The toxicological spectrum of the Bacillus cereus toxin cereulide points towards niche-specific specialisation.

Most microbes share their environmental niches with very different forms of life thereby engaging in specialised relationships to enable their persistence. The bacterium Bacillus cereus occurs ubiquitously in the environment with certain strain backgrounds causing foodborne and opportunistic infections in humans. The emetic lineage of B. cereus is capable of producing the toxin cereulide, which evokes emetic illnesses. Although food products favouring the accumulation of cereulide are known, the ecological role of cereulide and the environmental niche of emetic B. cereus remain elusive. To better understand the ecology of cereulide-producing B. cereus, we systematically assayed the toxicological spectrum of cereulide on a variety of organisms belonging to different kingdoms. As cereulide is a potassium ionophore, we further tested the effect of environmental potassium levels on the action of cereulide. We found that adverse effects of cereulide exposure are species-specific, which can be exacerbated with increased environmental potassium. Additionally, we demonstrate that cereulide is produced within an insect cadaver indicating its potential ecological function for a saprophytic lifestyle. Collectively, distinct cereulide susceptibilities of other organisms may reflect its role in enabling competitive niche specialization of emetic B. cereus.

Phylogenetic relationships and new genetic tools for the detection and discrimination of the three feline Demodex mites.

Two feline Demodex mite species have been described as causative agents of feline demodicosis, until recently a third species was detected. We provide an updated analysis on the phylogenetic relationship of Demodex mites. In addition, we present the first qPCR assay for the detection and differentiation of all three feline mite species in a single reaction. Specimen of Demodex cati, Demodex gatoi, and the recently discovered third species were collected from skin scrapings and fecal flotation for DNA extraction, conventional PCR, sequencing, and alignment. A total of 24 sequences of the partial 16S rRNA gene were used to estimate the evolutionary divergence in a p-distance model and a maximum likelihood phylogenetic tree. For the qPCR assay, new primers and fluorescent probes for the simultaneous detection of all three feline Demodex mites were designed. A consensus fragment of 351 bp was phylogenetically analyzed. The third species sequence of our study shares 98.6 % similarity to the available sequence in GenBank®. It is most similar to D. gatoi (82.41 %) and most distant to the canine Demodex injai (78.28 %). In contrast, D. gatoi is most similar to human Demodex brevis (87.01 %). The multiplex qPCR detected and discriminated the three different mite species in one reaction. The detection limit is ≤1.4 ng of mite DNA. The three feline Demodex species have distinct genotypes and did not cluster in one genetic clade. The species differentiation and assessment of evolutionary relationships will ultimately support correct diagnostics and treatment approaches.

Mosquitoes (Diptera: Culicidae) and their relevance as disease vectors in the city of Vienna, Austria.

Mosquitoes (Diptera: Culicidae) are important vectors for a wide range of pathogenic organisms. As large parts of the human population in developed countries live in cities, the occurrence of vector-borne diseases in urban areas is of particular interest for epidemiologists and public health authorities. In this study, we investigated the mosquito occurrence in the city of Vienna, Austria, in order to estimate the risk of transmission of mosquito-borne diseases. Mosquitoes were captured using different sampling techniques at 17 sites in the city of Vienna. Species belonging to the Culex pipiens complex (78.8 %) were most abundant, followed by Coquillettidia richiardii (10.2 %), Anopheles plumbeus (5.4 %), Aedes vexans (3.8 %), and Ochlerotatus sticticus (0.7 %). Individuals of the Cx. pipiens complex were found at 80.2 % of the trap sites, while 58.8 % of the trap sites were positive for Cq. richiardii and Ae. vexans. Oc. sticticus was captured at 35.3 % of the sites, and An. plumbeus only at 23.5 % of the trap sites. Cx. pipiens complex is known to be a potent vector and pathogens like West Nile virus (WNV), Usutu virus (USUV), Tahyna virus (TAHV), Sindbis virus (SINV), Plasmodium sp., and Dirofilaria repens can be transmitted by this species. Cq. richiardii is a known vector species for Batai virus (BATV), SINV, TAHV, and WNV, while Ae. vexans can transmit TAHV, USUV, WNV, and Dirofilaria repens. An. plumbeus and Oc. sticticus seem to play only a minor role in the transmission of vector-borne diseases in Vienna. WNV, which is already wide-spread in Europe, is likely to be the highest threat in Vienna as it can be transmitted by several of the most common species, has already been shown to pose a higher risk in cities, and has the possibility to cause severe illness.

