Heartworm-infected Dogs Research Articles

Heartworm (Dirofilaria immitis) Prevalence in Dogs Determined by In-House ELISA Based on Filaria-Specific Antibodies in Tropical and Temperate Regions of Mexico

Dirofilaria immitis is a mosquito-borne nematode of dogs, other carnivores and, occasionally, humans. Globally, D. immitis infection (which causes heartworm) is typically more prevalent in tropical than temperate regions. In this study, the seroprevalence of D. immitis was determined from a sample of 335 non-stray dogs from four municipalities, two each from the states of Puebla and Guerrero in Mexico, using polyclonal antibodies to detect serum antigens using an enzyme-linked immunosorbent assay (ELISA). The accuracy of the assay was compared with the modified Knott’s test. The polyclonal antibody used in the direct ELISA had a high sensitivity (100%) with variable specificity (98.2–98.8%) in the municipalities of Puebla and Guerrero. The area under the curve for the four municipalities was 1.0, indicating a high accuracy test, with a cut-off value ranging from 0.45 to 0.50. The overall prevalence of D. immitis infection was 17.56% (59 out of 335). The highest prevalence was in Acapulco (24.78%), followed by Chilpancingo (20.93%), Tecamachalco (10.81%) and Quecholac (8.06%). The highest percentage of positive samples was detected in tropical regions (23.12%) and the lowest in temperate regions (9.56%). This study demonstrates that polyclonal anti-D. immitis antibodies can successfully diagnose heartworm-infected dogs and be used to monitor prevalence effectively and develop prevention strategies against Dirofilaria infection.

Assessment of Thoracic Radiographic Alterations in Dogs with Heartworm and Their Correlation with Pulmonary Hypertension, Pre- and Post-Adulticide Treatment.

Pulmonary hypertension (PH) is a prevalent and severe complication in dogs infected with Dirofilaria immitis. This study aimed to elucidate the progression of PH by analyzing radiographic parameters and the Right Pulmonary Artery Distensibility (RPAD) Index at three key time points: diagnosis (day 0), discharge (day 90), and six months post-discharge (day 270). Fifty-two heartworm-infected dogs were divided into two groups: non-hypertensive and hypertensive. Radiographic measurements, including Vertebral Heart Size (VHS), CrPA/R4 ratio, and CdPA/R9 ratio, along with the RPAD Index, were assessed on Days 0, 90, and 270. Results indicated that, in Group A, the RPAD Index improved significantly from 42% on Day 0 to 43.16% on Day 90, with no significant change by Day 270 (42%). In contrast, hypertensive dogs exhibited a persistently low RPAD Index, averaging 17% throughout this study (p < 0.001). Radiographic parameters in hypertensive dogs showed continuous elevation compared to non-hypertensive dogs, with significant increases in VHS, CrPA/R4, and CdPA/R9 ratios on day 270 compared to day 0 (p < 0.05). The results confirmed that PH persisted in dogs with D. immitis after adulticide treatment, highlighting the importance of regular radiographic monitoring for assessing and managing long-term outcomes in dogs with PH during and after adulticide treatment. Continuous surveillance is thus essential for the effective post-treatment management of PH in dogs.

Association between Thoracic Radiographic Changes and Indicators of Pulmonary Hypertension in Dogs with Heartworm Disease.

Pulmonary hypertension (PH) is a consequence of pulmonary endarteritis during infection with Dirofilaria immitis in dogs. Echocardiography is the technique of choice but is not always accessible to all clinicians. This study aimed to evaluate the association of the radiological findings in dogs with heartworm disease and the presence or absence of echocardiographically characterised PH. The study included 62 heartworm-infected dogs that underwent thoracic radiographs and echocardiography. The studied dogs showed moderate to severe PH when the Right Pulmonary Artery Distensibility (RPAD) Index was <29.5%. The RPAD Index was used for comparison with thoracic radiographs. The Vertebral Heart Size (VHS), right cranial pulmonary artery passing through the fourth rib in the laterolateral projection (CrPA/R4) ratio, and right caudal pulmonary artery to the ninth rib in the dorsoventral projection (CdPA/R9) ratio showed significant differences between dogs with/without PH (p < 0.001). Sensitivity (sen) and specificity (sp) cut-off values were obtained: VHS ≥ 9.53 (sen 93.75%, sp 63.33%); CrPA/R4 ≥ 1.08 (sen 87.5%, sp 70%); and CdPA/R9 ≥ 1.10 (sen 96.88%, sp 76.66%). The CrPA/R4 and CdPA/R9 ratios showed potential as a preliminary screening tool for PH in heartworm-infected dogs, suggesting that they may reliably indicate the presence of PH and guide the decision for further diagnostic testing.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. This study evaluated platelet function in HWI dogs. Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/μL vs 0.267 ± 0.20 × 103/μL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Erythrocyte sedimentation rate in heartworm naturally infected dogs "with or without" Leishmania infantum seropositivity: an observational prospective study.

