Efficacy of a spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of Cercopithifilaria spp. and Onchocerca lupi microfilariae in naturally infected dogs from Portugal

Background Onchocerca lupi and Cercopithifilaria spp. are vector-borne filarioids of dogs, which harbour skin microfilariae (mfs), the former being of zoonotic concern. Proper treatment studies using compounds with microfilaricidal activity have not been performed. Therefore, this study aimed to assess the efficacy of a commercially available spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of O. lupi or Cercopithifilaria spp. skin-dwelling mfs in naturally infected dogs. Methods Privately owned dogs (n = 393) from southern Portugal were sampled via skin biopsies to identify and count mfs in 20 µl of skin sediment. A total of 22 mfs-positive dogs were allocated to treatment group (n = 11; G1) or left untreated as a control (n = 11; G2). As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with moxidectin 2.5%/imidacloprid 10% spot-on formulation on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2). Based on the negative results for both O. lupi and/or Cercopithifilaria spp. mfs of dogs in the pilot study from SD28 onwards, the remaining six dogs in G1 were treated at SD0 and assessed only at SD28. Results Of the 393 animals sampled, 78 (19.8%) scored positive for skin-dwelling mfs. At the pilot investigation, a mean number of 19.6 mfs for O. lupi was recorded among five infected dogs whereas no mfs were detected at SD28. At SD0, the mean number of Cercopithifilaria spp. larvae was 12.6 for G1 and 8.7 for G2. The mean number of mfs for G2 was 20.09. Conclusions Results herein obtained suggest that a single treatment with moxidectin 2.5%/imidacloprid 10% spot-on formulation is efficacious against skin-dwelling mfs in dogs. The microfilaricidal effect of moxidectin could also be useful in reducing the risk of O. lupi infection for humans.


Background
Vector-borne filarioids are transmitted by a wide range of arthropods (e.g., mosquitoes, fleas, blackflies, biting midges and ticks). About 12 species belonging to the genera Acanthocheilonema, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, and Onchocerca infect dogs [1][2][3][4]. Among these, the species Dirofilaria repens and Dirofilaria immitis are the best-known filarioids in veterinary parasitology for their worldwide distribution and their zoonotic potential [5][6][7]. The genus Onchocerca includes 30 species primarily infecting ungulates [8] and, to a lesser extent, carnivores and humans. In the last decade, the increased number of zoonotic cases of O. lupi has stimulated the interest of the scientific community in this little-known parasite [9][10][11]. Since the first molecularly confirmed case of infection by O. lupi in humans [9], to date, 22 patients have been diagnosed with this onchocercid species in Germany, Tunisia, Hungary, Greece, Turkey, Iran, and the USA [12][13][14][15]. Consequently, much more attention is currently given to the distribution and clinical significance of O. lupi in dogs. In the initial stage, canine onchocercosis is associated with nonspecific ocular discomfort (i.e. excessive lacrimation, photophobia, conjunctivitis, exophthalmos, and periorbital swelling), while in the chronic phase the typical nodules are detected on the external parts of the ocular apparatus (i.e. eyelids, nictitating membrane, conjunctiva, and sclera) [16]. However, dogs do not always display overt clinical signs when O. lupi adults localize in the retrobulbar space of the eye [17]. Moreover, adult nematodes may localise in other anatomical districts other than the ocular apparatus, given that microfilariae (mfs) were diagnosed in dogs with absence of nodules together with the lack of ocular lesions at ultrasound examination [18].
The arthropod species involved in the life cycle of O. lupi have not been identified so far. Blackflies of the species Simulium velutinum, Simulium reptans, and Simulium pseudequinum, which fed on an O. lupi-infected dog from Greece, did not demonstrate progression of nematode larval development [19]. However, O. lupi DNA has been found in Simulium tribulatum blackflies from the USA [20].
Dogs may also be infected by at least three species of Cercopithifilaria, namely Cercopithifilaria grassii, Cercopithifilaria sp. II sensu Otranto et al. 2013 (a formal taxonomic description of adults for this species is lacking), and C. bainae [21]. In particular, C. bainae is prevalent in canine and tick populations from Southern and Eastern Europe (i.e. Spain, Italy, Greece, Portugal, and Romania) [22][23][24]. In addition, the parasite has been reported in Australia, Brazil, Malaysia, South Africa, and the USA [25,26]. Generally, the parasite distribution overlaps that of its vector, the brown dog tick Rhipicephalus sanguineus sensu lato [27][28][29]. However, C. bainae is considered of minor importance in veterinary medicine with few reports of erythematous, papular, and pruritic dermatitis [21,26] and chronic polyarthritis [30] associated with the infection in dogs.
The diagnosis of canine onchocercosis and cercopithifilarioses is achieved by the microscopic detection and identification of mfs in skin biopsies [9,31,32] or PCRbased techniques [9,33,34]. Preliminary investigations to develop tools able to detect anti-O. lupi antibodies have also been carried out [35][36][37]. Treatment of clinical cases of canine onchocercosis has been attempted using combinations of melarsomine, ivermectin, topical and systemic antibiotics, and prednisone [15,16,38]. A single dog presenting erythematous lesions and affected by C. bainae was successfully treated with a spot-on formulation containing imidacloprid 10% and moxidectin 2.5% [26]. In addition, the administration of oxfendazole (50 mg/kg/PO for 5 or 10 consecutive days) in dogs was ineffective in reducing skin-dwelling O. lupi mfs and showed a not statistically significant reduction of 50% of ocular lesions in a 6-month follow-up period [39]. However, proper studies on the long-term outcomes of macro-and microfilaricidal therapies for treating both parasites have not yet been performed, and no indications for the best treatment protocol to use are available. We assessed the efficacy of a commercially available spoton formulation containing moxidectin and imidacloprid for the treatment of O. lupi or Cercopithifilaria spp. skindwelling mfs in naturally infected dogs.

