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Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Previous studies have shown the existence of an acute phase response in dogs with heartworm (Dirofilaria immitis), probably caused by the vascular inflammation that occurs during the pathogenesis of this disease. In addition, it has been seen that this acute phase response persists after finishing treatment, especially in dogs with pulmonary hypertension (PH). Furthermore, echocardiographic studies have shown that PH and endarteritis appear to persist for at least 10 months after completion of adulticide treatment, suggesting that the vascular changes in these dogs may not be reversible. Therefore, the objective of this study was to evaluate the serum concentrations of different positive acute phase proteins (APP) [C reactive-protein (CRP), haptoglobin and ferritin] and negative APP (albumin and paraoxonase-1 (PON-1)), and the usefulness of the endothelin-1 (ET-1) and adiponectin, in dogs infected by D. immitis to evaluate their usefulness as diagnostic biomarkers of vascular damage and PH and their progression throughout therapy up to 7 months after the end of adulticide treatment. Twenty-five heartworm-infected dogs received adulticide treatment, and serum measurements were performed on the day of diagnosis (day 0), day of discharge (day 90), and 6 months after discharge (day 270). In addition, presence or absence of PH was also echocardiographically determined using the Right Pulmonary Artery Distensibility Index. PH was present in 44% of the dogs on day 0 and day 90, and in 48% of dogs on day 270. Alterations were observed in the concentrations of all APP throughout the study, persisting the alterations in PON-1 and ferritin on day 270. Depending on the presence or absence of PH, CRP showed significant differences throughout the study, as did ET-1. On the other hand, adiponectin did not show variations throughout the study, so it did not seem a useful marker in this disease. These results could reflect the possible persistence of vascular inflammation up to 7 months after finishing treatment, whether or not there was PH, and consolidate the study of APP as useful markers in heartworm disease. Moreover, persistent PH could be the consequent clinical manifestation in dogs with more severe vascular alterations so the study of APP, especially CRP, and ET-1 could be especially advantageous in these patients in the early evaluation of the disease, as well as for the determination of disease severity, monitoring therapeutic responses, and predicting outcomes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Glomerulonephropathy associated with Dirofilaria immitis (heartworm) is relatively frequent in infected dogs. Given the importance and the scarcity of studies focused on its prevalence and diagnosis, the objective was to determine the prevalence of proteinuria and functional indicators of glomerular filtration rate in dogs with heartworm disease and discuss its utility in the detection of renal impairment. Sera and urine from 47 infected dogs were analyzed in a reference laboratory. Urea, creatinine, plasma proteins and serum symmetric dimethylarginine (SDMA) were analyzed in sera, while the UPC ratio was performed in urine. Dogs were further evaluated for the presence/absence of microfilariae, pulmonary and systemic hypertension, and the parasite burden was assessed. The results showed that 19.1 % of dogs showed proteinuria (UPC > 0.5) and 17 % showed borderline proteinuria (UPC 0.2-0.5). Creatinine and SDMA were high (>1.8 mg/dl and ≥18 μg/dl, respectively) in 4.2 % of dogs. UPC ratio was significantly increased in dogs with high parasite burden and in dogs with microfilariemia (p < 0.05). Dogs with pulmonary hypertension showed higher increases in proteinuria as well, which was probably due to the chronicity of the infection. No significant differences were found in serum and urine values regarding systemic blood pressure. Despite the limitations of this study, proteinuria/borderline proteinuria was present in 36.2 % of dogs with heartworm disease, and this may be due to glomerular disease. Therefore, the detection of proteinuria, along with other renal biomarkers in the diagnostic protocols, could help identify kidney alterations or risk of renal damage in heartworm disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The endosymbiont bacteria Wolbachia plays an important role in the pathogenesis and inflammatory immune response to heartworm (Dirofilaria immitis) infection in dogs. Doxycycline is used to reduce Wolbachia from all life stages of heartworm to avoid large releases of the bacteria during the death of the worms. However, the dose and duration currently recommended have been extrapolated from the treatment of other rickettsial infections. Therefore, the aim was to study the dynamics of Wolbachia IgG antibodies in heartworm-infected dogs under adulticide treatment using different dosages of doxycycline. Forty-nine