Seroprevalence and incidence of Puumala orthohantavirus in its bank vole (Myodes glareolus) host population in northeastern France: Between-site and seasonal variability.

Given the difficulty of measuring pathogen transmission in wildlife, epidemiological studies frequently rely on cross-sectional seroprevalence. However, seropositivity indicates only exposure to a pathogen at an unknown time. By allowing to obtain repeated test results from individuals sampled multiple times over an extended period, longitudinal data help reduce this uncertainty. We used capture-mark-recapture data on bank vole (Myodes glareolus) individuals collected at four sites over ten years in northeastern France to investigate the impact of environmental variables on seroprevalence and incidence of Puumala orthohantavirus (PUUV). PUUV causes a chronic infection without apparent symptoms, that may however impair survival of its rodent host in the wild. Viral transmission between rodents may occur through direct contact or via the environment. Principal component analysis was used to deal with multicollinearity among environmental variables. Incidence and seroprevalence were investigated with either generalized estimating equations or Poisson regression models depending on the number of observations for each season. In spring, only the factor site was found to be significant for seroprevalence, while a principal component including meteorological conditions of the previous winter and the normalized difference vegetation index (NDVI) of both the previous winter and spring had a significant effect on incidence. In autumn, only the factor site was significant for incidence, while two principal components, including either the meteorological conditions of the autumn and previous spring or NDVI of the autumn significantly affected seroprevalence. We discuss these results in light of the particular demography of small mammals. We encourage other researchers to investigate the relationships between demographic parameters of wild host populations and the environment, by using both incidence and seroprevalence.

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

Taxonomic assessment of Culicoides brunnicans, C. santonicus and C. vexans (Diptera: Ceratopogonidae) in France: Implications in systematics.

Culicoides brunnicans Edwards, 1939, Culicoides santonicus, Callot et al., 1966, and Culicoides vexans (Staeger, 1839) belong to the Vexans group of the subgenus Oecacta. These species had never been studied by molecular methods and their distribution in Western Europe overlapped. C. brunnicans and C. santonicus are two closely related species and their diagnoses are based on the wing pattern only. An integrative taxonomic approach was conducted on females of the Vexans group, Culicoides furens (the type species of the subgenus Oecacta), and Culicoides nubeculosus (as outgroup) using sequences of D1D2 rDNA domains, sequences of COI mtDNA, and wing morphometrics. Species of the Vexans group were discriminated by all traits, and were closely related in the phylogeny. Their taxonomic status and their relation to C. furens were re-evaluated.

Biting midges monitoring (Diptera: Ceratopogonidae: Culicoides Latreille) in the governate of Monastir (Tunisia): species composition and molecular investigations.

The results of entomological studies carried out in the governate of Monastir (Tunisia) in 2009-2010 (captures and emergences from muds) focusing on Culicoides species are presented in the present study. Identification of Culicoides at the species level is based on morphological characters, and a molecular study has been carried out based on mitochondrial DNA cytochrome C oxidase I gene (COI) and D1 and D2 domains of the 28S rDNA. The DNA sequences reported here are related to 10 species (on 25 known) of Culicoides described in Tunisia: Culicoides cataneii-gejgelensis, Culicoides circumscriptus, Culicoides imicola, Culicoides jumineri, Culicoides kingi, Culicoides langeroni, Culicoides newsteadi, Culicoides paolae, Culicoides puncticollis and Culicoides sahariensis. DNA sequencing of the COI gene and D1D2 domains discriminated all morphologically determined species. The choice of D1D2 domains considered as a conserved region is informative for Culicoides species identification. The molecular analyses of COI has grouped both C. circumscriptus, C. puncticollis within two clusters and C. newsteadi within five subclusters. However, C. newsteadi shows relatively deep intraspecific divergence using COI sequences.

Wing geometry as a tool for discrimination of Obsoletus group (Diptera: Ceratopogonidae: Culicoides) in France.

In Europe, Culicoides chiopterus, Culicoides dewulfi, Culicoides obsoletus and Culicoides scoticus, which belongs to the subgenus Avaritia and Obsoletus group are the most proficient Bluetongue and Schmallenberg vectors. Within this group, correct identification based on morphological traits is difficult but essential to assess disease transmission risk. The development of new tools has revolutionized taxonomy (i.e. geometric morphometrics and molecular biology). Wing morphology is of primary importance to entomologists interested in systematics. Here, we report phenotypic differentiation patterns among the species above mentioned using a landmark-based geometric morphometric approach that efficiently identified C. chiopterus and C. dewulfi. Wing shape of the C. scoticus sample exhibited large specific variability. Based on landmarks and phylogenetic analyses (Maximum Parsimony), we suggest that Obsoletus group in Europe includes only C. obsoletus and C. scoticus. C. dewulfi and C. chiopterus are clearly excluded. Their shape seems closer to C. obsoletus that is why we suggest that only these two species should be grouped in the Obsoletus group. In addition, the concordance between phenetic clusters and phylogenies inferred from molecular data based on a fragment of the mtDNA COI gene and rDNA 28S suggests the existence of a strong signal in wing shape. These findings encourage us to use this powerful tool in taxonomic studies.

