medicine, Nanjing Agricultural University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenqing","family":"Wang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05td3s095","id-type":"ROR","asserted-by":"publisher"}],"name":"Ministry of Agriculture Key Laboratory of Animal Bacteriology, the International Joint Laboratory of Animal Health and Food Safety, and College of Veterinary Medicine, Nanjing Agricultural University","place":["Nanjing, 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