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

BACKGROUND: 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. OBJECTIVE: This study evaluated platelet function in HWI dogs. METHODS: 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. RESULTS: 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. CONCLUSIONS: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial.

The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adult antigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.

Microfilariae infection by Acanthocheilonema reconditum and Dirofilaria immitis and their molecular detection in a dog with lymphoma: Case report.

Objective: Microfilariae parasites are common in tropical regions, and some species are reported as potentially zoonotic. The diagnosis of filarial infection in dogs by cytology or hematologic techniques showed lower sensibility and specificity, which may result in misdiagnosis. Thus, molecular techniques seem to be an alternative to identifying and detecting microfilariae infections. On the other hand, lymphoma is one of the main tumors in domestic animals, with a high prevalence in domestic canines. This study aims to report a mixed infection with microfilariae in a dog with lymphoma, emphasizing its diagnosis and the possible role of this infection in the development of the neoplasia. Materials and Methods: An 8-year-old male mixed breed dog was referred to consultation due to the presence of lethargy, recumbency, skin ulceration lesions, nonspecific pain manifestations, emesis, myoclonus in the left temporalis muscle, and seizures. Routine blood and biochemistry tests were normal, and cytology of the skin evidenced a microfilariae infection. The dog died due to a cardiorespiratory arrest, and tissue sampling was done for histopathology and molecular analysis at the necropsy examination. Results: Skin lesions were related to a microfilarial pyogranuloma related to Acanthocheilonema reconditum. Histopathology of the spleen and liver revealed a diffuse lymphoma composed of blast cells and large lymphocytes, distributed diffusely in the parenchyma and surrounding the vasculature. In the skin, microfilariae were seen in some superficial capillaries. Conclusion: This study describes a microfilariae mixed infection with A. reconditum and Dirofilaria immitis in a dog with a lymphoma and its molecular detection. To the knowledge of the authors, this is the first report of a mixed microfilariae infection in a tumor of a dog and highlights the use of molecular techniques, i.e., polymerase chain reaction, for an accurate diagnosis.

Diagnostic Accuracy of a Point-of-Care Immunoassay for Feline Immunodeficiency Virus Antibodies, Feline Leukemia Virus Antigen, and Dirofilaria immitis Antigen.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are retroviral infections of cats worldwide whose clinical manifestations range from mild to severe disease. In both cases, infected cats can live a long life with proper care and should be managed to prevent infection of other cats. Dirofilaria immitis, the nematode that causes heartworm disease, can infect cats in any region where dogs are infected. Though cats are more resistant to infection, clinical diseases in the form of heartworm-associated respiratory disease can cause death. Screening for these infectious diseases enables veterinarians to manage their cases and prevent the spread to other cats. We describe the diagnostic accuracy of a point-of-care immunoassay for FIV, FeLV, and heartworm, compared to reference methods commonly available through reference laboratories to the practicing veterinarian. For FIV, we report 100% sensitivity (95% confidence limits (CL): 96.2-100%) and 97.8% specificity (95% CL: 95.4-99.4%). For FeLV, we report 100% sensitivity (95% CL: 97.7-100%) and 99.2% specificity (95% CL: 97.1-99.9%). And for heartworm, we report 90.2% sensitivity (95% CL: 76.9-97.3%) and 100% specificity (95% CL: 98.3-100%). Veterinarians may expect this performance relative to the reference methods they use for confirmatory serological testing.

Clinical Characteristics of Human Pulmonary Dirofilariasis in Japan: An Uncommon Differential Diagnosis of a Solitary Pulmonary Nodule.

Human pulmonary dirofilariasis (HPD) is a zoonotic disease caused by Dirofilaria immitis. Most HPD cases are asymptomatic and are either detected during annual health checkups or incidentally identified during the investigation of other diseases, particularly primary or metastatic pulmonary lung cancers. However, the frequency and clinical features of Japanese patients with HPD remain unclear. We analyzed data from the Japanese Medical Abstract Society database and identified 69 cases between 1978 and 2022. The incidence of HPD increased until the 2000s but declined markedly in the 2010s. The incidence is higher in the southwestern region and lower in the northeastern region of Japan. Health checkups are the primary diagnostic opportunities. The Chugoku and Shikoku regions have had high incidence rates per population. The diagnosis of HPD using a noninvasive procedure is typically difficult because of the absence of specific clinical symptoms, and approximately 70% of the cases are detected using video-assisted thoracoscopic surgery. Climate change may increase the incidence of HPD in the northeastern region of Japan, and travel to countries with poor vector control may be a risk factor for HPD transmission. Physicians should consider this parasitic infectious disease when examining patients presenting with solitary lung nodules.

Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model.

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, non­stimulated 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.

Efficacy of oral, topical and extended-release injectable formulations of moxidectin combined with doxycycline in Dirofilaria immitis naturally infected dogs.

BACKGROUND: Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones and doxycycline against Dirofilaria immitis, showing that these protocols can be used as an alternative to melarsomine. The present study evaluated the efficacy of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy. METHODS: Thirty dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or extended release moxidectin injectable (G3) at enrolment and again at 6 months (Day 180). All treatment groups received doxycycline for the first 30 days. Microfilarial concentrations in 1 ml (mff/ml) blood were determined monthly for 9 months with the modified Knott's test. A clinical scoring system was employed for each dog enrolled in the study based on thoracic radiography and cardiac ultrasound (CU) examinations performed at Day - 15 (before treatment) and at Day 180. RESULTS: Results from the present study suggest that the majority of dogs from all treatment groups became antigen negative, as evaluated at Day 270: 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3. Improvement of radiographic alterations was observed in all treatment groups, and almost all dogs were cleared of pulmonary abnormalities by 6 months from the beginning of treatment (P = 0.000). Cardiac ultrasound examination showed a progressive improvement of cardiac function in a limited number of animals (4/30). CONCLUSIONS: The combination of doxycycline and three different formulations of moxidectin leads to antigen-negative status in naturally infected dogs.

First time identification of subconjunctival Dirofilaria immitis in Turkey: giant episcleral granuloma mimicking scleritis.

Dirofilariasis is a vector-borne disease that is present worldwide. This report describes a giant subconjunctival granuloma which mimics scleritis, caused by D. immitis. A 60-year-old man was referred with the complaints of irritation, redness, and swelling at the medial part of the right eye. He was living in Izmir province located in western Turkey. Slit-lamp examination showed a firm, immobile mass measuring 13.0 × 5.0 × 5.0 mm with yellowish creamy color. The mass was completely removed surgically under local anesthesia mainly for diagnosis. Histopathology revealed typical morphological features of a filarioid nematode in favor of Dirofilaria as characterized by the external smooth cuticular surface, cuticular layer, muscle layer, and intestinal tubule. Molecular study was performed using DNA isolated from paraffin-embedded tissue sections of the worm. PCR amplification and then DNA sequence analysis of cytochrome c oxidase subunit 1 (cox1) gene fragment confirmed that the worm was D. immitis. It is suggested that this may represent the first human case of D. immitis occurring in subconjunctival granuloma in Turkey. Although rare, D. immitis caused by ocular dirofilariasis in humans should be considered.

Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel for the prevention of heartworm disease in cats.

NexGard® Combo is a novel topical endectoparasiticide formulation for cats combining the insecticide/acaricide esafoxolaner, the nematodicide eprinomectin and the cestodicide praziquantel. The efficacy of this novel formulation for the prevention of heartworm disease in cats was tested in two experimental studies using an induced infection model and a randomized, blinded, placebo-controlled study design, and two USA isolates of Dirofilaria immitis. In each study, 20 naïve cats were each inoculated sub-cutaneously with 100 third-stage larvae of D. immitis 30 days before treatment. Following randomization to two treatment groups of ten cats, each cat was treated topically once, either with the minimum recommended dose of the novel formulation, or with an identical volume of placebo. Five months after treatment (6 months after infections), the cats were humanely euthanized for parasite recovery and count. Efficacy was calculated by comparison of the numbers of adult D. immitis recovered in the control and in the novel formulation groups. In the control groups of each study, D. immitis were recovered in seven and nine cats (respective worm counts ranges 1-7 and 1-16, respective geometric means 1.6 and 5.1). In both studies, none of the treated cats harbored any D. immitis at necropsy and the calculated efficacy of the novel formulation was 100%. There were no adverse reactions related to treatment with the novel formulation. The results of these two studies demonstrate that a topical NexGard® Combo application at the minimum label dose is well-tolerated and efficacious in preventing heartworm disease in cats.

