The tested enrichment materials differed considerably in their material type and source and also in their hygienic status. They were chosen due to their practical relevance and their commercial availability. With regard to practical relevance, hay and straw harvested on farms are widely used, but differ greatly in their quality due to weather, harvest, storage conditions, and other factors and are therefore not comparable. Thus, in this study hay, straw, and also maize silage were obtained from one example farm only. Those results are only intended to be a preliminary guide of what values can be expected and cannot be extrapolated to materials from other farms or material harvested at other points in time. However, in the commercially available materials with different standard processing steps that are done before they are sold, comparability would be desirable. In this context, one limitation of this study was the purchase of most of the materials from only one commercial source. For materials from other sources, results might differ, but as the manufacturing process is of high importance, this study provides the first results for materials produced under similar conditions, irrespective of the country of production. During production, more precisely during the pelleting process, the material is exposed to heat, pressure, shear forces, and water. Numerous studies have shown that pelleting reduces the contamination with vegetative microbes mainly due to a reduction in water activity [6]. Water activity characterizes the availability of free water in the material, which is essential for bacterial survival and growth.

Referring to the results in detail, due to the study design, only descriptive statistical analyses were performed. The study was intended as a pilot study to provide the first insights into this relevant topic. Regarding coliforms in general, the level of contamination in enrichment materials made of wood was below the median value of 281 wood shavings with 7.9 × 10⁵ cfu/g and 1.2 × 10³ cfu/g for TVC and coliform count, respectively [7]. It has been shown that wood, depending on several factors such as wood species and ambient temperature, can reduce the survival of bacterial species such as Escherichia coli [8] via hygroscopic effects and wood extractives. Furthermore, adherence of microorganisms to the surface of wood is discussed as a reason for reduced recovery. However, Vainio-Kaila et al. [9] demonstrated that this adherence can be avoided by vortexing the samples. Thus, adherence should not have a major influence on the results of this study. Kristula et al. [10] considered coliform counts in bedding material exceeding 10⁶ cfu/g as a potential cause of mastitis in dairy cows. Even though three of the loose straw and hay materials in this study had a higher coliform burden than the threshold of 10⁶ cfu/g described by Kristula et al. [10], it is questionable whether this threshold is applicable in pig husbandry. The total viable count in the maize silage, which was the highest in the present study, exceeded previously reported values [11]. Nevertheless, it was > 1 log₁₀ cfu lower than the counts 7 days after exposure to air [11].

Concerning the tested bacteria species, Escherichia coli is commonly used as a hygienic indicator for fecal contamination in different matrices such as meat [12]. Lynn et al. [13] found Escherichia coli in 30% of cattle feeds, which are partly comparable to the tested enrichment materials in this study. However, in our study, Escherichia coli was not detected.

In a longitudinal study concerning Belgian dairy farms, 8.2% of unused sawdust bedding samples were contaminated with K. pneumoniae [14]. In contrast, in our study Klebsiella spp. was not detected in any of the tested materials. The absence of Klebsiella spp. and coliforms on the maize pellets contradicts a previous study [15] in which 2.8 log₁₀ cfu/g of both microbes in unused pelleted corn cobs was detected.

Human yersiniosis is one of the most frequent foodborne infections in the EU, and pigs are an important reservoir for the main causative pathogen, Yersinia (Y.) enterocolitica [16]. Despite numerous studies on the subject, the infection source and farm environment impact, especially from living or inanimate vectors, remain unclear [16]. It is controversial whether the absence or sparse use of bedding material [17] or the presence of bedding material [18] is a risk factor for the prevalence of pathogenic Y. enterocolitica and antibodies to Y. enterocolitica in pigs. In our study, the analysis was not limited to Y. enterocolitica but was conducted with Yersinia spp. in general; still, there was no detection of Y. spp. in any enrichment material. This result is in accordance with Vilar et al. [17] who did not detect pathogenic Y. enterocolitica in straw, and a German study in which pathogenic Y. enterocolitica was not found in 830 environmental and feed samples [19].

Salmonella spp. is a common contaminant in feed raw materials, and therefore feed is one of the sources for introducing Salmonella into pig herds [20]. Control programmes for Salmonella have been implemented in several European countries and include financial penalties for herds with a high prevalence of Salmonella infection. Thus, it is important for pig farmers to minimise the introduction of Salmonella spp. onto farms. None of the tested enrichment materials contained Salmonella spp. in levels that could be detected by our methods even though a pre-enrichment step was included. Therefore, the tested materials do not represent a probable source of Salmonella infection.

