Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

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.

Emerging human pulmonary dirofilariasis in Hungary: a single center experience.

BACKGROUND: Human pulmonary dirofilariasis (HPD) is rare in Hungary, and it stems from Dirofilaria immitis, mainly transmitted through mosquito bites, with dogs as primary hosts. Despite its prevalence in veterinary settings, human cases are infrequent. Historically, Mediterranean countries report most HPD cases, but sporadic cases occur in temperate European regions. Radiologically, HPD often manifests in a non-specific manner, resembling pulmonary neoplasms, leading to unnecessary surgery and patient distress. METHODS: This study presents a notable case series from Hungary, encompassing a 12-year period, documenting 5 instances of HPD with the aim to provide baseline estimate of occurrence for future comparison. RESULTS: Among the patients studied, all were of middle age (median: 52 years, range: 37-69) and exhibited tumor-like lesions, primarily localized to the right lung, necessitating lobectomy or wedge resection. Histological examination consistently revealed a necrotizing granulomatous response characterized by remnants of helminths, without the presence of ovules. Furthermore, rigorous diagnostic procedures excluded other potential infectious agents through specialized staining techniques. Polymerase chain reaction analysis definitively confirmed the diagnosis of HPD in each case. CONCLUSIONS: This case series highlights HPD as a seldom zoonosis, with a probable escalation in its occurrence within temperate regions. Therefore, clinicians should maintain a heightened awareness of HPD in the differential diagnosis of pulmonary coin lesions. Early recognition and diagnosis are paramount for appropriate management and prevention of potential complications associated with this increasingly recognized infectious entity.

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.

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.

Microfilaruria of Dirofilaria immitis in a dog from Italy.

Dirofilaria immitis is a mosquito-borne nematode-causing canine heartworm disease, with adult worms localized in the pulmonary arteries and right heart. In rare cases, ectopic migration might occur, and adults and blood circulating microfilariae can be found in unusual organs or fluids (e.g., eyes, abdominal cavity, bone marrow, and urine). A 17-year-old mixed-breed female dog was presented in a private veterinary clinic in Italy for hematuria and dysuria. Physical examination showed cardiac mitral murmur with marked respiratory distress and cyanotic mucous membranes after handling. Abdominal ultrasounds revealed a non-specific chronic cystopathy, while the echocardiography showed enlargement of the right heart associated with tricuspid insufficiency and mitral regurgitation, with the presence of an adult filariae in the right ventricular chamber. Circulating microfilariae were observed in the blood smear and molecularly identified as D. immitis. Unusual microfilaruria was detected in the urine sediment. Data presented raise awareness about the occurrence of microfilariae in unusual locations, such as the bladder, suggesting the need of a thorough clinical and laboratory assessment where D. immitis is endemic.

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.

Dirofilaria immitis and Onchocercidae spp. in wild felids from Brazil.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca, Puma concolor, Herpailurus yagouaroundi, Leopardus geoffroyi, Leopardus guttulus, Leopardus pardalis, Leopardus wiedii, Leopardus munoai) from different locations in Brazil. Overall, 82 samples (n = 63 blood; n = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 (cox1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87-88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

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.

Heartworm disease in domestic dogs in Estonia: indication of local circulation of the zoonotic parasite Dirofilaria immitis farther north than previously reported.

BACKGROUND: The mosquito-borne zoonotic parasite Dirofilaria immitis continues to spread northwards in Europe. This parasite can cause potentially life-threatening heartworm disease in dogs and pulmonary dirofilariasis in humans and is, therefore, a major health concern in both the veterinary medicine and human medical fields. This is the first report of D. immitis infections and heartworm disease in the Baltic country Estonia. METHODS: Data on canine D. immitis infections and heartworm disease were collected from the electronic patient records database of the Small Animal Clinic of Estonian University of Life Sciences, the only university clinic in Estonia. The patient records of dogs with confirmed diagnosis of D. immitis infection or heartworm disease were reviewed and summarised. RESULTS: Six dogs had been diagnosed with confirmed D. immitis infection or heartworm disease at the university clinic in 2021-2022. The confirmed diagnoses had been reached following international guidelines, based on a combination of different tests. Molecular confirmation of the parasite species had not been performed. Two of the dogs had been imported while four had no travel history outside of the country. CONCLUSIONS: Four of the dogs with a confirmed D. immitis infection or heartworm disease had no history of being imported or travelling outside of the country, indicating autochthonous infections and, consequently, local circulation of the parasite in Estonia. These findings represent the new northernmost autochthonous cases of D. immitis infection and canine heartworm disease reported in the European Union.

