Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Enhancing arthropod occurrence in wheat cropping systems: the role of non-chemical pest management and nitrogen optimization.

This study analyzes arthropod biomass and abundance to track the changes in arthropod occurrence in relation to pesticide use in three winter wheat cropping systems managed at different intensities (organic, conventional, and hybrid). Arthropod occurrence was surveyed using three collection tools: sweeping nets, eclector traps, and yellow traps. Sampling was conducted over three years from 2020 to 2022 with 588 samples collected. The wet weight of the captured organisms was determined and arthropod abundance calculated. The application of a NOcsPS (no chemical-synthetic pesticides) strategy, a new hybrid cultivation method realized with optimized use of nitrogen fertilizers but without chemical-synthetic pesticides, showed a higher arthropod occurrence and performed more convincingly regarding produced arthropod biomass and abundance than the other cropping variants. The results also demonstrate a dependence of the obtained insect indices on the collection method. Although arthropod biomass and abundance correlated for all collection methods, the combination of various methods as well as multiple procedures of sample analysis gives a more realistic and comprehensive view of the impact of the wheat cultivation systems on the arthropod fauna than one-factor analyses.

'Candidatus Tisiphia' is a widespread Rickettsiaceae symbiont in the mosquito Anopheles plumbeus (Diptera: Culicidae).

Symbiotic bacteria can alter host biology by providing protection from natural enemies, or alter reproduction or vectoral competence. Symbiont-linked control of vector-borne disease in Anopheles has been hampered by a lack of symbioses that can establish stable vertical transmission in the host. Previous screening found the symbiont 'Candidatus Tisiphia' in Anopheles plumbeus, an aggressive biter and potential secondary vector of malaria parasites and West Nile virus. We screened samples collected over 10-years across Germany and used climate databases to assess environmental influence on incidence. We observed a 95% infection rate, and that the frequency of infection did not fluctuate with broad environmental factors. Maternal inheritance is indicated by presence in the ovaries through FISH microscopy. Finally, we assembled a high-quality 1.6 Mbp draft genome of 'Ca. Tisiphia' to explore its phylogeny and potential metabolic competence. The infection is closely related to strains found in Culicoides biting midges and shows similar patterns of metabolism, providing no evidence of the capacity to synthesize B-vitamins. This infection offers avenues for onward research in anopheline mosquito symbioses. Additionally, it provides future opportunity to study the impact of 'Ca. Tisiphia' on natural and transinfected hosts, especially in relation to reproductive fitness and vectorial competence and capacity.

Bacterial genome sequencing tracks the housefly-associated dispersal of fluoroquinolone- and cephalosporin-resistant Escherichia coli from a pig farm.

The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig faeces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, 'and TEM-1' was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2 km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man.

Impact of larvae of the Asian tiger mosquito Aedes albopictus on larvae of the Culex pipiens complex from Germany in laboratory co-breeding studies.

Since the first detection of the Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) eggs in Germany in 2007, several populations of this species have established in Germany. Although colloquially Ae. albopictus is called an 'invasive species', it is not considered 'invasive' and therefore to be controlled according to the European Union (EU) Environmental and Nature Protection Act since evidence of displacement of native species is missing. To test the competitive potential of Ae. albopictus towards mosquito species native to Germany, laboratory experiments were conducted with larvae of this species and indigenous Cx. pipiens complex species/biotypes. First instar larvae of Ae. albopictus and of one of the native taxa were exposed to different temperatures and fed with different food sources. The ratio of individuals developing into adults as well as the time the larvae needed for development were taken as a measure of competitive outcome. In addition, the size of emerging adults was compared between control and experimental groups. Regarding developmental time, no significant differences were found between treatments and controls while significant differences were found regarding developmental rate and average wing size of individuals. Because no evidence of competitive repression of the native species was found, Ae. albopictus cannot be included in the EU list of invasive species.

The microbial RNA metagenome of Aedes albopictus (Diptera: Culicidae) from Germany.

Aedes albopictus is a highly invasive mosquito species that has become widespread across the globe. In addition, it is an efficient vector of numerous pathogens of medical and veterinary importance, including dengue, chikungunya and Zika viruses. Among others, the vector potential of mosquitoes is influenced by their microbiome. However, this influence is very dynamic and can vary between individuals and life stages. To obtain a rough overview on the microbiome of Ae. albopictus populations in Germany, pooled female and pooled male individuals from seven German locations were investigated by total RNA sequencing. The mosquito specimens had been collected as larvae in the field and processed immediately after adult emergence, i.e. without females having fed on blood. RNA fragments with high degrees of identity to a large number of viruses and microorganisms were identified, including, for example, Wolbachia pipientis and Acinetobacter baumannii, with differences between male and female mosquitoes. Knowledge about the natural occurrence of microorganisms in mosquitoes may be translated into new approaches to vector control, for example W. pipientis can be exploited to manipulate mosquito reproduction and vector competence. The study results show how diverse the microbiome of Ae. albopictus can be, and the more so needs to be adequately analysed and interpreted.

