Helge Kampen

Identification of six sibling species of the Anopheles maculipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay

Abstract Until the eradication of malaria from Europe, members of the Anopheles maculipennis complex had been the major vectors for plasmodial parasites. With the possible reintroduction of Plasmodium species due to climate change and increased travel to and from countries where malaria is endemic, accurate identification of mosquito species will be essential for preventive studies. For this purpose, a diagnostic PCR system to differentiate between six of the seven A. maculipennis sibling species occurring in Europe was developed. The second internal transcribed spacer (ITS2) of the ribosomal DNA was amplified and sequenced for all six species. Based on differences in the nucleotide sequences, species-specific primers were constructed for PCR amplification of mosquito DNA that in combination with a universal primer generate amplification products of different length, each unique for one species.

Seroprevalence of Babesia Infections in Humans Exposed to Ticks in Midwestern Germany

ABSTRACT Babesiosis is considered to be an emerging tick-borne disease in humans worldwide. However, most studies on the epidemiology of human babesiosis to date have been carried out in North America, and there is little knowledge on the prevalence of infection and frequency of disease in other areas. The aim of this study was to investigate the prevalence of Babesia infections in a human population in Germany. A total of 467 sera collected between May and October 1999 from individuals living in the Rhein-Main area were tested for the presence of immunoglobulin G (IgG) and IgM antibodies to antigens of Babesia microti and Babesia divergens by indirect fluorescent-antibody (IFA) tests. These sera were derived from 84 Lyme borreliosis patients suffering from erythema migrans, 60 asymptomatic individuals with positive borreliosis serology, and 81 individuals with a history of tick bite. Cutoff values for discrimination between seronegative and seropositive results in the IFA tests were determined using sera from 120 healthy blood donors and 122 patients suffering from conditions other than tick-borne diseases (malaria, n = 40; toxoplasmosis, n = 22; syphilis, n = 20; Epstein-Barr virus infection, n = 20; and presence of antinuclear antibodies, n = 20). The overall specificities of the IFA tests for B. microti and B. divergens were estimated to be ≥97.5%. Positive IgG reactivity against B. microti antigen (titer, ≥1:64) or B. divergens antigen (titer, ≥1:128) was detected significantly more often (P < 0.05) in the group of patients exposed to ticks (26 of 225 individuals; 11.5%) than in the group of healthy blood donors (2 of 120 individuals; 1.7%). IgG antibody titers of ≥1:256 against at least one of the babesial antigens were found significantly more often (P < 0.05) in patients exposed to ticks (9 of 225) than in the control groups (1 of 242). In the human population investigated here, the overall seroprevalences for B. microti and B. divergens were 5.4% (25 of 467) and 3.6% (17 of 467), respectively. The results obtained here provide evidence for concurrent infections with Borrelia burgdorferi and Babesia species in humans exposed to ticks in midwestern Germany. They also suggest that infections with Babesia species in the German human population are more frequent than believed previously and should be considered in the differential diagnosis of febrile illness occurring after exposure to ticks or blood transfusions, in particular in immunocompromised patients.

European Surveillance for West Nile Virus in Mosquito Populations

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

An entomological review of invasive mosquitoes in Europe.

Among the invasive mosquitoes registered all over the world, Aedes species are particularly frequent and important. As several of them are potential vectors of disease, they present significant health concerns for 21st century Europe. Five species have established in mainland Europe, with two (Aedes albopictus and Aedes japonicus) becoming widespread and two (Ae. albopictus and Aedes aegypti) implicated in disease transmission to humans in Europe. The routes of importation and spread are often enigmatic, the ability to adapt to local environments and climates are rapid, and the biting nuisance and vector potential are both an ecomonic and public health concern. Europeans are used to cases of dengue and chikungunya in travellers returning from the tropics, but the threat to health and tourism in mainland Europe is substantive. Coupled to that are the emerging issues in the European overseas territorities and this paper is the first to consider the impacts in the remoter outposts of Europe. If entomologists and public health authorities are to address the spread of these mosquitoes and mitigate their health risks they must first be prepared to share information to better understand their biology and ecology, and share data on their distribution and control successes. This paper focusses in greater detail on the entomological and ecological aspects of these mosquitoes to assist with the risk assessment process, bringing together a large amount of information gathered through the ECDC VBORNET project.

Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany

BackgroundAs a result of globalization and climate change, Dirofilaria immitis and Dirofilaria repens, the causative agents of dirofilariosis in Europe, continue to spread from endemic areas in the Mediterranean to northern and northeastern regions of Europe where autochthonous cases of dirofilarial infections have increasingly been observed in dogs and humans. Whilst D. repens was recently reported from mosquitoes in putatively non-endemic areas, D. immitis has never been demonstrated in mosquitoes from Europe outside the Mediterranean.MethodsFrom 2011 to 2013, mosquitoes collected within the framework of a German national mosquito monitoring programme were screened for filarial nematodes using a newly designed filarioid-specific real-time PCR assay. Positive samples were further processed by conventional PCR amplification of the cytochrome c oxidase subunit I (COI) gene, amplicons were sequenced and sequences blasted against GenBank.ResultsApproximately 17,000 female mosquitoes were subjected to filarial screening. Out of 955 pools examined, nine tested positive for filariae. Two of the COI sequences indicated D. immitis, one D. repens and four Setaria tundra. Two sequences could not be assigned to a known species due to a lack of similar GenBank entries. Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium, D. repens was found in a single Anopheles daciae and all S. tundra were demonstrated in Aedes vexans. All positive mosquitoes were collected between mid-June and early September.ConclusionThe finding of dirofilariae in German mosquitoes implies the possibility of a local natural transmission cycle. While the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, potential culicid vectors and reservoir hosts must prospectively be identified and awareness among physicians, veterinarians and public health personnel be created. The health impact of S. tundra on the indigenous cervid fauna needs further investigation.

Individual cases of autochthonous malaria in Evros Province, northern Greece: serological aspects.

Abstract. Prompted by four autochthonous cases of malaria in 1994 and 1995 in Evros Province, northern Greece, we conducted an entomological study between 1997 and 1999 in Nipsa and Chandras, rural locations where two of the four cases had occurred, and in Feres where two additional autochthonous malaria cases had been diagnosed in 1998. In Nipsa and Chandras, we identified 29 Anopheles breeding sites and characterized them by physicochemical parameters. Larvae were collected both at these sites and in a brackish water breeding site near Feres in the Evros River delta. Adults were caught in sheds at all three locations. Morphology was used to classify larvae and adults as A. superpictus or as species belonging to the A. claviger or A. maculipennis species complexes. The latter were further identified by PCR as being A. maculipennis s.s., A. melanoon and A. sacharovi. Of the A.maculipennis complex larvae collected inland, approximately 94% were A. maculipennis s.s. and 6% A. melanoon, whereas all larvae collected in the coastal region were A. sacharovi. In contrast, the A.maculipennis adults were A. maculipennis s.s. and A. melanoon (both 47%), and A. sacharovi (6%). In the coastal region, no A. maculipennis s.s. adults were caught. The ratio of A. melanoon adults collected to A. sacharovi was about 3:1. As shown by a bloodmeal ELISA, only 5 of 266 fed females (1.9%) had ingested human blood, whereas 232 (87%) had fed on goats. Of the mosquitoes containing human blood, two were A. melanoon, one A. sacharovi and one A. maculipennis s.s. One human blood specimen could no longer be assigned to a particular mosquito.

Out of the bush: the Asian bush mosquito Aedes japonicus japonicus (Theobald, 1901) (Diptera, Culicidae) becomes invasive

The Asian bush or rock pool mosquito Aedes japonicus japonicus is one of the most expansive culicid species of the world. Being native to East Asia, this species was detected out of its original distribution range for the first time in the early 1990s in New Zealand where it could not establish, though. In 1998, established populations were reported from the eastern US, most likely as a result of introductions several years earlier. After a massive spread the mosquito is now widely distributed in eastern North America including Canada and two US states on the western coast. In the year 2000, it was demonstrated for the first time in Europe, continental France, but could be eliminated. A population that had appeared in Belgium in 2002 was not controlled until 2012 as it did not propagate. In 2008, immature developmental stages were discovered in a large area in northern Switzerland and bordering parts of Germany. Subsequent studies in Germany showed a wide distribution and several populations of the mosquito in various federal states. Also in 2011, the species was found in southeastern Austria (Styria) and neighbouring Slovenia. In 2013, a population was detected in the Central Netherlands, specimens were collected in southern Alsace, France, and the complete northeastern part of Slovenia was found colonized, with specimens also present across borders in adjacent Croatia. Apparently, at the end of 2013 a total of six populations occurred in Europe although it is not clear whether all of them are completely isolated. Similarly, it is not known whether these populations go back to the same number of introductions. While entry ports and long-distance continental migration routes are also obscure, it is likely that the international used tyre trade is the most important mode of intercontinental transportation of the mosquito. Aedes j. japonicus does not only display an aggressive biting behaviour but is suspected to be a vector of various disease agents and to displace indigenous culicid species. Therefore, Aedes j. japonicus might both cause public health problems in the future and have a significant impact on the biodiversity of the invaded territories.

Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of crude bacterial samples has been introduced as a very cost-efficient and rapid, yet highly informative tool to identify and classify bacteria. The potential of this approach to characterize whole animals, so far preferentially insects, is only evolving. Here, a simple protocol was developed to perform MALDI-MS analysis on extracts from whole ticks of 7 species and 4 developmental stages. Using commercially available software designed for the identification of bacteria, a reference database of spectra was constructed that allowed the species determination of ticks using larvae, nymphs, or adult individuals as starting material. Cluster analysis on the basis of MALDI mass spectra indicated that the primary determinant for the mass spectra was the species, followed by the developmental stages, which formed distinct clusters within the given species. With certain limitations, species identification was also possible using body parts and engorged animals. Spectra of developing Ixodes ricinus eggs showed dramatic changes with time, suggesting that, beyond its usefulness for species determination, MALDI-typing may have applications in developmental biology.

Approaches to passive mosquito surveillance in the EU

The recent emergence in Europe of invasive mosquitoes and mosquito-borne disease associated with both invasive and native mosquito species has prompted intensified mosquito vector research in most European countries. Central to the efforts are mosquito monitoring and surveillance activities in order to assess the current species occurrence, distribution and, when possible, abundance, in order to permit the early detection of invasive species and the spread of competent vectors. As active mosquito collection, e.g. by trapping adults, dipping preimaginal developmental stages or ovitrapping, is usually cost-, time- and labour-intensive and can cover only small parts of a country, passive data collection approaches are gradually being integrated into monitoring programmes. Thus, scientists in several EU member states have recently initiated programmes for mosquito data collection and analysis that make use of sources other than targeted mosquito collection. While some of them extract mosquito distribution data from zoological databases established in other contexts, community-based approaches built upon the recognition, reporting, collection and submission of mosquito specimens by citizens are becoming more and more popular and increasingly support scientific research. Based on such reports and submissions, new populations, extended or new distribution areas and temporal activity patterns of invasive and native mosquito species were found. In all cases, extensive media work and communication with the participating individuals or groups was fundamental for success. The presented projects demonstrate that passive approaches are powerful tools to survey the mosquito fauna in order to supplement active mosquito surveillance strategies and render them more focused. Their ability to continuously produce biological data permits the early recognition of changes in the mosquito fauna that may have an impact on biting nuisance and the risk of pathogen transmission associated with mosquitoes. International coordination to explore synergies and increase efficiency of passive surveillance programmes across borders needs to be established.

A new focus of Aedes japonicus japonicus (Theobald, 1901) (Diptera, Culicidae) distribution in Western Germany: rapid spread or a further introduction event?

BackgroundThe Asian bush mosquito, Aedes japonicus japonicus, a potential vector of several viruses, was first detected in Germany in 2008 on the Swiss-German border. In the following years, this invasive species apparently succeeded in establishing populations in southern Germany and in spreading northwards. In 2011, its distribution area already covered large areas of the federal state of Baden-Wurttemberg, and its northernmost German collection point was reported to be close to Stuttgart. Several independent submissions to our laboratories of Ae. j. japonicus specimens in July 2012, originating from the same area in the federal state of North Rhine-Westphalia, western Germany, prompted us to carry out an immediate surveillance in this region in the expectation of finding a further distribution focus of Ae. j. japonicus in Germany.MethodsAfter inspecting the places of residence of the collectors of the submitted mosquito specimens, all kinds of water containers in 123 cemeteries in surrounding towns and villages were checked for mosquito developmental stages. These were collected and kept to produce adults for morphological species identification. One specimen per collection site was identified genetically by COI sequence analysis.ResultsAedes j. japonicus adults and immature stages were found in 36 towns/villages that were checked (29%) over an area of approximately 2,000 km2 in southern North Rhine-Westphalia and northern Rhineland Palatinate. The species could not be demonstrated further south when monitoring towards the northernmost previous collection sites in southern Germany. It therefore remains to be elucidated whether the species has entered western Germany from the south, from Belgium in the west where it has been demonstrated to occur locally since 2002, or through a new introduction.ConclusionsAedes j. japonicus is obviously much more widely distributed in Germany than previously thought. It appears to be well adapted, to have a strong expansion tendency and to replace indigenous mosquito species. Thus, a further spread is anticipated and elimination seems hardly possible anymore. The vector potency of the species should be reason enough to thoroughly monitor its future development in Germany.