Molecular and morphological systematics of the sandfly Sergentomyia (Sintonius) clydei Sinton, 1928 and questions about its record in the Seychelles.

In the Phlebotomine sandflies, a few molecular studies related on the genus Sergentomyia have been published. The present study explored the genetic variability within Sergentomyia (Sintonius) clydei (Diptera, Psychodidae). The sampling included 15 populations originating from 12 countries. A morphological approach was coupled to the sequencing of two molecular markers (cytochrome b mtDNA and cacophony nuclear DNA). The most variable morphological characters resided in the cibarium of the females, especially (i) the pigment patch pattern and (ii) the number of cibarial teeth and denticles in the armature. However this morphological approach was unable to individualize any population within S. clydei. The NJ trees based on both molecular markers individualized the specimens from the Aldabra group of islands in the Seychelles. Surprisingly, cyt b variability was not compatible with the known data about the complete submersion of Aldabra occurring relatively recently some 125,000 years ago. The settlement of these islands by S. clydei from continental Africa, the Middle East or Asia, and the value of mtDNA markers are discussed.

Record of two species of Culicoides (Diptera, Ceratopogonidae) new for Madagascar and molecular study showing the paraphylies of the subgenus Oecacta and the Schultzei group.

Culicoides are vectors of diseases of Veterinary Medicine importance (bluetongue, African horse sickness, Schmallenberg virus) all over the world. In the present study, we report two species new for Madagascar: C. nevilli and C. enderleini. They belong to the Schultzei group which is sometimes classified in the subgenus Oecacta and sometimes in the subgenus Remmia, depending on authors. Consequently, we carried out a molecular cladistics of these groups based on cytochrome C oxidase subunit I mtDNA sequences. We processed the Malagasy specimens and some C. furens (the Oecacta type-species) caught in Florida and we analyzed their sequences and those available in Genbank: C. schultzei, C. oxystoma, C. festivipennis, C. brunnicans, C. kibunensis, C. truncorum and C. vexans. C. (Avaritia) imicola have been selected as an outgroup. The maximum parsimony analysis showed the paraphylies of the Schultzei group (=Remmia) and of the subgenus Oecacta if the first group is excluded from the latter. Our results underline the doubtful current classification and need to be validated by other molecular markers in the future.

Morphometric and molecular characterization of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) from the Ecuadorian Amazon Basin with description of a new species.

Several populations of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) were collected from the Yasuní National Park in the Ecuadorian Amazon region. The specimens comprised the species Psychodopygus geniculatus, Psychodopygus luisleoni and Psychodopygus corossoniensis. Within Ps. geniculatus, we observed two populations, one with a narrow paramere and relatively short genital filaments and the other characterized by a wider coxite and longer genital filaments. A multiple approach combining morphology, morphometry and DNA sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and the mitochondrial cytochrome b gene was carried out. Morphological, morphometric and molecular data strongly suggested the presence of two populations within Ps. geniculatus. The lack of intermediate forms within these populations supported the proposal of two sympatric species. This report describes Psychodopygus francoisleponti n. sp.

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.

Discrimination of Culicoides obsoletus and Culicoides scoticus, potential bluetongue vectors, by morphometrical and mitochondrial cytochrome oxidase subunit I analysis.

Biting midges of the Culicoides obsoletus Meigen species complex (Diptera: Ceratopogonidae) are increasingly suspected as vectors of the recent emergence of bluetongue virus in Europe. Within this complex, identification of the C. obsoletus and Culicoides scoticus females is considered as difficult or sometimes not possible while the identification of males is easy, based on genitalia observation. Nolan et al. (2007) concluded that the distinction of C. obsoletus and C. scoticus females is not possible according to morphology but require molecular analyses. In 2010, the identification of biting midges is done under a stereomicroscope without specific identification within the C. obsoletus species complex. However, such a specific identification distinguishing C. obsoletus s. str. and C. scoticus s. str. is crucial to identify the European competent vectors of the virus, their relative abundances and then accurately assess the risk. We performed morphometric analyses of head, genitalia and thorax of females combined with sequencing of the cytochrome oxidase I barcode fragment of mitochondrial DNA on 88 specimens in order to have a molecular identification of our sampled species. As we knew the actual species of individuals thanks to molecular results, we explored the discriminant power of 15 morphometric variables to distinguish the females according to their species. Multivariate analyses were performed on the morphometric measurements to identify and validate a combination of variables leading to an accurate species identification. It appears that females of C. obsoletus and C. scoticus can be accurately distinguished based on only four variables: width between chitinous plates, length and width of spermathecae1 and length of spermatheca2. This approach should improve the accuracy of morphologically-based species identification.