Bilateral lenticular Encephalitozoon cuniculi infection in a snow leopard (Panthera uncia).

Bilateral phacoclastic uveitis caused by lenticular infection with Encephalitozoon cuniculi is described in a snow leopard. The diagnosis was made on histopathological and immunohistological examination of both eyes submitted after postmortem examination. There was a positive antibody titer for E. cuniculi (1:320). Polymerase chain reaction (PCR) and sequence analysis of formalin-fixed, paraffin-embedded ocular tissue detected the DNA of E. cuniculi, strain III. No other systemic lesions attributable to the E. cuniculi infection were identified.

The detection of different Dirofilaria species using direct PCR technique.

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 µl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.

Immunology and pathogenesis of canine demodicosis.

Demodex mites colonized the hair follicles and sebaceous glands of mammals millions of years ago and have remained relatively unchanged in this protected ecologic niche since then. The host immune system detects and tolerates their presence. Toll-like receptor-2 of keratinocytes has been demonstrated to recognize mite chitin and to elicit an innate immune response. The subsequent acquired immune response is poorly understood at present, but there is experimental and clinical evidence that this is the main mechanism in the control of mite proliferation. A transgenic mouse model (STAT(-/-) /CD28(-/-) ) has demonstrated that the immune response is complex, probably involving both cellular and humoral mechanisms and requiring the role of co-stimulatory molecules (CD28). It is known that a genetic predisposition for developing canine juvenile generalized demodicosis exists; however, the primary defect leading to the disease remains unknown. Once the mite proliferation is advanced, dogs show a phenotype that is similar to the T-cell exhaustion characterized by low interleukin-2 production and high interleukin-10 and transforming growth factor-ß production by lymphocytes, as described in other viral and parasitic diseases. Acaricidal treatment (macrocyclic lactones) decreases the antigenic load and reverses T-cell exhaustion, leading to a clinical cure. Although in recent years there have been significant advances in the management and understanding of this important and complex canine disease, more research in areas such as the aetiology of the genetic predisposition and the immune control of the mite populations is clearly needed.

Indigenous Dirofilaria immitis in Bangladesh.

Dirofilaria immitis is a parasite of domestic and wild canids and felids in tropical, subtropical and temperate regions throughout the world. The canine heartworm (D. immitis) is the causative agent of canine and feline cardiopulmonary dirofilariasis. This parasite is known to cause a zoonotic disease, namely human pulmonary dirofilariasis. D. immitis is known to be endemic in several South and Southeast Asian countries (e.g. India and Malaysia), but there has previously been no information about the presence of this pathogen in Bangladesh. We present a case of canine dirofilariasis caused by D. immitis in rural southeastern Bangladesh. A male filaroid nematode (95 mm in length and 1.94 mm in width) was identified in the heart of a dog. Species classification was performed by microscopy and molecular tools. Sequence analysis revealed a 100 % identity within the mitochondrial cytochrome c oxidase I (CO1) gene to two Chinese and one Australian D. immitis samples. Usually, dogs stay outside overnight with a high risk to get infected with D. immitis via nocturnal mosquito vectors, which may lead to high prevalences of this pathogen in the canine population and thus increase the risk of human infections with this neglected parasitic disease.

The first case of Demodex gatoi in Austria, detected with fecal flotation.

