{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:14:31Z","timestamp":1787030071374,"version":"build-2736575974"},"reference-count":48,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,5,25]],"date-time":"2021-05-25T00:00:00Z","timestamp":1621900800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 104\u00a0Leptospira\/mL, and urine with pH \u2264 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 \u00b0C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.<\/jats:p>","DOI":"10.3390\/vetsci8060090","type":"journal-article","created":{"date-parts":[[2021,5,25]],"date-time":"2021-05-25T22:02:23Z","timestamp":1621980143000},"page":"90","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine"],"prefix":"10.3390","volume":"8","author":[{"given":"Romana","family":"Steinparzer","sequence":"first","affiliation":[{"name":"Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, 2340 M\u00f6dling, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamara","family":"Mair","sequence":"additional","affiliation":[{"name":"University Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4965-2088","authenticated-orcid":false,"given":"Christine","family":"Unterweger","sequence":"additional","affiliation":[{"name":"University Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7727-7508","authenticated-orcid":false,"given":"Adi","family":"Steinrigl","sequence":"additional","affiliation":[{"name":"Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, 2340 M\u00f6dling, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Friedrich","family":"Schmoll","sequence":"additional","affiliation":[{"name":"Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, 2340 M\u00f6dling, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,25]]},"reference":[{"key":"ref_1","unstructured":"Ellis, W.A. (2012). Diseases of Swine, Wiley-Blackwell. [10th ed.]."},{"key":"ref_2","unstructured":"Fain, S., Adler, B., Bloin, C., and Perolat, P. (1999). Leptospira and Leptospirosis, MediSci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.rvsc.2007.09.005","article-title":"The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue","volume":"85","author":"Fearnley","year":"2008","journal-title":"Res. Vet. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.1007\/s00284-010-9829-5","article-title":"Comparison of two PCR systems for the rapid detection of Leptospira spp. from kidney tissue","volume":"62","author":"Draeger","year":"2011","journal-title":"Curr. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1177\/1040638711407892","article-title":"Evaluation of a SYTO9 real-time polymerase chain reaction assay to detect and identify pathogenic Leptospira species in kidney tissue and urine of New Zealand farmed deer","volume":"23","author":"Subharat","year":"2011","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Xu, C., Loftis, A., Ahluwalia, S.K., Gao, D., Verma, A., Wang, C., and Kaltenboeck, B. (2014). Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0089507"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1177\/1040638715571360","article-title":"Evaluation of three 5\u2032 exonuclease-based real-time polymerase chain reaction assays for detection of pathogenic Leptospira species in canine urine","volume":"27","author":"Fink","year":"2015","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1177\/1040638715610378","article-title":"A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine","volume":"27","author":"Gentilini","year":"2015","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"584","DOI":"10.1016\/j.bjm.2017.09.004","article-title":"Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples","volume":"49","author":"Miotto","year":"2018","journal-title":"Braz. J. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"459","DOI":"10.5455\/javar.2018.e299","article-title":"Evaluation of LAMP for detection and\/or screening of Leptospira spp. infection among domestic animals in the Philippines","volume":"30","author":"Tubalinal","year":"2018","journal-title":"J. Adv. Vet. Anim. Res."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Hornsby, R.L., Alt, D.P., and Nally, J.E. (2020). Isolation and propagation of leptospires at 37 \u00b0C directly from the mammalian host. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-66526-4"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Weiss, S., Menezes, A., Woods, K., Chanthongthip, A., Dittrich, S., Opoku-Boateng, A., Kimuli, M., and Chalker, V. (2016). An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of Leptospira from Clinical Samples. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004996"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/S0034-5288(18)32489-5","article-title":"Serological and bacteriological survey of leptospiral infection in pigs in southern England","volume":"31","author":"Hathaway","year":"1981","journal-title":"Res. Vet. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1136\/vr.118.3.63","article-title":"Prevalence of Leptospira infection in aborted pigs in Northern Ireland","volume":"118","author":"Ellis","year":"1986","journal-title":"Vet. Rec."},{"key":"ref_15","first-page":"362","article-title":"The isolation and identification of Leptospira interrogans serovar bratislava from a pig in Germany","volume":"39","author":"Schmidt","year":"1992","journal-title":"Zentralbl. Vet. B."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1177\/104063879200400121","article-title":"Isolation of Leptospira interrogans serovars bratislava and hardjo from swine at slaughter","volume":"4","author":"Bolin","year":"1992","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1177\/104063879000200303","article-title":"Porcine leptospirosis in Iowa","volume":"2","author":"Miller","year":"1990","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1590\/S1517-83822008000300017","article-title":"Isolation and characterization of Leptospira interrogans from pigs slaughtered in S\u00e3o Paulo State, Brazil","volume":"39","author":"Miraglia","year":"2008","journal-title":"Braz. J. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1590\/S0100-736X2015000100002","article-title":"First isolation and characterization of Leptospira interrogans serogroup Australis from swine in Brazil","volume":"35","author":"Hamond","year":"2015","journal-title":"Pesq. Vet. Bras."},{"key":"ref_20","first-page":"Unit 12E.1","article-title":"Laboratory maintenance of pathogenic Leptospira","volume":"12","author":"Zuerner","year":"2005","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Chideroli, R.T., Gon\u00e7alves, D.D., Suphoronski, S.A., Alfieri, A.F., Alfieri, A.A., de Oliveira, A.G., de Freitas, J.C., and Pereira, U.P. (2017). Culture Strategies for Isolation of Fastidious Leptospira Serovar Hardjo and Molecular Differentiation of Genotypes Hardjobovis and Hardjoprajitno. Front. Microbiol., 8.","DOI":"10.3389\/fmicb.2017.02155"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1111\/j.1348-0421.2011.00347.x","article-title":"A novel combination of selective agents for isolation of Leptospira species","volume":"55","author":"Chakraborty","year":"2011","journal-title":"Microbiol. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1111\/lam.12501","article-title":"Usage of a selective media (EMJH-STAFF) in primary culturing of pathogenic leptospires from bovine clinical samples","volume":"61","author":"Loureiro","year":"2015","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1084\/jem.27.5.609","article-title":"The survival of leptospira (spirochaeatae) icterohaemorrhagiae in nature, observations concerning microchemical reactions and intermediary hosts","volume":"27","author":"Noguchi","year":"1918","journal-title":"J. Exp. Med."},{"key":"ref_25","first-page":"70","article-title":"Viability of Leptospira interrogans serotype grippotyphosa in swine urine and blood","volume":"68","author":"Nervig","year":"1978","journal-title":"Cornell Vet."},{"key":"ref_26","first-page":"35","article-title":"The effect of pH on the survival of leptospires in water","volume":"24","author":"Smith","year":"1961","journal-title":"Bull. World Health Organ."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.vetmic.2011.04.028","article-title":"Survival of a pathogenic Leptospira serovar in response to combined in vitro pH and temperature stresses","volume":"152","author":"Parker","year":"2011","journal-title":"Vet. Microbiol."},{"key":"ref_28","unstructured":"World Health Organisation (2003). Human Leptospirosis: Guidance for Diagnosis, Surveillance and Control, World Health Organisation (WHO)."},{"key":"ref_29","unstructured":"World Organisation of Animal Health (2018). Leptospirosis. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, World Organisation of Animal Health (OIE). [8th ed.]. Chapter 3.1.12."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"Unit 12E.5","DOI":"10.1002\/9780471729259.mc12e05s32","article-title":"Leptospirosis Serodiagnosis by the Microscopic Agglutination Test","volume":"32","author":"Goris","year":"2014","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Guglielmini, J., Bourhy, P., Schiettekatte, O., Zinini, F., Brisse, S., and Picardeau, M. (2019). Genus-wide Leptospira core genome multilocus sequence typing for strain taxonomy and global surveillance. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007374"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"913","DOI":"10.20506\/rst.35.3.2579","article-title":"Leptospirosis in domestic animals in France: Serological results from 1988 to 2007","volume":"35","year":"2016","journal-title":"Rev. Sci. Tech."