heartworm-infected dogs were recruited. On day 0 (diagnosis), monthly ivermectin (6 μg/kg) was prescribed, as well as daily doxycycline for 30 days, at 10 mg/kg/12 h (n = 13), 5 mg/kg/12 h (n = 19), and 10 mg/kg/24 h (n = 17). Dogs underwent adulticide treatment and blood samples were collected on days 0, 30, 90, and 120. All dogs had antibodies against recombinant Wolbachia surface protein (rWSP), confirming the important role of the bacteria in heartworm. No significant differences were found in anti-rWSP response by presence/absence of microfilariae, or by parasite burden on day 0. In all treated groups, the anti-rWSP antibody response was not significantly different between days 0 and 30 but was significantly lower between days 0 and 120 (p < 0.05). The results of the present study suggest that the administration of a lower dose than currently recommended is sufficient to achieve a significant reduction of Wolbachia in dogs infected by D. immitis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pulmonary endarteritis caused by Dirofilaria immitis and pulmonary hypertension (PH) are closely linked and the determination of PH could be validated to assess the severity and chronicity of the vascular damage, i.e. by the use of the Right Pulmonary Artery Distensibility (RPAD) Index. The aim was to evaluate the RPAD Index in dogs 10 months after the last dose of adulticide. The study included 23 client-owned dogs with heartworm brought for adulticide treatment. Echocardiographic exam was carried out to estimate worm burden, RPAD Index as well as other routine echocardiographic values on day 0 (diagnosis), day 120 (discharge), and 10 months after the last dose of adulticide. No significant differences were observed in the RPAD Index over time, neither when microfilaremic status and parasite burden were evaluated. An RPAD Index <29% was found in 52.2% of the dogs on day 0, day 120, and 10 months after the last dose of adulticide. Regarding other echocardiographic parameters, only significant differences were observed on tricuspid annular plane systolic excursion (TAPSE) values between day 0:day 120 (p = 0.008), and day 0:end of the study (p = 0.037). There was not significant improvement in pulmonary damage 10 months after the elimination of the parasites (p = 0.296), suggesting that, once the endarteritis has developed, the vascular changes are chronic and may not be reversible. The modifications of the TAPSE value suggest an improvement in the systolic function of the right ventricle after the disappearance of the worms, independently of the presence of PH. The endarteritis causes a decrease in the elasticity in the pulmonary vasculature and an increase in the resistance that, chronically, and depending on the severity, will cause the development of PH and later right heart failure. This complication of the disease is one of the most damaging and frequent, so it is necessary to adequately monitor PH in dogs undergoing adulticide treatment. Furthermore, knowing the pulmonary status could provide valuable information to help provide an objective prognosis and help assess the need to add additional treatments, once the worms have been eliminated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The treatment of canine heartworm has been modified over the years, adding improvements for greater efficacy, safeness and better prognosis. Currently, the recommended adulticidal protocol consists of the administration of three doses of melarsomine dihydrochloride, preceded by the administration of macrocyclic lactones over two to three months. The objective of this study was to evaluate a variation of the adulticide protocol of heartworm in 76 dogs infected by Dirofilaria immitis, which consists of the pre-administration of macrocyclic lactones (ivermectin) during a single month. On the day of diagnosis, presence of circulating microfilariae was determined and an echocardiography was performed to assess the parasite burden. Treatment began on day 0, with doxycycline for 30 days (10 mg/kg BID) and monthly ivermectin (6mcg/kg). On day 30, the first dose of melarsomine dihydrochloride was administered, followed by a second and third dose on days 60 and 61, respectively. On day 90, the dogs were examined and discharged. Six months after the last dose, all dogs were negative to the presence of antigens and amicrofilaremic. Also, 38.1% of animals were evaluated by echocardiography, showing absence of adult parasites. It is considered that the ineffectiveness of melarsomine against worms <4 months should be avoided by the previous administration of macrocyclic lactones for two to three months, killing larvae <2 months while older filariae are allowed to mature to be susceptible to melarsomine dihydrochloride. With this protocol, this gap would be covered for the 2nd and 3rd injections, when worms would be four months and older. In addition, there is evidence that melarsomine is effective against worms under four months and macrocyclic