Evelyne Picard-Meyer

Eliminating Rabies in Estonia

The compulsory vaccination of pets, the recommended vaccination of farm animals in grazing areas and the extermination of stray animals did not succeed in eliminating rabies in Estonia because the virus was maintained in two main wildlife reservoirs, foxes and raccoon dogs. These two species became a priority target therefore in order to control rabies. Supported by the European Community, successive oral vaccination (OV) campaigns were conducted twice a year using Rabigen® SAG2 baits, beginning in autumn 2005 in North Estonia. They were then extended to the whole territory from spring 2006. Following the vaccination campaigns, the incidence of rabies cases dramatically decreased, with 266 cases in 2005, 114 in 2006, four in 2007 and three in 2008. Since March 2008, no rabies cases have been detected in Estonia other than three cases reported in summer 2009 and one case in January 2011, all in areas close to the South-Eastern border with Russia. The bait uptake was satisfactory, with tetracycline positivity rates ranging from 85% to 93% in foxes and from 82% to 88% in raccoon dogs. Immunisation rates evaluated by ELISA ranged from 34% to 55% in foxes and from 38% to 55% in raccoon dogs. The rabies situation in Estonia was compared to that of the other two Baltic States, Latvia and Lithuania. Despite regular OV campaigns conducted throughout their territory since 2006, and an improvement in the epidemiological situation, rabies has still not been eradicated in these countries. An analysis of the number of baits distributed and the funding allocated by the European Commission showed that the strategy for rabies control is more cost-effective in Estonia than in Latvia and Lithuania.

Isolation of Bokeloh bat lyssavirus in Myotis nattereri in France

Bokeloh bat lyssavirus (BBLV) was found in Myotis nattereri for the first time in northeastern France in July 2012. The complete genome sequence of the virus from the infected Natterer’s bat was determined by whole-genome sequencing and compared to that of the first BBLV strain isolated in 2010 in Germany and with those of all currently identified lyssaviruses. The French isolate [KC169985] showed 98.7xa0% nucleotide sequence identity to the German BBLV strain [JF311903]. Several organs of the infected French bat were examined by classical rabies diagnostic methods: fluorescent antibody test, cell culture inoculation test and RT-qPCR. Antigen, infectious virus and high viral RNA levels were found in both the brain and salivary glands. Traces of genomic RNA were detected in the bladder, kidney and lung tissue. The results of an investigation of the distribution of lyssaviruses with the detection of infectious virus in the salivary glands suggest a possible mode of transmission of the virus.

Experimental infection of Foxes with European bat Lyssaviruses type-1 and 2

BackgroundSince 1954, there have been in excess of 800 cases of rabies as a result of European Bat Lyssaviruses types 1 and 2 (EBLV-1, EBLV-2) infection, mainly in Serotine and Myotis bats respectively. These viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in Denmark, a stone marten in Germany and a cat in France. The purpose of this study was to investigate the susceptibility of foxes to EBLVs using silver foxes (Vulpes vulpes) as a model.ResultsOur experimental studies have shown that the susceptibility of foxes to EBLVs is low by the intramuscular (IM) route, however, animals were sensitive to intracranial (IC) inoculation. Mortality was 100% for both EBLV-1 (~4.5 logs) and EBLV-2 (~3.0 logs) delivered by the IC route. Virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody (VNA) was limited (log(ED50) = 0.24–2.23 and 0.95–2.39 respectively for specific EBLV-1 and EBLV-2). Foxes were also susceptible, at a low level, to peripheral (IM) infection (~3.0 logs) with EBLV-1 but not EBLV-2. Three out of 21 (14.3%) foxes developed clinical signs between 14 and 24 days post-EBLV-1 infection. None of the animals given EBLV-2 developed clinical disease.ConclusionThese data suggest that the chance of a EBLV spill-over from bat to fox is low, but with a greater probability for EBLV-1 than for EBLV-2 and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection.

Vaccine-induced Rabies in a Red Fox (Vulpes vulpes): Isolation of Vaccine Virus in Brain Tissue and Salivary Glands

Abstract Oral vaccination campaigns to eliminate fox rabies were initiated in Slovenia in 1995. In May 2012, a young fox (Vulpes vulpes) with typical rabies signs was captured. Its brain and salivary gland tissues were found to contain vaccine strain SAD B19. The Basic Logical Alignment Search Tool alignment of 589 nucleotides determined from the N gene of the virus isolated from the brain and salivary glands of the affected fox was 100% identical to the GenBank reference SAD B19 strain. Sequence analysis of the N and M genes (4,351 nucleotides) showed two nucleotide modifications at position 1335 (N gene) and 3114 (M gene) in the KC522613 isolate identified in the fox compared to SAD B19.

