Renke Lühken

Repeated introduction of Aedes albopictus into Germany, July to October 2012

During a small-scale surveillance project to identify possible routes of entry for invasive mosquitoes into Germany, 14 adult Aedes (Stegomyia) albopictus (Skuse) were discovered between July and October 2012. They were trapped at three different service stations in Bavaria and Baden-Wuerttemberg located along two motorways that connect Germany with southern Europe. This indicates regular introduction of A. albopictus into Germany and highlights the need for a continuous surveillance and control programme.

Field evaluation of four widely used mosquito traps in Central Europe.

BackgroundTo monitor adult mosquitoes several trapping devices are available. These are differently constructed and use various mechanisms for mosquito attraction, thus resulting in different trapping sensitivities and efficacies for the various species. Mosquito monitoring and surveillance programs in Europe use various types of mosquito traps, but only a few comparisons have been conducted so far. This study compared the performance of four commercial trapping devices, which are commonly used in Europe.MethodsFour different traps, Biogents Sentinel trap (BG trap), Heavy Duty Encephalitis Vector Survey trap (EVS trap), Centres for Disease Control miniature light trap (CDC trap) and Mosquito Magnet Patriot Mosquito trap (MM trap) were compared in a 4 × 4 latin square study. In the years 2012 and 2013, more than seventy 24-hour trap comparisons were conducted at ten different locations in northern and southern Germany, representing urban, forest and floodplain biotopes.ResultsPer 24-hour trapping period, the BG trap caught the widest range of mosquito species, the highest number of individuals of the genus Culex as well as the highest number of individuals of the species Ochlerotatus cantans, Aedes cinereus/geminus, Oc. communis and Culex pipiens/torrentium. The CDC trap revealed best performance for Aedes vexans, whereas the MM trap was most efficient for mosquitoes of the genus Anopheles and the species Oc. geniculatus. The EVS trap did not catch more individuals of any genus or species compared to the other three trapping devices. The BG trap caught the highest number of individuals per trapping period in urban environments as well as in wet forest, while the CDC trap caught the highest number of individuals in the floodplain biotopes. Additionally, the BG trap was most efficient for the number of mosquito species in urban locations.ConclusionThe BG trap showed a significantly better or similar performance compared to the CDC, EVS or MM trap with regard to trapping efficacy for most common mosquito species in Germany, including diversity of mosquito species and number of mosquitoes per trapping period. Thus, the BG trap is probably the best solution for general monitoring or surveillance programs of adult mosquitoes in Central Europe.

Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa

ABSTRACT Usutu virus (USUV), one of the most neglected Old World encephalitic flaviviruses, causes epizootics among wild and captive birds and sporadic infection in humans. The dynamics of USUV spread and evolution in its natural hosts are unknown. Here, we present the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution. The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow. Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks. Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles. IMPORTANCE Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts. Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts.

Experimental transmission of Zika virus by mosquitoes from central Europe

Mosquitoes collected in Germany in 2016, including Culex pipiens pipiens biotype pipiens, Culex torrentium and Aedes albopictus, as well as Culex pipiens pipiens biotype molestus (in colony since 2011) were experimentally infected with Zika virus (ZIKV) at 18 °C or 27 °C. None of the Culex taxa showed vector competence for ZIKV. In contrast, Aedes albopictus were susceptible for ZIKV but only at 27 °C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

Widespread activity of multiple lineages of Usutu virus, western Europe, 2016.

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak.

The use of morphometric wing characters to discriminate female Culex pipiens and Culex torrentium

ABSTRACT: The reliability of the length of wing radial vein r2/3 as a character for the morphological discrimination of the two potential arbovirus vectors Culex pipiens s.s. and Cx. torrentium from Germany was reassessed, after this character had been neglected for more than 40 years. Additionally, multivariate morphometric analyses were applied to evaluate wing shape variation between both species. Although high-throughput molecular tools are now available to differentiate the two species, a simple, low-cost routine alternative may be useful in the absence of a molecular laboratory, such as under semi-field conditions. A thin-plate splines transformation confirmed that primarily the shrinkage of vein r2/3 is responsible for the wing differences between the two species. In the bivariate analysis, the r2/3/r3 indices of Cx. pipiens s.s. and Cx. torrentium were 0.185 and 0.289, respectively, resulting in a correct classification of more than 91% of all tested specimens. Using the absolute length of vein r2/3 alone still allowed for more than 90% accurate discrimination. Furthermore, classification accuracy of linear discriminant analysis exceeded 97%.

