Ocular Dirofilariasis in Migrant from Sri Lanka, Australia.

We describe a case of imported ocular dirofilariasis in Australia, linked to the Hong Kong genotype of Dirofilaria sp., in a migrant from Sri Lanka. Surgical extraction and mitochondrial sequences analyses confirmed this filarioid nematode as the causative agent and a Dirofilaria sp. not previously reported in Australia.

An unexpected case of a dog from Poland co-infected with Dirofilaria repens and Dirofilaria Immitis.

BACKGROUND: Dirofilariasis is a vector-borne disease caused by parasitic nematodes of the genus Dirofilaria spp., considered an emerging concern in both veterinary and human medicine. Climate changes and human activities, such as pet travel, contribute to the spread of diseases to new non-endemic regions. Poland is dominated by subcutaneous dirofilariasis caused by D. repens infections. Cardiopulmonary dirofilariasis, also known as a heartworm disease is much more rare with only single autochthonous cases reported so far. Also, imported infections are observed sporadically in dogs traveling to endemic countries. In this study, we report the first case of a dog in Poland, never having traveled abroad, co-infected with Dirofilaria repens and Dirofilaria immitis. CASE PRESENTATION: A 14-year-old mixed breed, an intact male dog with fever, lightly pale mucosal membranes, moderate abdominal pain, and a mild cough was presented in a veterinary clinic in Warsaw, Poland. The examination of the blood sample collected for complete morphology and biochemistry revealed the presence of live microfilariae. Presence of the DNA of both microfilariae species was detected using Real-Time PCR with species-specific primers. CONCLUSIONS: Since the remaining diagnostic methods like Knott's test, antigen test or echocardiography did not reveal the presence of D. immitis, we discussed the impact of microfilariae periodicity and low worm burden infections on the limited efficiency of these techniques. We strongly recommend using a mixed diagnostic approach for the most sensitive and specific diagnosis since the ideal diagnostic method does not exist, and several factors may contribute to misdiagnosis. Furthermore, we considered factors that contribute to the uncontrolled spread of dirofilariasis such as climate changes, introduction of new species of mosquitoes competent for the transmission of the disease, and wildlife animals as an important reservoir of this parasitosis. Given that Poland shares borders with countries classified as endemic and pre-endemic for D. immitis, such as Slovakia and Ukraine, it is reasonable to anticipate a rise in autochthonous heartworm infections and shifts in the epidemiological pattern of dirofilariasis in the coming years.

Whole genome amplification and sequencing of individual Dirofilaria immitis microfilariae.

Dirofilaria immitis is a filarial parasitic nematode of veterinary significance. With the emergence of drug-resistant isolates in the USA, it is imperative to determine the likelihood of resistance occurring in other regions of the world. One approach is to conduct population genetic studies across an extensive geographical range, and to sequence the genomes of individual worms to understand genome-wide genetic variation associated with resistance. The immature life stages of D. immitis found in the host blood are more accessible and less invasive to sample compared to extracting adult stages from the host heart. To assess the use of immature life stages for population genetic analyses, we have performed whole genome amplification and whole-genome sequencing on nine (n = 9) individual D. immitis microfilaria samples isolated from dog blood. On average, less than 1% of mapped reads aligned to each D. immitis genome (nuclear, mitochondrial, and Wolbachia endosymbiont). For the dog genome, an average of over 99% of mapped reads aligned to the nuclear genome and less than 1% aligned to the mitochondrial genome. The average coverage for all D. immitis genomes and the dog nuclear genome was less than 1, while the dog mitochondrial genome had an average coverage of 2.87. The overwhelming proportion of sequencing reads mapping to the dog host genome can be attributed to residual dog blood cells in the microfilariae samples. These results demonstrate the challenges of conducting genome-wide studies on individual immature parasite life stages, particularly in the presence of extraneous host DNA.

Dirofilariasis presenting as pleural effusion: a rare case report with unusual manifestations and treatment modalities.

BACKGROUND: We present an extremely rare manifestation of dirofilariasis in the pleural cavity. This is the first human pulmonary dirofilariasis reported in Lithuania; according to our knowledge, only two other patients were documented with this pathology in the world. CASE PRESENTATION: A 72-year-old woman was admitted to the hospital complaining of dyspnea, left-side chest pain, and a dry cough. She was a retiree living alone in the countryside without domestic pets (sometimes stray dogs appear) or a travel history. A complete blood count was within normal limits, with a CRP level of 16.8 mg/l and D-dimer concentration of 900 µg/l, which raised suspicion of pulmonary embolism. In chest computed tomography angiography, pulmonary embolism was excluded, and only left pleural effusion without abnormal lesions was confirmed. Left thoracocentesis was performed, and the pleural fluid was evaluated as an exudate with a predominance of eosinophils (59%), along with the presence of parasites. These parasites exhibited the morphology of Dirofilaria repens. Oral doxycycline (100 mg, twice daily) and albendazole (400 mg, twice daily) were prescribed for a 14-day course. A month later, there were no pathological findings on the chest X-ray, and the patient no longer had respiratory symptoms. However, the patient presented with an emerged, painful palpable right breastmass, where the rash was previously observed. Ultrasound imaging revealed a 1.5 × 2 cm nodule, which was surgically removed. Parasites consistent with Dirofilaria repens were suspected but not definitively identified. Pharmacological treatment for dirofilariasis was not further prescribed. CONCLUSIONS: This case encourages doctors to be more vigilant because the patient, who neither travelled nor kept any pets, contracted dirofilariasis. Diagnostic and treatment guidelines are lacking, necessitating further research. Treatment with doxycycline and albendazole yielded positive outcomes, suggesting potential efficacy for dirofilarial pleuritis.

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.

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.

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.

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.

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.

Microfilaremic Dirofilaria repens Infection in Patient from Serbia.

We report a case of Dirofilaria repens infection causing microfilaremia in a patient from Serbia. Serum samples tested positive for D. repens IgG by ELISA. Our findings and those of others suggest the parasite's progressive adaptation to humans. Clinicians should be aware that microfilaremia can develop during Dirofilaria spp. infections.

Dirofilaria immitis in Dog Imported from Venezuela to Chile.

We report a case of Dirofilaria immitis nematode infection in a dog imported from Venezuela that had been living for 2 years in Santiago, Chile, where this parasite had not been reported before. Our findings warrant surveillance for all dogs imported to Chile, given that suitable conditions exist for establishing this parasite.

Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

First molecular characterization of Dirofilaria Immitis in Cuba.

BACKGROUND: Dirofilarioses are widespread diseases caused by mosquito-borne nematodes of the family Onchocercidae, genus Dirofilaria. The major etiologic agent of canine dirofilariosis in the American continent is the zoonotic parasite Dirofilaria immitis. Existing reports of filarioid nematodes in Cuba are based solely on morphological and immunological analysis which do not allow unambiguous identification and/or direct detection of causal agents. RESULTS: Here we present the molecular characterization of filarioid nematodes found in a dog in Cuba. Based on the molecular and phylogenetic analysis of the 5.8S-ITS2-28S region and cox1 gene fragments, the worms were unambiguously classified as D. immitis. Sequence analysis showed high identity of the gene fragments in this study with others previously obtained from D. immitis found in dogs, wolfs and jackals but also from mosquito vectors of D. immitis. CONCLUSIONS: Further studies are guarantee to better understand the epidemiological impact of canine dirofilariosis in Cuba as well as the competence of different species of culicid mosquitoes as vectors of Dirofilaria in the country.

The first molecular confirmation of Culex pipiens complex as potential natural vectors of Dirofilaria immitis in Myanmar.

Dirofilariosis, known as one of the most widespread vector-borne zoonotic diseases, is caused by several different species of the nematodes of the genus Dirofilaria, which can be transmitted by Culex, Anopheles and Aedes mosquito vectors. In order to identify key vector mosquitoes of filarial parasites in Myanmar, mosquitoes were collected during three different seasons (summer, rainy and winter) in three townships in Nay Pyi Taw area, Myanmar. DNA extraction and polymerase chain reaction (PCR) analyses were conducted for 185 mosquito pools, with each pool containing 1-10 mosquitoes. Dirofilaria immitis was detected in 20 pools of Culex pipiens complex mosquitoes. The minimum infection rate of mosquitoes was found to be 16.33. The small subunit ribosomal RNA (12S rDNA) gene targeted PCR revealed that the sequences obtained were completely identical to the sequences of D. immitis derived from dogs in China, Brazil and France. The sequences obtained from mitochondrial cytochrome oxidase subunit I (COI) gene PCR exhibited 100% identity with the sequences of D. immitis derived from dogs in Bangladesh, Iran, Japan and Thailand, as well as humans in Iran and Thailand, and mosquitoes in Germany and Hungary. The findings of this study demonstrated that the mosquito species of Cx. pipiens complex are potential mosquito vectors for dirofilariosis in Myanmar.

In vitro maintenance of the endosymbiont Wolbachia of Dirofilaria immitis.

Wolbachia has an obligatory mutualistic relationship with many onchocercid nematodes of the subfamilies Dirofilariinae and Onchocercinae. Till date, no attempts have been made for the in vitro cultivation of this intracellular bacterium from the filarioid host. Hence, the current study attempted cell co-culture method using embryonic Drosophila S2 and the LD cell lines to cultivate Wolbachia from Dirofilaria immitis microfilariae (mfs) harvested from infected dogs. Microfilariae (mfs = 1500) were inoculated in shell vials supplemented with Schneider medium using both cell lines. The establishment and multiplication of the bacterium were observed during the initial inoculation, at day 0 and before every medium change (from days 14 to 115). An aliquot (50 µl) from each time point was tested by quantitative real-time PCR (qPCR). Comparing the average of Ct values, obtained by the tested parameters (i.e., LD/S2 cell lines and mfs with/without treatment), the S2 cell line without mechanical disruption of mfs provided the highest Wolbachia cell count by qPCR. Despite the maintenance of Wolbachia within both S2 and LD-based cell co-culture models for up to 115 days, a definitive conclusion is still far. Further trials using fluorescent microscopy and viable staining will help to demonstrate the cell line infection and viability of Wolbachia. Use of considerable amount of untreated mfs to inoculate the Drosophilia S2 cell lines, as well as the supplementation of the culture media with growth stimulants or pre-treated cells to increase their susceptibility for the infection and development of a filarioid-based cell line system are recommended for the future trials.

