JAHP_14_3_1209-1216

Introduction

Antimicrobial resistance is a critical factor that complicates mastitis treatment and often leads to therapeutic failure. Research indicates that microorganisms exhibit varying levels of resistance to antibiotics. Even with the introduction of advanced generations of antibiotics, the incidence of drug-resistant bacteria continues to grow (Wang et al., 2025). Moreover, recent studies stress the significance of antibiotic resistance, highlighting the urgent need for further research in this area (Naich et al., 2025). Staphylococcus aureus is a widespread commensal bacterium found in many mammalian species. Under certain conditions, however, it can act as an opportunistic pathogen, leading to infections that range from mild to severe, including mastitis. Consequently, S. aureus is considered one of the primary pathogens associated with both clinical and subclinical ovine mastitis worldwide (Gharaibeh et al., 2025; Mohsin and Allawe, 2025).

Mastitis is significant for three main reasons: its impact on sheep health, the economic losses it causes, and its implications for human health. Sheep milk is primarily used for traditional cheese production, making the control of mastitis and the maintenance of milk hygiene key objectives in sheep farming, alongside milk and lamb production. Although antimicrobials play an essential role in mastitis control programs, their use must be carefully managed (Saleem et al., 2021; Ahmed and Yousif, 2021; Ergene et al., 2025).

Essential oils consist of a complex blend of volatile compounds unique to each plant, which contribute to their diverse bioactivities, these compounds include alkaloids, carotenoids, flavonoids, isoflavones, phenolic acids, terpene hydrocarbons (oxygenated and non-oxygenated), and aldehydes (Tavares et al., 2022; Asaad, 2023). Their antimicrobial action involves several mechanisms, such as disrupting the cell wall and cytoplasmic membrane, causing cytoplasmic coagulation, inhibiting toxic bacterial metabolites, and blocking bacterial efflux systems. Nevertheless, the effectiveness of this activity depends on both the pathogen involved and the specific composition of the essential oil (Man et al., 2019; Salman et al., 2024).

The aim of this study was to assess the therapeutic efficacy of ethyl cellulose–based film-forming suspensions formulated with Thymus vulgaris and Mentha piperita essential oils (2% and 4%) against Staphylococcus aureus–induced mastitis in Awassi ewes.

Materials and Methods

Preparation of film-forming suspensions

The film-forming suspensions were prepared by simple dispersion. Ethyl cellulose (Sigma®, USA), xanthan gum (Kelco®, USA), and Tween 80 (Croda®, Singapore) were mixed with distilled water at 500 rpm at room temperature for 30 min using a mechanical stirrer. Then, the essential oil (EO) was dispersed at concentrations of 2% and 4% by adding it to the solution, which was subsequently stirred under the same conditions for 60 min. The formulation details are shown in Table 1 (Barreiros et al., 2022).

Evaluation of average droplet size and stability

The droplet size distribution of film-forming suspensions (2%, 4% of T. vulgaris and M. piperita suspensions) was analyzed via optical microscopy using Sudan IV dye (Merck@, Germany) and an Olympus BX41 microscope (1000×). Three images per sample were processed digitally to calculate mean droplet diameter and distribution. Stability was assessed after 30 days at room temperature by comparing droplet diameters on days 1 and 30 (Barreiros et al., 2022).

Table 1: Composition of T. vulgaris and M. piperita film-forming suspensions.

Concentrations/ Ingredients	T (2%)	T (4%)	M (2%)	M (4%)
Ethyl cellulose	2.5	2.5	2.5	2.5
Xanthan Gum	0.5	0.5	0.5	0.5
Tween 80	0.2	0.2	0.2	0.2
T. vulgaris	2.0	4.0	-	-
M. piperita	-	-	2.0	4.0
Distilled water	Up to the total volume of 100 ml

T: T. vulgaris; M: M. piperita

Experimental animal

The study was conducted at the University of Baghdad, College of Veterinary Medicine sheep sheds. Twenty-five healthy adult Awassi ewes (>2 years, 35–45 kg) were used. All underwent physical exams, milk analysis. Animals were acclimatized for one month with balanced feed and free access to water.