TITLE: Efficacité d'une nouvelle association topique d'esafoxolaner, d'éprinomectine et de praziquantel pour la prévention de la dirofilariose chez les chats. ABSTRACT: NexGard® Combo est une nouvelle formulation d'endectoparasiticide topique pour chats combinant l'insecticide/acaricide esafoxolaner, le nématodicide éprinomectine et le cestodicide praziquantel. L'efficacité de cette nouvelle formulation pour la prévention de la maladie du ver du cœur (dirofilariose) chez les chats a été testée dans deux études expérimentales utilisant un modèle d'infection induite et une conception d'étude randomisée, en aveugle et contrôlée par placebo, et deux isolats américains de Dirofilaria immitis. Dans chaque étude, vingt chats naïfs ont chacun été inoculés par voie sous-cutanée avec 100 larves de troisième stade de D. immitis 30 jours avant le traitement. Après randomisation dans deux groupes de traitement de dix chats, chaque chat a été traité par voie topique une fois, soit avec la dose minimale recommandée de la nouvelle formulation, soit avec un volume identique de placebo. Cinq mois après le traitement (6 mois après les infections), les chats ont été euthanasiés sans cruauté pour la récupération et le dénombrement des parasites. L'efficacité a été calculée en comparant les nombres de D. immitis adultes collectés dans le groupe contrôle et dans le groupe ayant reçu la nouvelle formulation. Dans les groupes témoins de chaque étude, D. immitis a été trouvé chez sept et neuf chats (les nombres de vers respectifs variaient de 1 à 7 et de 1 à 16, les moyennes géométriques respectives étaient 1,6 et 5,1). Dans les deux études, aucun des chats traités ne présentait de D. immitis lors de l'autopsie et l'efficacité calculée de la nouvelle formulation était de 100%. Il n'y a eu aucun effet indésirable lié au traitement avec la nouvelle formulation. Les résultats de ces deux études démontrent qu'une application topique de NexGard® Combo à la dose minimale indiquée sur l'étiquette est bien tolérée et efficace pour prévenir la dirofilariose chez les chats.

Infection with Dirofilaria immitis and Other Infections in Cats and Dogs from Rio de Janeiro, Brazil: The Need for Prophylactic Enforcement.

PURPOSE: Dirofilaria immitis, a mosquito-borne nematode that primarily infects dogs, can equally infect cats. Although there have been numerous studies on canine heartworm prevalence in Brazil, there have been few studies on feline infections. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are both life-threatening retroviruses transmitted directly between cats. Infections with Ehrlichia spp. and Anaplasma spp. are highly prevalent among dogs in Brazil, with Rhipicephalus sanguineus being the main vector for both bacteria. This study aimed to gather information on these infections among dogs and cats in the metropolitan area of Rio de Janeiro by performing rapid point-of-care tests for prophylactic enforcement. METHODS: Surplus samples of serum or plasma from private laboratories were tested by enzyme-linked immunosorbent assay (ELISA) (SNAP Feline Triple Test or SNAP 4Dx Plus Test). RESULTS: The prevalence of heartworm disease was 7% among dogs and 0.9% among cats, the latter being 12.9% of the former. The prevalence of FIV and FeLV was 4.3 and 11.9%, respectively. Among dogs, the seroprevalence of Ehrlichia spp. and Anaplasma spp. was 27.1 and 9.8%, respectively, and Borrelia burgdorferi was not detected. CONCLUSION: Given that such infections circulate among pets, prophylactic measures should be encouraged by small animal practitioners.

Detection of heartworm antigen without cross-reactivity to helminths and protozoa following heat treatment of canine serum.

BACKGROUND: Detection of Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference. METHODS: Canine sera (n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation. RESULTS: Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum microfilariae were confirmed in 22 dogs. Prevalence of heartworm infection confirmed by necropsy was 35.6% (58/163). In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp. CONCLUSIONS: These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs.

