[Orbital case of ocular dirofilariasis]. / Sluchaĭ orbital'nogo dirofiliarioza.

AIM: to report a clinical case of orbital dirofilariasis with attention to peculiarities of disease presentation, diagnostic measures, and treatment results. MATERIAL AND METHODS: A patient with a history of spontaneously subsiding recurrent oedema of the lower eyelid suspicious of parasitic cyst formation. The diagnosis was confirmed preoperatively by orbital ultrasound. Surgical intervention included orbitotomy with microsurgical excision of the parasitic cyst via a percutaneous approach through the lower eyelid skin crease. RESULTS: Ultrasonography of the orbits appeared to be the most informative diagnostic method in this case. Thorough surgical planning allowed us to avoid complications in the postoperative period and to achieve good cosmetic results. CONCLUSION: A painless solid mass lesion with recurrent perifocal oedema and hyperaemia without inflammatory infiltration of surrounding tissues is characteristic of parasitic cysts of the orbit. Amond other infectious diseases, a parasitic cyst stands out for symptomatic relapses and spontaneous regression, irrespective of the treatment received. The use of ultrasound allowed preoperative identification of the sac and the living helminth.

[Observation of dirofilariosis in the practical work of the otorhinolaryngologist].

A case of dirofilariosis is reported. The disease is believed to be caused by the contact with the cat that the patient brought in from Uzbekistan. The authors emphasize the importance of analysis of the medical history of the patients presenting with the pathology being considered, their medical examination, and correct interpretation of X-ray images of the paranasal sinuses taking into account the possibility of distribution of rare helminth species beyond their usual ranges.

Extensive Chorio-retinal Damage Due to Dirofilaria Repens- Report of a Case.

Purpose: To report a case of ocular dirofilariasis causing extensive chorio-retinal damage.Method: Surgical removal of the worm and identification of the same by microscopic examinationand polymerase chain reaction.Results: The worm was identified as Dirofilaria repens.Conclusion: Ocular dirofilariasis is an arthropod borne parasitic infestation affecting the subconjunctival tissue and anterior chamber. Vitreous involvement in Dirofilaria is very rare. Dirofilaria repens can cause extensive chorio-retinal damage resembling diffuse unilateralsubacute neuroretinitis.

High-resolution CT evaluation of bronchial lumen to vertebral body, pulmonary artery to vertebral body and bronchial lumen to pulmonary artery ratios in Dirofilaria immitis-infected cats with and without selamectin administration.

Objectives The bronchial lumen to pulmonary artery (BA) ratio is utilized to evaluate pulmonary pathology on CT images. The BA ratio may be unreliable when changes are present in bronchial and pulmonary arteries concurrently. Bronchial lumen to vertebral body (BV) and pulmonary artery to vertebral body (AV) ratios have been established in normal cats and may serve as an alternative. This study aimed to evaluate the BV, AV and BA ratios in cats before and after infection with Dirofilaria immitis, with and without selamectin administration, and to characterize the distribution of disease. Methods Archived CT images were reviewed from three groups of cats: D immitis-infected untreated (n = 6); infected pretreated with selamectin (n = 6); and uninfected untreated (n = 5). The BV, AV and BA ratios were calculated for all lung lobes for baseline (D0) and day 240 (D240) postinfection. Ratios and percentage change from baseline were compared between lobes and between groups. Results BV and AV ratios were more consistent in identifying abnormalities when disease was present in bronchial and arteries concurrently than BA ratios. Infected untreated cats had significant changes in both BV and AV ratios and percentage change from baseline. Abnormal BV and AV ratios were noted in the infected selamectin group, although less widely distributed. Conclusions and relevance The BV and AV ratios more accurately identified bronchial and pulmonary artery abnormalities in D immitis-infected cats. Both bronchial and pulmonary artery changes were present in infected cats, decreasing the diagnostic application of the BA ratio. Pulmonary artery changes were more widely distributed than bronchial changes in the lung. Heartworm-infected cats receiving selamectin had bronchial and pulmonary artery changes but to a lesser extent than untreated heartworm-infected cats. The CT-derived BV and AV ratios are a useful measure to evaluate lung disease of cats.