lactones have some efficacy against heartworms older than two months. This modification allows a faster elimination of heartworms and a better compliance from the owners of the infected dogs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pulmonary hypertension (pH) is a frequent and severe phenomenon in heartworm disease (Dirofilaria immitis). There is a lack of studies assessing the evolution of the proliferative endarteritis and pH caused by D. immitis after the death of the parasites, so this study evaluated the influence that the elimination of the worms exerts over the pulmonary pressure and therefore evolution of the endarteritis, through the evaluation of the Right Pulmonary Artery Distensibility (RPAD) Index and other echocardiographic measurements in 2D mode, M-mode and Doppler echocardiography in 34 dogs naturally infected by D. immitis on day 0, and one month after the last adulticide dose (day 120). pH, based on the determination of the RPAD Index, was present in 68% of the dogs (n=23) on day 0 and on day 120. No significant differences were observed between the RPAD Index between the two measurements, and only significant differences were found in pulmonary deceleration time, ejection time, and left ventricular internal diameter in telediastole when measurements from day 0 and day 120 were compared. There was not any worsening in the development of pH after the elimination of the parasites, independently of the parasite burden. During the adulticide treatment, the death of the worms causes thromboembolism and tends to worsen the vascular damage and presence of pH . It seems that following the adulticide protocol recommended by the American Heartworm Society with the previous elimination of Wolbachia and reduction of microfilariae followed by the stepped death of the worms did not cause a significant aggravation of the pulmonary damage of the treated dogs. Neither is present any significant improvement in the RPAD Index on day 120; probably, more time is needed before appreciating some positive changes after the elimination of the worms and Wolbachia from the vasculature and further studies are necessary., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cardiopulmonary dirofilariosis (heartworm) is a zoonotic vector borne disease caused by Dirofilaria immitis which affects domestic dogs and cats. Two of the seven Canary Islands are historically hyperendemic areas of dirofilariosis, although no epidemiological study has ever been carried out which includes the other islands. The aim of the study was to complete the epidemiological status of cardiopulmonary dirofilariosis in the canine and feline population throughout all the Canary Islands. 1643 client-owned dogs and 707 client-owned cats were tested for D. immitis antigens (dogs), and anti-D. immitis and anti-Wolbachia antibodies (cats). The prevalence of canine dirofilariosis in the Canary Islands was 15.7%, and the seroprevalence of feline dirofilariosis was 18.1%. A remarkable disparity was found when evaluating the results by island separately, which ranged from from 0% in Lanzarote and El Hierro, low prevalences and seroprevalences in Fuerteventura (1.8% and 2.5% in dogs and cats, respectively), to higher prevalences on the other 4 islands; ranging between 15.7% (dogs) and 14.3% (cats) in La Palma 22.5% (dogs) and 24.1% (cats) in Tenerife. In addition, prevalences and seroprevalences were very variable within each island, these differences being associated to local climate conditions. The distribution and prevalence of dirofilariosis in the Canary Islands is heterogeneous and related to climate, demographic factors and management of pets in the studied areas. Dirofilariosis remains hyperendemic in 4 of the 7 Islands. Since D. immitis is a zoonosis, veterinary and health authorities should be aware of the current prevalence and seroprevalence of animal dirofilariosis. The results show the need for awareness raising campaigns to promote the implementation of prophylactic measures in pets, in order to achieve a decrease in the prevalence of animal dirofilariosis in the Canary Islands., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Like other vector borne infections, the distribution of dirofilariosis caused by Dirofilaria immitis is influenced by climatic factors, which regulate the diversity and abundance of mosquito species able to transmit the parasite. Geographical Information Systems (GIS) can predict the distribution and epidemiological behavior of dirofilariosis based on temperature and humidity data. This study analyses the prevalence and current distribution of canine dirofilariosis in the province of Barcelona (Northeastern Spain), and uses a GIS model to evaluate the relationship between the spatial distribution of positive cases and different geo-environmental factors. Canine dirofilariosis is present in all the studied regions but unevenly distributed. The general prevalence is 2.4%, being located most of positive dogs in areas where the model predict both high number of annual generations of D. immitis in vectors and humidity, as a consequence of the presence of irrigated crops or the proximity to the sea. Furthermore, in the urban area of Barcelona, infected dogs were located in districts surrounded or close to parks and green areas. The model can be used as a tool to determine the need of implementation of prophylactic protocols in pets living in municipalities from these regions, based on the geo-environmental characteristics of the area., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

