{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T03:28:21Z","timestamp":1789702101989,"version":"4.0.1"},"reference-count":45,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,3,2]],"date-time":"2023-03-02T00:00:00Z","timestamp":1677715200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.<\/jats:p>","DOI":"10.3390\/v15030669","type":"journal-article","created":{"date-parts":[[2023,3,2]],"date-time":"2023-03-02T04:57:38Z","timestamp":1677733058000},"page":"669","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["Multiplex RT-qPCR Application in Early Detection of Bovine Respiratory Disease in Healthy Calves"],"prefix":"10.3390","volume":"15","author":[{"given":"Yusuke","family":"Goto","sequence":"first","affiliation":[{"name":"Central Iwate Prefectural Livestock Health and Hygiene Center, Takizawa 020-0605, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiro","family":"Fukunari","sequence":"additional","affiliation":[{"name":"Central Iwate Prefectural Livestock Health and Hygiene Center, Takizawa 020-0605, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3607-4787","authenticated-orcid":false,"given":"Tohru","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Division of Zoonosis Research and Division of Hygiene Management, Sapporo Research Station, National Institute of Animal Health, NARO, Sapporo  062-0045, Hokkaido, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1999","DOI":"10.2527\/jas.2006-046","article-title":"Bovine respiratory disease in feedlot cattle: Environmental, genetic, and economic factors","volume":"84","author":"Snowder","year":"2006","journal-title":"J. Anim. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/j.cvfa.2020.03.008","article-title":"The effect of market forces on bovine respiratory disease","volume":"36","author":"Peel","year":"2020","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"skaa005","DOI":"10.1093\/jas\/skaa005","article-title":"An evaluation of the economic effects of bovine respiratory disease on animal performance, carcass traits, and economic outcomes in feedlot cattle defined using four BRD diagnosis methods","volume":"98","author":"McMeniman","year":"2020","journal-title":"J. Anim. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/S0749-0720(15)30005-0","article-title":"The immunology of the bovine respiratory disease complex","volume":"17","author":"Ellis","year":"2001","journal-title":"Vet. Clin. North Am. Food Anim. Pract."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/j.cvfa.2010.04.001","article-title":"Pathogenesis and pathology of bovine pneumonia","volume":"26","author":"Panciera","year":"2010","journal-title":"Vet. Clin. Food Anim. Pract."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Murray, G.M., More, S.J., Clegg, T.A., Earley, B., O\u2019Neill, R.G., Johnston, D., Gilmore, J., Nosov, M., McElroy, M.C., and Inzana, T.J. (2018). Risk factors associated with exposure to bovine respiratory disease pathogens during the peri-weaning period in dairy bull calves. BMC Vet. Res., 14.","DOI":"10.1186\/s12917-018-1372-9"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.cvfa.2020.02.004","article-title":"Viruses in bovine respiratory disease in North America: Knowledge advances using genomic testing","volume":"36","author":"Fulton","year":"2020","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1016\/j.vetmic.2008.11.014","article-title":"Detection and characterization of bovine torovirus from the respiratory tract in Japanese cattle","volume":"136","author":"Ito","year":"2009","journal-title":"Vet. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5340","DOI":"10.1128\/JVI.00064-15","article-title":"A metagenomics and case-control study to identify viruses associated with bovine respiratory disease","volume":"89","author":"Ng","year":"2015","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5636","DOI":"10.1128\/JVI.03122-15","article-title":"Pathogenesis of influenza D virus in cattle","volume":"90","author":"Ferguson","year":"2016","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/j.cvfa.2010.04.004","article-title":"Bacterial pathogens of the bovine respiratory disease complex","volume":"26","author":"Griffin","year":"2010","journal-title":"Vet. Clin. Food Anim."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/s40168-020-00869-y","article-title":"Topography of the respiratory tract bacterial microbiota in cattle","volume":"8","author":"McMullen","year":"2020","journal-title":"Microbiome"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Bell, R.L., Turkington, H.L., and Cosby, S.L. (2021). The bacterial and viral agents of BRDC: Immune evasion and vaccine developments. Vaccines, 9.","DOI":"10.3390\/vaccines9040337"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1016\/j.cvfa.2020.03.005","article-title":"Bovine respiratory disease diagnosis: What progress has been made in infectious diagnosis?","volume":"36","author":"Pardon","year":"2020","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1128\/JCM.02485-13","article-title":"Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements","volume":"52","author":"Klima","year":"2014","journal-title":"J. Clin. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"101494","DOI":"10.1016\/j.cimid.2020.101494","article-title":"Microbial diversity involved in the etiology of a bovine respiratory disease outbreak in a dairy calf rearing unit","volume":"71","author":"Oliveira","year":"2020","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1093\/jas\/sky032","article-title":"Evaluating the microbiome of two sampling locations in the nasal cavity of cattle with bovine respiratory disease complex (BRDC)","volume":"96","author":"Mcdaneld","year":"2018","journal-title":"J. Anim. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/j.cvfa.2020.03.001","article-title":"Respiratory bacterial microbiota in cattle: From development to modulation to enhance respiratory health","volume":"36","author":"Timsit","year":"2020","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s42523-022-00167-y","article-title":"Identification of bovine respiratory disease through the nasal microbiome","volume":"4","author":"Glidden","year":"2022","journal-title":"Anim. Microbiome"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Thonur, L., Maley, M., Gilray, J., Crook, T., Laming, E., Turnbull, D., Nath, M., and Willoughby, K. (2012). One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. BMC Vet. Res., 8.","DOI":"10.1186\/1746-6148-8-37"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"902401","DOI":"10.3389\/fmolb.2022.902401","article-title":"Meta-analysis of qPCR for bovine respiratory disease based on MIQE guidelines","volume":"9","author":"Barnewall","year":"2022","journal-title":"Front. Mol. Biosci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/j.cvfa.2012.07.004","article-title":"Laboratory and postmortem diagnosis of bovine respiratory disease","volume":"28","author":"Caswell","year":"2012","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_23","first-page":"754","article-title":"Laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: Gold standards for diagnosis, do they exist?","volume":"53","author":"Fulton","year":"2012","journal-title":"Can. Vet. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1186\/s40168-017-0372-5","article-title":"Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves","volume":"5","author":"Nicola","year":"2017","journal-title":"Microbiome"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Zeineldin, M.M., Lowe, J.F., Grimmer, E.D., de Godoy, M.R.C., Ghanem, M.M., Abd El-Raof, Y.M., and Aldridge, B.M. (2017). Relationship between nasopharyngeal and bronchoalveolar microbial communities in clinically healthy feedlot cattle. BMC Microbiol., 17.","DOI":"10.1186\/s12866-017-1042-2"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s13567-021-01020-x","article-title":"Bovine respiratory microbiota of feedlot cattle and its association with disease","volume":"53","author":"Chai","year":"2022","journal-title":"Vet. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"832","DOI":"10.1111\/jam.14685","article-title":"Design of a multiplex quantitative reverse transcription-PCR system to simultaneously detect 16 pathogens associated with bovine respiratory and enteric diseases","volume":"129","author":"Goto","year":"2020","journal-title":"J. Appl. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"115","DOI":"10.20506\/rst.9.1.486","article-title":"The diagnosis of bovine virus diarrhoea-mucosal disease in cattle","volume":"9","author":"Edwards","year":"1990","journal-title":"Rev. Sci. Tech."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1292\/jvms.67.145","article-title":"Genetic and antigenic analyses of bovine respiratory syncytial virus detected in Japan","volume":"67","author":"Yaegashi","year":"2005","journal-title":"J. Vet. Med. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.cimid.2009.10.007","article-title":"Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes","volume":"34","author":"Minami","year":"2011","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Goto, Y., Yaegashi, G., Fukunari, K., and Suzuki, T. (2021). An importance of long-term clinical analysis to accurately diagnose calves persistently and acutely infected by bovine viral diarrhea virus 2. Viruses, 13.","DOI":"10.3390\/v13122431"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lovric, M. (2011). International Encyclopedia of Statistical Science, Springer.","DOI":"10.1007\/978-3-642-04898-2"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.cvfa.2020.03.002","article-title":"The immunology of bovine respiratory disease: Recent advancements","volume":"36","author":"McGill","year":"2020","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1186\/s13567-022-01086-1","article-title":"Understanding the mechanisms of viral and bacterial coinfections in bovine respiratory disease: A comprehensive literature review of experimental evidence","volume":"53","author":"Gaudino","year":"2022","journal-title":"Vet. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"674","DOI":"10.1177\/1040638719861686","article-title":"Coinfection of Swiss cattle with bovine parainfluenza virus 3 and Mycoplasma bovis at acute and chronic stages of bovine respiratory disease complex","volume":"31","author":"Mehinagic","year":"2019","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.vetmic.2006.10.001","article-title":"Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds","volume":"119","author":"Autio","year":"2007","journal-title":"Vet. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.cvfa.2009.10.003","article-title":"Bovine coronavirus associated syndromes","volume":"26","author":"Boileau","year":"2010","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.vetmic.2008.12.024","article-title":"Respiratory disease in calves: Microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response","volume":"137","author":"Angen","year":"2009","journal-title":"Vet. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1186\/s13567-020-00861-2","article-title":"Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: Actions and counteractions in the bacteria-host interactions","volume":"51","author":"Su","year":"2020","journal-title":"Vet. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"782872","DOI":"10.3389\/fvets.2021.782872","article-title":"The effects of ursolic acid treatment on immunopathogenesis following Mannheimia haemolytica infections","volume":"8","author":"Slate","year":"2021","journal-title":"Front. Vet. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.cvfa.2010.03.004","article-title":"Control, management, and prevention of bovine respiratory disease in dairy calves and cows","volume":"26","author":"Gorden","year":"2010","journal-title":"Vet. Clin. Food Anim."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Cirone, F., Padalino, B., Tullio, D., Capozza, P., Lo Surdo, M., Lanave, G., and Pratelli, A. (2019). Prevalence of pathogens related to bovine respiratory disease before and after transportation in beef steers: Preliminary results. Animals, 9.","DOI":"10.3390\/ani9121093"},{"key":"ref_43","first-page":"712","article-title":"Biosecurity practices in western Canadian cow-calf herds and their association with animal health","volume":"62","author":"Wennekamp","year":"2021","journal-title":"Can. Vet. J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1002\/cfg.474","article-title":"Effect of stress on viral-bacterial synergy in bovine respiratory disease: Novel mechanisms to regulate inflammation","volume":"6","author":"Hodgson","year":"2005","journal-title":"Comp. Funct. Genom."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1017\/S1466252307001326","article-title":"Immune evasion by pathogens of bovine respiratory disease complex","volume":"8","author":"Srikumaran","year":"2008","journal-title":"Anim. Health Res. Rev."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/3\/669\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:46:04Z","timestamp":1760121964000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/3\/669"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,2]]},"references-count":45,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["v15030669"],"URL":"https:\/\/doi.org\/10.3390\/v15030669","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,2]]}}}