Nine years of mosquito monitoring in Germany, 2011-2019, with an updated inventory of German culicid species.

Before the background of increasingly frequent outbreaks and cases of mosquito-borne diseases in various European countries, Germany recently realised the necessity of updating decade-old data on the occurrence and spatiotemporal distribution of culicid species. Starting in 2011, a mosquito monitoring programme was therefore launched with adult and immature mosquito stages being collected at numerous sites all over Germany both actively by trapping, netting, aspirating and dipping, and passively by the citizen science project 'Mueckenatlas'. Until the end of 2019, about 516,000 mosquito specimens were analysed, with 52 (probably 53) species belonging to seven genera found, including several species not reported for decades due to being extremely rare (Aedes refiki, Anopheles algeriensis, Culex martinii) or local (Culiseta alaskaensis, Cs. glaphyroptera, Cs. ochroptera). In addition to 43 (probably 44 including Cs. subochrea) out of 46 species previously described for Germany, nine species were collected that had never been documented before. These consisted of five species recently established (Ae. albopictus, Ae. japonicus, Ae. koreicus, An. petragnani, Cs. longiareolata), three species probably introduced on one single occasion only and not established (Ae. aegypti, Ae. berlandi, Ae. pulcritarsis), and a newly described cryptic species of the Anopheles maculipennis complex (An. daciae) that had probably always been present but not been differentiated from its siblings. Two species formerly listed for Germany could not be documented (Ae. cyprius, Ae. nigrinus), while presence is likely for another species (Cs. subochrea), which could not be demonstrated in the monitoring programme as it can neither morphologically nor genetically be reliably distinguished from a closely related species (Cs. annulata) in the female sex. While Cs. annulata males were collected in the present programme, this was not the case with Cs. subochrea. In summary, although some species regarded endemic could not be found during the last 9 years, the number of culicid species that must be considered firmly established in Germany has increased to 51 (assuming Cs. subochrea and Ae. nigrinus are still present) due to several newly emerged ones but also to one species (Ae. cyprius) that must be considered extinct after almost a century without documentation. Most likely, introduction and establishment of the new species are a consequence of globalisation and climate warming, as three of them are native to Asia (Ae. albopictus, Ae. japonicus, Ae. koreicus) and three (Ae. albopictus, An. petragnani, Cs. longiareolata) are relatively thermophilic. Another thermophilic species, Uranotaenia unguiculata, which had been described for southwestern Germany in 1994 and had since been found only at the very site of its first detection, was recently documented at additional localities in the northeastern part of the country. As several mosquito species found in Germany are serious pests or potential vectors of disease agents and should be kept under permanent observation or even be controlled immediately on emergence, the German mosquito monitoring programme has recently been institutionalised and perpetuated.

Can data from native mosquitoes support determining invasive species habitats? Modelling the climatic niche of Aedes japonicus japonicus (Diptera, Culicidae) in Germany.

Invasive mosquito species and the pathogens they transmit represent a serious health risk to both humans and animals. Thus, predictions on their potential geographic distribution are urgently needed. In the case of a recently invaded region, only a small number of occurrence data is typically available for analysis, and absence data are not reliable. To overcome this problem, we have tested whether it is possible to determine the climatic ecological niche of an invasive mosquito species by using both the occurrence data of other, native species and machine learning. The approach is based on a support vector machine and in this scenario applied to the Asian bush mosquito (Aedes japonicus japonicus) in Germany. Presence data for this species (recorded in the Germany since 2008) as well as for three native mosquito species were used to model the potential distribution of the invasive species. We trained the model with data collected from 2011 to 2014 and compared our predicted occurrence probabilities for 2015 with observations found in the field throughout 2015 to evaluate our approach. The prediction map showed a high degree of concordance with the field data. We applied the model to medium climate conditions at an early stage of the invasion (2011-2015), and developed an explanation for declining population densities in an area in northern Germany. In addition to the already known distribution areas, our model also indicates a possible spread to Saarland, southwestern Rhineland-Palatinate and in 2015 to southern Bavaria, where the species is now being increasingly detected. However, there is also evidence that the possible distribution area under the mean climate conditions was underestimated.

