Evaluation of acute phase proteins, adiponectin and endothelin-1 to determine vascular damage in dogs with heartworm disease (Dirofilaria immitis), before and after adulticide treatment.

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.

Evaluation of serum biomarkers and proteinuria for the early detection of renal damage in dogs with heartworm (Dirofilaria immitis).

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.

Evaluation of different dosages of doxycycline during the adulticide treatment of heartworm (Dirofilaria immitis) in dogs.

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.

Persistence of pulmonary endarteritis in canine heartworm infection 10 months after the eradication of adult parasites of Dirofilaria immitis.

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.

Variation of the adulticide protocol for the treatment of canine heartworm infection: Can it be shorter?

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.

Pulmonary hypertension in dogs with heartworm before and after the adulticide protocol recommended by the American Heartworm Society.

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.

Evaluation of pulmonary hypertension and clinical status in dogs with heartworm by Right Pulmonary Artery Distensibility Index and other echocardiographic parameters.

BACKGROUND: Pulmonary hypertension (PH) is a frequent and severe phenomenon in heartworm disease caused by Dirofilaria immitis, mainly caused by intimal proliferation of the arteries and pulmonary thromboembolisms. Transthoracic echocardiography is the method of choice for diagnosing PH in dogs although the diagnosis is often based on indirect and subjective parameters. The Right Pulmonary Artery Distensibility Index (RPAD Index) is a method that has been recently validated to estimate the presence and severity of PH in heartworm-infected dogs. This study compared some echocardiographic parameters commonly used to estimate PH in 93 dogs infected by D. immitis and evaluated the impact of the parasite burden, microfilaremia, sex or origin of the dog (client-owned/shelter). RESULTS: None of the studied echocardiographic variables seemed useful in the estimation of the evaluated clinical aspects, except for the PA/Ao ratio for parasite burden. The RPAD Index was determined in 88 of the dogs; of these, 70.4% had PH (mild: 37.5%, moderate: 19.3%, severe: 13.6%). This Index showed non-significant differences according to microfilaremia, sex, origin or parasite burden. Symptomatic dogs showed PH more often and displayed more severe PH, in addition the presence of symptoms was greater among dogs with high burden; on the other hand 64.4% of asymptomatic dogs had some degree of PH according to the RPAD Index. Apart from the PA/Ao ratio, the other evaluated echocardiographic variables were not useful in evaluating of the hypertensive status of the heartworm-infected dog compared to the RPAD Index. CONCLUSIONS: The estimation of most common indirect parameters is not useful in predicting PH in heartworm-infected dogs. The results confirm the RPAD Index as an objective and supportive test in the monitoring and evaluation of PH in the heartworm-infected dog, and show a potential diagnostic value for the detection of PH in asymptomatic animals.