Recurrence of Animal Rabies, Greece, 2012

To the Editor: Rabies is caused by 12 recognized virus species within the Lyssavirus genus (family Rhabdoviridae) (1) and each year causes 55,000 deaths worldwide among humans. In Europe, the main reservoir and vector of rabies is the red fox (Vulpes vulpes), followed by the raccoon dog (Nyctereutes procyonoides) in central and Baltic Europe (2). Among these virus species, the rabies virus (former genotype 1, classical rabies virus) is maintained in reservoir mammals, mainly carnivores. Despite the successful oral vaccination campaigns of wildlife that eliminated rabies in large parts of Europe (2), the disease still occurs in Europe; 4,884 cases in animals were recorded in 2012. n nUntil 2012, Greece had been free of rabies since 1987; the last case occurred in a dog. During 1971–1987, a total of 248 cases were recorded in domestic animals, of which only 6 occurred during 1981–1987 (3). The wide compulsory vaccination of dogs, along with control of stray dogs, were the main measures leading to the elimination of rabies in Greece. n nGreece shares land borders with Turkey, Former Yugoslav Republic of Macedonia (FYROM), and Bulgaria, where rabies is reported in wild and domestic animals (4; www.who-rabies-bulletin.org/). After the fox rabies case reported in FYROM in 2011, ≈0.3 km from Greece (4), rabies surveillance was improved along the northern and eastern land borders of Greece, and wild and domestic animals found dead or suspected of having rabies were collected. This program was approved by the European Commission. The first rabies case was diagnosed on October 19, 2012, in a red fox in Palaiokastro, 60 km from the Albanian border. The animal was wandering in the village during daytime and attacked a dog before being killed by local hunters and sent to the National Reference Laboratory for Animal Rabies (Athens, Greece) for rabies testing. The second case, isolated in Ieropigi, 4 km from the Albanian border, was a shepherd dog demonstrating aggressive behavior against dogs and sheep of the flock. Seven additional cases (6 foxes, 1 dog) were reported in December 2012 in 2 prefectures of northern Greece that share borders with FYROM (5). n nIn 2012, a total of 237 domestic and wild animals were submitted to the National Reference Laboratory for Animal Rabies for rabies testing. Samples were tested by the fluorescent antibody test, rabies tissue culture infection test, real-time reverse transcription PCR, and heminested reverse transcription PCR, as described (6,7). In brief, viral RNA was extracted (Viral RNA Mini Kit, QIAGEN, Hilden, Germany) from 140 μL of homogenized brain suspension supernatant and subjected to the partial nucleoprotein (N) gene amplification (positions 71–644 compared with PV strain genome). The PCR products were bidirectionally sequenced by using the same primers in a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The phylogenetic tree was constructed by using the neighbor-joining method with 1,000 replicates using MEGA5 (8). n nOf 237 animals tested, 9 (7 foxes, 2 dogs) were rabies positive by fluorescent antibody test, rabies tissue culture infection test, and PCR. Positive samples were subjected to sequencing analysis of the N gene. The sequence analysis of the first 567 nt of the N gene of the 3 isolates (GR64C/12, GR112C/12, GR238C/12) showed 99.8% nt identity with the N gene of a rabies virus (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JQ973884,term_id:409191659,term_text:JQ973884}}JQ973884) isolated from a red fox in FYROM in 2011. Nucleotide identity was 100% between the 6 isolates from Greece (GR177C/12, GR187C/12, GR217C/12, GR231C/12, GR236C/12, GR242C/12) and isolate {type:entrez-nucleotide,attrs:{text:JQ973884,term_id:409191659,term_text:JQ973884}}JQ973884, as well as a Serbia strain (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JF973785,term_id:337752481,term_text:JF973785}}JF973785). This perfect nucleotide identity shown with the Serbia isolate collected >15 years ago suggests the persistence of some viral strains over time in the Balkans, in accordance with previous studies (4,9). Amino acid identity was perfect among all 9 isolates from Greece. n nPhylogenetic analysis of the partial sequences of the N gene of isolates from Greece compared with representative sequences from the Balkans (Figure) showed that isolates from Greece resolved within the East Europe (EE) group of the cosmopolitan lineage. The EE group encompasses the 9 Greek isolates with referenced viral sequences from FYROM, Bulgaria, Serbia, Bosnia Herzegovina, and Montenegro. Within the EE group, <1.5%-nt divergence exists between all analyzed N gene sequences (567 nt). n n n nFigure n nNeighbor-joining phylogenetic tree comparing 9 isolates (7 red foxes, 2 dogs) from Greece with isolates from Former Yugoslav Republic of Macedonia, Bulgaria, and Serbia. All 9 samples were isolated in Greece during October 19, 2012–December 28, ... n n n nThe perfect nucleotide identity shown between the 6 isolates from Greece and 2 strains from Serbia and FYROM demonstrates a close genetic relationship between them. This finding supports the hypothesis of movement of rabies-infected hosts in the western Balkan countries. n nGreece was rabies free for 25 years. In the 2012 outbreak, the rabies cycle appears to be sylvatic, whereas until 1987, dogs were the main reservoir of rabies. Measures including public awareness campaigns and intensified vaccination of stray animals and shepherd dogs, combined with control of stray dogs and cats, already have been implemented. In accordance with the current European Commission recommendations, Greece continued rabies surveillance and eradication program throughout the country in 2013, including the implementation of an oral rabies vaccination program for foxes in autumn 2013 (10).