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

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

Frecuencia de dirofilariosis en caninos de la localidad 3 de Cartagena, Bolívar (Colombia) / Frequency of dirofilariosis in canines of the locality 3 of Cartagena, Bolívar (Colombia)

RESUMEN Objetivo: determinar la frecuencia de dirofilariosis en caninos de la localidad 3 de la ciudad de Cartagena. Materiales y métodos: estudio correlacional de corte transversal. Se tomaron muestras de sangre de 128 perros con el consentimiento previo e informado de sus dueños. Se registró la información sociodemográfica y los síntomas. A las muestras se les realizo observación directa, extendido de sangre periferia coloreado con Wright y la técnica de Knott. Resultados: se estableció que 19,5% de los perros se encontraban parasitados con Dirofilaria sp. Los signos sugestivos de dirofilariosis más frecuentes fueron pérdida de peso y disminución del apetito (10,9% cada uno). El 76% de los caninos eran mestizos. Conclusiones: la frecuencia de dirofilariosis se concentra principalmente en barrios de escasos recursos, los síntomas identificados son sugestivos de una dirofilariosis canina.

ABSTRACT Objective: to determine the frequency of dirofilariosis in canines from locality 3 of Cartagena city. Materials and methods: cross-sectional correlational study. Blood samples were taken for 128 dogs prior informed consent by their owners. Sociodemographic information and symptoms were registered. The samples have been analyzed using direct examination, Wright stained blood smear and Knott technique. Results: it was established that 19.5% of dogs were parasitized by Dirofilaria sp. Most common signs suggestive of Dirofilariosis were weight loss, and decreased appetite (10.9% each). 76% were mixed race dogs. Conclusions: the frequency of dirofilariosis focused on lower income neighborhoods. The identified symptoms are suggestive of canine dirofilariasis.

Preventive efficacy of four or six monthly oral doses of 24 µg/kg moxidectin compared to six monthly doses of Heartgard® Plus or Interceptor® Plus against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs.

BACKGROUND: Recent reports indicated that increasing the monthly oral dosage and the number of consecutive monthly doses of moxidectin improved the efficacy against macrocyclic lactone (ML)-resistant Dirofilaria immitis. The two laboratory studies reported here evaluated the efficacy of four or six monthly oral doses of 24 µg/kg moxidectin compared to six monthly doses of either Heartgard® Plus (ivermectin/pyrantel) or Interceptor® Plus (milbemycin oxime/praziquantel) against ML-resistant D. immitis strains. METHODS: Dogs were inoculated 30 days prior to first treatment with 50 third-stage (L3) larvae of a ML-resistant strain of D. immitis, ZoeLA or JYD-34. In each study, dogs (six per group) were randomized to treatment with six monthly doses of placebo, four or six monthly doses of 24 µg/kg moxidectin, or six monthly doses of Heartgard® Plus or Interceptor® Plus at their label dose rates. Efficacy was evaluated by adult heartworm counts approximately nine months after L3 inoculation. RESULTS: All negative-control dogs were infected with adult heartworms (geometric mean, 35.6; range, 24-41) for ZoeLA and (geometric mean, 32.9; range, 30-37) for JYD-34. Efficacies against ZoeLA for moxidectin, Heartgard® Plus and Interceptor® Plus were ≥ 96.1%, 18.7% and 21.2%, respectively. Adult counts for both moxidectin-treated groups were significantly lower than negative control (P < 0.0001), significantly lower than Heartgard® Plus and Interceptor® Plus (P < 0.0001), but not significantly different from each other (P = 0.5876). Counts for Heartgard® Plus and Interceptor® Plus were not significantly different than negative control (P ≥ 0.2471). Efficacies against JYD-34 were ≥ 95.9%, 63.9% and 54.6% for moxidectin, Heartgard® Plus and Interceptor® Plus, respectively. Counts for all groups were significantly lower than negative control (P ≤ 0.0001). Counts for six monthly doses of moxidectin were significantly lower than those for four monthly doses (P = 0.0470), and the counts for both moxidectin-treated groups were significantly lower than Heartgard® Plus and Interceptor® Plus (P ≤ 0.0002). CONCLUSIONS: Moxidectin administered orally at 24 µg/kg to dogs for four or six consecutive months was ≥ 95.9% effective in preventing the development of two ML-resistant heartworm strains and resulted in significantly fewer adult D. immitis than in dogs treated with Heartgard® Plus or Interceptor® Plus when administered for six consecutive months at their approved label dosages in two laboratory efficacy studies.

