Dirofilariosis is a vector-borne zoonotic disease whose distribution is linked to the presence of culicid mosquitoes. Spain and Portugal are considered endemic countries; however, the distribution of dirofilariosis is not uniform. Our aim was to develop a more accurate risk model of dirofilariosis transmission for the Iberian Peninsula (Spain and Portugal) and the Balearic Islands (Spain). To do this, we used a set of key variables related to parasite transmission: the potential distribution of suitable habitats for Culex pipiens calculated via an ecological niche model (ENM) and the potential number of Dirofilaria spp. generations. The resulting model was validated with the prevalence and geolocation of D. immitis-infected dogs from all provinces and districts. In addition, the impact of possible future climatic conditions was estimated. A quantitative estimate of the risk of infection by Dirofilaria spp. was obtained at a resolution of 1 km². The entire analyzed territory was susceptible to contact with the parasite. The highest risk of infection was found throughout the eastern coastal strip and the south of the Iberian Peninsula and the Balearic Islands, as well as in the areas surrounding the basins of the main rivers, and the lowest risk was located in the higher-altitude areas. We found a robust and positive relationship between the risk of dirofilariosis and the observed prevalence of infested dogs in the study area (β ± SE = 3.32 ± 1.43 p < 0.05). In 2080, the percentage of territory gain for Cx. pipiens will increase to 49.98%, which will increase the risk of infection. This new model provides a high predictive value for the current and predicted presence and risk and can serve as a tool for the management and control of dirofilariosis.

Background: Dirofilariosis is a vector-borne zoonotic disease primarily caused by the parasitic nematodes Dirofilaria immitis and D. repens . In Europe, the disease has expanded from traditionally endemic southern countries to central and northeastern regions, many of which are now also considered endemic. This study aimed to generate infection risk maps for dirofilariosis in Europe using ecoinformatic tools, at both annual and monthly scales, to serve as a prevention tool and contribute to more effective control of the disease, as well as helping to stop its spread. Methods: A habitat suitability map was generated for the two most important and widely distributed culicid vectors in Europe ( Culex pipiens and Aedes albopictus ). This map was weighted with the number of D. immitis generations in the vectors, both annually and monthly. The resulting annual risk map was validated with georeferenced records of D. immitis - and D. repens -infected dogs and cats. In addition, a future habitat suitability projection for both vector species was performed for the year 2100 under the Representative Concentration Pathway (RCP) 8.5 climate change scenario. Results: Dirofilariosis infection risk in Europe is highest in southern countries, where favorable climatic conditions and increased vector activity coincide. Central Europe showed medium- to high-risk values, while northern latitudes exhibited low or very low risk, correlating with lower average temperatures. Of the geolocated infected animals, 35.9%, 51% and 13% were located in high-, medium-, or low-risk areas, respectively. Infection risk appears to be very limited during winter, restricted mainly to Mediterranean coastal areas, the Canary Islands (Spain), and Madeira (Portugal); while in spring/summer it becomes high in these places and moderate across other parts of the range such us central and northeastern Europe. The 2100 projection predicts a 161.6% increase in habitat suitability for the vectors, particularly in northeastern regions, high-altitude areas, and northernmost countries. Conclusions: The combined use of habitat suitability for Culex pipiens and Aedes albopictus and the number of Dirofilaria spp. generations allowed the development of a more comprehensive color-coded dirofilariosis infection risk map than previously available. Monthly infection risk maps across Europe could help guide targeted prevention and control measures, disrupt disease establishment in specific areas and seasons, and raise awareness about infection risks in both animals and humans. Graphical

Heartworm disease caused by Dirofilaria immitis is a vector-borne zoonotic disease responsible for the infection of mainly domestic dogs and cats, or these are those for which the most data are known. Humans are an accidental host where a benign, asymptomatic pulmonary nodule may originate. Dirofilaria immitis also harbours the endosymbiont bacteria of the genus Wolbachia, which play a role in moulting, embryogenesis, inflammatory pathology, and immune response. When Wolbachia sp. is released into the bloodstream, endothelial and pulmonary damage is exacerbated, further encouraging thrombus formation and pulmonary hypertension, facilitating congestive heart failure and death of the animal. Previous studies have shown that parasite excretory/secretory products are able to activate the pro-angiogenic pathway (formation of new vessels) to facilitate parasite survival. The aim of this study was to analyse the role of Wolbachia sp. and its relationship with the cellular processes and the angiogenic pathway in a model of human endothelial cells in vitro. The use of recombinant Wolbachia Surface Protein (rWSP) showed that its stimulation exerted an anti-angiogenic effect by detecting an increase in the production of VEGFR-1/sFlt1 and sEndoglin and did not affect the production of VEGFR-2 and mEndoglin (pro-angiogenic molecules). Furthermore, it did not stimulate cell proliferation or migration, although it did negatively stimulate the formation of pseudocapillaries, slowing down this process. These cellular processes are directly related to the angiogenic pathway so, with these results, we can conclude that Wolbachia sp. is related to the stimulation of the anti-angiogenic pathway, not facilitating the survival of D. immitis in vascular endothelium.