Feline demodicosis is a rare parasitic condition caused by three different species of mites (Demodex cati, Demodex gatoi, and an unnamed species). D. gatoi inhabits the superficial skin layer (stratum corneum) and is easily transmitted between individual cats. A 2-year-old female spayed Cornish Rex was presented with alopecia and pruritus. The dermatological examination revealed bilateral alopecia and excoriations on trunk, limbs, and belly. The second cat in the household, a 3-year-old female spayed Thai, showed no clinical signs. Superficial and deep skin scrapings were performed and cellophane tapes applied, and living D. gatoi mites could be detected in both cats. Oral ivermectin (0.25 mg/kg every other day) was subscribed. Feces were collected from both cats and fecal flotation with sugar and zinc solutions performed. When compared to skin scrapings and cellophane tapes, D. gatoi was detected more frequently and in higher numbers in fecal samples. Our findings suggest that D. gatoi can be efficiently diagnosed with coproscopy, particularly in asymptomatic carrier animals. DNA was extracted from the flotation liquid, and a PCR protocol for the species verification was designed. A fragment targeting a 325-bp DNA fragment of the D. gatoi mitochondrial 16S rDNA gene was amplified with a 100% similarity to the D. gatoi entry in GenBank® (GI 421920216). We report the first finding of D. gatoi in Austria and propose fecal flotation as a valuable tool for mite detection. Fecal flotation liquid is suitable for DNA extraction and PCR-based species verification of D. gatoi.

Autochthonous Onchocerca lupi infection of a domestic dog in Austria.

Onchocerca lupi is an emerging canine ocular pathogen with zoonotic potential. In Europe, known endemic areas are the Iberian Peninsula and Greece, but the parasite has also been found in Romania, Hungary, and Germany. A 5-year-old Irish Wolfhound was presented in August 2021 with ocular discharge. A subconjunctival granulomatous nodule containing several nematode fragments was removed. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I gene confirmed the presence of O. lupi genotype 1. This is the first report of autochthonous O. lupi infection in a dog from Austria.

Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis.

The zoonotic nematode Thelazia callipaeda infects the eyes of domestic and wild animals and uses canids as primary hosts. It was originally described in Asia, but in the last 20 years it has been reported in many European countries, where it is mainly transmitted by the drosophilid fruit fly Phortica variegata. We report the autochthonous occurrence of T. callipaeda and its vector P. variegata in Austria. Nematodes were collected from clinical cases and fruit flies were caught using traps, netting, and from the conjunctival sac of one dog. Fruit flies and nematodes were morphologically identified and a section of the mitochondrial cytochrome c oxidase subunit I gene (COI) was analysed. A DNA haplotype network was calculated to visualize the relation of the obtained COI sequences to published sequences. Additionally, Phortica spp. were screened for the presence of DNA of T. callipaeda by polymerase chain reaction. Thelazia callipaeda and P. variegata were identified in Burgenland, Lower Austria, and Styria. Thelazia callipaeda was also documented in Vienna and P. variegata in Upper Austria and South Tyrol, Italy. All T. callipaeda corresponded to haplotype 1. Twenty-two different haplotypes of P. variegata were identified in the fruit flies. One sequence was distinctly different from those of Phortica variegata and was more closely related to those of Phortica chi and Phortica okadai. Thelazia callipaeda could not be detected in any of the Phortica specimens.

Diagnosis and successful treatment of an Austrian dog infected with Trypanosoma congolense forest type.

Canine African Trypanosomosis (CAT) is a rarely described disease with frequently lethal outcome. A 5-year-old female mongrel dog weighing 22 kg was presented in Austria due to unilateral uveitis, pancytopenia, and anorexia 4 months after return from a trip through Western Africa. Trypanosoma spp. flagellates were detected in a blood smear and identified as Trypanosoma congolense forest type by PCR. Initial treatment with imidocarb and miltefosine led to clinical improvement but only isometamidium chloride hydrochloride applied intramuscularly led to complete eradication of the pathogen from the dog's blood 4 months later.

