The study was carried out during the summer 2015 on 18 half-breed dogs (3 - 10 years old and body weight 15 - 20 kg), suffering from skin diseases, housed in a rescue shelter located in Messina (Southern Italy). The animals, fed with a commercial dry food and free of antibiotics, were selected based on evident skin lesions among 120 examined dogs.

The dogs affected by skin infections showed clinical signs such aspruritus, alopecia and/or erythema, presence of pustules or crusts, ulcers and/or erosions. The lesions were observed at the level of armpit, abdomen, groin and thigh. General symptoms included slightly raised body temperature, anorexia and depression. Diagnosis of skin infection in dogs was performed by clinical signs, cytological, bacteriological and mycological investigations and skin scrapings for parasites on cutaneous samples.

Dog skin samples were obtained from pustules and/or epidermal collarettes, using sterile swabs and plastic brushes, respectively for bacteriological and mycological surveys, and skin scrapings using sterile scalpels for cytological investigation and scrapings for parasites. The samples were transferred under sterile conditions and examined at the laboratory of infectious diseases of the University of Messina.

Samples of skin scrapings from affected dogs, taken with sterile disposable scalpels, were streaked onto slides, dried, stained with May Grunwald-Giemsa and microscopic examination was performed at high power under oil immersion (1000×).

Within 1 hour from collection, the skin swab samples were transferred into test tubes of Selenite-broth (Biolifes. r. l., Milano, Italy) and incubated to 37˚C for 24 hours. The next day, from each tube was removed the bacterial inoculum and swiped into the plates containing the following selective and differential media, SS-Salmonella-Shigella agar (Oxoid, Basingstoke, Hampshire, UK) e SM-Staphy- lococci 110 medium (Biolifes. r. l., Milano, Italy ), and then incubated to 37˚C for 24 hours, for the isolation of Gram-negative and Gram-positive bacteria respectively. Afterwards, the isolated colonies were transferred into tubes containing BHI-Brain Heart Infusion broth (Biolifes. r. l., Milano, Italy) for the identification and into tube slant containing Mueller Hinton II agar (Biolifes. r. l., Milano, Italy) for the conservation of the strains and incubated at 37˚C for 24 h. The isolated colonies were submitted to rapid bacterial identification by Mass Spectrometry VITEK MS (bioMérieux SA, Marcy l’Etoile, France). The samples for bacteriological investigations were performed before and 3, 5, 7 and 15 days after the treatment.

The bacterial isolates were identified by MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry), designed to provide an accurate and reliable bacterial identification rapidly. This procedure allows the measurement in an extremely accurate molecular weight of biological macromolecules and to determine their identity according to the mass/charge ratio. In particular, MALDI-TOF-MS is valid for the detection of high-abundance soluble proteins, including ribosomal and other structural proteins, directly from microbial cells, resulting in spectra that are analyzed with the VITEK MS system (bioMérieux SA, Marcy l’Etoile, France), using for reading the software Axima (Shimadzu Kyoto, Japan)-SARAMIS database (Spectral ARchive And Microbial Identification System) (AnagnosTec, Berlin, Germany). The spectra were acquired in positive linear mode in the range of m/z 2000 to 20,000. The isolated colonies were seeded in a 48-well metal plate with disposable loops, using as a reference strain Escherichia coli ATCC 8739.

The susceptibility of the isolated microorganisms to Thymus vulgaris EO was estimated by the in vitro bacteriological test using the disk diffusion method (Kirby-Bauer) in Mueller Hinton II agar (Biolifes. r. l., Milano, Italy) and measuring the diameter of the growth inhibition area, as recommended by CLSI (Clinical and Laboratory Standards Institute 2015) [34] . In particular, in vitro activity of Thymus vulgaris EO on the bacterial strains isolated from skin samples of dogs was evaluated in comparison to Citrus bergamia EO and some antimicrobial drugs widely used in veterinary medicine (amoxicillin-clavulanic acid, doxycycline, thiamphenicol and marbofloxacin) (Oxoid). Specifically, paper disks (6 mm diameter) were loaded with 20 µl (18 µg) of antimicrobial drugs, mixed with Tween 80 (Sigma Tau) (1:1 v/v) and then placed on the surface of the Muller Hinton II agar, inoculated with a suspension of the various isolated microorganisms (0.1 ml of 10⁸ CFU/ml). The plates were incubated at 37˚C for 24 h, subsequently the diameter (mm) of the growth inhibition areas were measured. All the assays were performed in duplicate. The microorganisms were considered very susceptible, susceptible, poorly susceptible and resistant when inhibited in vitro by concentrations of 0.56 µg/ml or less, 0.57 - 6.25 µg/ml, 6.25 - 12.5 µg/ml and greater than 12.5 µg/ml respectively.

Samples of skin scrapings and hairs were taken from the dogs using a sterile scalpel, then streaked on a specimen slide with the addition of a drop of KOH (10%) and sealed with coverslip and then observed microscopically (40× - 60×).

Afterwards, various cutaneous samples were collected from the dogs using sterile plastic brushes and were sealed in individual sterile bags. Subsequently, the brushes were impressed on Petri dishes containing Potato Dextrose agar (Biolife). The growth of fungal colonies was kept under observation for 7, 15, 21 and 30 days. Suspect colonies which developed (white, smooth, raised, not mucous) only in samples n. 3, 7 and 8 were seeded in Dermatophyte Selective Medium (Biolife).

The EOs tested (Thymus vulgaris L. and Citrus bergamia Risso & Poiteau) were purchased directly from the market (KOS srl, Lab. di Erboristeria, Prato, Italy).

The present investigation with an extra-label use of Thymus vulgaris essential oil in dogs was approved by Commission on Drug Experimentation of the University of Messina and carried out in accordance with local ethical regulations for veterinary practice [35] [36] [37] and with the prior informed consent of the kennel veterinarian.

The use of topical treatments on dogs were reported in the register of the rescue shelter “Millemusi” (Messina).

The dosage of T. vulgaris EO (20 ml/1000ml H₂O) was prepared in accordance with the manufacturer’s instructions. In particular, for this trial the use of a positive control group (treated with Citrus bergamia EO (20 ml/1000ml H₂O) rather than an untreated group was applied according to regulations for veterinary practice. Dogs were blocked in pairs and each animal assigned to one or other group at random. The two groups of animals were subjected to topical treatment using a sprayer.

In particular 4 ml of T. vulgaris EO (Group 1: n. 10 animals) and C. bergamiaEO (Group 2: n. 8 animals) respectively were sprayed in the areas affected by lesions for 7 days consecutively. Dogs with complete disappearance of clinical signs of skin infection were subjected to further preventive treatment for 10 days with T. vulgaris EO (Group 1). To prevent licking a collar was applied to each dog.

Effectiveness of essential oils was evaluated on the basis of the changes in the animal’s clinical signs, observed before treatment (T0) and 3, 5, 7 and 15 days after the drugs administrations, using a scores system [38] as follows:

・ pruritus (0 absent; 1 slight; 2 moderate; 3 marked).

・ alopecia and/or erythema (0 absent; 1 slight; 2 moderate; 3 marked).

・ pustules and/or crusts (0 absent; 1 slight; 2 moderate; 3 marked).

・ ulcers and/or erosion (0 absent; 1 slight; 2 moderate; 3 marked).

・ general health (0 good, 1 quite good, 2 fair, 3 poor).

The effectiveness of Thymus vulgaris EO (Group 1) and Citrus bergamia EO (Group 2) was evaluated by comparing the clinical signs in dogs and the microorganisms isolated from skin swab samples before and to 3, 5, 7 and 15 days after the treatment. All animals were kept under observation during the experiment to check for possible side effects.

The data were expressed as mean ± S.D. elaborated using an analysis of variance (ANOVA) InStat 3.0 software (GraphPad Software San Diego, CA, USA) was applied to assess the significance of the difference between the scores recorded before and 3, 5, 7, and 15 days after the treatment with Thymus vulgaris and Citrus bergamia EOs respectively. Difference with p < 0.05 were considered significant.

The cytological investigation of skin samples performed on 18 dogs with skin diseases highlighted the presence of inflammatory cells (neutrophils, lymphocytes, plasma cells and bacteria) (Figure 1). This cytological pattern, found in all the dogs, is indicative of the presence of a skin inflammation process.

The bacteria isolated by microbiological test from the skin swab samples of 18 dogs affected by bacterial skin disease were: Staphylococcus pseudintermedius (n. 7 samples), Staphylococcus sciuri (n. 4), ESBL Escherichia coli (n. 3) and

Proteus mirabilis (n. 4).

The in vitro susceptibility of the isolated microorganisms in dogs to Thymus vulgaris and Citrus bergamia EOs and other antimicrobials widely used in veterinary medicine amoxicillin-clavulanic acid, doxycycline, thiamphenicol and marbofloxacin) is reported in Table 1. Antimicrobial activity test in vitro showed that Thymus vulgaris EO is the most active antibacterial drug in the comparative analysis.

The bacteriological findings in the skin samples of dogs before (T0 baseline) and

+++ Highly susceptible (MIC ≤ 0.56 µg/ml); ++ Susceptible (MIC 0.57 - 6.25 µg/ml); +Poorly susceptible (MIC 6.26 - 12.5 µg/ml); - Resistant (MIC > 12.5 µg/ml).

3, 7 and 15 days after the end oftopical treatment for 7 days with Thymus vulgaris and Citrus bergamia EOs respectively are indicated in Table 2. The severity of the bacterial skin infection of dogs was expressed in the conventional manner (from + to ++++) in relation to the quantity of bacteria present (Table 1). No microorganisms were isolated in all skin samples of dogs at 7 and 15 days after the end of Thymus vulgaris EO treatment. No variation in the bacterial load, instead, was recorded in the animals treated with Citrus bergamia EO.

The skin scrapings for mites and mycological investigations for dermatophytes in all skin samples of dogs were negative.

Table 3 and Table 4 report the mean scores for clinical signs (pruritus, alopecia and/or erythema of the skin, presence of pustules or scabs, ulcers and/or erosions)

++++ = 10⁶ colony forming units (cfu); +++ = 10⁵ cfu; ++ = 10⁴ cfu; + = 10³ cfu.

*p < 0.01; **p < 0.05.

in dogs affected with skin infection before and at various time after the treatment with Thymus vulgaris and Citrus bergamia EOs respectively.

In Figure 2 is reported the trend of visible topical effect before, during and after the treatment with Thymus vulgaris EO (Group 1). The dogs with skin infection (Figure 2(a)) showed a moderate decrease of dermic clinical signs after 3 days but significant after 5 days (Figure 2(b)); an evident remission of disease it was observed after 7 days (Figure 2(c)), with complete disappearance of ulcers and erosions and initial regrowth of hair, followed by skin normalization. The treatment with Citrus bergamia EO (Group 2), instead, showed no significant effects in dogs with bacterial diseases of the skin and the clinical signs persisted markedly for the duration of our investigations.

No local or general signs of Thymus vulgaris EO intolerance were recorded in any treated dogs and no relapses occurred in the month following treatment.