Blog

Nov 30, 2023

Efficacy of oral, topical and extended

Parasites & Vectors

Parasites & Vectors volume 16, Article number: 54 (2023) Cite this article

1694 Accesses

1 Altmetric

Metrics details

Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones and doxycycline against Dirofilaria immitis, showing that these protocols can be used as an alternative to melarsomine. The present study evaluated the efficacy of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy.

Thirty dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or extended release moxidectin injectable (G3) at enrolment and again at 6 months (Day 180). All treatment groups received doxycycline for the first 30 days. Microfilarial concentrations in 1 ml (mff/ml) blood were determined monthly for 9 months with the modified Knott's test. A clinical scoring system was employed for each dog enrolled in the study based on thoracic radiography and cardiac ultrasound (CU) examinations performed at Day − 15 (before treatment) and at Day 180.

Results from the present study suggest that the majority of dogs from all treatment groups became antigen negative, as evaluated at Day 270: 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3. Improvement of radiographic alterations was observed in all treatment groups, and almost all dogs were cleared of pulmonary abnormalities by 6 months from the beginning of treatment (P = 0.000). Cardiac ultrasound examination showed a progressive improvement of cardiac function in a limited number of animals (4/30).

The combination of doxycycline and three different formulations of moxidectin leads to antigen-negative status in naturally infected dogs.

Canine heartworm disease (HWD) is caused by the filarial nematode Dirofilaria immitis, a vector-borne parasite transmitted by several mosquito species, and is endemic in many parts of the world [1]. The presence of adult worms in the pulmonary arteries of infected dogs causes changes in arterial structure and function that can lead to pulmonary hypertension and, eventually, to right-sided congestive heart failure [2, 3]. Melarsomine dihydrochloride is the only approved adulticidal drug for treatment of HWD. Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones (ML) and doxycycline against D. immitis, showing that these protocols are safe and effective [4,5,6,7,8,9,10,11]. Doxycycline targets the bacterial endosymbiont Wolbachia, whose reduction contributes to worm infertility and death [12, 13]. This activity, combined with the known detrimental effects of ML, may eliminate adult worms and lessens the inflammatory reaction against dead and dying worms [14]. The American Heartworm Society [15] and the European Society of Dirofilariosis and Angiostrongylosis [16] currently suggest that in cases where treatment with melarsomine is not possible or is contraindicated, a monthly treatment based on ML along with doxycycline for a 4-week period might be considered [11, 15, 16].

It has been shown that the combination of moxidectin/doxycycline has superior adulticide efficacy compared to ivermectin/doxycycline [17]. Grandi et al. [5] reported 73% efficacy 10 months after the beginning of doxycycline (daily for 1 month) combined with oral ivermectin every 15 days for 6 months. More recently, Savadelis et al. [8] reported a 95.9% adulticide efficacy in experimentally infected dogs after 1 month of doxycycline combined with 10 monthly treatments with a topical formulation of moxidectin. Genchi et al. [9] reported that 15/16 naturally infected dogs became antigen negative at 9 months with the same protocol. Paterson et al. [10] reported 93.0% of dogs treated with the same protocol were antigen-free at month 15. There is only one report on the efficacy of extended-release injectable moxidectin combined with doxycycline in naturally infected dogs, in which 90.0% of the dogs were antigen negative within 1 year of treatment with 1 month of doxycycline and administration of two doses of injectable moxidectin at 6 and 12 months [18]. To the authors' knowledge, there are no published data regarding the evaluation of oral moxidectin combined with doxycycline.

The aim of the present study is to evaluate the effect on antigen status of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy.

The study started in July 2020 and ended September 2021 (Fig. 1). The protocol of this study was approved by the ethics committee of animal experiments of the Department of Veterinary Medicine and Animal Production, University of Federico II Naples, Italy (approval number 0093204/2022). Shelter owner's consent was obtained prior to the start of the study.

Study design. DOXY = 10 mg/kg SID (Ronaxan®, Boehringer Ingelheim Animal Health, Italy); MOX ORAL = oral moxidectin (3 µg/kg SID; Afilaria®, Fatro-Italy); MOX SPOT-ON = topical 10% imidacloprid + 2.5% moxidectin (Advocate®, Elanco, Italy); MOX INJ = extended release moxidectin injectable (Afiliara SR®, Fatro, Italy)

Thirty dogs with naturally acquired D. immitis infection were enrolled from a municipal shelter located in southern Italy (Castel Volturno, Campania region; geographical coordinates 41°0′57″24 N and 13°56′49″56 E). Inclusion criteria were dogs of any breed/sex, weighing at least 1 kg body weight, being > 6 months of age and not having been treated within 2–3 months prior with any macrocyclic lactone or doxycycline.

Dogs were considered positive for infection when a circulating antigen test (PetChek® HTWM PF, IDEXX, USA) and/or microfilariae (mff; modified Knott's test) was positive for D. immitis. According to the manufacturer's instructions, all the samples were tested in duplicate. A sample was considered positive if it had more colour than the negative control (see "Parasitological evaluation" section for the scoring system, based on the intensity of the colour reaction).

