Exposure to (1? 3)-b-D-glucans in swine farms

Transcription

1 DOI /s ORIGINAL PAPER Exposure to (1? 3)-b-D-glucans in swine farms Marcin Cyprowski Alina Buczyńska Anna Kozajda Małgorzata Sowiak Karolina Bródka Irena Szadkowska-Stańczyk Received: 21 January 2011 / Accepted: 2 September 2011 Ó Springer Science+Business Media B.V Abstract The aim of this study was to assess the exposure to organic dust and (1? 3)-b-D-glucans in the buildings where an intensive breeding of swine is going on and evaluation of the impact of the breeding technical conditions on the observed levels of bioaerosols. The study was carried out in 30 swine farms differentiated by the size of the herd and technical conditions of breeding. In 35 randomly selected buildings, air samples were collected by stationary measurements to determine the concentrations of organic dust and (1? 3)-b-D-glucans in inhalable and respirable fractions. Furthermore, each of the investigated buildings was precisely characterized by means of a questionnaire for technical conditions and type of breeding. In each of the points, the microclimate parameters were measured, i.e., temperature, relative humidity, CO 2 concentration and air velocity. The analyzed levels of organic dust and (1? 3)-b-Dglucans were characterized by a wide range of concentrations. For inhalable fraction, they reached respectively: organic dust ( mg/m 3 ), (1? 3)- b-d-glucans (14 3,594 ng/m 3 ). For respirable fraction, the results were as follows: organic dust ( mg/ m 3 ), (1? 3)-b-D-glucans (1 703 ng/m 3 ). The concentrations of (1? 3)-b-D-glucans were positively M. Cyprowski (&) A. Buczyńska A. Kozajda M. Sowiak K. Bródka I. Szadkowska-Stańczyk Nofer Institute of Occupational Medicine, 8 Teresy St., Lodz, Poland macyp@o2.pl correlated with organic dust (r = 0.68; p \ 0.001). The most significant factor increasing the concentrations of organic dust and (1? 3)-b-D-glucans was the use of bedding in the form of cut straw. Additionally, the levels of (1? 3)-b-D-glucans were affected by manual forage feeding, mechanical manure disposal and the lack of the liquid manure container in breeding buildings. In view of the hazardous effects of biological agents on the health of swine-breeding workers, the swine management systems without beddings should be used, along with automated dosing techniques. Keywords Occupational exposure Organic dust (1? 3)-b-D-glucans Stationary measurements Swine breeding Technical conditions 1 Introduction Swine farm workers are exposed to biohazards at the workplace. Airborne biohazards (bioaerosols) in swine farms were evaluated mainly in the United States (Donham et al. 1995, 2000), the Netherlands (Vogelzang et al. 1998), Norway (Eduard et al. 2001, 2009) or South Korea (Kim et al. 2007). The analysis included viable microorganisms (bacteria, fungi), as well as bacterial endotoxins, suspended in dust, which may induce functional disorders of the respiratory system. Analyses of (1? 3)-b-D-glucans have been, so far, carried out occasionally (Eduard et al. 2001,

2 2009). (1? 3)-b-D-glucans are non-allergenic and hardly soluble in water glucose polymers, which consist of a part of the cellular wall of most fungi but also many plants and some bacteria. Owing to their presence in both viable and dead cells, they may be considered as a good indication of exposure to fungi (Douwes 2005). The swine breeding is characterized by variability of technical conditions applied depending on the farm s production profile. As some researchers have indicated, the breeding conditions may affect the concentrations of organic dust, microorganisms and endotoxins found at the workplace (Gustafsson 1999; Duchaine et al. 2000; Banhazi et al. 2008). Unfortunately, there are no similar analyses referring to (1? 3)-b-D-glucans. In connection with the above facts, an attempt was made to assess the exposure to organic dust and (1? 3)-b-D-glucans on swine farms in Poland as well as the impact of the technical conditions of breeding on the observed levels of these components of bioaerosol. 2 Materials and methods 2.1 Farms included into the study The study involved swine farms from central and north-western part of Poland, situated in a distance of km from the Institute. Thirty farms agreed to participate in the study of the list of 64 farms associated in unions of swine breeders and producers (participation at the level of 47%). The main reason of the other farms refusal was the apprehension of infecting the herds. Detailed characteristics of the swine farms and their buildings were based on visits to the farms and interviews with owners or their representatives. The interviews, in the form of questionnaires, comprised of the specificity, size and technical state of the buildings. Owners of the farms who did not participate in the study did not fill in the questionnaire. 