Evaluation of Real-Time PCR and Culturing for the Detection of Leptospires in Canine Samples


Validated real-time PCRs detecting leptospires for veterinary purposes are not readily available. This paper describes the prospective evaluation of a SYBR Green-based real-time PCR on serum samples collected from experimentally infected dogs. Compared to culturing, the assay had a diagnostic sensitivity and specificity of 91.7% and 90.0%, respectively. Culturing for part is complementary to PCR and preferably both should be applied for diagnosis and vaccine challenge experiments. In a subsequent prospective study on the dynamics of experimental infections with serovars Canicola and Copenhageni and serovar complex Bananal-Liangguang in young and adult dogs, the PCR was applied on serum samples in conjunction with culturing on blood, urine and kidney samples with the following results: 1) Leptospires persisted longer in the blood of young dogs than of adult ones; 2) Numbers of viable leptospires in the blood are rapidly reduced but DNA remains occasionally detectable up to 7 days post infection; 3) Appearance of viable leptospires in the urine follows a biphasic dynamics; 4) EDTA hampers effective culturing from blood samples; 5) Serovar Canicola persists longer in the blood of dogs than Copenhageni and Bananal-Liangguang. Together with a markedly higher recovery rate from kidney samples (71% compared to respectively 33% and 0% in young dogs), this probably reflects the adaptation power of Canicola to its canine maintenance host. Appearance of leptospires in the urine samples indicates that experimental infections have been successful. PCR presents a valuable adjunct to the diagnosis of veterinary leptospirosis and the follow-up in vaccine protection experiments.