The 30 dogs were allocated at random into three treatment groups (10 dogs in each group), based on the moxidectin formulations used for the treatment protocols as described below.

Group 1 (G1). Oral moxidectin (3 µg/kg SID; Afilaria®, Fatro-Italy) once a month for 9 consecutive months, together with doxycycline (10 mg/kg SID; Ronaxan®, Boehringer Ingelheim Animal Health, Italy) for the first 30 days.

Group 2 (G2). Topical 10% imidacloprid + 2.5% moxidectin (Advocate®, Elanco, Italy) once a month for 9 consecutive months, together with doxycycline (10 mg/kg SID; Ronaxan®) for the first 30 days.

Group 3 (G3). Extended release moxidectin injectable (Afiliara SR®, Fatro, Italy) every 6 months for two administrations, together with doxycycline (10 mg/kg SID; Ronaxan®, Boehringer Ingelheim Animal Health, Italy) for the first 30 days.

Blood samples were collected once a month for 9 months from all dogs enrolled in the study. Microfilarial concentration in 1 ml (mff/ml) blood was determined with the modified Knott's test [19] following the protocol by Genchi et al. [20]. Morphological identification of microfilariae was based on body length and width and the morphology of the head and tail [20]).

Serum samples were tested for circulating antigens using PetChek® HTWM PF (IDEXX, USA), following the manufacturer's instructions. A subjective scoring system, based on the intensity of the colour reaction, was used to express the results of each test as negative (−), weak positive (+) and strong positive (+ +). "First negative" was defined as a minimum of two consecutive negative tests, according to [10].

The radiological evaluation was performed at Day − 15 (before the treatment) and at Day 180 (after 6 months of treatment) by the same operator.

The thoracic radiographs included right-lateral, left-lateral and dorso-ventral views. All the radiographs were obtained on manually restrained and unsedated patients during peak inspiration. The following criteria were evaluated: size and shape of the pulmonary arteries and their branches (vascular pattern); dilatation of the main pulmonary artery, enlargement of the right atrium, enlargement of the left atrium, generalized cardiac enlargement evaluated both subjectively and objectively using the vertebral heart score (VHS) [21]. Furthermore, the presence and distribution of alterations affecting the pulmonary interstitium, alveolar space and bronchial walls were classified using common radiographic patterns: interstitial, alveolar and bronchial pattern, respectively, or mixed if two or more patterns were present [22].

Based on the presence and degree of extension of one or more of the radiographic alterations, a clinical scoring system was considered as follows: score 0 (normal, without radiographic alterations consistent for heartworm disease), score 1 (mild pulmonary and/or cardiac alterations), score 2 (moderate pulmonary and/or cardiac alterations) and score 3 (severe pulmonary and/or cardiac alterations, signs compatible with thrombo-embolism/pneumonia) [6, 23].

All the dogs underwent complete echocardiographic examination at Days − 15 and 180 by the same operator, including transthoracic 2D, M-mode and spectral and colour-flow Doppler echocardiographic evaluations using transducer arrays of 1–4 MHz and 3–8 MHz (ESAOTE, Italy).

Examinations were performed in conscious unsedated dogs during a period of quiet breathing, with the animals in left and right lateral recumbency. At least five consecutive cardiac cycles were acquired and stored for off-line measurements. A clinical scoring system was employed based on ultrasound findings, according to [6, 24, 25]. Briefly, pulsed wave (PW)-spectra signals for calculation of STIs were acquired from the right parasternal short axis view of the pulmonary artery with the sample volume at the valve level; the acceleration time (AT) of pulmonary artery (PA) flow was measured from the onset of the pulsed Doppler PA flow signal to peak flow velocity. The right ventricular ejection time (ET) was measured from the onset to the end of the Doppler PA flow signal; moreover, the AT/ET ratio was calculated. When tricuspid regurgitation and/or mitral regurgitation were present, CW spectra were acquired from the left apical cranial four-chamber views. Each dog was assigned a score from 0 to 3 for pulmonary hypertension, considering AT, ET, AT/ET and TRV [6].

Radiological and echocardiographic examinations were performed by two independent investigators that were blinded to dog identification and clinical and parasitological data.

Results were analysed using univariate statistical analyses (i.e. non-parametric Wilcoxon signed- rank test and t-test) to determine the level of significant differences between pre- (Day − 15) and post-treatment (at Day 180 and at Day 270) based on the antigen status, micofilarial counts and radiological and cardiac scores in the three treatment groups (G1, G2 and G3). Statistical analysis was performed using Windows SPSS® (version 17.0). The level of significance was set at a P value < 0.05.

Table 1 summarizes the results of Knott's test of all dogs at enrolment, while Table 2 reports results of antigen testing scores. All dogs except three were positive for circulating mff, with values ranging from 50 to 83,600 mff/ml blood. Circulating antigens were present in all but two dogs, who were however positive for circulating microfilariae. Antigen levels were comparable among dogs of the three groups.

Twenty-two/30 dogs (73.3%) were also positive for D. repens mff (range 100–78,200 mff/ml).

