Workshop on Alternatives for Leptospira Vaccine Testing

September 19-21, 2012
U.S. Department of Agriculture Center for Veterinary Biologics
National Centers for Animal Health
Ames, Iowa  USA

Workshop proceedings available: Biologicals Volume 41 Issue 5 (2013): view articles on Science Direct website

View workshop summary 

View final workshop program 

This workshop brought together over 80 international scientific experts from government, industry, and academia to review recent advances in science and technology, in addition to available methods and approaches for Leptospira vaccine potency testing. The main focus of the workshop was on methods and approaches that will provide improved accuracy, efficiency, and worker safety, and that are more humane and use fewer or no animals, with the goal of developing a strategy to achieve global acceptance and implementation of scientifically valid alternative methods.

Workshop Overview

Leptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus Leptospira. An estimated 500,000 human cases of leptospirosis occur worldwide each year with a fatality rate of up to 25% in some regions. Designated as a Neglected Tropical Disease by the NIH and a Neglected Zoonotic Disease by the World Health Organization, leptospirosis is a global research and public health priority. For example, the National Institute of Allergy and Infectious Diseases is currently funding several major leptospirosis research grants, which include investigations on mechanisms involved in the infectious cycle, enhanced tools for clinical diagnosis, and identification of immunogenic sites for improved immune response. In the United States, Leptospira vaccines are used in cattle, swine, and dogs to protect them from disease and to reduce the risk of animal-to-human transmission. Human vaccines are also available in some countries outside the United States. Manufacturers test the potency of vaccine lots prior to their release to ensure their effectiveness. However, methods currently used to test the potency of Leptospira vaccines involve large numbers of laboratory animals that experience significant pain and distress, accounting for over one third of the animals reported to the U.S. Department of Agriculture in this category.Read More

This workshop brought scientists from around the world together to consider improved methods and approaches for Leptospira vaccine potency testing that may also help reduce, refine, and replace animal use. The workshop was organized by NICEATM in collaboration with ICCVAM and partner organizations in the International Cooperation on Alternative Test Methods. The U.S. Department of Agriculture Center for Veterinary Biologics and the International Alliance for Biological Standardization were sponsors of the workshop.

Workshop Presentations

Please note that we have made some minor formatting changes to some of the presentations to improve accessibility, but none of the content of the presentations has been changed.

Opening Session

Welcoming Remarks and Introduction to ICATM Organizations
William Stokes, DVM, RADM, USPHS, NIEHS/NICEATM
Workshop Overview and Objectives
Richard McFarland, PhD, MD, Center for Biologics Evaluation and Research, U.S. FDA
View presentation 

Welcome and Introduction to CVB 
Richard Hill, DVM, MS, Center for Veterinary Biologics, USDA

Session 1: Overview of Public Health Needs, Regulatory Requirements, and Research Initiatives for Leptospira Vaccine Potency Testing
Session 2: In Vitro Replacement Alternatives for Potency Testing of Leptospira Vaccines: Development, Validation, and Implementation
Session 3: Reduction and Refinement Alternatives for Potency Testing of Leptospira Vaccines: Development, Validation, and Implementation

Poster Session

A poster session at the workshop featured presentations on current research, development, and validation of alternative methods for Leptospira vaccine potency testing. View abstracts:

