Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

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.

First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern.

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.

Canine angiostrongylosis in Italy: occurrence of Angiostrongylus vasorum in dogs with compatible clinical pictures.

This study evaluated the perception of veterinarians in including the emerging metastrongyloid Angiostrongylus vasorum in the differential diagnosis for dogs referred with appropriate signs. Veterinarians at teaching hospitals, private practices or kennels collected faecal samples from 606 symptomatic dogs in six different areas of Italy. The samples were examined with the Baermann technique. Fourteen dogs scored positive for A. vasorum, all showing different clinical pictures, with the most common reason for inclusion in the study being a combination of respiratory signs along with general signs. The most reported sign was cough, followed by aspecific distresses. Other respiratory signs were dyspnoea, tachypnoea and breathing sounds, while bleeding diathesis occurred in one dog. Gastrointestinal disorders were diarrhoea and vomitus, while no neurological signs were detected. Four key cases are herein described, with a special focus on their clinical description, findings at the collateral diagnostic imaging and therapeutic options with moxidectin. This survey provides new data on distribution of A. vasorum in Europe and on the clinical impact of the disease. The results confirm that clinical angiostrongylosis overlaps a series of other conditions of dogs. Awareness among vet practitioners is discussed along with the importance of including A. vasorum on the list of differential diagnoses in canine clinical practice and of performing appropriate diagnostic methods.

Nodular lesions due to infestation by Dirofilaria repens in dogs from Italy.

BACKGROUND: Canine subcutaneous dirofilariosis caused by Dirofilaria repens is an important cause of human dirofilariosis in the Old World. OBJECTIVES: To describe the clinical and pathological features of infestation by D. repens in dogs with cutaneous nodules and to discuss key diagnostic procedures. ANIMALS: Sixteen privately owned dogs of different breeds, ages and genders with subcutaneous nodules were positive for D. repens on at least one diagnostic test. METHODS: Diagnostic tests included cytological examination of fine-needle aspiration biopsies, detection of circulating larvae by Knott's test, morphological and molecular identification of adult parasites and microfilariae, and histopathological examination of excised nodules. RESULTS: Twelve animals were positive for D. repens microfilariae, using cytology and at least one other diagnostic test. Four of twelve dogs screened by Knott's test had circulating D. repens microfilariae, and three of them were co-infected with Dirofilaria immitis. In eight dogs, adult nematodes were morphologically identified as D. repens. Nodular panniculitis was observed in all cases, with nodules containing either one or several cross-sections of nematodes and microfilariae. The nematodes extracted were identified as D. repens by an RT-PCR on the second internal transcribed spacer (ITS-2). CONCLUSIONS AND CLINICAL IMPORTANCE: Adults and larvae of D. repens can induce skin nodules in dogs, and these lesions should be carefully evaluated in endemic areas. Clinicians should be aware that correct tissue sampling (including the storage of part of a sample in ethanol instead of formalin) is pivotal to achievement of a specific diagnosis. Cytology of fine-needle tissue aspirates was shown to be useful.

Right Pulmonary Artery Distensibility Index in Heartworm Infected Dogs: Are the Different Methods Leading to Same Results?

Canine Heartworm Disease (HWD) is mainly a pulmonary vascular disease. The reduction of the Pulmonary Artery Distensibility (PAD) is an early index of pulmonary vascular disease. Echocardiographic evaluation of the Right Pulmonary Artery Distensibility index (RPADi) is calculated as the percentage change in diameter of the right pulmonary artery (RPA) between systole and diastole. Historically, two main methods have been used for RPADi calculation: The Venco method and Visser method; however, different hybrid methods have also been used by other authors. Therefore, it could be difficult for a clinician to decide which method to apply and how to interpret the results based on the reference values reported. The aim of this study was to compare the RPADi obtained by five different techniques (Venco classic, Venco modified, Visser classic, Visser modified 1, and Visser modified 2). The study design was a retrospective, single center, observational study. Forty-seven client-owned dogs were included. The measurements were performed off-line as an average of three consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. The RPADi was satisfactorily obtained by all methods in all dogs. Intra-observer measurement variability was clinically acceptable both for systolic and diastolic measurements. Although the Bland-Altman test showed a statistical agreement between the various methods used to calculate the RPADi, these methods cannot be used interchangeably in a clinical setting. Instead, the measurement method and reference values should always be specified.

