Evaluation of Seroprevalence and Risk Factors of Heartworm Infection for Dogs in Rio de Janeiro with Access to Veterinary Care.

Heartworm infection is a chronic disease with clinical signs and effects ranging from an asymptomatic condition to severe disease and death. The prevalence of heartworm disease in the state of Rio de Janeiro has been reported to be high (21.3%). The present study was conducted to evaluate the seroprevalence and risk factors of heartworm infection for the canine population with access to veterinary services in different areas of the state of Rio de Janeiro, Brazil. A total of 1787 canine blood samples were obtained from 135 practices across 8 different areas of Rio de Janeiro state (Rio de Janeiro municipality, São Gonçalo municipality, Niterói municipality, Baixada Fluminense, and the northern, southern, eastern, and mountainous areas) and tested for the presence of Dirofilaria immitis antigens and antibodies against several tick-borne disease pathogens using a commercial immunochromatography technique (Vetscan® Flex 4 Rapid Test; Zoetis; NJ USA). Pet owners reported living conditions, physical characteristics, demographics, and clinical signs for evaluation of risk factors for heartworm infection. Only two evaluated risk factors were shown to enhance the risk for D. immitis infection, including having a short hair coat vs. having a medium or long hair coat (OR 2.62) or positive for antibodies to tick-borne disease parasites (OR 3.83). Clinical signs reported for dogs with heartworm disease were typical for that condition. The overall prevalence of heartworm disease in the state was 8.2%, ranging from 2.4% in the mountainous region to 29.4% in the eastern area. It could not be determined if veterinarians were not diligent about dispensing heartworm preventatives or if poor levels of compliance by dog owners were responsible for higher infection rates in some areas of the state.

A review of moxidectin vs. other macrocyclic lactones for prevention of heartworm disease in dogs with an appraisal of two commercial formulations.

Macrocyclic lactones (MLs) are the only drug class currently licensed for heartworm disease prophylaxis. Macrocyclic lactones kill third- and fourth-stage larvae of Dirofilaria immitis, thus preventing the development of adult worms in dogs, which are responsible for heartworm disease, a potentially life-threatening condition. Despite considerable overlap in terms of endectocide spectrum, several important differences distinguish moxidectin from other MLs. Moxidectin has beneficial pharmacokinetic characteristics, such as a longer half-life and greater tissue distribution compared to ivermectin. Additionally, moxidectin has a greater margin of safety compared to ivermectin in dogs with ABCB1 (previously MDR1) gene-defect, which is commonly recognized in collies and other breeds. Multiple laboratory studies have shown that moxidectin is more effective than other commonly used heartworm preventives against resistant strains of D. immitis. This improved efficacy benefits individual dogs and helps reduce the risk of spreading resistant strains within the community. Despite the presence of proven resistant strains in the United States, non-compliance with preventive measures remains a major factor contributing to the diagnosis of heartworm disease in dogs. In retrospective analyses, the oral moxidectin combination product Simparica Trio® (sarolaner, moxidectin, and pyrantel) was associated with increased compliance, resulting in more time of protection compared to dogs receiving flea/tick and heartworm preventive products separately. Compliance with the extended-release moxidectin injectables ProHeart® 6 and ProHeart® 12 was higher than with monthly heartworm preventives, as they provide 6 months or a full year of protection with one single injection, respectively, and revenues remain in the veterinary clinics as injectable moxidectin cannot be sourced through online retailers.

Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Wolbachia Promotes an Anti-Angiogenic Response Using an In Vitro Model of Vascular Endothelial Cells in Relation to Heartworm Disease.

Heartworm disease caused by Dirofilaria immitis is a vector-borne zoonotic disease responsible for the infection of mainly domestic dogs and cats, or these are those for which the most data are known. Humans are an accidental host where a benign, asymptomatic pulmonary nodule may originate. Dirofilaria immitis also harbours the endosymbiont bacteria of the genus Wolbachia, which play a role in moulting, embryogenesis, inflammatory pathology, and immune response. When Wolbachia sp. is released into the bloodstream, endothelial and pulmonary damage is exacerbated, further encouraging thrombus formation and pulmonary hypertension, facilitating congestive heart failure and death of the animal. Previous studies have shown that parasite excretory/secretory products are able to activate the pro-angiogenic pathway (formation of new vessels) to facilitate parasite survival. The aim of this study was to analyse the role of Wolbachia sp. and its relationship with the cellular processes and the angiogenic pathway in a model of human endothelial cells in vitro. The use of recombinant Wolbachia Surface Protein (rWSP) showed that its stimulation exerted an anti-angiogenic effect by detecting an increase in the production of VEGFR-1/sFlt1 and sEndoglin and did not affect the production of VEGFR-2 and mEndoglin (pro-angiogenic molecules). Furthermore, it did not stimulate cell proliferation or migration, although it did negatively stimulate the formation of pseudocapillaries, slowing down this process. These cellular processes are directly related to the angiogenic pathway so, with these results, we can conclude that Wolbachia sp. is related to the stimulation of the anti-angiogenic pathway, not facilitating the survival of D. immitis in vascular endothelium.

