1 Introduction

According to the Japanese Ministry of Health, Labour and Welfare, one in every two Japanese suffers from some form of allergy. Among these allergies, the rate of bronchial asthma is rapidly increasing, with childhood asthma having tripled in the past 20 years. Allergic diseases, such as asthma, significantly diminish the quality of life and have adverse effects on social activities, such as reduced labor productivity and increased medical costs (Ministry of Health, Labour and Welfare, 2011).

The hygiene hypothesis is widely known for allergic diseases. The hygiene hypothesis states that exposure to microorganisms during childhood contributes to the development of the immune system and protects against allergic diseases (Strachan, 1989). Conversely, exposure to microorganisms later in life can worsen symptoms such as asthma. In recent years, experimental and epidemiological findings supporting the hygiene hypothesis have been accumulated. These studies focused on endotoxins as substances associated with allergic diseases (Fahrlander et al., 2002; Mutius et al., 2000; Williams et al., 2005).

Endotoxins can cause lethal shock and fever when they enter the bloodstream. Therefore, it is closely monitored and strictly controlled in the medical, pharmaceutical, and food industries. Endotoxin is a cell wall component of gram-negative bacteria and is released when the cell wall breaks down (Mashimo, 2010; Tanamoto, 2008). Common examples of gram-negative bacteria include Escherichia coli, Salmonella, Helicobacter, Legionella, Enterobacteriaceae, and Acetobacter. Gram-negative bacteria are widespread in our living environment because they constitute a significant portion of the bacterial phylogeny (Tanamono, 2008; Mashimo, 2010). Endotoxin concentrations are particularly high in environments where animals are housed (Mutius et al., 2000).

Mutius et al. in rural areas of southern Germany and Switzerland found that endotoxin concentrations were highest in farm stables. In addition, endotoxin levels were significantly higher in mattresses and dust from kitchen floors in households where children had regular contact with farm animals, than in control subjects. Therefore, the authors proposed that the level of environmental exposure to endotoxins and other bacterial wall components plays a crucial role in protecting against the development of atopic diseases in childhood (Mutius et al., 2000).

Fahrlander et al. conducted a questionnaire survey on endotoxin levels, asthma, hay fever, and blood tests. The authors suggested that individual environmental exposure to endotoxins is a key factor in developing tolerance to common allergens found in natural environments (Fahrlander et al., 2002).

Williams et al. analyzed the existing literature. The results suggest that early endotoxin exposure may protect against the development of allergic sensitization and atopic asthma, particularly in children raised in rural European communities. However, endotoxin exposure may also contribute to nonatopic respiratory disorders and exacerbate disease in individuals with preexisting asthma (Williams et al., 2005).

In a population-based sample of US adults, endotoxin levels in homes were associated with a self-reported history of chronic bronchitis or emphysema (CBE) diagnosis and chronic bronchitis symptoms, with stronger associations among people sensitized to inhalant allergens (Mendy et al., 2018).

Although most studies on endotoxin concentrations have focused on the medical field, there have been several reports on studies conducted in indoor environments (Kim et al., 2016a, 2016b, 2016c; Kim et al., 2017; Lim and Kim, 2018; Shinohara et al., 2023). According to these reports, the average airborne concentration in office rooms was 0.14 EU/m3, and the maximum value was 1.0 EU/m3 (Kim et al., 2017). In the elderly care facility, the average indoor airborne concentration was 0.89 EU/m3, with a maximum value of 4.1 EU/m3 (Kim et al., 2017). Outdoor concentrations near offices and elderly care facilities were 0.4 and 0.3 EU/m3, respectively. Generally, the airborne concentrations were less than 1 EU/m3, but some locations reported values ranging from several to 70 EU/m3 (Kim et al., 2017).

Concentrations of dust in indoor air were reported to range from several hundred to tens of thousands of EU/g, with several thousand EU/g considered a typical value (Kim et al., 2016a, 2016b).

According to the latest research, endotoxin concentrations were higher in uninhabited houses than in inhabited houses (Shinohara et al., 2023).

Thus, while the status of endotoxin concentrations in the general environment is becoming clearer, there remains a scarcity of studies in environments with potentially higher concentrations.

In this study, we measured indoor endotoxin concentrations in buildings in Japan that are strongly associated with horses. The target buildings include a “Magariya,” an old Japanese house, an accommodation facility connected to a horse stable, and a stable, specifically designed for thoroughbreds.

Approximately, 50 years ago, horses were used for farming in Japan. In rural areas, horses were a familiar part of people’s daily lives. In addition, in modern times, accommodations that emphasize opportunities to interact with animals have emerged to obtain healing through animal therapy. The concept of animal therapy has become widespread, and interest in horseback riding is growing. Horses are larger than dogs, cats, and other animals kept as pets. Therefore, endotoxin concentrations are expected to be higher than in rooms where pets are housed. This study measured endotoxin concentrations in buildings strongly associated with horses.

