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.

Epidemiology and molecular diversity of rabies viruses in Bulgaria.

A health emergency situation occurred in Bulgaria in 2007 when positive rabies cases were notified in Sofia district in the central-western part of the country, suggesting a southward spread of the disease for the first time in the last 10 years. Phylogenetic analysis on 49 isolates sampled between 2009 and 2011 showed, for the first time, evidence of the existence of NEE and D clustered lineages in Bulgaria. Their geographical distribution clearly reveals the permeability of natural barriers, as already suggested by the disease spread that occurred across the Balkan mountain range in 2007. The monitoring and passive surveillance programmes conducted since the first 2009 oral vaccination campaign, the spatio-temporal evolution of the disease in the country since 2007, and the need for further investigation of the role of jackals in virus dispersion are discussed.

Molecular epidemiology of rabies in Ukraine.

To investigate the circulation of rabies virus in Ukraine, 78 rabies virus isolates were acquired from 14 states in 2002 and 2008-2010 for characterization. Partial sequences of nucleoprotein (359 nt) and glycoprotein (344 nt) genes were compared with those from neighbouring countries. The analysis identified 39 unique nucleoprotein genes and two geographically distinct RV variants belonging to the cosmopolitan lineage. The Ukrainian samples were similar to the North-East European lineage (NEE) (n = 19) and Russian group C (n = 20). The group C viruses were mainly isolated in Eastern Ukraine, from 9 regions, and from two other regions in Western Ukraine, suggesting the presence of group C throughout the country. These group C viruses are intermixed in bordering regions along the Dnieper River with viruses of group NEE, which were mainly isolated in six regions in Western Ukraine. Both nucleoprotein and glycoprotein gene analyses suggested evidence for cross-border movements of rabies virus.

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.

Efficacy of a square presentation of V-RG vaccine baits in red fox, domestic dog and raccoon dog.

Oral vaccination programmes conducted in rabies infected countries from Eastern Europe and Eurasia should not be restricted to foxes but should target other major rabies vectors such as dogs and raccoon dogs as well. The objective of this experimental trial was to assess the protection induced by the vaccine by challenging these different species, which had been previously vaccinated intramuscularly with the square V-RG baits (produced in the US). Different parameters were evaluated such as attractiveness of the baits and induction of neutralising antibodies as an indicator for immunogenicity and protection after rabies challenge. The acceptability of the square bait was satisfactory in dogs, foxes and raccoon dogs, confirming previous laboratory and field studies conducted with the rectangular baits. Only one vaccinated dog out of nine seroconverted after vaccination and among them one dog died of rabies. Eight of ten vaccinated foxes seroconverted after vaccination and survived the rabies challenge. All vaccinated raccoon dogs seroconverted after challenge and all survived the challenge. These trials demonstrated that the square presentation of the V-RG vaccine was attractive, immunogenic and efficacious.

Risk of rabies introduction by non-commercial movement of pets.

In response to a Commission request, EFSA has carried out a quantitative assessment of the risk of rabies introduction into the UK, Ireland, Sweden, and Malta due to the movement of pets incubating rabies at the time of movement. The risk that a pet is incubating rabies at the time of first vaccination is equal to the prevalence of rabies-incubating pets in the population of origin. Following induction of protective immunity by vaccination, animals already incubating rabies will still develop clinical disease as a function of time after vaccination (termed type A risk). A waiting period will reduce this risk. Afew animals may not be protected after single-shot primary vaccination. Such animals may become infected during the waiting period after vaccination. The risk of becoming infected after the first vaccination (termed type B risk) depends on the prevalence and efficiency of vaccination. Serological testing can be used to identify non-immune pets (depending on test specificity) and will therefore reduce this risk accordingly. The type A and B risks were modelled as a function of the waiting period after vaccination and fitted to a non-linear model incorporating vaccination efficiency and test specificity. The model can be used to quantify the risk of moving pets from rabies infected areas and also to investigate the effect of different control measures. In quantitative terms, the type A risk constitutes by far the major risk. Therefore, a waiting period (defined as the time spent between vaccination and pet movement to the destined country) is the major effective measure to mitigate the risk of rabies introduction due to an animal being infected before primo-vaccination. Serological testing will only add significantly to risk reduction when waiting periods exceed 100 days. Within the EU, the rabies prevalence in most countries is so low that the risk can be considered negligible. However, for some countries the risk is non-negligible.

