This study aimed at evaluating the neem oil antibacterial activity towards wound-contaminating-bacteria resulting from ectoparasites sting and bite. Mesophilic, thermophilic, E coli, E coli eae, hlya, EAST1 gene positive strains, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa were isolated by a non-invasive technique for microbiological sampling. Multiplex PCR assays using PMA™ dye and species specific primer pairs allowed the characterization of isolated bacteria and the detection of bacterial viable cell after neem oil treatments. The antibacterial activity was evaluated by standardized disc diffusion test and microdilution test. Neem oil showed antibacterial activity towards all isolates tested. The neem oil bacterial growth inhibition zone (mm) by disc diffusion test (100 μl neem oil) ranged between 22 ± 0.68 (P. Aeruginosa) and 30.21 ± 1.38 (E. Coli EAST1). The antibacterial activity was determined at lower neem oil dilutions (1:10 – 1:10,000) by microdilution method. The highest bacterial growth reduction (%) ranged between 81.51 ± 1.15 (S. Aureus sub sp. aureus, one of the reference strains considered in the experiment) and 95.51 ± 1.15 (E. Coli hlya) (100 μl neem oil and 50 μL of bacterial suspension, ca 1 × 106 CFU/ml). The effect of neem oil, Tween 20^® as well Tween 20^® and neem oil together at several dilutions (1:2 x 10²; 1:2 x 10³; 1:2 x 10⁴; 1:2 x 10⁵; 1:2 x 10⁶; v/v) towards goat peripheral blood mononucleate cells viability was investigated, too. Only 1:2 x 10² and 1:2 x 10³ dilutions of neem oil, Tween 20^® as well neem oil and Tween 20^® affect PBMC viability. On the basis of the obtained results neem oil should be used directly on wound or diluted in Tween 20^® for skin care.

Keywords: Wound-Contaminating-Bacteria; Antimicrobial Activity; Neem oil; Goat

Ectoparasites play a very detrimental role in terms of decreasing the productivity of sheep and goats. Common external sheep and goat parasites include ticks, lice, and mites. They cause restlessness and nuisance. Weight loss and reduction in milk production may occur as a result of nervousness and improper nutrition because animals spend less time eating. Bites can damage sensitive areas of skin (teats, vagina, eyes, etc.). Some parasites feed on blood causing blood-loss anemia, especially in young animals. The ectoparasite bite and sting let bacteria proliferate in wound from abrasions or lesions from scratching and cause levels of tissue reaction of different entities, super-infection and cervical lymphadenopathy. They also seriously damage sheep and goat skin resulting in the rejection or downgrading of the skins. This causes huge economic losses through skin damage rendering it unsuitable for the leather industry due to the decrease in skin quality. Wool is lowered in value, too. Pseudomonas aeruginosa wound infections are characterized by a change in the color of the skin around the wound area and formation of lesions [1]. The bacterium products and pigment cause a yellow discoloration of wool (http://www.infovets.com/books/smrm/A/A972.htm).

A major concern emerging in recent years in the field of bacterial infections, which stimulate the development of innovative molecules with antimicrobial activity, is represented by the problem of multiple antibiotic resistance, i.e. onset and rapid growing global dissemination of pathogens that have acquired resistance to multiple antibacterial drugs in use, capable of supporting common infections, potentially fatal for animals and humans. According to World Health Organization (WHO, 2002-2005) [2] medicinal plants would be the best source to obtain a variety of drugs. In fact, many plants produce secondary metabolites which act towards wound-contaminating-bacteria and parasites [3,4]. Plants are a good source of antioxidants and also cure various disorders associated with wound inflammation [5]. Identification and studies on active compounds from plants are now of major interest to the scientific community [6]. These include compounds such as diterpenoid from raw plant material which showed antibacterial activity towards Streptococcus pyogenes and some fungi. In addition, tannins, found in a number of plants used in wound healing were also found to have antibacterial activity [7].

