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%.     

Serologic prevalence of Dirofilaria immitis,Ehrlichia canis,and Borrelia burgdorferi infections in Brazil

Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs 19.8%). Only one dog tested positive for B. burgdorferi antibodies.     

Dermatitis associated with microfilariae (Filarioidea) in 10 dogs

Dermatitis associated with microfilariae of a nematode of the superfamily Filarioidea was diagnosed in 10 dogs from the western United States. Clinically, lesions were single or multiple papules and plaques with alopecia, scarring, erythema, ulceration, or crusting. Eight dogs had lesional pruritus. Microscopically, there was perivascular, periglandular, to interstitial inflammation, with many eosinophils and/or plasma cells, and scarring affecting the dermis and subcutis. Microgranulomas containing microfilaria were seen in six dogs. Microfilariae were noted in microgranulomas or free in the dermis or subcutis, but not in vessels. In one case, an adult female nematode emerged from a biopsy sample that was placed in physiologic saline. Study of this nematode revealed that it was a filarioid of the family Onchocercidae; it was identified as Acanthocheilonema sp. (syn: Dipetalonema ). Antigen tests of five dogs were negative for Dirofilaria immitis . The Knott tests and/or filter tests of nine dogs were negative for microfilariae. An indirect fluorescence antibody test of one dog was also negative for D. immitis . One dog was not evaluated for microfilariae.     

Canine Dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serodiagnostic methods

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.     

An evaluation of 4 commercially available ELISA kits for the diagnosis of Dirofilaria immitis infection in dogs

Serum samples from 100 pound dogs were used to evaluate 4 commercial ELISA kits available for the diagnosis of Dirofilaria immitis. The kits were assessed on sensitivity (the ability to identify infected dogs), specificity (the ability to identify uninfected dogs) and accuracy (sensitivity plus specificity). The kits varied in sensitivity from 36% to 86%, in specificity from 44% to 70%, and in accuracy from 53% to 65%. The sensitivity was not affected by the age of the dogs, nor by the number of circulating microfilariae. The kits were most specific when testing the youngest dogs (less than = 3 years). The problems associated with the serological diagnosis of D. immitis infection in practice are discussed.     

Immunoperoxidase staining characteristics of Dirofilaria immitis in the dog

The immunoperoxidase staining characteristics of Dirofilaria immitis and pulmonary tissues from infected dogs were studied by using the following sera: anti-fresh D immitis, anti-processed D immitis, anti-dog IgG, anti-dog IgG Fc, anti-dog IgM and anti-dog C3. Marked staining was observed using anti-fresh D immitis serum. Body cavity fluid and cuticle were strongly stained and hypodermis, muscle, lateral cord, testis, vas deferens, ovary, oviduct and uterus were moderately stained. Oesophagus and intestine were mildly stained. Degenerate worms were stained by all antisera. The intact and cut surfaces of microfilariae and eggs and sperm present in filariae were stained, but not their internal contents. Circulating and stored immotile microfilariae did not stain. Excreted eggs, presumed to be unfertilized and, or, degenerate, stained positively. Immunoperoxidase staining of routinely processed histological samples provides a means of assessing D immitis antigen.     

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.     

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.     

Immunological analysis of agglutination in Dirofilaria immitis microfllariae

The mechanism of agglutination phenomenon of Dirofilaria immitis microfilariae was analyzed. Circulating microfilariae were collected from a D. immitis-infected microfilaremic dog and cultured in the several kinds of sera from dogs and animals. The agglutination of D. immitis microfilariae is a specific phenomenon due to some immune complexes formed with the anti-microfilarial antibody, heat-instable factor(s) and excretory-secretory products of microfilariae. Only live microfilariae were agglutinated and the agglutinated microfilariae remained alive as long as 27 days in culture in vitro.     

Prevalence of Dirofilaria immitis and Dipetalonema reconditum infection in dogs: 805 cases (1980-1989).

Clinical records of 6,977 dogs examined at the small animal clinic of the University of Tennessee College of Veterinary Medicine from January 1980 through December 1989 were analyzed to determine the prevalence and changing frequency of Dirofilaria immitis and Dipetalonema reconditum infection. Using the Knott's test on these dogs, 805 (11.54%) tested positive for microfilariae, with D immitis in 430 dogs (6.16%), and Dip reconditum in 375 dogs (5.37%). Statistical analysis confirmed that the prevalence of D immitis and that of Dip reconditum were essentially equal in the population of dogs included in this study. There was a slight decrease in the prevalence of D immitis over the 10 years examined, but the prevalence of Dip reconditum remained constant. The results were not affected by year-to-year variability in the number of examinations performed. On the basis of our findings, in eastern Tennessee, those veterinarians who diagnose heartworm infection by the presence of microfilariae without differentiating the species involved, risk misdiagnosing 50% of the cases. If the patterns of prevalence seen in recent years continue, the chances of error may actually increase.     

Prevalence of Dirofilaria immitis infection among shelter cats

OBJECTIVE: To determine prevalence of Dirofilaria immitis infection among shelter cats. DESIGN: Cross-sectional study. ANIMALS: 239 cats euthanatized at an animal shelter in southeastern Michigan. PROCEDURE: A gross necropsy focusing on the thoracic cavity, heart, lungs, and pulmonary vessels was performed within 5 hours after cats were euthanatized. Blood was collected directly from the heart of cats found to be infected and tested, using a filter test for microfilariae. Serum was tested for D immitis antigens by use of 2 antigen detection kits and for D immitis-specific antibodies by use of 2 antibody detection kits. RESULTS: Cats ranged from approximately 4 months to 15 years old. Adult D immitis were found in 6 (2.5%) cats. Blood could not be recovered from 1 of the cats with heartworm infection. For the 5 other cats, results of the filter test were negative, and results of both antigen and both antibody tests were positive. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cats living in an urban area in the northern part of the United States have a low prevalence of adult D immitis infection. However, this is likely to be an underestimate of the true prevalence of infection, because no attempts were made to identify cats infected with larval or juvenile stages of D immitis.     

Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis

The present study determined the prevalence and geographical distribution of Dirofilaria immitis and other filariae, from dogs in littoral areas of Paraná state, in Brazil. This survey spanned eight months, between 1998 and 1999, and was also designed to compare the efficacy of different tests for diagnosis of heartworm infection in that area. Blood samples were collected from 256 native-owned dogs distributed along the Paraná coastal area. Five diagnostic procedures were used: direct smear examination, the Knott's modified test, filtration assay, and two heartworm antigen detection kits. A follow-up imaging exam was performed to support the heartworm diagnosis. The imaging diagnosis included radiographic and ultrasonographic exams of six dogs that had positive results for the heartworm antigen detection kits, but showed different microfilarial burdens. The presence and severity of radiographic and ultrasonographic signs were compared with the results obtained in microfilariae detection and antigen tests. Diagnostic parasitology results indicated that 31.25% of the dogs were microfilaremic. Three different microfilariae were recovered: D. immitis, Dipetalonema reconditum, and the third (mf3) was not identified. D. reconditum was the species with the highest prevalence: 22.6%. In general, D. immitis prevalence was 5.47% (28.57% occult infections), but it varied along the coast and the range was from 0 to 20%. No correlation could be established between the overall scores for microfilarial counts (small or large numbers) and the severity of radiographic results or the likelihood of detecting filariae in the pulmonary artery using echocardiography. The finding of a different type of microfilaria (mf) suggested the existence of a third species in Paraná state, whose prevalence was 4.68%. These results show that to obtain a reliable diagnosis of heartworm infection, antigen detection kits are indicated. Knott's test or filtration should be performed to confirm microfilaremia and not for diagnosis of heartworm infection. Imaging tests support parasitology exams and add more about severity of infection. The northern areas, specially Guaraque?aba and Ilha das Pe?as, presented the highest number of heartworm-infected dogs.     

Are antigen test kits efficient for detecting heartworm-infected dogs at the southern distribution limit of the parasite in South America? Preliminary results

The aim of this study was to evaluate the performance of commercial heartworm antigen tests in dogs harbouring Dirofilaria immitis microfilariae near its distribution limit in South America. A total of 4934 blood samples of adult dogs from Southern Greater Buenos Aires were examined to detect circulating microfilariae in the buffy coat interface between December 2005 and April 2006. Microfilariae were detected in 88 (1.8%) blood samples and all the microfilariae observed were identified as D. immitis by acid phosphatase stain technique. In a first trial, 69 (78.4%) out of the 88 serum were positive by Speed((R)) Diro. Then, a new test was performed over 25 microfilariae-positive serum samples randomly selected among the 88 previously tested samples and using simultaneously Speed((R)) Diro, Witness((R)) Dirofilaria and Snap((R)) 3dx. This second trial showed identical results for the three different tests, in which 19 (76%) samples were positive. Therefore, more than 20% of microfilaremic dogs were antigen negative. The main hypothesis that could explain our finding is a low worm burden in the study area. According to our preliminary results, it is highly recommendable the complementary use of antigen tests and other procedures to obtain an accurate diagnostic near the distribution limit of the parasite.     

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.     

Eurasian otter (Lutra lutra), a definitive host for Dirofilaria immitis.

A mature male and a mature female Dirofilaria immitis were found in the right ventricle of the heart of a naturally infected 2-yr-old male Eurasian otter (Lutra lutra) that had died of severe lung congestion at a zoo in South Korea. Both developing embryos and microfilariae were present in the uterus of the female D. immitis. Although circulating microfilariae were not detected in blood or tissue, the Eurasian otter may serve as a definitive host for D. immitis.     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.     

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.     

Epidemiology of canine heartworm in its southern distribution limit in South America: Risk factors, inter-annual trend and spatial patterns

This study was aimed at understanding some aspects of the canine heartworm epidemiology in the southern distribution limit of the parasite in South America. With this objective, 19,298 blood samples of owned dogs from 65 localities of 13 municipalities of Buenos Aires Province were tested for Dirofilaria immitis circulating microfilariae and/or female antigens. The overall heartworm prevalence was 1.63% by microhematocrit tube technique (n=19,136), 3.65% by modified Knott (n=713), and 14.41% by antigen test kit (n=118). Microfilaremic dogs showed a median of 1933 microfilariae per millilitre (q1=375, q3=5625, n=100). Male dogs belonging to breeds of short hair and large size recorded significantly higher prevalences than the other categories. Also, the prevalence increased significantly with the age and only dogs younger than 12 months were not found infected. A clear decreasing trend of the annual prevalence was observed during the whole study period, from 3.91% in 2001 to 1.17% in 2006. D. immitis-infected dogs were detected in 32 localities of 9 municipalities (prevalence range: 0.2-6.7%). Generalized linear models were used to assess associations between heartworm prevalence and environmental variables. The resulting significant models were univariate and included variables related with soil cover and human population density. The best model predicted maximum heartworm prevalences around middle values of bare soil cover, and lower at high and low covers. According to our analyses, canine heartworm infection in urban temperate Argentina could be described as relatively low, endemic, and spatially heterogeneous. Host and environmental factors affecting heartworm transmission at local level were identified and discussed.     

Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection

Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.