2.2 Sampling methods To determine the concentrations of biohazards (organic dust and (1? 3)-b-D-glucans) in breeding buildings, 35 stationary points were placed in randomly selected buildings in the farm areas. The number of selected stationary points in the farm area depended on the total number of buildings situated there, according to the following divisions: 1 10 buildings (small farms) 1 measurement point; buildings (medium-sized farms) 2 measurement points; buildings (big farms) 3 measurement points; [30 buildings (very big farms) 4 measurement points. Air samples were collected using two measurement kits, one of which was aimed to measure the inhalable fraction of organic dust, whereas the other the respirable fraction. The inhalable fraction sampling kit consisted of the following elements: pump of GilAir 5 type (Sensidine, USA) and head of the 7-hole aerosol sampler type (Casella, UK) in which a filter made of glass fibers GF/A was mounted (Whatman, UK), of 25 mm diameter. The kit worked with the 2 l/ min airflow. The respirable fraction sampling kit consisted of the following elements: pump of GilAir 5 type and cyclone separator of C2/03 type (Two-Met, Poland) where a filter made of glass fibers GF/A was mounted (Whatman, UK), of 37 mm diameter. The kit worked at the 1.9 l/min airflow. Both types of the measurement kits were precisely calibrated before each test, using the liquid calibrator Gillibrator 2 (Sensidine, USA). The measurement kits were placed on a 1.5-m high stand. The stand was situated in the central part of the breeding building/premises. The average sampling time was approximately 6 h. A gravimetric method was used to evaluate organic dust concentrations. Each filter was weighed at a laboratory before and after the measurements, in a temperature- and humidity-controlled room, using balance CP 225D (Sartorius, Germany), the d accuracy of which amounted to 0.01 mg. During these measurements, the average value of temperature reached 22.1 C, within the range C. In case of relative humidity, its average value amounted to 29.9% within the range %. The calculated coefficients of variation reached 7.60 and 15.3%, respectively, which indicates that microclimatic conditions were stable and extreme values acceptable. The weighed filters before analyses of (1? 3)-b-Dglucans were stored at -20 C. 2.3 Analysis of (1? 3)-b-D-glucans For the analysis of (1? 3)-b-D-glucans, the frozen filters were extracted with 10 ml of water LAL (Limulus Amebocyte Lysate) (Lonza, USA) with an

3 addition of 0.05% Tween 20 (Sigma, Poland). The samples were shaken on a platform shaker for 15 min, afterward they were centrifuged at 1,0009g for 15 min. From the obtained supernatant, 1.8 ml of eluate was taken for the determination of the watersoluble (WS) fraction of (1? 3)-b-D-glucans. To the remaining part of the supernatant, 10 M NaOH (Sigma, Poland) was added to obtain a solution of 0.3 M NaOH concentration. The prepared samples were shaken for 10 min at 4 C, then again centrifuged at 1,0009g for 15 min. The obtained supernatant was used for the determination of alkali-soluble (AS) fraction of (1? 3)-b-D-glucans. The analysis was carried out using Glucatell test in kinetic version (Associates of Cape Cod Inc., USA). The amounts of (1? 3)-b-D-glucans concentrations were determined using a spectrophotometric reader SpectraMax Plus384 (Molecular Devices, Sunnyvale, USA), with light waves of 405 and 490 nm length and constant temperature 37 C (the procedure suggested by the producer of the test). The results were obtained by comparing the samples with the standard curve within pg/ml, which was generated from twofold serial dilutions of (1? 3)-b-D-glucans standard. The final concentration of (1? 3)-b-D-glucans was the sum of the two determined fractions. The concentration values were presented in ng/m Analysis of microclimate parameters At each of the determined stationary measurement points in breeding buildings, a measurement of the basic microclimate parameters, i.e., temperature, relative humidity, CO 2 concentrations and air velocity was performed. The measurements were carried out using a multifunction meter of microclimate Testo (Testo AG, Germany), which was equipped with two connected probes: IAQ and thermal. The measurements were carried out 1.5 m above the floor surface, for 10 min. The values of parameters were read every minute, then the result was averaged for a given measurement point. 2.5 Statistical analysis of results The statistical inference was carried out at the 0.05 significance level. Two-sided tests were used for verification of statistical hypotheses. When modeling the factors affecting the magnitude of exposure (concentration of organic dust, concentration of (1? 