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.     

Subcutaneous and cardiopulmonary dirofilariasis in a dog

Canine subcutaneous dirofilariasis is rare and typically caused by Dirofilaria repens. An adult male, intact, mixed breed dog from South Carolina, USA, was examined because of physical trauma to the right maxilla. The dog died during hospitalization and was submitted for necropsy. Gross post-mortem changes included approximately 150 adult nematodes morphologically consistent with Dirofilaria spp. in the pulmonary artery, right atrium, and right ventricle. Histologically there was widespread proliferative pulmonary endarteritis with intraluminal nematodes morphologically consistent with Dirofilaria spp. Four similar nematodes were present in the subcutaneous tissue of the left medial thigh and tibial area. These nematodes were located within the fascia and skeletal muscles. They were surrounded by epithelioid macrophages and multinucleated giant cells, with some lymphocytes and plasma cells, and areas of mineralization (interpreted as mineralized cross sections of nematodes). Nematodes were morphologically identified as D. immitis. Subcutaneous dirofilariasis caused by D. immitis occurs when migrating nematode larvae develop into adults in the subcutaneous and skeletal muscle tissues.Key clinical message:Subcutaneous dirofilariasis in dogs is rare and most often associated with Dirofilaria repens. This case highlights the pathology findings of subcutaneous D. immitis in a dog.     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

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.     

Molecular and serological detection of animal and human vector-borne pathogens in the blood of dogs from Côte d’Ivoire

In Côte d’Ivoire, limited information are available on vector-borne pathogens, their prevalence and distribution. Here, we assess the occurrence and diversity of canine vector-borne diseases (CVBDs) in Abidjan and Yamoussoukro cities. Blood from a total of 123 dogs were tested for Leishmania infantum and Ehrlichia canis antibodies and screened for Leishmania and Trypanosoma spp., Piroplasmida, Filariidae and Anaplasmataceae by PCR and sequencing. Among dogs, 39 % were positive for at least one pathogen. Seroprevalences were: 15.4 % and 12.2 % for L. infantum and E. canis, respectively. DNA of L. infantum and T. congolense (4.1 %), Baabesia vogeli (1.6 %), Filariidae (Dirofilaria immitis, D. repens and Acanthocheilonema reconditum) (10.6 %) has been detected. Anaplasmataceae were detected in (17.1 %) and E. canis was the only identified specie. Co-infections were observed in 13.8 % of dogs: E. canis-L. infantum co-infection was the most prevalent (4.9 %). Age, breed and sex of dogs do not seem to influence infections. Village dogs were more susceptible to CVBDs than kennel dogs (PV = 0.0000008). This study reports for the first time the presence of L. infantum, B. vogeli, A. reconditum, D. immitis and D. repens in dogs from Côte d’Ivoire and determines the prevalence and diversity of CVBD pathogens. The results indicate that human and animal pathogens are abundant in Ivoirian dogs which requires attention of veterinarians, physicians and authorities against these diseases, especially against major zoonosis such as visceral leishmaniasis (L. infantum).     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

A Survey for the Microfilariae of the Canine Heartworm, Dirofilaria immitis, in the Calgary Region of Southern Alberta

A blood survey for the prevalence of the microfilariae of the canine heartworm, Dirofilaria immitis was conducted in the Calgary area of southern Alberta between November 1977 and August 1979. A total of 514 blood samples was examined by the modified Knott's test. All of the samples were negative for D. immitis microfilariae. Wright's stained blood smears taken from 19 animals at the Calgary Zoo also proved negative. One smear from a male two-toed sloth (Choloepus didactylus) contained sheathless microfilariae which were distinguishable from those of D. immitis. These results, as well as mail survey data indicate that D. immitis is not endemic in the Calgary area.

It is recommended that the modified Knott's test be used for similar large scale sampling studies. In addition, it is suggested that the testing of both native and exotic zoo animals which could serve as definitive hosts of D. immitis be continued. These animals may become local sources of infection or introduce other species of microfilariae which will have to be differentiated from those of D. immitis.