Population genetics of the invasive Asian bush mosquito Aedes japonicus (Diptera, Culicidae) in Germany-a re-evaluation in a time period of separate populations merging.

The Asian bush mosquito Aedes japonicus, endemic to East Asia, is one of the most expansive mosquito species in the world and has as yet established in 15 countries of Europe. Within Germany, the species has been spreading tremendously during the last years, and its four once geographically isolated populations were on the verge of merging in 2017. To reveal relationships and carry-over ways between the various populations, and thus, migration and displacement routes, the genetic make-up of Ae. japonicus from ten different locations throughout its German distribution area was investigated. For this purpose, a part of the mitochondrial DNA (nad4 gene) of collected specimens was sequenced and seven loci of short tandem repeats (microsatellites) were genotyped. When related to similar genetic studies carried out between 2012 and 2015, the results suggest that admixtures had since occurred, but no complete genetic mixture of populations had taken place. At the time of sampling for the present study, the western collection sites were still uniform in their genetic make-up; however, a carry-over of individuals from the southeastern to the northern and southwestern German populations was determined. Further introductions from abroad are possible. In summary, the genetic diversity of Ae. japonicus in Germany had grown considerably, thus increasing ecological variability and adaptability of the species. At this point (10 years after the first detection), it is not possible anymore to draw conclusions on the origins of the populations.

Molecular detection of vector-borne pathogens from mosquitoes collected in two zoological gardens in Germany.

In Germany, knowledge of disease agents transmitted by arthropods in zoological gardens is scarce. In the framework of ecological studies, mosquitoes were therefore collected in German zoological gardens and examined for mosquito-borne pathogen DNA and RNA. In total, 3840 mosquitoes were screened for filarial nematodes and three groups of viruses (orthobunyaviruses, flaviviruses, alphaviruses) while 405 mosquitoes were tested for avian malaria parasites. In addition to the filarial nematode species Dirofilaria repens (n = 1) and Setaria tundra (n = 8), Sindbis virus (n = 1) and the haemosporidian genera Haemoproteus (n = 8), Leucocytozoon (n = 10) and Plasmodium (n = 1) were demonstrated. Identified pathogens have the potential to cause disease in zoo and wild animals, but some of them also in humans. Positive mosquitoes were collected most often in July, indicating the highest infection risk during this month. Most of the pathogens were found in mosquito specimens of the Culex pipiens complex, suggesting that its members possibly act as the most important vectors in the surveyed zoos, although the mere demonstration of pathogen DNA/RNA in a homogenised complete mosquito is not finally indicative for a vector role. Outcomes of the study are not only significant for arthropod management in zoological gardens, but also for the general understanding of the occurrence and spread of mosquito-borne disease agents.

First record of Aedes koreicus (Diptera: Culicidae) in Germany.

Within the framework of a national mosquito monitoring programme, a mosquito specimen collected in mid-2015 in southern Germany was identified as Aedes koreicus, a non-endemic species originating from East Asia. After the Asian bush mosquito Aedes japonicus, which is already established in Germany and widely distributed, and the Asian tiger mosquito Aedes albopictus, which is increasingly often introduced from southern Europe, A. koreicus is the third demonstrated invasive mosquito species in Germany supposed to have significant vector potential for disease agents.

Schmallenberg virus in Germany 2011-2014: searching for the vectors.

Following the emergence of Schmallenberg virus (SBV) in 2011, 21,397 culicoid biting midges (Diptera: Ceratopogonidae) from targeted and non-targeted sampling activities carried out during the summer months of 2011 to 2013 and in late 2014 in various regions in Germany were analyzed for the virus by real-time RT-PCR. While no SBV was found in biting midges collected during 2011 and 2013, 2 out of 334 pools including 20 and 22 non-engorged females of the Obsoletus complex sampled in 2012 tested positive for the SBV S-segment with C(t) values of 42.46 and 35.45. In addition, 673 black flies (Diptera: Simuliidae) captured during the same studies were screened for the presence of SBV and proved negative. In late autumn 2014, biting midges were collected again in a limited study in eastern Germany after some cases of SBV infection had occurred in a quarantine station for cattle. Due to the unfavorable seasonal weather conditions, only few specimens were caught, and these were also negative for SBV. The German experience suggests that biting midge collections launched only after an outbreak and are not locally targeted may be ineffective as to virus detection. It rather might be advisable to collect biting midges at sentinel farms on a permanent basis so to have material available to be examined in the case of a disease outbreak.