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.cimid.2019.04.001","article-title":"Epidemiology of leptospirosis in North-Central Italy: Fifteen years of serological data (2002\u20132016)","volume":"65","author":"Bertelloni","year":"2019","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_34","unstructured":"Tagliabue, S., Figarolli, B.M., D\u2019Incau, M., Foschi, G., Gennero, M.S., Giordani, R., Natale, A., Papa, P., Ponti, N., and Scaltrito, D. (2016). Serological surveillance of Leptospirosis in Italy: Two-year national data (2010\u20132011). Vet. It., 52."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/s40813-018-0086-5","article-title":"Passive surveillance of Leptospira infection in swine in Germany","volume":"4","author":"Tschentscher","year":"2018","journal-title":"Porcine Health Manag."},{"key":"ref_36","first-page":"716","article-title":"A serological investigation of leptospirosis in sows from central Thailand","volume":"37","author":"Niwetpathomwat","year":"2006","journal-title":"Southeast Asian J. Trop. Med. Public Health"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Lee, H.S., Bui, V.N., Nguyen, H.X., Bui, A.N., Hoang, T.D., Nguyen-Viet, H., Grace Randolph, D., and Wieland, B. (2020). Seroprevalences of multi-pathogen and description of farm movement in pigs in two provinces in Vietnam. BMC Vet. Res., 16.","DOI":"10.1186\/s12917-020-2236-7"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1645","DOI":"10.1007\/s11250-019-01859-5","article-title":"Epidemiology of Leptospira infection in livestock species in Saint Kitts","volume":"51","author":"Shiokawa","year":"2019","journal-title":"Trop. Anim. Health Prod."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ngugi, J.N., F\u00e8vre, E.M., Mgode, G.F., Obonyo, M., Mhamphi, G.G., Otieno, C.A., and Cook, E.A.J. (2019). Seroprevalence and associated risk factors of leptospirosis in slaughter pigs; a neglected public health risk, western Kenya. BMC Vet. Res., 15.","DOI":"10.1186\/s12917-019-2159-3"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Boey, K., Shiokawa, K., and Rajeev, S. (2019). Leptospira infection in rats: A literature review of global prevalence and distribution. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007499"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Adler, B. (2014). Animal Leptospirosis. Leptospira and Leptospirosis, Springer. [1st ed.].","DOI":"10.1007\/978-3-662-45059-8"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Ettrup, K.S., Glud, A.N., Orlowski, D., Fitting, L.M., Meier, K., Soerensen, J.C., Bjarkam, C.R., and Olsen Alstrup, A.K. (2011). Basic Surgical Techniques in the G\u00f6ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion. J. Vis. Exp., 52.","DOI":"10.3791\/2652"},{"key":"ref_43","unstructured":"Adler, B. (2014). Leptospiral Structure, Physiology and Metabolism. Leptospira and Leptospirosis, Springer. [1st ed.]."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1099\/jmm.0.45860-0","article-title":"Detection of pathogenic leptospires by real-time quantitative PCR","volume":"54","author":"Levett","year":"2005","journal-title":"J. Med. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.diagmicrobio.2009.03.014","article-title":"Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the lipL32 gene","volume":"64","author":"Stoddard","year":"2009","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Barragan, V., Nieto, N., Keim, P., and Pearson, T. (2017). Meta-analysis to estimate the load of Leptospira excreted in urine: Beyond rats as important sources of transmission in low-income rural communities. BMC Res. Notes, 10.","DOI":"10.1186\/s13104-017-2384-4"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3067","DOI":"10.2527\/2003.81123067x","article-title":"Effects of dietary electrolyte balance on the chemistry of blood and urine in lactating sows and sow litter performance","volume":"81","author":"DeRouchey","year":"2003","journal-title":"J. Anim. Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1007\/s00284-015-0836-4","article-title":"Waterborne Leptospirosis: Survival and Preservation of the Virulence of Pathogenic Leptospira spp. in Fresh Water","volume":"71","author":"Aviat","year":"2015","journal-title":"Curr. Microbiol."}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/8\/6\/90\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:07:48Z","timestamp":1760162868000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/8\/6\/90"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,25]]},"references-count":48,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,6]]}},"alternative-id":["vetsci8060090"],"URL":"https:\/\/doi.org\/10.3390\/vetsci8060090","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,25]]}}}