2 Methods

2.1 Collection of endotoxins in air

A mixed cellulose ester membrane used for microbiological analysis was attached to a φ47-mm filter holder. In addition, 100 L of air was collected using a suction pump (MP-W5P; Sibata Scientific Technology Ltd.) for 30 min (3.3 L/min). Therefore, the effect of momentary wind speed fluctuations, etc., is small. For this measurement, one sample of data was collected per point. The measurement points were 4 or 5, so two measurements were performed using multiple suction pumps. The time required for the measurement was about 65 min, including filter replacement. During the measurement, care was taken to ensure that there were no major changes in the environment. The collected filter was mixed with distilled endotoxin-free water for injection and stirred with a vortex mixer for 90 s, and the supernatant liquid was used for analysis (Kim et al., 2018).

2.2 Preparation of dust samples

Dust samples were collected by attaching a collection filter to the suction port of a vacuum cleaner and suctioning approximately 1 m2 for 1 min. As with the air sample measurements, one sample of data was collected per point. The collected samples were weighed and diluted stepwise with distilled water at a predetermined ratio of dust weight to the analyzable range (Kim et al., 2018).

2.3 Analysis for endotoxins

A toxinometer (ET-5000; Wako Pure Chemical Corporation) was used to analyze endotoxin concentrations. The kinetic turbidimetric method was used to measure the changes in turbidity associated with endotoxin gelation of the lysate reagent. Quantification was based on a four-step concentration calibration curve (Kim et al., 2018).

3 Object of measurement

3.1 Japanese old houses

In traditional Japanese houses, horses used for farming are housed in one part of the entrance hall. In the Tono region of Iwate Prefecture, which was once the largest horse breeding area in Japan, the space designated for horse breeding was often connected to the human living space through an entrance hall. These houses are L-shaped and are called “Magariya.” It was common for horses to be kept near these old houses. Therefore, people caring for horses are likely to be exposed to high levels of endotoxins while maintaining their health. In this study, endotoxin concentrations in two Magariya buildings located in the Tono area were measured. These buildings are situated within a tourist facility that recreates a traditional mountain village. Because horses are tethered to the buildings, the conditions are considered to be close to those of a traditional Japanese living environment.

Figure 1 provides an overview of old house A, and Fig. 2 provides an overview of old house B. In both houses, the horse-keeping and human living spaces are connected through an entrance hall. Endotoxin concentrations were measured in the entrance hall, known as doma, the living room, known as Irori, and the living space with tatami mats. Horses are housed in both old houses; however, during the time of measurement in old house A, the horses were grazing outside. During the measurement period in old house B, the horses were stabled indoors. The entrances to both buildings were open at the time of measurement, but all other openings were closed. Measured objects A and B had been cleaned the day before the measurement.

Fig. 1
figure 1

Overview of Japanese old house A. The space designated for horse breeding is connected to the human living space through an entrance hall

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Fig. 2
figure 2

Overview of Japanese old house B. The layout is similar to old house A. These houses were inhabited by wealthy families

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3.2 Accommodation connected to horse stables

Accommodation C, located in Iwate Prefecture, Japan, was specifically built to coexist with horses and people. The property houses two horses (Haflinger breed) that freely graze in the pasture and stables. Notably, the guest rooms in accommodation C are directly connected to the stables through an entrance hall. Therefore, this accommodation can be considered a modern version of a “Magariya.”

Figure 3 provides an overview of accommodation C. On the first floor, the guest rooms and stables are connected via an entrance hall. On the second floor, there is a living space for the caretaker and an attic space in the stable area, with dried straw in the attic. The horses grazing on the property are managed in a way that is close to nature. Therefore, the horses are not brought back to the stables at night, resulting in their infrequent use. Guests are allowed to interact with the grazing horses, but sometimes the horses do not allow it. The facility fosters natural communication between horses and humans. In addition to the accommodations, there are spaces for the horses to exercise and a feeding area on the grounds. Measurements were taken in the guest rooms, entrance hall (outside air), stables, attic area, and caretaker’s living space. The two horses were not in the stables during the measurement periods. The measurement object C had been cleaned the day before the measurement.

Fig. 3
figure 3

Overview of accommodation C. On the first floor, the guest rooms and stables are connected via an entrance hall. This accommodation can be considered a modern version of a “Magariya”

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3.3 Racehorse stables

Japan is one of the most developed countries in horse racing. In Hokkaido, Japan, several racing thoroughbreds are produced each year. There are 24 racetracks in Japan, and horse races are held daily at one of these venues.