Canine heartworm disease by Dirofilaria immitis and canine leishmaniosis by Leishmania infantum (CanL) are both vector-borne diseases with frequently overlapping endemicity and able to trigger the acute phase response, being characterized by variations in acute phase proteins (APP). Recently, erythrocyte sedimentation rate (ESR), an indicator of inflammation, has gained attention in veterinary medicine, proving useful in several conditions that include CanL active forms in dogs. This study aims to evaluate ESR in heartworm-infected dogs, compare levels with heartworm-infected and L. infantum seropositive dogs as well as clinically healthy dogs, and assess correlations with other laboratory parameters. From October 2022 to January 2023, a prospective observational study was conducted enrolling heartworm-infected (Dirofilaria group) and heartworm-infected L. infantum seropositive (Dirofilaria/Leishmania group) animals subgrouped according to the CanL clinical form (Dirofilaria/Leishmania active and non-active groups). A group of clinically healthy dogs (control group) was also included. For each dog enrolled physical examination and laboratory tests (complete blood count, biochemical panel including APP, serum protein electrophoresis) were performed. Dirofilaria and Dirofilaria/Leishmania groups presented a significantly higher ESR level compared to healthy dogs. Dirofilaria/Leishmania active group had the highest ESR level among the groups considered. Dirofilaria/Leishmania non-active group had an ESR similar to the Dirofilaria group, but significantly higher and lower compared to the control and the Dirofilaria/Leishmania active group, respectively. A significant positive correlation between ESR and C-Reactive Protein has been found in all groups except for the Dirofilaria/Leishmania non-active group. In Dirofilaria/Leishmania active group a strong positive correlation between ESR and gamma globulins percentage as well as a strong negative correlation between ESR and albumin, albumin/globulins ratio were found. Overall, the ESR was confirmed to be an inflammation marker as well as a helpful disease index, being notably increased in heartworm-infected dogs affected by an active form of CanL.

Usefulness of Tissue Doppler Imaging for the Evaluation of Pulmonary Hypertension in Canine Heartworm Disease

Background: Dirofilaria immitis is a nematode that produces proliferative pulmonary endarteritis in dogs due to direct contact of the adult parasites with the intima layer of the pulmonary arteries, leading to irreversible severe structural damage and sustained pulmonary hypertension (PH), which can produce severe cardiorespiratory disorders. The purpose of this study was to assess the diagnostic value of the echocardiography tissue Doppler imaging (TDI) in determining the presence of PH in dogs with heartworm disease. Methods: There were 116 heartworm-infected dogs with PH and 33 healthy dogs included in the study. Based on the right pulmonary artery distensibility index (RPADi) &lt; 29.5%, PH was present in 47.4% of infected dogs. Additionally, the animals were evaluated using other standard alternative echocardiographic measures to estimate PH. Moreover, a total of eight echocardiographic measurements were analysed using the TDI to determine its usefulness in diagnosing PH (E′, A′, S, E′:A′, global TDI, HRI-IVCT, HRI-IVRT, R-TEI). Results: The TDI measurements showed significant differences between dogs with and without PH, demonstrating a positive correlation with respect to the RPADi. In addition, cut-off values for the detection of PH with excellent sensitivity and specificity were found for E′:A′, global TDI, HRI-IVCT, HRI-IVRT and R-TEI. Conclusions: The TDI mode may be useful as an adjunct diagnostic method for the determination of PH in dogs with Dirofilaria immitis.

Usefulness of NT-proBNP in dogs with heartworm: could this biomarker be useful to evaluate pulmonary hypertension?