3)-b-D-glucans) in piggeries, the linear regression model was used with random effects. The farm was considered as a random effect in this model. All dependent variables in the models related to exposure predictors were log-transformed. All statistical calculations were carried out using the statistical package R v (The R Foundation for Statistical Computing, GNU GPL Licence). 3 Results 3.1 Description of investigated farms and buildings The summarized description of the investigated farms and buildings is presented in Table 1. Of the surveyed farms, only 20% were of exclusively breeding profile, the aim of which was to breed animals for as pure genetic material as possible. Approximately 67% were the production farms dealing with fattening of animals, whereas the remaining 13% were mixed productionbreeding farms. The size of the herds varied a lot between the farms, from 78 to 17,500 adult animals. Of the investigated buildings, in 75% of cases, the non-bedded system was used, where animals stayed without any bedding, on a slatted base. In the other buildings (25%), the breeding was conducted in the bedded system, with straw used as bedding. A mechanical ventilation system (76%) dominated in the investigated buildings. In the remaining buildings (24%), only gravitational ventilation was functioning. The number of animals differed largely between the buildings, from 4 to approx. 1,650. On average, the herd consisted of 400 animals. 3.2 Concentrations of organic dust and (1? 3)- b-d-glucans in breeding buildings Average airborne concentrations of organic dust and (1? 3)-b-D-glucans, including the inhalable and respirable fractions, their ranges and standard deviations, are presented in Table 2. The concentrations of inhalable dust ranged from 0.43 mg/m 3 to 11.8 mg/m 3, with the geometric mean value equal to 2.18 mg/m 3. In case of respirable dust, this range was from 0.01 mg/m 3 to 4.69 mg/m 3, while the mean concentration value was 0.39 mg/m 3.

4 Table 1 General characteristics of farms (N = 30) and livestock buildings (N = 35) N % Size of farm: Small Medium 2 7 Big Very big 1 3 Type of farm: Breeding Production 6 20 Mixed 4 13 Ventilation: Gravitational 8 24 Mechanical Swine management system: Bedded 9 25 Non-bedded Forage feeding method: Manual Mechanical Manure disposal system: Mechanical 7 23 Slatted Sink basins: Present Missing 9 25 Liquid manure container: Present Missing 7 23 For (1? 3)-b-D-glucans, we could state that the determined concentrations were within 14 3,594 ng/ m 3 for inhalable fraction and ng/m 3 for respirable fraction. The average geometric concentrations of (1? 3)-b-D-glucans measured in livestock buildings amounted to 190 and 37 ng/m 3, respectively, for inhalable and respirable fractions. The calculated dispersion measures indicate that a higher variability of concentrations was observed in case of respirable fraction, as compared to inhalable fraction (GSD value, respectively 6.80 and 3.90). Owing to applied procedure of the analysis of water-soluble (WS) and alkali-soluble (AS) (1? 3)-b-D-glucans, it was indicated that the share of AS fraction amounted to 85 and 68%, respectively, for inhalable and respirable fractions. The concentrations of (1? 3)-b-D-glucans were positively correlated with organic dust, where for both fractions of dust the coefficient r reached 0.68 (p \ 0.001). 3.3 Microclimate parameters Microclimate parameters were characterized by a considerable differentiation between the investigated buildings. Microclimate in livestock buildings was characterized by moderate temperature (arithmetical mean (AM) = 18.6 C, standard deviation (SD) = 3.5, range: C). Moreover, in the investigated buildings were found: increased relative humidity (AM = 76.9%, SD = 11.8, range: %) and increased CO 2 content (AM = 2,054 ppm, SD = 1,023, range: 685 4,730 ppm). The average airflow velocity in the buildings reached 0.15 m/s; however, this parameter was characterized by a very high instability during measurements (SD = 0.09, range: m/s). Statistical analysis demonstrated a significant correlation only between temperature values and concentrations of (1? 3)-b-D-glucans in respirable fraction (r =-0.47, p \ 0.05). 3.4 Factors influencing the magnitude of exposure The analysis involved the factors that characterize the animal breeding, technical solutions applied in the premises and those connected with ventilation, accumulation and disposal of droppings, as well as animal feeding methods. Tables 3 and 4 present the results of statistical analysis for inhalable and respirable fractions of organic dust and (1? 3)-b-D-glucans, respectively. This analysis indicated that the factor that determined the concentrations of respirable dust but also (1? 3)-b-D-glucans was the swine management system. Breeding in buildings with bedding caused a significant increase in the airborne concentrations of the investigated agents. The mean concentration of respirable dust in the bedded system amounted to 0.62 mg/m 3, whereas in buildings without bedding it was 3 times lower (0.21 mg/m 3 ). In case of (1? 3)- b-d-glucans in respirable fraction, the concentrations were 15 times higher than in the non-bedded system. Furthermore, the analysis indicated that manual forage feeding was associated with an increased exposure to (1? 3)-b-D-glucans. In case of the inhalable