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.

Computed tomographic and magnetic resonance imaging features of canine cervical epidural dirofilariasis.

An 8-year-old neutered male Maltese dog presented with a month-long history of progressive nonambulatory tetraparesis. MRI revealed a well-defined, centrally nonenhanced, T1-weighted hypointense, extradural structure located in the vertebral canal at the level of the C5 vertebral body. CT demonstrated a hypoattenuating, space-occupying structure in the same area. Surgery revealed a long, narrow parasite in the epidural space. An adult Dirofilaria immitis was confirmed by multiplex polymerase chain reaction (PCR) assay. This is a novel report describing the MRI and CT features of cervical epidural dirofilariasis in a dog.

Loop-mediated isothermal amplification polymerase chain reaction in place of a modified Knott test in screening dogs for heartworm (Dirofilaria immitis) infection combined with antigen detection test.

OBJECTIVE: To improve the current recommendations for the diagnosis of canine heartworm (Dirofilaria immitis) disease. ANIMALS: Blood samples collected from 35 shelter dogs in the Republic of Korea. METHODS: Samples were tested for the presence of microfilaria using the modified Knott (MK) test and D immitis DNA using species-specific loop-mediated isothermal amplification (LAMP) PCR. The blood samples were additionally assessed for the presence of heartworm antigens using the Antigen Rapid Canine Heartworm AG Test Kit 2.0 (Bionote Co). The performance of the MK test and LAMP PCR was assessed through statistical analysis, with a paired McNemar test utilized for comparison. RESULTS: The heartworm antigen was detected in 28.5% of the subjects. Of the 10 positive animals, the MK test detected microfilaria in 4 of 35 (11.4%) animals, and LAMP PCR detected D immitis DNA in 6 of 35 (17.1%). The results of this study indicate that the LAMP PCR showed more positive results in samples compared to the conventional MK test. CLINICAL RELEVANCE: The D immitis-specific LAMP PCR assay has the potential to function as an alternative to current detection methods. It could complement the existing antigen detection tests in diagnosing canine heartworm infections.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse.

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Beyond Borders: Dirofilaria immitis Infection in Dogs Spreads to Previously Non-Enzootic Areas in Greece-A Serological Survey.

Although Dirofilaria immitis in dogs is considered enzootic in northern Greece, the available data on the occurrence of infection in southern parts of the country demonstrate its scarcity. The aim of this study was to update the current knowledge on D. immitis infection in dogs in areas of Greece previously considered non-enzootic (Central Greece, Attica, Peloponnese, North Aegean, South Aegean, Crete and the Ionian islands). In total, 1528 dog blood samples were collected from the aforementioned areas and examined by Dirochek® ELISA (Synbiotics). Additionally, data published until 2022 on the prevalence of infection in these areas were compared to the data of this study. The 'Wilson' Score interval method (Epitools) was employed. Overall, 10.8% of dogs were positive for D. immitis. In detail, the prevalence was 21.7, 13.7, 10.7, 5.4, 4.7, 6.2 and 17.0% for D. immitis, in Central Greece, Attica, Peloponnese, North Aegean, South Aegean, Crete and the Ionian islands, respectively. Infection with D. immitis is recorded for the first time in Crete. The probability of a dog becoming infected has increased 4.1 times since 2022 in previously non-enzootic areas. This study denotes the spread of D. immitis and highlights the necessity for preventive measures.

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.

Investigating Dirofilaria immitis isolates infecting domestic canines and their susceptibility/resistance patterns to macrocyclic lactones in the northern region of the Mississippi Delta area (southeast Missouri).