Nowadays, Neem (Azadirachta indica A. Juss) is considered an effective source of environmentally-powerful natural pesticide and considered to be one of the most promising pesticides. It is believed to be one of the trees of the 21st century for its great potential in pest management, environmental protection and medicine [8]. Among the many products obtained from the seeds, neem oil is the most commercially relevant (www.organicneem. com/why_parker_neem.html). More than four hundred substances were identified in the neem oil. The most biologically active are the nortriterpenoid limonoid. In particular, the azadirachtin A is the compound best known and studied for its properties that allows its use in agriculture as a natural insecticide, acaricide and nematocide, but it should not forget the salannin and nimbin, among other major limonoids. The insecticidal properties of neem oil are closely related to the larvicide, acaricide, nematicide, and repellent effects.

The antimicrobial activity of neem oil has been investigated [9,10] . In addition, it has been recently demonstrated the neem oil antibacterial activity towards several food spoilage bacteria [11] and strains of enteropathogenic Escherichia coli [12]. This study aimed at evaluating the neem oil antibacterial activity towards bacteria isolated from animal wounds resulting from ectoparasites sting or bite.

The microbiological, molecular biology, and Peripheral Blood Mononuclear Cells viability assays were carried out in the Biochemistry and Cell culture Laboratories of the Research Center for Animal Production, CRA PCM, Monterotondo (RM), Italy. Blood specimen for Peripheral Blood Mononuclear Cells viability assay were collected from goat reared in the Research Unit for the Extensive Animal Husbandry, CRA ZOE, Muro Lucano (PZ), Italy. High Performance Liquid Chromatography analysis was carried out in the Quality Control Laboratory of the University of Rome Sapienza, Dep. Environmental Biology, Rome, Italy.

A commercial neem oil produced by Neem Italia (Manerba (BS), Italy) was used as test starting material (0.35% azadirachtin A) determined by High Performance Layer Chromatography. It is a mixture of neem oil and emulsifying agents making it more fluid and less liable to hardening at cold temperatures and more suitable for inclusion in products or in cosmetics for animals. Neem oil was further diluted in Tween 20^® (1:1 v/v; VWR, PBI International, MI, Italy) under agitation and sterilised by filtration through a 0.22 μm Millipore express filter (Millex-GP, Bedford, OH, USA) before using in the experiment.

The neem oil metabolomic fingerprint was determined by High Performance Thin Layer Chromatography according the methodology previously developed [12,13].

Five goats reared in the Research Unit for the Extensive Animal Husbandry CRA ZOE, naturally infested by lice and showing wounds resulting from ectoparasites sting and bite, were randomly chosen for bacteria isolation. Bacteria isolation from complex infected skin lesions was made by a non-invasive technique for microbiological sampling performing the removal using flocked swabs (FLOQ Swabs™ patented technology, Copan, Brescia, Italy). Flocked swabs aimed to collect more sample volume than traditional swabs and automatically and completely release the sample immediately when in contact with liquid or surface. Three flocked swabs were used for each goat. ESwab and two selective enrichment broths for single organism screening: TSB salt broth and LIM broth (Copan, Brescia, Italy) were used respectively for S. aureus and Streptococcus spp . The isolation from ESwab, TSB salt and LIM broth are compliant with the Quality Control of Microbiological Transport Systems; Approved Standard, M40A2 (CLSI M40-A2, 2014) [14] as per guideline DIN 58942-4 Supplement 1 (DFNV, 2004) [15] and performed according to the manufacturer's instructions. The swab method was applied by taking three smears per sample on an area of 100 cm2 defined with a delimiter, adding 100 μL of isotonic eluent furnished by the Manufacturer for decimal dilutions and plating 1 mL per dilution on selected media using a sterile bacteriology loop to streak the primary inoculum across the surface of the second, third and fourth quadrants of the agar culture plate. Count determinations for mesophilic, thermophilic and coliform bacteria with Escherichia coli distinction and qualitative assays were also made to sample a wide range of microorganisms.

