Factors associated with dog rabies immunisation status in Bamako, Mali.

We conducted a cross-sectional survey in Bamako, Mali, to determine for the first time the seroprevalence of rabies virus antibodies in the dog population and people's knowledge, attitudes and practices (KAP) towards the disease and its control. Antibody detection was done with the fluorescent antibody virus neutralisation (FAVN) test, with a positivity threshold of 0.25IU/ml. We visited 2956 households in 2010 and 2011 and found 379 dogs in 279 households. Data were collected on 279 dog-owning households, on 1017 non-dog-owning households and on 311 dogs. A serum or plasma sample was collected from 98 dogs. For 26 dogs we had sufficient data to describe the antibody decline over time after rabies vaccination using a quadratic regression. Ninety percent of interviewed persons (95% CI: 85%-91%) knew about rabies. The majority of interviewees knew that rabies is transmitted from dogs to humans, and some of the characteristic clinical signs seen in rabid dogs (change of behaviour, biting, salivation) could be listed by the majority. When asked how people behave regarding a rabid dog, killing the animal was the most frequent answer (>70%). Most (65% of the non-dog-owners and 81% of the dog-owners) were aware that vaccination of dogs can prevent rabies, but only a minority of dog-owners could answer correctly at what age the dog should get a first rabies vaccination (i.e. at 3 months). There was also strong consensus among dog-owners that it is better to protect their dog from becoming rabid by vaccinating it rather than needing to treat a bitten person. Forty-five percent (n=306; 95% CI 38%-52%) of dogs were reported as vaccinated against rabies at least once, but less than half of these (59/136) had a valid vaccination card. When asked for reasons for non-vaccination, cost was the most frequent reason at 31% (95% CI: 21%-43%), while general negligence was mentioned by 15% (95% CI: 10%-24%). Approximately one third of dog-owners would not pay for vaccination. To reach a threshold of 70% of vaccinated owned dogs, vaccination should not cost more than 0.2€ (100 FCFA). The seroprevalence of rabies virus antibodies in the examined dog population was low: 24% (n=98; 95% CI 15%-36%) with titres ≥0.25IU/ml and was 46% (n=39; 95% CI 29%-63%) when only including those reported as vaccinated by their owners. A seroprevalence of 59% (n=18; 95% CI 33%-80%) was reached if the analysis included only dogs with a valid vaccination certificate. Interestingly 4/22 dogs showed titres ≥0.25IU/ml despite being reported as unvaccinated. The Rabisin® vaccine showed generally higher IU titres than the Dog Vac Rabia® vaccine. All animals after booster vaccination had titres ≥0.25IU/ml which was not the case in primo-vaccinated animals. For the Rabisin® vaccine, a Kaplan Meier estimate suggested that to maintain an antibody titre of ≥0.25IU/ml for 75% of owned dogs, revaccination should be done after not more than 2.5 years. This work contributes vital information towards planning an effective dog rabies control programme for the district of Bamako.

Evaluation of a rabies ELISA as an alternative method to seroneutralisation tests in the context of international trade of domestic carnivores.

For several years, international movements with pets have greatly increased. Most countries have relaxed their quarantine measures and adopted a scheme combining vaccination of pets against rabies followed by a serological test to check the efficacy of vaccination. This new scheme has been strongly supported by the OIE, WHO and the European Commission to facilitate the free movement of people and pets around the world. Currently, only two reference methods are recognised and prescribed (the FAVN test and the RFFIT) to measure rabies antibody levels in serum samples for international trade. They are reliable and valuable methods of assessing the efficacy of rabies vaccination but they are time-consuming and require well-trained people and specialised laboratory facilities. A few years ago, an ELISA (Platelia™ Rabies II kit ad usum Veterinarium) was developed for domestic carnivores and wildlife. To our knowledge, this ELISA is the only one certified and prescribed by the OIE. Following its marketing, one task of the EURL for rabies serology was to evaluate the performance of laboratories using this new kit. The results revealed that 26% of the participants, which were already approved laboratories for rabies serology, failed the inter-laboratory trial. Such unsatisfactory results have never been observed during any of the previous proficiency tests organised annually since 2000 by the EURL for rabies serology using reference methods. More investigations were undertaken through internal and collaborative studies to assess the performance of this newly marketed ELISA kit. The results of the internal study revealed that even with a specificity of 100%, the sensitivity evaluated on 593 samples of domestic carnivores came to 78.2%. An issue regarding the underestimation of serum titres was also revealed during the study. The results of a collaborative study involving 23 international laboratories reinforced the preliminary conclusions regarding lack of sensitivity. Indeed, only 5 laboratories out of the 23 obtained satisfactory results. We therefore suggest adopting a threshold of 0.3 EU/mL instead of 0.5 EU/mL to increase the sensitivity of the test.