Ecological niche modeling analysis (Cx. pipiens), potential risk and projection of Dirofilaria spp. infection in Greece.

Vector-borne diseases continue to increase worldwide. Dirofilariosis is one of the most common vector-borne zoonotic diseases, mainly caused by Dirofilaria spp. (D. immitis and D. repens) and spread by culicid mosquitoes of different species. Greece is one of the countries in southern Europe where it is traditionally endemic, and its distribution is not homogeneous. The aim of this study was to develop an environmental model for Greece that reflects the suitability of the ecological niche for Dirofilaria spp. infection risk and its projection until 2080. For this purpose, we used the potential distribution of suitable habitats for Culex pipiens calculated using an ecological niche model (ENM) and the potential number of generations of Dirofilaria spp. The ecological niche model of Cx. pipiens in Greece showed good predictive power (AUC=0.897) with the parasite at a resolution of 1 km2. The variables that contributed most to the model were mean annual temperature, rivers and human footprint. The highest risk of infection was found in coastal areas and in riverside areas of the main river basins, as well as in irrigated areas of the mainland and peninsular regions and in the whole territory of island areas, and the lowest risk was found in areas of higher altitude. A positive relationship was found between the risk of dirofilariosis and the location of infected dogs, with 86.65% located in very high and high risk areas. In 2080, the percentage of territory gained by Cx. pipiens will increase by 261.52%. This model provides a high predictive value, predicted presence, and risk of Dirofilaria spp. infection and can serve as a tool for the management and control of this disease.

Temperature-bounded development of Dirofilaria immitis larvae restricts the geographical distribution and seasonality of its transmission: case study and decision support system for canine heartworm management in Australia.

Dirofilaria immitis is the causative agent of canine heartworm disease. We used the established heartworm development unit (HDU) principle to map the extrinsic incubation period (EIP) of D. immitis in Australia using historical weather data from 2013-2022. We found weather conditions suitable for EIP completion showed substantial seasonality and geographical variability. Whilst a considerable percentage of the Australian territory showed suitable weather conditions to always support EIP completion (17%), only 2.7% of the 2021 Australian human population lived in this region. Therefore, 97% of the population lived in an area that changed its EIP suitability within the study period. EIP completion is required prior to D. immitis transmission, meaning that infection risk of D. immitis is seasonal and location-dependent, being disrupted each year for most of the human population's dogs. We developed an online, open access tool allowing us to visualise EIP completion across Australia historically and in near real-time. We aim to support veterinarians to make risk-based recommendations for dirofilariosis prevention by using the tool, available at https://heartworm-mapping.adelaide.edu.au/shiny/.

Atypical case of subcutaneous filariosis in a cat caused by Dirofilaria immitis.

Subcutaneous dirofilariosis is a well-known disease caused mainly by Dirofilaria repens and described in several mammalian species including humans, dogs, and cats. Additionally, early developing stages of the heartworm Dirofilaria immitis are rarely reported in subcutaneous localization from humans and dogs. To our knowledge, confirmed clinical evidence of this condition has not been described in the cats yet, even if the feline hosts can be affected either by the classic adult-related heartworm form or heartworm-associated respiratory disease (HARD) caused by immature stages. A 2 year old, spayed male cat was presented for three subcutaneous nodules on the head and trunk. The cat lived in Northern Italy and was regularly vaccinated and treated monthly with an antiparasitic spot on formulation containing selamectin. One of the three nodules was surgically excised and examined. Histology showed the presence of a nodular lesion in the subcutis characterized by a severe inflammatory infiltrate composed of macrophages, small lymphocytes, with fewer eosinophils, and mast cells, supported by a proliferation of mature fibroblasts (fibrosis). Inflammatory cells were multifocally surrounding sections of parasites identified as adult nematodes. Microscopic features were compatible with D. immitis, which has been molecularly confirmed (98.2% identity to D. immitis isolate OP107739). The cat tested negative for D. immitis antigenemia and the two remaining nodules disappeared spontaneously in a few months. In region where heartworm is prevalent, aberrant localization of D. immitis should be considered in the differential diagnoses of subcutaneous filarial worms in cats and dogs.

Zoonotic dirofilariases: one, no one, or more than one parasite.

