The adult stages of Dirofilaria immitis, a filarial nematode, and Angiostrongylus vasorum, a metastrongylid nematode, are both localized in the Arteria pulmonalis and the right heart of their definitive hosts. Dogs, foxes and some other carnivores are the definitive hosts of both parasites, while Culicidae and Gastropoda are the intermediate hosts of D. immitis and A. vasorum, respectively.
In Europe, D. immitis is present in coastal Mediterranean areas with expansion northwards, while in North America the parasite has expanded from the south-eastern coastal areas northwards and westwards up to Canada. A. vasorum was diagnosed for the first time in France in 1913, but it is only recently that this parasite has regained attention within the veterinary community[4, 5]. Its presence has been increasingly reported from several new areas in and outside Europe (reviewed in). Reports of an increasing number of cases of canine angiostrongylosis, as well as the development of new diagnostic tools such as ELISAs[7–10] or biomolecular techniques[11, 12] may have contributed and also incentivised epidemiological studies, confirming the presence of this parasite in dogs, foxes and snails throughout Europe. The Atlantic provinces of Newfoundland and Labrador are the only regions actually affected by A. vasorum in North America, with a potential for expansion to further regions[4, 13, 14]. Overlapping areas in large parts of southern Europe with the presence of both A. vasorum and D. immitis have therefore to be accounted for. Furthermore, in non-endemic areas of D. immitis, this agent has to be considered based on anamnestic information (travelling with pet dogs or imports) and differentiated from A. vasorum infections.
Fatal clinical consequences of D. immitis infection are usually prevented by the monthly use of macrocyclic lactones in known endemic areas[15–17], and treatment of dirofilariosis is based on the intramuscular application of the arsenic derivate melarsomine and/or, alternatively, by eliminating the endosymbiont Wolbachia with doxycycline supported by administration of macrocyclic lactones. Dogs infected with A. vasorum, instead, are treated using macrocyclic lactones such as moxidectin or milbemycin-oxime, or applying fenbendazole. Prophylactic treatment (with macrocyclic lactones) against potentially fatal canine angiostrongylosis is, as for dirofilariosis, recommended in highly endemic and well known areas.
The currently used diagnostic laboratory methods for the detection of these parasites are divergent. The diagnosis of D. immitis is based on the detection of microfilariae or circulating antigens released by mature adult female worms into the blood circulation, both being detectable starting from 6 months after infection. A variety of tests have been developed for the detection of circulating antigens, employing lateral flow immunochromatographic techniques, membrane ELISAs or conventional ELISAs[22–24]. Test evaluations showed that the sensitivity of heartworm antigen tests depends on the worm burden, and the sex and age of the parasites[24–28], while the specificity of the kits is regularly indicated to be very high, between 95% and 100%[23, 25, 26, 29, 30]. However, only occasionally were potential cross-reactions evaluated in animals with natural or experimental infections with other helminths, mainly against other filarial nematodes such as Dipetalonema reconditum[22, 31] or Dirofilaria repens, indicating that modern test kits may overcome cross-reactions detected in previously developed test kits for these parasites[32, 33], and, rarely, against intestinal parasites such as Ancylostoma caninum and Trichuris spp.[22, 34]. The most current diagnostic method for detection of A. vasorum infections in dogs is the isolation of first stage larvae (L1) from faecal samples, which are produced by the parasites approximately 6–7 weeks after infection. Larval migration techniques such as the Baermann-Wetzel method are commonly adopted. Furthermore, ELISAs for the detection of antibodies against A. vasorum have been described[7, 9], and, recently, tests for the detection of circulating antigen of A. vasorum have been developed. These latter ones have been evaluated for cross-reactions against Crenosoma vulpis[8, 10] and also against intestinal parasites (Toxocara canis, Ancylostoma caninum) and, importantly, against D. immitis, showing a high specificity (94-100%).
Due to their common localization within the definitive hosts, their considerable size and particularly the well documented production of circulating antigens[31, 36], it was argued that antigens of A. vasorum and D. immitis may share epitopes responsible for potential cross-reactions in antigen detection tests. This hypothesis has been confirmed during the development of the ELISA for the detection of circulating antigen for A. vasorum.
The aim of the study was to evaluate potential cross-reactions of sera from dogs experimentally infected with A. vasorum in six different commercially available tests for the detection of D. immitis antigen.