Mesophilic and thermophilic bacteria were counted on Dextrose Tryptone Agar (Oxoid, Garbagnate Milanese (MI), Italy), after incubation at 32°C for 72 hours and 55°C for 48 hours, respectively.

Coliforms with E. coli distinction were counted on Chromogenic Compact Dry and Chromogenic Compact Dry with Escherichia coli distinction (PBI, Milano, Italy) after incubation at 35°C for 20 hours [16, 17].

Baird Parker Agar (Acumedia, Neogen, lansing MI, USA) was used as selective medium for the isolation and presumptive identification of catalase-positive staphylococci. It contains 7.5% sodium chloride and thus selects for those bacteria which can tolerate high salt concentration.

Mannitol Salt Agar is both a selective and differential medium used in the isolation and distinction of S. aureus and S. epidermidis.

Catalase-negative Enterococcus spp. and Streptococcus spp. were isolated on Columbia Blood Agar Base, Streptococcus Selective Supplement and Defibrinated Horse Blood (OXOID, Basingstoke, UK) incubated plates at 37°C, either aerobically or anaerobically and examined after 18 to 24 hours incubation.

Pseudomonas Isolation Agar (Acumedia, Neogen Corporation, lansing, MI, USA) was used for the isolation of Pseudomonas aeruginosa and other Pseudomonas spp. under aerobic incubation at 35 ± 2°C and examined for growth after 18-48 hours. The medium includes Irgasan, a broad spectrum antibiotic not active towards Pseudomonas spp. This medium is selective and formulated to enhance formation of blue or blue-green pyocyanin pigment by Pseudomonas aeruginosa. The pigment diffuses into the medium surrounding growth.

Escherichia coli, American Type Culture Collection (ATCC)^® 51813^™, S. aureus subsp. aureus ATCC^® 6538™, E. faecalis ATCC^® 7088TM, and P. aeruginosa ATCC 27853 were used as reference strains.

To prepare working cultures, stock cultures were standardized through two successive 24 h growth cycles in Brain Heart Infusion broth (Difco, Buccinasco, MI, Italy) at 20° C without agitation.

The reference strains were grown on media and at the growth conditions as reported on products sheets.

The antibacterial activity of neem oil was assayed using standardized disc diffusion agar and microdilution methods. Whatman filter paper (no.1) discs (6 mm diameter) were impregnated with 100 μL of neem oil. Discs impregnated with 100 μL of sterile distilled water, Tween 20^®, as negative controls, and 10 μL (wt/v) of ciprofloxacin (1 mg/mL, Bayer, Milano, Italy), as positive control, were used. Two discs were prepared for each sample. They were evaporated at 37°C for 24 h.

Bacterial suspensions (100 μL), standardized by adjusting the optical density to OD 1 at 600 nm (Shimadzu UV-120-01 spectrophotometer, Shimadzu, Cinisello Balsamo, MI, Italy), were used to inoculate by flooding the surface of suitable agar media for each microorganism considered at their proper growth conditions. Excess liquid was air-dried under a sterile hood and the impregnated discs were applied at equidistant points on top of the agar medium. The plates were done in triplicates for each organism and the experiment was performed twice. Plates were checked after incubation at 35°C and 42°C for 48 h. Antimicrobial activity was measured as diameter (mm) of the inhibition zone around the disc. The results were recorded as means ± SD of the duplicate experiment.

