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Accurate diagnosis of canine filariosis is essential for choosing correct therapeutic approach. Therefore, reliable methods for discriminating among the different filarial infections in dogs are needed. The authors report simple and highly specific molecular methods that identify the three most common filarial nematodes of European dogs: Dirofilaria immitis, D. repens and Acanthocheilonema (syn. Dipetalonema) reconditum, based on (1) PCR amplifications of mitochondrial DNA (12S rDNA and coxI) with general filarial primers followed by digestion with restriction enzymes that generates band polymorphisms clearly discriminating the three species and (2) PCR amplifications with species-specific primers to support the restriction analysis, in particular in the case of multiple infections.

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 Guaraqueçaba and Ilha das Peças, presented the highest number of heartworm-infected dogs.

Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.

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.

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.

A survey on the prevalence of Dirofilaria immitis and Dipetalonema reconditum was conducted in 1,519 dogs from Maceió and two coastal areas in the State of Alagoas, Northeast Brazil, from 1995 to 1999, by testing for microfilariae in blood. All blood samples were from exclusively domiciled dogs with a known history, showing that the infections were autochthonous, confirming transmission of canine filariasis in these areas. In Greater Metropolitan Maceió, 15 (1.3%) microfilaremic dogs were detected with D. immitis and 15 (1,3%) with D. reconditum. In the southern coastal area there was an estimated prevalence of 12.7% for D. immitis. D. immitis and D. reconditum microfilaria were 298.1 micrometer and 249.2 micrometer long and 7.3 micrometer and 4.4 micrometer wide, respectively. A Witness immunotest that detects D. immitis antigen was used to confirm parasitological results and reveal occult dirofilariasis cases. Of the total 6,579 females examined, 8 (0.1%) Culex quinquefasciatus were observed to be naturally infected with D. immitis larvae. These results proved dirofilariasis transmission in Maceió and demonstrated D. reconditum in the same geographic area.

Using antigen capture and filter tests, 6,078 dogs throughout the state of Washington were examined for filariasis between July 1, 1997 and October 31, 1999. In western Washington, 791 males and 901 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 6/791 (0.8%) males and 7/901 (0.8%) females were infected with Dirofilaria immitis (D. immitis), and one (0.1%) male and one (0.1%) female were infected with Dipetalonema reconditum (D. reconditum). There were also 392 males and 362 females examined that were outdoors, not on prophylaxis, and had not traveled out of western Washington. One (0.1%) female was infected with D. immitis, and two (0.5%) males and one (0.2%) female were infected with D. reconditum. In eastern Washington, 707 males and 826 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 9/707 (1.0%) males and 4/826 (0.5%) females were infected with D. immitis, and no D. reconditum was found. There were also 376 males and 412 females examined that were outdoors, not on prophylaxis, and had not traveled out of the state. Three (0.8%) males and three (0.7%) females had D. immitis. One (0.2%) female had D. reconditum. Distribution of D. immitis-infected, nontravel dogs in eastern Washington was only found between 120 degrees and 119 degrees west longitude in the communities of Richland, Moses Lake, Okanogan, and Omak. Enzootic transmission of D. immitis and D. reconditum is occurring in both eastern and western Washington.

Field and laboratory studies were performed in order to assess the degree of canine dirofilariasis caused by Dirofilaria immitis (Leidy) in the Baix Llobregat region, a fluvial area near Barcelona, Spain. A total of 188 dogs were sampled between May and August of 1994. Three main areas were chosen: the Western Delta, the Eastern Delta and the Northern zone. Simultaneously, a mosquito sampling programme was carried out with CO2 light traps, to search for infective larvae (L3) of D. immitis. Of the 188 dogs sampled, 38 were positive for at least one of the three filaria found: D. immitis 12.8%, Dipetalonema reconditum (Grassi) 3.7% and Dipetalonema dracunculoides (Cobbold) 2.7%. Only 1.1% showed a mixed infection of both D. immitis and D. dracunculoides. Although Dirofilaria repens Raillet et Henry has been found in Spain, it was not found in this study. Comparing the three zones of the Baix Llobregat, the Eastern Delta showed the highest level of D. immitis (35.3%), probably due to the presence of Aedes caspius (Pallas). Despite the effort in sampling the mosquito population, D. immitis was not found in any of the 2001 females dissected, belonging to 5 species.