Can rabies be eradicated?

Rabies, an acute progressive encephalitis, is an ancient zoonosis. Its distribution encompasses all continents, except Antarctica. Agents consist of at least 11 species orgenotypes of rhabdoviruses, in the Genus Lyssavirus. Susceptible natural hosts include all mammals. Primary reservoirs reside in the Orders Carnivora and Chiroptera. A plethora of variants, maintained by a diversity of abundant hosts, presents a challenge to a strict concept of true eradication. Globally, the domestic dog remains the most significant species for viral transmission, responsible for millions of suspect human exposures and tens of thousands of fatalities. As such, this single major target provides an ideal opportunity for focused intervention programmes in humane disease prevention and control, driven by laboratory-based surveillance and guided via modern epidemiological insights. Historically, substantial technical progress throughout the 20th century led to the development of safe, affordable and efficacious animal and human vaccines, resulting in declining disease burdens in selected developed and developing countries. Regional and local disease resurgence occurs, due in part to a combination of political and economic instability, environmental perturbations, and shifting government priorities. Society must recall that despite the recent recognition of other important emerging infectious diseases, none exceed the case fatality rate of rabies. Given the clear relevance of rabies in public health, agriculture, and conservation biology, substantive international progress must continue towards enhanced public awareness, human rabies prevention, wildlife rabies control, and canine rabies elimination, with renewed collaborative vigour.

Epidemiology and pathogenicity of African bat lyssaviruses.

Lyssaviruses belonging to all four known African Lyssavirus genotypes (gts) have been reported and isolated from SouthAfrica over the past few decades. These are: (1) Duvenhage virus (gt4), isolated again in 2006 from a human fatality; (2) Mokola virus (gt3), isolated irregularly, mostly from cats; (3) Lagos bat virus (gt2) continually isolated over the past four years from Epomophorus fruit bats and from incidental terrestrial animals and (4) Rabies virus (gt1) - with two virus biotypes endemic in mongoose and in canid species (mostly domestic dogs, jackals and bat-eared foxes), respectively. Only two of these are associated with bats in Southern Africa, viz. Duvenhage virus and Lagos bat virus (gts 4 and 2). For both these genotypes the authors have embarked on a programme of comparative study of molecular epidemiology. Duvenhage virus nucleoprotein nucleotide sequence analysis indicated a very low nucleotide diversity even though isolates were isolated decades apart. In contrast, individual isolates of Lagos bat virus were found to differ significantly with respectto nucleoprotein gene nucleotide sequence diversity as well as in pathogenicity profiles.

Experimental infection of foxes with European bat lyssaviruses type-1 and -2.

Experimental studies have been undertaken to assess the susceptibility of silver foxes to bat variants of rabies virus, namely European Bat Lyssaviruses (EBLVs). Both EBLV-1 and EBLV-2 have been isolated in European bats since 1954, in Eptesicus serotinus and Myotis species, respectively. Since 2000, the number of reported cases has increased largely due to the improvement of the surveillance of bat rabies virus throughout Europe. Although over >800 EBLVs cases have been reported in bats in Europe, EBLV-1 and -2 viruses are rarely reported to infect humans and terrestrial animals. The study presented here shows that the sensitivity of silver foxes is low when infected with EBLVs via the intramuscular route; in contrast all animals infected via intracranial inoculation succumbed to the experimental challenge. The mortality rate was 100% for both EBLV-1 (approximately 4.5 log) and EBLV-2 (approximately 3.0 log). This data suggests that the susceptibility of foxes to EBLV-1 and EBLV-2 is low and that the transmission (spillover) and adaptation of EBLVs from a bat to a fox may be theoretically possible but unlikely.