During the adulticide treatment of Dirofilaria immitis the worms die releasing fragments of parasites and causing pulmonary thromboembolisms which could exacerbate the clinical condition. To determine the utility of acute phase proteins (APPs) to monitor the progression of the treatment, different positive [C-reactive protein (CRP), haptoglobin (hp)] and negative [albumin, paraoxonase-1(PON-1)] APPs were measured in 15 heartworm-infected dogs (5 with high and 10 with low parasite burden) following adulticide treatment. The results showed increased concentrations of CRP, decreased concentrations of haptoglobin and PON-1 in infected dogs before starting the treatment. Progressive but not significant increases were observed in PON-1 activity and albumin concentration along the treatment. After the treatment with doxycycline and ivermectine a decrease in CRP and Hp levels was experienced, which could reflect a reduction of the vascular inflammation caused by the elimination of Wolbachia and reduction of microfilariae. Fifteen days after the first melarsomine injection, marked increases in CRP and Hp were observed, which could be due to pulmonary inflammation and thromboembolism caused by the post-adulticide death of the worms. This increase was greater in dogs with high parasite burden. As the pathology disappeared, there was an improvement in the concentrations of CRP and Hp, returning into reference values in dogs with low parasite burden at the end of the treatment. The measurement of CRP and Hp could be a resource of support to evaluate the magnitude of the post-adulticide complications during the adulticide treatment of D. immitis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Adulticide treatment of dogs with canine heartworm disease causes the death of the adult Dirofilaria immitis lodged in the vascular system of the host. During the death of the worms, pulmonary thromboembolisms (PTE), pulmonary inflammation, congestive heart failure, or renal disease are possible consequences. The aim of this study was to evaluate cardiopulmonary biomarkers and renal parameters during adulticide treatment of canine heartworm to compare the classic two-injection treatment protocol versus the American Heartworm Society (AHS) recommended protocol. Fourteen heartworm-infected dogs with high parasite burdens were divided in three groups and allocated to adulticide protocols as follows: Group A (n=5) was treated with the classic two-injection protocol; group B (n=5) was treated using the AHS recommended protocol, and group C (n=4) was treated as for group B but received diminishing anti-inflammatory doses of prednisone. To assess cardiorespiratory status, cardiac troponin I (cTnI), myoglobin, and D-dimer were measured. Renal function was evaluated by measuring urea, creatinine, and urine protein:creatinine (UP:C). Serum and urine samples were collected day 0 (day of diagnosis), 7 and 14 days after the first dose of adulticide, and 1 month after the last adulticide injection. Dogs that received classic treatment presented pathologic concentrations of D-dimer more frequently and showed higher average D-dimer levels, which may indicate the presence of more severe PTE. Group C showed the highest levels of D-dimer during treatment, which may be due to an exacerbation of PTE caused by the administration of prednisone. CTnI and myoglobin values remained above reference values in all groups during the study but reached the lowest values 1 month after the last injection. Levels of urea and creatinine were within normal ranges in all groups, and 28.5% of the dogs were proteinuric on day 0, progressing to better UP:C values at the end of the treatment, except in group A. The results of this study justify the treatment of canine heartworm disease using the AHS recommended adulticide guidelines and recommends re-evaluation of the role of glucocorticosteroids in the prevention and treatment of PTE., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The prognosis and success of adulticide treatment depends on the baseline severity of infection with Dirofilaria immitis in dogs and can influence the therapeutic protocol to choose. A study was conducted to assess the utility of the cardiopulmonary biomarkers N-terminal of the prohormone brain natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), myoglobin, and D-dimer, as well as the biomarker of inflammation C-reactive protein (CRP) to