Ethics statement
The trial was a negative control, and clinical efficacy study partially blinded using a randomized block design, conducted according to the standards of Good Scientific Practice (GSP) and the national animal welfare requirements. Privately owned dogs, which lived in an area endemic for O. lupi and Cercopithifilaria spp. of the Algarve region (southern Portugal), were enrolled [39,40]. At the end of the study all dogs were treated with Advocate ® spot-on (Bayer Animal Health) and all screened dogs were dewormed with Drontal ® Plus (Bayer Animal Health).  (Fig. 1). Inclusion criteria were living outdoors or having regular outdoor activities and being at least 8 weeks of age and weighing > 1 kg. Dogs were excluded when they were treated with macrocyclic lactones 6 weeks before study start or when they were pregnant and/or lactating. All skin samples were collected using a 4-mm-diameter skin punch. Before the biopsy, the skin was disinfected with 70% alcohol and a 4% chlorhexidine solution followed by local anaesthesia with 2% lidocaine hydrochloride. Biopsies were taken from the interscapular regions of the dogs and soaked in 2 ml saline solution (NaCl 0.9%) for at least 12 h. After the procedure, the wounds on the dogs' skin were closed with Histoacryl ® (Braun) or a Sterile Disposable Skin Stapler as per the owners' preferences. Dogs were enrolled when O. lupi or Cercopithifilaria spp. mfs were detected in 20 µl of skin sediment and their number was recorded in individual forms.

Sampling procedure, enrolment and follow-up
On study day (SD) 0, dogs were weighed and their body weight recorded with the purpose of dosing the pharmaceutical product used. Twenty-two dogs meeting the inclusion criteria were included in the study and allocated to treatment group (n = 11; G1) or left untreated as control (n = 11; G2) following a random treatment allocation plan on the basis of an inclusion sequence. As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with Advocate ® spoton (Bayer Animal Health), on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2), at the minimum recommended dose rate of 2.5 mg moxidectin/kg body weight (BW) and 10 mg imidacloprid/kg BW corresponding to ≥ 0.1 ml spoton formulation per kg BW. Dogs were treated with the appropriate pipette size according to their weight. Based on the negative results for both O. lupi or Cercopithifilaria spp. mfs obtained in animals treated in the pilot study, from SD28 onwards, the remaining six dogs in G1 were treated and assessed only once, at SD0 and SD28, respectively. The 11 animals in the control group (G2) were left untreated, but samples for diagnosis were taken at the same time point of the six animals left in G1.

Molecular identification
Microfilariae were isolated and genomic DNA extracted using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen, Germany) in accordance with the manufacturer's instructions. Samples were molecularly processed for specific amplification and sequencing of the partial cytochrome oxidase subunit 1 (cox1) gene (~ 689 bp), following procedures described elsewhere using generic primers for filarioids [21]. Amplicons obtained from the skin sediments were purified using Ultrafree-DA columns (Amicon, Millipore, USA) and sequenced directly with the Taq DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems, USA) in an automated sequencer (ABI-PRISM 377, Applied Biosystems).