Figure 2a and Table 3 report the results for reduction of circulating D. immitis microfilariae induced by the three treatment protocols evaluated in the study. In general, mff loads decreased markedly in all dogs from all groups at Day 30, and all but one dog were negative at Day 60. The one positive dog remaining was from the injectable formulation group (G3) and became negative at Day 90. Interestingly, dog no. 3 from the oral formulation group (G1) became negative at Day 60 (from 2900 mff/ml at enrolment) and then became once again positive for mff at Day 90 (100 mff/ml). The dog was negative at all subsequent time points.

Reduction of circulating microfilariae of D. immitis (a) and D. repens (b) in the three treatment groups

All dogs with D. repens mff were negative at Day 90 (Fig. 2b).

Table 4 reports the results for antigen testing for each group at each time point until Day 270. There was a clear reduction of antigen concentration for all treatment groups, beginning as early as Day 60. Overall, negative antigen results (at least two consecutive negative tests) were observed at Day 60 for 2 dogs; at Day 150 for 1 dog; at Day 180 for 4 dogs; at Day 210 for 10 dogs; at Day 240 for 4 dogs. At Day 270, 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3 had at least two consecutive negative tests. Several dogs from each group reverted to positive antigen status at different time points following negativization.

The results of statistical analyses showed a significant difference in antigen status before and after treatment in all three groups (G1: P = 0.015; G2: P = 0.006; G3: P = 0.020). However, the paired sample t-test revealed no significant difference among the three groups after treatment on Day 270 (P > 0.05). In addition, there was a significant difference between pre- and post-treatment in terms of mff counts in all three groups starting at Day 30 through Day 270 (P < 0.05).

Table 5 reports the radiological and cardiopulmonary scores obtained by all the dogs in the three treatment groups before treatment (Day − 15) and after treatment (Day 180). A more detailed description of pulmonary and/or cardiac alterations can be found as Additional file 1: Table S1.

The results of radiological examination showed that at Day − 15, 5/30 (16.6%) were assigned a score of 0 (2 dogs from G1; 1 dog from G2; 2 dogs from G3), 12/30 (40%) a score of 1 (four dogs from G1; three dogs from G2; five dogs from G3) and 13/30 (43.4%) a score of 2 (four dogs from G1; six dogs from G2; three dogs from G3). No dog was assigned to score 3.

At Day 180, all the dogs with score 0 at day − 15 were assigned the same score. Furthermore, 9/12 (75%) dogs with score 1 at Day − 15 showed substantial improvement at Day 180 (Fig. 3) and the remaining three were stable (one dog from G1 and two dogs from G3; Fig. 4). The 13 dogs with score 2 at day − 15 showed radiographic improvement of pulmonary and/or cardiac conditions. Indeed, 4/13 (30.7%) dogs had a partial resolution of the radiological alterations and were reduced to score 1 (one dog from G1; one dog from G2 and two dogs from G3) and 9/13 (69.3%) had a substantial improvement and therefore were moved from score 2 into score 0 (three dogs from G1; five dogs from G2 and one dog from G3).

Right-lateral radiographs pre- (A) and post-treatment (B) of the same dog. A A diffuse mixed alveolar/unstructured interstitial pattern. This increase in pulmonary opacity involves especially the caudal lung lobes where multiple air bronchograms are visible. In B, there is a partial resolution of the radiographic alterations involving the lung lobes, although there is a residual unstructured interstitial pattern associated with mild to moderate thickening of the bronchial walls. This dog was initially assigned in score 2, but after the 6 months of therapy was assigned a score of 1

Dorsoventral (DV) radiographs pre- (A) and post-treatment (B) of the same dog. In both views, there is a mild, diffuse, unstructured interstitial pattern associated with mild dilatation of the right atrium (black arrowheads). Furthermore, several caudal lobar arteries (white arrows) have a convoluted shape. This dog was assigned a pre-treatment score of 1 and, after 6 months of therapy, was re-assigned in the same score. Legend: R = right, L = left

Cardiac ultrasound examination showed that at the beginning of the study (Day − 15), 11/30 (36.7%) dogs showed altered pulmonary blood flow, ranging from score 1 (three dogs from G1; three dogs from G2; four dogs from G3) to score 2 (one dog from G3). Moreover, 19/30 (63.3%) dogs were assigned score 0 (six dogs from G1; seven dogs from G2 and six dogs from G3). In particular, 12/30 (40%) dogs were classified with chronic degenerative mitral disease (CDMD), representing a comorbidity for the enrolled animals [26]. Specifically, two of the dogs classified with score 1, one dog with score 2 and three dogs with score 0 showed enlarged left atrium and concomitant haemodynamic significant mitral regurgitation and were classified in stage B2 of CDMD. Moreover, stage B1 of CDMD was recorded in five dogs classified in score 0 and in three dogs classified in score 1. Six dogs with CDMD in stage B2 (6/30) received oral administration of benazepril 0.25 mg/kg SID.

Subsequent echocardiographs carried out at day 180 did not show any significant variation of previously assigned scores, except for 4/11 dogs that showed a progressive improvement. In particular, three dogs with slight alteration assigned in score 1 became normal (one dog from each group) while one dog (from G1) with moderate alteration assigned a score of 2 showed slight modifications and was assigned a score of 1 (Fig. 4). However, according to the American College of Veterinary Internal Medicine (ACVIM) guidelines [27], the echocardiographic probability of pulmonary hypertension (PH) was low (only one dog staged in score 2) considering that peak tricuspid regurgitation velocity was ≤ 3.0. During the second echocardiographic control, PH score and peak tricuspid regurgitation velocity were reduced.