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.

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.

[Ultrasonography as an effective diagnostic method for human subcutaneous dirofilariasis].

The diagnosis of human dirofilariasis is difficult because of the absence of laboratory tests. Up-to-date ultrasound devices have a high resolution that can visualize this subcutaneous helminth. Ultrasonography can improve the timely diagnosis of this helminthism, as demonstrated by the given clinical example.

Elevated endothelin-1 expression in dogs with heartworm disease.

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.

Smooth muscle proliferation in chronically injured canine pulmonary arteries is reduced by a potent platelet aggregation inhibitor U-53,059.

Dirofilaria immitis (DI) infection chronically injures canine pulmonary arteries. This injury produces endothelial cell loss, platelet/leukocyte adhesion, and smooth muscle proliferation. In the present study we assessed the effect of the cyclooxygenase inhibitor, U-53,059, on platelet function, platelet kinetics, coagulation, and smooth muscle proliferation in DI infected dogs. Platelet aggregation to the combination of arachidonic acid/ADP was significantly inhibited by U-53,059. Coagulation and hematologic parameters were not effected by either DI infection or U-53,059 treatment. Platelet survival and the number of platelet dense granules were reduced in DI infection. Quantification of the lesions demonstrated that U-53,059 reduced both severity and density compared to non-treated dogs. U-53,059 is a potent and effective inhibitor of platelet aggregation which modifies smooth muscle proliferation produced by chronic vascular injury.

Hemodynamics of heartworm infection.

To determine the hemodynamic responsiveness of dogs testing positive for heartworms (direct blood smear), we compared the response of control and heartworm-infected animals to stellate stimulation (SS). Using the anesthetized, open-chest model, twenty dogs (10 control and 10 heartworm-positive) were instrumented to measure left ventricular pressure (LVP), arterial pressure (AP), and coronary flow velocity (CFV) of the left circumflex coronary artery. CFV was converted to flow per gram tissue (CBFG) by weighing the perfusion area of the circumflex artery. The average wet weight of the worm mass infecting the dogs was 1.0 +/- 0.1 g, which represented an average of 5% of the right ventricular free wall weight. Overall heart size and right ventricular weights were greater in the heartworm-positive animals, as well as heart weight to body weight ratios. Prior to SS, LVP was the same for both groups. However, dP/dt and CBFG were 21% and 19% lower, respectively, in the heartworm-positive animals. Stellate stimulation sufficient to produce a 30-mmHg rise in AP led to 25%, 13%, and 40% increases in LVP, dP/dt, and CBFG, respectively, in the control animals. Increases of 22%, 10%, and 52% in the same parameters were observed in the heartworm-positive animals. Although control dP/dt and CBFG in the heartworm-positive animals were lower than in the heartworm-negative animals, heartworm infection at this level did not hinder myocardial responsiveness to SS.

Quantitative analysis of microfilarial periodicity of Dirofilaria immitis in cats.

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.

Relaxing and contracting activities of heartworm extract on isolated canine abdominal aorta.

Effect of adult heartworm (HW) crude extract on isolated canine abdominal aortic strips precontracted with noradrenaline was examined by recording isometric changes in tension. HW extract caused contraction of the aortic strip at a low concentration (LC) and its relaxation at a high concentration (HC). In aortic strips without endothelium, LC extract elicited a contraction similar to that in the strips with endothelium, whereas HC extract failed to produce any relaxation but instead produced a contraction. The relaxing effect of HC extract was blocked after treatment with 300 microM NG-nitro-L-arginine methyl ester hydrochloride, with reversal by additional treatment with 3 mM L-arginine. It was also markedly reduced or abolished after treatment with 3 microM oxyhemoglobin or 1 microM methylene blue. Fractionation of HW extract by high-performance liquid chromatography revealed that the relaxing and contracting activities are due to different substances in the extract. The results indicate that HW extract contains 2 different vasoactive substances, 1 causing contraction of canine abdominal aorta via a direct action on the smooth muscle, and the other its relaxation indirectly by releasing nitric oxide from endothelial cells. These vasoactive substances might play a role in HW extract-induced shock in dogs, and in the pathogenesis of HW infection.

