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Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease–free area.

Abstract Background Dirofilaria immitis is responsible for heartworm disease in dogs in endemic areas worldwide. Screening for this infection is done by blood tests. Antigen testing is the most sensitive method to detect an infection with adult (female) worms. Microscopic examination of a blood smear or Knott’s test can be used to detect circulating microfilariae, the infective larvae. To increase the sensitivity of the antigen test by decreasing the false negative test results, heating of the blood sample has been recommended in recent guidelines. Heating is believed to remove blocking immune-complexes. Circulating microfilariae are not specific findings for heartworm infection, as other nematodes (among others, Acanthocheilonema dracunculoides) can also result in microfilaremia. Although the type of microfilariae cannot be determined by microscopy alone, real-time PCR can reliably identify the infecting nematode species. Correct identification of the parasite is of major importance, as an infection with D. immitis requires antiparasitic therapy, whereas A. dracunculoides is thought to be a clinically irrelevant coincidental finding. The present case report describes a microfilaremic dog where the initial antigen test for D. immitis turned positive after heat treatment, whereas real-time PCR revealed that the microfilariae were A. dracunculoides (syn. Dipetalonema dracunculoides). Results A circa 5-year old, asymptomatic Spanish mastiff dog was referred for heartworm therapy because microfilariae were found via a screening blood test. The dog was recently imported to the Netherlands from Spain, where it had been a stray dog. Antigen tests on a plasma sample for D. immitis were performed with three different test kits, which all turned out to be negative. However, heat treatment of two of these samples were carried out and both of them led to a positive antigen test result. Real-time PCR showed that the circulating microfilariae belonged to A. dracunculoides species. Three administrations of moxidectin spot-on at monthly intervals resulted in a negative antigen and a negative Knott’s tests one month after the last treatment. Conclusions We conclude that heat treatment of initially negative blood samples for D. immitis could lead to false positive antigen test results if the dog is infected with A. dracunculoides.

Abstract Background Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis. Results Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females. Conclusions Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors’ knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.

Abstract Background Heartworm antigen testing is considered sensitive and specific. Currently available tests are reported as detecting a glycoprotein found predominantly in the reproductive tract of the female worm and can reach specificity close to 100%. Main concerns regard sensitivity in the case of light infections, the presence of immature females or cases of all-male infections. Research and development have been aimed at increasing sensitivity. Recently, heat treatment of serum prior to antigen testing has been shown to result in an increase in positive antigen test results, presumably due to disruption of natural antigen–antibody complexes. Cross-reactions in dogs with both natural and experimental infections with Angiostrongylus vasorum and Spirocerca lupi have been reported, but cross-reactions with other helminths have not been extensively studied. In order to evaluate potential cross-reactivity with other canine and feline parasites, two studies were performed. Study 1: Live adults of Dirofilaria immitis, Dirofilaria repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis and Mesocestoides spp. larvae were washed and incubated in tubes with saline solution. All worms were alive at the time of removal from the saline. Saline solutions containing excretory/secretory antigens were then tested for heartworm with six different, commercially available antigen tests. All results were evaluated blind by three of the authors. Study 2: Sera from dogs with natural infections by A. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same tests before and after heat treatment (103 °C for 10 min). Results Results suggest that antigens detected by currently available tests are not specific for D. immitis. They may give positive results through detection of different parasites’ antigens that are normally not released into the bloodstream or released in a low amount and/or bound to antibodies. Tests may even detect antigens released by male D. immitis adult worms. D. repens appears to release more detectable antigens than the other worms studied. Conclusions Cross-reaction with A. vasorum and D. repens does occur in the field and could potentially occur with other helminths. Heat treatment decreases specificity by enhancing cross-reactivity.

Abstract Background The parasitic nematode Angiostrongylus vasorum causes severe clinical signs in dogs. The disease is often challenging because infected animals are often presented with clinical signs overlapping those of other diseases. Methods The present article describes six angiostrongylosis cases (Cases 1-6) that represent key examples of how canine angiostrongylosis may be extremely confounding. The six animals presented clinical signs compatible with canine angiostrongylosis but they were subjected to clinical examinations for other diseases (e.g. dirofilariosis or immune-mediated disorders) before achieving a correct diagnosis. Results In Case 1 clinical, radiographic and ultrasound examinations' results resembled a lung neoplasia. Case 2 was a dog with a mixed infection caused by A. vasorum and Dirofilaria immitis. Case 3 was a critically ill dog presented in emergency for an acute onset of dyspnoea caused by lungworm infection. The dog died a few hours after presentation despite support and etiologic therapy. Case 4 was a dog presented for chronic hemorrhages and ecchymoses caused by thrombocytopenia of unknown origin, thought to have an inherited, immune-mediated or infective cause. Case 5 was referred for neurological signs due to a suspected discospondylitis. Case 6 was erroneously diagnosed infected only with D. immitis although the dog was infected only with A. vasorum. A timely administration of an anthelmintic (mostly moxidectin) showed to be effective in treating the infection in those dogs (i.e. Cases 1,2, 4 and 5) that did not suffer with severe lung haemorrhages yet. Conclusions Dogs 1-5 were referred in two regions of Italy that are considered non-endemic for A. vasorum. These findings indicate that veterinarians should include angiostrongylosis in the differential diagnosis of cardio-respiratory distress also in non-endemic regions and should perform appropriate diagnostics in the presence of compatible signs even if the clinical picture is atypical.

A female Rottweiler dog was presented with a history of intermittent vomiting and diarrhoea, dysorexia, weakness, and weight loss. Haemocytometry and biochemistry values were within normal ranges except for electrolyte analyses, that demonstrated hyponatremia and hyperkalemia with a decreased sodium/potassium ratio. A diagnosis of hypoadrenocorticism was suspected. Basal and post-ACTH stimulation cortisolemia were within the normal values. Electrocardiography was normal, and thoracic radiography showed no significant modifications. On abdominal ultrasonography, adrenal glands appeared normal, while the bowel was distended, and several thin linear hyperechoic objects floating in the lumen were observed. Two adult female whipworms (Trichuris vulpis) were collected following bowel irrigation. Anthelmintic treatment against the parasite was curative.