## 1 Introduction  
Bovine digital dermatitis (DD) is a claw disease in cattle which often causes lameness due to painful lesions. These lesions are characterized by erosive ulcerations often presented as moist, circular, strawberry-like structures with long hair surrounding them [1, 2]. The reduced cattle welfare is reflected in decreased milk yield, lower body condition scores, poor reproductive performances and increased losses due to early culling [3, 4]. DD has a multifactorial etiology. Causative factors, leading to pre-damage of the skin are manifold. These are e.g., unhygienic stable conditions, slatted floors without scargers, as well as standing in wet manure, which allow the infiltration of pathogens [2, 5, 6]. In particular, pathogenic bacteria, including _Treponema_ spp., _Fusobacterium necproformum_, _Porphyromonas levii_, Mycoplasmaaceae, _Escherichia coli_ and staphylococci are considered to play an important role in the development of DD [2, 6, 7, 8, 9, 10]. For decades, the treatment of DD has been a major challenge. Research is still being conducted on treatment alternatives to the current commonly used systemic or topical antibiotic applications. Oxytetracycline or limomycin are often used as a topical therapy whereas erythromycin is frequently used for group treatments with footbaths [2, 11, 12]. In addition, the use of antibiotics causes economic losses for farmers, due to the transient prohibition of milk delivery and meat production [13]. Novel insights into the treatment of DD show that non-antibiotic agents also have a beneficial effect on the lesions. These include formaline, copper sulfate, dragonhyde or phenytoin, but these chemicals often have environmentally damaging potentials [13; 14; 15]. It Is important to reduce the existing pathogenic organisms involved in DD to achieve prevention or effective healing of the specific lesions. Plasma-activated liquids (PAL) may provide an antibiotic-free option to minimize pathogens and prevent or at least mitigate DD. A wide variety of PALs are now being produced and studied for their antimicrobial activity. Plasma-activated water (PAW) is made from pure distilled water, whereas various buffers are used to produce plasma-activated buffers for example phosphate buffered saline (PBS) or citrate-phosphate buffer [16; 17]. Nowadays, PAW in particular is used in a variety of ways, for example in microbiology for bacterial reduction or in medicine for wound healing, treatment of skin diseases or cancer therapy [18; 19; 20; 21], even insecticidal effects of PAW have been demonstrated [22]. PAW contains various reactive oxygen species (ROS), as well as reactive nitrogen species (RNS). ROS include hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), hydroxyl radicals (OH\({}^{-}\)) or superoxides (O\({}_{2}\)\({}^{-}\)), whereas peroxynitrite (ONOO\({}^{-}\)) and nitrogen dioxide (NO\({}_{2}\)\({}^{-}\)) radicals belong to the RNS [23; 24; 25]. It has been widely documented that these reactive species can damage the microorganisms at various cell components and thus inactivate them. For example, [PERSON] et al. [26] found that oxidative stress can break the cell wall due to the destruction of peptide-glycan bonds. Similarly, it was shown by [PERSON] et al. [27] hat after intracellular entry of reactive species, both DNA and proteins in the bacterial cell were destroyed. In this study, we investigated the influence of a TRIS-buffered PAW (Tb-PAW) on Gram-positive and Gram-negative aerobic, as well as anaerobic microorganisms associated with DD and frequently found on the claw or in the stable, using an in vitro model. These investigations were followed by an analysis of specific factors influencing the antimicrobial effectivity of the Tb-PAW, such as proteins, temperature or storability. The aim of the research was to evaluate the microbicidal activity of Tb-PAW for its applicability in an agricultural facility to reduce pathogenic microorganisms under various conditions, thus providing an alternative therapy or prophylaxis for the management of DD in the stable.