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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.

Dirofilaria immitis , also known as canine heartworm, contains an endosymbiont, Wolbachia , in all life stages. The antibiotic, doxycycline, has been incorporated into heartworm treatment protocols to eliminate Wolbachia . Previous studies indicate that subsequent infection cannot be established using viable third-stage larvae (L3) developed from doxycycline-treated microfilariae (mf). The stages in which the development of larvae is impacted by doxycycline remain unknown. We examined the impact of doxycycline on the third-stage to fourth-stage larval molt, as it is the first molt of D. immitis after it invades the vertebrate host. Microfilaremic blood was collected weekly from D. immitis– infected dogs with or without doxycycline treatment at 10 mg/kg as recommended by the American Heartworm Society. Blood was collected weekly until the end of doxycycline treatment. The blood was used for L3 production and mf isolation. Wolbachia levels in mf and L3 were measured using real-time quantitative PCR. L3 were cultured in vitro for 9 days to assess whether molting occurred. The Fisher’s exact test and Bonferroni correction were used for statistical analysis. The molting of L3 from the doxycycline-treated groups did not show a significant difference compared to the L3 from the control group at weeks 0, 1, 2, 3, and 4. The Wolbachia levels in mf and L3 decreased starting from 7 days post-treatment and remained less than five percent of controls throughout the treatment. Doxycycline treatment can eliminate Wolbachia in both mf and subsequently developed L3. The molts of the mf to L3 in the mosquito and the L3 to L4 molt in vitro do not appear to be impacted by the reduction or elimination of Wolbachia .

Dirofilarioses are widespread diseases caused by filarioid nematodes (superfamily Filarioidea) of the genus Dirofilaria, which are transmitted by a plethora of mosquito species. The principal agent of canine dirofilariosis in the Americas is Dirofilaria immitis, which may also occasionally infest humans, resulting in pulmonary nodules that may be confounded with malignant lung tumours. Because human cases of dirofilariosis by D. immitis are relatively frequent in the Americas and rare in Europe and other eastern countries, where Dirofilaria repens is the main causative agent, the existence of a more virulent strain of D. immitis in the Americas has been speculated. Recently, a case of human ocular infestation by Dirofilaria sp. was diagnosed in Pará State, northern Brazil, where canine heartworm dirofilariosis is endemic. The nematode was shown to be morphologically and phylogenetically related to D. immitis but it was genetically distinct from reference sequences, including those of D. immitis infesting dogs in the same geographical area. This finding raised questions regarding the aetiology of human dirofilariosis in the Americas, since information on the genetic makeup of filarioids infesting dogs and humans is meagre. Further studies would be needed to better characterize filarioids infesting dogs, wild animals, and humans in the Americas and to assess the existence of a more virulent D. immitis strain in this continent. Finally, the competence of different culicid species/strains from Europe and the Americas as vectors of Dirofilaria species should be investigated. Such studies would help us to understand possible variations in transmission patterns and even to predict possible scenarios that may emerge in the future, with the introduction of non-endemic Dirofilaria species/strains in free areas through importation of infested animals, vectors, or both.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca , Puma concolor , Herpailurus yagouaroundi , Leopardus geoffroyi , Leopardus guttulus , Leopardus pardalis , Leopardus wiedii , Leopardus munoai ) from different locations in Brazil. Overall, 82 samples ( n  = 63 blood; n  = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 ( cox 1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87–88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

Background Dirofilaria immitis , the agent of canine and feline heartworm disease, and Dirofilaria repens , the agent of subcutaneous dirofilariosis, are widespread mosquito-borne helminths. The present study is aimed at updating current knowledge of the composition of potential vector species in the northern region of Emilia-Romagna, a traditionally endemic area for D. immitis and D. repens . Methods Mosquitoes were collected in 2022 and 2023 as part of the regional surveillance plan for West Nile Virus (WNV). The capture zones included peri-urban and rural areas and mosquitoes were captured with CDC-CO 2 traps. DNA from approximately 30% of female mosquitoes of each captured species ( Culex pipiens , Aedes caspius , Aedes vexans , Aedes albopictus ) was extracted and analyzed for the presence of D. immitis and D. repens. Results A total of 140 pools (~ 20 mosquitoes each) in 2022 and 133 in 2023 have been analyzed. DNA of D. immitis was identified in 14 pools in 2022 and in 15 pools in 2023. None of the pools was positive for D. repens . In 2022, about 85% of the positive pools belonged to Ae. caspius species (11/13) and the other three pools to Ae. vexans . In 2023, 73% of the positive pools belonged to Ae. caspius , followed by Ae. vexans and Ae. albopictus (both 13.3%). A significant overlap emerged from the same traps positioned in Ferrara and Bologna provinces, which tested positive for D. immitis in both 2022 and 2023. Conclusions These data highlight how, despite the abundance of Cx. pipiens captured, the most receptive species for D. immitis appear to be Ae. caspius and Ae. vexans . Furthermore, the geographical data highlights how the areas of the province of Ferrara and Bologna are the main geographical reservoirs of the parasite. Graphical abstract

