A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus).

BACKGROUND: The Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent. METHODS: Jird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry. RESULTS: In vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry. CONCLUSIONS: These results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Dirofilaria immitis possesses molecules with anticoagulant properties in its excretory/secretory antigens.

Dirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.

Infrared light photobiostimulation mediates periodicity in Dirofilaria immitis microfilariae.

Microfilariae (Mfs) of filarial nematode parasites exhibit nocturnal periodicity, with their numbers in peripheral blood peaking at night and decreasing during the day. However, the reason for their appearance at night remains unknown. In this study, in vitro photobiostimulation experiments showed that Mfs exhibited positive phototaxis toward infrared light with lower photon flux densities of infrared light at wavelengths of 890 and 700 nm, in particular, mediating paradoxically higher velocity than intense ones. Microarray analysis revealed that infrared light stimulation influenced gene expression in Mfs and induced significant upregulation of genes, with phosphorylation- and neurogenesis-related genes being highly enriched. Weaker natural infrared beams from the atmosphere only at midnight may induce microfilaria periodicity, and the nature of the periodic pattern is innate and plastic, as demonstrated by artificially changing the light-dark cycle. This is the first report of positive phototaxis toward infrared light in Dirofilaria immitis Mfs. The notable finding is that they moved in union despite the lack of a fluid current inside the container, indicating that infrared light appears to control nocturnal periodicity in D. immitis Mfs. The newly developed culture medium and the adoption of charge-coupled device (CCD) camera and time-lapse VHS videocassette recorder used in this study made possible to be a long observation.

The application of proteomic methods (MALDI-toff MS) for studying protein profiles of some nematodes (dirofilaria and ascaris) for differentiating species.

INTRODUCTION: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-toff MS) is a reliable method for diagnosing a number of bacterial and fungal infections. It is also effective as a method of rapid diagnosis of several parasitic agents. We used MALDI-toff MS to study the protein profiles of four nematodes: Dirofilaria repens, Dirofilaria. immitis, Ascaris suum and Ascaris lumbricoides. METHODS: We studied the protein profiles of dirofilaria (five of each species: D. repens and D. immitis) and ascaris (five of each species: A. suum and A. lumbricoides), using a proteomic analysis based on MALDI-toff MS. RESULTS: Analysis of protein extracts of dirofilaria and ascaris showed spectra with high-intensity peaks in the range of 2-20 kDa. The quality of the spectra (clear graphical reflection of mass/charge to luminous intensity, consistent in repeated analyzes) and the intensity of the spectral peaks were consistent in all samples of the same species. The spectra profiles of D. repens and D. immitis differed in eight major peaks which makes it possible to differentiate species according to the protein profile. The spectra profiles obtained from A. suum and A. lumbricoides proteins differed slightly in 3 major peaks in both species and were discovered in m/z 13000; 13400 and 14400. The protein peaks in diapason 3000 kD-7300 kD specific for all genus ascaris are constant. CONCLUSIONS: MALDI-toff MS-based proteomic analysis can serve as an effective taxonomic tool for parasitological studies.

G-protein-coupled receptor genes of Dirofilaria immitis.

The diversity and uniqueness of nematode heterotrimeric G-protein-coupled receptors (GPCRs) provides impetus for identifying ligands that can be used as therapeutics for treating diseases caused by parasitic nematode infections. In human medicine, GPCRs have represented the largest group of 'drugable' targets exploited in the market today. In the filarial nematode Dirofilaria immitis, which causes heartworm disease, the macrocyclic lactones (ML) have been used as the sole preventatives for more than 25 years and now there is confirmed ML resistance in this parasite. A novel anthelmintic emodepside, with antifilarial activity, can act on a GPCR. In view of the ML resistance, there is an urgent need to identify new drug targets and GPCRs of D. immitis may be promising receptors. Knowledge of polymorphism within the GPCR superfamily is of interest. A total of 127 GPCR genes have been identified, so far, in the genome of D. immitis. Whole genome sequencing data from four ML susceptible and four ML loss of efficacy populations was used to identify 393 polymorphic loci in 35 D. immitis GPCR genes. Out of 57 SNPs in exonic regions, 36 of them caused a change in an amino acid, out of which 2 changed the predicted secondary structure of the protein. Knowledge about GPCR genes and their polymorphism is valuable information for drug design processes. Further studies need to be carried out to more fully understand the implications of each of the SNPs identified by this study.