Preventive efficacy of oral moxidectin at various doses and dosage regimens against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs.

BACKGROUND: Moxidectin has previously shown limited efficacy (≤ 44.4%) against confirmed macrocyclic lactone (ML)-resistant Dirofilaria immitis strains at 3 µg/kg after single and multiple oral dosages. Three studies were conducted to evaluate higher oral moxidectin doses for efficacy against confirmed ML-resistant D. immitis strains. METHODS: Dogs were inoculated with 50 D. immitis L3 and randomly allocated to treatments. Study 1: 6 groups of dogs (n = 8) were inoculated with JYD-34 (Day - 30) and treated as follows: T01, negative control; T02-T05, moxidectin at 3, 6, 12 or 24 µg/kg, respectively, on Day 0 only; T06, moxidectin at 3 µg/kg on Days 0, 30 and 60. Study 2: 10 groups of dogs (n = 5) were inoculated (Day - 30) with either JYD-34 (T01, T03-05) or ZoeLA (T02, T06-T10) and treated as follows: T01 and T02, negative controls; T03-T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56; T06 and T09, moxidectin at 3 or 60 µg/kg on Day 0 only; T07, T08 and T10, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. Study 3: 5 groups of dogs (n = 5) were inoculated with ZoeMO (Day - 28) and treated as follows: T01, negative control; T02, moxidectin at 3 µg/kg moxidectin on Day 0 only; T03-T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. All dogs were necropsied for adult heartworm recovery ~ 4-5 months post-inoculation. RESULTS: All moxidectin-treated dogs showed significantly lower worm counts than controls. The efficacy of moxidectin administered once at 3 µg/kg was 19% (JYD-34), 44.4% (ZoeLA) and 82.1% (ZoeMO). Increasing both the dose and the number of dosages of moxidectin improved efficacy, with 100% protection obtained using three dosages of moxidectin at either 40 µg/kg (JYD-34, ZoeMO) or 60 µg/kg (ZoeLA). Three dosages of 24 µg/kg were also highly effective, providing ≥ 98.8% efficacy for all three strains. CONCLUSIONS: Increasing both the dose and number of consecutive monthly dosages of moxidectin improved the efficacy against ML-resistant heartworms. Based on these data and other technical considerations, the 24 µg/kg dose was considered the optimal dose for further commercial development.

Laboratory and field studies to investigate the efficacy of a novel, orally administered combination product containing moxidectin, sarolaner and pyrantel for the prevention of heartworm disease (Dirofilaria immitis) in dogs.

BACKGROUND: Dirofilaria immitis is a filarial parasite of dogs that can cause serious or fatal cardiopulmonary disease. Three studies were conducted to evaluate the efficacy and safety of monthly treatment with moxidectin in a chewable tablet product in combination with sarolaner and pyrantel to prevent heartworm disease in dogs after experimental challenge and in a clinical field study in the USA. METHODS: In two laboratory studies, dogs (8 per group) that had been inoculated 30 days prior with 50 third-stage D. immitis larvae were randomized to treatment on Day 0 with placebo or combination product, at the minimum dose of 24 µg/kg moxidectin, 2 mg/kg sarolaner and 5 mg/kg pyrantel (as pamoate salt). Study 2 also included groups treated with tablets containing moxidectin-alone (24 µg/kg) or sarolaner-alone (2 mg/kg). Efficacy was evaluated ~ 5 months after inoculation by adult heartworm counts at necropsy. In the field study, 410 dogs ≥ 8 weeks-old from 23 USA veterinary clinics were treated for 11 months with either combination product at 24-48 µg/kg moxidectin, 2-4 mg/kg sarolaner and 5-10 mg/kg pyrantel (n = 272) or Heartgard® Plus (ivermectin/pyrantel) at the label recommended dose rate (n = 138). Efficacy was evaluated on Day 330 using antigen and microfilaria testing to assess adult heartworm infection. RESULTS: In the laboratory studies, there were no heartworms recovered from any dog treated with the combination product or moxidectin alone and all dogs treated with placebo or sarolaner-alone were infected with 20-44 adult heartworms. In the field study, all dogs treated with the combination product tested negative for heartworm infection on Day 330, whereas two dogs treated with Heartgard® Plus tested positive. The Heartgard® Plus-treated dogs that tested heartworm positive were from the lower Mississippi River Valley region, where heartworm resistance has been confirmed to occur. The combination product was well tolerated in all studies. CONCLUSIONS: In laboratory studies, no heartworms were recovered from dogs treated with a single dose of the novel combination product containing moxidectin, sarolaner and pyrantel. Additionally, in the field study no dog tested positive for adult heartworm infection when dosed with the combination product monthly for 11 months, while two dogs treated with Heartgard® Plus tested positive.