Results of statistical analysis of radiological scores obtained before treatment (Day − 15) and after treatment (Day 180) showed that treatment with moxidectin/doxycycline was effective for all the animals in the three treatment groups (P = 0.000). There was no significant difference among the three groups (P = 0.564) post-treatment at Day 180. Regarding the cardiac evaluation, the Wilcoxon test showed statistically significantly higher rank of post-treatment scores than the pre-treatment rank scores (P = 0.045).

In the present study we evaluated the efficacy of different formulations of moxidectin combined with doxycycline to induce negative antigen status in dogs naturally infected with D. immitis. According to [28], even though melarsomine is still widely used by veterinary practitioners in Italy, a monthly macrocyclic lactone together with doxycycline is currently being used by over 30% of surveyed veterinary facilities. The use of moxidectin is a valid alternative to ivermectin, as shown by several studies [8,9,10, 17, 29].

Microfilarial counts were not considered when forming the different treatment groups. It is known that the intensity of microfilaraemia is not correlated with the adult worm burden [30].

The adulticidal efficacy (i.e. two consecutive negative antigen tests) of the injectable formulation of moxidectin was 90% at 9 months, slightly higher compared to the Spot-On formulation (80%) and the oral formulation (60%). This may be due to its pharmacokinetics. Lok et al. [31], in an early pharmacokinetic study of the formulation used here (0.17 mg moxidectin/kg), reported that effective serum moxidectin levels peak 8 days after injection and remain at this level for 6 months. McCall et al. [32] studied the retroactive activity of moxidectin extended release on immature worms and reported 85.9% efficacy against 4-month-old D. immitis infections and efficacy was even higher (97.2%) when a second treatment was given 6 months later. Only one previous study assessed the efficacy of an extended-release injectable formulation of moxidectin combined with doxycycline, in which two doses 6 months apart resulted in 90% of dogs becoming antigen negative [18].

The Spot-On formulation was efficacious in 80% of dogs at 9 months. Bowman et al. [33] reported that topical moxidectin reaches steady-state serum concentrations at levels that are much higher than those needed for heartworm prophylaxis. The exposure of adult parasites to high concentrations of moxidectin likely contributes to the efficacy of this protocol.

Oral moxidectin combined with doxycycline gave the lowest percentage of dogs (60%) that became negative for circulating antigens by 9 months. It has been reported that, when compared to oral ivermectin, moxidectin has lower total body clearance and higher volume of distribution, which results in a prolonged elimination half-life [34]. However, it may be that oral administration is not always followed by optimal gastrointestinal absorption, leading to under-dosing and lack of efficacy. It is well known that bioavailability of orally administered drugs depends on a multitude of factors, including gastric pH and emptying time, small intestinal fluid properties, changes in gastrointestinal integrity, etc. [35].

The concentration of circulating antigens was variable from month to month in all treatment groups, as reported previously by others [8,9,10], and the authors of the present study defined "first negative" as a minimum of two consecutive negative tests, according to [10]. The reason for transitory return to antigen-positive status is not clear, but may be due to various factors including variable antigen concentrations due to gradual death of parasites or the arrival of migrating worms to the pulmonary artery during the study period. This phenomenon, however, may not be unique to alternative protocols using doxycycline and macrocyclic lactones. Paterson et al. [10], who compared doxycycline/moxidectin to melarsomine, reported variations in the results of monthly antigen testing in both groups. In a similar study [9] authors reported a return to positive antigen status in one dog treated with melarsomine at 12 months post-treatment. To the authors’ knowledge, no other studies have evaluated monthly antigen status in dogs treated with melarsomine. Early studies only evaluated dogs after 4, 8 or 12 months following treatment and also reported persistent antigenemia in some dogs [36,37,38]. It would be interesting to conduct studies on the monthly trend of antigen status following the diverse adulticide regimens.

It has also been reported that immune-complex formation can lead to false-negative antigen test results and that this happens frequently in dogs treated with doxycycline and macrocyclic lactones. Pre-heating serum samples can disrupt immune-complexes, resulting in a positive test result [39, 40]. The authors of the present study chose not to preheat the serum samples. Over 70% of dogs were also infected with D. repens, and it has been reported that pre-heating serum samples from D. repens mono-infected dogs leads to false-positive antigen tests for D. immitis [41, 42]. Correct interpretation of conversion to positive antigen status in these dogs would have been impossible. Furthermore, according to the American Heartworm Society guidelines for canine heartworm disease, heat treatment should be considered only when antigen test is negative, but mff are circulating [15]: in the present study only two dogs were mff positive and antigen negative and both were positive for D. repens.

Treatment regimens were also effective against D. repens mff, with no differences observed among the three formulations used (data not shown). However, there is no current test to verify adulticide efficacy against D. repens, and the clearance of circulating mff observed in the present study is not necessarily indicative of adult worm death. However, Petry et al. [43] reported the adulticide effect of the same topical formulation used in the present study in D. repens experimentally infected dogs.