Aedes albopictus breeding in southern Germany, 2014.

Larvae, pupae and eggs of the Asian tiger mosquito Aedes albopictus were found in Freiburg, southern Germany, after submission of an adult mosquito specimen from that area to the 'Mückenatlas', a German instrument of passive mosquito surveillance. While previously collected Ae. albopictus in Germany were trapped on, or close to, service stations on motorways, suggesting introduction by vehicles from southern Europe, these new specimens were out of flight distance from the motorway on the one hand and indicate local reproduction on the other. The findings call for a thorough active and passive surveillance in exposed geographic regions such as the relatively warm German Upper Rhine Valley to prevent Ae. albopictus from establishing.

A comparison of four light traps for collecting Culicoides biting midges.

Epidemiological analyses of vector-associated diseases such as bluetongue (BT), African horse sickness, or epizootic hemorrhagic disease require substantiated data on the species diversity and activity patterns of vector species. To this end, Spain and Italy implemented extensive Culicoides biting midge monitoring programs since 2000, as several other countries did after the arrival of BT in northern Europe in 2006. The seasonal occurrence, spatial distribution, and abundance of Culicoides species, as the major results of such monitoring programs, are used as parameters for assessing the risk of virus introduction and transmission in a given area. However, the quality of entomological monitoring results fundamentally depends on the collection techniques. In this publication, we describe a Latin Square design trial carried out in Germany under field conditions in 2009/2010 to compare the efficacy of four commonly used light baited/suction traps in collecting Culicoides. A total of 2651 Culicoides were caught over 18 nights. In both years, the Onderstepoort and BG-Sentinel traps caught significantly more Culicoides than the Rieb and the CDC trap. Most specimens were caught by the Onderstepoort trap (1246, i.e., 76 % in 2009 and 819, i.e., 82 % in 2010). Most were classified as midges of the Culicoides obsoletus group.

[The recurring necessity of mosquito surveillance and research]. / Die wiederkehrende Notwendigkeit von Stechmücken-Surveillance und -Forschung.

Hematophagous arthropods and the diseases associated with them represent a growing threat to human and animal health in Europe. After the eradication of endemic malaria from Europe in the middle of the last century, there has been a resurgence of mosquitoes as significant vectors of disease agents under the influence of continuing globalisation, as exotic species and mosquito-borne pathogens are being introduced with increasing frequency. At present, southern Europe is particularly affected by disease outbreaks and cases, but invasive mosquito species, including efficient vectors, have also emerged in Germany. While there is considerable knowledge on the vector potential of many tropical and subtropical mosquito species, corresponding data on the indigenous mosquito species are scarce. Exceptions are the Anopheles species, which were already vectors of malaria parasites in historic Europe. It must be assumed, however, that many further indigenous species are able to transmit pathogens under certain conditions and will by all means gain vector competence under a scenario of climate warming. Thus, the permanent surveillance of mosquitoes and mosquito-borne disease agents is paramount for the purposes of conducting risk analyses and modelling, in addition to research work addressing the conditions of the spread of vectors and pathogens and of pathogen transmission. Only ample data can facilitate taking appropriate prophylactic action and designing control strategies. International health organizations have realised this and started to promote data collection on mosquitoes and mosquito-borne diseases in the EU. At a national levels, authorities are more reluctant, although, similar to other fields of health, it has been shown for mosquito-borne diseases that preventive measures are more cost-saving than disease case management and the coverage of follow-up costs. The present article is intended to illustrate the necessity of the re-intensification of mosquito surveillance and research in Germany and other European countries.

Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

PCR identification and distribution of Anopheles daciae (Diptera, Culicidae) in Germany.

Based primarily on nucleotide polymorphisms in the internal transcribed spacer 2 (ITS2) of the ribosomal DNA, Anopheles daciae was recently described as an additional member of the Maculipennis Group of species, separate from Anopheles messeae with which it had previously been confused due to morphological and genetic similarity. Species differentiation between A. messeae and A. daciae was possible only by ITS2 polymerase chain reaction (PCR) amplification followed by DNA sequencing or RFLP analysis. In addition to its siblings, Anopheles maculipennis, Anopheles atroparvus and A. messeae, A. daciae has been shown to occur in Germany, although with limited distribution. We here describe additional collection sites for this species in Germany, showing concentrations in East Germany and the northern Upper Rhine Valley in Southwest Germany. A species-specific multiplex PCR assay is presented that is able to differentiate the four Maculipennis Group sibling species occurring in Germany plus Anopheles sacharovi, Anopheles melanoon and Anopheles labranchiae. The correct identification and detailed knowledge of the biology of A. daciae are of relevance since it might be a vector of disease agents, as suggested by the vector potential of its siblings and the recent finding of an A. daciae female infected with Dirofilaria repens in southern Germany.

