The vectorial role of blood-sucking arthropods, other than mosquitoes, has been explored for Dirofilaria immitis and Dirofilaria repens which are filarioids of increasing interest due to their zoonotic potential. Given the expansion of canine heartworm infection in southern Europe due to D. immitis and the report of the infection in Linosa Island (Sicily, Italy), we investigated and focused on the potential involvement of Culicoides spp. in Dirofilaria spp. transmission as well as the blood-meal source in Dirofilaria spp. molecularly positive biting midges. Out of 1791 specimens collected 1773 were morphologically and molecularly identified as Culicoides paolae and 18 as Culicoides obsoletus complex. Of the 1596 females, 4 (0.26%) parous C. paolae tested positive for D. immitis and D. repens (i.e. 2 Culicoides specimens for each filarial species), of which D. immitis-positive samples fed on both humans and dogs and D. repens-positive samples fed only on humans. The detection of D. immitis and D. repens DNA in Culicoides midges suggests their potential involvement in the epidemiology of the filarial nematodes, which may be of medical and veterinary importance in hyper-endemic areas. Findings of this study shed new light on the epidemiology of Dirofilaria spp. infections indicating that further investigations are needed.

Dirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.

Dirofilariosis caused by Dirofilaria immitis (heartworm) is a zoonosis, considered an endemic disease of dogs and cats in several countries of Western Europe, including Portugal. This study assesses the levels of D. immitis exposure in humans from Northern Portugal, to which end, 668 inhabitants of several districts belonging to two different climate areas (Csa: Bragança, Vila Real and Csb: Aveiro, Braga, Porto, Viseu) were tested for anti-D. immitis and anti-Wolbachia surface proteins (WSP) antibodies. The overall prevalence of seropositivity to both anti-D. immitis and WSP antibodies was 6.1%, which demonstrated the risk of infection with D. immitis in humans living in Northern Portugal. This study, carried out in a Western European country, contributes to the characterisation of the risk of infection with D. immitis among human population in this region of the continent. From a One Health point of view, the results of the current work also support the close relationship between dogs and people as a risk factor for human infection

Prevention of heartworm disease caused by Dirofilaria immitis in domestic dogs and cats relies on a single drug class, the macrocyclic lactones (MLs). Recently, it has been demonstrated that ML-resistant D. immitis are circulating in the Mississippi Delta region of the USA, but the prevalence and impact of these resistant parasites remains unknown. We review published studies that demonstrated resistance in D.immitis, along with our current understanding of its mechanisms. Efforts to develop in vitro tests for resistance have not yet yielded a suitable assay, so testing infected animals for microfilariae that persist in the face of ML treatment may be the best current option. Since the vast majority of D. immitis populations continue to be drug-sensitive, protected dogs are likely to be infected with only a few parasites and experience relatively mild disease. In cats, infection with small numbers of worms can cause severe disease and so the clinical consequences of drug resistance may be more severe. Since melarsomine dihydrochloride, the drug used to remove adult worms, is not an ML, the ML-resistance should have no impact on our ability to treat diseased animals. A large refugium of heartworms that are not exposed to drugs exists in unprotected dogs and in wild canids, which may limit the development and spread of resistance alleles.

Third-stage and fourth-stage Dirofilaria immitis larvae exhibited positive thermotaxis when placed in a thermal gradient. Negative thermotaxis was not observed. Positive thermotaxis may be important for the successful transmission and for directing third and fourth-stage larval migration toward predilection sites in the Host.

Evidence has been presented showing two kinds of acidic protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum. A haemoglobinolytic activity without adding any SH-containing compounds was maximal at pH 3.5, 2.5, 30 and 3.5 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The inhibitor studies demonstrated that this activity is ascribable to carboxyl protease(s). In the presence of dithiothreitol, activity on Azocoll (azo-dye coupled hide powder) was maximal at pH 4.6, 4.6, 3.5 and 5.6 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The effects of inhibitors demonstrated that this activity belongs to the thiol protease class. The intraspecific distribution of the protease activities was studied in some of the nematodes from which the organs could be anatomically separated. The distribution patterns of the haemoglobinolytic and azocollytic activities were quite different in An. cantonensis and much the same in As. suum. Based on the present results, acidic haemoglobinolytic activities reported in adult S. mansoni by other authors are thought to be due to carboxyl and thiol protease(s) respectively.

