Dirofilaria immitis and Dirofilaria repens in mosquitoes from Corsica Island, France.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are the main causative agents of heartworm disease and subcutaneous dirofilariasis in domestic and wild canids, respectively. Both pathogens have zoonotic potential and are transmitted by mosquitoes. The present study aimed to determine the transmission period, prevalence and diversity of Dirofilaria spp. vectors from endemic areas of Corsica (France). METHODS: A monthly point data model based on average temperature recorded by four meteorological stations during 2017 was used to calculate the Dirofilaria transmission period. From June to September 2017, female mosquitoes (n = 1802) were captured using Biogents® Sentinel 2 traps lured with carbon dioxide and BG-Lure™ or octanol. Mosquitoes were identified to species level, pooled accordingly, and screened using multiplex real-time qPCR to detect D. immitis and D. repens. RESULTS: The monthly point data model showed the possible transmission of Dirofilaria spp. from the third week in May to the last week in October in the studied area. Mosquitoes were identified as Ochlerotatus caspius (n = 1432), Aedes albopictus (n = 199), Culex pipiens sensu lato (n = 165) and Aedes vexans (n = 6) and were grouped into 109 pools (from 1 to 27 specimens, mean 11.4 ± 0.7), of which 16 scored positive for Dirofilaria spp. (i.e., n = 13; estimated infection rate [EIR] = 1.1% for D. immitis and n = 3; EIR = 0.2% for D. repens). Specifically, 6 (i.e., EIR = 3.8%) of 15 pools of Ae. albopictus were positive for D. immitis, 2 of 14 of Cx. pipiens s.l. were positive for D. immitis and D. repens, respectively, and 8 of 77 pools of Oc. caspius were positive for D. immitis (i.e., n = 6; EIR = 0.4%) and D. repens (i.e., 2; EIR = 0.1%). The highest mosquito infection rate was recorded in July (EIR = 2.5%), then in June (EIR = 1.3%) and September (EIR = 0.6%). CONCLUSIONS: The data suggest that both Dirofilaria species are endemic and occur possibly in sympatry in the studied area in Corsica, highlighting the need to implement preventive chemoprophylaxis and vector control strategies to reduce the risk of these filarioids in dog and human populations.

The prevalence of Dirofilaria immitis and D. repens in the Old World.

Dirofilaria immitis and D. repens are endemic throughout Europe and southern eastern regions of Asia and reported with increasing frequency in Africa. Nevertheless, the increased awareness of veterinary practitioners, even in countries where the prevalence is low, has led to a decrease D. immitis prevalence in dogs, especially in previously endemic/hyper-endemic areas. Prevalence has significantly increased, however, in areas where heartworm has apparently spread more recently, such as Central and North Eastern Europe. Furthermore, autochthonous cases have been observed in Siberia. Low seroprevalence has been reported in Croatia, while in Romania it has reached 14%. In Greece, the prevalence ranges between 0.7% and 25% whilst in Turkey is 0-18%. Data for canine dirofilariosis in Africa is scarce, and most are case reports. Overall, the dominant species is Achanthocheilonema dracunculoides, although both D. immitis and D. repens have been reported from some countries. In the Far East, the prevalence ranges from 2% to 15% in northeastern of China. In Hong Kong a novel species has been found in dogs and humans (Candidatus Dirofilaria hongkongensis). In India, the prevalence ranges from 4.7%-29.5% in Northeastern states. The main factors that have influenced the spreading of Dirofilaria infections are the climate changes and the introduction of new, invasive, competent mosquito species such as Aedes albopictus and Ae. koreicus. Other factors include relocation and insufficient prevention in dogs, manly in the new areas of colonization. Feline heartworm infection has been diagnosed in every European country when diagnosed either by the Knott test or by serology for circulating antibodies and antigens of the parasite. However, prevalence is much lower than in dogs. In spite of the continuing spreading of heartworm infection, D. repens is the main concern in Europe, mostly for physicians, while the infection is nearly always asymptomatic in dogs. The infection is spreading from Portugal to the Southeastern regions of Finland and Siberia, and in some areas its prevalence overlaps that of D. immitis. Many reasons make more difficult the control of D. repens than D. immitis: the frequent lack of clinical symptoms of suspicion, the specific diagnosis being possible only by blood examination and the inefficacy of some macrocyclic lactones.

