Maintenance of rabies-free areas.

Rabies is endemic in many parts of the world and is maintained in particular host species. Surveillance activities in areas that have remained rabies free or that have succeeded in eliminating the rabies virus (RABV) must continue, so as to effectively detect any infected animals in a timely manner. Rabies should be classified as a notifiable disease both in animals and in humans, irrespective of the rabies status of the country, and the establishment of a legal framework for biting animals is of the utmost importance. The maintenance of rabies-free status depends, in part, on the geographical situation of a country. A wide range of measures have proven effective, such as the maintenance of the mass vaccination of dogs, the establishment of cordons sanitaires to prevent the reincursion of sylvatic (wildlife) rabies, the application of human prophylaxis, the implementation of strict measures for travelling with companion animals (mainly domestic dogs and cats) and risk-based surveillance. Awareness regarding rabies among personnel working at border entry points as well as among the general public, veterinarians and general practitioners is also a major factor in ensuring the effectiveness of the surveillance network. Cross-border threats, even for islands, strengthen the need for regional cooperation, irrespective of the rabies status of the countries involved. Another important measure is the maintenance of adequate laboratory capacity for rapid and reliable diagnosis.

La rage est endémique dans de nombreuses régions du monde où certaines espèces hôtes assurent la persistance du virus. Les activités de surveillance doivent être poursuivies dans les zones demeurées indemnes de rage ou qui ont réussi à éliminer le virus rabique afin de pouvoir détecter rapidement tout animal infecté. La rage doit être inscrite sur la liste des maladies humaines et animales à déclaration obligatoire, indépendamment du statut du pays à l'égard de la rage ; de même, il est absolument primordial de mettre en place une réglementation sur les animaux mordeurs. Le maintien du statut indemne de rage dépend en partie de la situation géographique du pays. Un large éventail de mesures ont fait preuve de leur efficacité, notamment la vaccination massive et continue des chiens, la mise en place de cordons sanitaires afin d'empêcher toute nouvelle incursion de la rage sylvatique (affectant la faune sauvage), le recours à la prophylaxie chez l'homme, la mise en oeuvre d'une réglementation rigoureuse sur les voyages avec des animaux de compagnie (chiens et chats principalement) et la surveillance basée sur le risque. La sensibilisation des personnels chargés des contrôles aux frontières, du grand public, des vétérinaires et des médecins généralistes au problème de la rage est également un facteur crucial pour garantir l'efficacité du réseau de surveillance. Les menaces transfrontalières (même dans un contexte insulaire) renforcent l'impératif d'une coopération régionale quel que soit le statut des pays participants au regard de la rage. Une autre mesure importante est de s'assurer que les laboratoires sont dotés des capacités appropriées pour réaliser un diagnostic rapide et fiable.

La rabia, enfermedad endémica en muchas partes del mundo, se perpetúa en determinadas especies que ejercen de anfitrión. Es imperativo que en aquellas zonas que se han mantenido libres de rabia o donde se ha logrado eliminar el virus rábico se sigan efectuando labores de vigilancia, a fin de detectar eficazmente y con prontitud la presencia de todo animal infectado. La rabia debe ser considerada enfermedad de declaración obligatoria tanto en animales como en personas, independientemente del estatuto del país con respecto a la enfermedad. En este sentido, es de la mayor importancia instituir un ordenamiento jurídico aplicable a los animales mordedores. El mantenimiento del estatuto de «libre de rabia¼ depende en parte de la situación geográfica de cada país. Hay toda una panoplia de medidas probadamente eficaces, como el mantenimiento de la vacunación masiva de perros, la creación de cordones sanitarios para impedir toda nueva incursión de rabia selvática (en la fauna silvestre), la administración de profilaxis a las personas, la aplicación de estrictas medidas de control de los viajes con animales de compañía (principalmente perros y gatos domésticos) y la vigilancia basada en el riesgo. La sensibilización acerca de la rabia del personal empleado en puntos de entrada fronterizos, así como del gran público, los veterinarios y el personal médico en general también es un factor básico para que la red de vigilancia sea eficaz. Las amenazas transfronterizas, incluso en territorios insulares, hacen tanto más necesaria la cooperación regional, sea cual sea el estatuto con respecto a la rabia de los países en cuestión. Otra medida importante consiste en mantener una adecuada capacidad de laboratorio para efectuar diagnósticos rápidos y fiables.

