A randomised, double-blind, controlled efficacy trial of the LiESP/QA-21 vaccine in naïve dogs exposed to two leishmania infantum transmission seasons

Abstract

Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.

Figure 1. Classification of the infection status of the dogs.

This system of classification was used at each full clinical assessment and at the end of the study to determine the status of each dog.

Figure 2. Progression in log-transformed anti-ESP IgG1 and IgG2 reciprocal titres (Panel A) and anti-PSA IgG1 and IgG2 reciprocal titres (Panel B) in vaccinated dogs during the vaccinal period.

The technique was performed using serial three-fold dilutions of the serum to be tested, from 1/150 to 1/12150. Dogs were considered as negative when the titre was inferior to 1/450. Dogs with a negative result were regarded as having a result of zero after log conversion of the reciprocal titres to allow this to be represented on the charts. Error bars represent the standard error of the mean.

Figure 3. Longitudinal record of the infection status of each dog between months 9 and 24.

The dogs were classified at each time point using a system based on PCR, culture and clinical signs and clinicopathological parameters (the details of this system are given in figure 1). Points at which subpatent dogs converted back to a Leishmania-free (PCR negative) status are circled.

Figure 4. Clinical findings, laboratory abnormalities and IFAT titres seen in dogs diagnosed with symptomatic infections at the time of the diagnosis and at the end of the study.

Figure 5. Comparison of the proportion of dogs in each group that progressed to active infection by the end of the 2 year study.

Active infection was determined by positive results with PCR and biphasic medium culture with or without clinical signs.

Figure 6. Comparison of the proportion of dogs in each group that progressed to symptomatic CanL by the end of the 2 year study.

Symptomatic CanL was defined as active infection with the presence of a clinical score>3.

Figure 7. Summary of the dogs' Leishmania status at month 24 after two years of exposure in a highly endemic environment.

Figure 8. Progression of the proportion of dogs with active infection as assessed by a survival curve analysis.

The status of the dogs was assessed at months 9, 15, 18, 21 and 24 using the classification system in figure 1. Active infection was identified by positive PCR and positive culture. The two groups were significantly different (p = 0.0265).

Figure 9. Progression of the proportion of dogs with symptomatic infection as assessed by a survival curve analysis.

The status of the dogs was assessed at months 9, 15, 18, 21 and 24 using the classification system in figure 1. Symptomatic infection was identified by positive PCR and positive culture associated with a clinical score>3. The two groups were significantly different (p = 0.0466).

Figure 10. Geometric means of the bone marrow parasite loads over the course of the study.

The bone marrow parasite loads were assessed by quantitative RT-PCR targeting a 200 bp fragment of the kinetoplast DNA performed at months 0, 9, 15, 18, 21 and 24. When a dog died from leishmaniasis before M21 or M24 (natural death or euthanasia for ethical reasons), the last result available was reported until M24. The number of parasites per ml of bone marrow could thus be calculated, and the results are presented as the geometric mean for each group. Samples were considered as negative if the parasite load was inferior to 40 parasites per ml bone marrow. Error bars represent the SEM. The two groups were significantly differently over the duration of the study (p = 0.035).