Preliminary findings on the efficacy of selamectin in the treatment of dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariosis caused by Dirofilaria repens is common in dogs and it is an emerging helminthozoonosis in Europe, Asia, Africa and also in Hungary. Macrocyclic lactones are used for preventing the infection; however, their activity against the microfilariae and mature stages of this species is questionable. Selamectin is widely used for the prophylaxis of heartworm (D. immitis) infection. The objective of the present study was to test the microfilaricidal efficacy of the topical formulation of selamectin in dogs naturally infected with D. repens . A total of 78 Beagle dogs were examined for the presence of circulating microfilariae by Knott's test. Twenty-three of the microfilaraemic dogs were divided into four groups and included in the trial. The dogs received monthly or biweekly selamectin treatment and were subjected to monthly blood testing for a period of 252 or 336 days. At the end of the study, 65% of the dogs were not microfilaraemic and the rest had low number of microfilariae in their blood. These results indicate that chronic spot-on selamectin treatment may be a useful tool also in the control of canine subcutaneous dirofilariosis.     

Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectin

Therapy of canine dirofilariois due to Dirofilaria repens is indicated for dogs suffering from clinical signs of this disease, such as dermal swelling, sub-cutaneous nodules and pruritus. It is also important in order to decrease the risk of infection to other dogs and humans in the vicinity of the infected animal when suitable mosquito vectors are present. Combined therapy with the arsenic adulticide melarsomine and the avermectin microfilaricidal doramectin was effective in clearing infection with D. repens in a dog. The number of microfilariae dropped from 17 microl(-1) blood pre-treatment to 7 microl(-1) following the first adulticide injection and reached 0 a day after the microfilaricidal administration. The dog remained negative for D. repens microfilaremia during a follow-up period of 90 days. Euthanasia and necropsy performed 3 months after the initiation of therapy due to a progressive neoplastic disease revealed no evidence of filariae.     

Autochthonous Dirofilaria (Nochtiella) repens infection in dogs in Kuwait

Human dirofilariasis caused by Dirofilaria repens is a rare but increasingly reported zoonosis in the Middle East. However, eligible animal reservoirs seldom have been investigated in the area until now. Seven (1.8 percent) out of 381 dogs examined in Kuwait carried D. repens microfilariae showing compatible cutaneous clinical signs and positive response to the adulticide and microfilaricide therapies. This study indicates that dogs from Kuwait are natural hosts of D. repens and therefore act as an animal reservoir for the zoonotic condition.     

A prevalence survey and risk analysis of filariosis in dogs from the Mt. Vesuvius area of southern Italy.

A dog microfilariae prevalence and risk factor survey was conducted in 51 contiguous municipalities of the Mt. Vesuvius area (Campania region, southern Italy) in order to add data to the limited epidemiological information available regarding filarial worms in this zone. Between May 1999 and June 2000, blood samples were collected from 351 asymptomatic dogs. Blood samples were examined using a modified Knott's technique and histochemical staining in order to count and identify microfilariae. The results were subjected to statistical analysis and choroplethic municipal maps (MMs) were drawn by a geographical information system (GIS) software. Microfilariae were detected in 63 of the 351 dogs surveyed, constituting a total filarial prevalence of 17.9%. In particular, 56 dogs (15.9%) showed only microfilariae of Dipetalonema reconditum; three dogs (0.8%) only microfilariae of Dirofilaria repens; two dogs (0.6%) microfilariae of both D. reconditum and D. repens and two dogs (0.6%) microfilariae of both Dirofilaria immitis and D. repens. High D. reconditum prevalence was associated with hunting practice, masculine gender and older dogs. There was also a tendency to find high prevalence in dogs sampled in the afternoon.In conclusion, the presence of microfilariae of D. reconditum in 92% of microfilaraemic dogs indicates that this filarial worm was the predominant filarial species in dogs in the Mt. Vesuvius area.In addition, the general trends of the MMs showed that D. immitis and D. repens were present only in a few municipalities, whereas D. reconditum was widely and homogeneously spread throughout the entire study area.     

Discrimination between six species of canine microfilariae by a single polymerase chain reaction

Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.     