Production and calibration of the second batch of OIE anti-rabies positive reference serum.

The Biological Standards Commission of the World Organisation for Animal Health (OIE) oversees the preparation and validation of OIE-approved International Reference Standards for use in serological assays for detecting infectious diseases of animals or the adequacy of their immune response following vaccination against those diseases. The principal use of OIE-approved International Reference Standards is to harmonise serological testing and to promote the mutual recognition of test results for international trade. In the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, the organisation recommends the use of the OIE anti-rabies positive reference serum of dog origin to titrate serum samples in international units (IU)/ml for use in rabies serological tests. The first batch of OIE reference serum of dog origin was produced in1991 and was used internationally until the beginning of 2010. The preparation of the new batch began in 2012 and, in contrast to the previous batch, three commercial inactivated rabies vaccines based on the most frequently used vaccine strains (Pasteur Virus and Flury Low Egg Passage) were selected for the immunisation of dogs in accordance with OIE guidelines. In 2013, calibration was completed through an inter-laboratory test involving five OIE Reference Laboratories for Rabies with the Second World Health Organization (WHO) International Standard for Anti-Rabies Immunoglobulin being used as a reference standard in this calibration. After statistical analysis of the results, the consensus titre was established as 5.59 IU/ml. The technical and statistical data were submitted to the OIE for assessment. In February 2014, the OIE Biological Standards Commission adopted this serum as an OIE-approved standard reagent for rabies serology.

La Commission des normes biologiques de l'Organisation mondiale de la santé animale (OIE) supervise la préparation et la validation de réactifs internationaux de référence approuvés par l'OIE et destinés aux épreuves sérologiques ayant pour objet le diagnostic des maladies infectieuses des animaux ou le suivi de l'effet protecteur obtenu par la vaccination contre ces maladies. Les réactifs internationaux de référence approuvés par l'OIE sont principalement utilisés pour harmoniser les tests sérologiques et permettre la reconnaissance mutuelle des résultats des tests dans le cadre des échanges internationaux. Le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres de l'OIE recommande d'utiliser le sérum de référence antirabique positif d'origine canine de l'OIE pour exprimer le titre des échantillons de sérum analysés en unités internationales (UI)/ml lors des épreuves sérologiques. Le premier lot de sérum de référence d'origine canine de l'OIE, produit en 1991, a été utilisé à l'échelle internationale jusqu'au début de l'année 2010. La préparation d'un nouveau lot a commencé en 2012 et, contrairement au lot précédent, trois vaccins antirabiques inactivés disponibles dans le commerce, basés sur les souches vaccinales les plus utilisées dans le monde (souche Pasteur et souche Flury Low Egg Passage) ont été choisis pour l'immunisation des chiens, conformément aux lignes directrices de l'OIE. L'étalonnage s'est achevé en 2013 lors d'un essai inter-laboratoires auquel ont participé cinq Laboratoires de référence de l'OIE pour la rage ; le second étalon international pour l'immunoglobuline antirabique de l'Organisation mondiale de la santé (OMS) a été utilisé en tant que réactif de référence pour cet étalonnage. Après analyse statistique des résultats, le titre consensuel obtenu est de 5,59 UI/ml. Les données techniques et statistiques ont été soumises à l'OIE pour évaluation. En février 2014, la Commission des normes biologiques de l'OIE a adopté ce sérum en tant qu'étalon de référence approuvé par l'OIE pour la sérologie de la rage.