Background For dogs and cats, chemoprophylaxis with macrocyclic lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers. Methods MiSeq technology was used to identify allele frequencies with the 42 genetic markers previously reported. Microfilaria from ten well-characterized new isolates called ZoeKY, ZoeMI, ZoeGCFL, ZoeAL, ZoeMP3, ZoeMO, ZoeAMAL, ZoeLA, ZoeJYD-34, and Metairie were extracted from fresh blood from dogs. DNA were extracted and sequenced with MiSeq technology. Allele frequencies were calculated and compared with the previously reported susceptible, LOE, and resistant D. immitis populations. Results The allele frequencies identified in the current resistant and susceptible isolates were in accordance with the allele frequencies previously reported in related phenotypes. The ZoeMO population, a subset of the ZoeJYD-34 population, showed a genetic profile that was consistent with some reversion towards susceptibility compared with the parental ZoeJYD-34 population. The Random Forest algorithm was used to create a predictive model using different SNPs. The model with a combination of three SNPs (NODE_42411_RC, NODE_21554_RC, and NODE_45689) appears to be suitable for future monitoring. Conclusions MiSeq technology provided a suitable methodology to work with the microfilarial samples. The list of SNPs that showed good predictability for ML resistance was narrowed. Additional phenotypically well characterized D. immitis isolates are required to finalize the best set of SNPs to be used for large scale ML resistance screening.

BACKGROUND: Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott’s technique. METHODS: Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs. RESULTS: The mean length and width of D. immitis mf from 60 dogs were 301.77±6.29 μm and 6.30±0.26 μm, of D. repens mf from 171 dogs 369.44±10.76 μm 8.87±0.58 μm, of A. dracunculoides mf from 133 dogs 259.43±6.69 μm and 5.09±0.47 μm and of A. reconditum mf from 13 dogs 264.83±5.47 μm and 4.63±0.52 μm. For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott’s technique was shown to be stable over 105 days. CONCLUSIONS: The Knott’s test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.

Background Dirofilariosis is an emerging mosquito-borne disease that particularly affects domestic dogs worldwide but also causes symptoms in humans. Monitoring the distribution of emerging pathogens is essential for understanding the environmental and ecological factors influencing their transmission, which can be used to develop better prevention strategies. Methods We applied both community science and molecular xenomonitoring to assess the occurrence of Dirofilaria immitis in domestic dogs and mosquitoes. Results As part of the community science approach, we collected infection data from 1491 dogs from owners across Hungary, using a questionnaire survey. We found that 321 dogs (21.5%) tested positive for current or past dirofilariosis infection, with the highest prevalence observed in the southeastern (47.8%) and the eastern regions (43.4%) of the country. Age and living conditions affected infection status, with older dogs (aged 5–10 years and over 10 years) and those kept exclusively outdoors showing significantly higher infection rates. Molecular xenomonitoring revealed D. immitis infection in Aedes albopictus , Aedes koreicus , and Aedes vexans , with the highest minimum infection rate (MIR) in Ae. koreicus (28.5). Similar to community science results, the highest infection rates were observed in the southeastern and eastern regions (MIR: 14.9 and 11.6, respectively), but the two approaches generally provided overall similar geographical patterns. Conclusions While xenomonitoring did not detect infections in Central Transdanubia, community science successfully provided host infection data, demonstrating its usefulness in assessing the presence and distribution of the disease. Finally, we emphasize the value of using an integrative approach, combining community science and xenomonitoring for monitoring dirofilariosis, especially in areas where direct pathogen screening is unavailable. Graphical Abstract

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. Graphical Abstract

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 .