The antibacterial activity of neem oil towards the bacteria was also evaluated in microdilution assays using conventional sterile polystyrene microplates. Each well of the microplate was filled with 100 μL of sterile suitable liquid media for each microorganism considered, 50 μL of inoculums (see Table 4) and amounts of extract at lower concentrations (1:10 to 1:100,000) were added. Control treatment without neem oil was used in the experiment. The microplates were incubated at 37°C for 24 h. Bacterial growth was determined by OD 0.02 reading at 630 nm/10 mm path length with an ELISA microplate reader (Dynatech ML-3000, Pina de Ebro, España). Bacterial cell concentration was transformed to cells/mL using the reference curve equation. The reference curve was constructed by diluting 1:100 each bacterial species. Counting the number of bacterial cells of an aliquot of this dilution was done using a Neubauer chamber (Celeromics, Vedano al Lambro, MI, Italy). Finally, cell concentrations were transformed to a percentage of bacterial inhibition. The percentage of bacterial growth reduction (GR%) was estimated using as reference the control treatment (without neem oil) as:

GR% = C – T/C x 100

Where, 'C' is the cell concentrations under the control treatment and 'T' is the cell concentrations under the neem oil treatment.

Three replicates were considered. The results were recorded as means ± SD of the duplicate experiment. Differences between means of data were compared by Least Significant Difference (LSD) calculated using the Statistical Analysis System (S.A.S., Institute, Inc. Cary, NC, USA).

Molecular biology analysis was used either to identify and characterize isolated microorganisms or together with PMA^™ dye (Biotium, Hayward, CA, USA) to detect bacteria viable cells after neem oil treatment in vitro.

The dye (PMA^™ Biotium Inc. Hayward, CA, US) is a photoreactive dye with high affinity for DNA. It intercalates into DNA and forms a covalent linkage upon exposure to intense visible light. It is cell membrane impermeable. When a sample comprising both live and dead bacteria is treated with PMA™, only dead cells are susceptible to DNA modification due to their compromised cell membranes. Thus, it permits selective detection of the sole live cells.

DNA extraction was performed using ChargeSwitch^® gDNA Mini Bacteria Kit (Life Technologies Italia, Monza, MB, Italy) following manufacturer's instructions.

The molecular identification and characterization was made using specific primer pairs for each isolated bacterium as mentioned in the section microbiological analysis, mixture and reaction conditions as reported in literature (Table 2 list A and list B).

Two primer pairs that amplify specific E. coli 16S rRNA sequences and fourteen primer pairs that specifically amplify target gene coding for virulence factors (adhesins and toxins) were employed to characterize the E. coli isolates [18].

The detection and identification of Staphylococcus spp. were performed by multiplex PCR with primer pairs that amplify variable sequences of the nuc gene, specific for Staphylococcus genus and expressing thermo nuclease enzyme, mixtures and reaction conditions as reported in the literature [19].

Enterococci isolates were identified using Internal Transcribed Spacer region to identify the majority of enterococci and the ddl genes encoding D-alanine-D-alanine ligase specific for the individual species [20,21].

P. aeruginosa is an opportunistic pathogen capable of infecting virtually all tissues. Its molecular detection and identification was performed using PCR amplification of two specific outer bacterial membrane proteins: I lipoprotein (oprI) and L lipoprotein (oprL) using two sets of primers (fPS1/rPS2 and fPAL1/rPAL2) were used in multiplex PCR [22,23].

The amplified products were revealed by electrophoresis in agarose (2%) gel, stained with GelGreen^™ Nucleic Acid Gel Stains (Biotium, Inc., Hayward, CA, USA) and visualized by blue light transilluminator UltraBright-LED (Syngene Europe, Cambridge, UK) using a 100 bp DNA ladder (Thermo Fisher Scientific. Milano, Italy).

Water without templates and the reference strains were considered as negative and positive controls.

Tween^®20 as well Tween^® 80 is non ionic surfactants employed as emulsifier and stabilizer. They are accustomed in human foodstuffs, cosmetic, pharmaceutical preparations, and in many research methodologies. It has been recently reported that Tween®20 can induce decrease growth of treated cells (A459 cells and human umbilical vein endothelial cells) and apoptosis [24]. Since both surfactants were used in previous studies on the antibacterial activity of neem cake extract and neem oil

[25,26,27] the effect of neem oil in association with Tween 20® on goat's Peripheral Blood Mononucleate Cells (PMBC) viability was investigated, too.

