Eﬃ cacy of ozonized sunﬂ ower oil as treatment of canine generalized demodicosis

Background: The acaricidal action of Ozonized sunﬂ ower oil (OSO) has demonstrated in diﬀ erent clinical cases of diﬀ erent animal species, such as psoroptic rabbits and pig sarcoptic scabies and recently in demodesic goat. Objectives: This study evaluated the eﬀ ectiveness of OSO as treatment for generalized demodicosis mange in dogs. Animals: Twenty dogs of diﬀ erent breeds, between six months and one year of age with generalized demodicosic mange that attended to Veterinary Clinic “José Luis Callejas” Havana, Cuba, during the year 2015, were included in the study. Methods: The demodicosis diagnostic was based on its clinical history, the mite’s presence by deep scraping and clinical signs. All cases had a history of receiving before conventional treatments without solution. OSO treatment was topically applied daily, every 12 hours, after shaving all aﬀ ected areas. Monitoring of clinical signs, mite counting on scaling, pruritus and capillary regrowth (7, 14, 28, 56 and 84 days) were performed. Results: The results showed a signiﬁ cant reduction of mite counts, clinical signs and pruritus since the 7 th day of application. The animals recovered the fur on more than 90% of the body surface. All the animals (100%) recovered from generalized demodesic mange in 84 days of treatment. Conclusion: The eﬀ ectiveness and safety of OSO as election treatment of generalized demodicosis mange in dogs was demonstrated.

and secondary bacterial infections are very frequent [3]. The diagnosis is typically based on clinical signs and is con irmed by the presence of mites in deep skin scrapings. Finding more than one mite is strongly suggestive of clinical demodicosis [3,4].
Amitraz, as a rinse or sponge-on, has approved for the treatment of canine generalized demodicosis in many countries for decades. Several amitraz-based protocols was described at various concentrations and frequencies [7]. Protocols based on daily to weekly oral or subcutaneous injections of macrocyclic lactones including ivermectin, doramectin, and moxidectin have been published but represent off-label use with potential for toxicity, especially in dogs mutated for MDR-1 (P-glycoprotein de iciency), especially including colley breeds [4,7,8]. Also daily oral milbemycin oxime at a dose of 500 mg/kg is registered in many countries for the treatment of canine demodicosis [1]. Whatever the choice of the antiparasitic drug, the duration of the treatment of demodicosis usually requires three months or more [9]. Certifect ® is a spot-on formulation that combines ipronil, amitraz, and (S)-methoprene. The addition of amitraz to ipronil has shown to signi icantly potentiate the acaricidal effects of ipronil [10,11]. The product has been recently demonstrated to be active against sarcoptic mange [12]. Fluralaner (Bravecto ® , Merk Animal Health; Madison NJ, USA) is a long acting systemic insecticide and acaricide belonging to the isoxazoline class of parasiticides with selective inhibition of arthropod gamma-aminobutyric acid and L-glutamate gated chloride channels. It is an inhibitor of the arthropod nervous system. An oral single dose of luralaner proved to be > 99% effective against leas and ticks over a period of 12 weeks [13,14]. Two studies reported the ef icacy of luralaner in canine generalized demodicosis, both showing that luralaner can decrease the number of demodex mites by 100% after 2 months [15][16][17]. Isoxazolines are safe for dogs with the MDR-1) mutation [18].
Ozonized Sun lower oil (OleoVet) have been well physicochemically characterized . In addition, its antibacterial activity was demonstrating on in vitro studies [23][24][25]. This pharmaceutical product has demonstrated a bene icial effect as treatment of different dermatological parasitic diseases in domestic animal, such as in rabbits psoroptic scab [26], pigs sarcoptic mange [27] and localized demodesic scab in Goat species [28].
Additionally, antiparasitic action of OSO was evidence too. The in vitro antigiardiasic action of OSO was demonstrated [29] and in vivo [30] studies. Recently a clinical trial demonstrated its effectiveness as a treatment of giardiasis in beagle dogs [31].
Based on these indings, the purpose of the present study was to determine the ef icacy of topical administration of OSO in dogs with generalized demodicosis.