Previous reports of macrocyclic lactone (ML) resistance in Dirofilaria immitis, the parasitic nematode which causes heartworm disease, have mainly been from the southern Mississippi Delta region. Southeast Missouri (SEMO), forming the northern boundary of this region, has not previously been well studied. The area is an ideal propagation region for heartworm infection and possibly for the spread of ML resistance. To assess whether D. immitis isolates infecting domestic canines in SEMO exhibit evidence of resistance to MLs, domestic canines, presented to veterinary facilities testing positive for heartworms through antigen and microfilariae (MF) examination, were utilized in the study. Using a descriptive epidemiological cross-sectional study, from March 2021 through February 2022, blood sample collection from 96 canines living in SEMO testing positive for heartworms were analyzed. MiSeq technology was utilized to sequence specific genetic markers associated with susceptibility/resistance for MLs in D. immitis isolates. Genomic data revealed most D. immitis isolates had genotypic profiles consistent with resistance to MLs. Of the 96 samples tested, 91 (94.8%) had a resistant genotype, 4 (4.2%) had a mixed genotype, and 1 sample (1%) genotyped as susceptible. While detailed and reliable medical histories were not available for most canines, detailed medical history from 2 canines indicated evidence of phenotypic resistance that was consistent with their genotypes. However, in vivo preventive tests are needed to confirm a high frequency of phenotypic ML resistance in D. immitis from this region. Increasing resistance patterns to MLs indicate the approach to heartworm prevention/treatment protocol should be reconsidered. New measures may be required to stop heartworm disease.

First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern.

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

Current State of Canine Heartworm in Portugal.

The favourable geo-climatic conditions in Portugal have made it highly conducive to the development of Dirofilaria immitis in dogs, leading to its identification as an endemic region. This nematode is rapidly spreading across Europe, particularly in northeastern countries. The objective of this study was to provide an updated assessment of the prevalence of this disease in Portuguese dogs, analysing the results in relation to epidemiological and geo-environmental factors, and to identify potential risk factors. A total of 1367 dogs from all continental and insular districts were included in the study and tested for D. immitis antigens. The overall prevalence was found to be 5.9%. It was observed that the disease is spreading northward, with previously unaffected districts now reporting cases, and that the prevalence in coastal districts exceeded that of inland ones. Notably, the Aveiro district exhibited a significant increase in D. immitis prevalence, while in certain districts such as Setúbal, Santarém, Madeira, or Faro, a stabilisation or decrease in prevalence was noted. Furthermore, outdoor and older dogs were found to be at a higher risk of infection. No positive cases were detected in the Azores. Most of the infected dogs were located in areas of high and medium risk of infection. This study underscores the importance of implementing pharmacological prophylaxis, vector control strategies, and public awareness programs to control the spread of this zoonotic disease.

Epidemiological analysis of Dirofilaria immitis (Spirurida: Onchocercidae) infecting pet dogs (Canis lupus familiaris, Linnaeus, 1758) in Baixada Fluminense, Rio de Janeiro.

Dirofilaria immitis infection is routinely detected in dogs during veterinary care in tropical and subtropical regions worldwide. Parasitological tests for the detection of this infection are routinely performed only in areas with a high prevalence. Baixada Fluminense, a region in Rio de Janeiro, was considered heartworm-free until local veterinarians began to receive blood exams results indicating the presence of microfilariae (MF). A laboratory database was hence used to collect data from 2017 to 2020 to understand the extent of spread of the parasite in this area. The results of complete blood count analysis and MF or heartworm antigen detection tests conducted on canine samples sent from veterinary clinics in Baixada Fluminense (Magé, Duque de Caxias, Guapimirim, Nova Iguaçu, and São João de Meriti municipalities) were included. In total, the results of 16,314 hematological tests were considered. The overall prevalence of D. immitis was 3.4% (554/16,314), considering that only one test result was obtained per animal on the same day. This study is highly relevant because it indicates the spreading geographic distribution of the worms, heightens awareness among local health professionals and the general population, and encourages compliance with prophylactic measures to prevent further spread of parasite.

Efficacy of moxidectin, using various dose regimens, against JYD-34, a macrocyclic lactone resistant isolate of Dirofilaria immitis.

BACKGROUND: Macrocyclic lactones (MLs) are the only class of drugs currently commercially available that are effective for preventing heartworm disease. The data presented in this article provide information on the efficacy of oral moxidectin against JYD-34, a known ML-resistant Dirofilaria immitis isolate, when dogs are treated under various dosing regimens. METHODS: Fifty-two purpose-bred Beagle dogs were used in five laboratory studies. All dogs were inoculated with 50 D. immitis third-stage larvae (L3) (JYD-34 isolate) 30 days prior to the first treatment. Dogs were randomized to treatment (four to five animals in each group) with one, three, or five monthly doses of oral moxidectin ranging from 6 to 100 µg/kg body weight. In each study, control dogs were not treated. Five to 6 months after L3 inoculation, dogs were euthanized, and adult worms were counted to evaluate efficacy of the dosing regimens. RESULTS: Adult heartworms were recovered from all control dogs, with an overall geometric mean of 29.7 worms (range 15.2 to 38.0, individual counts ranged from 8 to 51). Five monthly doses of 6 µg/kg provided 83.3% and 90.2%, efficacy, and the same number of monthly doses of 9 µg/kg demonstrated 98.8% and 94.1% efficacy. Three monthly doses of 30 and 50 µg/kg demonstrated 97.9% and 99.0% efficacy, respectively, while a single dose of 100 µg/kg demonstrated 91.1% efficacy. CONCLUSIONS: Five monthly doses of 9 µg/kg provided similar or only marginally lower efficacy against JYD-34, a known ML-resistant isolate, compared to substantially higher doses administered for 3 months. This underscores the importance of duration of exposure to moxidectin when facing ML-resistant isolates. Repeated administration of lower doses of moxidectin are an alternative to higher doses in the prevention of heartworm disease associated with less susceptible or resistant isolates.