The heartworm (Dirofilaria immitis) seems to be able to metabolize organochlorine pesticides and polychlorinated biphenyls: A case-control study in dogs.

It has been described that the co-existence of parasite infection and chemical exposure has various effects on the accumulation of persistent organochlorine pollutants (POPs) in the host. Certain parasites are not only able to accumulate POPs but also seem to have the ability to metabolize certain compounds. We have designed a case-control study aimed to disclose the role of Dirofilaria immitis in the bioavailability of POPs in dogs trying to know whether these parasites store or metabolize the POPs. A total of 40 common POPs (18 polychlorinated biphenyls congeners (PCBs) and 22 organochlorine pesticides were quantified in dog serum. The study included three groups of dogs prospectively recruited in the island of Gran Canaria (Canary Islands, Spain): a) control animals, non-parasitized (serologically tested negative, n=24); b) a group constituted by dogs tested positive for heartworm disease (D. immitis) and negative for other parasites (n=25); and c) the same group of parasitized dogs after the treatment against the parasite (n=25). The presence of D. immitis was strongly associated with lower serum levels of a wide range of pollutant in their hosts (PCB congeners 28, 105, 118, 123, 138, 153, 167 and 180; hexachlorobenzene, lindane, aldrin, dieldrin, and methoxychlor). The serum levels of these substances remained at very low levels after the treatment against the parasite, suggesting that D. immitis do not simply store such compounds, but they probably have some ability to metabolize these pollutants. We encourage the use of the parasite infestation status as a cofactor that needs to be taken into account in studies aimed to evaluate the serum levels of POPs.

Glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis participate in heartworm disease endarteritis via plasminogen/plasmin system.

The interaction between parasitic protozoa and helminths, both in the blood and in tissues and the fibrinolytic system of their hosts is usually considered as a survival parasite mechanism since this system is the physiological route responsible for degrading fibrin clots. The broad-range proteolytic activity of plasmin, the final enzyme of the route, implies that its recruitment by these parasites is an important mechanism that mediates their invasion and establishment in the hosts. However, recent studies have proposed a dual role for plasmin by linking its over-production with pathological mechanisms at vascular level. Most of these studies have been conducted in Dirofilaria immitis, a blood-borne parasite that survives in the pulmonary arteries of its host for years while it produces a chronic inflammatory disease, whose main pathogenic mechanism is the appearance of proliferative endarteritis. Recently, the participation of two proteins from D. immitis, glyceraldehyde 3-phosphate dehydrogenase (DiGAPDH) and galectin (DiGAL), in the activation of the fibrinolytic system of its host has been demonstrated, which has been a priori associated with parasite survival mechanisms. The aim of the present paper was to study the role of plasmin generated by these proteins in the emergence of proliferative endarteritis. An in vitro model of canine endothelial and smooth muscle cells, as well as the two parasitic recombinant proteins were employed. The results show that DiGAPDH and DiGAL stimulate the proliferation and migration of both cell types, as well as the degradation of the extracellular matrix (ECM) via plasminogen (PLG)/plasmin system, being all of these mechanisms related to the appearance of proliferative endarteritis. Due to the high degree of evolutionary conservation of these antigens, these data support the hypothesis of the survival/pathology ambivalence in the interactions between parasites and the fibrinolytic system of their hosts and represent an advance in the knowledge of the mechanisms involved in the emergence of proliferative endarteritis in the cardiopulmonary dirofilariosis (heartworm disease).

Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

BACKGROUND: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues. Generally, due to their small size, tissue dissection of human-infecting filarial nematodes remains extremely challenging. However, canine heartworm disease is caused by a closely related and much larger filarial nematode, Dirofilaria immitis. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont present in the hypodermis and developing oocytes within the uterus. Here, we describe the first concurrent tissue-specific transcriptomic and proteomic profiling of a filarial nematode (D. immitis) and its Wolbachia (wDi) in order to better understand tissue functions and identify tissue-specific antigens that may be used for the development of new diagnostic and therapeutic tools. METHODS: Adult D. immitis worms were dissected into female body wall (FBW), female uterus (FU), female intestine (FI), female head (FH), male body wall (MBW), male testis (MT), male intestine (MI), male head (MH) and 10.1186/s12864-015-2083-2 male spicule (MS) and used to prepare transcriptomic and proteomic libraries. RESULTS: Transcriptomic and proteomic analysis of several D. immitis tissues identified many biological functions enriched within certain tissues. Hierarchical clustering of the D. immitis tissue transcriptomes, along with the recently published whole-worm adult male and female D. immitis transcriptomes, revealed that the whole-worm transcriptome is typically dominated by transcripts originating from reproductive tissue. The uterus appeared to have the most variable transcriptome, possibly due to age. Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis. Interestingly, wDi gene expression in the male and female body wall is fairly similar, yet slightly different to that of Wolbachia gene expression in the uterus. Proteomic methods verified 32 % of the predicted D. immitis proteome, including over 700 hypothetical proteins of D. immitis. Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function. CONCLUSIONS: The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus.

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I-V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.

Can the activation of plasminogen/plasmin system of the host by metabolic products of Dirofilaria immitis participate in heartworm disease endarteritis?

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.

Prevalence of Dirofilaria immitis antigen and antibodies to Leishmania infantum in cats from southern Portugal.

Vector-borne diseases (VBD) are caused by a range of pathogens transmitted by arthropods and have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary medical importance, cats play a central role in the transmission cycles of some VBD agents by acting as reservoirs, amplifying hosts or sentinels. The aim of this study was to determine the prevalence of Dirofilaria immitis antigen and of antibodies to Leishmania infantum in a sample of 271 cats from southern Portugal. Thirteen (4.8%) cats were positive to D. immitis, while antibodies to L. infantum were detected in 10 (3.7%) animals. The prevalence of D. immitis and L. infantum in the feline population from southern Portugal should alert for the need to implement control measures to protect animals and people from these zoonotic parasites. Furthermore, both parasitoses must be included in the differential diagnosis in feline clinical practice.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria.

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

Proteomic analysis of the somatic and surface compartments from Dirofilaria immitis adult worms.

Dirofilaria immitis (hearthworm) is a filarial roundworm transmitted by mosquitoes to different vertebrate hosts (dogs, cats and humans, among others), causing dirofilariosis. The adult worms reside in the pulmonary arteries affecting vessels and tissues and resulting in different pathological manifestations. Worms migrate to the heart and surrounding major vessels in heavy infections. Dirofilariosis can result in serious damage to affected hosts. In the last few years, a re-emergence of the disease driven by the climate change has been pointed out. Very recently, the knowledge at molecular level of this parasite has been extended by the published studies on its genome and transcriptome. Nevertheless, studies on the expression of defined protein sets in different parasite compartments and the corresponding role of those proteins in the host-parasite relationship have been relatively scarce to date. These include the description of the adult worm secretome, and some of the proteins eliciting humoural immune responses and those related with plasminogen binding in secreted and surface extracts of the parasite. Here, we investigate by proteomics the somatic and surface compartments of the D. immitis adult worm, adding new information on protein expression and localization that would facilitate a deeper understanding of the host-parasite relationships in dirofilariosis.

Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.

The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host's innate immune system.

Human and animal dirofilariasis: the emergence of a zoonotic mosaic.