Relevant References

General Information
  • Hein B and Freudenstein H. Immunohistochemical investigations of Leptospira-infected Golden hamsters in potency tests. Zentralbl Bakteriol Mikrobiol Hyg A 1984;257:521-525. [View abstract on PubMed ]
  • Goodman SA. USDA: progress toward in vitro tests and other trends. Dev Biol Stand 1996;86:41-47. [View abstract on PubMed ]
  • Ruby KW, Walden DM, Wannemuehler. Development of an in vitro assay for measuring the relative potency of Leptospiral bacterins containing serovar Canicola and its correlation to the hamster potency assay. Dev Biol Stand 1996;86:341. [View citation on PubMed ]
  • Castle P, Coune C, Ellis W, Esposito-farese M-E, Spieser J-M, LeTarnec C, editors. Alternatives to animal challenge tests in the batch control of Leptospira vaccines for veterinary use. Pharmeuropa Special Issue. Strasbourg: Council of Europe, EDQM; 1999. BIO 99e2.
    Available to subscribers only; please visit the Pharmeuropa website for more information
  • Ruby KW. Development of in vitro assay for measuring relative potencies of Leptospiral bacterins. Pharmeuropa Special Issue, Bio 1999;99-2:35-45.
    Available to subscribers only; please visit the Pharmeuropa website for more information
  • Reed N, Varney WC, Goddard RD, Wyeth PJ. The maintenance of challenge strains used in the potency test for canine Leptospira vaccines. Biologicals 2000;28:25-28. [View abstract on PubMed]
  • Halder M, Hendriksen C, Cussler K, Balls M. ECVAM’s contributions to the implementation of the three Rs in the production and quality control of biologicals. ATLA 2002;30:93-108. [View abstract on PubMed ]
  • Berlioz-Arthaud A. Evaluating reagents for serological diagnosis of Leptospirosis: EPINET 1 workshop (Guam, December 2001).
  • Naiman BM, Alt D, Bolin CA, Zuerner R, Baldwin CL. 2001. Protective killed Leptospira borgpetersenii vaccine induces potent Th 1 immunity comprising responses by CD4 and gammadelta T lymphocytes. Infect Immun 69(12):7550-7558. [View abstract on PubMed ]
  • Brown RA, Blumerman S, Gay C, Bolin C, Duby R, Baldwin CL. 2003. Comparison of three different leptospiral vaccines for induction of a type 1 immune respeonse to Leptospira borgpetersenii serovar hardjo. Vaccine 21:4448-4458. [View abstract on PubMed ]
  • Sambsiava RR, Naveen G, Bhalla P, Agarwal SK. 2003. Leptospirosis in India and the rest of the world. Braz J Infect Dis 7(3):178-193. [View abstract on PubMed ]
  • Hartskeerl RA. 2005. International Leptospirosis Society: objectives and achievements. Rev Cubana Med Trop 57(1):7-10. [View citation on PubMed ]
  • Hattskeerl RA. Leptospirosis: current status and future trends. Indian J Med Microbiol 2006;24(4):309. [View citation on PubMed ]
  • Sehgal SC. Epidemiological patterns of leptospirosis. 2006. Indian J Med Microbiol 24(4):310-311. [View citation on PubMed ]
  • Srivastava SK. 2006. Prospects of developing Leptospiral vaccines for animals. Indian J Med Microbiol 24(4):331-336. [View citation on PubMed ]
  • Terpstra WJ. Historical perspectives in leptospirosis. 2006. Indian J Med Microbiol 24(4):316-320. [View citation on PubMed ]
  • Vijayachari SC, Sehgal SC. 2006. Recent advances in the laboratory diagnosis of leptospirosis and characterization of Leptospires. Indian J Med Microbiol 24(4):320-322. [View citation on PubMed ]
  • Stokes JE, Kaneene JB, Schall WD, Kruger JM, Miller RA, Kaiser L, Bolin CA. 2007. Prevalence of serum antibodies against six Leptospira serovars in healthy dogs. JAVMA 230(11):1657-1664. [View abstract on PubMed ]
  • Wang Z, Jin, L, Wegrzyn A. 2007. Leptospirosis vaccines. Microb Cell Fact 6:39-48. [View abstract on PubMed ]
  • Cooper J and Jennings M. Advancing animal welfare and the 3Rs in the batch testing of veterinary vaccines. Royal Society for the Prevention of Cruelty to Animals. 2008. [View on RSPCA website ]
  • Guerra MA. 2009. Leptospirosis. JAVMA 234(4):472-478. [View abstract on PubMed ]
  • Victoriano AFB, Smythe LD, Gloriani-Barzaga N, Cavinta LL, Kasai T, Limpakarnjanarat K, Ong BL, et al. 2009. Leptospirosis in the Asia Pacific region. BMC Infect Dis 9:147-155. [View abstract on PubMed ]