Pulmonary arteriography and hemodynamics during feline heartworm disease. Effect of aspirin.

The ability of aspirin to block arterial disease and thromboembolism of the pulmonary arteries was studied in heartworm-infected cats. Three groups of cats were transplanted with four heartworms per cat and studied. One group of eight cats (aspirin group) received aspirin (97.5 mg, twice a week) for the five-month infection and another group of eight cats served as the nontreated control group (nontreated group). Based upon the results of the first two groups, the third group (adjusted aspirin group) of six cats was studied in which the aspirin dosage was adjusted in order to maintain an inhibition of in vitro platelet aggregation. Cats were studied by nonselective pulmonary arteriograms before heartworm transplantation and by selective arteriograms, aortograms, and pulmonary hemodynamics five months after heartworm transplant. Pulmonary hypertension, (mean pulmonary artery pressures greater than 16 mmHg), was discovered in three cats with one cat in each group. There were no differences in the mean pulmonary artery pressure or vascular resistance between the groups. Many of the arterial diameters for the nontreated and aspirin groups were greater after the five-month infection than before heartworm infection. All of the postinfection caudal arteries were tortuous and had aneurysms. Some of the caudal lung lobes had perfused areas that appeared to have a hypervascular microvasculature. The proportion of obstructed right and left distal caudal pulmonary arteries and the resulting nonperfused area of the caudal lung lobe in the nontreated and aspirin treated groups were each greater than in the adjusted aspirin group.(ABSTRACT TRUNCATED AT 250 WORDS)

A clinical study on velocity patterns of pulmonary venous flow in canine heartworm disease.

In this study, we evaluated methods of determining the velocity patterns of pulmonary venous flow (PVF) in dogs and then investigated the relationship of the patterns to cardiac functions in heartworm disease (HD) by transthoracic echocardiography (TTE). The results revealed that there was a good correlation between PVF patterns determined by transesophageal echocardiography (TEE) and TTE in animals lying on their left sides. The measurement of S and D wave velocities (PVS and PVD) by TTE was shown to allow clinical determination of the velocity patterns of PVF in dogs. The HD groups showed significant increases in PVS and PVD, and S and D wave time-velocity integrals (S-TVI and D-TVI) of the right cranial lobe PVF, when compared with the normal group, as determined by TTE (P<0.05). In contrast, the HD groups produced significant decreases in PVD and D-TVI of the right caudal lobe PVF compared with the normal group (P<0.05), and a significant increase in the ratio of S-TVI to (S-TVI + D-TVI) (P<0.05). It is, therefore, suggested that measurement of the velocity patterns of the right cranial and caudal lobe PVF could be one method of assessing the stages of obstructive lesions in the pulmonary artery.

Relationship between pulmonary arterial pressure and pulmonary thromboembolism associated with dead worms in canine heartworm disease.