5 Table 2 Concentrations of organic dust and (1? 3)-b- D-glucans in livestock buildings (N = 35) AM arithmetic mean, SD standard deviation, GM geometric mean, GSD geometric standard deviation AM SD GM GSD Range Organic dust (mg/m 3 ) Inhalable fraction Respirable fraction (1? 3)-b-D-glucans (ng/m 3 ) Inhalable fraction Total ,594 WS water-soluble AS alkali-soluble ,400 Respirable fraction Total WS water-soluble AS alkali-soluble fraction, the mean geometric concentration with the manual forage feeding amounted to 277 ng/m 3, which was almost 4 times more than in automated feeding (72 ng/m 3 ). The high levels of (1? 3)-b-D-glucans were also determined by mechanical manure disposal and the lack of the liquid manure container. 4 Discussion The determined values of organic dust concentrations were compared with the Polish MAC (maximum allowable concentration) values mandatory for respective fractions of dust (total dust 4 mg/m 3, respirable dust 2 mg/m 3 ). Analysis of dust demonstrated exceeded MAC values in 18% and 5% of the investigated samples for concentrations of inhalable and respirable dust, respectively. The values of organic dust concentrations inhalable and respirable fractions are comparable with the levels described in literature (Duchaine et al. 2000; Kim et al. 2007; Banhazi et al. 2008; Thorne et al. 2009). The analysis showed that higher concentrations of both fractions of organic dust were found where the bedded system was used. The same relationship has also been mentioned by Kim et al. (2007) and Banhazi et al. (2008). In this case, generated higher concentrations of organic dust are probably associated with its accumulation in the bedding material and particle rising in the air when the animals are moving. The microclimate parameter that significantly affected (1? 3)-b-D-glucans in respirable fraction was the temperature; however, there are no data in literature, which would allow to compare the obtained results. Analysis of available literature shows that the project s detailed analysis of the exposure to airborne (1? 3)-b-D-glucans during the works connected with intensive swine breeding is one of very few in the world. We should explicitly emphasize that so far no reference values have been proposed, which would allow for a hygienic evaluation of the obtained results of the study. Other researchers reports about the exposure to (1? 3)-b-D-glucans in piggeries are extremely scarce. Actually, presently two articles dealing with this issue can be mentioned, both referring to farmers in Norway. The first (Eduard et al. 2001) investigated the exposure of 106 farmers representing different profiles of vegetable production (crops, potatoes and onions) and livestock production (breeding of the cattle, swine and poultry). Unfortunately, the presented results of exposure assessment do not differentiate the analyzed groups of farmers. The obtained 8-h average weighed concentration of (1? 3)-b-D-glucans amounted to 0.82 lg/m 3 (geometric mean). That value was almost 4 times higher than the mean value obtained in our study (GM = 0.22 lg/m 3 ). In the latest article, Eduard et al. (2009) evaluated more than 4,700 Norwegian farmers, regarding to different types of agricultural production. Almost 9% of the studied population were the farmers specialized only in swine production. The exposure assessment in this group of farmers demonstrated the concentrations of (1? 3)-b-D-glucans at the level of 16 lg/m 3 (geometric mean). Two factors may affect such significant differences between the