New steps in the control of canine rabies in India.

In India, about 20,000 people die of rabies every year. The dog is the main reservoir and transmitter of the disease. A pilot rabies control programme was launched in five Indian federal states in February, 2007. This initiative is led by the Animal Welfare Board of India (AWBI) federating many animal welfare organizations and the Ministry of Agriculture. It aims at creating a "Rabies Free India." The programme combines parenteral vaccination of accessible owned and stray dogs, spaying/neutering followed by parenteral vaccination and oral vaccination of inaccessible dogs. The freeze-dried vaccine SAG2, including the bait casing, was registered in India following successful evaluation of vaccine-bait safety and efficacy (by survival after virulent challenge) in captive Indian stray dogs in the Bhopal High Security Animal Disease Laboratory. Furthermore, bait acceptance was tested under both experimental and field conditions.

Infection of foxes by Echinococcocus multilocularis in urban and suburban areas of Nancy, France: influence of feeding habits and environment.

This study evaluated the impact of biological and environmental factors on the infection of red foxes (Vulpes vulpes) by Echinococcus multilocularis in an endemic area of north-east France. From January 2004 to April 2006, 127 foxes were examined for E. multilocularis and their stomach contents analysed. The effect of year, season, age, sex and urbanisation level on E. multilocularis presence was estimated using a General Linear Model (GLM) with logit link, (i.e. logistic regression). Urbanisation level was the only influencing factor, with a decreasing gradient from rural [54%, CI 95% (40-68)] to peri-urban [31%, CI 95% (15-52)] and urban area [4%, CI 95% (0.7-15)]. The consumption of Arvicola terrestris and Microtus sp., grassland species, the main presumed intermediate hosts of E. multilocularis, was studied by the same approach. The two species were consumed less in the urban area and more in autumn than in spring. Anthropogenic food consumption was linked to urbanisation and to age. The frequency of anthropogenic food consumption decreased in the rural area. A global model explaining the presence of E. multilocularis and including urbanisation level and diet was then elaborated. Independently of urbanisation, there was a suggestion of less E. multilocularis infection with anthropogenic food consumption. Red foxes consuming Microtus sp. and A. terrestris had higher worm burden than those that did not. The results suggest that the decreasing gradient observed from rural to urban area is linked to behaviour and feeding habits.

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.

Use of filter paper (FTA) technology for sampling, recovery and molecular characterisation of rabies viruses.

This study evaluates the feasibility of the use of the FTA Gene Guard System (a commercial product consisting of filter paper impregnated with patented chemicals supplied by the Whatman company) for the shipment, storage and detection of RNA rabies viruses by a simplified hemi-nested reverse transcriptase polymerase chain reaction. HnRT-PCR of the rabies virus nucleoprotein gene with specific primers showed that viral RNA extracted from crude infected tissues remained stable after fixation on the filter paper under diverse environmental conditions for at least 35 days. The sequence analysis of the products amplified from five out of the seven known genotypes of Lyssaviruses showed the stability of viral RNA viruses after fixation on the filter paper. Furthermore, the sensitivity of the hnRT-PCR following RNA fixation on the filter paper was equivalent to that of standard hnRT-PCR. In conclusion, the stability of viral RNA and the inactivation of infectivity make the FTA technology useful for the storage, transport, collection and subsequent molecular analysis of viral rabies RNA, facilitating epidemiological investigations in the field.

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.

Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores.

To prevent any introduction of rabies, many rabies-free countries have adopted a scheme requiring the rabies vaccination of pets associated with a serological test. FAVN test and RFFIT are the current OIE prescribed techniques to perform this assay. A qualitative indirect ELISA (Serelisa) test has been recently described as a screening test to monitor the effectiveness of rabies vaccination of pets. A lack of sensitivity requires ELISA negative samples to be retested using an OIE confirmatory test. This raised the question whether this new test could be reasonably proposed as an alternative tool in the context of international trades of pets. The Community Reference Institute of Nancy organized a short trial to answer this question. In this study, 16 laboratories tested a panel of their own samples with FAVN test/RFFIT and the Serelisa. The comparison of results revealed that the performance of the Serelisa is highly heterogeneous. A lack of sensitivity was detected in 50% of participants, when 25% of laboratories obtained a significant rate of false positive results. This last point questions the pertinence of using the Serelisa in the context of international trades by preventing any movements of insufficiently or non-protected animals.