Transjugular occlusion of large patent ductus arteriosus with an Amplatzer™ muscular ventricular septal defect occluder in a 3-month-old dog.

Background: Cardiologists close most patent ductus arteriosus (PDA) defects in dogs using the Amplatz® canine duct occluder via a transarterial approach. However, this approach can be problematic in small dogs due to the small femoral artery diameter. In such cases, cardiologists have opted to use coils or vascular plugs deployed from a transvenous approach. However, in small dogs with large PDA, the risk of device protrusion into the pulmonary artery, incomplete closure, or device embolization, often leads to surgical PDA closure via thoracotomy. Case Description: The present report describes a 3-month-old male 6 kg Border collie with a large, PDA which was successfully occluded using the Amplatzer™ muscular ventricular septal defect (mVSD) device from a transvenous approach after closure attempts with an Amplatzer™ vascular plug II failed. Conclusion: This is the first case report in veterinary medicine of PDA closure with an Amplatzer™ mVSD occluder device. This approach, described in PDA closure in people, could be considered in minimally invasive PDA closure in small dogs with challenging anatomy.

Linguatula serrata (Pentastomida: Linguatulidae) infection in a paucisymptomatic greyhound imported from Romania to Italy: A case report and literature overview.

Background: Linguatula serrata is a pentastomid zoonotic parasite with worldwide distribution. Although some cases of L. serrata infection have been reported in dogs, the epidemiology of this parasite remains largely unknown in developed countries. In recent years, canine linguatulosis has been repeatedly linked to cases of imported infections. This study aims to focus attention on this uncommon parasite through the presentation of a case report and an overview of the literature. Case Description: A 1-year-old intact female Borzoi imported from Romania to Italy sneezed spontaneously a worm-like parasite specimen. Morphological and molecular diagnosis identified the parasite as a female of the zoonotic pentastomid L. serrata (so-called European tongue worm) that lives in the nasopharyngeal tract of canids. Eggs of Linguatula were detected in the feces. Molecular identification (99%-100% homology) was based on DNA extraction, polymerase chain reaction of a 700-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene, and alignment with BLAST analysis. Due to the possibility of other adult or juvenile specimens of the parasite still occurring in the dog, a treatment attempt with a combination of febantel/pyrantel/praziquantel was made. No parasite eggs were detected in fecal samples after the drug was administered. Endoscopy confirmed the absence of adult parasites and slight pathological changes. A follow-up examination conducted 3 months after the treatment did not reveal any clinical and laboratory abnormalities. Conclusion: Linguatula serrata appears to be currently prevalent in some European countries, but there are no recent extensive studies on the prevalence of canine linguatulosis, so the parasite frequently remains undetected and unreported in dogs as the diagnosis is often overlooked. Parasites not commonly found such as L. serrata can become increasingly prevalent and may be detected in imported dogs. Therefore, veterinarians must be aware of the possible presence of uncommon and exotic pathogens in these dogs, be able to recognize the relevant clinical signs, and diagnose the infection quickly. This will improve the prognosis in individual dogs, reduces the risk of possible public health implications, and reduces the risk of uncommon and exotic pathogens establishing new endemic foci.

How Italian practitioners manage dirofilariosis in dogs and cats? Data of a second national survey.