BackgroundIn recent years, the usefulness of echocardiography and serum biomarkers in the diagnosis of pulmonary hypertension (PH) in dogs with heartworm disease has been studied. Previously, N-terminal pro B-type natriuretic peptide (NT-proBNP) has shown high concentrations in dogs with heart disease and/or PH as well as its usefulness as a prognostic indicator, but it has never been evaluated in the diagnosis and prognosis of PH in dogs with heartworm disease. The aim was to evaluate the serum concentrations of NT-proBNP in dogs infected by Dirofilaria immitis to determine its usefulness as a tool to detect precapillary PH.MethodsNT-proBNP was determined in 50 heartworm-infected dogs. Presence/absence of PH was determined echocardiographically, using the Right Pulmonary Artery Distensibility Index (RPAD Index) and the systolic flow of tricuspid regurgitation mainly, together with other echocardiographic measurements following the guidelines of the American College of Veterinary Internal Medicine (ACVIM) for the diagnosis of PH. Other epidemiological parameters (breed, age, sex, status: client-owned or shelter dogs) and clinical parameters (microfilaremia, parasite burden, presence of symptoms, body condition) were collected as well.ResultsModerate-severe PH was present in 40% of the dogs (RPAD Index < 29.5%), NT-proBNP concentrations being significantly higher compared with dogs that did not have PH. A cutoff for NT-proBNP of ≥ 1178.45 pmol/l showed a sensitivity of 64.3% and a specificity of 95.5% for the presence of moderate-severe PH. Older dogs and dogs from shelters showed significantly higher NT-proBNP concentrations. Dogs with symptoms and low body condition presented significantly higher NT-proBNP concentrations as well.ConclusionsThe determination of NT-pro-BNP concentration can be a useful tool in the diagnostic work-up of dogs with heartworm disease and associated PH and can help to identify animals in the more advanced stage of this disorder.Graphical abstract

Fluralaner (Bravecto®) treatment kills Aedes aegypti after feeding on Dirofilaria immitis-infected dogs

BackgroundTransmission of canine heartworm (Dirofilaria immitis) from infected to naïve dogs is dependent on successful mosquito feeding and survival.MethodsTo determine whether treating heartworm-infected dogs with fluralaner (Bravecto®) limits the survival of infected mosquitoes, and potentially the transmission of D. immitis, we allowed female mosquitoes to feed on microfilaremic dogs and evaluated mosquito survival and infection with D. immitis. Eight dogs were experimentally infected with D. immitis. On day 0 (~ 11 months post-infection), four microfilaremic dogs were treated with fluralaner according to label directions while the other four were non-treated controls. Mosquitoes (Aedes aegypti Liverpool) were allowed to feed on each dog on days −7, 2, 30, 56, and 84. Fed mosquitoes were collected, and the number of live mosquitoes determined at 6 h, 24 h, 48 h, and 72 h post-feeding. Surviving mosquitoes held for 2 weeks were dissected to confirm third-stage D. immitis larvae; PCR (12S rRNA gene) was performed post-dissection to identify D. immitis in mosquitoes.ResultsPrior to treatment, 98.4%, 85.1%, 60.7%, and 40.3% of mosquitoes fed on microfilaremic dogs were alive at 6 h, 24 h, 48 h, and 72 h post-feeding, respectively. Similarly, mosquitoes fed on microfilaremic, non-treated dogs were alive 6 h post-feeding (98.5–100%) throughout the study. In contrast, mosquitoes fed on fluralaner-treated dogs 2 days after treatment were dead or severely moribund by 6 h post-feeding. At 30 and 56 days post-treatment, > 99% of mosquitoes fed on treated dogs were dead by 24 h. At 84 days post-treatment, 98.4% of mosquitoes fed on treated dogs were dead by 24 h. Before treatment, third-stage larvae of D. immitis were recovered from 15.5% of Ae. aegypti 2 weeks after feeding, and 72.4% were positive for D. immitis by PCR. Similarly, 17.7% of mosquitoes fed on non-treated dogs had D. immitis third-stage larvae 2 weeks after feeding, and 88.2% were positive by PCR. Five mosquitoes fed on fluralaner-treated dogs survived 2 weeks post-feeding, and 4/5 were from day 84. None had third-stage larvae at dissection, and all were PCR-negative.ConclusionThe data indicate that fluralaner treatment of dogs kills mosquitoes and thus would be expected to reduce transmission of heartworm in the surrounding community.Graphical

Safety of Simparica Trio® (sarolaner, pyrantel, moxidectin) in heartworm-infected dogs