Dirofilaria and Wolbachia in mosquitoes (Diptera: Culicidae) in central European Russia and on the Black Sea coast.

Dirofilariasis is endemic in Russia, as well as in many other European countries. The aim of this study was to assess the ability of mosquitoes to transfer Dirofilaria immitis and Dirofilaria repens in regions with temperate and subtropical climates. The possible impact of the symbiotic bacterium Wolbachia on Dirofilaria transmission was also investigated. 5333 female mosquitoes were collected at 11 points in central European Russia and on the Black Sea coast during the period 2013-2017. Out of 20 mosquito species examined, 14 were infected with D. repens and 13 with D. immitis. Both species of Dirofilaria were found in different climatic regions. The total Dirofilaria spp. estimated infection rate (EIR) in the central part of Russia varied from 3.1% to 3.7% and, in the southern region, from 1.1% to 3.0%. The highest estimated infection rate was found in Anopheles messeae, the lowest in Culex pipiens. The greatest epidemiological danger was represented by Aedes aegypti, Ae. geniculatus, An. messeae and Ae. communis. Six out of 20 mosquito species were infected with Wolbachia. Pools of Aedes albopictus, Cx. pipiens and Coquillettidia richiardii were simultaneously infected with Dirofilaria and Wolbachia. After checking mosquitoes individually, it was found that there was no development of Dirofilaria to the infective larval stage in specimens infected with Wolbachia. Twenty-two Dirofilaria-infective pools were Wolbachia-free and only two mosquito pools were Wolbachia-infected. The potential for transmission of Dirofilaria in mosquito species naturally uninfected with the symbiotic bacterium Wolbachia is higher than in species infected with the bacterium.

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.

Peripheral venous vs. capillary microfilariaemia in a dog co-infected with Dirofilaria repens and D. immitis: A comparative approach using triatomine bugs for blood collection.

Dirofilaria immitis and D. repens are mosquito-borne nematodes, primarily infecting dogs, but also other species of carnivores and even humans. Given their impact on animal and human health, the transmission of these filarioids has been widely studied. The microfilariaemia has been shown to have a circadian variation for both Dirofilaria species infecting dogs. Due to methodological difficulties, the periodicity was only studied using venous blood samples, while the mosquitoes feed, in fact, on capillary blood. In this context, the present study aimed to test the feasibility of using triatomine bugs for the collection of capillary blood and to comparatively evaluate the level of microfilariaemia and its circadian variation in capillary blood vs. peripheral venous blood in a dog naturally co-infected with D. immitis and D. repens. The results showed a feeding success of 50%, with variations in the blood meal volume that the bugs ingested. The relative values of microfilariaemia (mf/bug) were strongly correlated with the volume of blood recovered: the more blood recovered from each bug, the higher values of microfilariaemia in the evening samples while the opposite results were obtained for the morning samples. The counting of microfilariae revealed a dominance of D. immitis in all the samples, but with significantly higher microfilariaemia in the venous blood. Meanwhile, for D. repens, the situation was opposite, with higher counts in the capillary blood samples. Our study showed that triatomine bugs can be used as a model for the collection and study of microfilariaemia in the capillary blood in mammals.

Subcutaneous dirofilariosis (Dirofilaria repens): an infection spreading throughout the old world.

