Determination of an infectious dose of leptospira for the performance of challenge test in assessing the efficacy of leptospira vaccines

The efficacy of vaccines against leptospiral disease can be determined objectively by challenge test in experimental animals. Selection of suitable leptospiral challenge strains and determination of an optimal challenge dose to prove exactly that the given vaccine Leptospira serotype induces protective immunity in vaccinated dogs is a critical point in performing challenge experiments. The aim of our study was to verify and determine an appropriate challenge dose for efficacy tests in dogs for the following Leptospira serovars: L. grippotyphosa, L. icterohaemorrhagiae and L. canicola. The appropriate challenge dose was determined on the basis of pathognomonic symptoms of infection, Leptospira capture at cultivation and pathological changes in dogs infected experimentally with various doses (5 × 104 5 × 108) of Leptospira serovars. A dose of 5 × 106 of each respective serovar administered intraperitoneally was determined to be a suitable challenge dose. The dogs infected with the selected dose showed the typical symptoms of the disease and met the requirements of an objective and standard evaluation of the vaccine efficacy according to the pharmacopoeial monograph. A study of such extent was done for the first time in the Czech Republic. Vaccine against leptospirosis, determination of efficacy, challenge test, determination of infectious dose Leptospirosis is an infectious disease of animals and humans caused by spirochaetes of the genus Leptospira. It is a worldwide zoonotic infection with a much greater incidence in tropical regions and has now been identified as one of the emerging infectious diseases (Vinetz 2001; Levet 2001; Roca 2006; Vijayachari et al. 2008). The bacteria cause a polyfactorial disease from asymptomatic infection, subclinical cases, up to acute infections with a fatal outcome. One of the ways of protecting animals from leptospirosis is vaccination, which may reduce the incidence and severity of the disease. Currently, not only proven whole-cell inactivated vaccines are used, but also new vaccines are being tested: recombinant vaccines based on outer membrane proteins (OMP), lipopolysaccharide vaccines, DNA vaccines, etc. (Srivastava 2006; Wang et al. 2007). To increase the immune response, a mineral adjuvant (aluminum hydroxide) is mainly used. Regarding the fact that Leptospira is a weak immunogen, vaccinations with a subsequent revaccination are used at the start of immunoprophylaxis to create an antibody content. Sufficient titre of antibodies is then maintained by regular annual revaccination. The efficacy of Leptospira vaccines was formerly tested in hamsters. The laboratory hamster (Mesocricetus auratus) is regarded a very sensitive and suitable model animal for testing of Leptospira infections and has been used for a long time (Randall and Cooper 1944). Using of hamsters in testing of efficacy of Leptospira vaccines was mentioned in earlier editions of the pharmacopoeia. The efficacy test using the challenge in dogs appeared in the monograph of Czech Pharmacopoeia (2005), which corresponds to the fifth edition of the European Pharmacopoeia (EP). The preceding pharmacopoeia ČL 2002 monograph “Vaccinum leptospirosis ad usum veterinarium”, described the challenge test only in hamsters. Currently, Leptospira canicola, L. icterohaemorrhagiae, L. sejroe, L. grippotyphosa, L. bratislava and L. pomona are the most important serovars that cause infections in dogs ACTA VET. BRNO 2011, 80: 263–268; doi:10.2754/avb201180030263 Address for correspondence: MVDr. Jiří Nepeřený Bioveta, a.s., Komenského 212 683 23 Ivanovice na Hané, Czech Republic Phone: +420 517 318 553 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm (Boutilier et al. 2003; Schreiber et al. 2005). An efficient way of preventing leptospirosis in dogs is immunoprophylaxis with effective, especially multivalent vaccines (Klaasen et al. 2003; Schreiber et al. 2005). Protective efficacy against each Leptospira serovar contained in the vaccine must be demonstrated by a challenge test in dogs before registering and using these immunobiological preparations in practice (European Pharmacopoeia, Article 0447). The critical point in performing of challenge experiments is to determine optimal challenge doses of individual Leptospira serovars so