BackgroundAssessment of the safety of heartworm preventatives in dogs with pre-existing patent heartworm (Dirofilaria immitis) infections is necessary because rapid adult worm and microfilarial death can lead to severe clinical complications, including thromboembolism and anaphylactic shock in dogs. The aim of this study was to determine the clinical safety of Simparica Trio® (sarolaner, pyrantel, moxidectin) in heartworm-infected dogs and the degree of microfilaricidal and adulticidal activity of three consecutive monthly treatments of Simparica Trio.MethodsTwenty-four laboratory Beagle dogs were implanted with 10 male and 10 female D. immitis (ZoeKY isolate), and once infection was patent, they were randomized equally among three groups to receive no treatment, 1× or 3× the maximum recommended label dose of Simparica Trio. Dogs in the treated groups received Simparica Trio on days 0, 28 and 56. In-life assessments included body weight, physical examinations, clinical observations, daily general health observations, a quantitative estimate of food consumption and blood collections for pharmacokinetic (PK) analysis, microfilariae (MF) counts and D. immitis antigen testing. At the end of the study the heart, lungs and pleural and peritoneal cavities were examined for adult D. immitis worms.ResultsSimparica Trio was generally well tolerated. Emesis occurred at low frequency in all groups including control. Abnormal stool occurred occasionally in the 1× and 3× groups throughout the 3-month study. Fever (> 104 °F/40 °C) was recorded in one 1× and one 3× dog 1 day after the first dose and resolved by the following day. No severe hypersensitivity reactions occurred. The mean number of circulating microfilariae (MF) counts in the control group increased from 12,000/ml at study start (Day 0) to > 20,000/ml at Day 28 and remained > 20,000/ml for the duration of the study. The least squares means of circulating MF were reduced by 69.8% on Day 1 and 97.4% on Day 7 for the 1× group and remained at > 99% lower than the control group for the remainder of the study. Similarly, least squares means of circulating MF were reduced by 85.3% on Day 1 and 93.9% on Day 7 for the 3× group and remained > 98% lower than the control group for the remainder of the study. At the end of the study, the mean number of implanted adult worms recovered was < 10 per sex in all groups with 90%, 85% and 75% of live adult heartworms recovered in control, 1× and 3× treatment groups, respectively. Low numbers of dead adult worms were recovered in 1× and 3×, with none in control. Following each dose, the moxidectin and sarolaner AUC and Cmax had close to dose proportional increases.ConclusionsThis study demonstrated that Simparica Trio (sarolaner, pyrantel, moxidectin) was well tolerated when administered to heartworm-positive dogs at 1× and 3× the maximum recommended dose at 28-day intervals for 3 consecutive months. Simparica Trio significantly reduced microfilaria counts in both treatment groups, without significant clinical consequences. At the doses administered, Simparica Trio had minor adulticidal activity but resulted in no clinical sequelae.Graphical

Interaction between Wolbachia pipientis and Leishmania infantum in heartworm infected dogs

BackgroundWolbachia is a Gram-negative endosymbiont associated with several species of arthropods and filarioid nematodes, including Dirofilaria immitis. This endosymbiont may elicit a Th1 response, which is a component of the immunity against Leishmania infantum.MethodsTo investigate the interactions between Wolbachia of D. immitis and L. infantum in naturally infected dogs and cytokine circulation, dogs without clinical signs (n = 187) were selected. Dogs were tested for microfilariae (mfs) by Knott, for female antigens of D. immitis by SNAP, and for anti-L. infantum antibodies by IFAT and assigned to four groups. Dogs of group 1 (G1) and 2 (G2) were positive for D. immitis and positive or negative to L. infantum, respectively. Dogs of group 3 (G3) and 4 (G4) were negative to D. immitis and positive or negative to L. infantum, respectively. Wolbachia and L. infantum DNA was quantified by real-time PCR (qPCR) in dog blood samples. A subset of dogs (n = 65) was examined to assess pro- and anti-inflammatory cytokine production using an ELISA test.ResultsOf 93 dogs positive to D. immitis with circulating mfs, 85% were positive to Wolbachia, with the highest amount of DNA detected in G1 and the lowest in dogs with low mfs load in G1 and G2. Among dogs positive to L. infantum, 66% from G1 showed low antibody titer, while 48.9% from G3 had the highest antibody titer. Of 37 dogs positive to Wolbachia from G1, 26 (70.3%) had low antibody titers to L. infantum (1:160). Among cytokines, TNFα showed the highest mean concentration in G1 (246.5 pg/ml), IFNγ being the one most represented (64.3%). IL-10 (1809.5 pg/ml) and IL-6 (123.5 pg/ml) showed the highest mean concentration in dogs from G1. A lower percentage of dogs producing IL-4 was observed in all groups examined, with the highest mean concentration (2794 pg/ml) recorded in G2.ConclusionResults show the association of D. immitis and Wolbachia with the lower antibody titers of L. infantum in co-infected dogs, suggesting the hypothesis that the endosymbiont may affect the development of the patent leishmaniosis. However, due to the limitations associated with the heterogeneity of naturally infected dogs in field conditions, results should be validated by investigation on experimental models.Graphical

Right Pulmonary Artery Distensibility Index in Heartworm Infected Dogs: Are the Different Methods Leading to Same Results?