BACKGROUND: Two main Dirofilaria species infect dogs: D. immitis and D. repens. While D. immitis has a worldwide distribution, D. repens is currently found only in Europe, Asia, and Africa. Adult D. repens are located in subcutaneous tissues of natural hosts where they survive for long periods of time. First-stage larvae, microfilariae, circulate in the peripheral bloodstream, where they are taken up by the mosquito intermediate hosts. Infected mosquitoes then transmit infective third-stage (L3) larvae to new hosts through the blood meal. In dogs, most infections are asymptomatic, although cutaneous disorders such as pruritus, dermal swelling, subcutaneous nodules, and ocular conjunctivitis can be observed. Currently, two factors have increased the concerns about this parasitic infection 1) its spread throughout the European countries and to other continents and its prevalence in dog populations, where in some cases it has overcome D. immitis; and 2) its zoonotic potential, which is much greater than that of D. immitis. RESULTS: Different hypotheses can be put forward to explain these concerns. First, climate change has allowed more favorable conditions for survival of culicid vectors. Second, accidental hosts such as humans may have a less efficient immune reaction against a parasite that is located in subcutaneous tissues, and thus less exposed to the host's immune response than, for instance, D. immitis. Furthermore, the absence of clinical signs in the majority of canine infections and the difficulty in diagnosing the infection, due to the lack of serologic tests and thus the reliance on the identification of microfilariae and differentiation from D. immitis to confirm the presence of the parasite, favor the further spread of this species. Finally, among the macrocyclic lactones currently used to prevent heartworm infection, only moxidectin has been found to be fully effective against the infective larvae transmitted by mosquitoes and partially effective (efficacy 96%) against adult D. repens in experimental studies. CONCLUSIONS: Dirofilaria repens infection is much more difficult than D. immitis to diagnose and control in the reservoir population (microfilaremic dogs). In addition, lack of familiarity with D. repens infection could lead to lack of vigilance underestimation for this parasite. The number of human cases in Europe and Asia is currently a serious public health concern. Medical doctors and veterinarians must collaborate closely for better control and surveillance of D. repens infection.

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. METHODS: Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott's test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. RESULTS: The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. CONCLUSIONS: Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus.

BACKGROUND: The mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes. METHODS: For this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates. RESULTS: In field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species. CONCLUSIONS: To our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

Molecular Study of Dirofilaria immitis and Dirofilaria repens in Dogs from Tunisia.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne nematodes which infect primarily dogs as their main definitive hosts. They cause cardiopulmonary (D. immitis) or cutaneous (D. repens) dirofilariasis in canids and other carnivores and can accidentally be transmitted to humans where they can induce a variety of clinical outcomes depending on organ localization. Dirofilaria spp. infection in dogs was assessed using molecular methods (PCR and sequencing) to identify the different Dirofilaria species occurring in 200 dogs from Northern and Central Tunisia. The overall molecular prevalence of Dirofilaria spp. was 17.5% (35/200). The prevalence of D. immitis (14.5%) was significantly higher than for D. repens (3%). Molecular prevalence of D. immitis was significantly higher in suburban compared to urban and rural regions. There was no difference in molecular prevalence of D. immitis or D. repens according to the dogs' (sex or use). Dirofilaria immitis amplicons (accession numbers KR676386) fall into the same clade with D. immitis from China, India and Taiwan. Comparison of the partial sequences of D. repensITS2 rDNA gene (KR676387) revealed 99.6% similarity with D. repens reported in dogs from USA. It had also 97.6% similarity with D. repens from mosquitoes in Czech Republic. High dog parasite burdens should motivate both medical doctors and veterinarians to consider these frequent infections.

Morphological and molecular detection of canine dirofilarial species of veterinary and medical importance in Morogoro municipality, Tanzania.

A descriptive cross-sectional survey of Dirofilaria infections in dogs was carried out from January to March 2015 in Morogoro municipality, Tanzania. One hundred and fifty two blood samples were collected from healthy dogs aged more than 6 months living in different areas of Morogoro, and analyzed by modified Knott's technique for circulating microfilariae. Microfilaraemic samples were further analyzed by Polymerase chain reaction (PCR) and PCR products were sequenced for molecular identification. Microfilariae were detected by microscopy in 9 samples (5.92%), of which 6 tested positive by PCR. The 5.8S-internal transcribed spacer 2 (ITS2)-28S ribosomal DNA (rDNA) sequences generated were 97% identical to Dirofilaria immitis and 86% to 90% identical to D. repens, confirming the presence of D. immitis in Tanzania and showing the presence of D. repens, not previously observed.

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).

A Rare Human Case of Dirofilaria repens Infection in the Subcutaneous Posterior Thorax with Molecular Identification.

The emergence of Dirofilarial infections in Asia including Vietnam is a clinically significant threat to the community. We here report a rare case of subcutaneous Dirofilaria repens infection on the posterior thoracic wall in a young woman presenting a painful, itchy, and palpable nodule. The adult worm was identified by mitochondrial cox1 and nuclear ITS-2 sequence determination. The diagnosis was additionally confirmed by 16S rRNA sequencing of the endosymbiont Wolbachia pipientis commonly co-existing with D. repens. This is a rare case of subcutaneous human infection on the posterior thoracic region caused by D. repens.