Dirofilaria spp. are vector-borne filarial nematodes that affect a variety of animal species, including humans. Dirofilaria immitis and Dirofilaria repens are the two main zoonotic species, but also other wildlife-associated Dirofilaria species are occasionally reported as causative agents of human dirofilariasis, including Dirofilaria striata, Dirofilaria tenuis, Dirofilaria ursi, Dirofilaria spectans, and Dirofilaria magnilarvata. Since the etiological identity of most of the species mentioned here is arguable, we summarized and critically discussed data concerning infections in humans, focusing on the reliability of Dirofilaria species identification. We advocate the importance of combined morphological and genomic approaches to provide unequivocal evidence for their zoonotic potential and pathogenicity.

Is the European badger a new host for Dirofilaria immitis? The first records in Greece.

Dirofilaria immitis is a ubiquitous nematode parasite with zoonotic potential, transmitted by mosquitoes, that causes heartworm disease in various animal species. Dogs are the parasite's typical final host, and wild carnivores represent the parasite's reservoir in nature. Studies on D. immitis infections in wild animals are essential to assess infection pressure for domestic animals, and until now, there has been only one infection case reported in a European badger (Meles meles). The current report describes the first two European badger cases with cardiovascular dirofilariosis in Greece. Two adult male badgers were rescued in Heraklion and Chania, Crete Island, and admitted to "ANIMA -Wildlife Rehabilitation Centre" in Athens. The detailed clinical examination revealed that the first badger suffered from severe broncho-pneumonitis while the second one displayed clinical signs associated with severe brain trauma. Blood samples were taken for haematology and biochemistry analyses during their short hospitalisation period. In addition, different routine diagnostic tests were carried out, including heartworm antigen testing (ELISA) and the modified Knott's test for microfilariae. Both badgers were positive in both tests. The animals died a few hours after their admission and the detailed necropsies followed, revealed the presence of three parasites in each animal's right heart, morphologically identified as adults of D. immitis. These findings add the European badger in the list of additional potential reservoir hosts for D. immitis and highlight the potential role of wildlife for companion animals and human health.

Genetic variability of Dirofilaria repens isolates from humans and dogs in Italy.

Dirofilaria repens is a paradigmatic example of an emerging vector-borne pathogen (VBP) in both human and veterinary fields. The spatial expansion and the increasing zoonotic impact of this VBP can be related to several drivers including the genetic structure of parasite populations. Italy is one of the European countries traditionally endemic with the highest incidence of canine and human cases of subcutaneous dirofilariosis. The present study aimed to assess the genetic identity and variability of D. repens isolates of human and canine origin from areas of Central Italy, compared with those isolated from different areas of Europe by sequence analysis of mtDNA genes (i.e., 12 S rDNA and cox1). A total of twenty isolates of D. repens were obtained from biopsies of subcutaneous and ocular cases of dirofilariosis occurring in 10 dogs and 10 humans. The sequence analysis of 12 S rDNA showed that all the sequences obtained clustered as a monophyletic group with a strong nodal support, indicating that all sequence types represented D. repens. The cox1 and the 12 S sequence analysis did not show host-related polymorphisms between human and dog-derived specimens. The sequence analysis of cox1 was performed including 8 additional sequences previously obtained from human and canine isolates in the same areas. Out of the 28 sequences analyzed, 20 were grouped in a haplogroup comprising 15 haplotypes (i.e., DR1, DR2, DR4, DR5, DR7, DR8, DR10-DR18), 2 sequences matched to DR9, reported for the first time in Italy, and 6 showed peculiar polymorphisms that were not previously described. The results obtained have implications for a better understanding of the epidemiology and phylogeography of this emerging vector-borne zoonotic parasite.

Vector-borne parasites in dogs from Ukraine translocated to Poland following Russian invasion in 2022.

INTRODUCTION: Since 24 February 2022, the day the Russian aggression against Ukraine began, millions of refugees and thousands of pets crossed the Polish-Ukrainian border. Additionally, an unknown number of shelter and stray dogs and cats were rescued and translocated to Poland by private persons and non-profit organizations. The aim of the present study was to examine rescued dogs and cats for presence of canine vector-borne parasites to determine the role of armed conflict in spreading these parasites. METHODS: In July 2022 blood samples were collected from two animal shelters in central Poland hosting dogs and cats rescued from Ukraine. Animals were imported from various regions of Ukraine, including eastern and southeastern Ukraine (military conflict area). Fifty-three dogs (51 from two shelters and two owned ones) and one shelter cat were examined by molecular methods (PCR and sequencing) for the presence of Babesia/Theileria spp., Dirofilaria spp. and Hepatozoon spp. DNA. RESULTS: We detected Dirofilaria immitis, a parasite species non-endemic in Poland, in two dogs translocated from Ukraine (2/53 = 3.8% [95% CI 0.5-13.0%]). One dog had a history of previous heartworm infection. High prevalence of Hepatozoon canis (51% [95% CI 36.8-64.9%]) was noted among translocated dogs. Prevalence of Dirofilaria repens in Ukrainian dogs (18.9% [95% CI 9.4-32.0%) was similar to prevalence in dogs from central Poland (12%). Co-infection of D. repens and D. immitis was found in two dogs and six dogs were co-infected by D. repens and H. canis. Hepatozoon canis infection was also identified in an imported cat. CONCLUSION: We confirmed that this military conflict has facilitated the spread of canine vector-borne parasites, including zoonotic species.