Canine Heartworm Disease (HWD) is mainly a pulmonary vascular disease. The reduction of the Pulmonary Artery Distensibility (PAD) is an early index of pulmonary vascular disease. Echocardiographic evaluation of the Right Pulmonary Artery Distensibility index (RPADi) is calculated as the percentage change in diameter of the right pulmonary artery (RPA) between systole and diastole. Historically, two main methods have been used for RPADi calculation: The Venco method and Visser method; however, different hybrid methods have also been used by other authors. Therefore, it could be difficult for a clinician to decide which method to apply and how to interpret the results based on the reference values reported. The aim of this study was to compare the RPADi obtained by five different techniques (Venco classic, Venco modified, Visser classic, Visser modified 1, and Visser modified 2). The study design was a retrospective, single center, observational study. Forty-seven client-owned dogs were included. The measurements were performed off-line as an average of three consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. The RPADi was satisfactorily obtained by all methods in all dogs. Intra-observer measurement variability was clinically acceptable both for systolic and diastolic measurements. Although the Bland-Altman test showed a statistical agreement between the various methods used to calculate the RPADi, these methods cannot be used interchangeably in a clinical setting. Instead, the measurement method and reference values should always be specified.

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 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.

Comparison of acid- versus heat-treatment for immune complex dissociation and detection of Dirofilaria immitis antigen in canine plasma

Annual antigen testing is a mainstay for diagnosing infection with Dirofiliaria immitis in dogs; yet, it has been documented that some heartworm-infected dogs and cats test false-negative for antigen due to the presence of antigen-antibody complexes. Several studies have reported the use of heat as a reliable means of immune-complex dissociation (ICD) in recent years; however, the data regarding the use of acid as a reliable method of ICD for D. immitis detection are limited. The objective of this study was to compare an acid-based form of ICD to the more established and evaluated method of heat-based ICD in experimentally infected and non-infected dogs. Plasma from class A dogs experimentally infected ∼4 months prior with D. immitis (infected; n = 24) and dogs reared indoors with no history of exposure to mosquitoes (non-infected; n = 75) were evaluated for presence of D. immitis antigen (DiroCHEK® Heartworm antigen test kit). Each sample was divided into three aliquots for testing: [1] Control plasma (no acid- or heat-treatment), [2] acid-treated plasma (trichloroacetic acid (TCA), incubation, centrifugation for 5 min at 16,000 X g, buffer), and [3] heat-treated plasma (104 °C followed by centrifugation at 16,000 X g). Treatments for each aliquot were performed and tested in triplicate; results were determined both visually (color change) and by spectrophotometric analysis (optical density [OD] value). Of the 24 infected dogs, 0/24 tested positive for antigen in the absence of acid- or heat-treatment. Those same plasma samples following processing by either acid- or heat-treatment yielded 18/24 (75.0%) and 19/24 (79.2%) antigen-positive results, respectively. Of the 75 plasma samples from non-infected dogs, neither acid- nor heat-treatment of plasma caused any false-positive color changes or spectrophotometric values. These results indicate that acid as a means of ICD reliably allowed for the detection of D. immitis antigen in infected plasma while not inducing false-positive results in non-infected plasma samples.

Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both

BackgroundCombination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis.ResultsQuantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females.ConclusionsFurther studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors’ knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.

Mosquito abundance in a Dirofilaria immitis hotspot in the eastern state of Rio de Janeiro, Brazil.