Induction of experimental mastitis

An overnight inoculated trypticase soy agar with S. aureus was utilized to prepare 7 x 102 CFU/ml bacterial suspension in phosphate buffer saline; twenty ewes were served as a model for experimental mastitis. The previously milked-out of the udder was infused with 1 ml of bacterial suspension (each 12 hr. per day) by sterilized stainless blunt end needle (Kerro et al., 2020).

Experimental design

Twenty-five ewes were used in this experiment. The animals were randomly allocated into five experimental groups (n = 5 per group), viz.:

Group A: Healthy ewes that received no treatment and served as the negative control.

Group B: Infected ewes treated with 10 ml of suspension free essential oil, serving as the positive control.

Group C: Infected ewes treated with 10 ml of 2% T. vulgaris suspension.

Group D: Infected ewes treated with 10 ml of 2% M. piperita suspension.

Group E: Infected ewes treated with amoxicillin (10 ml; 31.25 mg/kg) according to Machado and Bicalho (2018).

All treatments were administered via intramammary injection once daily for three consecutive days.

Somatic cell count

Milk samples were taken before infection, after infection, and after treatment. The first three streams of milk were discarded, and approximately five ml of milk were collected from each quarter into sterile test tubes before being delivered to the laboratory. For the direct examination of the somatic cell count, the freshly collected milk samples were thoroughly mixed. Then, 0.01 ml (10 μl) of milk was spread evenly over a 1 cm2 area on a clean slide. The slides were dried on a flat horizontal surface without the application of heat. Finally, they were stained with Newman-Lambert stain (Buswell, 1995).

Milk yield

Milk yield per ewe was evaluated before infection, after infection, and following treatment. For quality, the first three milk jets were discarded, after which milk samples were manually collected into individual sterile bottles for total bacterial count (TC) analysis (Jaguezeski et al., 2018).

Milk fat content (%)

Ten ml of sulphuric acid were transferred into the butyrometer, and 10 ml of milk sample were added at 24 °C. One ml of isoamyl alcohol was added, and the lock stopper was inserted securely. The butyrometer was shaken until the curd was completely digested. It was then placed in a water bath at 60-63°C for 5 minutes, centrifuged for 4 minutes, and the fat percentage was recorded (Shah et al., 2015).

Measurements of interleukin 8 and 12

The concentrations of cytokines in quarter milk samples were determined by ELISA using kits (Cloud-clone®, Houston, USA) specific for IL-8 and IL-12. All procedures were performed in accordance with the manufacturer’s instructions. Absorbance readings were recorded using an ELx800 automatic microplate reader at 450 nm, with 630 nm serving as the reference wavelength. The inter- and intra-assay coefficients of variation for all examined cytokines were found to be less than 10%, indicating high procedural precision and reproducibility (Caroprese et al., 2006; Touma et al., 2021; Mahmoud and Yassein, 2024).

Statistical analysis

The Statistical Package for the Social Sciences (SPSS, 2019) was used to detect the effects of different groups and periods on the study parameters. A two-way Analysis of Variance (ANOVA) followed by the Least Significant Difference (LSD) test was employed to compare the means across the different experimental groups.

Results

Droplet size and stability of film-forming suspensions

As shown in Figure 1-4, increasing the concentration of T. vulgaris and M. piperita suspensions from 2% to 4% resulted in a significant increase in average droplet diameter. After 30 days, droplet sizes further increased for both suspensions at both concentrations, indicating reduced stability. The M. piperita suspension consistently exhibited larger average droplet diameters compared to the T. vulgaris suspension.