Dirofilaria immitis infection in dogs in Algodoal Island, Brazilian Amazon / Infecção por Dirofilaria immitis em cães na Ilha de Algodoal, Amazônia brasileira

Dirofilaria immitis, a parasite that mainly infects domestic or wild canids, but can infect felines or humans as well, is frequent in many Brazilian areas. The main objective of this research was to determine the prevalence of natural canine infection at the Algodoal-Maiandeua Island complex, in the coastal region of the state of Pará, Brazil. A total of 67 dogs were sampled for blood microfilariae detection and for D. immitis DNA detection. Microfilaria and D. immitis DNA could be detected in 35.8% (24/67) of the animals. In one dog's sample no microfilariae were detected, but the PCR was positive, suggesting that either larvae recently were eliminated or adults died shortly before sample collecting. Therefore, it can be concluded that the occurrence of D. immitis is a health threat for domestic and wild canids at the Island of Algodoal, as well as for feline or human health.(AU)

Dirofilaria immitis, um parasito que infecta principalmente canídeos domésticos ou selvagens, embora também possa infectar felinos e humanos, é frequente em muitas áreas do Brasil. O objetivo deste estudo foi determinar a ocorrência da infecção natural em cães provenientes do complexo da Ilha de Algodoal-Maiandeua, região litorânea do estado do Pará, Brasil. Um total de 67 cães tiveram o sangue coletado para detecção de microfilárias de D. immitis e seu DNA. Microfilárias e o DNA de D. immitis foram detectados em 35,8% (24/67) dos animais. Na amostra de um animal, não foram observadas microfilárias, mas o seu DNA foi detectado, sugerindo que as larvas tenham sido recentemente eliminadas ou os adultos tenham morrido antes da coleta da amostra. Portanto, pode-se concluir que a ocorrência de D. immitis é uma ameaça à saúde de canídeos domésticos no complexo da Ilha de Algodoal-Maiandeua, bem como para felinos e seres humanos.(AU)

Thirteen cases of pulmonary dirofilariasis in a single institution in Okinawa Island.

Pulmonary dirofilariasis is an infection caused by Dirofilaria immitis, which is an endemic parasite in Japan. We experienced 13 surgical cases of pulmonary dirofilariasis in our hospital. Of the 13 patients, 61.5% were men. The responsible lesions were located in the right lung in all cases, and 76.9% of them were in the lower lobe. Histologically, 12 cases showed necrotic nodules with peripheral granuloma with worms inside the pulmonary artery. One case did not show a necrotic nodule but showed only thickening and hyalinization of the pulmonary artery wall with a degenerated worm inside. Eosinophils were found histologically in all cases. Thirteen cases of dirofilariasis in one institution seem to be the largest number in Japan, based on previous reports. One reason for this increased prevalence may be the hot and humid climate of our prefecture considering the ecology of the mosquito as a vector. Elastic staining and eosinophils in peripheral granulomatous areas can contribute to the diagnosis when the worms are degenerated.

Molecular Characterization of a Dirofilaria immitis Cysteine Protease Inhibitor (Cystatin) and Its Possible Role in Filarial Immune Evasion.

Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.