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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.
On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric.
Eight stocks of Aedes aegypti-4 geographic stocks and 4 selected stocks-were tested for their susceptibility to infection with Dirofilaria immitis and D. repens. Four of the stocks were found to be susceptible and the other 4 were refractory of infection with D. immitis. All the stocks of Ae. aegypti that were susceptible to D. immitis were also susceptible to D. repens but the stocks that were refractory to D. immitis were all partially susceptible to D. repens.
The differential epidemiology of D. repens and D. immitis is still poorly understood due to the lack of a diagnostic method which would make possible the routine identification of these parasites as developing larvae either in the vector or in unsuitable hosts, including man. The PCR-based method here described allows: i) the unambiguous identification of mature and immature adult worms in bioptic material, of microfilariae in blood samples and of developing larvae in mosquito vectors; ii) the analysis of samples stored either dry or in various preservation media, with the exception of formalin. The high specificity and sensitivity of the diagnosis improve the perspectives for comparative epidemiological investigations on D. repens and D. immitis in areas where the two nematodes are sympatric.
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Cuticular markings of five species of Dirofilaria were examined by SEM. Variations in the patterns on the en face view were observed among the species. Transverse striations were observed on the cuticles of all five species, but longitudinal ridges were lacking on Dirofilaria immitis, the type species of the subgenus Dirofilaria. They were present on the other four species, Dirofilaria repens, Dirofilaria tenuis, Dirofilaria corynodes, and Dirofilaria magnilarvatum, members of the subgenus Nochtiella. Longitudinal ridges and other cuticular structures and patterns were seen to vary among the species and with the portion and aspect of the worm examined. It is suggested that SEM would be a good tool for a detailed study of specific cuticular morphology of all species of Dirofilaria, thus contributing toward their differentiation and recognition.
The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.
Two new species of Diorfilaria, Dirofilaria macrodemos and Dirofilaria panamensis, are described from the subserosa of Central and South American sloths. Dirofilaria macrodemos, described from the three-toed sloth, Bradypus tridactylus, in Guyana, is characterized and differentiated from other dirofilarias by its length (female 214mm, male 64mm), relatively long tail (female 140 micron, male 110 micron), and the number and distribution of the caudal papillae (7 pairs) in the male. Dirofilaria panamensis, parasite of the two-toed sloth, Cholopeus hoffmanni, in Panama, was previously described as Dirofilaria incrassata by Caballero (1947), but can be distinguished from both D. macrodemos and D. incrassata on the basis of body size (female 66 mm long by 360 micron wide, male 34 mm long by 250 micron wide), tail length (female 80 micron, male 80 micron), and number (6 pairs) of caudal papillae in the male.
Human dirofilariasis caused by Dirofilaria repens D. repens is a common zoonosis in the Mediterranean countries and parts of South Asia. During the last decade, it has been reported from countries previously considered non-endemic. This is likely due to the increased awareness regarding Dirofilaria infection. In some such cases however, dirofilariasis correlated with the travel of the patient to the endemic areas. We present the case of a Saudi male who had D. repens infection in the subcutaneous tissues of the forearm. The patient had traveled to Iraq, Syria, United Arab Emirates and Kuwait in the last 2 years. However, the known Dirofilaria endemic countries were not visited. Any prior occurrence of the human dirofilariasis in the Kingdom of Saudi Arabia is not documented. Lack of epidemiological studies or incidence reports in the Arabian region precludes any factual evaluation of Dirofilaria prevalence, which requires increased awareness amongst health workers regarding its potential occurrence in the native population.
Dirofilaria cancrivori sp. n. is described from subcutaneous tissues of the crabdog, Procyon cancrivorus, in Guyana, South America. The filarid is morphologically distinct from Dirofilaria tenuis, parasite of a related host, the raccoon, in the southern United States, and all other species of Dirofilaria. The parasite can be distinguished from other dirofilarias based on a combination of morphological features including its size, number, and arrangement of caudal papillae on the male tail, size and shape of the spicules, the presence of longitudinal ridges and transverse striations on the cuticle, and the microfilaria.