The isolation of goat Peripheral Blood Mononucleate Cells procedure, Peripheral Blood Mononucleate Cells culture conditions, and their quantification by viable cells WST-1 test used in the experiment were those reported by De Matties et al. [27]. Neem oil and Tween 20^® were used singularly as controls.

The neem oil metabolomic fingerprint shows characteristic sequence of metabolites according to the polarity of constituents. The identification of the raw material was assured by the presence of salannin (Rf = 0.42), that is a typical marker of neem. In comparison with the spot of azadirachtin (Rf = 0.23), salannin appear as the main limonoid spot. Spots concerning lipids are present at Rf values, at ca. 0.80, due to unsaturated fatty acids and fatty alcohols, and at Rf ca. 0.50, due to saturated and unsaturated triglycerides.

The most interesting feature of the plate concerns the presence of compounds with high fluorescent reaction at between Rf 0.55- 0.66, that are perfectly visible at 366 nm after derivatization with anisaldehyde. These spots can be attributed to compounds with high conjugated unsaturation in polycyclic aromatic structures, very different from those of the nortriterpenes limonoids, so far considered as responsible for the activities investigated in our studies (Figure 1).

The microbiological analysis allowed the isolation of coliform,

mesophilic, thermophilic bacteria, E. coli, Staphylococcus spp., Enterococcus spp., and Pseudomonas aeruginosa from goat's wound resulting from ectoparasites bite and sting. The enumeration counts of isolated microorganisms are reported in Table 1 as means of five samples each of three repetitions (3 wounds / goat).

The molecular biology analysis allows the identification and characterization of three strains of E. coli. They were intimin (eae), hemolysin (hlyA) and heat-stable enterotoxin (EAST1) gene positive E. coli. Furthermore, Enterococcus

faecalis, Staphylococcus aureus and P. aeruginosa were detected. Amplicons of the expected size were obtained from DNA extracted from each bacterium investigated as showed in list A and List B of Table 1.

Amplicons were also obtained from DNA extracted from American Type Culture Collection reference strain of each tested bacterium (positive controls) while amplified products were never obtained from reaction mixtures without templates (negative controls) (Figure 2).

The neem oil antibacterial activity was assayed towards six bacterial isolates and four reference strains as control. The neem oil average Growth Inhibition Zone (mm) ranges 22.68 ± 0.55 to 30.21 ± 1.38. The highest neem oil Growth Inhibition Zone was revealed towards E. coli strains being, respectively, E. coli eae 28.71 ± 1.29, E. coli hly A 29.59 ± 1.17 and E. coli EAST1 30.21 ± 1.38. S. aureus isolate results the less susceptible among all tested

bacteria by the disc diffusion method. The ciprofloxacin Growth Inhibition Zone ranges 23.62 ± 1.00 - 31.42 ± 1.39. There was significant difference between the neem oil antibacterial activity and ciprofloxacin antibacterial activity towards E. coli eae E. coli hly A and EAST1 (Table 3).

As shown in Table 4, the percent bacterial Growth Reduction revealed at 100 μL, 10 μL, 1 μL and 0.1 μL of neem oil ranged, respectively, 74.51 ± 1.15 - 95.51 ± 1.15; 59.61 ± 0.58 - 75.61 ± 1.00; 48.10 ± 0.10 -61.67± 1.35 and 32.63 ± 1.73 - 32.63 ± 1.73. There was significant difference among the antibacterial activity at the mentioned neem oil dilutions.

The highest percent bacterial Growth Reductions were detected at 100 μL neem oil and they concerned mainly pathogenic E. coli isolates as follows: E. coli hlyA 95.51 ± 1.15, E. coli eae 91.25 ± 1.43, E. coli EAST1 89.65 ± 1.53 (Table 4).