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.

Means used for terrestrial rabies elimination in France and policy for rabies surveillance in case of re-emergence.

In France, the first case of wildlife rabies was detected in 1968, with the red fox (Vulpes vulpes) as reservoir and vector of terrestrial rabies. The last case was reported at the end of 1998. The maximum infected area amounted to 140,000 km2 in 1989 with a record number of 4,213 infected wild and domestic animals. The contaminated areas included various landscapes such as low populated farming areas and low mountainous areas as well as very densely populated areas and industrial areas contiguously urbanised. Oral vaccination of wildlife against rabies was implemented in 1986 and 1987 on limited areas treated by hand distribution and then helicopter distribution was implemented in 1988. The control of wildlife rabies was centralised at the country level in one institute for elaboration of baiting strategies, rabies surveillance network, laboratory investigations such as tetracycline and serological testing, vaccine titration and rabies diagnosis. Oral vaccination campaigns were organised in spring and in autumn by dropping annually 40 baits/km2. The following vaccine baits were used: SAD B19 from 1986 to 1992, SAG1/SAG2 from 1990 to 2003 and VRG from 1989 to 2003. A cost analysis study of the system of oral vaccination implemented in France demonstrated that it is beneficial compared to the traditional expenses of rabies control. Animal rabies prophylaxis has been progressively adapted to the disease free status of the country; the surveillance network for rabies is still working and an emergency procedure is in place in the event of a re-emergence of the disease in the context of high density fox populations.

Rabies diagnosis.

A reliable diagnosis of rabies can only be made based on laboratory examination of specimens collected from suspected animals. Without data from diagnostic tests, it is not possible to establish rabies control policies. These data are also necessary for evaluating the impact of control measures on the disease and to adapt veterinary and human health policies to the rabies situation. This paper presents the routine laboratory techniques for rabies diagnosis that are published and recommended by both the OIE and WHO and which form the basis of rabies surveillance. They are presented along with more recent techniques which have proved useful in epidemiological and phylogenetic studies. The first step in controlling a disease is to be able to identify it consistently. A reliable and sensitive diagnostic test is therefore essential for use in epidemiological studies and disease control. Reliable diagnosis is also needed by official medical and veterinary services to decide how to deal with infected humans or animals. Without knowing where rabies is present in a country, it is difficult to establish a solid, long-term control policy. A viral disease may be diagnosed on the basis of clinical signs or after laboratory examination. Clinical diagnosis is based on the observation of symptoms and on observation of specific or at least highly evocative signs. Laboratory tests reveal the presence of infection using either indirect or direct methods. The most frequently used indirect methods are based on serological testing. Direct methods aim to detect the virus itself, its proteins, genetic material or traces of its replication in tissues.

First oral vaccination of wildlife against rabies in Estonia.

Estonia is a small country, with many islands, that covers a total area of 45,000 km2 in the Baltic region. Rabies is widely distributed all over the country, even on some islands. The disease is maintained by two species: red foxes and raccoon dogs. In 2003, 813 cases of rabies were recorded with 315 cases in foxes and 362 cases in raccoon dogs; since 2002 the latter species has had the highest level of rabies infection among all animal species in Estonia. The measure used to control rabies is vaccination of dogs and cats, as they are the source on infection of humans. In Spring 2004, a limited trial of oral vaccination of wildlife was conducted. The goal of this was to train the actors who will be implicated in larger future campaigns. This trial was conducted on Vormsi island (92 km2), located 10 km away from the west coast. Oral vaccination was performed manually over three days with SAG2 baits (Virbac company, France), by three teams.