The zoonotic nematodes Dirofilaria immitis and Dirofilaria repens are endemic in many European countries and their distribution is changing. A correct diagnosis is the starting point to reduce their spread and veterinary practitioners represent the first line of defense against these two vector-borne diseases in pets and humans. With the aim to better understand current knowledge by veterinarians and to compare them with the previous survey, an online questionnaire was sent to Italian companion animal veterinary facilities. The overall response rate was 22.3%. Approximately 50% and 5% of facilities have diagnosed at least one case of Dirofilaria spp. in the last year in dogs and cats, respectively. In agreement with international guidelines, of the 665 facilities that responded to the questionnaire, 16.8% used Knott's test in combination with serology (Antigenic test-Ag-) to diagnose heartworm infection in dogs. For the diagnosis of D. repens infection, 13.8% used Knott's test in dogs and cats, and 10.5% used Knott's test only in dogs. Most facilities recommended beginning prevention of canine heartworm disease in April and ending in November. Injectable moxidectin and oral ivermectin were the two most commonly used preventives for D. immitis in dogs, while selamectin was most frequently used in cats. The drug most commonly used for heartworm adulticide treatment in dogs was melarsomine followed by the combined ivermectin-doxycycline treatment. The results of this study suggest that, on the one hand, veterinarians are aware of the recent developments in adulticide therapy, while on the other hand, diagnosis is not always made according to what is recommended by international guidelines and that the start and end times of prevention may not be optimal. Furthermore, the data show an increase in cases of coinfection compared to the previous survey.

Detection of a characteristic beta-2 peak in serum by capillary zone electrophoresis in dogs with Angiostrongylus vasorum.

BACKGROUND: Angiostrongylosis is an emerging canine parasitic disease that often causes polyclonal hyperglobulinemia. In the authors' experience, we have seen what could be a typical serum protein electrophoretic pattern characterized by a large, symmetrical beta-2 peak in dogs with angiostrongylosis. OBJECTIVES: The purpose of this study was to evaluate the frequency of this pattern in dogs infected with Angiostrongylus vasorum compared with a randomly selected canine population. METHODS: Serum protein capillary zone electrophoreses (CZEs) from dogs with angiostrongylosis were assigned to Group 1. The CZE pattern was classified as normal, nonspecific polyclonal, or polyclonal with a typical beta-2 peak. The frequency of this latter pattern in Group 1 was compared with the frequency of similar findings in a randomly selected canine sera group (Group 2). RESULTS: Groups 1 and 2 included 22 and 3687 cases, respectively. Thirteen of 22 (59.1%) dogs in Group 1 had this peak in the beta-2 region. A similar CZE pattern was observed in 43 dogs in Group 2 (1.2%). The remaining dogs in Group 1 had a nonspecific polyclonal hyperglobulinemia (six cases), a normal tracing (one case), or an ambiguous tracing (two cases). CONCLUSIONS: Dogs with angiostrongylosis commonly have polyclonal hyperglobulinemia, frequently characterized by a large and symmetrical beta-2 peak on CZE, which is uncommon in the canine population. Additional studies are needed to identify the protein composition of this peculiar beta fraction.

Abnormalities of External Jugular Veins in Bulldogs with Pulmonary Valve Stenosis.

Two French bulldogs and one English bulldog affected by pulmonary valve stenosis and referred for pulmonary balloon valvuloplasty were diagnosed with different abnormalities of the external jugular veins. The diagnosis included unilateral absence of the right external jugular vein and bilateral hypoplasia of the external jugular veins, associated with persistent left cranial vena cava. Vascular ultrasound and computed tomography were used for the diagnosis. Jugular vein anomalies can affect decision-making regarding the central venous catheterization needed to perform procedures such as pulmonary balloon valvuloplasty or central catheter placement. Based on the results of the present case series, anomalies of external jugular veins should be considered in French and English bulldogs when the external jugular veins cannot be palpated or when echocardiography shows coronary sinus dilation. Vascular ultrasound or computed tomography may help identify jugular venous anomalies and should be considered in the preoperative evaluation of bulldogs that need to undergo interventional procedures requiring transjugular catheterization.

