Current trends in canine dirofilariosis in Austria-do we face a pre-endemic status?

A retrospective study based on cases of canine dirofilariosis presented to the University of Veterinary Medicine, Vienna or diagnosed by private practitioners throughout Austria, from 1998 to 2018 was conducted to investigate the long-term development and current state of canine dirofilarial infections in Austria. Included in this study were 146 dogs which were tested positive for D. immitis and/or D. repens. The most commonly used diagnostic methods and the probable geographical origins of the infections were evaluated and the treatment protocols applied were compared with each other and with the literature. The results show that most infections were found due to screening for common travel infections using antigen-ELISA or PCR-testing, or by the incidental finding of microfilariae. Remarkably, only 24.3% of all cases presented showed clinical signs indicating canine dirofilariosis. Regarding the origin and travel history of the dogs, thirteen different countries could be identified. The three treatment protocols used showed a similar outcome after 8 months of treatment and minor side effects, which is consistent with the literature. An alarming increase in reported infections with both D. immitis and D. repens in Austria was noted since 2014. The number of documented cases had almost tripled by 2018, raising severe concerns about the threat of it becoming endemic in Austria. Therefore, the existing recommendations in current guidelines regarding canine dirofilariosis should be widely publicised and more strictly enforced. Prophylactic measures for dogs travelling abroad and diagnostic and therapeutic strategies for dogs imported from endemic countries should be obligatorily established throughout Europe, to reduce the risk of further spread of canine filarial infections to non-endemic regions.

Human seroprevalence data indicate other factors than climatic conditions influencing dirofilariosis transmission in the Russian Federation.

The species of the genus Dirofilaria are filarial parasites causing zoonotic infections in humans with an increasing incidence in temperate and tropical areas of the world. Due to its classification as a vector-borne disease, the most important factors influencing dirofilariosis transmission are those related to climate, such as temperature and humidity. However, other factors linked with human behaviour can influence the distribution of the parasite. Although the Russian Federation could be considered as a non-suitable area for Dirofilaria spp. transmission due to its climatic conditions, one third of the human cases of dirofilariosis have been declared in this country. Here, seroepidemiological data on human dirofilariosis for five different regions distributed throughout the Russian Federation (Rostov, Moscow, Ekaterinburg, Yakutia and Khabarovsk) were obtained. A total of 940 serum samples from totally random donors living in these areas were analysed by enzyme-linked immunosorbent assay for the detection of anti-Dirofilaria immitis immunoglobulin G antibodies. Similar seroprevalence data ranging from 3.41% in Yakutia to 6.95% in Khabarovsk, with no significant correlation with climatic data of yearly average temperature and rainfall from these regions were found. These results suggest that other factors probably related to human behaviour, and not only climatic conditions, might be facilitating the spread of human dirofilariosis in these areas.

Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

Two cases of subcutaneous dirofilariasis in Barcelona, Spain.

In recent years, the number of reported cases of human dirofilariasis in Europe has increased and the circulation of Dirofilaria spp. in mosquitoes in several European countries has been proven. We report here two likely autochthonous cases of subcutaneous human dirofilariasis from Barcelona, Spain, caused by Dirofilaria repens. The potential for an increase in human infection is high given the number of cases published recently and the ability of vectors to spread through the Mediterranean basin.

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

[(HUMAN DIROFILARIASIS: CLINICAL DIAGNOSTIC FEATURES ASSOCIATED WITH DIFFERENT DEVELOPMENTAL STAGES OF THE PATHOGEN)].

To seek medical advice is due to the time when a person has become infected during the infection transmission season (July-August) and the duration of development of the pathogen Dirofilaria (N.) repens in his/her body Clinical manifes- tations occurred in 61% of the patients 6-10 months after infection, as confirmed by the maximum body sizes (125-160 mm) of removed females which have reached puberty. PCR-based diagnosis in conjunction with microscopic studies improves the efficiency of identifying the patients and the species of the pathogens D.repens and D.immitis. The use of these methods for the first time in 2016 could confirm D.immitis infestation in a 14-month-old infant living in the Solnechnogorsk District, Moscow Region.

Molecular detection of Wolbachia pipientis in natural populations of mosquito vectors of Dirofilaria immitis from continental Portugal: first detection in Culex theileri.