Materials and methods
The study was conducted as a nonblinded, controlled open label trial. Pet owners provided written consent and the study was approved by the Animals Care and Use Committee of the National Center for Scienti ic Research.

Ozonized sunfl ower oil obtain
The sun lower oil was placed in a jacketed bubbling reactor at a controlled temperature of 25 ± 0.1 °C. A mixture of O 3 /O 2 was continuously bubbled into the solution through a diffuser placed at the bottom of the reactor. The ozone was generated from pure oxygen in an Trailigaz OZAT CFS-7, France, ozone generator and its concentration was determined by measuring the absorbance at 254 nm in an Anseros ozone analyzer, Germany.

Inclusion criteria
Participants were client-owned dogs diagnosed with generalized demodicosis between January 2015 and march 2016. Medical records, including previous acaricidal treatments and underlying diseases (for adult-onset cases) were documented. Demographic data recorded, including age between sex months to one year, breed and gender (males and females). Breed distributions were not compared to the general hospital populations to ascertain breed speci ic risk for disease.

Exclusion criteria
Puppies less than 6-weeks-old and dogs weighing less than 2 kg, pregnancy animals were excluded from the study.
Following all this inclusion and exclusion criteria, the study was performed with twenty dogs of different breeds (males and females) weighing from 6 to 19 kg and being at least 1-year old. The owner give the consentient to participate on the study. All had clinical signs of Demodex infestation such as erythema, hair loss, seborrhea, follicular casts, scales, and crust in at least ive spots or on an entire body region. All animals included in the study has received traditional treatment for more than two times before, without diseases solution.

Clinical methods
Generalized demodicosis was de ined as more than four small areas (< 100 cm 2 ) on the body affecting by demodex, at least one < 100 cm 2 , or at least one paw. Five deep skin scrapings, from ive affecting areas, were taken in each dog exhibiting at least ive lesion areas. The surface of each lesion skin scraping was four cm 2 , using a blade until capillary oozing occurred. The diagnosis of demodicosis was based on inding more than three live adults Demodex canis mites from at least three or more scrapings, or from lesions > 100 cm 2 or from a lesional paw [32]. The scrapings were mixed with oil and examined under a stereomicroscope. https://doi.org/10.29328/journal.ivs.1001030 The average of percentage reduction in mite counts was calculated by:

Reduction % = GM inclusion -GM post-treatment/GM inclusion X 100
Where GM Inclusion = the geometric Mean (GM) of the Inclusion (pre-treatment) mite counts, and GM posttreatment = the geometric mean of the post-treatment mite counts.
Clinical signs and the extent of demodectic lesions on each dog were assessed on the days when scraping were made [33]. At each clinical examination, the clinician determined as Skin Lesion Score (SLS) based upon the principles of the Canine Atopic Dermatitis Extent and severity Index, CADESI [34], but adapted for the lesions of generalized demodicosis. This SLS evaluates lesions including erythema, erosions, comedones/ papules/pustules, scales/crust at least ive spots or on an entire body region from zero (normal) to ive (severe).
A semi-quantitative assessment of hair regrowth was made. The skin surface of the dogs on which hair regrowth occurred, compared to pretreatment observations, was occurred as 1 if < 50% hair regrowth, 2 if 50% -90% regrowth occurred, and 3 if > 90% hair regrowth took place [33].
The owners rated the current pruritus on the day of consultation on a visual score from 0 (no pruritus visible) to 10 (severe pruritus), using a validated pruritus scale [35,36]. Furthermore, owners rated the overall condition before and after treatment as improved, deteriorated or unchanged.
The treatment of OSO was as a ine line on the lesions areas applied every 12 hours (at mornings and at night) previously the lesions areas were shaved. The owner of the dogs were the responsible for the treatment. All the dogs were evaluated every two weeks for 12 weeks: four skin scraping were taken from four initially effected areas and the clinical evaluation of CADESI, hair regrowth and pruritus scale were registered every two weeks for 12 weeks. Side effects were recorded during the study period.
In this study, the ef icacy of OSO was de ined as the absence of demodex canis mites in any life stage at monthly scraping for eight consecutive weeks. These dogs received no other treatment against ectoparasites during the study period.