Figure 2 A shows the effect on percent viability of Peripheral Blood Mononucleate Cells grew in Roswell Park Memorial Institute medium and treated with neem oil at different dilutions (1:2x10², 1:2x10³, 1:2x10⁴; 1:2 x10⁵; 1:2 x10⁶). There was significant difference between percent of Peripheral Blood Mononucleate Cells viability detected at neem oil dilution 1:2x10² and untreated control, only. This was revealed in Peripheral Blood Mononucleate Cells culture of 24 h and 48 h incubation periods according to the data reported by De Matteis, et al. [27]. While, Figure 2 B shows the effect of Tween^®20 on percent viability of Peripheral Blood Mononucleate Cells grew in Roswell Park Memorial Institute medium at the same dilutions. The percent of Peripheral Blood Mononucleate Cells viability was significantly different between Tween^®20 dilutions 1:2 x10² and 1:2 x10³ (42% up 7% decrease of cell viability) and untreated

Peripheral Blood Mononucleate Cells viability. This was revealed in Peripheral Blood Mononucleate Cells culture of 24h and 48h incubation periods. Figure 2 C represents the effect of neem oil and Tween^®20 (1:1 v/v) on percent viability of Peripheral Blood Mononucleate Cells grew in Roswell Park Memorial Institute medium at different dilutions (1:2x10², 1:2x10³, 1:2x10⁴; 1:2 x10⁵; 1:2 x10⁶). If Tween^®20 is used with neem oil the adverse effect increases at 1:2 x102 dilution (28% cell viability) while it remains similar at the dilution 1:2 x103 (6% cell viability) respect the treatment with only Tween^®20. This was revealed either in Roswell Park Memorial Institute medium culture of 24h or 48 h incubation periods.

There is significant difference between the adverse effect of neem oil, Tween^®20, Tween^®20 and neem oil treatments at dilution 1:2 x102 on percent Peripheral Blood Mononucleate Cells viability. Furthermore, there is no significant difference between Tween^®20 and Tween^®20 and neem oil treatments at dilution 1:2 x103. While there is significant difference between neem oil and Tween^®20 as well Tween^®20 and neem oil treatments at the same dilution (Figure 3).

High Performance Thin Layer Chromatography, the last evolution of planar chromatography, allows to evidence most of the constituents of an extract in an identifying track, named fingerprint. Plates can be visualized and derivatized in several ways, obtaining multiple information [28]. Identification of constituents in the fingerprint can be obtained by standards. Therefore, neem products are subjected to great variation in composition, due to pre and post-harvesting factors [29].

The phytochemical studies and bio-assay guided fractionation showed that the insecticidal activity are correlated with the tetranortriterpenoids, such as those already mentioned as the

meliatetraolenone, but also aromatic compounds [2,6-bis- (1, 1-dimethylethyl) -4-methylphenol, 2-(phenylmethylene)-octanal, 1,2,4-trimethoxy-5-(1Z-propenyl) benzene], benzopiranoidi (3,4-dihydro-4,4,5, 8-tetramethyl coumarin, 3,4-dihydro-4,4,7,8- tetramethyl coumarin-6-ol, 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8- hexamethyl-cyclopenta[g]-2-benzopyran); sesquiterpene (methyl-3,7,11-trimethyl-2E, 6E, 10-dodecatrienoate); fatty acid esters (methyl 14-methyl-pentadecanoate, ethyl hexadecanoate, ethyl 9Z-octadecenoate) and monoterpene (3,7-dimethyl-1-octen-7-ol). Despite all phytochemicals studies already made and the large amount of constituents identified and reported in special databases, these studies show that, although the limonoids are to be considered important for the insecticidal activity, other oil components of neem, or other preparations of neem, are to be considered important and necessary for other biological activities. So far, the phytocomplex of neem oil can be considered fundamental for the obtained results [13].