To assess the current status of Dirofilaria immitis infection and to determine whether there were other canine filarial infections in north Taiwan, postmortem examination was conducted in 180 stray dogs more than 12 months old. Blood and serum samples were examined using a modified Knott's test and an antigen-detecting enzyme-linked immunosorbent assay (ELISA) kit, respectively. Filarial infection was found in 60.6% of the dogs: 55% with D. immitis and 12.2% Dipetalonema reconditum. Moreover, the ELISA was determined to be more sensitive than the Knott's test. Although canine heartworm infection in Taiwan has been attributed to the unrestricted import of dogs from endemic areas, the results of this study indicate that transmission of D. immitis and Dip. reconditum may occur indigenously in the local canine population. This study is also the first record of Dip. reconditum in Taiwan.

Between June 1993 and May 1996 infections with filariae were diagnosed in 80 dogs. Five animals were infected with Dirofilaria repens and three other dogs with Dipetalonema reconditum. One of the D. reconditum positive dogs was also infected with Dirofilaria immitis. An epidemiological analysis of the cases was possible for three D. repens and two D. reconditum infected dogs. Two of the dogs with D. repens infections had been imported from Italy or Greece, the third dog had been taken by its owner to Hungary and former Yugoslavia. The dogs infected with D. reconditum were imported from Corse or Spain. Regarding the other 72 dogs with filariae, 45 animals were infected with D. immitis. For 27 dogs being negative in the D. immitis ELISA, microfilariae have been demonstrated. However, a histochemical species differentiation of the microfilariae was not possible because fresh blood smears were not available.

The prevalence of filariae in wild raccoons trapped in southeast Georgia was determined. Examination of blood samples revealed that 74 of 113 raccoons (66%) trapped in 6 southeastern Georgia counties were infected. Seventy-three of these raccoons (65%) were infected with Mansonella llewellyni and this parasite was observed in raccoons from every location examined. Dirofilaria tenuis was found in 22 raccoons (20%) and was observed in only 3 of the 6 counties surveyed. An adult specimen of Acanthocheilonema procyonis was found in the subcutaneous tissues of 1 of 5 necropsied raccoons. This is the first record of filariae in raccoons from Georgia. In addition, Dirofilaria-like larvae were found in Aedes taeniorhyncus mosquitoes collected in Liberty County.

A field survey aimed at updating knowledge of the distribution and prevalence of filarial infections in the Piedmont region was undertaken. Blood samples were collected from 2628 dogs (10-15 dogs/100 km2) which had remained in the local area, were kept in the open overnight, were more than 2 years old and had not previously been treated with microfilaricidal drugs. Infection was diagnosed by a modified Knott test. Comparison of the results with those from a similar survey carried out 25 years ago showed a 3-fold increase in the size of the endemic area, with the colonization of new habitats, and a 4-fold increase in the prevalence of Dirofilaria immitis infection.

Blood samples from 50 dogs with episodic weakness and 50 clinically normal dogs, all belonging to hunting breeds, were examined for microfilariae using the modified Knott's method. The prevalence of microfilaraemia in dogs belonging to the episodic weakness group was 10% for Dirofilaria immitis, 30% for Dirofilaria repens and 8% for Acanthocheilonema reconditum. In the clinically normal group, the figures were 14% for Dirofilaria repens and 4% for Acanthocheilonema reconditum. The overall percentage of dogs with microfilaremia in the episodic weakness group was 44 and that of the clinically normal group 16, with a statistically significant difference (P < 0.01). The prevalence of microfilaremia due to the nonpathogenic filarial species (Dirofilaria repens, Acanthocheilonema reconditum) was 34% and 16% for the episodic weakness group and the clinically normal dogs, respectively, with no significant difference.

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.

The prevalence of Dirofilaria immitis and Dipetalonema reconditum infections in Missouri dogs was investigated by means of a statewide veterinary mail survey and a study of shelter dogs from central Missouri and Mississippi River communities. The veterinary survey revealed microfilariae of D immitis in 447 of 11,823 (3.8%) dogs, microfilariae of D reconditum in 14 (0.1%), and microfilariae of both species in 8 (less than 0.1%). Of 493 shelter dogs examined, using the modified Knott test, 23 (4.7%) were positive for D immitis, 23 (4.7%) for D reconditum, and 2 (0.4%) for both helminths. The prevalence of infection with both D immitis (9.4%) and D reconditum (6.3%) was higher in shelter dogs from the Mississippi River communities than in those from central Missouri (1.7% for D immitis, 3.7% for D reconditum). The microfilariae of D immitis in shelter dogs ranged in length from 255 to 343 micrometer (mean, 314 micrometer) and in width from 5.0 to 7.5 micrometer (mean, 6.5 micrometer); those of D reconditum ranged in length from 238 to 295 micrometer (mean, 257 micrometer) and in width from 3.2 to 6.5 micrometer (mean, 4.9 micrometer). Buttonhook tails were seen in 41% of D reconditum. In the final analysis, the shape of the anterior end was considered the most reliable criterion for differentiation.

