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Abstract Background Ascaris roundworms are the parasitic nematodes responsible for causing human and porcine ascariasis. Whereas A. lumbricoides is the most common soil-transmitted helminth infecting humans in the world, A. suum causes important economic losses in the porcine industry. The latter has been proposed as a model for the study of A. lumbricoides since both species are closely related. The third larval stage of these parasites carries out an intriguing and complex hepatopulmonary route through the bloodstream of its hosts. This allows the interaction between larvae and the physiological mechanisms of the hosts circulatory system, such as the fibrinolytic system. Parasite migration has been widely linked to the activation of this system by pathogens that are able to bind plasminogen and enhance plasmin generation. Therefore, the aim of this study was to examine the interaction between the infective third larval stage of A. suum and the host fibrinolytic system as a model of the host-Ascaris spp. relationships. Methods Infective larvae were obtained after incubating and hatching fertile eggs of A. suum in order to extract their cuticle and excretory/secretory antigens. The ability of both extracts to bind and activate plasminogen, as well as promote plasmin generation were assayed by ELISA and western blot. The location of plasminogen binding on the larval surface was revealed by immunofluorescence. The plasminogen-binding proteins from both antigenic extracts were revealed by two-dimensional electrophoresis and plasminogen-ligand blotting, and identified by mass spectrometry. Results Cuticle and excretory/secretory antigens from infective larvae of A. suum were able to bind plasminogen and promote plasmin generation in the presence of plasminogen activators. Plasminogen binding was located on the larval surface. Twelve plasminogen-binding proteins were identified in both antigenic extracts. Conclusions To the best of our knowledge, the present results showed for the first time, the pro-fibrinolytic potential of infective larvae of Ascaris spp., which suggests a novel parasite survival mechanism by facilitating the migration through host tissues.

Abstract Background Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. Methods The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. Results First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. Conclusions The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Abstract Background Dirofilaria immitis causes heartworm disease, a chronic and potentially fatal cardiopulmonary disease which mainly affects dogs and cats. It is present in most of Spain, due to favourable climatic factors. Madrid, located in the centre of the Iberian Peninsula, is the most highly populated city in the country. There is a lack of current data on canine heartworm and there are no published epidemiological data regarding feline heartworm in this region, therefore the aim of this study was to assess the prevalence and current distribution of canine and feline dirofilariosis in the province of Madrid. Methods Serum samples from 1716 dogs and 531 cats, from animals living in the metropolitan area of Madrid and adjacent areas, were studied. All the samples, either from cats and dogs, were tested for circulating D. immitis antigens using a commercial immunochromatographic test kit. Furthermore, to establish the seroprevalence of heartworm infection in cats, serological techniques for anti-D. immitis and anti-Wolbachia antibody detection were used. Results Prevalence of D. immitis in the canine population of Madrid was 3%, showing an increase in comparison to previous data. The presence of heartworm in the city centre could be influenced by the presence of Urban Heat Islands, while the positive dogs from metropolitan and adjacent areas were mainly located under the influence of rivers. Regarding cats, 0.2% were positive to the antigens test and 7.3% were seropositive to both anti-D. immitis and Wolbachia surface protein antibodies, which demonstrate the presence of feline heartworm in Madrid. Seropositive cats were present in the same areas where positive dogs were found. Indoor/outdoor cats showed the highest seroprevalence whereas the lowest corresponded to indoor cats, demonstrating that prophylactic treatments should be carried out regardless of lifestyle. Infection was found in 2.2% of dogs and 6.7% of the cats

Abstract Background Proliferative endarteritis is one of the key pathological mechanisms of cardiopulmonary dirofilariosis, a cosmopolitan parasitosis caused by Dirofilaria immitis affecting dogs and cats around the world. It has been shown that the excretory/secretory antigens from D. immitis adult worms (DiES) bind plasminogen (PLG) and activate fibrinolysis, which can lead to a survival mechanism for the parasite in its intravascular environment. However, overproduction of plasmin (final product of the route) has been related to pathological processes similar to those described in proliferative endarteritis. The aim of this study is to relate the appearance of this pathological condition with the activation of the PLG/plasmin system of the host by DiES. Methods Cell proliferation through the crystal violet technique, cell migration by wound healing assay and degradation of the extracellular matrix by measuring collagen degradation and levels of matrix metalloproteinases were studied in an “in vitro” model using canine vascular endothelial and smooth muscle cells. These cells were treated with a mixture of DiES + PLG. Untreated cells, cells only stimulated with DiES or with PLG, or with a mixture of DiES + PLG + εACA (an inhibitor of the PLG-plasmin conversion) were employed as controls. In addition, the effect of DiES on the expression of the fibrinolytic activators tPA and uPA, the inhibitor PAI-1 and the PLG receptor Annexin A2 was analyzed in both types of cultures by western blot. Results Plasmin generated by DiES + PLG binding produced a significant increase in the cell proliferation and migration of the endothelial and smooth muscle cells, as well as an increase in the destruction of the extracellular matrix based on a further degradation of Type I Collagen and an increased level of matrix metalloproteinase-2. DiES also induce an increase in the expression of tPA and uPA in endothelial cells in culture, as well as a decrease in the expression of PAI-1 in both types of cells. Conclusions Our study reports an interrelationship between plasmin caused by fibrinolysis activation by metabolic products of D. immitis and the appearance of pathological events similar to those described in the emergence of proliferative endarteritis in the cardiopulmonary dirofilariosis.

Abstract Background The metropolitan area of Barcelona is the most densely populated metropolitan area on the Mediterranean coast. Several studies have reported the presence of canine heartworm disease in this region; however, there are no published epidemiological data regarding feline heartworm in this region and the prevalence in this species remains unknown. Methods Serum samples from 758 cats living in the metropolitan area of Barcelona (Spain) were collected between 2012 and 2013. To establish the seroprevalence of heartworm infection in cats, serological techniques for anti-D.immitis and anti-Wolbachia antibody detection were used while a commercial ELISA test kit was used to detect circulating D.immitis antigens. Results Of these samples, 11.47% were positive to D.immitis and Wolbachia surface protein antibodies and 0.26% were positive to D.immitis antigens. The higher antibody seroprevalences were found in the areas that follow the courses of the rivers Llobregat and Anoia (Baix Llobregat 11.5%, Vallés Occidental 13.2%; Barcelonés 11.7%) where humidity and vegetation favour the development of the mosquito vectors. High antibody seroprevalences were also found in the urban areas (Barcelona city 13.1%; Sabadell 15.5%), which demonstrates that city cats are also at risk from D.immitis infection. Conclusions Generally, in Spain cats do not receive prophylactic treatment and therefore the risk of infection is higher in this species than in dogs. Adequate prophylactic plans should be implemented in the feline population. This is the first epidemiologic study on feline heartworm infection to be carried out in continental Spain.