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A. Ahmed, H. L. B. M. Klaasen, M. van der Veen, H. van der Linden, M. G. A. Goris and R. A. Hartskeerl, "Evaluation of Real-Time PCR and Culturing for the Detection of Leptospires in Canine Samples," Advances in Microbiology, Vol. 2 No. 2, 2012, pp. 162-170. doi: 10.4236/aim.2012.22021.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] W. A. Ellis, “Control of Canine Leptospirosis in Europe: Time for a Change?” Veterinary Record, Vol. 167, No. 16, 2010, pp. 602-605.doi:10.1136/vr.c4965
[2] S. Faine, B. Adler, S. P. Gale, et al., “Leptospira and Leptospirosis,” MediSci, Melbourne, 1999.
[3] C. Fischer-Tenhagen, C. Hamblin, S. Quandt, et al., “Serosurvey for Selected Infectious Disease Agents in Free-Ranging Black and White Rhinoceros in Africa,” Journal of Wildlife Diseases, Vol. 36, No. 2, 2000, pp. 316-323.
[4] P. N. Levett, “Leptospirosis,” Clinical Microbiology Reviews, Vol. 14, No. 2, 2001, pp. 296-326. doi:10.1128/CMR.14.2.296-326.2001
[5] World Organization for Animal Health (OIE), “OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,” 15th Edition, Office International des Epizooties, 2004.
[6] G. M. Cerqueira and M. Picardeau, “A Century of Leptospira Strain Typing,” Infection, Genetics and Evolution, Vol. 9, No. 5, 2009, pp. 760-768. doi:10.1016/j.meegid.2009.06.009
[7] B. Adler and M. A. de la Pena, “Leptospira and Leptospirosis,” Vet.Microbiol., Vol. 140, No. 3-4, 2010, pp. 287-296.doi:10.1016/j.vetmic.2009.03.012
[8] R. Bharadwaj, A. M. Bal, S. A. Joshi, et al., “An Urban Outbreak of Leptospirosis in Mumbai, India,” Japanese Journal of Infectious Diseases, Vol. 55, No. 6, 2002, pp. 194-196.
[9] A. R. Bharti, J. E. Nally, J. N. Ricaldi, et al., “Leptospirosis: A Zoonotic Disease of Global Importance,” The Lancet Infectious Diseases, Vol. 3, No. 12, 2003, pp. 757-771.doi:10.1016/S1473-3099(03)00830-2
[10] S. B. Agampodi, S. J. Peacock, V. Thevanesam, et al., “Lep- tospirosis Outbreak in Sri Lanka in 2008: Lessons for Assessing the Global Burden of Disease,” The American Journal of Tropical Medicine and Hygiene, Vol. 85, No. 3, 2011, pp. 471-478.doi:10.4269/ajtmh.2011.11-0276
[11] A. M. Monahan, I. S. Miller and J. E. Nally, “Leptospirosis: Risks during Recreational Activities,” Journal of Applied Microbiology, Vol. 107, No. 3, 2009, pp. 707-716. doi:10.1111/j.1365-2672.2009.04220.x
[12] B. Abela-Ridder, R. Sikkema and R. A. Hartskeerl, “Estimating the Burden of human Leptospirosis,” International Journal of Antimicrobial Agents, Vol. 36, Suppl. 1, 2010, pp. S5-S7.doi:10.1016/j.ijantimicag.2010.06.012
[13] E. G. Hartman, P. Franken, B. A. Bokhout, et al., “Leptospirosis in Cattle; Milker’s Fever in Cattle Farmers,” Tijdschrift Voor Diergeneeskunde, Vol. 114, No. 3, 1989, pp. 131-135.
[14] J. F. Prescott, D. Key and M. Osuch, “Leptospirosis in Dogs,” The Canadian Veterinary Journal, Vol. 40, No. 6, 1999. pp. 430-431.
[15] M. Ribotta, M. Fortin, R. Higgins, et al., “Canine Leptospirosis: Serology,” The Canadian Veterinary Journal, Vol. 41, No. 6, 2000, pp. 494-495.
[16] J. E. Sykes, K. Hartmann, K. F. Lunn, et al., “2010 AC-VIM Small Animal Consensus Statement on Leptospirosis: Diagnosis, Epidemiology, Treatment, and Prevention,” Journal of Veterinary Internal Medicine, Vol. 25, No. 1, 2011, pp. 1-13.doi:10.1111/j.1939-1676.2010.0654.x
[17] S. Rahelinirina, A. Leon, R. A. Hartskeerl, et al., “First Isolation and Direct Evidence for the Existence of Large Small-Mammal Reservoirs of Leptospira sp. in Madagascar,” PLoS One, Vol. 5, No. 11, 2010, p. e14111. doi:10.1371/journal.pone.0014111
[18] F. Merien, P. Amouriaux, P. Perolat, et al., “Polymerase Chain Reaction for Detection of Leptospira spp. in Clinical Samples,” Journal of Clinical Microbiology, Vol. 30, No. 9, 1992, pp. 2219-2224.
[19] A. M. Bal, “Unusual Clinical Manifestations of Leptospirosis,” Journal of Postgraduate Medicine, Vol. 51, No. 3, 2005, pp. 179-183.
[20] R. U. Palaniappan, Y. F. Chang, C. F. Chang, et al., “Evaluation of Lig-Based Conventional and Real Time PCR for the Detection of Pathogenic Leptospires,” Molecular and Cellular Probes, Vol. 19, No. 2, 2005, pp. 111-117. doi:10.1016/j.mcp.2004.10.002
[21] A. Ahmed, M. F. Engelberts, K. R. Boer, et al., “Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials,” PLoS One, Vol. 4, No. 9, 2009, p. e7093. doi:10.1371/journal.pone.0007093
[22] R. A. Stoddard, J. E. Gee, P. P. Wilkins, et al., “Detection of Pathogenic Leptospira spp. through TaqMan Polymerase Chain Reaction Targeting the LipL32 Gene,” Diagnostic Microbiology and Infectious Disease, Vol. 64, No. 3, 2009, pp. 247-255. doi:10.1016/j.diagmicrobio.2009.03.014
[23] J. Thaipadungpanit, W. Chierakul, V. Wuthiekanun, et al., “Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipL32 Genes for Human Leptospirosis in Thailand: A Case-Control Study,” PLoS One, Vol. 6, No. 1, 2011, p. e16236. doi:10.1371/journal.pone.0016236
[24] B. Victoria, A. Ahmed, R. L. Zuerner, et al., “Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira,” PLoS One, Vol. 3, No. 7, 2008, p. e2752.doi:10.1371/journal.pone.0002752
[25] H. L. Klaasen, M. J. Molkenboer, M. P. Vrijenhoek, et al., “Duration of Immunity in Dogs Vaccinated against Leptospirosis with a Bivalent Inactivated Vaccine,” Veterinary Microbiology, Vol. 95, No. 1-2, 2003, pp. 121-132. doi:10.1016/S0378-1135(03)00152-4
[26] S. Subharat, P. R. Wilson, C. Heuer, et al., “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,” Journal of Veterinary Diagnostic Investigation, Vol. 23, No. 4, 2001, pp. 743-752.
[27] E. S. Broughton and J. Scarnell, “Prevention of Renal Carriage of leptospirosis in dogs by vaccination,” Veterinary Record, Vol. 117, No. 12, 1985, pp. 307-311. doi:10.1136/vr.117.12.307
[28] P. Schreiber, V. Martin, W. Najbar, et al., “Prevention of Renal Infection and Urinary Shedding in Dogs by a Leptospira Vaccination,” Veterinary Microbiology, Vol. 108, No. 1-2, 2005, pp. 113-118. doi:10.1016/j.vetmic.2005.03.007
[29] R. M. Nervig and H. C. Ellinghausen Jr., “Viability of Leptospira interrogans Serotype Grippotyphosa in Swine Urine and Blood,” Cornell Veterinary, Vol. 68, No. 1, 1978, pp. 70-77.
[30] J. M. Minke, R. Bey, J. P. Tronel, et al., “Onset and Duration of Protective Immunity against Clinical Disease and Renal Carriage in Dogs Provided by a Bi-Valent Inactivated Leptospirosis Vaccine,” Veterinary Microbiology, Vol. 137, No. 1-2, 2009, pp. 137-145. doi:10.1016/j.vetmic.2008.12.021
[31] W. I. Morrison and N. G. Wright, “Canine Leptospirosis: An Immunopathological Study of Interstitial Nephritis Due to Leptospira Canicola,” The Journal of Pathology, Vol. 120, No. 2, 1976, pp. 83-89. doi:10.1002/path.1711200204
[32] N. F. Cheville, R. Huhn and R. C. Cutlip, “Ultrastructure of Renal Lesions in Pigs with Acute Leptospirosis Caused by Leptospira Pomona,” Veterinary Pathology, Vol. 17, No. 3, 1980. pp. 338-351.
[33] V. Sitprija, V. Pipatanagul, K. Mertowidjojo, et al., “Pathogenesis of Renal Disease in Leptospirosis: Clinical and Experimental Studies,” Kidney International, Vol. 17, No. 6, 1980. pp. 827-836.doi:10.1038/ki.1980.95
[34] M. G. Goris, J. F. Wagenaar, R. A. Hartskeerl, et al., “Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood,” PLoS One, Vol. 6, No. 3, 2011, p. e18279.doi:10.1371/journal.pone.0018279
[35] S. Faine, “Leptospirosis,” In: L. Collier, A. Balows and M. Sussman, Eds., Topley and Wilson’s Microbiology and Microbial Infections, Arnold, London, 1998, pp. 854-856.

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