IVD GmbH, School of Veterinary Medicine Hannover, Germany

Development of a novel ELISA for serodiagnosis of Leptospirosis and additional detection of pathogenic Leptospira by polymerase chain reaction for veterinary routine diagnostics

Dr. Dimitrios Theodoridis,¹ Dr. Jan Böhmer,¹ Dr. Matthias Homuth¹ y Dr. Katrin Strutzberg-Minder²

Summary

A multi-serovar ELISA based on the outer membrane Lipoprotein L41 (LipL41) of pathogenic Leptospira was developed to increase sensitivity by using a single test antigen. A sensitivity of 99 % and a specifity of 92 % could be achieved. The established diagnostic polymerase chain reaction is also able to detect fast and reliably pathogenic Leptospira in different clinical samples.

Key words: Leptospirosis, leptospira, ELISA, PCR, diagnosis.

 

Since a reliable clinical diagnosis is frequently not possible because of unspecific symptoms and the identification of the etiologic agent by culture is very difficult, serological diagnosis is of great importance. Different serological methods are able to detect antibodies directed against the test antigen of the corresponding serovar only. In addition they do not achieve the sensitivity of the MAT,^1-5 the reference method, that allows a serovar diagnosis. A fast and sensitive identification of Leptospira spp. is possible with different PCR methods, that are based on the amplification of segments of the 16S- and 23S-RNA sequences; however, these PCR methods can not always assure specifity for pathogenic Leptospira spp. Only.^2,3

A multi-serovar ELISA based on the outer membrane Lipoprotein L41 (LipL41) of pathogenic Leptospira was developed to increase sensitivity by using a single test antigen. The ELISA was evaluated with 343 Sera from pigs in comparison to MAT results.

A sensitivity of 99 % (Cut-off 5) and a specifity of 92 % (Cut-off 30) could be achieved. Additional Evaluation with 28 Sera from experimentally infected and 28 Sera from not infected mongolian Gerbils by using the state of infection as reference showed 100% sensitivity and specifity dependent on stage of infection. For the specific detection of the lipl41-gene of pathogenic Leptospira PCR primers were selected and PCR was successfully performed with clinical samples. LipL41⁴ shows to be an interesting candidate for diagnostics of Leptospirosis by ELISA-techniques. The established diagnostic PCR based on the lipl41 gene sequence occurring only in pathogenic Leptospira spp.⁴ is able to detect fast and reliably pathogenic Leptospira in different clinical samples and represents an alternative to the time-consuming and difficult identification of the causative agent by culture.

 

Desarrollo de un ELISA novedoso para el serodiagnóstico de Leptospirosis y la detección adicional de Leptospira patogénica mediante la reacción en cadena de la polimerasa para diagnóstico veterinario de rutina

Resumen

Se desarrolló un ELISA multiserovar basado en la lipoproteína de membrana externa L41 (LipL 41) de Leptospira patogénica para incrementar la sensibilidad mediante el uso de un antígeno de prueba. Se logró 99 % de sensibilidad y 92 % de especificidad. La reacción en cadena de la polimerasa diagnóstica establecida es capaz de detectar de una manera rápida y confiable la Leptospira patogénica en diferentes muestras clínicas.

Palabras clave: Leptospirosis, leptospira, ELISA, RCP, diagnóstico.

 

References

1. Ellis WA, O'Brien JJ, Neill SD, Hanna J. Bovine leptospirosis: serological findings in aborting cows. Vet Rec 1982; 110:178-80.
2. Faber NA, Crawford M, Lefebvre RB, Buyukmihci NC, Madigan JE, Willits NH. Detection of Leptospira spp. in the aqueous humor of horses with naturally acquired recurrent uveitis. J Clin Microbiol 2000;38:2731-3.
3. Merien F, Amouriaux P, Perolat P, Baranton G, Saint G. Polymerase chain reaction for detection of Leptospira spp. in clinical samples. J Clin Microbiol 1992;30:2219-24.
4. Shang ES, Summers TA, Haake DA. Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species. Infect Immun 1996;64:2322-30.
5. Zochowski WJ, Palmer MF, Coleman TJ. An evaluation of three commercial kits for use as screening methods for the detection of leptospiral antibodies in the UK. J Clin Pathol 2001;54:25-30.

Recibido: 27 de diciembre de 2004. Aprobado: 10 de marzo de 2005.
Dr. Dimitrios Theodoridis. IVD GmbH, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany; Tel. +49 - 511 / 856 - 7501 FAX: - 7672. E-mail: [e-mail]

¹ Doctor en Ciencias. Doctor en Medicina Veterinaria.
² Máster en Ciencias. Doctor en Ciencias. Doctor en Recursos Naturales.