It has been reported that dogs treated with doxycycline can have gastrointestinal upset, while coughing has also been reported in dogs treated with the ML/doxycyline protocol [10]. In the present study, all treatment regimens were well tolerated. Only six dogs were treated with furosemide and benazepril for cough and mitral regurgitation during the entire study.

Radiographic alterations occurring during natural heartworm infection are related to the parasite load and time elapsed since the infection, with radiographic findings ranging from subclinical disease without apparent alterations in the lung fields and pulmonary vasculature to severe pulmonary and cardio-circulatory impairment [44,45,46]. In our study, the most frequent radiographic findings in dogs with score 1 were a diffuse interstitial pattern and pulmonary vascular changes, similar to those reported by Mavrapoulou et al. and Genchi et al. [6, 9]. These findings are considered common in cases of heartworm disease, and the alterations in the lung parenchyma are attributed to eosinophilic bronchopneumonia, fibrotic changes and focal pulmonary consolidation [44, 47].

The dogs classified as moderate (score 2) had dilatation of the right atrium, among the most frequent alterations, likely related to infection with a higher or long-lasting parasite load and a more severe pulmonary interstitial pattern. Moreover, there were many dogs with enlargement of the left atrium or an overall increase in cardiac size, most likely related to concurrent mitral valve disease.

In our study, there was no evidence of worsening of the radiographic findings at the follow-up after 6 months. Moreover, four of the dogs initially classified in score 2 showed a partial improvement and nine an almost complete resolution. Similarly, all the dogs with score 1 improved. Overall, the treatment with moxidectin and doxycycline combination was effective and almost all the dogs from the treatment groups were cleared of pulmonary abnormalities by 6 months from the beginning of treatment. The combination of moxy/doxy was previously reported in experimentally treated animals to induce the reduction of pro-inflammatory antigen mass [14]. Similarly, Genchi et al. [9] reported that no dogs showed worsening of pulmonary patterns 12 to 24 months after the treatment with the same topical formulation of moxidectin combined with doxycycline for the first 30 days in dogs naturally infected by D. immitis.

Although echocardiography represents a valid option for measurement of pulmonary artery pressure (PAP), limits of the method include variability and imprecision in individual dogs. Therefore, evaluation of echocardiographic Doppler parameters is useful for assessing the probability, rather than the diagnosis, of pulmonary hypertension (PH), as shown by Reinero et al. [27]. However, in our study, echocardiography gave important information regarding the diagnostic profile and the therapeutic follow-up. Echocardiography showed a progressive improvement of cardiac function in a limited number of animals (4/30). Notably, we observed CDMD as a comorbidity associated with heartworm disease in 40% of dogs enrolled and this could have influenced assignment of echocardiographic scores [48].

In conclusion, doxycycline/moxidectin combination treatment for HWD has been reported as being safe and effective. Results from the present study suggest that efficacy may be related to the moxidectin formulation.

All data obtained are shown in the manuscript.

Genchi C, Bowman D, Drake J. Canine heartworm disease (Dirofilaria immitis) in Western Europe: survey of veterinary awareness and perceptions. Parasit Vectors. 2014;7:206. https://doi.org/10.1186/1756-3305-7-206.

Article Google Scholar

Balbo T, Panichi M. La filariasi del cane. La Nuova Veterinaria 1968; 44:18 (Article in Italian).

Bowman DD, Atkins CE. Heartworm biology, treatment, and control. Vet Clin North Am Small Anim Pract. 2009;39:1127–58. https://doi.org/10.1016/j.cvsm.2009.06.003.

Article Google Scholar

Bazzocchi C, Mortarino M, Grandi G, Kramer LH, Genchi C, Bandi C, et al. Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. Int J Parasitol. 2008;38:1401–10. https://doi.org/10.1016/j.ijpara.2008.03.002. (Epub 2008 Mar 21).

Article CAS Google Scholar

Grandi G, Quintavalla C, Mavropoulou A, Genchi M, Gnudi G, Bertoni G, Kramer L. A combination of doxycycline and ivermectin is adulticidal in dogs with naturally acquired heartworm disease (Dirofilaria immitis). Vet Parasitol. 2010;169(3–4):347–51. doi: https://doi.org/10.1016/j.vetpar.2010.01.025. Epub 2010 Jan 25. Erratum in: Vet Parasitol. 2011;177(1–2):196. Dosage error in published abstract; MEDLINE/PubMed abstract corrected; Dosage error in article text..

Mavropoulou A, Gnudi G, Grandi G, Volta A, Kramer LH, Quintavalla C. Clinical assessment of post-adulticide complications in Dirofilaria immitis-naturally infected dogs treated with doxycycline and ivermectin. Vet Parasitol. 2014;205:211–5. https://doi.org/10.1016/j.vetpar.2014.06.014. (Epub 2014 Jun 17).

Article CAS Google Scholar

Bendas AJR, Mendes-de-Almeida F, Von Simson C, Labarthe N. Heat pretreatment of canine samples to evaluate efficacy of imidacloprid + moxidectin and doxycycline in heartworm treatment. Parasit Vectors. 2017;10:246. https://doi.org/10.1186/s13071-017-2189-2.