Wolbachia pipientis (Rickettsiales: Rickettsiaceae) protects mosquitoes from infections with arboviruses and parasites. However, the effect of its co-infection on vector competence for Dirofilaria immitis (Spirurida: Onchocercidae) in the wild has not been investigated. This study aimed to screen vectors of D. immitis for wPip, to characterize these, and to investigate a possible association between the occurrence of W. pipientis and that of the nematode. The presence of W. pipientis was assessed in the five mosquito potential vectors of D. immitis in Portugal. Polymerase chain reaction (PCR) products were sequenced, and wPip haplotypes were determined by PCR-restricted fragment length polymorphism (RFLP). Results showed that wPip was detected in 61.5% of Culex pipiens (Diptera: Culicidae) pools and 6.3% of Culex theileri pools. wPip 16s rRNA sequences found in Cx. theileri exactly match those from Cx. pipiens, confirming a mosquito origin, rather than a nematode origin, as some specimens were infected with D. immitis. Only wPip haplotype I was found. No association was found between the presence of wPip and D. immitis in mosquitoes and hence a role for this endosymbiont in influencing vectorial competence is yet to be identified. This study contributes to understanding of wPip distribution in mosquito populations and, to the best of the authors' knowledge, is the first report of natural infections by wPip in Cx. theileri.

Detection of Dirofilaria repens and Dirofilaria immitis DNA in mosquitoes from Belarus.

During the last two decades, Belarus faces an increase of human cases of Dirofilaria (Nematoda, Spirurida, Onchocercidae) infections. However, comprehensive analyses explaining this development and the identification of mosquito vector species are missing. Here, we present results using temperature data from Belarus and show that the annual number of human Dirofilaria cases is significantly correlated with the yearly average temperatures (Spearman's rho = 0.49, p < 0.05) and the average sum of potential Dirofilaria transmission days (Spearman's rho = 0.46, p < 0.05), suggesting that autochthonous transmission is at least in part responsible for the increasing number of clinical Dirofilaria cases in the country. In addition, 467 female mosquitoes were collected from different sampling sites in the regions of Brest and Minsk, which were analyzed by molecular methods for the presence of Dirofilaria repens and Dirofilaria immitis DNA, respectively. Two pools (5.56 %) were tested positive for Dirofilaria (estimated infection rate per 100 specimens = 0.44, 95 % confidence interval = 0.08-1.43), comprising one Anopheles claviger s.l. pool that was positive for D. repens and one Culex pipiens s.l./Culex torrentium pool positive for D. immitis DNA. This, to our knowledge, is the first molecular evidence for the presence of Dirofilaria in mosquitoes from Belarus, suggesting a high probability of autochthonous Dirofilaria transmission in the country.

[CURRENT IDEAS ON OBLIGATE AND FACULTATIVE RELATIONSHIPS BETWEEN MAN AND THE DIROFILARIASIS PATHOGEN DIROFILARIA (N.) REPENS].

The ability of D. repens to complete its ontogenesis in man points to their obligate, rather than facultative rela- tionships. The fact that microfilariae are rarely found in human blood or are absent there may be associated with the removal of developing dirofilariae from humans in earlier than they achieve sexual maturity. Facultative ecological relationships to mosquitoes may be one of the reasons for limitation of human invasion cases. However, in long-standing microfilaremia in man (an obligate host), D.repens may take part in the epidemiological chain of dirofilariasis as a source of invasion.

[CIRCULATION OF DIROFILARIA REPENS (RAILLIET ET HENRY, 1911) IN THE ARID ZONE OF SOUTHERN RUSSIA].

The prevalence of dirofilariasis was studied in dogs from the urban and rural areas in Southern Russia. The high prevalence of Dirofilaria repens infection were established among the dogs in the Republic of Kalmykia. The blood samples from 328 dogs were tested; D. repens infection was detected in 23.6 and 15.5% of the rural and urban dogs, respectively. The highest prevalence of D. repens infection was noted in of 4-6-year-old dogs. Dogs aged 0, 1-3, 4-6 7-9, and more 10 years were infected in 0, 26.3, 33.3, 29.4, and 28.5% of cases, respectively.

[INFECTION OF BLOOD-SUCKING MOSQUITOES (DIPTERA: CULICIDAE) WITH DIROFILARIAE (SPIRURIDA, ONCHOCERCIDAE) IN THE TULA REGION].