Calculation of the efficacy and statistical analyses
The percentage (%) of reduction [t] of mfs was calculated as follows: [t] = [(Ct0-Ct)/Ct0] × 100, where Ct0 is the baseline count before treatment and Ct was the count at any time point (t). Moreover, efficacy (%) = [(Ct-T)/ Ct] × 100, where Ct is the mean count of mfs of the control group at X time and T is the mean count of mfs of the treated animal groups at X time, was calculated and demonstrated by Fisher's exact test on contingency tables. Fisher's exact test was also performed to compare and assess the risk of mf infection according to the age and sex of the animals. Statistical analysis was planned and conducted in compliance with current guidelines [41]. Statistical calculations and randomization were performed with SPSS ® statistical package for Windows, version 13.0, and nQuery + nTerim 3.0 (StatSols), Statistical Solutions ® Ltd. 2014, Microsoft.  (Fig. 2). Risk estimates for the infection demonstrated that males are more prone to be infected by Cercopithifilaria spp. mfs than females (p = 0.01; odds ratio: 2.1; 95% CI 1.2-3.8). In contrast, the age of the animals evaluated was not statistically significant for mfs infection (p > 0.05).

Discussion
A single administration of moxidectin/imidacloprid spot-on formulation (Advocate ® spot-on) was shown to be efficacious for the treatment of skin-dwelling mfs in naturally infected dogs. This has been assessed through counting mfs in the sediment of skin biopsy punches obtained pre-and post-treatment by comparison with untreated control animals positive for Cercopithifilaria spp. mfs. Based on the preliminary results in 5 animals, the remaining 17 animals were checked for mfs only on SD28, also considering the reluctancy of animal owners to authorize collection of multiple skin biopsies, which is a rather invasive procedure.
The efficacy of a single administration of a moxidectin/ imidacloprid spot-on formulation is expected because the product is currently licensed in Europe for the hemoprophylaxis of D. immitis and D. repens. Furthermore, the product is labelled for microfilaria treatment based on the microfilaricidal efficacy of moxidectin against bloodcirculating mfs. This has been demonstrated herein also against skin-dwelling mfs of Cercopithifilaria spp. and it is suggestive also for O. lupi. Nonetheless, since reinfection may be an issue, in endemic areas, the treatment should be kept monthly to avoid new infection.
Overall, to our knowledge this is the first evidencebased protocol for cleaning O. lupi mfs infections since efficacy of drugs has been mainly assessed for curing nodules [42].
Indeed, avermectins have been used for treating O. lupi nodules in dogs in different formulations and timing. For example, other treatment protocols previously adopted included moxidectin (i.e. 0.22 mg/kg subcutaneous injection administered, followed by two additional injections at monthly intervals), doxycycline (5 mg/kg orally twice daily for 3 months), and following monthly recheck examinations and a final moxidectin injection, and prophylactic oral heartworm therapy (400 µg/kg orally every day for 30 days) [16]. Also, ivermectin administered at 150 μg/kg/dose every 3 months was successfully used for curing nodules [43]. While previous studies conducted in Greece demonstrated that ivermectin cured and prevented infection in 23 dogs with subconjunctival onchocerciasis [44], 16 ivermectin-treated dogs in the USA showed a 67% infection recurrence [38]. This could be due to the strain of O. lupi circulating in the USA or to specific epidemiological conditions that are yet unknown [45].
Similarly, the moxidectin/imidacloprid spot-on formulation herein tested was shown to be efficacious against mfs of Cercopithifilaria spp. This study confirms a previous report which suggested the efficacy of the imidacloprid/moxidectin spot-on formulation in association with prednisone in treating C. bainae-caused dermatitis in the USA [26]. The prevalence of infection by O. lupi at the initial screening (i.e. 1.5%) was lower than that recorded in a previous study in the same geographical area (i.e. 8% [40]; 17.1% [39]). However, due to the knowledge gaps in the life cycle of this filarioid (e.g. the identity of the vector is unknown), the epidemiology of the infection remains rather obscure. On the other hand, for Cercopithifilaria spp. the overall prevalence herein detected (19.8%) is similar to that previously reported in the same area in Portugal (i.e. 23.5%; [23]) and in central Spain (i.e. 21.6%; [21]). Particularly, C. bainae was the most prevalent species, followed by Cercopithifilaria sp. II and C. grassii as reported in the same geographical area [23].

Conclusions
Results obtained in this study indicate that moxidectin/imidacloprid spot-on formulation seems to be efficacious for the treatment of O. lupi and Cercopithifilaria spp. skin-dwelling mfs in infected dogs in a single administration. Therefore, this treatment may prove useful for the prevention of the infection caused by these little-known filarioids. Finally, assessing the microfilaricidal efficacy in dogs could also be useful in reducing the risk of O. lupi infection for humans.