Authors carried out dose determination studies and field study on parasiticidal activity of neem oil for the evaluation of its efficacy according the guidelines of World Association for the Advancement of Veterinary Parasitology. The neem oil parasiticidal activity was evaluated towards goat (Capra hircus L.) lice (Linognathus stenopsis and Damalinia caprae). The results confirmed that neem oil is an effective parasiticide that control two lice (Linognathus stenopsis and Damalinia caprae) biological cycles with one neem oil dose administration. Furthermore, they evaluated skin reactions during the experimentation using Draize Scoring System [30] and demonstrated neem oil does not cause irritation of skin. It was always obtained a score equal zero regardless of the dose used (manuscript submitted and under revision). Due the possibility of neem oil absorption through skin, a methodology was set up to evaluate the effect of neem oil on goat blood cells [27]. Neem oil was efficacy in containing louse with no side effects on treated goats.

Many studies carried out so far demonstrated the microbial inhibitory effects of different phytoconstituents of A. indica. Neem has been reported to exhibit antibacterial as well as antifungal activities depending from which part of the plant and how extracts were obtained. Margolone (12-methyl-7-oxopodocarpa-8,11,13-triene-13-carboxylic acid), margolonone (2-Phenanthrenecarboxylicacid, 4b, 5, 6, 7, 8, 8a, 9, 10-octahydro-3, 4b, 8, 8-tetramethyl-7, 10-dioxo-, (4bS,8aR)- ) and isomargolonone ("3-Phenanthrenecarboxylic acid, 4b, 5,6,7,8,8a, 9,10-octahydro- 2,4b, 8,8- tetramethyl-7,10-dioxo-,(4bS-trans)-") are tricyclic diterpenoids isolated from stem bark, nimbidin (Methyl (2R, 3aR, 4aS, 5R, 5aR, 6R, 9aR, 10S, 10aR)-5-(acetyloxy)-2-(furan- 3-yl)-10-(2-methoxy-2-oxoethyl)-1,6,9a,10a-tetramethyl-9- oxo-3,3a,4a,5,5a,6,9,9a,10,10a-decahydro-2H-cyclopenta[b] naphtho[2,3-d]furan-6-carboxylate ) and nimbolide (4α,5, α 6 α,7 α,15,17 α )-7,15:21,23-diepoxy-6-hydroxy-4,8-dimethyl- 1-oxo-18,24-dinor-11,12-secochola-2,13,20,22-tetraene-4,11- dicarboxylic acid α-lactone methyl ester] from seed oil, as well azadirachtins (dimethyl (2aR,3S,4S,R,S,7aS,8S,10R,10aS,10 bR)- 10-(acetyloxy)- 3,5-dihydroxy- 4-[(1S,2S,6S,8S,9R,11S)- 2-hydroxy- 11-methyl- 5,7,10-trioxatetracyclo[6.3.1.0^2,6.0^9,11]dodec- 3-en- 9-yl]- 4-methyl- 8-{[(2E)- 2-methylbut- 2-enoyl]oxy} octahydro- 1H-furo[3',4':4,4a]naphtho[1,8-bc]furan- 5,10a(8H)- dicarboxylate), quercetin (3,3',4',5,7-pentahydroxyflavone) and β- sitosterol (17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl- 2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a] phenanthren-3-ol) from leaves, all show several biological activity, among them antimicrobial activity, too [31-35].

This study confirms neem oil antibacterial activity towards wound-contaminating-bacteria resulting from ectoparasites sting and bite, too. Therefore, the neem oil should be considered as a safe and ready to use innovative topical solution for the care of wounds.

The authors declare that there is no conflict of interests regarding the publication of this paper. This work was carried out in the frame of the collaborative research between the Council for Agricultural Research and Economics CRA, the Animal Production Research Centre CRA-PCM, and the University of Rome Sapienza, Department of Environmental Biology on: "Herbal remedies for the animal welfare and the improvement of livestock productivity"

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