Somatic cell count

Somatic cell count (SCC) increased significantly (p ≤ 0.05) 48 h after infection compared with the pre-infection values (Figure 5). After 72 h of treatment, SCC decreased significantly (p ≤ 0.05) in the treated groups compared with the positive control. The lowest SCC was recorded in the M. piperita-treated group (63.11 ± 2.79 ×10³ cells/ml), followed by the T. vulgaris group (78.43 ± 3.08 ×10³ cells/ml) and the amoxicillin-treated group (91.13 ± 4.52 ×10³ cells/ml).

Milk yields

As shown in Figure 6, milk yield decreased significantly (p ≤ 0.05) 48 h after infection compared with the pre-infection values. After 72 h of treatment, milk yield increased significantly (p ≤ 0.05) in the treated groups compared with the positive control. The highest milk yield was observed in the M. piperita-treated group (453 ± 21.6 ml), followed by the T. vulgaris group (433 ± 17.3 ml) and the amoxicillin-treated group (406 ± 15.2 ml).

Milk fat percentage

Milk fat percentage decreased significantly (p ≤ 0.05) 48 h after infection compared with the pre-infection values (Figure 7). After 72 h of treatment, milk fat percentage increased significantly (p ≤ 0.05) in the treated groups compared with the positive control. The highest milk fat percentage was recorded in the M. piperita-treated group (6.47 ± 0.79%), followed by the T. vulgaris group (6.31 ± 0.51%) and the amoxicillin-treated group (6.01 ± 0.37%).

IL-8 concentration in milk

As presented in Figure 8, milk IL-8 concentration increased significantly (p ≤ 0.05) after 48 h of infection in all infected groups compared with the pre-infection values. After 72 h of treatment, IL-8 levels decreased significantly (p ≤ 0.05) in the treated groups compared with the positive control. The lowest IL-8 concentration was observed in the M. piperita-treated group (131.65 ± 4.93 pg/mL), followed by the T. vulgaris group (179.77 ± 8.77 pg/mL) and the amoxicillin-treated group (193.88 ± 8.35 pg/mL).

IL-12 concentration in milk

Milk IL-12 concentration increased significantly (p ≤ 0.05) after 48 h of infection in all infected groups compared with the pre-infection values (Figure 9). After 72 h of treatment, IL-12 levels decreased significantly (p ≤ 0.05) in the treated groups compared with the positive control. The lowest IL-12 concentration was recorded in the T. vulgaris-treated group (211.13 ± 9.13 pg/mL), followed by the M. piperita group (248.19 ± 10.86 pg/mL) and the amoxicillin-treated group (282.58 ± 26.05 pg/mL).

Discussion

Droplet size and stability are key determinants of film-forming suspension performance. In this study, both T. vulgaris and M. piperita suspensions showed droplet size depended on EO concentration and storage time. At 2% EO, droplet sizes were smaller than at 4%. M. piperita consistently produced larger droplets, likely due to its higher viscosity and interfacial tension. These results align with previous findings that higher EO content increases droplet size and reduces dispersion efficiency (McClements, 2012; Weiss et al., 2009; Barreiros et al., 2022).

After 30 days of storage, all formulations exhibited increased droplet size, indicating instability from coalescence or phase separation. The effect was more pronounced in 4% EO formulations, confirming that higher oil content yields larger, less stable droplets (Anton and Vandamme, 2011; McClements, 2012).

Somatic cell count (SCC) is a primary indicator of udder health and mastitis in small ruminants. In healthy sheep milk, SCC is usually below 500,000 cells/ml but rises sharply during infection due to immune cell infiltration (Paape et al., 2007; Abood, 2010; Albenzio and Ciliberti, 2018).

Infection caused a marked rise in SCC (892.22 ± 26.52 and 984.66 ± 29.86 after 48 h), confirming acute mastitis, consistent with previous reports of pathogen-induced leukocyte infiltration (Zhang et al., 2021). Post-treatment SCC significantly declined in both T. vulgaris gel and M. piperita suspension groups, with the greatest reduction in M. piperita (63.11 ± 2.79), followed by T. vulgaris (67.43 ± 2.66), compared to amoxicillin (91.13 ± 4.52). These results indicate that essential oils effectively suppress inflammation and bacterial growth, enhancing udder recovery beyond antibiotic therapy (Bassolé and Juliani, 2012).