Article CAS Google Scholar

Savadelis MD, Ohmes CM, Hostetler JA, Settje TL, Zolynas R, Dzimianski MT, et al. Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi® for dogs (10% imidacloprid + 2.5% moxidectin) and doxycycline. Parasit Vectors. 2017;10:245. https://doi.org/10.1186/s13071-017-2190-9.

Article CAS Google Scholar

Genchi M, Vismarra A, Lucchetti C, Viglietti A, Crosara S, Gnudi G, et al. Efficacy of imidacloprid 10%/moxidectin 2.5% spot on (Advocate®, Advantage Multi®) and doxycycline for the treatment of natural Dirofilaria immitis infections in dogs. Vet Parasitol. 2019;273:11–6. https://doi.org/10.1016/j.vetpar.2019.07.011. (Epub 2019 Jul 27).

Article CAS Google Scholar

Paterson T, Fernandez C, Burnett PJ, Lessey L, Hockley T, Hagen R, et al. Heartworm control in Grenada, West Indies: Results of a field study using imidacloprid 10% + moxidectin 2.5% and doxycycline for naturally-acquired Dirofilaria immitis infections. Vet Parasitol. 2020;284:109194. https://doi.org/10.1016/j.vetpar.2020.109194. (Epub 2020 Aug 15).

Article CAS Google Scholar

Vörös K, Becker Z, Kónya R, Arany-Tóth A, Farkas R. Application of moxidectin and ultrasound-aided injection of melarsomine during the american heartworm society recommended treatment protocol in dirofilaria immitis infected dogs. Vector Borne Zoonotic Dis. 2022;22:382–90. https://doi.org/10.1089/vbz.2021.0108. (Epub 2022 Jun 10).

Article Google Scholar

Krame NM, McCall JW, Grandi G, Genchi C. Wolbachia and its importance in veterinary filariasis. Issues Infect Dis. 2007;5:124.

Google Scholar

Louzada-Flores VN, Kramer L, Brianti E, Napoli E, Mendoza-Roldan JA, Bezerra-Santos MA, et al. Treatment with doxycycline is associated with complete clearance of circulating Wolbachia DNA in Dirofilaria immitis-naturally infected dogs. Acta Trop. 2022;232:106513. https://doi.org/10.1016/j.actatropica.2022.106513. (Epub 2022 May 19).

Article CAS Google Scholar

Kramer L, Grandi G, Passeri B, Gianelli P, Genchi M, Dzimianski MT, et al. Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride. Vet Parasitol. 2011;176:357–60. https://doi.org/10.1016/j.vetpar.2011.01.021. (Epub 2011 Jan 19).

Article CAS Google Scholar

American Heartworm Society, 2018. The AHS guidelines, update. https://www.heartwormsociety.org/veterinary-resources/veterinary-education/ahs-board-speaks-out/510-the-ahs-guidelines-2018-updates.

ESDA 2017. European society of dirofilariosis and angiostrongylosis. Guidelines for Clinical Management of Canine Heartworm Disease; 2017. https://www.esda.vet/wp-content/uploads/2017/11/Guidelines-for-clinical-management-of-canine-heartworm-disease.pdf.

Jacobson LS, DiGangi BA. An Accessible Alternative to Melarsomine: "Moxi-Doxy" for Treatment of Adult Heartworm Infection in Dogs. Front Vet Sci. 2021;8:702018. https://doi.org/10.3389/fvets.2021.702018.

Article Google Scholar

Alberigi B, Fernandes JI, Paiva JP, Mendes-de-Almeida F, Knackfuss F, Merlo A, et al. Efficacy of semi-annual therapy of an extended-release injectable moxidectin suspension and oral doxycycline in Dirofilaria immitis naturally infected dogs. Parasit Vectors. 2020;13:503. https://doi.org/10.1186/s13071-020-04380-z.

Article CAS Google Scholar

Knott J. A method for making microfilarial surveys on day blood Trans. Royal Soc Trop Med Hyg. 1939;33:191.

Article Google Scholar

Genchi M, Ciuca L, Vismarra A, Ciccone E, Cringoli G, Kramer L, et al. Evaluation of alternative reagents on the performance of the modified Knott's test. Vet Parasitol. 2021;298:109555. https://doi.org/10.1016/j.vetpar.2021.109555.

Article CAS Google Scholar

Buchanan JW. Vertebral scale system to measure heart size in radiographs. Vet Clin North Am Small Anim Pract. 2000;30:379–93.

Article CAS Google Scholar

Thrall DE. Principles of Radiographic Interpretation of the Thorax. In: Elsevier Saunders, editors. Veterinary Diagnostic Radiology. 2018. p. 568–582.

Thrall DE, Badertscher RR, Lewis RE, McCall JW, Lonsonsky JM. Radiographic changes associated with developing dirofilariasis in experimentally infected dogs. Am J Vet Res. 1980;41:81–90.