Evaluation of an ELISA to detect rabies antibodies in orally vaccinated foxes and raccoon dogs sampled in the field.

The assessment of the efficacy of oral vaccination in wildlife is based on detection in the teeth of a biomarker (tetracycline) which is incorporated in the vaccine bait, and the quantification of rabies antibodies. A blocking ELISA was evaluated and compared with the FAVN test and a validated in-house ELISA, using sera from foxes and raccoon dogs collected following oral vaccination campaigns in France and Estonia. Specificity reached 100% in sera from naïve animals. A high concordance (95%) was observed between the BioPro ELISA and the FAVN test, which was similar in sera from red foxes and raccoon dogs. Concordance between the BioPro ELISA and the in-house ELISA reached 96.5% for sera from red foxes. The agreement with tetracycline results was excellent in the fox for both the BioPro ELISA (95.9%) and the FAVN test (91.8%). Concordance was slightly lower in the raccoon dog, with a value of 82.8% for the BioPro ELISA and 78.4% for the FAVN test. Rabies antibodies were detected with the BioPro ELISA in animals vaccinated with different types of vaccines and in highly haemolysed sera. The BioPro ELISA is a valuable test to assess the efficacy of oral vaccination in foxes and raccoon dogs.

A quantitative indirect ELISA to monitor the effectiveness of rabies vaccination in domestic and wild carnivores.

This paper reports a new ELISA to measure the level of rabies anti-glycoprotein G antibodies after vaccination. The Platelia Rabies II kit was evaluated on different populations of dogs, cats and foxes. For each target species, sera from naive, unvaccinated and vaccinated animals were tested. Platelia Rabies II results were compared to the reference fluorescent antibody virus neutralisation test (for dogs and cats) and to a published in house ELISA test (for foxes). The Platelia Rabies II test was found to be highly specific whatever the species (more than 98%) using a cut-off value of 0.5 EU/ml. The index of sensitivity was between 92.4% and 94.5% for fox samples, and reached 83% for domestic carnivores. Data collected by testing field samples revealed that the rate of false negative results ranged between 8.9% and 11.1% and the rate of false positive results ranged between 1% and 2% for the dog/cat population. Therefore, the Platelia Rabies II test described here would be a good candidate for routine detection of rabies antibodies not only in domestic carnivores (within the framework of international trade) but also in foxes for the follow up of rabies oral vaccination programs.

Neutralising antibody titration in 25,000 sera of dogs and cats vaccinated against rabies in France, in the framework of the new regulations that offer an alternative to quarantine.

Regulations governing international movements of domestic carnivores from rabies-infected to rabies-free countries have recently been loosened, with the adoption of a system that combines vaccination against rabies and serological surveillance (neutralising antibody titration test with a threshold of 0.5 UI/ml). Since 1993, the Research Laboratory for Rabies and Wild Animal Pathology in Nancy, France, has analysed over 25,000 sera from dogs and cats using a viral seroneutralisation technique. The statistical analyses performed during this time show that cats respond better than dogs. Although no significant difference in titres was observed between primovaccinated and repeat-vaccinated cats, repeat-vaccinated dogs had titres above 0.5 IU/ml more frequently. In primovaccinated dogs, monovalent vaccines offered a better serological conversion rate than multivalent ones. Finally, the results of these analyses showed a strong correlation between antibody counts and the time that elapsed between the last vaccination and the blood sampling.

ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields.

The assessment of the efficacy of rabies oral vaccination campaigns requires the titration of specific antibodies in the target species. Unfortunately, in Continental Europe, most fox serum samples are in fact "body fluids" taken from cadavers and the lack of a validated titration method for these poor quality sera made it impossible to survey and compare the efficacy of various oral vaccination protocols used by the different European teams. By using ready to use microplates sensitised with rabies virus glycoprotein purchased from a manufacturer and applying a simple and rapid ELISA technique on field fox sera, we obtained antibody quantitation highly correlated with seroneutralising antibody titres measured with a seroneutralisation test on cell culture. We obtained, with fox sera sampled in the same area, the same distribution of high, medium and low titres within all categories of serum quality (from high to very poor quality) and therefore conclude that this ELISA test allows a reliable titration even with highly contaminated body fluids. This test was shown to be equally capable of detecting rabies antibodies in serum samples taken from foxes vaccinated with an highly attenuated rabies virus (the SAG2 double mutant of the Street Alabama Dufferin strain) or with the VRG, the Vaccinia recombinant glycoprotein. Additionally, a strong correlation was demonstrated between titres given by this ELISA (or by the seroneutralisation test) and protection against challenge of foxes orally vaccinated with SAG2 vaccine baits. In view of this validation, this simple and reliable test is proposed for sero-surveying foxes following rabies oral vaccination campaigns.