Recent studies support these findings; Omid et al. (2023) reported that phytogenic extracts in ewes significantly reduced SCC and improved milk quality, surpassing some antibiotics. The antimicrobial and anti-inflammatory effects of thymol and carvacrol (in T. vulgaris) and menthol (in M. piperita) are well established, as they disrupt bacterial membranes, inhibit biofilms, and modulate immune responses (Fathi et al., 2022; Salehi et al., 2021).

Milk yield decreased significantly (P ≤ 0.05) 48 hours after infection, indicating the detrimental effect of mastitis on milk production. This decline can be attributed to inflammatory damage in mammary tissue, impaired alveolar secretion, and reduced glandular activity, consistent with previous findings (Bannerman, 2019; Islam et al., 2020).

After 72 hours of treatment, milk yield improved significantly in all groups. The M. piperita suspension group showed the highest recovery (453 ± 21.6 ml), followed by T. vulgaris suspension (433 ± 17.3 ml) and amoxicillin (406 ± 15.2 ml). The superior effect of M. piperita may be linked to menthol’s antimicrobial and anti-inflammatory properties, which promote mammary tissue repair and restore normal milk secretion (Kumar et al., 2019; Fathi et al., 2022). These findings highlight the potential of essential oil-based formulations as effective natural alternatives for improving milk yield during mastitis recovery.

Milk fat content significantly decreased (P ≤ 0.05) 48 hours after infection, likely due to impaired lipid synthesis and secretion resulting from mammary inflammation (Auldist and Hubble, 1998; Silanikove et al., 2014).

Following 72 hours of treatment, milk fat percentage increased markedly, reaching 6.47 ± 0.79% in the M. piperita group, compared with 6.31 ± 0.51% in T. vulgaris and 6.01 ± 0.37% in amoxicillin-treated ewes. The improvement may result from the antioxidant and anti-inflammatory actions of menthol and thymol, which enhance mammary gland recovery and lipid metabolism (Kumar et al., 2019; Shakeel et al., 2019).

The significant elevation of milk IL-8 and IL-12 concentrations within 48 h post-infection confirms activation of the innate immune response following S. aureus challenge. Bacterial components such as lipoteichoic acid and peptidoglycan are recognized primarily by Toll-like receptor 2 (TLR2) expressed on mammary epithelial cells and macrophages, triggering MyD88-dependent signaling and activation of NF-κB, which induces transcription of pro-inflammatory cytokines (Bannerman, 2009).

IL-8 upregulation reflects enhanced neutrophil chemotaxis toward the mammary gland. As a potent chemokine, IL-8 mediates rapid recruitment of polymorphonuclear leukocytes during mastitis; however, excessive neutrophil infiltration may contribute to tissue injury via reactive oxygen species and proteolytic enzyme release (Bannerman, 2019; Caroprese et al., 2006). Therefore, the markedly elevated IL-8 concentrations observed during early infection are consistent with acute inflammatory activation.

Similarly, the sharp increase in IL-12 levels suggests activation of antigen-presenting cells and polarization toward a Th1-type immune response. IL-12 promotes IFN-γ production and enhances macrophage bactericidal activity, facilitating intracellular pathogen clearance (El-Sayed and Kamel, 2020). However, sustained IL-12 expression has been associated with exacerbated inflammatory responses and tissue damage in bovine mastitis (Bannerman, 2019).