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.

Spatial and temporal patterning of bank vole demography and the epidemiology of the Puumala hantavirus in northeastern France.

Epidemiological data from bank voles, Myodes glareolus, naturally infected by the hantavirus Puumala (PUUV) were collected by a capture-mark-recapture protocol from 2000 to 2002 in the French department of Ardennes. Four monitored trapping sites were established in two forests located in two cantons (Flize and Monthermé). We captured 912 bank voles corresponding to 557 different individuals during 8820 trapping nights for an overall trapping success of 10.34%. The average PUUV seroprevalence was 22.4%. Characteristics of the system reported in North European countries are confirmed in France. PUUV seroprevalence and abundance of rodents appeared weakly linked. Adult voles were more frequently antibody-positive, but no difference between sexes was established. Anti-PUUV seropositive voles were captured and high seroprevalence was observed from both forests, without human infection reported in Flize canton during the study. One site among the four exhibited peculiar infection dynamics, where vole weight and infection risk were negatively correlated.

[Molecular and morphological characterisation of two closely related species belonging to the subgenus Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 et P. riouxi Depaquit, Killick-Kendrick & Léger, 1998 (Diptera: Psychodidae)]. / Caractérisation moléculaire et morphologique de deux espèces affines de Paraphlebotomus: Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 et P. riouxi Depaquit, Killick-Kendrick & Léger, 1998 (Diptera: Psychodidae).

In 1998, the description of Phlebotomus riouxi emphasised the difficulty to differentiate the female from the closely related P. chabaudi, a suspected vector of Leishmania killicki in several foci in Tunisia. In order to be able to distinguish the females of these two species, we started a molecular study based on 37 Algerian and Tunisian specimens. The alignment of the sequences of the cytochrome b of the mitochondrial DNA and their analysis using Neighbor-Joining, maximum likelihood and maximum parsimony shows the individualisation of two species including an intraspecific variability. Following a morphological approach, it is not possible to distinguish the females on the basis of their spermathecae. A new character is proposed: the presence of anterolateral teeth of the pharyngeal armature for P. chabaudi, never observed in P. riouxi. However, a molecular typing is necessary at the present time for a sure identification of the females.

Dynamics of Puumala virus infection in bank voles in Ardennes department (France).

The hantaviruses (genus Hantavirus, family Bunyaviridae) include human pathogens and occur worldwide. In Western and Central Europe, the predominant serotype is Puumala (PUU) virus, which causes epidemic nephropathy. Voles are considered to be the main reservoir and the vector of PUU virus. A total of 719 rodents (mainly Clethrionomys glareolus, Apodemus sp.) trapped by capture-mark-recapture (CMR) in four sites in Ardennes department (France) between April 2004 and October 2005 were tested for the presence of PUU virus antibodies by enzyme-linked immunosorbent assay (ELISA). The predominant species, C. glareolus (86.5% [622 of 719]), also had the highest antibody prevalence (37.6% [291 of 773]). In C. glareolus, the antibody prevalence rate increased with age (weight) in site A, B and D, reaching more than 50% in the heaviest weight, and suggesting that horizontal infection may be important.

Detection of Echinococcus multilocularis in wild boars in France using PCR techniques against larval form.

Recently, new data have been collected on the distribution and ecology of Echinococcus multilocularis in European countries. Different ungulates species such as pig, goat, sheep, cattle and horse are known to host incomplete development of larval E. multilocularis. We report a case of E. multilocularis portage in two wild boars from a high endemic area in France (Department of Jura). Histological examination was performed and the DNA was isolated from hepatic lesions then amplified by using three PCR methods in two distinct institutes. Molecular characterisation of PCR products revealed 99% nucleotide sequence homology with the specific sequence of the U1 sn RNA gene of E. multilocularis, 99 and 99.9% nucleotide sequence homology with the specific sequence of the cytochrome oxydase gene of Echinococcus genus and 99.9% nucleotide sequence homology with a genomic DNA sequence of Echinococcus genus for the first and the second wild boar, respectively.