Stables where thoroughbreds are bred for racing are often built on the grounds of racetracks. In addition, numerous stables are designed for rest, training, and riding purposes. This study focused on measuring stables specifically designated for racehorses. The workers at the racehorse stables are responsible for work in stalls and caring for the horses. As a result, some people develop allergic diseases shortly after starting work and are compelled to leave the company. Some jockeys are allergic to horses. These jockeys ride in races but attempt to minimize contact with horses as much as possible. In addition, the upper floors of the stables and adjacent spaces may be occupied by trainers and their families. These residences may significantly differ from typical living conditions. In this study, measurements were performed at two different stables located in two different areas. Stable D is located on a racetrack in the urban region of Tokyo. Stable D is built on reclaimed land in the bay area, surrounded by parks, shopping centers, and housing complexes. Figure 4 provides an overview of stable D. The first floor of stable D consists of stables, and the second floor consists of living space. During the measurement period, the trainer and his family resided there. Measurements were performed near the center of the corridor in the stables on the first floor and in the reception room, bedroom, and Japanese-style room in the living space on the second floor. Airborne concentration measurements are performed before and after work in the stalls. Work in the stalls includes replacing the dried straw in the stalls and removing horse excrement. This means that work in the stalls is likely to cause air quality to deteriorate. Therefore, it is expected that the endotoxin concentrations before and after work would be different. Measurements were conducted immediately after work in the stall was completed. In other words, measurements after the end of the work are average values up to 30 min after the end of work. The purpose of the after-work measurements is to determine the environment the worker is exposed to. Although an air-conditioning system was installed in stable D, it was not used during the measurement period. In addition, no mechanical ventilation system was installed. During the measurement period, all stalls (12 stalls) were occupied by horses.

Fig. 4
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Overview of racing stable D. Wet straw is routinely dried outside the stables. There are 34 stables built in total, and the distance from the neighboring stable is 4.5 m

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Stable E is located within a horse race track in the mountains of Iwate Prefecture. Because stable E is located within a horse racing track that was built by carving out a mountain, it is surrounded by mountain forests. Figure 5 provides an overview of the stable E. The first floor of stable E consists of stables and a resting area for the stable staff, and the second floor is the attic area. Measurements were performed in the corridor and rest area on the first floor and in the attic on the second floor. No air-conditioning or ventilation system was installed in stable E. During the measurement periods, all the stables (20 stalls) were occupied by horses.

Fig. 5
figure 5

Overview of racing stable E. As in the stable D, wet straw is routinely dried outside the stables. There are 16 stables built in total, and the distance from the neighboring stable is 12.5 m

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Table 1 provides an overview of the measured object. Measurements A to C are connected to one or two horses, but the number of horses in the stables (D and E) is very large (Table 1).

Table 1 Overview of measurement objects. The enrollment density of horses is higher in racehorse stables D and E
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4 Results

4.1 Japanese old houses

First, we examined the concentration of endotoxins in the air. In old house A, where horses were not present during the measurement period, the outdoor air concentration was 1.1 EU/m3 (Table 2). Measurements taken at three indoor locations yielded concentrations lower than those of the outdoor air. It is possible that there was an effect of the two separate airborne concentration measurements. However, since both outdoor and indoor concentrations were low, indoor concentrations were considered to be almost the same as outdoor concentrations. In contrast, the concentration in the entrance hall of house B, where horses were stabled during the measurement period, was as high as 11.0 EU/m3 (Table 2). The living room near the entrance hall recorded a concentration of 6.4 EU/m3, and the tatami mat room farthest from the entrance hall recorded a concentration of 1.1 EU/m3, both of which exceeded the outdoor air concentration of 0.5 EU/m3. The measurement targets A and B are only about 100 m apart. They also have the same building orientation, floor plan, and cleaning frequency, which may affect endotoxin concentrations. These results indicate that the presence/absence of horses has a significant effect on airborne concentration. Takano et al. reported that endotoxin concentrations in the air before rearing horses were 10 times higher than that after rearing in a newly constructed stable (Takano et al., 2019). In addition, a part of the entrance hall of old house B is used as a stable. Therefore, the airborne concentration in the entrance hall was high, and the concentration tended to decrease as one moved away from the entrance hall (Table 2). This suggests that the stables where the horses are present are a source of endotoxin.

Table 2 Results of endotoxin concentration measurements. The results for airborne and dust concentration
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Furthermore, we focused on the concentration of endotoxins in the dust. In both old houses A and B, the highest concentrations were found in the entrance hall near the stables, followed by the tatami mat room and the living room. Thus, the results for dust concentration differed from those for airborne concentration. The concentration of endotoxin in dust being higher in the tatami mat room than in the living room could be due to the floor material used. Tatami is a traditional flooring material used in Japan.