Statistical methods
All the dates were analysis by One-way analysis of variance, using for normal distributed variables with Tukey´s multiple comparison test. All the statistical analysis were performed by GraphPad Prism Version 5 program. Continuous data are represented as means ± standard desviation. Statistical signi icant was set at p ≤ 0.05.

Results
The primary assessment variable in the study was the decrease in total number of mites counted in skin scrapings following OSO treatment. The OSO treatment applied by topical way reduced signi icantly the mean mite number present in skin scrapings at 28, 56 and 84 days of treatment, representing (25. 4, 3.9 and 0 mite, respectively) compared to mite counted before treatment (54.3 mites), representing 54, 93 and 100% of reduction, respectively ( Figure 1).
Generally, improvement become evident in all dogs included in the study, within two weeks (14 days) after OSO treatment started, by decreased score lesion skin (SLS) severity of affected areas (Figure 2). OSO reduced signi icantly (62. 9, 86 and 100% reduction) the SLS at +7, +14 and +56 days, respectively after treatment, compared to before treatment. The SLS was totally reduce at 84 days of treatment. The dog´s owner reported no side effects during the treatment.
The presence of pruritus was another clinical signs registered in this study. Surprisingly, the OSO reduced pruritus score signi icantly in all dogs included in the study, with reduction percent of 51% and 85% at 7 and 14 days of treatment, respectively ( Figure 3). The pruritus reduction, as a main signs of allergic-in lammatory reaction from the skin parasite with demodex canis, it was in correspondence to the reduction of the SLS at 7 and 14 days of treatment with OSO ( Figure 2). Hair regrowth compared to the proportion of the body covered by hair prior to treatment is in igure 4 summarized. Up to the 56 th days of treatment with OSO, the animals recovered in more than 50% capillary regrowth, while at the 84 days of treatment achieves it in more than 90%. One of the most important observation, was during this evaluation time of the skin dermatitis, the animals experimented irstly between seven and fourteen days an alopecic sign due to the OSO application. Such alopecia was found in correspondence with the reduction of SLS. Alopecia reversibility was demonstrated after 56 days of OSO treatment, and to 84 days, the hair regrowth was observed until 90% of affected body surface (Figure 4). These results could be seen with better certainty in the igure 6 CD and EF, one of the animals selected.
The SLS decrease was shown too by a representative pictures from one of the cases include in the study before the treatment ( Figure 5A,C). Both pictures show, the mean clinical skin changes characteristic of generalized demodicosis, as granulomatous chronic in lammation, erosions, dryness and licheni ication, accompanied by alopecia and an opaque appearance in the hair. OSO treatment applied every 12 hours (mornings/nights) during 56 consecutive days evidenced the improvement of the skin damage of the animals with generalized demodicosis. Those are representing in the igure 5 B,D, where show in this case no lesions in the skin and the recovery of hair regrowth in more than 90% of the body surface. Figure 6 A,B, shows the animal´s face area before start the treatment, where erosions, scabs, with edematous and granulomatous appearance, as lesions typical of demodesic dermatitis clearly showed. After 21 days of OSO treatment in igure 6 C,D, are shown (left and right, respectively). The reversion of the skin lesions, previously observed in igures. A and B. However, it would be note that this skin healing aspect areas of the animal was accompanied by an obvious alopecia in the affected areas treated with OSO during the 21. This experimental alopecia observed in the animals was reverted since 56 days of treatment applied, where capillary regrowth in more than 90% was observed ( Figure 6E,F). Actually, the healing mechanism demonstrated for Ozonized oils (OOs), were based on that peroxides presents into the OOs, activated NF kB expression [48], increase the initial in lammatory response [39], thought out the ibroblast in iltration and collagen synthesis, without phases of the process alterations [39]. Therefore, this healing mechanism was accompanied by the increment of growth tissue factors expressions, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) [49]. However, knowing that this latter factor (TGF-β), is linked to the immunosuppression experienced in demodicosis [50], it is suggested that this factor, according to the favorable results achieved, was not directly linked in. The mechanism of the healing process with the treatment of OSO in cases of generalized demodicosis in canines, and if the rest of the growth factors (PDGF), vascular endothelial growth factor (VEGF).
The capillary regrowth is an important aspect to consider, due to the applied treatment, for the resolution of infestation by demodex canis and lesions caused by demodesic dermatitis. However, during this recovery process of animal's skin, it must emphasize that there was induced alopecia experience, due to application-evaluated product. Such alopecia, took place from 28 days of application product ( Figure 6, C,D). The alopecia reversibility seen at 56 days of OSO treatment. In an experimental study demonstrated, that topical application of ozonized olive oil (AOO) in mice produces apoptosis of the matrix of hair follicle cells at 6 days of application, reversing this effect at 35 days of evolution [51].
In general, the effectiveness and safety of OSO applied every 12 hours during 84 days as treatment of generalized demodicosis in canines was demonstrated. Suggesting that irstly, OSO eliminated mites that are parasitizing both hair follicles and sebaceous glands. Together to its proin lammatory/anti-in lammatory action, OSO reverses the immunosuppression process induced by the mites itself, activating the initial in lammatory response (neutrophil and ibroblasts in iltration), important for degrading the detritus produced by the mite. Then, detritus degraded lead to granulomas formation and increase growth factors expression, favoring the healing process. On the other hand, OSO increases the endogenous antioxidant enzymatic activity in the damaged skin, thus reversing the oxidative damage generated by the parasitosis itself and in this way favors the resolution of the generalized demodicosis in canines.
The results indicated as conclusion, that ozonized sun lower oil could be used as choice therapy for generalized demodicosis treatment for dogs.