Cercarial productivity of Fasciola hepatica in Lymnaea truncatula during usual and unusual development of redial generations.

Single-miracidium infections of Lymnaea truncatula by Fasciola hepatica were carried out to determine the cercarial productivity of each redial generation when the first-appearing mother redia (R1a redia) normally developed (usual development of redial generations) or quickly died (unusual development). In both developmental patterns of infection, the number of morulae was significantly higher in the second-appearing mother rediae (R1b group) than in either the daughter rediae from R1a rediae (R2a group) or the rediae from subsequent groups (R2b/R3a). The live R1a group did not produce cercariae. The percentages of cercariae produced by the R1b, R2a, and R2b/R3a groups were 28.2%, 62.8%, and 8.9% when the development of redial generations was usual. In contrast, in an unusual development, they were 69.3%, 20.3%, and 10.2%, respectively. Cercarial production until day 63 post-exposure was essentially provided by the R2a rediae, when the development of redial generations was usual, and by the R1b rediae when it was unusual.

Fasciola hepatica: an unusual development of redial generations in an isolate of Lymnaea truncatula.

Single-miracidium infections of Lymnaea truncatula by Fasciola hepatica were experimentally carried out to identify the redial generations of this trematode when the larval development was unusual (when the first-appearing mother redia, or R1a redia, died after its exit from the sporocyst). Four parameters were measured in the body and pharyngeal region at weekly intervals. At day 49 post-exposure at 20°C, the body of the second mother rediae (R1b) was significantly longer than that of the subsequent generations, R2a and R2b/R3a (a mean of 3.0 mm instead of 1.0 and 0.9 mm, respectively). The body was significantly wider in the R1b and R2a groups than in the R2b/R3a rediae. The pharyngeal lumen was significantly wider in the R1b group than in the R2a and R2b/R3a rediae (a mean of 48.6 µm instead of 10.8 and 3.3 µm at day 49). The thickness of the pharyngeal wall did not differ in the R1b and R2b/R3a groups, but was significantly lower in the R2a group (19.5 µm instead of 23.0-23.6 at day 49). There was better development of R1b and R2b/R3a rediae in the snails when the R1a redia died, compared with normal larval development (with a living R1a redia).

Characterization of Fasciola hepatica redial generations by morphometry and chaetotaxy under experimental conditions.

Morphometric and chaetotactic studies were carried out on the body and cephalic regions of the rediae of Fasciola hepatica (Trematoda: Fasciolidae) in order to precisely identify the different redial generations of this trematode in Lymnaea truncatula under experimental infection. At day 49 post-exposure at 20 degreesC, the length of the redia was significantly higher in the first group of the first generation (R1a) compared with successive generations, R1b, R2a and R2b/R3a. The width of the body was similar in the R1a, R1b, and R2a rediae, but was significantly lower in the R2b/R3a groups. The intrapharyngeal cavity of R1a rediae was significantly wider compared with the R1b, R2a, and R2b/R3a groups, whereas the pharyngeal wall was significantly thicker in the R2b/R3a rediae compared with the R1b and R2a groups. Four other measurements, namely the maximum length and width of the pharynx, diameter of the mouth, and width of intestine, also showed significant variations in relation to pharyngeal morphology and age of infection. Discriminant analysis based on these measurements demonstrated that 98% of the rediae were readily categorized into the four groups identified. The number of perioral sensillae ranged from 126 to 160 but a significant difference was only noted between the mean values of the first generation and those of the group R2b/R3a. From these parameters, the maximum width of the pharyngeal lumen was found to be the best characteristic in the identification of the redial generations.

Fasciola hepatica: in vitro production of daughter rediae and cercariae from first- and second-generation rediae.

Fasciola hepatica mother rediae belonging to the first generation and the first cohort of the second generation were extracted from their snail intermediate host (Lymnaea truncatula) and maintained in vitro for 16 days to determine the quantity of daughter rediae and cercariae produced by each generation. L-15 Leibovitz medium with a saline fraction adjusted to that of the L. truncatula hemolymph was used for experiments. A total of 35 daughter rediae exited from the 21 mother rediae of the first and second generations, which were 14-32 days old at the onset of culture. Microscope examination of the mother rediae demonstrated the presence of numerous redial embryos and differentiating daughter rediae, whereas no cercaria was seen within their body. A total of 49 cercariae exited from the 17 mother rediae of the first and second generations, which were 29-50 days old at the onset of culture. No differentiating daughter redia was seen in these last mother rediae. The productivity of the first or second generation (first cohort) was nearly identical in terms of cercariae.