Assessment of the abundance of Culicoides chiopterus and Culicoides dewulfi in bovine dung: A comparison of larvae extraction techniques and emergence traps

We compared two larvae extraction methods involving sugar-flotation and an adapted Berlese funnel-extraction with emergence traps. This was done in order to analyse the colonisation of cowpats by Culicoides chiopterus (Meigen 1830) and Culicoides dewulfi (Goetghebuer 1936) (Diptera: Ceratopogonidae) and to gain information on the comparability and efficacy of these three methods. With all three methods, a considerable number of individuals was obtained. Significantly more individuals were obtained via sugar-flotation and Berlese funnel-extraction compared to the emergence traps. These differences, likely due to natural mortality and sample processing are discussed. We recommend Berlese as an efficacious method for extracting Culicoides larvae from bovine dung. It produces data rapidly and extracted larvae are viable. In comparison with Berlese, slightly more larvae were obtained by sugar-flotation, but this method was very labour intensive.

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking.

Distance from the stable affects trapping of biting midges (Diptera, Ceratopogonidae)

Several species of Culicoides biting midges (Diptera, Ceratopogonidae) are competent vectors of important veterinary pathogens in Europe. Bluetongue virus (BTV) has emerged in Europe since the late 1990s and is transmitted by several species of the genus Culicoides (Caracappa et al. 2003, De Liberato et al. 2005). African horse sickness (AHS) was observed at the end of the last century on the Iberian Peninsula (Mellor and Hamblin 2004) and since the end of 2011 there has been evidence that biting midges are important vectors of the Schmallenberg Virus (SBV) emerging in Central Europe (Hoffmann et al. 2012). SBV causes considerable congenital damage, premature births, and reproductive disorders in ruminants. Monitoring programs for biting midges are generally based on UV-light/suction trap sampling (Mehlhorn et al. 2009, Nielsen et al. 2010). Results of the sampling and trapping protocols could be significantly affected by uncontrollable environmental factors like wind and temperature (Bishop et al. 2000, Carpenter et al. 2008) and/or even the phase of the moon (Bishop et al. 2000). In contrast, there are controllable factors that could be standardized and would result in more comparable results from entomological surveillance programs. Firstly, there is the standardization of the trapping method, which could have different specificity for abundance, taxa, sex, or feeding stadium (Holbrook 1985, Anderson and Linhares 1989, Venter et al. 2009a, Viennet et al. 2011). Secondly, some authors mentioned that the positions of the traps have a significant impact on the sampling results, such as height (Venter et al. 2009b) or habitat (Bishop et al. 1994, Bishop et al. 1995). In this study, we were interested in the impact of the distance to the stable on the trapping result. Due to a positive correlation between biting midge abundance and the presence and abundance of hosts (Bellis and Reid 1996, Baylis et al. 2010, Garcia-Saenz et al. 2011, Viennet et al. 2011), we expected a decreasing number of biting midges to occur in traps with increasing distance to the stable. The study was conducted between May and June, 2009 at one livestock farm that is surrounded by meadows in northern Germany. This site represents a typical dairy farm with 220 dairy cattle and is located in a typical agricultural region in the marshland of Lower Saxony. The grazing density of cattle was approximately 7.3 individuals per hectare, but the cattle were in the stables during the night. Sampling of Culicoides spp. was conducted with BGSentinelTM light traps fitted with ultraviolet light. Each of the three traps was placed in concentric circles in distances of 25, 50, 100, and 200 m (variable DISTANCE) around one trap in direct proximity to the stable (Figure 1a). All traps were installed at 1.5 m above the ground and operated with one car battery each per sampling period. A photo sensor was installed in the immediate vicinity of each trap. The photo sensor was adjusted to illuminate the ultraviolet light from sunset to dawn, because the activity of Culicoides spp. was expected to be highest in this time period (Kettle et al. 1998). The traps were sampled five times, every seventh day (variable SAMPLINGPERIOD). Insects were collected in 70% ethanol and stored in separate containers. The samples were presorted in order to separate biting midges from other insects and to differentiate members of the C. obsoletus and C. pulicaris group (also C. obsoletus or C. pulicaris in the subsequent text) from other Ceratopogonidae. All data analyses and graphs were made with the program R (R Development Core Team 2011). Due to overdispersion in the data, collection data of females of the Culicoides obsoletus and Culicoides pulicaris group were analyzed with negative binomial generalized linear models through the function glm. nb from the package MASS (Venables and Ripley 2002). The variables DISTANCE, DIRECTION, SAMPLINGPERIOD, and all interactions between the variables were implemented in the model. The value of the variable DIRECTION for each trap tr was calculated as:

Distribution of Usutu Virus in Germany and Its Effect on Breeding Bird Populations

Usutu virus (USUV) is an emerging mosquitoborne flavivirus with an increasing number of reports from several countries in Europe, where USUV infection has caused high avian mortality rates. However, 20 years after the first observed outbreak of USUV in Europe, there is still no reliable assessment of the large-scale impact of USUV outbreaks on bird populations. In this study, we identified the areas suitable for USUV circulation in Germany and analyzed the effects of USUV on breeding bird populations. We calculated the USUV-associated additional decline of common blackbird (Turdus merula) populations as 15.7% inside USUV-suitable areas but found no significant effect for the other 14 common bird species investigated. Our results show that the emergence of USUV is a further threat for birds in Europe and that the large-scale impact on population levels, at least for common blackbirds, must be considered.