6 Table 3 Evaluation of concentrations of organic dust in inhalable and respirable fraction, considering the technical conditions of livestock buildings GM geometric mean, GSD geometric standard deviation, ns not significant Type of factor Inhalable fraction (mg/m 3 ) Respirable fraction (mg/m 3 ) GM GSD p GM GSD p Type of farm Breeding ns ns Production Mixed Ventilation Gravitational ns ns Mechanical Swine management system Bedded ns \0.05 Non-bedded Forage feeding method Manual ns ns Mechanical Manure disposal system Mechanical ns ns Slatted Sink basins Present ns ns Missing Liquid manure container Present ns ns Missing results of our study and those mentioned above. First, the analytical technique for the determination of (1? 3)-b-D-glucans was different. In both mentioned items of literature, the immunoenzymatic method ELISA was used, which measures the content of epitopes sensitive to the antibody used in the analysis. Our study used the method with the LAL enzymatic test containing factor G, specific to (1? 3)-b-D-glucans, which measures the level of activity of (1? 3)-b-D-glucans. As indicated by Iossifova et al. (2008), both methods are characterized by a completely different sensitivity level, which for the LAL method amounts to pg/ml, whereas for the ELISA method to 250 ng/ml. Heldal et al. (2003) indicate that the comparison of the results from air samples obtained with the use of the above methods might lead to false conclusions. Another factor that hampers the comparison of these results might be the fact that the mentioned studies carried out by Eduard et al. (2001, 2009) were based on personal sampling, while in our study the measurements were carried out in a stationary way. Actually, it was known that in the situation when the swine workers moved from one building to another during their work, the results from personal sampling not always reflected the actual concentrations of the investigated agents in the buildings selected for the study. Therefore, the stationary measurements were more adequate for fulfillment of the aim of our study, i.e., indicating the factors that determine the investigated concentrations in breeding houses. Summarizing the results obtained by the LAL method but in different occupational environments, we can state that the concentration values obtained in our study are quite high. In the study carried out by Rylander and Carvalheiro (2006) on poultry farms, the average concentrations of (1? 3)-b-D-glucans at the level of 270 and 20 ng/m 3 were obtained, respectively, for the alkali-soluble (AS) and water-soluble (WS) fractions. According to that study, the percentage participation of the water-soluble fraction reached approximately 10% of all determined (1? 3)-b-Dglucans, so it was slightly lower than the result obtained on swine farms (approx. 15%). In the studies

7 Table 4 Evaluation of concentrations of (1? 3)- b-d-glucans in inhalable and respirable fraction, considering the technical conditions of livestock buildings GM geometric mean, GSD geometric standard deviation, ns not significant Type of factor Inhalable fraction (ng/m 3 ) Respirable fraction (ng/m 3 ) GM GSD p GM GSD p Type of farm Breeding ns ns Production Mixed Ventilation Gravitational ns ns Mechanical Swine management system Bedded \ \0.01 Non-bedded Forage feeding method Manual \ \0.01 Mechanical Manure disposal system Mechanical \ \0.01 Slatted Sink basins Present ns ns Missing Liquid manure container Present \ \0.01 Missing investigating the municipal wastes treatment plant workers, the concentrations at the level of approx. 40 ng/m 3 were found (Gladding et al. 2003; Heldal et al. 2003). Instead, during the work in sawmills, the concentration of (1? 3)-b-D-glucans was at the level of approx. 3 ng/m 3 (Mandryk et al. 2000). Our study indicates quite a high correlation of (1? 3)-b-D-glucans and organic dust for inhalable fraction (r = 0.68), which corresponds with the results obtained by Eduard et al. (2009). Unfortunately, there is no literature analyzing the impact of technical parameters of swine-raising in breeding houses on the concentrations of (1? 3)-b-D-glucans. Our study indicates a significant correlation between the forage feeding method and airborne concentration of (1? 3)-b-D-glucans on swine farms. This probably results from the fact that during the manual forage feeding more dust is released than when this procedure is performed mechanically (automation). Besides, the latest reports indicate that new types of forage more and more often contain (1? 3)-b-Dglucans as immunostimulators, which help to maintain a better condition of the animals and decrease their susceptibility to infections of the alimentary tract and respiratory system (Szymańska-Czerwińska et al. 2007). It was demonstrated, among others, that (1? 3)-b-D-glucans could significantly protect piglets against infections with enterotoxic strains of Escherichia coli (Stuyven et al. 2009). However, our study did not analyze the content of (1? 3)-b-Dglucans in the forage with which the animals are fed. As it was indicated in the study, the type of the manure disposal technique and presence/absence of a liquid manure container may be also defined as the variables, which indirectly point to the animal management system. Mechanical disposal of the manure usually takes place in the bedded system of breeding. The same is also proven by the lack of the liquid manure container. 5 Conclusions The study confirms that the swine breeding is characterized by high concentrations of (1? 3)-b-D-

