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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.

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

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: 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 Dirofilaria immitis and D. repens are mosquito-borne filaroids that primarily infect dogs but also cats. Diagnosing feline dirofilariosis is challenging because of the low parasitic burdens and transient or absent microfilaremia. To improve detection of antibodies against Dirofilaria spp. in cats, an indirect enzyme-linked immunosorbent assay (ELISA) using somatic antigens of D. immitis was standardized. Methods Serum samples from cats positive for D. immitis , D. repens , bronchopulmonary metastrongylids and gastrointestinal helminths as well as negative sera were tested to evaluate the sensitivity (Se) and specificity (Sp). Three different antigen concentrations (2, 3 and 4 μg/ml) and two concentrations of horseradish peroxidase (HRP) IgG anti-cat conjugate (1:10,000 and 1:20,000) were used to screen the optimal titration of the test. Once the best conditions were established, Se and Sp were assessed by testing 151 serum samples positive for D. immitis , D. repens and other parasites (i.e. feline lungworms, gastrointestinal helminths) or negative. Results The optimized ELISA showed 89% Se and 98% Sp in detecting D. immitis infections, presenting a cross-reactivity with D. repens . A cut-off point of 1.150 was established to classify positive and negative samples. Conclusions The developed ELISA may improve the detection of antibodies against Dirofilaria spp. in cats, also in epidemiological contexts characterized by multiple helminth infections. Future efforts will focus on reducing the cross-reactivity with D. repens , therefore increasing ELISA Sp. Graphical Abstract

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.

Background The increasing population of stray dogs and cats in Ukraine poses an important risk of the transmission of vector-borne parasites, particularly Dirofilaria repens , Dirofilaria immitis and Acanthocheilonema reconditum , all of which are zoonotic and may affect human health. Despite numerous reports of human dirofilariosis in Ukraine, epidemiological data on these filarial parasites in companion animals remain limited. The aim of the study was to conduct a molecular epidemiological survey to assess the prevalence of filarial infections in stray dogs and cats across Ukraine and evaluate factors associated with infection in dogs. In collaboration with the European Scientific Counsel Companion Animal Parasites (ESCCAP) and local non-governmental organizations (NGOs), a total of 457 blood samples (233 dogs and 224 cats) were collected between March and December 2023 from Berdychiv, Lviv, Kharkiv, Sumy and Zvenyhorodka. Molecular detection of D. repens , D. immitis , and A. reconditum was performed using a two-step quantitative PCR (qPCR) assay with novel species-specific primers. Results The method demonstrated high sensitivity and specificity, capable of detecting DNA from a single microfilaria, with no evidence of cross-reactivity among target species. Among the canine samples, 66 (28.3%) tested positive for at least one filarial species, including cases of both mono- and co-infection. In contrast, only 8 feline samples (3.6%) were positive for D. repens or D. immitis . Statistical analysis revealed a higher prevalence among male dogs and those weighing over 10 kg, while the lowest prevalence was observed in the youngest age group (< 3 years). Conclusions These findings provide the first molecular evidence of Dirofilaria spp. and A. reconditum infections in stray animals from several under-studied areas of Ukraine, highlighting the relevance of the One Health approach in mitigating the risk of zoonotic transmission.

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 The extraordinary Galapagos Islands, with an impressive number of endemic and native species, maintain the interest and curiosity for researchers from all over the world. The native species are known to be vulnerable to new pathogens, cointroduced with their invasive hosts. In the case of invasive parasitic arthropods, their evolutionary success is related to the association with other invasive hosts (such as domestic animals). These associations could become a significant driver of change, as occasionally they can seek another hosts and have the capacity to transmit pathogens between domestic and wild animals. The current study aims to identify the distribution and abundance of canine vector-borne parasites in the Galapagos Islands based on the possibility that some of them could spill over to endemic mammals. Methods A total of 1221 blood samples were randomly collected from privately owned dogs on San Cristóbal, Isabela, Santa Cruz, and Floreana Islands during the years 2021 and 2022. All samples were examined for vector-borne pathogens using the modified Knott’s test and conventional, nested, and multiplex polymerase chain reactions (PCRs), followed by sequencing. Results The PCR and Knott tests confirmed the presence of Dirofilaria immitis (2%, 25/1221) in all islands. While molecular analyses showed heartworm DNA only in dogs from the San Cristóbal (3.3%) and Isabela (2.4%) Islands. Moreover, other pathogens Babesia vogeli (3%, 37/1221) and Hepatozoon canis (0.2%, 2/1221) were detected for the first time by molecular analyses. Conclusions Dogs from Galapagos are hosts to various pathogens, of which some are potentially zoonotic while some other could be spill-over to endemic endangered carnivores, such as sea lions. To understand and limit their impact, long-term surveillance, control, and awareness is needed. Graphical abstract

Background Dogs in the US are commonly infected with vector-borne pathogens, including heartworm and tick-borne disease agents. The geographic distribution of both arthropod vectors and the pathogens they transmit continues to expand. Methods To describe the current geographic distribution and prevalence of antigen of Dirofilaria immitis and antibody to Borrelia burgdorferi , Ehrlichia spp., and Anaplasma spp. in dogs, we summarized over 144 million test results from 2013 to 2019, inclusive, by county, state, and region. Canine seroprevalence by state was compared to population-adjusted human reports of tick-borne diseases. Results Results varied regionally, with D. immitis antigen and Ehrlichia spp. antibodies more frequently detected in the Southeast (2.6% and 5.2%, respectively) and antibody to B. burgdorferi and Anaplasma spp. most common in the Northeast (12.1% and 7.3%, respectively). Overall, percent positive test results to D. immitis decreased in the Southeast by 33.3% when compared to earlier summaries using the same strategy (from 3.9 to 2.6%). Geographic expansion of areas where dogs commonly test positive for Ehrlichia spp. was evident, likely because of a change in the test made in 2012 to allow detection of antibodies to E. ewingii concomitant with expansion of vector tick populations. Percent positive test results to Ehrlichia spp. increased in every region; this shift was particularly pronounced in the Southeast, where percent positive test results increased fourfold (from 1.3 to 5.2%). Continued geographic expansion of B. burgdorferi and A. phagocytophilum was apparent in the Northeast, Midwest, and Upper South, although canine seroprevalence of antibody to B. burgdorferi was much lower than prior surveys in many Lyme-endemic areas. Annual reports of human cases of Lyme disease, ehrlichiosis, and anaplasmosis were associated with percent positive canine results by state for the three tick-borne disease agents ( R 2  = 0.812, 0.521, and 0.546, respectively). Within endemic areas, percent positive test results for all three tick-borne agents demonstrated evidence of geographic expansion. Conclusions Large scale analysis of results from screening dogs in practice for evidence of vector-borne infections, including those with zoonotic importance, continues to be a valuable strategy for understanding geographic trends in infection risk over time. Graphical Abstract