Transjugular Patent Ductus Arteriosus Occlusion in Seven Dogs Using the Amplatzer Vascular Plug II.

Although vascular plugs for the closure of patent ductus arteriosus (PDA) have been validated in dogs, studies are lacking on its use as a first-choice device with a transjugular approach. The present case series describes the transvenous right jugular embolization of PDA using an Amplatzer Vascular Plug II in seven dogs of different ages, breeds, and body weights. Complete closure of the PDA was demonstrated in all cases. All dogs showed significant hemodynamic reduction of pulmonary overcirculation and left heart size after the procedure and at following echocardiographic check-ups. Transjugular PDA occlusion using an Amplatzer Vascular Plug II can thus be considered as a safe alternative to the arterial or venous femoral approach using an Amplatzer canine ductal occluder (ACDO), particularly in puppies with small femoral vessels.

Monitoring and detection of new endemic foci of canine leishmaniosis in northern continental Italy: An update from a study involving five regions (2018-2019).

Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission.

Successful treatment of pleural and peritoneal larval mesocestoidosis with fenbendazole in two dogs.

Mesocestoides spp. are common intestinal tapeworms of dogs and cats. The presence of numerous larval stages in the pleural or peritoneal cavities causes larval mesocestoidosis, that is a potentially life-threatening disease in massive infection. In vivo diagnosis of larval mesocestoidosis is challenging and confirmed diagnosis is often made post-mortem. For this reason, therapeutic recommendations are empirical and there is currently no data on the best treatment options in dogs and cats. Both fenbendazole and praziquantel have been used to treat canine larval mesocestoidosis but failure of complete clearance of larvae has been described for both. Treatment with fenbendazole at 100 mg/kg orally twice a day, for 1-3 months has been reported as being effective. However, it is known that administration of high, off-label doses of fenbendazole can lead to adverse reactions. Here, we report the efficacy of fenbendazole at a lower dosage (50 mg/kg twice a day) in two dogs with pleural and peritoneal mesocestoidosis.

Dosage variability of veterinary drug products, containing furosemide, linked to tablet splitting.

Background: Furosemide is a potent diuretic drug widely used to treat congestive heart failure in dogs and cats, but it shows remarkable variability in bioavailability and efficacy when administered orally. In particular, a different diuretic effect can be detected after repeated administrations of the same medicinal product in the same animal. For this reason, we investigate the possible reasons for this peculiar behavior. Drug products for veterinary andhuman use are compared in terms of variability for tablet splitting, in vitro dissolution profiles (in different fluids that could simulate the gastrointestinal environment of pets), and drug distribution uniformity. Aim: To study the in vitro performances of drug products in terms of variability. Methods: Five veterinary products and five products for human use, containing different furosemide doses, are characterized. Tablets splitting uniformity, in vitro dissolution profiles in different fluids that could simulate the gastrointestinal environment of the different species, and drug content distribution, were tested. Results: The in vitro dissolution profiles of the different medicines are comparable but confirm a different dissolution rate as a function of the medium pH and volume. Many of the products considered show wide variability in the division performances of the scored tablets, and this problem could lead to the detected fluctuations in the diuretic effect. The four-leaf clover shape of a veterinary product appears to give rise to more uniform fractions. A uniform distribution of the drug in the tablets and their fractions is confirmed for all the products considered. Conclusion: The possibility of tablets splitting allows considerable dosage flexibility, but a non-uniform break of the tablets to obtain the dosage suitable for the pet's weight, can cause dangerous over-or sub-dosing condition, especially in critical pathologies and in small breed pets.

First Autochthonous Infection of a Cat with Dirofilaria immitis in Austria.

