First isolation of a rabid bat infected with European bat lyssavirus in Luxembourg.

Rabid bats are regularly reported in Europe, especially in countries that have implemented a bat surveillance network. In May 2013, bat rabies was evidenced for the first time in Luxembourg (southern city of Differdange). The rabies virus, an EBLV-1b strain, was diagnosed in a serotine bat that bit a 29-year-old male person while he was asleep. The man received rapidly a post-exposure RABV treatment and was put under strict medical supervision.

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.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

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.

International interlaboratory trials on rabies diagnosis: an overview of results and variation in reference diagnosis techniques (fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques.

Interlaboratory trials on rabies diagnosis were organised in 2009 and in 2010 by the European Union Reference Laboratory (EURL) for rabies. In 2009, two panels of virus samples were sent to participating laboratories to compare results on reference diagnosis techniques and on RT-PCR. A single panel was sent in 2010 to test FAT (fluorescent antibody test), RTCIT (rabies tissue culture infection test) and RT-PCR techniques. The virus panels included the RABV, EBLV-1, EBLV-2 and ABLV strains. Results revealed that laboratories produced the highest proportion of concordant results using RT-PCR (90.5%) and FAT (87.1%), followed by RTCIT (70.0%) and MIT (35.0%) in 2009 and in FAT (85.0%) and RT-PCR (80.6%) followed by RTCIT (77.3%) in 2010. Errors were only observed in bat strains (i.e. none in the RABV strain) for the RT-PCR or FAT techniques, highlighting the need to improve diagnosis most specifically in such strains. RT-PCR was the technique showing the lowest rate of false negative results in either trial year, while RTCIT and MIT (performed in 2009 only) were the techniques with the lowest proportion of false positive results. Nevertheless, the FAT technique represented a good compromise with both satisfactory sensitivity and specificity, as only a few false positive (1.6% in 2009, 5.8% in 2010) and false negative results (1.6% in both 2009 and 2010) were detected. The analysis of technical questionnaires describing the protocols used by participating laboratories revealed variation in the methods used that may induce inconsistencies in the results. In this study, the number of readers for FAT slide examination was identified as a factor affecting significantly the results of laboratories, suggesting that two independent readers are necessary for routine rabies diagnosis. Our findings highlight the need for all rabies diagnostic laboratories to improve harmonisation of procedures.

Fox defecation behaviour in relation to spatial distribution of voles in an urbanised area: An increasing risk of transmission of Echinococcus multilocularis?

Urbanisation of alveolar echinococcosis is a new phenomenon that has been highlighted during the last few decades. It has thus become necessary to understand the dynamics of transmission of Echinococcus multilocularis in urbanised areas. Spatial heterogeneity of infection by E. multilocularis has been explained as the result of a multifactorial dependence of the transmission in which the factors depend on the scale of the investigation. The aim of this study was to assess, in an urbanised area, the effect of such environmental factors as season, habitat type and the level of urbanisation, on the availability of two major intermediate hosts (Microtus spp. and Arvicola terrestris), the distribution of red fox faeces and the distribution of E. multilocularis as determined by detection of coproantigens in faeces. Results of the study revealed higher densities of Microtus spp. in rural than in peri-urban areas. Moreover this species was highly aggregated in urban wasteland. Arvicola terrestris densities did not appear to be linked to the level of urbanisation or to the type of habitat studied. Distribution of faeces was positively linked to distance walked and to Microtus spp. and A. terrestris distributions whatever the level of urbanisation. Such a distribution pattern could enhance the transmission cycle in urban areas. The Copro-ELISA test results on faeces collected in the field revealed that ODs were significantly negatively correlated with the abundance of A. terrestris. The larger population densities of Microtus spp. found in urban wastelands and the well known predominance of Microtus spp. in the red fox diet in the region suggest that Microtus spp. may play a key role in urban transmission of the parasite in the study area.

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.