Coastal lowlands in the state of Rio de Janeiro, Brazil, support high numbers of heartworm-infected dogs. Microfilariae of heartworm need to be ingested by a potencial mosquito vector in order to develop into infective larvae and infect a new host. Ochlerotatus taeniorhynchus and Ochlerotatus scapularis are the primary vector species in the coastal lowlands of metropolitan Rio de Janeiro; thus, the aim of this study was to investigate whether these two species were abundant enough at the heartworm hotspot in the eastern area of the state to be important to the local parasite's life cycle. The study was conducted at the Massambaba sandbank (22°55'45″S;42°18'51″W), where canine heartworm prevalence was 53.1%. Mosquitoes were captured monthly using two traps with CO2, as well as eventual human landing. A total of 6118 mosquitoes of 16 species were collected. Ochlerotatus taeniorhynchus (37.5%), Culex quinquefasciatus (19.3%), Oc. scapularis (18.2%), and Coquillettidia sp. (10.5%) were the most abundant species. The monthly variation in frequency was marked for Oc. scapularis and Cx. quinquefasciatus and steadier for Oc. taeniorhynchus and Coquillettidia sp. The abundance of the two Ochlerotatus species reaffirms that they are the primary natural vectors for D. immitis in the coastal Rio de Janeiro state lowlands.

Doxycycline treatment for Dirofilaria immitis in dogs: impact on Staphylococcus aureus and Enterococcusantimicrobial resistance.

Doxycycline is an antibiotic that, in addition to the classic antibacterial use, is also prescribed to fight parasitic diseases, like heartworm disease in dogs. Despite the concern that the overuse of this antibiotic may decrease susceptibility of clinically important bacteria, the consequences of the prolonged doxycycline therapy in heartworm-infected dogs have never been studied before. We have analyzed the impact of this therapy on Staphylococcus aureus and Enterococcus antimicrobial resistance. In this study, 17 heartworm-infected dogs (10 that had completed the doxycycline treatment and 7 dogs that had not yet begun) were included. Twenty-four isolates of Staphylococcus aureus were obtained from two locations of each dog. After treatment, 73.3% of isolates were resistant to at least one antibiotic but only 22.2% of isolates before treatment. Most of doxycycline resistant isolates were obtained from dogs that have received treatment. Erythromycin resistance or intermediate susceptibility was detected in 45.6% of isolates, most of them from dogs after treatment. For Enterococci, 48 isolates were obtained from fecal samples (25 before treatment and 23 after treatment). Before treatment, 32% of isolates were resistant at least to one antibiotic while after, this data increase up to 65%. Comparing isolates before and after treatment, a clear increase in resistance to doxycycline (12% against 21.74%) and erythromycin (20% against 39.13%) was observed. Although the present work is a preliminary research, the results encourages the development of further studies to determinate the effect of prolonged doxycycline therapy on antimicrobial resistance.

Acute phase proteins and markers of oxidative stress to assess the severity of the pulmonary hypertension in heartworm-infected dogs

BackgroundCanine heartworm infection is characterized by pulmonary endarteritis and pulmonary hypertension (PH). The aim of the present study was to evaluate the relationship between PH with the concentrations of different positive (C-reactive protein [CRP] and haptoglobin [Hp]) and negative (albumin and paraoxonase-1 [PON-1]) acute phase proteins (APP), as well as the oxidative stress, by measuring glutathione peroxidase (GPx) and total antioxidant capacity (TAC) in 27 heartworm-infected dogs on Day 0 (diagnosis) and Day 120 (1 month after the last adulticide injection). Presence/absence of PH was determined by the Right Pulmonary Artery Distensibility (RPAD) Index.ResultsOn Day 0, 62.9% of the dogs showed PH. Concentrations of CRP and Hp were higher in dogs with PH, especially in dogs with moderate-severe PH (P < 0.005 and P < 0.05, respectively). Albumin and PON-1 concentrations were higher in dogs without PH (P < 0.05 for albumin). On Day 120, 59.2% of the dogs presented with PH; CRP decreased while Hp increased (P < 0.005 and P < 0.05, respectively). Also, albumin and PON-1 rose, especially in absence of PH. There were not significant changes in the serum values of GPx and TAC.ConclusionsCRP and Hp have a potential prognostic role in dogs with dirofilariasis because increases in positive APP correlated with presence and severity of PH. CRP decreased, but Hp persisted at an elevated level in dogs with PH 1 month after the end of the adulticide treatment. CRP and Hp could work as early biomarkers of PH and be useful to stage the disease and to monitor the evolution of the patient and indirectly evaluate the persistence of arterial damage after the parasites have been eliminated. Albumin and PON-1 also showed potential value as markers of PH, although further research is necessary to determine its utility.

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

BackgroundPulmonary 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).ResultsNone 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.ConclusionsThe 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.

Acute phase protein response in heartworm-infected dogs after adulticide treatment

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.