{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T13:09:16Z","timestamp":1788959356042,"version":"build-2803163510"},"reference-count":26,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Project of the Special Program for Technological Innovation, Application and Development of Chongqing Municipality","award":["CSTB2025TIAD-KPX0079"],"award-info":[{"award-number":["CSTB2025TIAD-KPX0079"]}]},{"name":"Agricultural Fundamental & Long-term Scientific Observation and Research","award":["MARA-ORS261-AH024"],"award-info":[{"award-number":["MARA-ORS261-AH024"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Mannheimia haemolytica is a primary pathogen causing bacterial pneumonia in cattle and sheep. Multiple PCR targets exist for its identification, but no standardized primer pairs have been widely adopted. This study evaluated the universality of six reported primers (including rpt2) using clinical M. haemolytica isolates. Genus specificity was tested against standard strains of common ruminant pathogenic\/commensal bacteria and other Mannheimia species. Four primer sets (gcp, artJ-lktC, lktD, sodA) failed to distinguish M. haemolytica from other Mannheimia species. The rpt2 primer showed suboptimal universality, with 3 of 22 tested isolates yielding negative results. The rpoB primer had favorable universality and specificity, but its 136 bp amplicon was unsuitable for constructing multiplex PCR with M. haemolytica serotyping primers. We identified design flaws in the original rpt2 primer and developed a novel rpt2-N primer. Evaluation confirmed its strong universality, specificity, and sensitivity, as well as compatibility with serotyping primers for multiplex PCR assays. Field testing on three goat farms in the Chongqing region showed markedly higher M. haemolytica positivity rates after sharp temperature drops and transportation. Serotype A2 was confirmed as the dominant serotype in goats in Chongqing. This study systematically assessed multiple PCR identification primers, confirmed the feasibility of rpoB, and resolved the universality limitation of rpt2, providing technical support for rapid and accurate M. haemolytica detection.<\/jats:p>","DOI":"10.3390\/microorganisms14091998","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T12:26:11Z","timestamp":1788956771000},"page":"1998","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Construction and Comparison of Molecular Identification Methods for Mannheimia haemolytica"],"prefix":"10.3390","volume":"14","author":[{"given":"Zhen","family":"Yang","sequence":"first","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongqiang","family":"Miao","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Mou","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suhui","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liu","family":"Yang","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dengfeng","family":"Xu","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3329-8152","authenticated-orcid":false,"given":"Lizhi","family":"Fu","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kefei","family":"Shen","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziqi","family":"Li","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Southwest University, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7136-4951","authenticated-orcid":false,"given":"Long","family":"Zhao","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuandi","family":"Yu","sequence":"additional","affiliation":[{"name":"National Technology Innovation Center for Pigs, Chongqing 402460, China"},{"name":"Chongqing Academy of Animal Sciences, Chongqing 402460, China"},{"name":"Rongchang Field Scientific Observation and Research Station for Animal Diseases, Ministry of Agriculture and Rural Affairs, Chongqing 402460, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1051\/vetres:2004065","article-title":"How Mannheimia haemolytica defeats host defence through a kiss of death mechanism","volume":"36","author":"Zecchinon","year":"2005","journal-title":"Vet. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.nmni.2015.10.005","article-title":"Genome sequence and description of Mannheimia massilioguelmaensis sp. nov","volume":"8","author":"Hadjadj","year":"2015","journal-title":"New Microbes New Infect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e0120025","DOI":"10.1128\/spectrum.01200-25","article-title":"Comparison of virulence and resistance genes in Mannheimia haemolytica and Pasteurella multocida from dairy cattle with and without bovine respiratory disease","volume":"13","author":"Garzon","year":"2025","journal-title":"Microbiol. Spectr."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"978","DOI":"10.14202\/vetworld.2019.978-983","article-title":"Association between Mannheimia haemolytica infection with reproductive physiology and performance in small ruminants: A review","volume":"12","author":"Jesse","year":"2019","journal-title":"Vet. World"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1177\/03009858241235393","article-title":"Mannheimia haemolytica-associated fibrinonecrotizing abomasitis in lambs","volume":"61","author":"Uzal","year":"2024","journal-title":"Vet. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.vetmic.2011.03.024","article-title":"The role of Mannheimia species in ovine mastitis","volume":"153","author":"Omaleki","year":"2011","journal-title":"Vet. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3419","DOI":"10.1128\/JCM.01145-10","article-title":"Mannheimia species associated with ovine mastitis","volume":"48","author":"Omaleki","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5605552","DOI":"10.1155\/2024\/5605552","article-title":"Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia","volume":"2024","author":"Abdulkadir","year":"2024","journal-title":"Sci. World J."