Dermatitis associated with Dirofilaria (Nochtiella) repens microfilariae in dogs from central Italy

Pruritic dermatitis associated with Dirofilaria (Nochtiella) repens microfilariae in the blood was diagnosed in 22 dogs from Fermo (Central Italy). According to the history, previous unsuccessful treatments with corticosteroids, antibiotics, restricted diet, flea control, levamisole and ivermectin were recorded in 17 dogs (77.3%). The combined filtration tests and antigen tests, performed during the study, were negative for Dirofilaria immitis and Acanthocheilonema reconditum in each case. Dermatological lesions included erythema, papules, single or multifocal alopecia, eczema, lichenification, crusting and nodules. All dogs had pruritus. Concurrent babesiosis was diagnosed in the blood smears of each case (100%), and 60% of the dogs were found to be carriers of canine granulocytic ehrlichiosis (CGE). Three dogs (13.6%) were positive for leishmaniosis. Eradication of the concurrent infections followed by specific macro- and microfilaricide treatment led to complete recovery from the dermatological syndrome. The main conclusion of the study is that D. repens infection can be more pathogenic than is currently considered, and it is apparently an opportunistic disease with serious dermatological consequences.     

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.     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

Cutaneous lesions in dogs with Dirofilaria (Nochtiella) repens infestation and concurrent tick-borne transmitted diseases

A pruritic dermatitis characterized by the presence of erythema, papules, focal or multifocal alopecia, crusting and nodules was seen in 28 dogs with Dirofilaria repens microfilariae infestation in an endemic area in north-west Italy. Previous unsuccessful nonspecific antipruritic treatments, flea control and restricted diet were recorded in 53.6% of the patients. Both the Knott and the antigen tests were negative for Dirofilaria immitis and Acanthocheilonema reconditum. Concurrent babesiosis and/or canine granulocytic ehrlichiosis was also diagnosed in many affected dogs. Preliminary treatment of the concurrent diseases was followed by specific filaricide treatment. The cutaneous lesions, although slightly improved with the initial treatment, resolved completely with macro- and microfilaricide treatment. Although D. repens may be an opportunistic pathogen, this parasite should not be considered as harmless as previously thought and its potential pathogenic role in causing cutaneous lesions in dogs should be considered. Furthermore, it has a zoonotic importance as human cases have been reported worldwide.     

Dirofilaria repens in a cat with acute liver failure

Acute liver failure was diagnosed in a 12-year-old cat. Fine needle aspirate cytology revealed high numbers of unsheathed microfilariae and a hepatocellular reaction with no evidence of bacterial infection. The microfilariae were identified as those of Dirofilaria repens by acid phosphatase staining. The high number of microfilariae seen in both the blood and the liver aspirate samples as well as the favourable response to ivermectin amongst other drugs administered, is suggestive that D. repens was the cause of the liver insult. A positive result obtained with an antigen-capture ELISA (Dirochek) for Dirofilaria immitis antigen was interpreted as false. This is the 1st report of Dirofilaria repens for South Africa.     

Subcutaneous dirofilariosis in South-East Serbia--case report

Human dirofilariosis is a relatively rare infection caused by filarial worms of the genus Dirofilaria. We herein report the first case of human subcutaneous dirofilariosis in the southeastern part of Serbia. A complete alive nematode was removed from a nodule in the periorbital region of a 50-year-old woman. The nematode was morphologically identified as a D. repens-like immature female. The diagnosis was confirmed with molecular methods. The patient was probably infected in the South-East Serbia as she had not travelled abroad, nor in other parts of Serbia such as Vojvodina, recently identified as a hyperendemic area for D. repens infection of dogs.     

Prevalence of Dirofilaria immitis infection in living raccoon dogs assessed by hematological examination

The prevalence of Dirofilaria immitis in free-ranging raccoon dogs (Nyctereutes procyonoides viverrinus) was examined in the southeast region of Kanagawa Prefecture, Japan, using a rapid immunomigration (RIM) test kit. Between April 2007 and March 2010, we examined 108 raccoon dogs rescued and housed by the Kanazawa Zoological Garden. D. immitis infection was found in 8 (7.4%) raccoon dogs. This is the first report to reveal the prevalence of D. immitis infection in living raccoon dogs. The prevalence of the infection was lower than previously reported values obtained on postmortem examination. One reason might be that the present study included young raccoon dogs infected with immature worms. Significant high-risk areas of D. immitis infection in the raccoon dogs were not observed.     

Autochthonous canine Dirofilaria repens in the vicinity of Warsaw

In 2009 microfilaremia was recognized in ten dogs living in five distinct districts located near Warsaw, the capital of Poland. Based on PCR results, all the animals were found to be infected with Dirofilaria repens. Nine of the infected dogs have never travelled outside the country and it was assumed that the cases were native. Monitoring the infection in the European countries should be introduced to establish the actual geographic range of dirofilariosis.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Canine dirofilariosis in two cities of southeastern Spain