4.2 Accommodation connected to horse stables

Airborne endotoxin concentrations were below 1.0 EU/m3 at all measurement points (Table 2). This is similar to the results for old house A, where horses were absent during the measurement period. Endotoxin concentrations in the dust were above 10,000 EU/g at all measurement points (Table 2). The highest value was found in the attic area, exceeding 50,000 EU/g (Table 2). This could be due to the large amount of dry straw in the attic area. After the attic, the next highest concentrations were found in the caretaker’s living area near the attic. The lowest values were found in the guest room. The guest room was the most frequently cleaned of the locations measured. The guest rooms were well-cleaned and maintained in a clean condition during the measurement period. The measured value for the guest room was 11,100 EU/g (Table 2), which is approximately the same as the average value for a typical house with pets.

4.3 Racehorse stables

First, we focused on the concentration of endotoxins in the air within stable D. Before the start of work in the stalls, the concentration in the building was 110.1 EU/m3 (Table 2), which is a very high value compared with the values in old houses A and B and accommodation facility C. The high concentration could be due to the high density of horses in the room and the presence of numerous sleeping straws in the space, which is a habitat for many microorganisms. In addition, stables are not subject to extensive human activity. Therefore, the cleanliness of the stable was low, and the lack of systematic mechanical ventilation further contributed to this situation. The airborne concentration in stable D after work in the stalls was 274.2 EU/m3 (Table 2), which was more than twice the value before work in the stalls. This increase could be due to the large amount of endotoxin contained in the straw that was dispersed into the air, while the straw was replaced in the stalls. Thus, people working in a stable environment are exposed to very high concentrations of endotoxins. The outdoor air concentration in stable D was 61.0 EU/m3, indicating a high concentration. This could be due to the wet straw being routinely dried outside the stables (Fig. 4). Furthermore, the air concentrations in the corridor portion of stable E were 135.3 and 208.8 EU/m3, which were similar to those of stable D (Table 2). The air concentrations in the outdoor environment with stable E were 9.7 EU/m3 (Table 2), which is higher than that in the general environment. However, it was lower than stable D. Similar to stable D, straw drying was routinely performed outside in the same manner as in stable E. In the case of stable D, several stables were densely built in an urban area. In contrast, stable E is located in a mountainous area and has sufficient distance from neighboring stables. Thus, the density of the stables could affect the air concentration.

Furthermore, we focused on dust concentration measurements. The dust concentration in stable D was 129,105 EU/g (Table 2), which is a very high value. The concentration in the reception room on the second floor of stable D was 21,698 EU/g (Table 2), which is lower than that on the first floor of the stable portion of the building. The tatami mat room on the second floor of stable D had a concentration of 9323 EU/g (Table 2). This was approximately half the value of the adjacent reception room but similar to the tatami mat room in old house A. This discrepancy could be due to the frequent use of the room. Trainers use the reception room to meet with horse owners. In addition, trainers perform clerical work in the reception room; therefore, the reception room is used frequently. In contrast, the tatami mat room is not used daily. When trainers frequently move in and out of the stables and reception room, it is possible that sources of endotoxin could be brought into the reception room through the trainer. The dust concentrations in stable E ranged from 34,654 to 58,741 EU/g (Table 2), which were high.

In summary, the enrollment density of horses tended to be higher, and endotoxin concentrations were generally higher in racehorse stables.

5 Conclusions

In this study, we focused on endotoxins, which play an important role in the development of allergen tolerance and performed measurements in horse-related facilities, where their concentrations may be higher than those in the general environment. Measurements were performed at an old Japanese house called “Magariya,” an accommodation facility connected to a horse and racehorse stable. As a result, the following conclusions were obtained. Buildings with horses present had higher air concentrations than those without horses. This suggested that the presence/absence of horses has a significant effect on airborne concentration. Additionally, airborne concentrations in stables where many horses were kept were higher than in old houses and accommodations where fewer horses were kept. Therefore, as the density of horses increases, endotoxin concentrations also tend to increase. Dust concentration had different values in different rooms even in the same building. These results suggest that dust concentrations may be affected by floor materials, frequency of cleaning, and frequency of human traffic from areas of high concentrations. Air concentrations measured immediately after work in stalls were very high. Therefore, workers are likely to be exposed to air with very high endotoxin concentrations during work in stalls.

These results indicate that the presence of horses in indoor environments involves in the increase of endotoxin concentrations. In addition, factors affecting endotoxin concentrations were inferred. In future work, it is desirable to conduct measurements for a wide range of building applications, including general housing, offices, and childcare facilities.