Dirofilaria immitis is the causal agent of canine and feline cardiopulmonary dirofilariasis. Moreover, the existence of canine dirofilariasis implies a risk for human populations living in an endemic area in which, the parasite can cause pulmonary dirofilariasis. The Spanish epidemiological situation is not well understood, lacking data from many central and Northern provinces. In our study, epidemiological data on canine and human dirofilariasis for La Rioja (Northern Spain) have been obtained for the first time. The overall prevalence of D. immitis in dogs was 12% (9% of patent and 3% of occult infections), being 11·6% the seroprevalence of human residents in this province. The geographic distribution of both canine and human D. immitis infections in La Rioja is restricted to humid and irrigated areas near the Ebro Valley River, being absent in the rest of the province where hills and mountains predominate.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 k Da antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a λ gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consider of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34 400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.

Dogs, naturally infected with Dirofilaria immitis, were treated with the residues of the alcoholic extracts of the rhizomes of Zingiber officinale (ginger). Twelve subcutaneous injections of the extract given at 100 mg/kg reduced microfilarial concentration in blood by a maximum of 98%. Fifty five days after the last injection there was 83% reduction in microfilarial concentration suggesting partial destruction of adult worms. Half of the treated dogs showed some lethargy at the beginning of treatment possibly due to the mass annihilation of microfilariae in blood.

A previously reported cDNA clone encoding 34 kDa antigenic polypeptide of Dirofilaria immitis (λ cD34) was studied to elucidate the mechanism of stage-specific gene expression. The 34 kDa polypeptide was a larva-specific antigen and the mRNA was detectable in microfilariae but not in adult worms and eggs. The λ cD34 gene was not sex linked and was contained in the genome of D. immitis at each stage. The stage-specific expression of the developmentally regulated gene in D. immitis may be controlled primarily at the mRNA level.

The presence of circulating immune complexes (CIC) in the sera of dogs infected with Dirofilaria immitis was detected by using a Clq-binding enzyme-linked immunosorbent assay. Specificity of this assay with different concentrations of heat-aggravated canine IgG (ACG) was observed, i.e., the ELISA readings, expressed as ug equivalents ACG/ml, increased with increasing amounts of ACG. The intra-assay variability was below 10%. The CIC levels of infected and uninfected dogs were 177–0± 104–7 ug/ml and 22–8±45–8 ng/ml (mean±SD), respectively. The highest level was observed in 12 dogs with amicrofilaraemic infection. Age distribution of CIC levels in the 23 infected dogs also showed a significant positive correlation. These findings suggested that the CIC are present in the sera of dogs with dirofilariasis and may relate to canine glomerulonephritis.

The percentage composition and spatial distribution of embryogenic stages in the uteri of female Dirofilaria immitis were examined at various times after treatment with a microfilaricidal dose of ivermectin and compared to nontreated parasites. Worms sampled 42 days post-treatment (PT) exhibited an increased proportion of stretched microfilariae in the distal portion of the uterus. A decreased proportion of developed embryos was noted in the mid body region of worms sampled 42 days PT, and these forms were completely absent from the proximal area of the uterus. Relative numbers and spatial distribution of other stages remained virtually identical to controls. Radical changes in the composition and spatial distribution of embryogenic forms were noted in the uteri of a single worm sampled 80 days PT. Unlike nontreated parasites and worms sampled 42 days PT, stretched crofilariae constituted the predominant form in the distal uterus of this worm, and these stages were found in decreasing numbers throughout the proximal segments. Also, the intermediate embryogenic stages were either rare or absent.

An enzyme-linked immunosorbent assay (ELISA) using antigenic β-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-β-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.