as to demonstrate exactly that the given vaccine Leptospira serotype induces protective immunity in vaccinated dogs. The maintenance of challenge Leptospira strains for testing the efficacy of vaccines against canine leptospirosis is described by Reed et al. (2000). The performance and results of challenge tests in dogs have been described recently in a series of papers (Greenlee et al. 2004; Schreiber et al. 2005a; Schreiber et al. 2005b; Greenlee et al. 2005). Most authors evaluate the efficacy of the vaccine Leptospira component serologically, mainly using the agglutination reaction lysis (MAL), when specific agglutination-lytic antibodies are determined in vaccinated animals. However, only one part of the immune response is assessed like this. It has been proved experimentally that the cell-mediated immunity (CMI) plays an important role at the onset of immunity and in the protection against leptospirosis (Koizumi and Watanabe 2005). During the development of inactivated veterinary vaccines against canine leptospirosis it is important to ensure that the registration dossier includes all the required validations and testing as required, in particular: Special requirements for veterinary immunobiological products (Directive 92/18/EEC/1992) in the European Union, any new product shall comply with the European Pharmacopoeia monograph and its supplements issued by the Council of Europe, any new product shall meet the recommendations of international organizations (WHO, OIE), any new product shall meet specific and additional requirements of the state where it is registered. The aim of our study was to determine the optimal challenge dose of leptospira strains for the efficacy testing of vaccine against leptospirosis in dogs using challenge test in accordance with the guidelines of the European Pharmacopoeia monograph 01/2005:0447. Materials and Methods Fifteen eight-week-old female Beagle dogs, without detectable antibodies against L. grippotyphosa, L. icterohaemorrhagiae and L. canicola by microscopic agglutination test (MAL), from a commercial breed of laboratory animals (Biotest Konárovice, Czech Republic) were used in this study. Animals were randomly divided into 3 groups of 5 dogs. One group received the serovar L. grippotyphosa, the second group received the serovar L. icterohaemorrhagiae and the third group received the serovar L. canicola. Challenge strains of Leptospira used in the test must be different from the strains used for vaccine production. The strains L. grippotyphosa, L. icterohaemorrhagiae and L. canicola were acquired from the American Type Culture Collection (ATCC) via LGC Promochem (United Kingdom). In the selection of strains, emphasis was laid to their pathogenicity. All Leptospira strains were kept in liquid nitrogen at -196 °C on a long-term basis. The selected Leptospira strains were cultured in liquid culture medium Stuart with an addition of 10% rabbit serum SIGMA (R4505) at 28 °C. The number of Leptospirae in the culture was counted by means of Petroff Hausser counting chamber (Electron Microscopy Sciences, cat. 63512-21) in a dark field of microscope. The concentration of Leptospira of each serovar was adjusted by dilution with sterile saline so that 1 ml of culture contained 104, 105, 106, 107 and 108 Leptospirae. The infectious doses of individual Leptospira strains used for infection in dogs are described in table No. 1 An infectious dose for each animal was 5.0 ml and was administered intraperitoneally after a seven-day acclimatization period. All animals were observed for 28 days. During the whole experiment, body temperature and clinical status of experimental animals were recorded daily. Body weight of animals was recorded before and after the experiment. The following pathognomonic signs of leptospirosis infection were selected as critical for determining an appropriate challenge dose of Leptospira: 1) Culture detection of Leptospira in the blood before the trial and on days 2, 3, 4, 5, 8 and 11 post infection by microscopic evaluation, 2) Culture detection of Leptospira in the urine before the trial and on days 3, 5, 8, 11, 14, 21 and 28 post infection by microscopic evaluation, 3) Culture detection of Leptospira in the kidney, liver and gall bladder by microscopic evaluation at dissection on day 28 post infection or on the day of death, 4) Clinical symptoms of leptospirosis during the test period in particular: 264