evaluate the severity of canine heartworm disease in different stages. Serum samples were collected from 20 heartworm-infected dogs for measurement of these biomarkers. Dogs were assigned to four groups (n=5/group) according to the severity of the disease: Class I and Class II dogs showed concentrations of biomarkers generally within normal ranges for healthy dogs, except CRP in dogs classified as Class II, which showed values slightly above laboratory reference values (19.46±5.69mg/l). Dogs of Class III showed elevated levels of NT-proBNP (1220.12±465.18pmol/l) (p<0.05), cTnI (0.99±0.39ng/ml), CRP (33.98±7.99mg/l) (p<0.05), and 40% of these dogs (2/5) presented pathological values of D-dimer and myoglobin (p<0.05). Dogs of Class IV (caval syndrome) presented elevated levels of NT-proBNP (>2530.8pmol/l), cTnI (1.99±0.32ng/ml), and CRP (80.24±47.69mg/l); all Class IV dogs showed pathological elevations of D-dimer (0.81±0.46ng/ml), and 60% (3/5) of these dogs showed pathological elevations of myoglobin (significant elevations [p<0.05] for all biomarkers with respect to reference values and values for dogs in Classes I, II, and III). In canine heartworm disease, chronic presence of D. immitis causes a proliferative endoarteritis, thromboembolisms, pulmonary hypertension, and right-sided congestive heart failure. As the severity of the disease worsens, serum values of cTnI, myoglobin, and NT-proBNP increase, indicating significant cardiac damage. The finding of pathological concentrations of D-dimer suggests the presence of thromboembolism and/or disseminated intravascular coagulation. CRP increases according to the severity of the disease, indicating inflammatory processes that could contribute to the progression of the disease. These preliminary results demonstrate the utility of cardiopulmonary and inflammation biomarkers to assist in the establishment of the severity of canine heartworm disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this study was to determine concentrations of different positive and negative acute phase proteins (C-reactive protein, haptoglobin, albumin and paraoxonase-1) in dogs naturally infected with Dirofilaria immitis at the time of diagnosis. 194 dogs were included in the study. All were evaluated for the presence or absence of D. immitis circulating antigens and for the presence or absence of microfilariae and a clinical examination was carried out. 38 dogs were negative and 156 dogs were positive for circulating D. immitis antigens. A significant increase in C-reactive protein and significant decreases in albumin and paraoxonase-1 activity were observed in positive dogs. These changes appeared with independence of the presence/absence of microfilariae or clinical signs. C-reactive protein was the only acute phase protein that showed significant differences between asymptomatic and symptomatic dogs. Interestingly, the increases seen in C-reactive protein values were not accompanied by increases in haptoglobin, and haptoglobin even decreased in the dogs with microfilaria. This could be due to the hemolytic anemia which can be produced in dirofilariasis. In conclusion, there is an acute phase response (with increases in C-reactive protein and decreases in albumin and paraoxonase-1) and a divergence in the behaviour between C-reactive protein and haptoglobin in dogs with D. immitis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Canine cardiopulmonary dirofilariosis caused by Dirofilaria immitis habitually develops as a chronic disease affecting pulmonary arteries, lung parenchyma and heart. Other organs like kidneys can also be involved. Renal pathology is a consequence of glomerulonephritis whose main sign is proteinuria. The aim of the present work is to identify proteins excreted in the urine of D. immitis infected dogs showing proteinuria, and the possible contribution of their loss to heartworm disease. Proteinuria is higher in microfilaremic (mf+) than in amicrofilaremic (mf-) dogs. Using bidimensional electrophoresis and mass spectrometry 9 different proteins from Canis lupus familiaris in the urine of both mf- and mf+ dogs were identified (serotransferrin isoform 6, serum albumin precursor, albumin, immunoglobulin gamma heavy chain D, apolipoprotein A-I, immunoglobulin lambda-like polypeptide 5-like, arginine esterase precursor, inmunoglobulin gamma heavy chain B and hemoglobin subunit alpha). Furthermore, 3 additional proteins were identified only in the urine of mf+ dogs, corresponding to dog fibrinogen alpha chain and immunoglobulin gamma heavy chain A and actin 2 homologous to a protein of Brugia malayi. The loss of these proteins and other in the urine of D. immitis infected dogs could affect the general condition of parasitized dogs through the interference in the cholesterol metabolism and O₂ transport, among other mechanisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Zoonotic filarioses caused by Dirofilaria immitis and Dirofilaria repens are transmitted by culicid