CAS Google Scholar

Kellum HB, Stepien RL. Sildenafil citrate therapy in 22 dogs with pulmonary hypertension. J Vet Intern Med. 2007;21:1258–64. https://doi.org/10.1892/07-006.1.

Article Google Scholar

Schober KE, Baade H. Doppler echocardiographic prediction of pulmonary hypertension in West Highland white terriers with chronic pulmonary disease. J Vet Intern Med. 2006;20:912–20. https://doi.org/10.1892/0891-6640(2006)20[912:depoph]2.0.co;2.

Article Google Scholar

Keene BW, Atkins CE, Bonagura JD, Fox PR, Häggström J, Fuentes VL, et al. ACVIM consensus guidelines for the diagnosis and treatment of myxomatous mitral valve disease in dogs. J Vet Intern Med. 2019;33:1127–40. https://doi.org/10.1111/jvim.15488. (Epub 2019 Apr 11).

Article Google Scholar

Reinero C, Visser LC, Kellihan HB, Masseau I, Rozanski E, Clercx C, et al. ACVIM consensus statement guidelines for the diagnosis, classification, treatment, and monitoring of pulmonary hypertension in dogs. J Vet Intern Med. 2020;34:549–73.

Article Google Scholar

Genchi M, Rinaldi L, Venco L, Cringoli G, Vismarra A, Kramer L. Dirofilaria immitis and D. repens in dog and cat: A questionnaire study in Italy. Vet Parasitol. 2019;267:26–31. https://doi.org/10.1016/j.vetpar.2019.01.014. (Epub 2019 Feb 13).

Article Google Scholar

Kryda K, Holzmer S, Everett WR, McCall JW, Mahabir SP, McTier TL, et al. Preventive efficacy of four or six monthly oral doses of 24 µg/kg moxidectin compared to six monthly doses of Heartgard® Plus or Interceptor® Plus against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs. Parasit Vectors. 2020;13:339. https://doi.org/10.1186/s13071-020-04178-z.

Article CAS Google Scholar

McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm Disease in Animals and Humans. In: Rollinson D, Hay SI, editors. Advances in parasitology. Berlin: Springer; 2008. p. 193–285.

Google Scholar

Lok JB, Washabau RJ, Heaney K, Nolan TJ, Hendrick MJ, Neumann NR, et al. Six-month prophylactic efficacy of moxidectin sustained release (SR) injectable for dogs against experimental heartworm infection in growing puppies. Vet Parasitol. 2005;133:233–41. https://doi.org/10.1016/j.vetpar.2005.06.019.

Article CAS Google Scholar

McCall JW, Supakorndej P, Dzimianski MT, Supakorndej N, Mansour AE, Juni JJ, et al., et al. Evaluation of retroactive and adulticidal activity of moxidectin canine sr (sustained release) injectable formulation against Dirofilaria immitis in Beagles. In: Seward HL, et al., editors. Recent Advances in Heartworm Disease—Symposium ’01. Batavia: American Heartworm Society; 2001. p. 165–72.

Google Scholar

Bowman DD, Grazette AR, Basel C, Wang Y, Hostetler JA. Protection of dogs against canine heartworm infection 28 days after four monthly treatments with Advantage Multi® for Dogs. Parasit Vectors. 2016;9:12. https://doi.org/10.1186/s13071-016-1293-z.

Article CAS Google Scholar

Al-Azzam SI, Fleckenstein L, Cheng KJ, Dzimianski MT, McCall JW. Comparison of the pharmacokinetics of moxidectin and ivermectin after oral administration to beagle dogs. Biopharm Drug Dispos. 2007;28:431–8. https://doi.org/10.1002/bdd.572.

Article CAS Google Scholar

Vinarov Z, Abdallah M, Agundez JAG, Allegaert K, Basit AW, Braeckmans M, et al. Impact of gastrointestinal tract variability on oral drug absorption and pharmacokinetics: An UNGAP review. Eur J Pharm Sci. 2021;162:105812. https://doi.org/10.1016/j.ejps.2021.105812. (Epub 2021 Mar 20).

Article CAS Google Scholar

Dzimianski MT, McCall JW, McTier TL, Keister M, Strickland J. Preliminary Results of the Efficacy of RM 340 Administered Seasonally to Heartworm Antigen- and Microfilaria-Positive Dogs Living Outdoors in a Heartworm Endemic Area. In: Proceedings of the Heartworm Symposium '92 Austin, Texas March 27–29,1992

Vezzoni A, Genchi C, Raynaud JP. Adulticide Efficacy of RM 340 in Dogs with Mild and Severe Natural Infections. In: Proceedings of the Heartworm Symposium '92 Austin, Texas March 27–29,1992

Knight DH. Persistent Post adulticide Antigenemia. In: Proceedings of the Heartworm Symposium '95 Auburn, Alabama March 31-April 2, 1995.

Drake J, Gruntmeir J, Merritt H, Allen L, Little SE. False negative antigen tests in dogs infected with heartworm and placed on macrocyclic lactone preventives. Parasit Vectors. 2015;4:68.

Article Google Scholar

Ciucă L, Genchi M, Kramer L, Mangia C, Miron LD, Prete LD, et al. Heat treatment of serum samples from stray dogs naturally exposed to Dirofilaria immitis and Dirofilaria repens in Romania. Vet Parasitol. 2016;225:81. https://doi.org/10.1016/j.vetpar.2016.05.032.

