First molecular detection of Dirofilaria immitis (Spirurida: Onchocercidae) infection in dogs from Northern Algeria

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioid nematodes that affect dogs and other domestic and wild carnivores, causing heartworm disease and subcutaneous dirofilariosis, respectively. In Algeria, the data about the epidemiology of these infections is largely unknown. The present study was designed to establish the occurrence of D. immitis and D. repens in dogs in Algeria using molecular tools.In 2014 and 2015, a total of 209 dogs over one year of age of different breed and sex, living in Northern Algeria, were examined and blood samples were collected from each dog. The presence of D. immitis and D. repens in these samples was detected by real-time PCR followed by standard PCR and sequencing. Overall, the blood of 209 dogs from two departments was collected and only 3 (1.4%) of the blood samples were found positive for D. immitis DNA. Sequencing of the corresponding amplicon displayed a 99.8% identity to D. immitis, confirming the presence of this mosquito-borne nematode in Algeria. Furthermore, all tested samples were negative for D. repens.     

Molecular survey of Dirofilaria species in stray dogs,red foxes and golden jackals from Vojvodina,Serbia

Cardiopulmonary dirofilariosis in dogs and other carnivores is caused by Dirofilaria immitis, while Dirofilaria repens usually causes a subcutaneous infection. The importance of red foxes and golden jackals in the epidemiology of dirofilariosis remains unknown. Thus, the aim of this study was to conduct a cross-sectional molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from the endemic region of Vojvodina, Serbia, in order to determine and update data on their prevalence and provide insight into the epidemiological importance of wild canids.A total of 59 blood samples from stray dogs, 94 from red foxes and 32 from golden jackals were collected and screened by real-time PCR targeting a 115-bp fragment of the mitochondrial 12S gene of filarioids and by conventional PCR assay targeting a 484–524-bp fragment of 5.8S-ITS2-28S locus of filarioids.The cross-sectional molecular survey detected the filarioid mitochondrial 12S gene fragment in stray dogs (27.1 %), red foxes (8.5 %) and golden jackals (6.3 %) in the same endemic region of Vojvodina, Serbia. Only D. immitis was detected in stray dogs, while both D. immitis and D. repens were detected in populations of red foxes and golden jackals. These results outline a possible interaction of D. immitis infection between the dog population and the wild canid populations, while D. repens was found to circulate mostly in golden jackals and red foxes populations.     

Unusual localization of Dirofilaria repens (Spirurida: Onchocercidae) infection in the testicle of a dog

This report describes an uncommon localization of Dirofilaria repens in the testicle of a nine-year-old dog from southern Italy. The dog underwent neutring and three adult nematodes were found in the tunica vaginalis. At gross anatomy, no pathological changes were observed on the tunica, in the testicle or epididymis. The parasites, one female and two males, were morphologically and molecularly identified as D. repens. This is the first report of D. repens in the canine testicle in Italy. This case report highlights the asymptomatic nature of D. repens infection, suggesting that dogs living in endemic areas may act as silent carriers. Careful screening and an effective chemoprophylaxis should be ensured for all animals potentially exposed to D. repens infection in order to reduce the risk of infection to humans in areas where the parasite is endemic.     

Dirofilaria repens infection in a dog imported to Norway

Dirofilaria repens infection was diagnosed in a dog that had been imported to Norway from Hungary three years previously. The dog was a four-year-old castrated male mixed-breed dog and presented for examination of two masses on the right thoracic wall. Fine needle sampling from the subcutaneous nodules and subsequent cytological examination revealed a high number of microfilariae and a pyogranulomatous inflammation. At re-examination approximately 3 weeks later, both masses had apparently disappeared spontaneously, based on both inspection and palpation. However, examination of peripheral blood by a modified Knott’s test revealed a high number of unsheathed microfilariae with mean length of 360 μm and mean width of 6-7 μm, often with the classic umbrella handle appearance of D. repens. Polymerase chain reaction and sequencing confirmed the D. repens diagnosis. Subcutaneous dirofilariosis caused by D. repens is probably the most common cause of human zoonotic dirofilariosis in Europe, but currently is rarely encountered in northern countries such as Norway. However, travelling, import and relocation of dogs have increased, and thus the geographical range of these parasites is likely to increase from traditionally endemic southern regions. Increasing numbers of autochthonous cases of D. repens infections in dogs have been reported in eastern and central Europe. Although infection with D. repens often induces only mild signs or subclinical infections in dogs, they nevertheless represent a reservoir for zoonotic transmission and thus a public health concern, and, in addition, due to the long prepatent period and the high frequency of subclinical infections or infections with unspecific clinical signs, could easily be missed. Lack of experience and expectation of these parasites may mean that infection is underdiagnosed in veterinary clinics in northern countries. Also, predicted climate changes suggest that conditions in some countries where this infection is currently not endemic are likely to become more suitable for development in the intermediate host, and thus the establishment of the infection in new areas.     