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.

Microfilaremic infection in canine filariosis in Colombia: a challenge in morphological and molecular diagnostics.

Canine filariosis is caused by filiform nematodes and affects several species of animals as well as humans. The disease produces a wide range of symptoms that can often be confused with other diseases, which increases the complexity of its diagnosis. The search for methodologies to facilitate its diagnosis is a challenge, and specific and differential identification of the parasite species causing the disease holds key to a successful diagnosis. In Colombia, there is a problem of underdiagnosis of filariosis in microfilaremic dogs infected by Dirofilaria immitis and Acanthocheilonema reconditum, and of microfilaremias not related to heartworm disease. The highest prevalences have been reported for D. immitis infections, although new cases of A. reconditum infections are beginning to appear. The aim of this study was to differentiate the microfilariae infections caused by D. immitis and A. reconditum by a morphological and molecular characterization of microfilariae so as to facilitate an accurate diagnosis of canine filariosis in the metropolitan area of Bucaramanga (Colombia). For this purpose, 400 blood samples with anticoagulants were collected from the dogs and analyzed with the help of a commercial immunochromatography kit for the detection of D. immitis circulating antigen. The Woo, Knott, and polymerase chain reaction (PCR) techniques were employed for determining the parasite count, morphological observation, and molecular identification of microfilariae present in the dogs respectively. The prevalence of microfilaremic dogs in Bucaramanga metropolitan area was 18.75% (75/400). The prevalence of dogs that tested positive for D. immitis in the antigen and in PCR tests was 1.25% (5/400) and 1% (4/400), respectively. Furthermore, the PCR test revealed that 17.75% of the microfilaremic dogs tested positive for A. reconditum (71/400) (first report in the metropolitan area of Bucaramanga), with one animal co-infected by both species, and 0% for D. repens (0/400). However, by morphological characterization, 4% of the microfilariae (3/75) corresponded to D. immitis, 20% (15/75) to D. repens, and 76% (57/75) to A. reconditum. The use of molecular diagnostic methods such as PCR aids in the specific identification of the parasite, thus making it a more accurate method than the morphological characterization of microfilariae. The identification of the parasites by PCR helps improve the veterinary diagnosis of canine filariosis in Colombia, which would lead to the establishment of an appropriate treatment protocol for each species of filaria and also to the generation of reliable data to be used at the clinical and epidemiological levels.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR.

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease") primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Filarial Nematodes in Dogs from the Northeast Region of Brazil.

PURPOSE: Medical and veterinary filarial nematodes are transmitted by blood-feeding vectors. In dogs, these parasites are mainly represented by nematodes in which microfilariae dwell in the blood (Dirofilaria spp. and Acanthocheilonema spp.) or skin (Cercopithifilaria spp. and Onchocerca lupi). The aim of this study was to determine the prevalence of these filarial infections in dogs residing in a touristic, heavily populated location in the northeastern region of Brazil. METHODS: Blood samples (n = 245) were assessed by a modified Knott test, followed by a qualitative ELISA test (SNAP® 4Dx® Plus, IDEXX Laboratory, Westbrook, Maine, USA) for the detection of antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and antigens of Dirofilaria immitis. Skin samples (n = 71) were microscopically examined and molecularly assessed through a PCR targeting the 12 S rRNA gene. RESULTS: Microfilariae and antigen of D. immitis were detected simultaneously in 15 (6.1%; 95% CI = 3.7-9.8) animals. Nine animals (3.6%; 95% CI = 1.9-6.8) were D. immitis antigen positive but microfilariae negative and nine other animals (3.6%; 95% CI = 1.9-6.8) were microfilariae positive but D. immitis antigen negative. D. immitis positive dogs were found in four different municipalities. No filarioids were detected in the skin after microscopical and molecular analyses. CONCLUSION: Data from this study demonstrate that D. immitis is the main filarial nematode infecting dogs in coastal areas in northeastern Brazil. Based on the potential risk of infection in which animals are submitted, it is essential to perform tests to detect microfilariae and D. immitis antigen. Preventive measures must be adopted by using microfilaricidal compounds and anti-feeding insecticides to prevent canine infection.