Article Google Scholar

Venco L, Manzocchi S, Genchi M, Kramer LH. Heat treatment and false-positive heartworm antigen testing in ex vivo parasites and dogs naturally infected by Dirofilaria repens and Angiostrongylus vasorum. Parasit Vectors. 2017;10:476. https://doi.org/10.1186/s13071-017-2444-6.

Article CAS Google Scholar

Sobotyk C, Savadelis MD, Verocai GG. Detection and cross-reaction of Dirofilaria repens using a commercial heartworm antigen test kit. Vet Parasitol. 2021;289:109302. https://doi.org/10.1016/j.vetpar.2020.109302. (Epub 2020 Dec 10).

Article CAS Google Scholar

Petry G, Genchi M, Schmidt H, Schaper R, Lawrenz B, Genchi C. Evaluation of the Adulticidal Efficacy of Imidacloprid 10 %/Moxidectin 25 % (w/v) Spot-on (Advocate®, Advantage® Multi) against Dirofilaria repens in Experimentally Infected Dogs. Parasitol Res. 2015;114:S131–44. https://doi.org/10.1007/s00436-015-4519-7.

Article Google Scholar

Venco L, McCall JW, Guerrero J, Genchi C. Efficacy of long-term monthly administration of ivermectin on the progress of naturally acquired heartworm infections in dogs. Vet Parasitol. 2004;124:259–68. https://doi.org/10.1016/j.vetpar.2004.06.024.

Article CAS Google Scholar

Losonsky J, Thrall D, Lewis R. Thoracic radiographic abnormalities in 200 dogs with spontaneous heartworm infestation. Vet Radiol. 2005;2005:120.

Google Scholar

Savadelis MD, Coleman AE, Rapoport GS, Sharma A, Sakamoto K, Keys DA, et al. Clinical assessment of heartworm-infected Beagles treated with a combination of imidacloprid/moxidectin and doxycycline, or untreated. J Vet Intern Med. 2020;34:1734–45. https://doi.org/10.1111/jvim.15853. (Epub 2020 Jul 21).

Article Google Scholar

McCall JW, Arther R, Davis W, Settje T. Safety and efficacy of 10% imidacloprid+2.5% moxidectin for the treatment of Dirofilaria immitis circulating microfilariae in experimentally infected dogs. Vet Parasitol. 2014;206:86–92. https://doi.org/10.1016/j.vetpar.2014.09.011. (Epub 2014 Sep 23).

Article CAS Google Scholar

Tai TC, Huang HP. Echocardiographic assessment of right heart indices in dogs with elevated pulmonary artery pressure associated with chronic respiratory disorders, heartworm disease, and chronic degenerative mitral valvular disease. Vet Med. 2013;58:613.

Article Google Scholar

Download references

The authors thank to the staff of the dog shelter for their valuable help in many critical points of the present study (Gabriela Guadagno, Roberta Grillo and Dr. Francesco Russo).

This research was supported by EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT).

Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Via Federico Delpino 1, 80137, Naples, Italy

Lavinia Ciuca, Dario Constanza, Antonio Di Loria, Leonardo Meomartino, Paolo Ciaramella, Giuseppe Cringoli & Laura Rinaldi

Department of Veterinary Medicine Sciences, University of Parma, Strada del Taglio, 10, 43126, Parma, Italy

Alice Vismarra, Marco Genchi & Laura Kramer

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

LC, LK, MG, LR: Conceptualization; LC, DC, AD, AV: Data curation; LC, AV, LK: Formal analysis; LC, DC, AD: Investigation; LC, DC, AD: Methodology; LC, LK, LR: Project administration; LK, LR, MG, GC, PC, LM: Supervision; LC, LK, AV, DC, AD: Writing—original draft; LK, LR, MG, GC, PC, LM, AD, DC, AV, LC: Writing—review and editing. All authors read and approved the final manuscript.

Correspondence to Alice Vismarra.

The protocol of this study was approved by the ethical committee of animal experiments of the Department of Veterinary Medicine and Animal Production, University of Federico II Naples, Italy (approval number 0093204/2022).

All the authors read and agree with the publication of the manuscript in the present form.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Results of pulmonary and/or cardiac alterations (left atrial enlargement; pulmonary arteries enlargement; interstitial pattern; cardiac enlargement; right atrial enlargement; bronchial pattern; main pulmonary artery enlargement; alveolar pattern) for both radiographic evaluations (day – 15 and day 180) for each dog from all the treatment-groups (oral, Spot-On, injectable).

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

Ciuca, L., Vismarra, A., Constanza, D. et al. Efficacy of oral, topical and extended-release injectable formulations of moxidectin combined with doxycycline in Dirofilaria immitis naturally infected dogs. Parasites Vectors 16, 54 (2023). https://doi.org/10.1186/s13071-023-05673-9

Download citation

Received: 02 November 2022

Accepted: 16 January 2023

Published: 06 February 2023

DOI: https://doi.org/10.1186/s13071-023-05673-9

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative