Canine rabies continues to be a major threat in many countries especially in Asia and in Africa [1]. The disease is endemic in all provinces of Morocco except the southern desert region, with the domestic dog being the main reservoir and vector [2345] of the virus. Since 1986, about 22 human deaths have been reported yearly [6] and since 2000, an average of 376 animal cases have been recorded annually, mainly in dogs and in livestock, especially cattle [7]. The major element of rabies control strategies is regular application of injectable vaccine to reach and maintain sufficient vaccination coverage in the field enough to stop rabies virus transmission. Moroccan authorities have set up several rabies eradication plans since 1986, but to date rabies remains a serious health problem in Morocco [8]. Mass dog parenteral vaccination is an integral component of the rabies control measures [9], using an inactivated adjuvanted cell culture veterinary rabies vaccine produced locally since 1986 [2].

The mass annual vaccination campaigns are conducted in suburban and rural areas and organized locally by each district, with a vaccinator team visiting each house (door to door model) or present at several central points [10]. The dog vaccination campaigns are free of charge for dog owners and cover all the country. In urban settlements, parenteral vaccination is ensured by private veterinarians only, based on the ownership responsibility.

In view of the current epidemiological situation and of the fact that prophylactic efforts did not lead to the expected results, it appeared necessary to assess the efficacy of the vaccine in laboratory controlled conditions and also in the field. The World Health Organization (WHO) [11] recommends assessing mass dog vaccination campaigns efficacy by using well-designed serological monitoring, aiming to evaluate the vaccine potency in field conditions and also the vaccination coverage of dog population in vaccinated areas. The humoral response to rabies parenteral vaccination shows a classic profile with a latent phase, an exponential phase after first vaccination and a plateau and then a decrease in the antibody titers [12]. In primary vaccinated dogs, the seroconversion occurs generally between 4 and 6 weeks [13] and it has been shown that seroconversion is an indicator of protection against rabies [12]. In the present study, we evaluated the efficacy of the locally produced vaccine to protect field dogs in experimental conditions against a field dog rabies virus challenge. The immunogenicity of the vaccine was also investigated to evaluate vaccine effectiveness in field conditions. A blood test was performed thirty days after rabies vaccination of field dogs in eight Moroccan districts and the immunological response was measured with a WHO/World Organization of Animal Health (OIE) reference antibody virus neutralization test [14] to check seroconversion rates.

In Morocco, many efforts are deployed in infected areas to fight against the disease based essentially on mass canine vaccination campaigns and dog population control [3]. The parenteral dog vaccination using injectable vaccines is the gold standard tool recommended by OIE [15] and WHO for effective rabies control in dogs [1011]. A vaccination coverage of 70% of the dog population is considered to be a critical threshold to achieve this goal [1123]. The rabies vaccine to be used should be approved by the national regulatory authorities according to relevant high quality international standards [111516].

Rabivac is a locally produced inactivated rabies vaccine in use in mass dog vaccination campaigns in Morocco. In this study, its efficacy was assessed in experimental conditions comparable to those of the European Pharmacopeia monograph [16], which requires a virulent challenge of the target species following vaccination. In addition, a field experiment organized in eight Moroccan districts was undertaken to check the immunogenicity of the vaccine in owned dogs.

All dogs maintained under experimental conditions were collected in the field at early age (between 3 and 6 months old), housed in an experimental kennel and kept for a long quarantine period (9 months) prior to be vaccinated since their health status as regards rabies was unknown. All dogs had negative rabies serology (<0.24 IU/mL) at D0 except dogs Nos. 4 and 11 that had a titer ≤1.15 IU/mL. The serum samples were pre-diluted prior to serological testing because of insufficient volume, hence the exact value of the titer was impossible to calculate and the immunological status (positive or negative) was therefore unknown. All vaccinated dogs seroconverted with titers above 0.24 IU/mL as early as 14 days post-vaccination (geometric mean, 3.34 IU/mL). This is similar to the findings of other authors [2425] who tested another commercial vaccine produced with the same virus strain and who found a mean antibody value of 2.53 IU/mL [26] and 5.0 IU/mL [24] 2 weeks post-vaccination in laboratory dogs. The same finding was demonstrated 1 month after vaccination, just before the boost, with a mean antibody levels of 3.10 UI/mL in our study compared with 2.03 UI/mL in the study of Minke et al. [26] and 2.8 IU/mL in the study of Kallel et al. [24] on laboratory dogs. Two weeks after the boost vaccination (D49), all dogs had high serology titers with a maximum of 41.59 IU/mL indicating an adequate immunization. The mean serological titer of the vaccinated dogs 4 months after vaccination (D119) was 0.57 IU/mL, with a high number of dogs still positive (5/8) for rabies antibody detection, contrary to previous studies demonstrating a decrease in time in rabies antibody levels in laboratory and owned field dogs primary vaccinated with one dose of commercial vaccines [242526272829]. The longer persistence of detectable rabies antibodies in this study is due to the boost one month after vaccination. After the rabies virus inoculation, one dog (No. 4) died without any symptoms or necropsy findings related to rabies. The kinetics of the rabies antibody response of this dog was similar to that of other dogs. Rabies diagnosis using FAT and MIT provided negative results. The death was not due to rabies but to a torsion mesenteric accident observed during the necropsy of the animal. All other vaccinated dogs survived, demonstrating the ability of the vaccine to protect dogs against a challenge virus of dog origin, as it would happen in field conditions.