Blood-sucking mosquitoes (n = 2277) collected in Tula and its Region in 2013-2014 were examined using a PCR assay for dirofilariae. A total of 12 species from 4 genera (Culiseta, Aedes, Ochlerotatus [foreign character] Culex) out of 18 found mosquito species were infected with Dirofilaria immitis and D. repens. The proportion of the infected mosquitoes was 2.5% (D. immitis, 1.5%; D.repens, 1%). According to preliminary data, the most efficient Dirofilaria vectors, in the Tula Region may be Ae. vexans, Ae. geniculatus, Och. cantans, and Cx. pipiens.

Potential mosquito (Diptera:Culicidae) vector of Dirofilaria repens and Dirofilaria immitis in urban areas of Eastern Slovakia.

This paper follows the study from 2013 focused on the molecular screening of mosquitoes as vectors of Dirofilaria spp. which provided the information on Aedes vexans as a potential vector of Dirofilaria repens in Slovakia. Current entomological and molecular research indicates that Ae. vexans can participate also in the transmission of Dirofilaria immitis within the region. Using the standard PCR method, we examined 10,500 mosquitoes (Ae. vexans, Ae. rossicus, Anopheles maculipennis s.l., Ochlerotatus sticticus, Ochlerotatus cantans, Ochlerotatus caspius, Culex pipiens/Culex torrentium, Coquillettidia richiardii), collected using CO2-baited traps at six locations in the Eastern Slovakia. Out of 105 pools, 6 pools of mosquitoes Ae. vexans were positive for D. repens DNA (minimum infective rate in Ae. vexas was 6:6.900, i.e. 0.8 per 1.000 mosquitoes), within which 4 were concurrently positive for D. immitis (minimum infective rate in Ae. vexans was 4:6.900 i.e. 0.5 per 1.000 mosquitoes).

Risk factors for human heartworm infections (dirofilariasis) in the South.

Human pulmonary dirofilariasis (HPD) is caused by the transmission of infective third stage larvae of the canine heartworm, Dirofilaria immitis, during blood-feeding by several species of infected mosquitoes. Since humans are incidental hosts and cannot support the parasite's life cycle, infective larvae die after migrating to the pulmonary vascular bed, where an initial subclinical inflammatory reaction is typically followed by a single pulmonary granuloma. The resulting nodular granuloma is described radiographically as a "coin lesion" that resembles a neoplastic lesion, which must be ruled out by invasive lung biopsy. Since HPD cases have been reported mainly from regions with high canine heartworm prevalence, such as the southern United States (US), the objectives of this review were (1) to describe the microbiology of the parasite; (2) to resolve any misconceptions regarding the pathophysiology and outcomes of canine versus human heartworm infections; (3) to describe the prevalence and parasite burden of canine dirofilariasis in the South compared to other areas; (4) to describe the prevalence of HPD in the South; (5) to identify the most important species of mosquito vectors of dirofilariasis based on seroprevalence rates of infection and transmission efficiency; (6) to identify the key risk factors for HPD in the South; and (7) to recommend new strategies for the diagnosis, management, control, and prevention of HPD. Future investigations should focus on targeting specific mosquito species for improved vector control of D. immitis transmission and on developing new immunologic and molecular methods for diagnosing HPD and eliminating the need for invasive diagnostics for differential diagnosis of innocuous, parasitic "coin lesions".

[BLOODSUCKING MOSQUITOES (DIPTERA: CULICIDAE) IN, THE TULA REGION ARE POTENTIAL VECTORS OF DIROFILARIAS].

Bloodsucking mosquitoes were collected in Tula and its Region in May to August 2013-2014. The fauna included 17 species from 5 genera in the subfamily Culicinae and Anopheles maculipennis complex in the subsystem Anophelinae. Ochlerotatus cantans was a dominant species in the collections. The dominant species also included Aedes einereus, Ae. vexans, Ae. geniculatus, Och. diantaeus, Och. intrudens, Och. Cataphylla, and Culex pipiens. The possible value of different mosquito species Dirofilaria repens and D. immitis as vectors of dirofilarasis was discussed.

Comparison of the vector potential of different mosquito species for the transmission of heartworm, Dirofilaria immitis, in rural and urban areas in and surrounding Stillwater, Oklahoma, U.S.A.