Nodular presentation of Dirofilaria repens infection in a cat mimicking a fibrosarcoma

A cat with multiple subcutaneous nodules suggesting a soft tissue sarcoma by physical and computed tomographic examination was diagnosed as being affected by subcutaneous filariosis based on cytologic and ultrasonographic assessments. Nodules were surgically removed and extracted nematodes were identified by PCR as Dirofilaria repens. Furthermore, DNA of Dipetalonema dracunculoides (syn. Acantocheilonema dracunculoides) was detected by PCR, with no evidence of circulating microfilariae. To the best of the authors' knowledge, this represents the first report describing adults of D repens in multiple subcutaneous nodules in a cat. Cytopathologic examination allowed characterization of the parasitic nature of the nodules. Veterinary practitioners should be aware of the possible nodular presentation of D repens in cats and should include D repens in the differential diagnosis of subcutaneous neoformations in the cat.     

Climate and Dirofilaria infection in Europe

Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of several vector borne parasites like Dirofilaria, and in the risk of infection for animals and humans. The present paper reviews the effects of climate and other global drivers on Dirofilaria immitis and Dirofilaria repens infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes. In the last several years, growing degree day (GDD)-based forecast models, which use wide or local scale temperature data, have been developed to predict the occurrence and seasonality of Dirofilaria in different parts of the world. All these models are based on the fact that: there is a threshold of 14 °C below which Dirofilaria development will not proceed; and there is a requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito. The output of these models predicts that the summer temperatures (with peaks in July) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. These factors not only favour incubation of Dirofilaria, but also impact on mosquito species. Recent findings have also demonstrated that Aedes albopictus is now considered to be an important, competent vector of Dirofilaria infections. This mosquito species could spread from southern to northern European countries in the near future, changing the epidemiological patterns of dirofilariosis both in humans and animals.     

Dirofilaria repens—An etiological factor or an incidental finding in cytologic and histopathologic biopsies from dogs

Dirofilaria repens is an endemic, zoonotic parasite of carnivores, causing subcutaneous dirofilariasis, which is mostly asymptomatic. The aim of this study was to describe 22 cases of canine subcutaneous dirofilariasis. The cytologic and histopathologic samples were collected from dogs, which presented with various clinical signs such as cutaneous/subcutaneous nodules, hydropericardium, ascites, and lymphadenomegaly. The cytologic or histopathologic examination revealed purulent, pyogranulomatous, granulomatous or eosinophilic dermatitis/panniculitis, and the presence of D repens microfilariae or adults. The microfilariae or adults were also found incidentally in neoplastic cutaneous or subcutaneous tumors and in a sialocele. For the first time, microfilariae were also detected and described in pericardial and abdominal effusions and in enlarged reactive lymph nodes. Although it is well known that D repens can cause dermatitis and panniculitis in dogs, no previous reports of D repens microfilariae in body cavity fluids were found. The role of this parasite in the accumulation of body cavity fluid or in reactive lymphadenomegaly requires further investigation. Due to its zoonotic potential, and apparently underestimated pathogenicity, each case of canine subcutaneous dirofilariasis should be treated.     

Aberrant heartworm migration to the abdominal aorta and systemic arteriolitis in a dog presenting with vomiting and hemorrhagic diarrhea

A 2-year-old Dachshund was presented for vomiting and diarrhea. Abdominal ultrasound revealed Dirofilaria immitis in the abdominal aorta and an avascular segment of small intestine. The dog was euthanized. Necropsy revealed D. immitis in the abdominal aorta and widespread necrotizing arteriolitis. This is a unique presentation of aberrant migration of D. immitis.     

Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

Canine dirofilariasis is a common tropical parasitic disease of companion animals, caused by infestation of Dirofilaria immitis filarids within the pulmonary arteries and extending into the right heart. Increased reports of adverse reactions elicited by current microfilaricidal agents against D. immitis such as neurological disorders, circulatory collapse and potential resistance against these agents, warrant the search for new agents in forms of plant extracts. The use of plant extracts in therapeutic medicine is commonly met with scepticism by the veterinary community, thus the lack of focus on its medical potential. This study evaluated the presence of microfilaricidal activities of the aqueous extracts of Zingiber officinale, Andrographis paniculata and Tinospora crispa Miers on D. immitisin vitro at different concentrations; 10 mg/ml, 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml within 24 h, by evaluation of relative microfilarial motility as a measure of microfilaricidal activity. All extracts showed microfilaricidal activity with Z. officinale exhibiting the strongest activity overall, followed by A. paniculata and T. crispa Miers. It is speculated that the microfilaricidal mechanism exhibited by these extracts is via spastic paralysis based upon direct observation of the microfilarial motility.     