  • Adler B and de la Peña Moctezuma A. 2010. Leptospira and leptospirosis. Veterinary Microbiology 140:287-296. [View abstract on PubMed ]
  • Jennings M, Morton DB, Charton E, Cooper J, Hendriksen C, Martin S, Pearce MC, Price S, Redhead K, Reed N, Simmons H, Spencer S, Willingale H. 2010. Application of the three Rs to challenge assays used in vaccine testing: tenth report of the BVAAWF/FRAME/RSPCA/UFAW joint working group in refinement. Biologicals 38:684-695. [View abstract on PubMed ]
  • Lim VK. 2011. Leptospirosis: a re-emerging infection. Malays J Pathol 33(1):1-5. [View abstract on PubMed ]
  • Milne C, Buchheit K-H. 2012. EDQM's 3R Activities in the Field of Quality Control of Vaccines. ALTEX Proceedings 1(1):65-69.
  • Casey W, Schmitt M, McFarland R, et al. 2011. Improving animal welfare and reducing animal use for human vaccine potency testing: state-of-the-science and future directions. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 33-46.
  • Claassen I. 2011. Case study of development, validation, and acceptance of a non-animal method for assessing veterinary vaccine potency. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 175-183.
  • Draayer H. 2011. Overview of currently approved veterinary vaccine potency testing methods and methods in development that do not require animal use. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 171-174.
  • Hill R. 2011. Alternative methods to reduce, refine, and replace the use of animals in the development and testing of veterinary biologics in the United States; a strategic priority. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 141-145.
  • Kulpa-Eddy J, Srinivas G, Halder M, et al. 2011. Non-animal replacement methods for veterinary vaccine potency testing: state of the science and future directions. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 60-83.
  • McFarland R, Verthelyi D, Casey W, et al. 2011. Non-animal replacement methods for human vaccine potency testing: state of the science and future directions. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 16-32.
  • Stokes WS, Brown K, Kulpa-Eddy J, et al. 2011. Improving animal welfare and reducing animal use for veterinary vaccine potency testing: state-of-the-science and future directions. In: International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions (Kulpa-Eddy J, McFarland R, Stokes WS, eds). Procedia Vaccinol 5: 84-105.
Antigen
  • Velineni S, Asuthkar S, Sritharan M. 2006. Iron limitation and expression of immunoreactive outer membrane proteins in Leptospira Interrogans serovar Icterohaemorrhagiae strain Lai. Indian J Med Microbiol 24(4):339-342. [View citation on PubMed ]
  • Thongboonkerd V. 2008. Proteomics in leptospirosis research: towards molecular diagnostics and vaccine development. Expert Rev Mol Diagn 8(1):53-612. [View abstract on PubMed ]
  • Seixas FK, da Silva É F, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, Dellagostin OA. 2007. Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine 26:88-95. [View abstract on PubMed ]
  • Felix SR, Silva ÉF, Jouglard SDD, Hartmann DM, Grassmann AA, Dellagostin OA. 2009. Leptospirosis vaccine: search for subunit candidates. Proc Vaccinol 1:110-114. [View article on journal website ]
  • Lottersberger J, Guerrero SA, Tonarelli GG, Frank R, Tarabla H, Vanasco NB. 2009. Epitope mapping of pathogenic Leptospira LipL32. Lett Applied Microbiol 49:641-645. [View abstract on PubMed ]
  • Pinne M, Haake DA. 2009. A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans. PLos ONE 4(6):e6071. [View abstract on PubMed ]
  • Zhao W, Chen CY, Zhang WY, Lai WQ, Hu BY, Zhao GP, Qin JH, Guo XK. 2009. Molecular characterization of the pL40 protein in Leptospira interrogans. Can J Microbiol 55:739-749. [View abstract on PubMed ]
  • Atzingen MV, Gonçales AP, de Morais ZM, Araújo ER, De Brito T, Vasconcellos SA, Nascimento AL. 2010. Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans. J Med Microbiol 59:1005-1015. [View abstract on PubMed ]