Large-Scale Cytochrome C Oxidase Subunit I Gene Data Analysis for the Development of a Multiplex Polymerase Chain Reaction Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of the Culicoides Subgenus Avaritia Fox, 1955.

The emergence of culicoid-transmitted bluetongue and Schmallenberg viruses in several European countries demonstrated the ability of indigenous biting midge species to transmit pathogens. Entomologic research programs identified members of the Obsoletus Group (Culicoides subgenus Avaritia) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors is challenging due to the recent discovery of new genetic variants (haplotypes) of C. obsoletus sensu stricto (s.s.), forming distinct clades. In this study, 4422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenus Avaritia members of the genus Culicoides were analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and clades of the Western Palearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three C. obsoletus clades represent independent species, whereas C. montanus should rather be regarded as a genetic variant of C. obsoletus s.s. Based on these findings, specific primers were designed and validated with DNA material from field-caught biting midges which achieved very high diagnostic sensitivity (100%) when compared to an established reference PCR (82.6%).

Improvements of weaned pigs barn hygiene to reduce the spread of antimicrobial resistance.

The spread of antimicrobial resistance (AMR) in animal husbandry is usually attributed to the use of antibiotics and poor hygiene and biosecurity. We therefore conducted experimental trials to improve hygiene management in weaned pig houses and assessed the impact on the spread. For each of the two groups examined, the experimental group (EG) and the control group (CG), three replicate batches of piglets from the same pig breeder, kept in pre-cleaned flat decks, were analyzed. In the flat decks of the experimental groups, the hygiene conditions (cleaning, disinfection, dust removal and fly control) were improved, while regular hygiene measures were carried out in the control groups. The occurrence and spread of AMR were determined in Escherichia coli (E. coli; resistance indicator) using cultivation-dependent (CFU) and -independent (qPCR) methods as well as whole genome sequencing of isolates in samples of various origins, including feces, flies, feed, dust and swabs. Surprisingly, there were no significant differences (p > 0.05) in the prevalence of resistant E. coli between the flat decks managed with conventional techniques and those managed with improved techniques. Selective cultivation delivered ampicillin- and sulfonamide-resistant E. coli proportions of up to 100% and 1.2%, respectively. While 0.5% E. coli resistant to cefotaxime and no ciprofloxacin resistance were detected. There was a significant difference (p < 0.01) in the abundance of the blaTEM-1 gene in fecal samples between EG and CG groups. The colonization of piglets with resistant pathogens before arrival, the movement of flies in the barn and the treatment of bacterial infections with antibiotics obscured the effects of hygiene improvement. Biocide tolerance tests showed no development of resistance to the farm regular disinfectant. Managing hygiene alone was insufficient for reducing antimicrobial resistances in piglet rearing. We conclude that the complex factors contributing to the presence and distribution of AMR in piglet barns underscore the necessity for a comprehensive management strategy.

The further spread of Aedes japonicus japonicus (Diptera, Culicidae) towards northern Germany.

After its first detection in 2008 in the south German federal state of Baden-Wuerttemberg, another distinct population of the invasive Asian bush mosquito Aedes japonicus japonicus was unexpectedly found in western Germany in 2012. Range expansion had already been observed for the southern German population and was anticipated for the western German one. Here, we report on a third, apparently independent and even more northerly German colonization area of Aedes j. japonicus in southern Lower Saxony and northeastern North Rhine-Westphalia, which was discovered in spring 2013. In a snapshot study, intended to determine the presence or absence of Aedes j. japonicus in an area close to Hanover, the capital of the northern German federal state of Lower Saxony, where a specimen had been collected in late 2012, central water basins of cemeteries were checked for pre-imaginal mosquito stages at the beginning of the mosquito season 2013. Almost 20% of the inspected cemeteries were found positive (25 out of 129), with many of them being located in towns and villages close to the motorways A2 and A7. Being of Far Eastern origin, the Asian bush mosquito is well adapted to moderate climates and appears to be further expanding its distribution area in Central Europe. As it is a proven laboratory vector of several mosquito-borne disease agents, its present and future distribution areas should be carefully monitored.