Comparison of Three Protocols to Preserve Leptospira spp. in Cat Urine for Efficient DNA Extraction and PCR Amplification

Carolina Trochmann Cordeiro, Jéssica Damiana Marinho Valente, Leonardo Gaspareto dos Santos, Rafael Felipe da Costa Vieira, Thálitha Samih Wischral Jayme Vieira, Simone Tostes de Oliveira Stedile

Abstract


Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recent studies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine may inhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processing to optimize the detection of pathogenic leptospires in cat urine.

Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested. Aliquots of standard concentration of L interrogans serovar Canicola culture were added to urine samples to achieve concentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by the addition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS was added to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B, PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24 and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Samples were then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets were discarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and the pellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20 min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºC for 10 min. DNA was stored at -20ºC until the molecular analysis. The PCR detection limit was evaluated. In samples from protocol A, leptospires were detected in concentrations up to 1×103 (4 h) and 1×104 (24 and 48 h). In protocol C, leptospires were detected in concentrations up to 1×104 (4 h) and 1×105 (24 and 48 h). No leptospiral DNA was detected in samples from protocol B.

Discussion:   Leptospires are sensitive to acid conditions, at pH 6.8 or lower and the urine pH of cats may vary from 5 to 7. In the present study, we found best results for DNA amplification with the addition of PBS immediately after urine collection (protocol A). Previous studies have shown the importance of neutralizing urine samples immediately after collection to avoid loss of bacterial DNA during the extraction process. However, protocols B and C may be an alternative in clinical practice, when PBS cannot be added immediately after collection. The delay after urine collection before DNA extraction is one more factor that may interfere with the PCR sensitivity. This was observed in the samples from protocol A, because although these samples were neutralized immediately, there was a 10-fold decrease in the detection limit of the test at 24 and 48 h. Leptospires rapidly lose their integrity in urine and the detection limit declines considerably over time, so prompt extraction is essential. These results show that the in-house method of preserving cat urine is useful to maintain the viability of leptospiral DNA extraction. In addition, this study highlights the importance of neutralizing urine samples immediately after collection and the need for prompt DNA extraction to improve PCR detection limit. However, if PBS cannot be added to the collected sample immediately, it is better to process the sample without PBS and extract DNA as soon as possible to minimize the risk of false-negative results.


Full Text:

PDF

References


Agunloye C.A. & Nash A.S. 1996. Investigation of possible leptospiral infection in cats in Scotland. Journal of Small Animal Practice. 37(3): 126-129

Arbour J., Blais M-C., Carioto L. & Sylvestre D. 2012. Clinical Leptospirosis in Three Cats (2001-2009). Journal of the American Animal Hospital Association. 48(4): 256-260.

Beaudu-lange C. & Lange E. 2014 Unusual clinical presentation of leptospirosis in a cat. Revue Vétérinaire Clinique. 49(3): 115-122.

Chan K.W., Hsu Y.H., Hu, W.L., Pan M.J., Lai J.M., Huang K.C. & Chou S.J. 2014. Serological and PCR detection of feline leptospira in southern Taiwan. Vector borne zoonotic diseases. 14(1): 118-123.

Faine S., Adler B., Bolin C. & Perolat P. 1999. Leptospira and leptospirosis. 2nd edn. Melbourne: MedScience, 272p.

Fenimore A., Carter K. & Lunn K.F. 2012. Detection of Leptospiruria in Shelter cats in Colorado. [abstract ID-3] In: ACVIM Forum 2012 (New Orleans, USA). Journal of Veterinary Internal Medicine. 26(3): 783.

Holt P.E. 2008. Urological disorders of the dog and cat - investigation, diagnosis and treatment. London: Manson Publishing Ltd.,176p.

Larsson C.E., Rosa C.A.S., Larsson M.H., Birgel E.H., Fernandes W.R., Paim G.V. & Santa Rosa C.A. 1985. Laboratory and clinical features of experimental feline leptospirosis. International Journal of Zoonoses. 12(2): 111-119.

Lévesque B., De Serres G., Higgins R., D’Halewyn M.A., Artsob H. & Grondin J., Major M., Garvie M. & Duval B. 1995. Seroepidemiologic study of three zoonoses (leptospirosis, Q fever, and tularemia) among trappers in Québec, Canada. Clinical and Diagnostic Laboratory Immunology. 2(4): 496-498.

Lucchesi P.M.A, Arroyo G.H., Etcheverría A.I., Parma A.E. & Seijo A.C. 2004. Recommendations for the detection of Leptospira in urine by PCR. Revista da Sociedade Brasileira de Medicina Tropical. 37(2): 131-134.

Millán J., Candela M.G., López-Bao J.V., Pereira M., Jiménez M.A. & León-Vizcaíno L. 2009. Leptospirosis in wild and domestic carnivores in natural areas in Andalusia, Spain. Vector Borne Zoonotic Diseases. 9(5): 549-554.

Rodriguez J., Blais M.C., Lapointe C., Arsenault J., Carioto L. & Harel J. 2014. Serologic and urinary PCR survey of leptospirosis in healthy cats and in cats with kidney disease. Journal of Veterinary Internal Medicine. 28(2): 284-293.

Weis S., Rettinger A., Bergmann M., Llewellyn J.R., Pantchev N., Straubinger R.K. & Hartmann K. 2016. Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany. Journal of Feline Medicine and Surgery. 19(4): 470-476.




DOI: https://doi.org/10.22456/1679-9216.95720

Copyright (c) 2019 Carolina Trochmann Cordeiro, Jéssica Damiana Marinho Valente, Leonardo Gaspareto dos Santos, Rafael Felipe da Costa Vieira, Thálitha Samih Wischral Jayme Vieira, Simone Tostes de Oliveira Stedile

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.