Dirofilaria immitis Leidy (Spirurida: Onchocercidae), or heartworm, is a mosquito-borne nematode that causes a fatal disease in carnivores. Although infection is preventable through prophylactic drugs, compliance and the spectre of resistance suggest vector control is a viable alternative. There were two main objectives in this study: (a) to evaluate the relationships between landscape and social factors and the number and species of heartworm-positive mosquitoes, with a specific focus on the importance of the invasive Asian tiger mosquito, Aedes albopictus (Skuse) (Stegomyia albopicta) (Diptera: Culicidae), and (b) to test the hypothesis that dog heartworm is more prevalent in suburban than in rural areas. To achieve these objectives, mosquitoes were collected from May to November 2010 at 16 rural and 16 urban locations in Payne County, Oklahoma, U.S.A. using three trapping methods that utilized, respectively, resting boxes, carbon dioxide traps and BG Sentinel traps. Urban areas showed greater numbers of Ae. albopictus and a higher overall likelihood of infection with D. immitis. Because many species of mosquito are responsible for heartworm transmission, current prophylactic treatment remains the best method of controlling this parasite.

Dirofilaria repens and Dirofilaria immitis DNA findings in mosquitoes in Germany: temperature data allow autochthonous extrinsic development.

After the repeated demonstration of Dirofilaria repens infections in German dogs, D. repens and Dirofilaria immitis DNA was detected in mosquitoes trapped in 2011, 2012 and 2013 in northeastern and southwestern Germany within the framework of culicid monitoring projects. As temperature is the most important factor dictating the extrinsic development of dirofilarial larvae in the potential vector, data of the German Weather Service (DWD) were analysed for the regions where the positive mosquitoes had been collected. Based on the mean daily temperatures recorded by weather stations most closely to the collection sites of the mosquitoes, it can be concluded that the mosquitoes were trapped in time periods that allowed for the completion of the developmental cycle of the worms in the mosquitoes and a subsequent transmission to a vertebrate host. The results of this study confirm the principal climatic suitability of certain German regions for the establishment of natural dirofilarial transmission cycles. Moreover, the theoretical climatic considerations, together with findings of D. repens infections in German dogs and mosquitoes, strongly suggest that the continuing spread of at least D. repens from its traditional habitats in the Mediterranean has reached southwestern and northeastern Germany.

Zoonotic Dirofilaria repens (Nematoda: Filarioidea) in Aedes vexans mosquitoes, Czech Republic.

The surveillance of vectors for arthropod-borne pathogens is nowadays an important tool in surveillance programmes throughout Europe. Whereas many studies have been performed to screen arthropods for viruses or bacterial pathogens, only limited information is available concerning the geographical distribution and vector range of pathogenic filariae in Central Europe. To consider the prevalence of filarial parasites in mosquito vectors, we performed a molecular survey of mosquitoes for filarial DNA. Mosquito collection was conducted at six study sites in the South Moravian region (Czech Republic) close to the borders with Slovakia and Austria from 2009 to 2011. Molecular screening of mosquitoes was conducted using conventional PCR with primers designed to amplify the mitochondrial cytochromoxidase subunit I gene as well as the partial 5.8S ribosomal RNA gene. A total of 13,222 mosquitoes belonging to six species were captured and distributed into 237 pools with different numbers of individuals. Overall, four pools were positive for Dirofilaria repens (a minimum infection rate 0.03%) at two study sites (both natural and urban). Another filarial parasite detected during a study into Aedes vexans mosquitoes revealed the closest homology to Setaria spp. We detected specific D. repens DNA in Ae. vexans mosquitoes for the first time in the Czech Republic and confirmed the circulation of Dirofilaria spp. in a natural focus of infection providing an epidemiological link between autochthonous canine cases and mosquito vectors in the area studied.

[Entomological monitoring of an area to assess Dirofilaria transmission risk].

As the basis for entomological monitoring, the authors first propose to investigate the structure of a season of D. repens invasion transmission in the carrier in relation to the ambient temperature, such as onset of a transmission season, terminaton of mosquito infection, the number of invasion circulations. A calculating procedure has been developed. It is shown that there is a need for entomological monitoring of each specific area irrespective of the latitude to study a risk for local dirofilariasis cases.