Potentially zoonotic bartonella in bats from France and Spain

We detected Bartonella in 11 of 109 insectivorous bats from France and 1 of 26 bats from Spain. These genetic variants are closely related to bat-associated Bartonella described in Finland and the United Kingdom and to B. mayotimonensis, the agent of a human endocarditis case in the United States.

Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R 2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981u2009copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22u2009copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

Vaccine-induced rabies case in a cow (Bos taurus): Molecular characterisation of vaccine strain in brain tissue.

Rabies is a fatal neuropathogenic zoonosis caused by the rabies virus of the Lyssavirus genus, Rhabdoviridae family. The oral vaccination of foxes - the main reservoir of rabies in Europe - using a live attenuated rabies virus vaccine was successfully conducted in many Western European countries. In July 2015, a rabies vaccine strain was isolated from the brain tissues of a clinically suspect cow (Bos taurus) in Romania. The nucleotide analysis of both N and G gene sequences showed 100% identity between the rabid animal, the GenBank reference SAD B19 strain and five rabies vaccine batches used for the national oral vaccination campaign targeting foxes.

Rabies in the Baltic States: Decoding a Process of Control and Elimination

Rabies is a fatal zoonosis that still causes nearly 70, 000 human deaths every year. In Europe, the oral rabies vaccination (ORV) of red foxes (Vulpes vulpes) was developed in the late 1970s and has demonstrated its effectiveness in the eradication of the disease in Western and some Central European countries. Following the accession of the three Baltic countries—Estonia, Latvia and Lithuania—to the European Union in 2004, subsequent financial support has allowed the implementation of regular ORV campaigns since 2005–2006. This paper reviews ten years of surveillance efforts and ORV campaigns in these countries resulting in the near eradication of the disease. The various factors that may have influenced the results of vaccination monitoring were assessed using generalized linear models (GLMs) on bait uptake and on herd immunity. As shown in previous studies, juveniles had lower bait uptake level than adults. For the first time, raccoon dogs (Nyctereutes procyonoides) were shown to have significantly lower bait uptake proportion compared with red foxes. This result suggests potentially altered ORV effectiveness in this invasive species compared to the red foxes. An extensive phylogenetic analysis demonstrated that the North-East European (NEE) rabies phylogroup is endemic in all three Baltic countries. Although successive oral vaccination campaigns have substantially reduced the number of detected rabies cases, sporadic detection of the C lineage (European part of Russian phylogroup) underlines the risk of reintroduction via westward spread from bordering countries. Vaccine induced cases were also reported for the first time in non-target species (Martes martes and Meles meles).

Molecular characterisation of rabies virus strains in the Republic of Macedonia.

Rabies, a worldwide zoonosis, remains a public-health concern despite oral wildlife vaccination in Europe. After a ten-year break, Macedonia reported eight rabies cases in 2011-2012. Two countries (Serbia and Bulgaria) bordering Macedonia are reporting cases in domestic and wild animals. This report describes the genetic characterisation of eight isolates from Macedonia compared with representative samples from neighbouring countries. All of the isolates tested belong to the Eastern European group, with a high degree of nucleotide sequence identity in the nucleoprotein gene. The close genetic relationship between isolates from the three bordering countries suggests that wildlife is responsible for rabies movements in the region.