mosquitoes. Therefore Dirofilaria transmission depends on climatic factors like temperature and humidity. In spite of the dry climate of most of the Spanish territory, there are extensive irrigated crops areas providing moist habitats favourable for mosquito breeding. A GIS model to predict the risk of Dirofilaria transmission in Spain, based on temperatures and rainfall data as well as in the distribution of irrigated crops areas, is constructed. The model predicts that potential risk of Dirofilaria transmission exists in all the Spanish territory. Highest transmission risk exists in several areas of Andalucía, Extremadura, Castilla-La Mancha, Murcia, Valencia, Aragón and Cataluña, where moderate/high temperatures coincide with extensive irrigated crops. High risk in Balearic Islands and in some points of Canary Islands, is also predicted. The lowest risk is predicted in Northern cold and scarcely or non-irrigated dry Southeastern areas. The existence of irrigations locally increases transmission risk in low rainfall areas of the Spanish territory. The model can contribute to implement rational preventive therapy guidelines in accordance with the transmission characteristics of each local area. Moreover, the use of humidity-related factors could be of interest in future predictions to be performed in countries with similar environmental characteristics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Heartworm disease (Dirofilaria immitis) is a parasitic disease of dogs and other carnivores, characterized by the presence of adult worms in the pulmonary arteries and right ventricle, leading to pulmonary hypertension which may progress to congestive heart failure. Cardiac biomarkers are biological parameters that can be objectively measured as indicators of pathological processes, or to assess the response to therapeutic interventions. To evaluate the myocardial damage during the adulticide treatment in 15 heartworm-infected dogs with ivermectin, doxycycline and melarsomine, measurements of cardiac troponin I (cTnI), myoglobin, MB isoenzyme of creatine kinase (CK-MB) and aspartate aminotransferase (AST) were carried out on days 0, 60 and afterwards weekly on days 67, 75, 82, 91, 106, 113 and 120. Dogs were divided by low parasite burden (n=9) and high parasite burden (n=6). On day 0, dogs with high worm burden showed increased cTnI concentrations (3.62 ± 4.78 ng/ml) while dogs with low worm burden had concentrations similar to those of healthy dogs (0.78 ± 0.22 ng/ml), CK-MB concentrations were increased only in dogs with high parasite burden as well (54.4 ± 54.2U/l) and 26.6% (4/15) of the dogs showed pathological concentrations of myoglobin. On day 91, most dogs showed pathological concentrations of myoglobin, CK-MB and AST, probably due to the myositis associated to the intramuscular injection of melarsomine. The rest of the measurements made in the study, the biomarkers concentrations were within normal values, except for cTnI in dogs with high parasite burden, which remained above reference concentrations for healthy dogs during all the study. The evaluation of cardiac biomarkers seems to be a helpful test in the assessment of the myocardium in dogs with heartworm disease during the adulticide treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cardiopulmonary dirofilariosis (Dirofilaria immitis) is characterized by apparent contradictory events, like the long-term survival of adult worms in the circulatory system of the infected hosts and the development of life-threatening events like thromboembolisms and others. Thus parasite mechanisms, like the activation of fibrinolytic system, are key to the survival of both the worms and the host. The aim of this study was to investigate the interaction between D. immitis adult worms surface-associated antigens (DiSAA) and the fibrinolytic system of the host. We demonstrate that DiSAA extract is able to bind plasminogen and generate plasmin, with the latter occurring in a tissue plasminogen activator (t-PA) dependent manner. Additionally, 11 plasminogen-binding proteins from DiSAA extract were identified by proteomics and mass spectrometry (MS) (actin-5C, actin-1, enolase, fructose-bisphosphate aldolase, GAPDH, MSP domain protein, MSP 2, beta-galactosidase-binding-lectin, galectin, immunoglobulin I-set domain-containing protein and cyclophilin Ovcyp-2). Because in a previous work we have shown the positive interaction between the excretory/secretory antigens of D. immitis (DiES) and the host fibrinolytic system and many of the molecules identified here are shared by both antigens, we hypothesize that DiSAA cooperate in host fibrinolytic system activation promoting the fibrin clot lysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Heartworm associated respiratory disease is a pulmonary syndrome in cats that results from the vascular and parenchymal inflammatory response associated with the arrival and death of Dirofilaria immitis worms into the distal pulmonary arteries. This