Compatibility between a rabies vaccine and two canine combined vaccines against canine distemper, adenovirosis, parvovirosis, parainfluenza virus disease and leptospirosis, with or without canine coronavirus.

In many countries, vaccination programs still require dogs to be vaccinated against rabies in addition to Canine distemper virus (CDV), adenovirus (CAV), parvovirus (CPV), parainfluenza virus (CPiV), Leptospira (L) or Canine coronavirus (CCV= Cv). Few vaccines containing all these antigens are commercially available and, unless compatibility between the vaccines was demonstrated, concurrent administration of a DAPPi-L(Cv) vaccine and a vaccine against rabies should not be recommended. This may be of concern for practitioners who wish to vaccinate dogs with all components on the same day. This study aimed at evaluating immunological compatibility between a monovalent rabies vaccine (Rabisin™) and two large combination vaccines against CDV, CAV, CPV, CPiV with 2 leptospira components +Cv (Recombitek® C6/Cv) or with 4 Leptospira components (Recombitek® C8), when injected concomitantly at two separate injection sites. Fourteen days after administration of the rabies vaccine, with or without concomitant administration of combo vaccines, all dogs had seroconverted against rabies and maintained protective titers over the duration of the study. In addition, 100% of the puppies vaccinated with one or the other combo vaccines seroconverted against CDV, CAV, CPV, CPiV (CCV) and Leptospira, whatever the vaccination group. Lack of immunological interference between Rabisin™ and all components of the Recombitek® C6/Cv or Recombitek® C8 Combo vaccines was demonstrated by non-inferiority analysis, except for CDV in the Recombitek®C8+ Rabisin™ group. Based on these results, a concomitant administration of Rabisin™ with Recombitek® C6/Cv or Recombitek® C8 can be recommended in daily practice, which can be essential for facilitating vaccination compliance.

Quantitative Real-Time PCR Assays for the Detection of Pathogenic Leptospira Species in Urine and Blood Samples in Canine Vaccine Clinical Studies: a Rapid Alternative to Classical Culture Methods.

Leptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of Leptospira canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of Leptospira, followed by detection of Leptospira in blood and urine. We assessed the consistency between results obtained for urine and blood samples from clinical studies with species-specific real-time quantitative PCR (qPCR) targeting the lipL32 gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic Leptospira and for several canine viruses, bacteria, and parasites. The results from the two methods were compared using McNemar's test, kappa coefficient (κ), and percentage of agreement analyses. The results for numbers of positive and negative dogs were similar, with no false-negative results with the qPCR assay. For both blood and urine, there was strong agreement between the culture method and qPCR results (κ = 0.68 [95% confidence interval (CI), 0.62 to 0.74] and κ = 0.65 [95% CI, 0.59 to 0.71], respectively). However, there was a statistically significant difference between blood samples (P < 0.001) and urine samples (P = 0.028). The negative percentage agreements were 97% and 84% and the positive percentage agreements were 68% and 83% for blood and urine samples, respectively. Although the cell culture method is the recommended gold standard, our results show that qPCR assay is a valid alternative method for the rapid and specific detection of pathogenic Leptospira spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.

Successful Leptospira genotyping strategy on DNA extracted from canine biological samples.

Leptospirosis is an emerging worldwide zoonosis with a changing epidemiology responsible for an acute disease in humans and dogs. A better knowledge of the responsible bacterium Leptospira and in particular its various serovars and serogroups prevalence is essential for better diagnosis and prevention of the disease. The gold standard for leptospirosis diagnosis is the Microscopic Agglutination Test (MAT) but it requires long and fastidious laboratory work and sometimes results in controversial data. For these reasons, PCR-based techniques for detection of pathogenic leptospiral DNA in biological samples are currently replacing the MAT. However, these strategies do not provide any information regarding the infecting serovar or serogroup. In this study, an optimized genotyping method is described to allow the identification of Leptospira ssp. directly at serovars level using DNA extracted from canine blood and urine. 16S rDNA, Variable Number Tandem Repeat (VNTR) and Multispacer Sequence Typing (MST) protocols were adapted to biological samples. Eighty-eight DNA samples were analyzed from 72 different European canine clinical cases of leptospirosis confirmed by real-time PCR. 92% of DNA samples with Ct values below 34 were fully typed, and typing success decreased to about 30% for the other samples. Typing failure also showed a specie-specific correlation, with 63% of complete typing for L. interrogans and only 40% for L. kirschneri. Additionally, an exact match was observed between serological and molecular data for the few investigated cases where MAT data were available. This methodology is a suitable alternative to the MAT for determining the infecting serovar when Leptospira DNA from blood or urine is detected at Ct values below 34, contributing to clinical surveillance of leptospirosis.