     

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.     

Canine vector-borne disease in travelled dogs in Germany--a retrospective evaluation of laboratory data from the years 2004-2008

When importing dogs from various Mediterranean countries into Western Europe canine vector-borne infections are often considered as a major issue. Several diseases including babesiosis, leishmaniosis, hepatozoonosis, canine heartworm disease or ehrlichiosis can potentially be endemic in this region and pose a potential health risk for travelling dogs. Information on such infections in travelled dogs is scarce and therefore this study has been undertaken to examine the frequency of vector-borne infections in travelled dogs from the years 2004-2008. A total of 997 samples were screened by direct and/or indirect methods. Total seroprevalence was 7.5% with individual seroprevalence for the 3 species Leishmania spp., Ehrlichia canis and Babesia canis spp. ranging from 3.1 to 4.9%. Total detection rate for pathogens by direct methods was 3.5%. Ninteen Giemsa-stained blood smears were positive for large Babesia. None of the samples screened for microfilariae by Knott's test or for Dirofilaria immitis antigen by DiroChek^® were positive. Using PCR methods Leishmania-DNA was detected in 1/42 samples but none of 59 animals screened for E. canis-DNA was positive. The prevalence values as established by indirect and direct pathogen detection are considered as rather low.     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

The prevalence of Dirofilaria immitis in dogs in Sydney

Four hundred and four dogs from 9 pounds in Sydney were examined for circulating microfilariae and antigens of Dirofilaria immitis. One hundred of these were also examined post mortem for adult heartworms. The prevalence of infection in the 404 dogs as shown by serology was 11.4%, and 5.9% had circulating microfilariae of D immitis. Adult heartworms were present in 15 of 100 dogs. Dipetalonema reconditum microfilariae were present in 3.7% of dogs. Dirofilariosis is still a common and important parasite of dogs in the Sydney region and chemoprophylaxis is recommended.     

The incidence of canine haematozoa in peninsular Malaysia

In 3 urban areas in Selangor, Peninsular Malaysia between 1973 and 1981, blood from 4080 dogs was examined for haematozoa. The following frequencies were found: Babesia gibsoni 17.7%; microfilariae of Dirofilaria immitis 9.6%; Hepatozoon canis 1.2%; B. canis 1.1%; Ehrlichia canis 0.2%; Trypanosoma evansi 0.1%. A detailed examination of B. gibsoni infections and microfilariasis due to D. immitis with regards to monthly distribution, breed frequency, sex and age, revealed that pedigree and non-pedigree dogs were equally susceptible to Babesia and microfilariae infections.     

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.     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

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.     

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.     

Immunohistological observations on pulmonary tissues from dogs infected withDirofilaria immitis

Pulmonary tissues from non-infected dogs, naturallyDirofilaria immitis-infected dogs and experimentally infected puppies, selectively necropsied after infection, were assessed using peroxidase-antiperoxidase (PAP) staining technology. Sequential sections of pulmonary tissue were PAP stained with anti-freshD. immitis serum, anti-paraffin processedD. immitis serum, anti-dog immunoglobulin (IgG, IgG Fc, IgM) sera and anti-dog complement (C₃) serum, and examined for antibody, complement and forD. immitis antigen. The extent of alveolar septal thickening was positively correlated with infection status. Cellular infiltration was most evident surrounding obstructed areas whereD. immitis werein situ. Antigenic material (microfilariae, eggs, fragmented filariae) labelled by PAP was identified in the pulmonary arteries, alveolar capillaries and alveolar septa. Deposits of complement and IgG, presumably associated with immune complex formation, were also observed in some of the infected dogs. Identification of antigen, antibodies and complement associated with alveolar septal pathology suggested that immune mechanisms were active in its development.Abbreviations FW fresh worm - PAP peroxidase-antiperoxidase - PW processed worm     

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.     

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.