Blood samples from 331 greyhounds in the Hunter Valley and nearby coastal areas of New South Wales were examined for microfilariae using a filtration technique. Species were identified by histochemical staining; 10.9% of the greyhounds were infected with Dirofilaria immitis and 3.6% with Dipetalonema reconditum. The prevalence of infection of both species was significantly greater in summer than in winter (p = less than 0.05). Infection with D. immitis was correlated with differences in age, sex, bodyweight and coat colour, and a reported lack of stamina and the presence of a cough. No significant association was found. Diethylcarbamazine citrate was used for prophylaxis in 8.8% of all the greyhounds examined.

Screening of 25,822 dog blood samples indicated approximately a 1% infection rate with Dirofilaria immitis. Dipetalonema reconditum microfilariae were found in approximately 2% of 1,876 feral dogs examined. Laboratory experimentation indicated that 6 of 10 local mosquito species examined allowed successful extrinsic incubation of D immitis. Indices of experimental infection indicated Aedes triseriatus and Anopheles quadrimaculatus were excellent hosts for D immitis, but other factors considered, Aedes vexans probably served as the primary vector of D immitis. Coquillettidia perturbans and Culex pipiens-restuans group were considered unsuitable hosts and thus unlikely vectors of canine heartworm. Natural filarial infections were found in An quadrimaculatus and Ae vexans.

A house-to-house survey of dogs in a 26-km2 area of Pleasants Valley (Northern California) was done to determine the prevalence of the filariids Dirofilaria immitis Leidy and Dipetalonema reconditum Grassi in the canine population. Blood samples were taken from the cephalic vein of 97 dogs (greater than or equal to 5 months of age) and were tested for the presence of microfilariae, using a modification of the Knott technique. Two dogs with autochthonous D immitis (2.1% of the dogs examined) were discovered. These dogs were outdoor-housed working or sporting dogs, 5 to 8 years old, 1 female and 1 male. Five male dogs, greater than 2 years of age (5.1%), were positive for Dip reconditum microfilariae. Mixed infections were not detected.

In August 1986, 133 dogs at the Veterinary Service of the Netherlands Antilles and the SPCA of Curaçao were examined for microfilaremia and for evidence of gastrointestinal parasitism. Microfilariae of Dipetalonema reconditum were present in 27.8% of the dogs examined with no significant difference in the infection rate between domestic and feral dogs. Microfilariae of the canine heartworm Dirofilaria immitis were found in 9% of the dogs with a significantly higher rate of infection in domestic (pet) dogs (13.5%) than in feral dogs (3.4%). Of the intestinal parasites observed Ancylostoma sp. was present in the highest percentage of dogs (68.4%) followed by Toxocara sp. (7.5%). Other parasites were present in less than 5% of the dogs examined and included, in decreasing order of prevalence, Spirocerca sp., Giardia sp., coccidia, Taenia sp. and Trichuris sp. The present paper presents the first evidence of Di. reconditum on Curaçao and suggests the introduction of D. immitis to the island within the 10 years preceeding this report. The persistently high rate of infection with Ancylostoma underscores the continuing risk of cutaneous larva migrans to human beings in the region.

Ninety dogs naturally infected with Dirofilaria immitis and 103 noninfected dogs, as determined by necropsy, were used to compare the sensitivity and specificity of the cuticular and somatic reactions of the indirect fluorescent antibody test (IFA-C and IFA-S, respectively) and 2 enzyme-linked immunosorbent assays (ELISA). In microfilaremic and amicrofilaremic heartworm-infected dogs, negative results were common for all serotests. In dogs without adult heartworms at necropsy, 32% to 49% were positive, using 1 ELISA, 27% to 29% were positive with the other ELISA, 15% to 36% were positive with the IFA-S, and 0% to 1% were positive using the IFA-C, depending on the classification of borderline reactions. The prevalence of false positive serotests was probably not due to the detection of precardial stages of D immitis in dogs obtained from areas of low endemicity. Until the causes of the false-positive tests are resolved, the use of currently available serotests for routine diagnostic screening or as criteria for instituting treatment is not recommended.