A novel snapback primer probe assay for the detection and discrimination of sympatric Haemonchus species using DNA melting analysis.

Different sympatric species of Haemonchus parasites infecting ruminants and camels can be distinguished morphologically, but involves tedious microscopic examinations, measurements and several other limitations. Information on internal transcribed spacer-2 (ITS-2) sequence provides confirmatory differentiation of sympatric Haemonchus species. The present study introduces a novel, snapback primer probe based, real time PCR assay for the differentiation of three sympatric Haemonchus species, H. contortus (Hco), H. placei (Hpl) and H. longistipes (Hlo). The assay was designed to amplify a region of 130bp within the ITS-2 gene that included three diagnostic mutational sites capable of discriminating Hco, Hpl and Hlo. Following melt curve analysis, species-specific diagnostic melt peaks were obtained for Hco, Hpl and Hlo with a mean melting temperature of 56.6±0.3°C, 64.4±0.1°C and 54.4±0.1°C respectively. The test for analytical sensitivity revealed the ability of the assay to detect up to 5 copies per reaction. To evaluate the discriminating power of the assay, 174 samples from adult worms and 3rd stage larvae belonging to different Haemonchus species and various other nematode species including Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, and Teladorsagia circumcincta were tested. Additionally, DNA extracted from 25 fecal egg samples was also tested and the specificity of the assay was verified by sequencing the ITS-2 gene of all the Haemonchus positive and non-Haemonchus samples. The assay worked accurately with 100% specificity in at least three real time PCR platforms. The assay is an effective alternative to the sequencing approach and is expected to be helpful for the screening of individual adult and larval Haemonchus parasites. However, caution needs to be applied while interpreting the results from fecal egg samples due to varying levels of sympatric co-infections from different Haemonchus species. The present study is the first report on the application of snapback primer probe methodology for the differentiation of nematode parasites.

Dirofilaria in Humans, Dogs, and Vectors in Austria (1978-2014)-From Imported Pathogens to the Endemicity of Dirofilaria repens.

BACKGROUND: Dirofilaria repens and D. immitis are filarioid helminths with domestic and wild canids as main hosts and mosquitoes as vectors. Both species are known to cause zoonotic diseases, primarily pulmonary (D. immitis), ocular (D. repens), and subcutaneous (D. repens) dirofilariosis. Both D. immitis and D. repens are known as invasive species, and their distribution seems associated with climate change. Until very recently, both species were known to be nonendemic in Austria. METHODOLOGY AND PRINCIPAL FINDINGS: Metadata on introduced and possibly autochthonous cases of infection with Dirofilaria sp. in dogs and humans in Austria are analysed, together with analyses of mosquito populations from Austria in ongoing studies. In Austria, most cases of Dirofilaria sp. in humans (30 cases of D. repens-six ocular and 24 subcutaneous) and dogs (approximately 50 cases-both D. immitis and D. repens) were most likely imported. However, occasionally infections with D. repens were discussed to be autochthonous (one human case and seven in dogs). The introduction of D. repens to Austria was confirmed very recently, as the parasite was detected in Burgenland (eastern Austria) for the first time in mosquito vectors during a surveillance program. For D. immitis, this could not be confirmed yet, but data from Germany suggest that the successful establishment of this nematode species in Austria is a credible scenario for the near future. CONCLUSIONS: The first findings of D. repens in mosquito vectors indicate that D. repens presumably invaded in eastern Austria. Climate analyses from central Europe indicate that D. immitis also has the capacity to establish itself in the lowland regions of Austria, given that both canid and culicid hosts are present.

Detection, Prevalence and Phylogenetic Relationships of Demodex spp and further Skin Prostigmata Mites (Acari, Arachnida) in Wild and Domestic Mammals.