This case report is about a seven-year-old male neutered European Shorthair cat infected by Dirofilaria immitis as the first reported autochthonous Dirofilaria immitis infection in Austria. There was no history of periods abroad. Echocardiography showed suspected D. immitis in the right cardiac chamber with increased pulmonary pressure and ascites. Surgical removal of the heartworms was performed. Twenty adult heartworms were removed by transvenous jugular approach under general anesthesia and stored in 4% formalin. Five out of 20 specimens were examined via light and stereomicroscopy and feline heartworm infection was confirmed. Amplification of a 203 bp or 724 bp fragment of the cytochrome c oxidase subunit I gene was unsuccessful. After surgery the cat developed acute renal failure but recovered quickly. One year later, the cat underwent a control examination including echocardiography and blood work. There were no more D. immitis detectable at echocardiography. Lung pressure was mildly increased. Complete blood count and creatinine were unremarkable. The Knott's test and Dirofilaria-Antigen-test produced negative results. The cat did not show any clinical signs during the follow-up period. The aim of this case report is to highlight the growing risk of acquiring infection with D. immitis not only for Austrian dogs, but also for cats. This case report represents the first report of autochthonous D. immitis infection in Austria. Moreover, even if the prognosis in cats with caval syndrome due to feline heartworm disease is guarded to poor, surgical removal of the filariae can be a successful treatment option.

False positive antigen test for Dirofilaria immitis after heat treatment of the blood sample in a microfilaremic dog infected with Acanthocheilonema dracunculoides.

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.

Optimized protocol for DNA/RNA co-extraction from adults of Dirofilaria immitis.

Dirofilaria immitis, the etiologic agent of canine heartworm disease, like several other filarial nematodes, harbors the bacterial endosymbiont Wolbachia. To investigate metabolic and functional pathways of D. immitis and Wolbachia individually, along with their interactions, the use of both transcriptomic and genome analysis has becoming increasingly popular. Although several commercial kits are available for the single extraction of either DNA or RNA, no specific protocol has been described for simultaneous extraction of DNA and RNA from such a large organism like an adult D. immitis, where female worms generally reach ∼25 cm in length. More importantly, adult worms of D. immitis can only be obtained either through necropsy of experimentally infected dogs or by minimally-invasive surgical heartworm removal of naturally infected dogs. This makes each individual worm sample extremely important. Thus, in the context of a project aimed at the evaluation of both gene expression analysis and Wolbachia population assessment following different treatments, an optimized protocol for co-extraction of DNA and RNA from a single sample of adult D. immitis has been developed. •An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol® reagent.•Allows maximum exploitation of unique samples as adults of D. immitis.

Dirofilaria immitis and D. repens in dog and cat: A questionnaire study in Italy.

Dirofilaria immitis and D. repens are vector-borne parasites of dogs and cats, with zoonotic potential, endemic in many parts of Europe, including Italy. Control and prevention of dirofilariosis are essential points to reduce their diffusion in animals and humans and veterinarians are the main subjects involved in this "battle". With the aim to better understand current practice by veterinarians, an online questionnaire on different aspects of D. immitis and D. repens was sent to companion animal veterinary facilities registered with the Italian Veterinary chamber. The overall response rate was 25%. Approximately 47% had diagnosed at least one case of Dirofilaria spp. in dogs in the last year and approximately 6% had diagnosed at least one case of Dirofilaria spp. in cats. Of the 662 facilities surveyed, 83.8% used serology to diagnose heartworm infection. For the diagnosis of D. repens infection in dogs and cats, a high percentage of facilities relied on an external laboratory. Most recommended beginning prevention of canine heartworm disease in April-May. Topical and injectable moxidectin and oral ivermectin were the two most commonly used preventives for D. immitis in dogs. The drug most commonly used for heartworm adulticide treatment in dogs was melarsomine. This study show that veterinary facilities ians working in an endemic area for D. immitis and D. repens are aware of recent developments in adulticide therapy. Results would suggest however that diagnosis is not always carried out according to what is currently recommended by international guidelines and that the timing for starting prevention may not be optimal.

Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both.

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.

Highly modified and immunoactive N-glycans of the canine heartworm.

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF-MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.