Dirofilariasis represents a zoonotic mosaic, which includes two main filarial species (Dirofilaria immitis and D. repens) that have adapted to canine, feline, and human hosts with distinct biological and clinical implications. At the same time, both D. immitis and D. repens are themselves hosts to symbiotic bacteria of the genus Wolbachia, the study of which has resulted in a profound shift in the understanding of filarial biology, the mechanisms of the pathologies that they produce in their hosts, and issues related to dirofilariasis treatment. Moreover, because dirofilariasis is a vector-borne transmitted disease, their distribution and infection rates have undergone significant modifications influenced by global climate change. Despite advances in our knowledge of D. immitis and D. repens and the pathologies that they inflict on different hosts, there are still many unknown aspects of dirofilariasis. This review is focused on human and animal dirofilariasis, including the basic morphology, biology, protein composition, and metabolism of Dirofilaria species; the climate and human behavioral factors that influence distribution dynamics; the disease pathology; the host-parasite relationship; the mechanisms involved in parasite survival; the immune response and pathogenesis; and the clinical management of human and animal infections.

First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

BACKGROUND: The characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce. METHODS: The proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis. RESULTS: This study identified 110 proteins. Of these proteins, 52 were unique to D. immitis. A total of 23 (44%) were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes. CONCLUSION: The present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

Excretory/secretory antigens from Dirofilaria immitis adult worms interact with the host fibrinolytic system involving the vascular endothelium.

Dirofilaria immitis is the causative agent of canine and feline heartworm disease. The parasite can survive for long periods of time (7 years or more) in the circulatory system of immunocompetent reservoirs, producing usually a chronic inflammatory vascular disease. In addition, the simultaneous death of groups of adult worms can trigger an acute disease characterized by the exacerbation of inflammatory reactions and the emergence of serious thromboembolic events. In the context of the D. immitis/host relationships, the aim of this study was to investigate the interaction between the excretory/secretory antigens from D. immitis adult worms (DiES) and the fibrinolytic system of the host. Using an enzyme-linked immunosorbent assay we showed that DiES extract is able to bind plasminogen and generate plasmin, although this fact requires the presence of the tissue plasminogen activator (t-PA). Moreover, we established that DiES extract enhances t-PA expression in cultured vascular endothelial cells. Additionally, 10 plasminogen-binding proteins from DiES extract were identified by mass spectrometry (HSP60, actin-1/3, actin, actin 4, transglutaminase, GAPDH, Ov87, LOAG_14743, galectin and P22U). The data suggest that DiES antigens interact with the environment of the parasite regulating the activation of the fibrinolytic system of the host with involvement of the vascular endothelium in the process.

Adult Dirofilaria immitis excretory/secretory antigens upregulate the production of prostaglandin E2 and downregulate monocyte transmigration in an "in vitro" model of vascular endothelial cell cultures.

Canine and feline cardiopulmonary dirofilariosis caused by Dirofilaria immitis is a chronic and potentially fatal disease. Adult worms live in the pulmonary arteries of infected immunocompetent hosts for years. The aim of the present study is the identification of the influence of the metabolic products (excretory/secretory antigens, DiE/S) of D. immitis on the vascular endothelial cells, because the vascular endothelium interplays in a direct manner with the parasite and their products. For this purpose, HAAE-1 vascular endothelial cells were treated with DiE/S, using non-treated cultures as negative controls. Significant increases in the COX-2, 5-LO expression and PGE(2) level were detected in the treated cells compared with the control cells. Moreover, DiE/S decreases monocyte transmigrations across vascular endothelial cell monolayers. Treatment with DiE/S does not have a cytotoxic effect and do not alter apoptosis, necrosis or cell cycle of vascular endothelial cells. These results suggest that the DiE/S stimulates the production of mediators and mechanisms that favor the survival of the parasite, in vascular endothelial cells, contributing to restrict vascular and lung damages in the infected host, without altering the basic physiologic processes of endothelial cells.