parasite harbors intracellular Wolbachia, an endosymbiont bacteria. The association between the parasite and the bacteria is obligatory. Some studies suggest the involvement of Wolbachia in the development of the inflammatory reaction and in the polarization of the host immune response against the parasite. Barometric whole-body plethysmography is a non-invasive pulmonary function test that allows a dynamic study of breathing patterns and is useful to study airway disease and the response to different treatments. The aim of this prospective non-blinded study was to compare the influence of Wolbachia upon the respiratory function variables in a population of cats seropositive to D. immitis by use of Barometric whole-body plethysmography. Fourteen seropositive cats to Wolbachia and eight seronegative cats were put into the plethysmograph chamber and different respiratory variables were measured. The results were analyzed and compared between the two groups of animals. Significant differences were found for bronchoconstriction index variables PAU (pause) (P-value<0.05) and Penh (enhanced pause) (P-value<0.05). The results obtained in our study suggest that Wolbachia seems to produce a greater acute inflammatory response at bronchial, vascular and parenchymal level worsening the state of broncho-reactivity associated with the presence of seropositivity to D. immitis in cats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dirofilaria immitis adult worms lodged in the pulmonary arteries are the causative agent of heartworm disease, characterized by endarteritis and obstruction of the blood flow. Dying worms form embolic fragments which trigger thrombosis. Thus, pulmonary thromboembolism is an inevitable consequence of successful adulticide therapy. Clinical signs of pulmonary thromboembolisms are highly variable and non-specific, and its diagnosis is often difficult to obtain. The D-dimer assay is considered the marker of choice for dogs with a suspicion of pulmonary thromboembolism. The aim of the present study was to evaluate the variation of D-dimer and platelets in 15 heartworm-infected dogs during the adulticide treatment with ivermectin, doxycycline and melarsomine. Nine dogs were considered to have a low parasite burden and 6 had a high burden. Measurements were carried out on days 0, 60, and afterwards weekly on days 67, 75, 82, 91, 98, 106, 113, 120, and finally on day 271 after treatment. 40% of dogs showed pathological concentrations of D-dimer and 40% showed slight or mild thrombocytopenia on day 0. The levels of D-dimer decreased by day 60, probably due to the joint action of the ivermectin, doxycycline and exercise restriction. All dogs with high parasite burden showed elevated values of D-dimer on several occasions during the treatment, reaching the highest values the first and second week after the injections of melarsomine. Only 33.3% of the dogs with low parasite burden showed elevated D-dimer levels at some point during the treatment, and all of them showed undetectable levels from the third week after the first injection of melarsomine. On day 271, all dogs showed undetectable levels of D-dimer. There was no correlation between thrombocytopenia and levels of D-dimer during the treatment. The evaluation of serum D-dimer appears to be a supportive test in the assessment and monitoring of pulmonary thromboembolism in dogs with heartworm disease during the adulticide treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It has been reported that dogs with heartworm disease (Dirofilaria immitis) show increased plasma levels of D-dimer, a fibrin degradation product present in the blood after a blood clot is degraded by fibrinolysis. In the present study the authors show that, in dogs with both experimental and natural infections with D. immitis, D-dimer deposits in lungs and kidneys are associated with pulmonary thromboembolism and microfilariemic status, as well as there was a clear association between increased plasma values of D-dimer and positive staining in immunohistochemistry. Results suggest that the monitoring of D-dimer levels in infected dogs could be useful in evaluating the presence of pulmonary thromboembolism in the lungs and that microfilariae may induce microthrombosis in kidneys, thus contributing to renal pathology., (Copyright © 2012 Elsevier B.V. All rights reserved.)