  • Coutinho ML, Choy HA, Kelley MM, Matsunaga J, Babbitt JT, Lewis MS, Aleixo JAG, Haake DA. 2011. A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans. PLos Negl Trop Dis 5(12)e1422. [View abstract on PubMed ]
  • Félix SR, Hartwig DD, Argondizzo APC, Silva EF, et al. 2011. Subunit approach to evaluation of the immune protective potential of Leptospiral antigens. Clin and Vaccine Immunol 18(12):2026-2030. [View abstract on PubMed ]
  • Lucas DSD, Cullen PA, Lo M, Srikram A. Recombinant LipL32 and LigA from Leptospira are unable to stimulate protective immunity against leptospirosis in the hamster model. 2011. Vaccine 29:3413-3418. [View abstract on PubMed ]
  • Monte LG, Conceição FR, Coutinho ML, Sexias FK, da Silva ÉF, Vasconcellos FA, deCastro LAS, Hartleben CP, Dellagostin OA, Aleixo JAG. 2011. Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions. Comp Immunol Microbiol Infect Dis 34:441-446. [View abstract on PubMed ]
  • Humphryes PC, Weeks ME, Gielbert A, Thomson G, Coldham NG. 2012. Analysis of multiple Leptospira interrogans serovar canicola vaccine proteomes and identification of LipL32 as a biomarker for potency. Clinical and Vaccine Immunol 19(4):587-593. [View abstract on PubMed ]
Serology
  • Huhn RG, Baldwin CD, Cardella MA. 1975. Immunity to leptospirosis: bacterins in dogs and hamsters. Am J Vet Res 36(1):71-74. [View abstract on PubMed ]
  • Huhn RG, Hanson LE, Killinger AH, Cardella MA. Immunity to leptospirosis: leptospira interrogans serotype pomona bacterins in cattle. Am J Vet Res 1975;36(1):59-65. [View abstract on PubMed ]
  • Whyte PBD, Ratclift RM, Cargill C, Dobson KJ. 1982. Protection of pregnant swine by vaccination against leptospira infection. Aust Vet J 59(2):41-45. [View abstract on PubMed ]
  • Sanford SE Morris PJ. 1990. Serological titers to various leptospiral serovars before and after vaccinating gilts with three commercial vaccines. Can Vet J 31:277-279. [View abstract on PubMed ]
  • Ebert E. 1999. Guinea pig serology as an alternative to the hamster challenge test for potency testing of Leptospira hardjo vaccines. Pharmeuropa 102e10.
    Available to subscribers only; please visit the Pharmeuropa website for more information
  • Hotka ML, Wilson MA, Anderson TM, Tichenor CL, Miller DA. 2007. Comparative serological study of Leptospira serovar hardjo genotypes for use in the microscopic agglutination test. J Vet Diagn Invest 19:84-87. [View abstract on PubMed ]
Human Vaccines
  • Nardone A, Capek I, Baranton G, Campèse C, Postic D, Vaillant V, Liènard, Desenclos JC. 2004. Risk factors for leptospirosis in metropolitan France: results of a national case-control study, 1999-2000. Clin Infect Dis 39:751-753. [View abstract on PubMed ]
  • Gonzàlez M, Martinez R, de la Paz RC, Bourzac JFI, et al. Vax-Sprial®. Trivalent antileptospirosis vaccine for human use; research, development and impact on the disease in Cuba. Medic Review Vaccination & Immunization in Cuba 2004 [View webpage at MEDICC.org]
  • Bracho G, Varela E, Fernández R, Ordaz B, et al. 2010. Large-scale application of highly-diluted bacteria for leptospirosis epidemic control. Homeopathy 99:156-166. [View abstract on PubMed ]
  • Dellagostin OA, Grassman AA, Hartwig DD, Félix SR, da Silva ÉF, McBride AJA. 2011. Recombinant vaccines against leptospirosis. Human Vaccines 7(11):1215-1224. [View abstract on PubMed ]
U.S. Regulations: Veterinary Vaccines
European Pharmacopoeia
  • European Pharmacopoeia. Monograph 01/2008:0447. Canine Leptospirosis vaccine (inactivated). 7th ed. Strasbourg, France: European Department for the Quality of Medicines within the Council of Europe; 2011.
  • European Pharmacopoeia. Monograph 01/2008:1939. Bovine leptospirosis vaccine (inactivated). 7th ed. Strasbourg, France: European Department for the Quality of Medicines within the Council of Europe; 2011.
VICH
  • International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). Stability testing of new biotechnological/biological veterinary medicinal products. 2000. [View on VICH website ]

Additional Information

Questions discussed in breakout groups

Draft summary document: "The Economic Burden of Leptospirosis"