This study was conceived to detect skin mites in social mammals through real-time qPCR, and to estimate taxonomic Demodex and further Prostigmata mite relationships in different host species by comparing sequences from two genes: mitochondrial 16S rRNA and nuclear 18S rRNA. We determined the mite prevalence in the hair follicles of marmots (13%) and bats (17%). The high prevalence found in marmots and bats by sampling only one site on the body may indicate that mites are common inhabitants of their skin. Since we found three different mites (Neuchelacheles sp, Myobia sp and Penthaleus sp) in three bat species (Miotis yumanensis, Miotis californicus and Corynorhinus townsendii) and two different mites (both inferred to be members of the Prostigmata order) in one marmot species (Marmota flaviventris), we tentatively concluded that these skin mites 1) cannot be assigned to the same genus based only on a common host, and 2) seem to evolve according to the specific habitat and/or specific hair and sebaceous gland of the mammalian host. Moreover, two M. yumanensis bats harbored identical Neuchelacheles mites, indicating the possibility of interspecific cross-infection within a colony. However, some skin mites species are less restricted by host species than previously thought. Specifically, Demodex canis seems to be more transmissible across species than other skin mites. D. canis have been found mostly in dogs but also in cats and captive bats. In addition, we report the first case of D. canis infestation in a domestic ferret (Mustela putorius). All these mammalian hosts are related to human activities, and D. canis evolution may be a consequence of this relationship. The monophyletic Demodex clade showing closely related dog and human Demodex sequences also supports this likely hypothesis.

Screening blood-fed mosquitoes for the diagnosis of filarioid helminths and avian malaria.

BACKGROUND: Both Dirofilaria repens and recently D. immitis are known to be endemic in Hungary. As one of several recent cases, the fatal case of a dog infested with D. immitis in Szeged, Southern Hungary, received attention from the media. Hence it was decided to catch mosquitoes in the garden where the dog lived to screen for filarioid helminths and Plasmodium spp. using molecular tools. METHODS: Mosquitoes were caught in Szeged, in the garden where the infected dog was kept, in July 2013 with M-360 electric mosquito traps and were stored in ethanol until further procedure. Female mosquitoes were classified to genus level by morphology. Each mosquito was homogenized and analyzed for filarioid helminths and avian malaria using standardized PCR techniques. Positive mosquito samples were further identified to species level by comparing a section of the mitochondrial COI gene to GenBank® entries. RESULTS: In this study, 267 blood-fed mosquitoes were caught in July 2013 in Szeged. Subsequent molecular screening revealed that not only D. immitis was present in the analyzed specimens but also DNA of D. repens, Setaria tundra and Plasmodium spp. was confirmed. CONCLUSIONS: The analysis of blood-fed mosquitoes for the diagnosis of Dirofilaria spp. and other mosquito-borne pathogens seems to be an adequate technique to evaluate if filarioid helminths are present in a certain area. Usually only unfed female mosquitoes are analyzed for epidemiological studies. However, blood-fed mosquitoes can only be used for screening if a pathogen is present because the role of the mosquito as vector cannot be classified (blood of bitten host). Furthermore, Setaria tundra was confirmed for the first time in Hungary.

Trypanosomosis: potential driver of selection in African cattle.