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.

Occurrence of Dirofilaria repens in wild carnivores in Poland.

Dirofilaria repens is an expanding vector-borne zoonotic parasite of canines and other carnivores. Sub-clinically infected dogs constitute the most important reservoir of the parasite and the source of infection for its mosquito vectors. However, occurrence of D. repens infection in wild animals may contribute to the transmission of the parasite to humans and may explain the endemicity of filariae in newly invaded regions. The aim of the current study was to determine the occurrence of D. repens in 511 blood and spleen samples from seven species of wild carnivores (wolves, red foxes, Eurasian badgers, raccoons, raccoon dogs, stone martens, and pine martens) from different regions of Poland by means of a PCR protocol targeting the 12S rDNA gene. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodeships in four of the seven regions of Poland: Masovia, Lesser Poland, Pomerania and Warmia-Masuria. The highest prevalence was found in Masovia region (8%), coinciding with the highest previously recorded prevalence in dogs in Central Poland. The DNA of Dirofilaria was detected in 16 samples of three species (total prevalence 3.13%). A low and similar percentage of positive samples (1.9%, 4.2% and 4.8%) was recorded among badgers, red foxes, and wolves, respectively. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodships. Based on detection in different voivodeships, D. repens-positive animals were recorded in four out of the seven regions of Poland: in Masovia, Lesser Poland, Pomerania, and Warmia-Masuria. The highest prevalence of filariae was found in Masovia region (8%), reflecting the highest previously recorded prevalence in dogs (12-50%) in Central Poland. In summary, we conducted the first comprehensive study on the epidemiology of D. repens in seven species of wild hosts in all seven regions of Poland and identified the first case of D. repens infection in Eurasian badgers in Poland and the second in Europe.

Dirofilaria immitis conquering the regions in Slovakia previously endemic for D. repens.

The present study was focused on the current state of Dirofilaria species distribution in the territory of the Slovak Danubian Lowland, a region previously identified endemic for Dirofilaria repens. For the research, blood samples of 330 dogs tested positive for dirofilariosis using concentration tests or "rapid heartworm tests" were sent by private veterinary practitioners for further DNA analyses and species determination. The results revealed an unquestionable change in the pattern of Dirofilaria species distribution with Dirofilaria immitis, diagnosed as the agent of mono- and co-infections with D. repens, responsible for 60.00% of all cases. The results showed that in the course of the last 5 years, D. immitis has spread significantly in Slovakia and has become the dominant causal agent of dirofilariosis in the former endemic areas of D. repens distribution, which increases infection risk for both dogs and humans.

Filariasis in dogs brought to the Veterinary Teaching Hospital, University of Peradeniya, Sri Lanka.

Dirofilariasis is the predominant emerging zoonotic filariasis in the world. The two most frequent filarial worms that infect dogs are Dirofilaria repens and Dirofilaria immitis. This study reports filariasis among dogs brought to the Veterinary Teaching Hospital (VTH) at the University of Peradeniya and signifies the first molecular characterization of D. repens, responsible for an emerging zoonotic filarial disease in Sri Lanka. Blood samples were collected and were morphologically analyzed using Modified Knott's Technique, followed by molecular analyses. The difference in filariasis prevalence among gender, breed, and age categories was analyzed using a chi-square test. Infection intensities were analyzed using the Mann-Whitney U test and the Kruskal Wallis test. The dogs were brought to the clinic for either vaccination and/or for a regular checkup, and most were sick having non-specific clinical signs. Among the 87 dogs tested, 27.6% were positive for Dirofilaria. Conventional PCR and bi-directional sequencing of genomic DNA of microscopically tested positive samples revealed that the species in Sri Lanka was D. repens. The infection was significantly higher in males (39.1%) than in females (14.6%; χ2 = 0.447, p = 0.011), though it is not significant between puppies (age < 1 year) and adult dogs. More crossbred dogs were infected compared to older and purebred dogs. There was no difference in intensity of infection based on their gender, age, or breed. Sequences obtained from the current study were unique and were only 63% identical to those of D. repens reported from South India. The high number of Dirofilaria infections in domestic dogs indicates a potential reservoir for emerging human dirofilariasis cases in Sri Lanka. Thus, morphological and molecular diagnosis, along with epidemiological assessment of these zoonoses, is critical for the formulation of effective public health programs and control mechanisms.

Unusual cause of unilateral facial oedema: intramuscular dirofilariasis.

INTRODUCTION: Unilateral facial oedema may occur due to inflammatory, infective, or cystic pathology; patients often seek medical help at an early stage. CASE REPORT: We report one such case caused mimicking a parotid abscess, caused by dirofilariasis. CONCLUSION: Dirofilariasis is an emerging zoonosis and should be considered a differential diagnosis of atypical facial swelling. It is equally important for clinicians, radiologists, and pathologists to be familiar with the diagnostic characteristics to avoid misdiagnosis.