Marked subcutaneous mast cell and eosinophilic infiltration associated with the presence of multiple Dirofilaria repens microfilariae in 4 dogs

Dirofilaria repens is a parasitic nematode in the subcutaneous tissue of carnivores, including dogs and cats, transmitted by mosquitoes. Human beings may be accidental hosts. Infection of a dog with D repens was first reported in Palestine in 1934, and 2 additional cases were reported in dogs in Israel to date. This report describes D repens infection in 4 dogs in Israel that presented with subcutaneous masses, which were cytologically characterized by marked mast cell and eosinophil infiltration. In 3 cases, multiple microfilariae were present in the lesions; rare microfilariae were present in the 4^th case. In all 4 dogs, PCR of fine‐needle aspirates from the lesions were positive for D repens. The mast cells observed in all lesions were uniform and highly granulated, and with the presence of the microfilariae, a mast cell tumor was considered less likely. This report suggests that D repens infection‐associated subcutaneous lesions, characterized cytologically by massive mast cell and eosinophil infiltration, should be considered a differential diagnosis for mast cell tumor, especially in geographic locations endemic for this nematode. Notably, all 4 dogs were infected with D repens despite a routine prophylactic doramectin therapy administered every 3 months, probably due to the relatively long time interval between treatments.     

Serological update and molecular characterization of Dirofilaria immitis in dogs,South Korea

Eighty-one dogs in the Chungnam province were tested for heartworm (Dirofilaria immitis) infection by ELISA (SNAP test, IDEXX Laboratories, Maine, USA). Seventeen (20.9%) of the 81 samples were found to be positive and further analyzed by 16S rRNA sequencing. In this study, all dogs tested lived outdoors. Using the χ² test and Fisher’s exact test, no significant differences in the prevalence of dirofilariasis were observed among different gender and age groups, although the prevalence of this disease among dogs 2–4 yrs of age remains highest. Sequence analysis revealed that the species prevalent in Chungnam province were genetically distinct from the type strain of D. immitis based on the nucleotide deletion found at position nt 276 (cytosine) and nucleotide substitution at position 428 (G to A) of the partial 16S rRNA sequence of the type strain. Furthermore, phylogenetic analysis suggests at least 2 groups of D. immitis circulating in the Chungnam area between the year 2007 and 2008.     

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.     

Pulmonary artery dissection causing haemothorax in a cat: potential role of Dirofilaria immitis infection and literature review

A 7-year-old male castrated domestic short-haired cat suddenly died. Gross examination revealed severe right-sided haemothorax with blood clots, four adult filarial nematodes in the blood clots and the caudal vena cava and haemorrhage dissecting into the tunica media of the right pulmonary artery. Histopathological investigation showed fibrosis of the tunica intima and disorganization/fragmentation of the elastic fibres accompanied by fibrous tissue deposition in the tunica media of both branches of pulmonary artery. Degenerative vasculopathy (intimal fibromuscular hyperplasia and medial hypertrophy/hyperplasia) involving pulmonary arteries was also observed. The polymerase chain reaction amplification and sequencing confirmed the identification of the parasite as Dirofilaria immitis. A diagnosis of pulmonary artery dissection with haemothorax and concomitant heartworm disease was formulated. Degenerative processes of the tunica media have been reported to cause pulmonary artery dissection in both humans and animals. Pulmonary artery remodelling induced by heartworms may be considered the underlying cause in the first case of feline pulmonary artery dissection, herein described.     

D-dimer deposits in lungs and kidneys suggest its use as a marker in the clinical workup of dogs with heartworm (Dirofilaria immitis) disease

It has been reported that dogs with heartworm disease (Dirofilaria immitis) show increased plasma levels of D-dimer, a fibrin degradation product present in the blood after a blood clot is degraded by fibrinolysis. In the present study the authors show that, in dogs with both experimental and natural infections with D. immitis, D-dimer deposits in lungs and kidneys are associated with pulmonary thromboembolism and microfilariemic status, as well as there was a clear association between increased plasma values of D-dimer and positive staining in immunohistochemistry. Results suggest that the monitoring of D-dimer levels in infected dogs could be useful in evaluating the presence of pulmonary thromboembolism in the lungs and that microfilariae may induce microthrombosis in kidneys, thus contributing to renal pathology.     

Autochthonous Dirofilaria immitis infection in a ferret with aberrant larval migration in Europe

A two-year-old male ferret (Mustela putorius furo) was presented to the Faculty of Veterinary Science, Szent István University, for investigation of somnolence. Following unsuccessful therapeutic attempts, the ferret was euthanased and a male Dirofilaria immitis worm was found in the pulmonary artery and a female D. immitis specimen in the subdural space of the cranial cavity. To the authors’ knowledge, this is the first European record of D. immitis infection in a ferret, and the first case in which aberrant larval migration and consequent central nervous system signs were observed in a ferret in the course of D. immitis infection.     

Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe

A five‐year‐old, entire female mixed‐breed dog was presented with corneal oedema and episcleral hyperaemia in the left eye. The ophthalmological examination revealed the presence of a free‐swimming nematode in the anterior chamber. Circulating microfilariae were not observed by a modified Knott test nor were adult antigens detected in serum by a commercial ELISA. The parasite was surgically removed from the dog's eye, but its anterior end was damaged during the surgery. Based on the morphology of the posterior end, the nematode was preliminarily identified as a male Dirofilaria immitis. The species identification was confirmed by PCR amplification and sequencing of the mitochondrial coxI and 12S rDNA genes, using a DNA barcoding approach. Although other cases of ocular dirofilariosis by D. immitis have been previously recorded in Australia and the United States, the case reported herein is the first in a dog from Europ.     

Efficacy of the avermectins against filarial parasites: A short review

The avermectins are macrocyclic lactones produced byStreptomyces avermitilis. One of them has been chemically modified and given the non-proprietary name invermectin. The compounds have shown efficacy against various stages of filarial parasites. With respect to pre-adult stages in the mammalian host, activity has been observed againstDirofilaria immitis in ferrets and dogs, andDipetalonema viteae andLitomosoides carinii in jirds (Meriones); but activity has not been observed against the developing stages ofBrugia spp. in jirds orOnchocerca spp. in cattle. The compounds have not shown activity against adult filarial worms, except in the case ofSetaria equina in horses and possiblyDipetalonema viteae in jirds. With respect to the first stage larva (microfilaria) in the mammalian host, efficacy has been observed againstDirofilaria immitis in dogs,Brugia malayi in multimammate rats (Mastomys),Dipetalonema viteae andLitomosoides carinii in jirds andOnchocerca spp. in horses and cattle; but activity has not been observed against microfilariae ofBrugia pahangi in jirds or cats. Efficacy against filarial parasites has been observed following oral or parenteral administration of drug, sometimes at extremely low dosage. For example, a single oral dosage of 0.05 mg/kg was highly active against the third and fourth larval stages and microfilariae ofD. immitis in dogs.     

What is happening outside North America regarding human dirofilariasis?

The etiologic agents of human dirofilariasis in the Old World are Dirofilaria immitis, which cause pulmonary and subcutaneous nodules, and Dirofilaria repens, which cause ocular lesions. Although reports of new cases of dirofilariasis are sporadic in other parts of the world, a considerable amount of information is generated in Europe regarding human dirofilariasis. Most cases have been detected in the Mediterranean countries, Ukraine, and Russia; however, isolated or short series of cases have been reported in the Balkan Republics and central and northern European countries. Seroepidemiologic studies have provided evidence that humans living in endemic areas present rates of infection similar to those of the autochthonous canine populations. Antibodies against endosymbiont Wolbachia bacteria have been demonstrated recently in human Dirofilaria infections. During D. immitis infections, preadult worms and third- and fourth-stage larvae are often destroyed by the host reaction, releasing a considerable amount of Wolbachia, and a Th1-type response against Wolbachia and/or filarial antigens is mounted. On the contrary, infections with D. repens, in which worms frequently remain intact, no Th1-type response has been observed. As humans are resistant hosts, the Th1-response could have a role in the resistance against parasites. The causes for the rise in the incidence of human dirofilariasis as well as the possible application of Wolbachia antigens in the serodiagnosis of human infections are discussed.     

Subconjunctival Dirofilaria repens infection in a dog resident in the UK

Dirofilaria repens infection was diagnosed in a 5‐year‐old female German shepherd crossbreed, originally from Romania but brought into the UK in February 2014. The dog presented with conjunctivitis in March 2014 and then again 2 months later with additional ocular and nasal mucopurulent discharge. Bacterial cultures from the nasolacrimal duct were negative for bacterial growth. The case was referred in August 2014 for ophthalmic examination, which revealed abnormalities in both eyes, especially the left. They included mild palpebral conjunctival hyperaemia and marked follicular conjunctivitis, as well as a dorsonasal bulbar conjunctival mass. Serum biochemistry was unremarkable and a conjunctival biopsy taken from the dorsonasal bulbar conjunctival mass revealed eosinophilic/lymphoplasmacytic conjunctivitis. At re‐examination, nematodes were found in the area of the previous biopsy site and in the ventral palpebral conjunctival fornix. Polymerase chain reaction and sequencing confirmed these to be D. repens. Treatment with 10% imidacloprid and 2·5% moxidectin (Advocate Spot‐On) was successful, and clinical signs resolved over a 6‐week period. This case report indicates that D. repens infection should be considered as a possible aetiological cause of ocular lesions in dogs in the UK, especially those with a history of foreign travel. Implications for establishment and spread of D. repens in the UK are discussed.