8 glucans. Their levels in livestock buildings vary largely due to applied technical solutions. Considering the hazardous effects of such biological agents on the health of the swine-breeding workers, the non-bedded systems of the animal management and automated forage feeding techniques should be aimed at. In case of the bedded system of breeding, the animal servicing workers respiratory tract should be protected. Acknowledgments We are grateful to Wojciech Sobala, MSc who carried out the statistical analysis of the results. The study has been financed by the Ministry of Science and Higher Education grant No N /1987. Grant manager Irena Szadkowska-Stańczyk, PhD, MD. References Banhazi, T. M., Seedorf, J., Rutley, D. L., & Pitchford, W. S. (2008). Identification of risk factors for sub-optimal housing conditions in Australian piggeries: Part 2. Airborne pollutants. Journal of Agricultural Safety and Health, 14, Donham, K. J., Cumro, D., Reynolds, S. J., & Merchant, J. A. (2000). Dose-response relationship between occupational aerosol exposures and cross-shift declines of lung function in poultry workers: Recommendations for exposure limits. Journal of Occupational and Environmental Medicine, 42, Donham, K. J., Reynolds, S. J., Whitten, P., Merchant, J. A., Burmeister, L., & Popendorf, W. J. (1995). Respiratory dysfunction in swine production facility workers: Doseresponse relationships of environmental exposures and pulmonary function. American Journal of Industrial Medicine, 27, Douwes, J. (2005). (1? 3)-b-D-glucans and respiratory health: A review of the scientific evidence. Indoor Air, 15, Duchaine, C., Grimard, Y., & Cormier, Y. (2000). Influence of building maintenance, environmental factors, and seasons on airborne contaminants of swine confinement buildings. American Industrial Hygiene Association Journal, 61, Eduard, W., Douwes, J., Mehl, R., Heederik, D., & Melbostad, E. (2001). Short term exposure to airborne microbial agents during farm work: Exposure-response relations with eye and respiratory symptoms. Occupational and Environmental Medicine, 58, Eduard, W., Pearce, N., & Douwes, J. (2009). Chronic bronchitis, COPD, and lung function in farmers. The role of biological agents. Chest, 136, Gladding, T., Thorn, J., & Stott, D. (2003). Organic dust exposure and work-related effects among recycling workers. American Journal of Industrial Medicine, 43, Gustafsson, G. (1999). Factors affecting the release and concentration of dust in pig houses. Journal of Agricultural Engineering Research, 74, Heldal, K. K., Halstensen, A. S., Thorn, J., Djupesland, P., Wouters, I., Eduard, W., et al. (2003). Upper airway inflammation in waste handlers exposed to bioaerosols. Occupational and Environmental Medicine, 60, Iossifova, Y., Reponen, T., Daines, M., Levin, L., & Khurana Hershey, G. K. (2008). Comparison of two analytical methods for detecting (1 3)-b-D-glucan in pure fungal cultures and in house dust samples. The Open Allergy Journal, 1, Kim, K. Y., Ko, H. J., Kim, H. T., Kim, Y. S., Roh, Y. M., Lee, C. M., et al. (2007). Influence of extreme seasons on airborne pollutant levels in a pig-confinement building. Archives of Environmental and Occupational Health, 62, Mandryk, J., Alwis, K. U., & Hocking, A. D. (2000). Effects of personal exposures on pulmonary function and workrelated symptoms among sawmill workers. Annals of Occupational Hygiene, 44, Rylander, R., & Carvalheiro, M. F. (2006). Airway inflammation among workers in poultry houses. International Archives of Occupational and Environmental Health, 79, Stuyven, E., Cox, E., Vancaeneghem, S., Arnouts, S., Deprez, P., & Goddeeris, B. M. (2009). Effect of beta-glucans on and ETEC infection in piglets. Veterinary Immunology and Immunopathology, 128, Szymańska-Czerwińska M., Bednarek D., & Kowalski C. (2007). Effect of prebiotic additives on interleukin 1 activity and alternations of peripheral blood leukocyte subpopulations in calves. Medycyna Weterynaryjna, 63, [in Polish]. Thorne, P. S., Ansley, A. C., & Spencer Perry, S. (2009). Concentrations of bioaerosols, odors, and hydrogen sulfide Inside and downwind from two types of swine livestock operations. Journal of Occupational and Environmental Hygiene, 6, Vogelzang, P. F. J., van der Gulden, J. W. J., Folgering, H., Kolk, J. J., Heederik, D., Preller, L., et al. (1998). Endotoxin exposure as a major determinants of lungs function decline in pig farmers. American Journal of Respiratory and Critical Care Medicine, 157,