It has recently been reported that dogs affected by canine heartworm disease (Dirofilaria immitis) can show an increase in plasma levels of myoglobin and cardiac troponin I, two markers of muscle/myocardial injury. In order to determine if this increase is due to myocardial damage, the right ventricle of 24 naturally infected dogs was examined by routine histology and immunohistochemistry with anti-myoglobin and anti-cardiac troponin I antibodies. Microscopic lesions included necrosis and myocyte vacuolization, and were associated with loss of staining for one or both proteins. Results confirm that increased levels of myoglobin and cardiac troponin I are indicative of myocardial damage in dogs affected by heartworm disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Heartworm Associated Respiratory Disease (HARD) is a pulmonary syndrome that results from the vascular and parenchymal inflammatory response associated with the arrival and death of Dirofilaria immitis in the distal pulmonary arteries. Barometric whole-body plethysmography (BWBP) is a non-invasive pulmonary function test (PFT) that allows a dynamic study of breathing patterns and is useful to study airway disease and the response to different treatments. The aim of this prospective non-blinded study was to compare respiratory function variables between healthy cats and HARD cats (seropositive to D. immitis) by use of BWBP. Twenty-five healthy cats and six HARD cats were put into the plethysmograph chamber and different respiratory variables were measured. The results were analyzed and compared between the two groups of animals. There were significant differences for bronchoconstriction index variables Pause (P-value<0.001) and enhanced pause (P-value<0.001), minute volume (P-value<0.05) and tidal volume (P-value<0.05) between healthy and HARD cats. There were no significant differences in respiratory rate and inspiratory and expiratory times between both groups of animals. The results obtained in our study support that HARD cats show significant differences in pulmonary function variables obtained by BWBP due to an acute inflammatory response at bronchial, vascular and parenchymal level. This PFT could be a useful method to facilitate the diagnosis of pathological states of bronchoconstriction in HARD cats., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The island of Gran Canaria is a hyperendemic area for canine dirofilariasis. The aim of the present study was to provide data on Dirofilaria immitis in dogs, cats, and humans on this island in 2010. The data confirms the prevalence in the overall canine population (19%), with a considerably higher prevalence (43%) in the autochthonous breed of Canarian Warren hound. The prevalence in the feline population (33%) is higher than that of the canine population, and the existence of specific D. immitis antibodies in the inhabitants of the island of Gran Canaria (12%) is confirmed. In both cats and humans, the prevalence, according to the different climate areas on the island, is related to the prevalence of D. immitis in dogs in the same area, which shows the key epidemiological role played by the canine host with regard to the transmission to other hosts of D. immitis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cardiopulmonary biomarkers are biological parameters that can be objectively measured and quantified as indicators of pathogenic processes (heartworm disease) or as indicators of response to therapeutic intervention. To determine levels of cardiopulmonary biomarkers in canine dirofilariasis, measurements of cardiac troponin T, cardiac troponin I, myoglobin, and D-dimer concentrations were performed for dogs with and without evidence of adult heartworm infection. The results showed that levels of cardiac troponin T were undetectable in all dogs studied while levels of cardiac troponin I were higher in dogs infected with Dirofilaria immitis. In healthy dogs, levels of myoglobin and D-dimer were below detection limits of the instrument and were significantly higher in heartworm-infected dogs, notably in microfilaremic dogs. The results suggest the possibility of using troponin I and myoglobin as markers for cardiac damage and the D-dimer as a supportive tool for a diagnosis of pulmonary thromboembolism in dogs with cardiopulmonary dirofilariasis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Canine cardiopulmonary dirofilariosis caused by Dirofilaria immitis is endemic on the island of Gran Canaria. Epidemiological surveys have shown differences in the prevalence of heartworm disease in the canine population of the island between 1989 and 1998. The aim of the present study was to follow-up the prevalence of D. immitis in both the canine population as a whole and the Canarian Warren Hound population on Gran Canaria between 2000 and 2008. Prevalences observed were always significantly higher in the Canarian Warren Hound population than in the entire canine population. A significant decrease in the prevalence (from 30.19% to 19.36%) in the whole population was observed. In the Canarian Warren Hound the prevalence decreased slightly between 2000 and 2007 (40.42-34.65%), rising in 2008 to values higher than those observed at the beginning of follow-up study (41.6%). These changes are mainly attributed to lack of preventive chemotherapy and the prolonged exposure of the Canarian Warren Hound to mosquito bites. As this breed can act as a natural reservoir in Gran Canaria, regular surveillance is needed to detect changes in the epidemiological picture on this island., (Copyright 2010 Elsevier B.V. All rights reserved.)