},{"key":"ref_9","first-page":"239","article-title":"A study on ovine pneumonic pasteurellosis: Isolation and identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia","volume":"9","author":"Anijajo","year":"2023","journal-title":"BMC Vet. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1177\/10406387211068447","article-title":"Development and validation of an insulated isothermal PCR assay for the rapid detection of Mannheimia haemolytica","volume":"34","author":"Dao","year":"2022","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"105576","DOI":"10.1016\/j.tvjl.2020.105576","article-title":"Emergence of fatal Mannheimia haemolytica infections in cattle in the Netherlands","volume":"268","author":"Biesheuvel","year":"2021","journal-title":"Vet. J."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Clawson, M.L., Murray, R.W., Sweeney, M.T., Apley, M.D., DeDonder, K.D., Capik, S.F., Larson, R.L., Lubbers, B.V., White, B.J., and Kalbfleisch, T.S. (2016). Genomic signatures of Mannheimia haemolytica that associate with the lungs of cattle with respiratory disease, an integrative conjugative element, and antibiotic resistance genes. BMC Genom., 17.","DOI":"10.1186\/s12864-016-3316-8"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Legesse, A., Abayneh, T., Mamo, G., Gelaye, E., Tesfaw, L., Yami, M., and Belay, A. (2018). Molecular characterization of Mannheimia haemolytica isolates associated with pneumonic cases of sheep in selected areas of Central Ethiopia. BMC Microbiol., 18.","DOI":"10.1186\/s12866-018-1338-x"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"109930","DOI":"10.1016\/j.vetmic.2023.109930","article-title":"Molecular identification and characterisation of Mannheimia haemolytica","volume":"288","author":"Kayal","year":"2024","journal-title":"Vet. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Wang, C., Bai, X., Wang, J., Ye, D., Dou, L., and Yang, Z. (2025). Exploring the diagnostic landscape of Mannheimia haemolytica: Technologies, applications, and perspectives. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1680478"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1292\/jvms.16-0489","article-title":"Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex","volume":"79","author":"Kishimoto","year":"2017","journal-title":"J. Vet. Med. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1556\/004.2017.032","article-title":"Development of a multiplex PCR for simultaneous detection of Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes","volume":"65","author":"Zhang","year":"2017","journal-title":"Acta Vet. Hung."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.14202\/vetworld.2015.1073-1077","article-title":"Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture","volume":"8","author":"Kumar","year":"2015","journal-title":"Vet. World"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.vetmic.2008.01.001","article-title":"A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis","volume":"130","author":"Alexander","year":"2008","journal-title":"Vet. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1128\/AEM.02086-09","article-title":"Bibersteinia trehalosi inhibits the growth of Mannheimia haemolytica by a proximity-dependent mechanism","volume":"76","author":"Dassanayake","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.mimet.2017.05.010","article-title":"A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6","volume":"139","author":"Klima","year":"2017","journal-title":"J. Microbiol. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Mason, C., Errington, J., Foster, G., Thacker, J., Grace, O., and Baxter-Smith, K. (2022). Mannheimia haemolytica serovars associated with respiratory disease in cattle in Great Britain. BMC Vet. Res., 18.","DOI":"10.1186\/s12917-021-03121-3"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1007\/s12088-016-0611-7","article-title":"Mannheimia haemolytica and Bibersteinia trehalosi Serotypes Isolated from Merino Breed Lambs in Extremadura (Southwestern Spain)","volume":"56","author":"Galapero","year":"2016","journal-title":"Indian J. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1803","DOI":"10.1002\/vms3.1166","article-title":"Isolation and identification of Mannheimia haemolytica and Pasteurella multocida from symptomatic and asymptomatic sheep and their antibiotic susceptibility patterns in three selected districts of north Gondar zone, Gondar Ethiopia","volume":"9","author":"Abate","year":"2023","journal-title":"Vet. Med. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"589","DOI":"10.3168\/jds.2022-21929","article-title":"Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in Northeast China","volume":"106","author":"Zhou","year":"2023","journal-title":"J. Dairy Sci."},{"key":"ref_26","unstructured":"Guo, Y.C. (2022). Comparison of PCR Methods for Detection of Mannheimia haemolytica and Investigation of Mannheimia Infection in Goats from Sichuan Province. [Master\u2019s Thesis, Southwest Minzu University]. (In Chinese)."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1998\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T12:55:44Z","timestamp":1788958544000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1998"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,9]]},"references-count":26,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["microorganisms14091998"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14091998","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,9]]}}}