Climate suitability for the transmission of Dirofilaria immitis and D. repens in Germany.

Recently concerns are increasing that dirofilarial nematodes may spread from endemic areas in southern, eastern and central Europe to countries in northern regions of Europe. The increasing number of autochthonous cases of canine Dirofilaria repens infections in Germany indicates that worms of this genus may invade new areas, and climate change may be a key factor in this scenario. Thus analysis of long term development of regional temperature is a pivotal factor in risk analysis related to transmission of these worms. Such information is important for suggestions of counteracting strategies, such as definition of periods of increased transmission risk and, consequently, time slots most suited for preventative measures. In this study, mean daily temperature data from 34 geographical clustered weather stations representing all parts of Germany were analyzed. It is concluded that the increasing trend for average daily temperatures observed in the period from 1984 to 2013 has led to climatic conditions that allow the completion of dirofilarial life cycles in large parts of Germany between May and October. Autochthonous infection with D. repens is already established in some regions and targeted diagnosis and medical prophylaxis is advisable for dogs assumedly exposed during risk of transmission periods. It appears likely that global warming will support further spread of D. repens. Furthermore for the population of dogs the spread of the more pathogenic species D. immitis in hitherto non-endemic Germany is a potential risk if mean temperatures rise to a level suitable for parasite development in the abundant vector mosquitoes during the warmer seasons.

Human Dirofilaria repens infection of the zygomatico-temporal region.

A 40-year old patient developed a painless swelling in the left zygomatico-temporal region. Magnetic resonance images and computed tomograms showed a non-specific soft tissue mass in the tumour region, but no invasion of bone. After application of antibiotics, the tumour reduced in size but a firm mass of about 3 cm in maximum diameter persisted under therapy. Surgical exploration revealed a distinct firm mass adhering to the superficial layer of the temporal muscle. Histological and molecular biological investigations demonstrated fragments of Dirofilaria repens in the centre of the lesion. Clinical follow-up was uneventful and additional investigations excluded further manifestations of the parasite. D. repens infections are extremely rare in northern Europe, but recent reports about the increase of human dirofilariasis in northern parts of Europe should alert the clinician to include helminthoses in the differential diagnosis of atypical space-occupying lesions of the maxillofacial regions.

Is Dirofilaria repens endemic in the Havelland district in Brandenburg, Germany?

In Brandenburg, northeastern Germany, an outbreak of zoonotic Dirofilaria repens in a sledge dog kennel was detected in January, 2012. Using the modified Knott test, 8 out of 28 adult dogs were microfilaria positive. A D. repens-specific PCR based on the internal transcribed spacer 2 region detected filarial DNA in 11 of these dogs. Northeastern Germany is currently considered to be free of Dirofilaria. The 39% prevalence within the kennel and the fact that the dogs transiently stayed in endemic areas only during the winter suggest that autochthonous infections occurred. Analysis of weather data shows that extrinsic development of Dirofilaria was continuously possible in the summers of 1994-2012, thus allowing active transmission within the area.

[Dirofilariasis in the Nizhny Novgorod Region].

The paper presents the results of clinical, epidemiological, and epizootological analyses of local cases of human dirofilariasis in the Nizhny Novgorod Region, which suggest that natural and climatic changes, namely the abnormally hot summer in 2010-2011 and increasing migratory processes among human beings and animals, open up possibilities for forming foci ofdirofilariasis outside habitats of Dirofilaria repens.

Qualitative risk assessment for the endemisation of Dirofilaria repens in the state of Brandenburg (Germany) based on temperature-dependent vector competence.

Climate change with an increase in average temperature may be responsible for propagation of temperature-dependent vectors and/or vector-associated pathogens in regions that were previously not endemic. The analysis of climate data of Brandenburg state (Germany) had showed that the extrinsic development of Dirofilaria repens, the causative agent of canine subcutaneous dirofilariosis, was possible from the years 2001 to 2012. This finding, associated to the movement of infected dogs and their relocation from endemic European areas to Germany makes possible a rapid endemization of the infection and highlight the risk for human health, being D. repens a frequent cause of zoonotic infections.