All dogs of group C had negative serology during all the experiment except dog No. 11 who had a titer ≤1.15 IU/mL at D0. The serum was pre-diluted prior to serological testing as its volume was insufficient, as for the serum D0 of dog No. 4 (see above). Therefore, the status (positive or negative) could not be determined. All other antibody titers of this dog during the experiment were clearly below 0.24 IU/mL. This dog died of rabies 17 days after the challenge, i.e., in the same time range than other control dogs, suggesting this dog was presumably not vaccinated. A percentage of 80% (4/5) of control dogs died from rabies between 17 and 27 days after challenge, which corresponded to the classic rabies incubation period generally reported in experimentally infected dogs [173031]. The observed symptoms were in concordance with rabies clinical signs described by the OIE [15]. The 80% mortality rate observed in the control group is in conformity with the requirements of WHO, OIE, and European Pharmacopeia [111516] for validating the assay. The results of other studies using the same rabies vaccine strain [25] showed a comparable level of protection after a rabies virus challenge of vaccinated dogs. Our results demonstrate the ability of the tested vaccine to induce a high specific humoral response and full protection of field dogs against a dog rabies virus challenge.

Of 919 serologically field owned dogs sampled in urban, suburban, and rural areas, an overall of 24% harbored a positive serological response before vaccination, likely as a result of residual antibody level of a previous vaccination. Unfortunately, the dates of the last vaccinations before D0 were unknown. At D0, depending on districts, seropositive percentages varied with the highest levels obtained at Settat, Khemisset, and Oujda (50%, 48%, and 28%, respectively) and the lowest levels found at Sidi Kacem, Casablanca, and Choutka (7%, 14%, and 16%, respectively). The vaccination coverage in rural (20%) and suburban areas (17%) were statistically similar while it was statistically higher in suburban areas (29%); however, these results suggest globally that no significant difference seems occurring between the geographical status of the regions on the seropositive results obtained. The Sidi Kacem province seemed to be less covered by mass parenteral vaccination campaigns at the date of the experiment. It should be noted that the cumulative numbers of human and animal rabies cases in this province are among the highest within the country [37]. The seroprevalence percentages determined before vaccination provide a picture at a certain time of the level of detectable residual antibodies in dogs sampled in the different areas. The time elapsed between the last vaccination of each dog and the blood sampling at D0 was unknown. The curve of rabies antibodies after primary vaccination of laboratory or field dogs has been largely described (for review, see [12]) and consists in a rapid production of specific antibodies after the first vaccination followed by a regular decrease then a new rise after a booster to obtain a higher level then a new decrease but with a higher level than that previously achieved. The peak of rabies antibodies is generally observed between 3 and 6 weeks post-vaccination [121326]. A detailed review analysis of the significance of seroconversion results coupled to those of the probability of surviving a challenge has clearly demonstrated that animals seroconverting at a certain time after vaccination have a very high probability of surviving in case of rabies infection, even if they have no more detectable antibodies at the time or before the challenge [12]. Therefore, the interpretation of seroprevalence results in a field dog population sampled at different times after vaccination has a number of limitations. The percentage of 24% of seropositive dogs at D0 obtained in our study is an indicator suggesting that dogs have been previously vaccinated, and probably higher vaccination coverage of dog populations existed as these dogs had seroconverted before the D0 sampling. This value corresponds to those which have been published in similar countries [53233].

At D30 post-vaccination, an overall percentage of 92% of dogs were tested positive for rabies antibodies demonstrating the vaccine was highly effective in field conditions. The vaccination coverage was estimated at the best period, i.e., 30 days after the vaccination of dogs older than 3 months of age, and results showed in all regions significant increases in rabies antibody levels before and after vaccination. In Casablanca, where 22% of dogs were seronegative at D30, of which 9% had rabies antibodies levels less than 0.1 IU/mL, demonstrating a total absence of antibody response, and the weakest levels of rabies antibodies among different regions tested were observed (geometrical mean titer, 1.21 IU/mL). The reasons for that cannot be attributed to the vaccine since a better efficacy was achieved in other regions but could be linked to human deficiencies during the vaccination and sampling steps at the date of the experiment. The negative results could also be due to unhealthy living conditions as dogs were not dewormed and maybe did not receive sufficient food. It is known that alimentation and parasitism may influence the production of antibodies [12]. Another explanation could be that the immune response of certain dogs did not already reach the 0.24 IU/mL threshold 30 days after vaccination.

The serological monitoring of dogs following the parenteral mass vaccination campaigns is no longer recommended by WHO on a routine basis [11] because of the kinetics of rabies antibodies showing a rapid decrease particularly in primary vaccinated dogs, which represent >30% of the population because of the high turnover of the dog population [533]. Field well–designed seroprevalence studies are now recommended, as done here, for assessing the efficacy of a novel vaccine or if some failures are suspected during the campaigns, such as vaccine potency or cold chain maintenance [11], at the best period after vaccination to check seroconversion dates.

In view of these results obtained on field dogs and on the literature available on this topic, it is hypothesized that a booster with Rabivac undertaken 1 month after the primary vaccination is probably not justified. A single vaccine injection carried out each year on all dogs, including puppies, as currently done in other places with available comparable commercial vaccines, should be sufficient to ensure adequate immunization of dogs. Studies aiming at estimating the dog population should be organized also in Morocco to update the knowledge of the dynamics of the population, estimated in the 1990s at 1.6 to 2 million [5].

These results demonstrated that Rabivac induced a complete protection in experimental dogs after a virulent dog rabies virus challenge and a satisfactory humoral response in field dogs living in different conditions. This vaccine is therefore adequate for mass dog vaccination campaigns against rabies in Morocco.