Climatic and various socio-geographical variables have significantly influenced the global spread of Dirofilaria immitis. The coastal district of Aveiro, Portugal, marked by its unique hydrographic structure, Ria de Aveiro, and a concerning rise in heartworm disease, was the focus of our study. We aimed to update the prevalence of D. immitis in dogs and the seroprevalence in cats and humans, correlating these data with epidemiological information. A total of 430 dogs were sampled for D. immitis antigens, and 426 cats and 398 humans for D. immitis and Wolbachia sp. antibodies. In addition, we developed and validated an infection risk map for D. immitis with the geolocation of positive samples. Our results indicate a canine prevalence of 4.7%, peaking at 16.7% in Vagos. Feline and human seroprevalences were 8.9% (26.7% in Espinho) and 3.0% (Vagos presented the most alarming results), respectively. Positive samples were found in both high- and low-risk areas highlighting the need for chemoprophylaxis in all municipalities. Risk factors identified included lack of vaccination and internal deworming in cats, while dogs faced risks from inadequate vaccination and outdoor exposure. Our study identifies Aveiro as an endemic area, with a need for control measures to address this public health threat.

Heartworm disease is caused by Dirofilaria immitis, which mainly affects canids and felids. Adult D. immitis worms are located between the heart’s right ventricle and the pulmonary artery. These parasites produce an inflammatory and hypoxic process in the vascular endothelium. It has been demonstrated that D. immitis excretory/secretory antigens are able to stimulate the angiogenic process as a survival mechanism of D. immitis in the vascular endothelium, stimulating the proangiogenic pathway and related cellular processes. Our goal was to study the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) (proteins of D. immitis excretory/secretory antigens) plus vascular endothelial growth factor isoform A (VEGF-A) in the angiogenic process and their relationship with three cellular processes (cell proliferation, cell migration, and pseudocapillary formation) in an in vitro model of vascular endothelial cells. Cell viability and cytotoxicity were analyzed by live cell analysis and a commercial kit, respectively. VEGF-A, sVEGFR-2, VEGFR-1/sFlt, soluble endoglin, and membrane endoglin were analyzed by commercial ELISA kits. Cell proliferation, cell migration, and pseudocapillary formation were analyzed by MTT-based assay, the wound healing technique, and counting cell connections and cell clusters, respectively. rDiGAPDH+VEGF-A and rDiGAL+VEGF-A significantly increased the expression of sVEGFR-2, mEndoglin, and VEGF-A compared to cultures treated with only the proteins (rDiGAPDH and rDiGAL), VEGF-A, or unstimulated cultures. In addition, they also produced a significant increase in cell proliferation, cell migration, and pseudocapillary formation. Therefore, these proteins together with VEGF-A can activate the proangiogenic pathway and could be related to D. immitis survival in the circulatory system.

Heartworm disease (Dirofilaria immitis) is an important zoonotic infection of major clinical importance in dogs widespread, and transmitted by culicid vectors. Although D. immitis mostly affects dogs with an overall low incidence, some islands of the Atlantic archipelagos such as the island of Gran Canaria (Canary Islands, Spain) had one of the highest historical prevalence/seroprevalence values in dogs, cats and humans. Molecular tools allow us to perform species identification diagnosis, phylogeographic and population genetics analysis that can provide key information about the factors making the disease still a threat (uncover untreated range of hosts, putative origin, etc.). In this study, we have optimized primers to amplify mitochondrial (COI, 12S) and nuclear (ITS) molecular markers from adult D. immitis worms. The genetic diversity and structure of D. immitis at the global level is limited, especially when compared with results obtained for other species of the same genus, such as D. repens. New minor haplotypes in the mitochondrial COI marker have been identified from adult D. immitis worms from infected dogs from the hyperendemic island of Gran Canaria, suggesting that the disease may have originated locally or may have been introduced from the mainland in historical times and evolved in isolation. To obtain a more complete understanding of its evolutionary history, structure and genomic diversity, comparative studies using next-generation sequencing data from endemic areas are needed, which will help in the long term to implement monitoring and control measures in a given area and to better understand its global phylogeographic history.