A Dirofilaria immitis protein allergen was purified; it had a molecular weight of 15,000-20,000 and a carbohydrate content of 2%. The allergenic activity of adult Dirofilaria extracts was assayed by passive cutaneous anaphylaxis (PCA) in rats using mouse sera obtained by immunization with various fractions. The mouse-Dirofilaria system was used to study the degree of purification of allergen. The purified Dirofilaria allergen appeared as one band after sodium dodecyl sulphate polyacrylamide gel (SDS-gel) electrophoresis and one precipitin arc by immunoelectrophoresis (IEP). It was inclined to aggregate in buffered solution. The results suggested that the allergen--reagin axis was a simple single antigen-antibody interaction. Immunological responses to the purified allergen were compared among four inbred strains, two hybrid strains and two outbred strains of mice. They produced relatively high titres of reaginic antibody but did not produce detectable indirect haemagglutinating test (IHA) antibody. Among the strains tested, BALB/c was a high responder and also contained to produce the reaginic antibody for longer than the other strains.
In Europe, zoonotic filarial infections in humans are caused by two species, Dirofilaria immitis and Dirofilaria repens. These parasites are associated mainly with embolic infarcts of the pulmonary artery and subcutaneous nodules, respectively. An unusual dirofilarial infection in a Greek patient who showed marked eosinophilia and microfilaremia is presented. Although the identification of Dirofilaria species is not conclusive, this report is the first on a case of microfilaremia from a Dirofilaria infection in an immunocompetent patient.
The mosquito-borne filarial worm, Dirofilaria immitis, causes heartworm disease in dogs. Detection of this parasite in its mosquito intermediate host currently involves dissection and microscopic examination for larval stages. Although this method is used commonly as a screening tool for epidemiological surveys, it lacks both sensitivity and specificity. In this study, a more efficient PCR- and probe-based diagnostic assay was developed. The target selected for this assay is a segment of the 16 S rRNA gene. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, equivalent to DNA derived from one third stage larva (L(3)), but does not detect 100 ng (10 000-fold excess) of the purified DNA from several other filarial nematodes, including Dirofilaria striata, Dirofilaria tenuis, Dipetalonema reconditum, Wuchereria bancroftii, Brugia pahangi, B. malayi, Onchocerca volvulus or Loa loa. This assay also detects one L(3)of D. immitis, the minimal biological unit of infection, in a pool of 200 mosquito heads. This assay can serve as a highly specific and sensitive tool for efficiently screening the large numbers of mosquitoes to determine, with statistical validity the seasonal transmission pattern of D. immitis in a locality prior to designing a rational preventive medication program for that parasite.
Dirofilaria immitis, the etiological agent for heartworm disease, is a zoonotic, vector-borne parasite. The dog is the most common natural host. Heartworm disease is present in all US states, as well as in South and Central America, Asia, Europe, Africa and Australia [. Prevalence of feline heartworm disease - a global review. In: Proceedings of the Heartworm Symposium'95, Auburn, Alabama, USA, 31 March-2 April, 1995. American Heartworm Society, Batavia, USA, pp. 79-86]. According to Menda [Menda, J.A., 1989. Transplacental migration of Dirofilaria immitus microfilariae. Companion Anim. Pract. 19 (6-7), 18-20], 18% of the canine population in the city of Santo Domingo tested positive for heartworm using the Filar Assay Kit. In the year 2000, clinical veterinarians from the Samana Peninsula in the Dominican Republic stated that the incidence of heartworm was minimal (Goodman, personal communication). In this small epidemiological study performed during the summer of 2001, 104 dogs from the Samana Peninsula were tested for heartworm antigen with the WITNESS HW Test and a wet mount evaluation for microfilaria under light microscopy. It was found that 18.2% of the canine population tested positive according to either one or both of the diagnostic methods. It would be advisable to apply prevention measures to control Dirofilaria immitis infection in the canine population of the Samana Peninsula in the Dominican Republic.
Canine heartworm or Dirofilaria immitis can occasionally infect man. We present the case of a 36-year-old Balkan woman referred to us for a thoracoscopic biopsy of a well defined pulmonary mass. The latter was thoracoscopically resected and proved to be due to Dirofilaria immitis on histopathological examination. To our knowledge this is the second reported case in Western Australia, the first being that reported by Brine et al. The prevalence of the disease in Australia was recognized in 1969 in one study in the state of Queensland, where 12% of 761 dogs at one veterinary clinic and 20% of 296 dogs at another were shown to be infected. Dirofilaria immitis resides in the right ventricle and pulmonary arteries of dogs. The microfilariae are released into the blood stream and are transmitted to secondary hosts by mosquitoes.
Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.