Various catecholamines and catecholamine antagonists have been examined for their effects on the third larval moult of the parasitic nematode, Dirofilaria immitis, cultured in vitro. The non-selective α and β agonist, noradrenaline, and the β agonist, isoprenaline, had no effect on the timing of the third stage moult when used at a concentration of 10−5M. The α-adrenergic antagonist. phentolamine, resulted in worm mortality at 10−5M. At 10−7M, both phentolamine and the β-antagonist, propranolol caused a significant reduction in the numbers of larvae capable of completing the third stage moult. Idazoxan, an a2-antagonist, at 10−5M did not affect worm mortality but did completely prevent ecdysis. The potential of these compounds as possible filaricides is discussed.

Dirofilaria immitis (the dog heartworm) microfilarial periodicity was determined hourly for five days in an infected dog from Kambai village in Muheza district Tanzania. Maximal microfilarial counts were found at 1100 h and minimal at 2200 h. This finding represents the first record of D. immitis microfilarial periodicity in Tanzania.

The effects of the insect hormones, ecdysone and 20-hydroxyecdysone, certain non-steroidal ecdysteroid agonists (RH compounds) and the inhibitor, azadirachtin, on the timing of the 3rd-stage moult of Dirofilaria immitis were investigated. 20-Hydroxyecdysone and RH 5849 when used at a concentration of 10−5 M, resulted in a premature timing of this moult. Azadiracthin, at a similar concentration, prevented moulting of most of the larvae to the 4th stage. The results are discussed in relation to the possibility of a hormonal role for ecdysteroids and neuropeptide-like compounds in the control of ecdysis in filarial nematodes, that maybe somewhat comparable to the system which is found in insects.

A qualitative and quantitative analysis was made of the release of surface-associated molecules from developing Dirofilaria immitis infective-stage larvae (L3). D. immitis L3s were labelled with 125I using an Iodogen catalysed reaction and either maintained in culture or placed in chambers that were implanted into Lewis rats. The larvae released 10–20% of the labelled material each day during the first 4 days of in vitro and in vivo development. The loss of surface-labelled peptides from developing larvae corresponded with an increase in the amount of trichloroacetic acid-precipitable radioactivity found in the culture medium. SDS—PAGE analysis of the labelled material showed that the same 35 and 6 kDa components found in larval extracts were shed into culture medium by the developing parasites. Metabolic labelling studies and experiments in which larvae were labelled after different times in culture indicated that, once released, the surfaceassociated molecules were not replaced, and that this net loss of surface peptides resulted in a reduction in the antigenic potential of the cuticular surface. Antibodies from both immunized rabbits and naturally infected dogs immunoprecipitated the 35 kDa component. In contrast, the 6 kDa molecule was not recognized by the antibodies in any of the sera tested. Shedding of surface peptides and reducing surface antigenicity may represent mechanisms by which D. immitis infective-stage larvae evade immune attack.

Dirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis. Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6·5 months post-infection or later) and therefore fail to detect pre-patent infections. Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvDSB) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively. Previous studies have suggested that a homologue exists in D. immitis (DiT33), which may have potential in diagnosis of heartworm infection. In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described.‡ This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26·4 kDa-protein with a high level of similarity (87–89%) to other filarial members of the family. DJT33 was over-expressed in E. coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera. The findings of this study indicate that DiT33 is a promising antigen for the early detection of D. immitis and may be a valuable tool in the control and management of heartworm infection.

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.

Phase-interference microscopic examination of the infective, post-infective 3rd-stage and 4th-stage larvae of Dirofilaria immitis has identified a single cell body in each of the paired lateral amphidial nerves which undergoes characteristic morphological change during the development from 3rd- to 4th-stage larvae. Acetaldehyde—fuchsin staining of worm sections revealed fuchsinophilic material in the precise location of the amphidial nerve-cell bodies observed by phaseinterference microscopy. This material was found in 70% of infective larvae recovered from mosquitoes and in 100% of larvae recovered from micropore chambers 24 h after implantation into BALB/C mice. In pre-moult larvae recovered at 42 h (Experiment 1) and at 48 h (Experiment 2) fuchsinophilic material was demonstrable, but no staining was observed in those larvae in which separation of the 3rd- and 4th-stage cuticles had occurred. No such material was observed in 4thstage larvae recovered from chambers after 74 h, and in these larvae the amphidial nerve-cell bodies were not discernible. These cytological observations are consistent with a cycle of elaboration and release of neurosecretion associated with moulting in D. immitis.