Several cases of human subcutaneous/ocular dirofilariosis caused by Dirofilaria repens from an area in the southeastern Spain where previous epidemiological studies have shown a very low prevalence of this species in dogs, have been studied in our laboratory. Since the prevalence of this species in dogs did not correspond to the incidence of human cases in the zone studied, a preliminary epidemiological survey was carried out on 114 dog blood samples from two kennels and one veterinary clinic. Knott, polymerase chain reaction (PCR) with specific primers for Dirofilaria immitis and D. repens, and ELISA with adult E/S D. immitis and adult somatic D. repens antigens for the detection of specific IgG, were used. Fifty-three out of the 114 samples analyzed were positive for Knott and/or PCR to D. immitis or to D. repens. D. repens was the species with the highest prevalence, 84.6 and 37.1%, respectively, in each kennel. IgG antibodies against D. immitis and D. repens, were detected in 11 samples which gave negative results to both Knott and PCR. These results demonstrate a very high prevalence of D. repens that could be associated with the increasing incidence of human subcutaneous/ocular cases recently detected in this zone.     

Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.     

Comparison of the acid-phosphatase staining and polymerase chain reaction for detection of Dirofilaria repens infection in dogs in Korea

This study was performed to compare acid-phosphatase staining with polymerase chain reaction (PCR) analysis for the diagnosis of Dirofilaria repens infection. The infection of D. repens was confirmed in Korean reared German shepherd dogs. Knott's tests were carried out for the detection of microfilaria in 543 Korean reared German shepherd dogs (255 females and 288 males). Eighty four of the 543 dogs (15.5%) showed microfilaria-positive reactions with the modified Knott's test, and the test-positive microfilariae were then examined by both acid phosphatase staining and PCR analysis. Six (7.1%) and 17 (20.2%) of the 84 microfilaria-positive samples, by the Knott's tests were positive to D. repens by acid-phosphatase staining and in D. repens-specific PCR analysis, respectively. All samples found to be positive by the acid-phosphatase staining were also found to be positive by PCR analysis. Therefore, we conclude that PCR analysis (20.2%) is more valuable for the diagnosis of D. repens infection than acid-phosphatase staining (7.1%) (p<0.001).     

Dermatitis associated with Dirofilaria repens microfilariae in three dogs in Saudi Arabia

The presence of microfilariae of the zoonotic nematode Dirofilaria repens is reported in three dogs from Saudi Arabia, manifesting as pruritic dermatitis with signs including erythema, alopecia, papules, crusting and hyperkeratosis. All dogs were found to have concurrent babesiosis. Specific treatments against the two conditions led to complete clinical recovery and disappearance of microfilariae from the blood one month later.     

The incrimination of Aedes (stegomyia) aegypti as the vector of Dirofilaria repens in Nigeria

Six local species of culicides were identified as the common mosquitoes in Zaria, out of 15 species captured using various adult and larval collection methods. These common culicides are Culex pipiens fatigans, Anopheles gambiae grp., Mansonia africana, Culex pipiens pipiens, Aedes (stegomyia) aegypti and Aedes vittatus. They were each fed directly on a local dog naturally infected with Dirofilaria repens to evaluate their refractoriness/susceptibility to dirofilarial infection. In a number of donor-feeding trials, 39. 4% Culex pipiens fatigans; 58.9% An gambiae grp.; 60.5% Mansonia africana; 1.8% of Culex pipiens pipiens; 23.4% Ae aegypti and 3.3% of Ae vittatus successfully fed on the microfilaraemic host. Only Aedes aegypti was susceptible to the infection as all 40 (100%) Ae aegypti reaching 10-14 day post-blood meal had infective (L(3)) larvae of D. repens. The remaining five species were refractory. The microfilariae in the five non-susceptible mosquitoes were always found trapped in the blood meal in the insects midgut (stomach). These trapped microfilaria were dead by the 2nd day in the insect's midgut. However, in the susceptible Ae aegypti, the microfilariae were set free from the blood meal in the midgut and within 24h migrated to the malpighian tubules (MT) of the mosquitoes. All Ae aegypti dissected 5-7 day post-infective blood meal showed the typical quiescent sausage stage (L(2)) larvae in the malpighian tubules. At day-10 post-blood meal, relatively active infective (L(3)) larvae of D. repens were found in the MT; and by day 12-14, highly motile infective larvae had reached the insect's head and proboscis, with infective larvae occasionally oozing out during dissection through the tip of the proboscis. The rate of development of D. repens to infective larvae was faster in mosquitoes infected in July when the environmental temperature was 24.5 degrees C than those infected in November when the temperature was 22.5 degrees C. The latter were delayed for 4 days. The breeding sources of Ae aegypti, the local vector implicated were also identified. As no particular vector of this zoonotic filaria has been identified previously in Nigeria, these findings could make any control programme more focussed and easier.