La Comisión de Normas Biológicas de la Organización Mundial de Sanidad Animal (OIE) supervisa la preparación y validación de patrones de referencia internacional aprobados por la OIE para su utilización en ensayos serológicos destinados a detectar enfermedades animales infecciosas o a valorar la idoneidad de la respuesta inmunitaria de un animal al ser vacunado contra una u otra enfermedad. Dichos patrones sirven sobre todo para armonizar la realización de pruebas serológicas y promover el reconocimiento mutuo de los resultados de las pruebas con fines de comercio internacional. En su Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres, la OIE recomienda el empleo del suero positivo antirrábico de referencia de la OIE, de origen canino, para titular muestras de suero en unidades internacionales (UI)/ml y utilizarlas en pruebas serológicas de detección de la rabia. El primer lote de suero de referencia de la OIE procedente de perros fue elaborado en 1991 y estuvo en uso a nivel internacional hasta principios de 2010. La preparación del nuevo lote dio comienzo en 2012 y, a diferencia del lote anterior, para la inmunización del perro se seleccionaron tres vacunas inactivadas comerciales basadas en las cepas vacunales utilizadas con más frecuencia (virus Pasteur y cepa Flury Low Egg Passage), de conformidad con las directrices de la OIE. En 2013 culminó el proceso de calibración con una prueba interlaboratorios en la que intervinieron cinco Laboratorios de Referencia de la OIE para la rabia. En esta calibración se utilizó como patrón de referencia el segundo patrón internacional de inmunoglobulina antirrábica de la Organización Mundial de la Salud (OMS). Tras el análisis estadístico de los resultados, el título de consenso quedó fijado en 5,59 UI/ml. Los datos técnicos y estadísticos fueron sometidos a la valoración de la OIE, cuya Comisión de Normas Biológicas, en febrero de 2014, aprobó este suero como reactivo de referencia aprobado por la OIE para pruebas serológicas de detección de la rabia.

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.

First international collaborative study to evaluate rabies antibody detection method for use in monitoring the effectiveness of oral vaccination programmes in fox and raccoon dog in Europe.

The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97.2% and 97.5%, respectively. The overall agreement values obtained for the four marketed oral vaccines used in Europe were all equal to or greater than 95%. The coefficients of concordance obtained by laboratories ranged from 87.2% to 100%. The results of this collaborative study show good robustness and reproducibility of the BioPro ELISA Rabies Ab kit.

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.

Comparison of antibody responses after vaccination with two inactivated rabies vaccines.

Thirty laboratory dogs were randomly assigned to two groups (A and B) of 15 dogs and subcutaneously vaccinated with a single dose of one of two commercially available monovalent inactivated rabies vaccines: RABISIN (Merial, France) (group A) and NOBIVAC Rabies (Intervet International) (group B). Rabies antibodies were measured over a period of 4 months using the fluorescent antibody virus neutralization (FAVN) test. The two vaccines performed differently in terms of magnitude and persistence of rabies antibodies titers in dogs. Two weeks after vaccination, average rabies antibody titers peaked at 2.53 IU/mL (range, 0.17-13.77 IU/mL) and 1.26 IU/mL (range, 0.50-4.56 IU/mL) in groups A and B dogs, respectively. The average FAVN antibody titres against rabies on D28, D56, D84, D112 and D120 were significantly higher in group A than in group B. Although all dogs from group B serologically responded to vaccination, the proportion of dogs with antibody titres >or=0.5 IU/mL dropped significantly after D28 and was statistically significantly lower on D56, D84 and D112 compared to group A dogs. In conclusion, in the context of international trade, the choice of the vaccine and the timing of blood tests are critical factors in achieving successful serological test results after rabies vaccination. RABISIN induces high and sustained antibody titres against rabies, increasing the flexibility for the time of blood sampling after primo-vaccination.

Tools for rabies serology to monitor the effectiveness of rabies vaccination in domestic and wild carnivores.

Serology remains the only way to monitor the effectiveness of vaccination of humans and animals against rabies. Many techniques for determining the level of rabies antibodies have been described, including seroneutralisation techniques such as tests for fluorescent antibody virus neutralisation (FAVN) and rapid fluorescent focus inhibition (RFFIT), enzyme-linked immunosorbent assay (ELISA), and in-vivo tests (the mouse neutralisation test, MNT). The need to verify the effectiveness of rabies vaccination has become widespread, particularly in the context of international trading of domestic carnivores from infected to rabies-free territories. The standardisation of serological techniques, approval of laboratories and proficiency tests are key concepts to ensure the practicability of such systems. Serological tests for rabies are also often used by laboratories in infected territories to assess the efficacy of campaigns aimed at the eradication of the disease via oral vaccination of wildlife. The adaptation of these methods should provide the means to titrate specific antibodies in dogs during mass parenteral vaccination in countries infected by canine rabies. However, in most cases these serological tests are carried without any standardised procedure. On the basis of our experience in rabies serology and its harmonisation throughout laboratories worldwide, we propose here an adapted standard technique for the serological monitoring for rabies in wildlife at the European level. Such harmonisation would allow the monitoring of vaccination campaigns to be enhanced by increasing the exchange of epidemiological data, with the ultimate goal being the eradication of rabies in Europe.

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.