Trypanosomosis is a serious cause of reduction in productivity of cattle in tsetse-fly infested areas. Baoule and other local Taurine cattle breeds in Burkina Faso are trypanotolerant. Zebuine cattle, which are also kept there are susceptible to trypanosomosis but bigger in body size. Farmers have continuously been intercrossing Baoule and Zebu animals to increase production and disease tolerance. The aim of this study was to compare levels of zebuine and taurine admixture in genomic regions potentially involved in trypanotolerance with background admixture of composites to identify differences in allelic frequencies of tolerant and non-tolerant animals. The study was conducted on 214 animals (90 Baoule, 90 Zebu, and 34 composites), genotyped with 25 microsatellites across the genome and with 155 SNPs in 23 candidate regions. Degrees of admixture of composites were analyzed for microsatellite and SNP data separately. Average Baoule admixture based on microsatellites across the genomes of the Baoule- Zebu composites was 0.31, which was smaller than the average Baoule admixture in the trypanosomosis candidate regions of 0.37 (P = 0.15). Fixation index F ST measured in the overall genome based on microsatellites or with SNPs from candidate regions indicates strong differentiation between breeds. Nine out of 23 regions had F ST ≥ 0.20 calculated from haplotypes or individual SNPs. The levels of admixture were significantly different from background admixture, as revealed by microsatellite data, for six out of the nine regions. Five out of the six regions showed an excess of Baoule ancestry. Information about best levels of breed composition would be useful for future breeding ctivities, aiming at trypanotolerant animals with higher productive capacity.

Proteomics elucidates key molecules involved in exsheathment in vitro in Oesophagostomum dentatum.

We explored molecules involved in in vitro exsheathment of Oesophagostomum dentatum L3s using a proteomic-transcriptomic-bioinformatic approach. Analysis of L3s before, during and after exsheathment identified 11 proteins that were over-expressed exclusively during exsheathment. These proteins (including key enzymes, heat shock, structural and nematode-specific proteins) were inferred to be involved in development, metabolism, structure, motility and/or host-parasite interactions. Some of these molecules represented homologues linked to entry into and exit from the dauer stage in the free-living nematode Caenorhabditis elegans. The approach established here provides a basis for investigations of ecdysis in other strongylid nematodes.

Autochthonous Dirofilaria repens in Austria.

BACKGROUND: In Europe animal and human infections due to Dirofilaria repens are increasing. FINDINGS: In a nationwide screening for filarioid parasites in Austria, 7,632 mosquitoes were collected from June till October 2012 and divided into 437 pools according to same trapping date and sight and mosquito species. For the molecular detection, a real-time PCR approach was followed by conventional PCR. D. repens was detected in the villages Moerbisch and Rust, Burgenland in one Anopheles maculipennis group and one Anopheles algeriensis species pool, respectively. CONCLUSIONS: The geographical distribution of the two positive pools points to the invasion of D. repens from Eastern neighboring countries. The finding of D. repens in mosquito vectors suggests the occurrence of the causative agent for cutaneous dirofilariosis in Austria.

A novel qPCR assay for the detection of African animal trypanosomosis in trypanotolerant and trypanosusceptible cattle breeds.

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource.

Detection of Plasmodium sp.-infested Anopheles hyrcanus (Pallas 1771) (Diptera: Culicidae) in Austria, 2012.

On July 15, 2012, adult Anopheles hyrcanus (Pallas 1771) mosquitoes were caught next to a farm barn near Rust, Burgenland, close to Lake Neusiedl National Park in eastern Austria. Six weeks later, adults of this invasive species were also found in a sheep shelter outside the village of Oggau and another 2 weeks later, in a horse barn in Mörbisch. The morphological typing was confirmed genetically by amplification and sequencing of a 1,404-bp-long fragment within the 5.8S ribosomal RNA gene, the internal transcribed spacer 2, and the 28S ribosomal RNA gene. Out of two A. hyrcanus pools analyzed, one was found positive for Plasmodium sp. A 460-bp-long sequence within the mitochondrial cytochrome b region revealed 100 % identity to a sequence of a Plasmodium parasite identified in a New Zealand bellbird (Anthornis melanura). The Austrian finding sites are close to the Hungarian border. In Hungary, the occurrence of A. hyrcanus was already reported in 1963. A. hyrcanus is considered the most important potential vector of malaria in southern France today. In Austria, sporadic autochthonous malaria cases could emerge, caused by immigration from malaria-endemic countries and heavy tourism. However, the broad population coverage of the Austrian health care system makes the reestablishment of endemic areas for malaria unlikely.