Canine parvovirus type 2 (CPV2) emerged in 1978 as a highly contagious and very serious disease in dogs. The characterization of CPV2 antigenic types is exclusively based on the identification of the amino acid residue at position 426 of the capsid protein VP2. Currently, three antigenic types CPV-2a (asparagine N(426)), CPV-2b (aspartic acid D(426)) and CPV-2c (glutamic acid E(426)) are circulating worldwide. In Tunisia, despite the fact that many clinical and few serological investigations clearly indicate that CPV is widespread and of major concerns in the local dog population, no molecular and antigenic type characterization of circulating variants has been carried out. This investigation showed that most of clinically presumed CPV infections were confirmed by classical or real-time PCR. When no real-time PCR facilities were affordable, classical PCR as reported here in association with restriction fragment length polymorphism (RFLP) with MboI and MboII can be very useful for screening and diagnosing CPV infections. A total of 50 variants were characterized by sequencing and an almost even representation of the different antigenic types, including CPV-2c and slightly more type 2b, were evidenced. Characterization of the Tunisian variants by MGB probe assays as reported was inefficient for most of CPV-2a variants because of their typical nucleotide mutation C(1269). Phylogenetic analysis showed that the Tunisian variants underwent evolution for a relatively long period of time inside the country. The analysis also showed some crossings of the different antigenic types, leaving both genotypic and phenotypic characteristic mutations.

In Tunisia, rabies continues to be considered as a serious public health concern. Very costly mass vaccination of dogs against rabies and expensive post-exposure prophylaxis are prerequisites to maintain a low level of human rabies cases. In Tunisia, the implementation of mass vaccination campaigns at the national level has undoubtedly contributed to the drop of rabies endemicity, but the overall outcome is rather suboptimal. In this investigation, we wanted to estimate the extent of the vaccination coverage in dogs in three Governorates (Manouba, Kassrine and Mednine), by collecting data through questionnaires and interviews relevant to 1470 owned dogs. When the campaign is correctly applied, as in Manouba, almost all the targeted dog population can be reached by parenteral vaccination and an almost elimination of the disease can be evidenced. However, in Kasserine and especially in Medenine, where the vaccination coverage is lower than 31%, a reservoir for the disease can be maintained. To match the official figures of vaccination coverage, we should update the statistics of the size of dog population which seems to be bigger than what is assumed. In addition, we wanted to assess the level of involvement of the local population with the vaccination campaigns by marking vaccinated dogs with necklaces and establishing later on the vaccination coverage accordingly. The highest levels of vaccination coverage can be reached in the rural regions. Therefore, the low vaccination coverage in rural areas, reported at the national level, is more attributable to the lack of human and financial resources to reach remote regions. We think that rabies control programmes in Tunisia can be successful if vaccination coverage can reach 70% in all parts of the country. The achieved vaccination coverage should be estimated after random visits in many parts of the country and by checking valid vaccination certificates.

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BackgroundDuring the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage.MethodsWe have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES) domain.ResultsThe monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient in the design of multivalent plasmids.ConclusionsIn a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.