To examine effects of thromboemboli due to dead worms on pulmonary arterial pressure (PAP), 20 to 50 dead heartworms were inserted into the pulmonary arteries of 4 heartworm uninfected dogs (uninfected group) and 11 dogs infected with heartworms (infected group). In the uninfected group, the mean PAP rose 1 week after worm insertion (10.9 to 166. mmHg), but it recovered by the 4th week. Clinical signs, hemodynamics and blood gas findings also deteriorated at the 1st week, but recovered at the 4th week. Angiographic and pathological findings indicated that blood flow recovered through the spaces between thromboemboli and vessel walls at the 4th week. The infected dogs were divided into three groups. In the infected-I group (5 dogs), the intimal lesions of the pulmonary arteries were slight, and clinical and laboratory findings showed changes similar to those of the uninfected group. In the infected-II group (4 dogs), the pulmonary arterial lesions were severe and the mean PAP was higher (25.7 mmHg) than in the uninfected group before worm insertion. An increase in PAP (34.1 mmHg) and worsening of clinical and laboratory findings were noticed till the 4th week. Thromboemboli adhered extensively to the vessel walls. Two dogs in the infected-III group died of severe dyspnea on the 9th and 10th day, and the mean PAP rose remarkably at the 1st week (from 19.4 to 28.2 mmHg). Severe pulmonary parenchymal lesions with edema or perforation were observed. From the above results, it was clarified that effects of dead worms on PAP and clinical signs depended on the severity of pulmonary arterial lesions before worm insertion.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressure-flow relationship and longitudinal distribution of pulmonary vascular resistance in heartworm-infected dogs.

The pressure-flow relationships and the longitudinal distributions of pulmonary vascular resistance in normal and heartworm-infected (HWI) dogs were compared in an isolated, blood perfused preparation. The pulmonary circulation was partitioned into pulmonary arterial, middle, and venous segment based on the concept of a five element lumped model. The pulmonary arterial pressure-flow relationships were found to be non-linear and convex to the pressure axis in both normal and HWI lungs. The pressure-flow relationships of the pulmonary arterial and venous segment were linear and these slopes in the HWI lungs were significantly higher than the normal lungs. The pressure gradient of the middle segment was increased as flow increased at lower flow range, however, it was not increased during higher perfusion range in both lungs. At higher flow, the pressure gradient of the middle segment in the HWI lungs was significantly higher than the normal lungs. These results suggest that the ohmic resistance was almost equal to the sum of the two slopes of the pressure-flow relationships of the pulmonary arterial and venous segment because the pressure gradient of the middle segment was not altered as flow increased during higher perfusion rate. Because the slopes of the pressure-flow relationships of the pulmonary arterial and venous segment were increased with heartworm infection, the ohmic resistance of HWI lungs would be higher than normal lungs. The intercept pressure on the pressure axis of the linear portion of the pulmonary arterial pressure-flow relationship, a critical closing pressure, was regarded as pressure gradient of the middle segment during higher perfusing rate because the intercept pressures of pressure-flow relationships of pulmonary arterial and venous segment were almost equal to zero. Therefore, the critical closing pressure of HWI lungs would be higher than normal lungs. The pulmonary hypertension of filariasis appears to be due to an increase in ohmic resistance and elevated critical closing pressure.

Plasma renin activities, angiotensin II concentrations, atrial natriuretic peptide concentrations and cardiopulmonary function values in dogs with severe heartworm disease.

Relationships among plasma renin activities (PRA), plasma angiotensin II (ATII) concentrations, atrial natriuretic peptide (ANP) concentrations and cardiopulmonary function values were examined in dogs with ascitic pulmonary heartworm disease and acute- and chronic-vena caval syndrome (CS). PRA, plasma ATII concentration and plasma ANP concentration tended to be higher or were significantly higher in dogs with ascites, acute- and chronic-CS. PRA correlated significantly with plasma ATII concentration, WBC count, ALP activity, plasma concentrations of urea nitrogen, creatinine, sodium, potassium, and chloride, right ventricular endodiastolic pressure and right atrial pressure. Plasma ATII concentration correlated significantly with WBC count, plasma concentrations of urea nitrogen, sodium, and potassium, right ventricular endodiastolic pressure and right atrial pressure. Plasma ANP concentration did not correlate with PRA or ATII concentration, but correlated significantly only with pulmonary arterial pressure.

Blood gas analysis in dogs with pulmonary heartworm disease.