With regard to antimicrobial resistance, MRSA is a major problem for human healthcare, and intensively reared pigs are one of the main reservoirs of livestock-associated MRSA [21]. Antibiotic resistant bacteria in addition to antibiotics and resistance genes can reach crop fields by land application of manure [22]. Furthermore, MRSA is regularly found in farm environments of positive herds and the air and soil outside the barns [23]. The risk of enrichment material contamination by these potential routes can be considered as low and did not lead to MRSA findings in the tested materials.

The only detected specific pathogens in the material tested in this study were M. smegmatis, M. avium, and M. vulneris in the hemp straw and peat. M. smegmatis belongs to the rapidly growing mycobacteria, which are saprophytes found in environments such as soil, water, and dust [24]. It is potentially pathogenic to humans and animals and can cause, for instance, cardiac-related infections, pulmonary diseases, or ulcerative skin lesions [24]. Due to the widespread occurrence of M. smegmatis in the environment and the rare cases of clinical disease, the presence of this microorganism in hemp straw should not be overestimated. M. avium and M. vulneris both belong to the M. avium complex [25]. M. vulneris can cause lymphadenitis and abscesses in immunocompetent patients [26]. M. avium is classified into four subspecies: (1) avium; (2) hominissuis; (3) paratuberculosis; and (4) silvaticum, each showing distinct host species and pathogenicity [25]. The detection of M. avium in unused peat is in accordance with previous studies, and peat is assumed to be a source of infection for pigs [26, 27]. In a Spanish study, M. avium was found in unused sawdust samples [28], which is neither supported by Agdestein et al. [27] nor by our present results. The most prevalent species in the mentioned studies is M. avium subsp. hominissuis, a microorganism that causes granulomatous lesions in pigs, leading to carcass condemnation at the abattoir and financial losses for farmers [28]. Matlova et al. [26] tested various decontamination methods for peat and demonstrated that steam treatment at 100 °C for 10 min effectively reduced mycobacterial contamination, though not completely eradicating the mycobacteria. The peat tested in our study had been heated at 65 to 70 °C for at least 10 min as certified by the producer. Obviously, this treatment is not sufficient to decontaminate peat, and therefore this commercial peat designated for piglets poses a risk for infecting animals with mycobacteria. It is debatable whether the infectious and granulomatous lesions should be interpreted as jeopardising the health in the legal sense as stipulated in the Council Directive 2008/120/EC [3]. Peat is a very heterogeneous material but is generally characterised by an acidic pH and humic substances [29]. The presence of microorganisms is crucial for the formation of peat; concurrently, some of them produce antibiotics [29]. All of these factors make peat a difficult environment for microbes. However, the aforementioned detection of mycobacteria is a major health risk, thus excluding the use of peat as a safe and hygienic enrichment material. As moulds are considered ubiquitous, the detected mould contamination is not remarkable. However, deterioration of organic material by moulds can minimise the palatability, which might result in reduced acceptance of the material, and most importantly, cause mycotoxin production [30]. Thus, further investigations on mycotoxins should be carried out. The hay from the farm had the highest mould count of all materials. Different hays can vary widely in their hygienic status, and a TVC of up to log₁₀ 6.47 cfu/g and a mould count of up to log₁₀ 6.67 have previously been reported [31]. The contamination in the present study is comparable to these studies, but to the authors’ knowledge, there is no evidence that this kind of contamination poses a health risk for pigs.

There are no microbiological thresholds set for enrichment material in pig husbandry to date. However, the thresholds for feeding material give good orientation even though the intake of enrichment material by the animals is assumed to be minor despite there being no exact data available on this [2]. Moreover, to standardize microbiological testing of enrichment material, proof of the sensitivity and the specificity of applied methods are important to avoid false negative results.

Analyses of other groups of potential pathogenic agents such as parasites or viruses were not part of this study. Nevertheless, transmission via the vector fomites and personnel of porcine reproductive and respiratory syndrome virus, one of the most important viruses in pig husbandry, has already been demonstrated [32]. Thus, introduction of other microorganisms through contaminated enrichment material might also be possible and scientific research in this field should be conducted.