Discussion
The present study demonstrates that OSO treatment with for almost for three months, twice a day, resulted in a rapid reduction in mite numbers and a marked improvement in clinical signs, determined as Skin Lesions Score (SLS), in all dogs with generalized demodicosis.
The acaricide OSO action by signi icant reduction of mites count on parasitize site infection sampled was demonstrated. The decrease of mites account achieved by OSO treatment was in correspondence to the SLS reduction (62%) after 7 days application and the 100% of reduction at 56 days. OSO acaricidal action was demonstrated as the main pharmacological effect in different studies both in vitro and in vivo, as in the case of rabbit psoroptic [27] and pig sarcoptic mange [26]. Recently, the effectiveness of OSO was demonstrated as a treatment for localized demodesic mange in goats [37].
Pruritus represent a characteristic clinical sign of the hypersensitivity allergic reaction, induced by the presence of the mite. The pruritus was signi icantly reduced (51%), after 7 days of OSO application. These results constitute the irst evidence on the effectiveness and safety of AGO treatment in generalized canine demodicosis.
Taking these results into account, the acaricidal action of the OSO was considered the irst line of pharmacological action. Additionally, the anti-in lammatory activity of OSO is evidenced in our study by reducing SLS (edema, erythema) and pruritus, corresponding to previously reported experimental results where OSO reduced the edema formation and in lammatory in iltrated cells [38]. The results of experimental studies designed for this purpose [39], as well as a clinical study in horses [40], canines [41]. Associated with the stimulation of endogenous antioxidant enzymes in skin damaged by UV radiation [21] and experimental studies (suggesting the great potential of OSO as a treatment of choice for generalized demodesic mange in dogs. Oxidative stress (OS) has been implicated in various mite's infestations including canine demodicosis [42,43]. Numerous cytokines and growth factors have been described to generate ROS to transmit information upon binding to their receptors [44]. Therefore, shifting of oxidant/antioxidant balance in demodicosis dogs toward oxidative status could be involved in the progression of canine demodicosis [45]. Considering, another bene icial pharmacological action of OSO, as stimulator of some antioxidant enzymes activity (SOD and GPx). Action that was in different experimental models demonstrated, like orthokeratosis and UV damage skin in rats [21,46]. Recently, was demonstrated that SOD activity, as antioxidant enzyme increase in mice skin damage was associated to ozonized oils (OOs) healing action [47]. Thus, it suggests, OSO could be able reverts oxidative stress, involved in skin demodicosis infection too.