Following treatment, the significant reduction in IL-8 and IL-12 concentrations in the plant extract–treated groups indicates modulation of inflammatory signaling pathways in addition to antibacterial activity. The superior immunomodulatory effect observed in the T. vulgaris group may be attributed to thymol and carvacrol, which inhibit NF-κB activation and suppress pro-inflammatory cytokine expression (Fathi et al., 2022). These compounds have been shown to interfere with TLR-mediated signaling cascades, thereby attenuating cytokine overproduction.

M. piperita exhibited moderate immunoregulatory activity, likely mediated by menthol, which has demonstrated inhibitory effects on inflammatory mediator synthesis and oxidative stress pathways (Kumar et al., 2019). In contrast, although amoxicillin reduces bacterial load, it does not directly suppress host inflammatory signaling, which may explain the comparatively higher residual cytokine levels. Moreover, S. aureus persistence through biofilm formation and intracellular survival mechanisms can limit antibiotic efficacy (Atalla et al., 2008).

Collectively, these findings support a dual mechanism of action for the tested essential oils: Direct antimicrobial disruption of bacterial cell membranes combined with immunomodulatory suppression of NF-κB–dependent cytokine production. This integrated mechanism may explain the more pronounced reduction in inflammatory markers compared with antibiotic treatment alone.

Limitations

Despite the promising findings of the present study, several limitations should be acknowledged:

First, GC–MS characterization of the tested oil was not performed within this study. However, the extraction procedure, chemical profiling by GC–MS, and antimicrobial susceptibility testing were comprehensively described in our previously published study in the same journal. As the present investigation represents a direct continuation of that work and utilized the same oil preparation protocol, repetition of the chemical characterization was intentionally avoided to prevent methodological redundancy. Nevertheless, future studies may re-confirm chemical composition when using newly extracted batches to ensure consistency and reproducibility.
Second, post-treatment bacterial assessment following oil removal was not conducted. Therefore, the persistence of the antibacterial effect and the potential for bacterial regrowth after discontinuation of exposure were not evaluated. Future research should incorporate post-treatment monitoring to determine the duration and sustainability of the antimicrobial effect.
Third, histological integrity testing was not included in the current experimental design. Although the study focused primarily on evaluating antibacterial efficacy, the absence of histopathological or cytotoxicity assessment limits conclusions regarding tissue compatibility and structural safety. Further investigations involving histological analysis are recommended to confirm the safety profile and potential translational applicability of the tested oil.
Collectively, addressing these limitations in future studies will strengthen the mechanistic understanding, safety validation, and clinical relevance of the findings.

Conclusions

The essential oils demonstrated positive antimicrobial activity against pathogens responsible for subclinical mastitis in sheep. This study highlighted the antibacterial and anti-inflammatory activities of T. vulgaris and M. piperita suspensions and their potential use in treating animal infectious diseases caused by S. aureus, including drug-resistant and biofilm-forming strains. These results indicate that essential oils may serve as a promising natural alternative for controlling veterinary pathogens. The findings highlight the potential of essential oil particularly in the context of the growing problem of antibiotic resistance

Acknowledgements

The authors would like to thank members of the Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq, Veterinary medicine, Baghdad university, Iraq for their collaboration.

Novelty Statement

This study introduces ethyl cellulose-based film-forming suspensions containing Thymus vulgaris and Mentha piperita essential oils as a novel intramammary treatment for experimental ovine mastitis. The formulations demonstrated superior therapeutic efficacy by reducing inflammation and somatic cell count while improving milk production and quality, highlighting their potential as natural alternatives to conventional antibiotic therapy

Authors’ Contribution

HMA and OMS designed the study. HMA conducted the experimental work and collected the samples. NAR performed the statistical analysis and interpretation of data. HMA wrote the manuscript. All authors read and approved the final version of the manuscript. The final manuscript draft was reviewed by all authors.

Ethical approval

This study received ethical approval in accordance with the Animal Utilization Protocol Certification (No. 924/PG), issued by the Research Ethics Committee of the College of Veterinary Medicine, University of Baghdad.

Funding

The authors received no specific funding for this work.

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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