Dirofilaria immitis in pet dogs from the metropolitan area of the Colombian Caribbean.

A cross-sectional study was carried out to determine the frequency and factors associated with Dirofilaria immitis infection in pet dogs in the metropolitan area of the Colombian Caribbean (northern Colombia). A total of 173 dogs were analyzed by a commercial rapid immunochromatographic test (RIT) and a nested PCR of the cytochrome oxidase subunit I gene, in parallel. Ninety-two (53.2%) of the dogs showed positive results to the RIT, while 59 (34.1%) animals had D. immitis DNA by PCR. Positivity to one or both diagnostic techniques was detected in 104 (60.1%; CI95%: 53.8-67.4) of the sampled dogs. In PCR-positive dogs, phylogenetic analyses evidenced high nucleotide identity (100%) with sequences previously obtained from mosquitoes, dogs and other mammals in different countries. Exercise intolerance (p = 0.002; OR: 2.33; CI95%: 1.37-3.96) and thrombocytopenia (p = < 0.001; OR: 1.95; CI95%: 1.11-3.43) were the main factors associated with D. immitis infection in dogs. The high frequency of D. immitis in dogs indicates a wide distribution of this parasite in the metropolitan area of the Colombian Caribbean, which can be of animal and public health concern. Our results highlight the need to combine different methods to increase the diagnostic accuracy of D. immitis.

"Slow kill" treatment reduces DNA damage in leukocytes of dogs naturally infected with Dirofilaria immitis.

Parasitic diseases are considered to be a cause of oxidative stress which leads to oxidative damage of various molecules including DNA. This can result in mutations, replication errors, and genome instability. Therefore, aim of this study was to measure DNA damage induced by Dirofilaria immitis in the single cells such as dogs' leukocytes using the comet assay. Also, we monitored the effects of antiparasitic treatment on mitigation of sensitivity to DNA damage in leukocytes treated with H2O2 using the in vivo and ex vivo comet assay. The whole blood samples from 34 dogs from Serbia were used, both males and females, from one to 13 years old, both pure and mixed-breeds. A rapid immunochromatographic test (Antigen Rapid Heartworm Ag 2.0 Test Kit, Bionote, Minnesota, USA) was used for the detection of D. immitis antigens. The modified Knott's test and PCR were used in the aim of detecting D. immitis microfilariae in dogs' blood, and evaluating the number of circulating microfilariae during the treatment. The genotoxicity evaluation showed that D. immitis infection resulted in DNA damage in naturally infected dogs, with the highest DNA damage occurring in the group of dogs with severe clinical signs. Treatment with ivermectin and doxycycline decreased DNA damage in leukocytes of dogs in all groups, as the intensity of infection decreased due to applied therapy. Ex vivo comet assay results showed that leukocytes exhibited decreased sensitivity to H2O2-induced DNA damage during treatment. The results of the modified Knott's test and PCR in our study showed that treatment with ivermectin and doxycycline was successful in decreasing the average number of microfilariae during the time and at the end eliminating them from the dogs' blood.

The first report of Dirofilaria repens infection in dogs from Colombia.

Dirofilariasis is a mosquito-borne disease caused by Dirofilaria parasites, affecting both wild and domestic animals, including humans considered as accidental hosts. Dirofilaria repens is the principal causative agent of dirofilariasis in the Old World, with increasing reports of the parasite in countries where it has not been previously identified, due to several factors such as the expansion of mosquito vectors' geographical distribution. By utilizing newly designed primers for molecular detection and confirming through next-generation sequencing, here, we report the first plausible cases of D. repens in dogs from Colombia. Our results support the classification of this species as an emergent pathogen in the Americas. Finally, we encourage an increase in diagnostic and surveillance efforts to prevent and control the current and future dirofilariasis cases in this region.