Blood gases were analyzed in dogs with pulmonary heartworm (HW) disease. The arterial oxygen tension (PaO2) in dogs with mild signs of dirofilariasis (mildly affected group, n = 48, 85.7 +/- 8.2 mmHg) and in dogs with signs of right heart failure (severely affected group, n = 13, 76.4 +/- 11.6 mmHg) was lower (p < 0.01) than in dogs without HW infection (HW-free group, n = 19, 91.5 +/- 7.3 mmHg). Only 2 dogs in the severely affected group had a PaO2 less than 60 mmHg. The arterial carbon dioxide tension (PaCO2, p < 0.01) and mixed venous O2 (p < 0.01) and CO2 (p < 0.01) tensions were lower, and alveolar-arterial oxygen difference (AaDO2, p < 0.01) was greater in the severely affected group than in the HW-free and mildly affected groups. Arterial pH and bicarbonate (HCO3-) concentrations were lower (p < 0.01) in both affected groups than in the HW-free groups. The anion gap level was not different among the 3 groups. Serum lactic acid level in the severely affected group was higher (p < 0.01) than in the HW-free and mildly affected groups. However, a slightly higher serum lactic acid concentration was found only in 2 dogs of the severely affected group (3.84 mmol/l and 3.82 mmol/l). The PaO2 (r = -0.62) and AaDO2 (r = 0.66) correlated significantly (p < 0.01) with mean pulmonary arterial pressure. One week after HW removal, blood gases, pH and HCO3- concentration remained unchanged in the mildly affected group. In the severely affected group, blood gas values were the same, but pH and HCO3- concentration improved slightly.

Cardiopulmonary values in dogs with artificial model of caval syndrome in heartworm disease.

Cardiopulmonary values were determined in dogs with an artificial model of heartworm caval syndrome, which was produced by insertion of heartworm-like silicone tubes into the tricuspid valve orifice and right atrium. Fifteen to 25 tubes with some knots were inserted in 6 dogs (knot group), and 7 to 11 tubes (small-number group) or 29 to 37 tubes (large-number group) without a knot in 3 dogs, respectively. After tube insertion, angiographic contrast medium infused into the right ventricle regurgitated to the right atrium in all cases, and the regurgitation was the most severe in the large-number group. On electrocardiographic findings, the atrial and/or ventricular premature beat developed. The height of a- and v-wave of right atrial pressure curves elevated in all groups. The elevation in v-wave was obvious in the large-number group. The pulmonary arterial pressure tended to fall or to elevate slightly, and total pulmonary resistance increased in all groups. The right cardiac output decreased significantly in all cases. The right heart hemodynamics of the model might resemble those in spontaneous cases without disturbed pulmonary circulation.

Relationship between pulmonary arterial pressure and lesions in the pulmonary arteries and parenchyma, and cardiac valves in canine dirofilariasis.

The relationship between pulmonary hypertension and lesions was examined in 41 dogs infested naturally with heartworms, which consisted of 28 cases with pulmonary heartworm disease and 13 cases with caval syndrome. Pulmonary arterial pressure (PAP) was measured before and 1 or 7 days after heartworm removal with a flexible alligator forceps. In these dogs, lesions were examined after the last measurement of PAP. The mean PAP was 28.2 +/- 16.0 mmHg (10.9 to 81.4 mmHg in range) at post-removal phase. Pulmonary arterial intimal lesions, pulmonary thromboemboli, pneumonic lesions, tricuspid valvular lesions and mitral valvular lesions were macroscopically recognized in 95, 59, 39, 54 and 56% of cases, respectively. These lesions were classified by severity and the relationship with PAP was examined by the multiple correlation analysis. The multiple coefficient correlation was found the highest between PAP and thromboemboli, followed by mitral valvular lesion, tricuspid valvular lesion, and pneumonic lesion. There was no significant correlation between PAP and intimal lesions. The coefficient of determination showed the highest value in thromboemboli when one variable was used, and increased only very slightly when a variable of thromboemboli was added to those of other lesions. The cases with high PAP had fresh thromboemboli in large pulmonary arteries. From these evidences, it was concluded that thromboemboli following natural death of heartworm was the most important factor causing an increase in PAP and developing clinical signs in canine heartworm disease.