Development of antibody ELISA specific of Leptospira interrogans serovar Grippotyphosa, Canicola, and Icterohaemorrhagiae to monitor vaccine immunogenicity.

The antibody response after primary vaccination and annual revaccination with a multivalent DAPPi-L vaccine was assessed respectively in SPF dogs and in client owned dogs against the Grippotyphosa (Lg), Canicola (Lc) and Icterohaemorrhagiae (Li) Leptospira serovars. To overcome limitations of the microscopic agglutination test (MAT), we developed serovar-specific and sensitive blocking ELISA assays. Serovar-specific antibodies against Lg, Lc and Li were detected in 100%, 45% and 91% of dogs, respectively, after the first dose of vaccine, and in 100% of dogs for all serovars after the second dose. In addition, mean ELISA antibody titers increased 14 days after annual revaccination with most dogs remaining ELISA antibody positive against Lg (85.3%), Lc (90%) and Li (100%). Parallel testing of sera from the annual revaccination study in the MAT and ELISA assays resulted in an overall agreement of 72%, 67%, 77% of samples for Lg, Lc and Li serovars, respectively. More sera tested positive by ELISA than by MAT, suggesting that the ELISA assay is more sensitive than the MAT. These three new antibody-based assays are the first suitable and reliable ELISA assays for the assessment of the canine antibody response following vaccination and an attractive alternative to the MAT.

Multivariate analysis of the immune response to a vaccine as an alternative to the repetition of animal challenge studies for vaccines with demonstrated efficacy.

The assessment of vaccine combinations, or the evaluation of the impact of minor modifications of one component in well-established vaccines, requires animal challenges in the absence of previously validated correlates of protection. As an alternative, we propose conducting a multivariate analysis of the specific immune response to the vaccine. This approach is consistent with the principles of the 3Rs (Refinement, Reduction and Replacement) and avoids repeating efficacy studies based on infectious challenges in vivo. To validate this approach, a set of nine immunological parameters was selected in order to characterize B and T lymphocyte responses against canine rabies virus and to evaluate the compatibility between two canine vaccines, an inactivated rabies vaccine (RABISIN®) and a combined vaccine (EURICAN® DAPPi-Lmulti) injected at two different sites in the same animals. The analysis was focused on the magnitude and quality of the immune response. The multi-dimensional picture given by this 'immune fingerprint' was used to assess the impact of the concomitant injection of the combined vaccine on the immunogenicity of the rabies vaccine. A principal component analysis fully discriminated the control group from the groups vaccinated with RABISIN® alone or RABISIN®+EURICAN® DAPPi-Lmulti and confirmed the compatibility between the rabies vaccines. This study suggests that determining the immune fingerprint, combined with a multivariate statistical analysis, is a promising approach to characterizing the immunogenicity of a vaccine with an established record of efficacy. It may also avoid the need to repeat efficacy studies involving challenge infection in case of minor modifications of the vaccine or for compatibility studies.

Impact of plasmid supercoiling on the efficacy of a rabies DNA vaccine to protect cats.

As of today, most DNA vaccination trials have been performed with plasmid preparations highly enriched in supercoiled molecules (sc) and the importance of supercoiled versus open circular (oc) plasmid isoforms for vaccine immunogenicity has only received limited attention. This study demonstrated that a single rabies DNA vaccination fully protected cats against a lethal rabies challenge as early as 3 weeks post vaccination provided that the proportion of supercoiled isoform in the vaccinal solution is at least 48%. In contrast, vaccination with a plasmid containing only 20% of supercoiled molecules induced significant but only partial protection. Further, a single rabies DNA vaccination with plasmids containing at least 70% of supercoiled molecules triggered statistically significant specific antibody titers and specific Th-1 oriented cell-based immunity as early as 2 and 3 weeks post vaccination, respectively. It is concluded that the oc isoforms are less efficient than supercoiled isoforms at inducing a complete profile of immune responses. Therefore, it is proposed that the target threshold of supercoiling that must be met by a rabies DNA vaccine to guarantee optimal immune responses and protection, be set at 70% of supercoiled molecules in the vaccine solution.