Hatchery workers´ exposure to airborne bacteria and development of immunofluorescence based methods for detection of antibody responses

Abstract

Structural changes in livestock farming also led to densification of poultry egg production in commercial hatcheries. In consequence work conditions altered significantly and hatchery workers are frequently exposed to bioaerosols that are insufficiently characterized. Moreover, etiology of occupational bioaerosol related respiratory disorders is not well understood. In this doctoral thesis quantitative and qualitative culture dependent and independent methods were applied to analyze and to compare in particular airborne bacterial communities in different poultry hatcheries. Furthermore, the source of airborne bacteria in hatcheries was investigated. Considering that bioaerosol-related health effects and disorders often share immunological features this study also aimed at development of adequate serological methods for detection of workers specific immunoreactions to workplace antigens. Determination of the microbial exposure in a chicken and a turkey hatchery during different tasks with handling of poults or eggshells revealed high concentrations of airborne microorganisms (up to 5.3 x 106 cells m-3). Analysis of bacterial community compositions by construction of clone libraries displayed a low species diversity but an extensive similarity on the genus level in both hatcheries, revealing Enterococcus, Clostridium, Staphylococcus and Acinetobacter as predominant genera. Moreover, identification of bacterial and fungal isolates confirmed occurrence of various pathogens which are classified as risk group 2 microorganisms (German technical rule for biological agents (TRBA) 460, 466). Furthermore, the bacterial community present on eggshells samples was analyzed. For the first time it was documented that eggshell fragments facilitate excessive bacterial growth under hatchery incubator conditions and multiplication clearly contributes to potential harmful bioaerosol formation. Determination of workers antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, this technique was used to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, a tool for automated image analysis was developed and validated. The Fluorolyzer soſtware is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers to bacterial isolates from duck hatchery air. Moreover, in addition to established assays with pure bacterial cultures, a new approach utilized complex bioaerosol samples fordetection of anti-microbial antibodies in human sera by determination of percentages of antibody-bound cells in different serum dilutions. Mean titers in sera from hatchery workers and a non-exposed control group did not display significant differences for most tested isolates and application of comprehensive cluster analysis to entire titer data revealed no structure reflecting workers and controls group. Furthermore, determination of immunoreactivity to the complete microbial community in workplace air displayed similar proportions of antibody-bound cells in both groups. Although no general differences in immunoreaction patterns were observed, mean titers to a Proteus mirabilis isolate and to 3 of 4 distinct Acinetobacter baumannii isolates were higher in the group of hatchery workers than in the reference group indicating a need for further investigations regarding potential involvement in pathogenesis of occupational respiratory disorders. A novel approach presented here aimed at identification of immunogenic bacteria in complex bioaerosol samples. For this purpose a protocol for separation of bacterial cells from complex bioaerosol samples by Laser Microdissection and Pressure Catapulting (LMPC) was developed. Although technical key issues like an appropriate base material or mode of cell lysis have been established successfully, PCR based amplification of 16S rRNA genes from microdissected cells could not be accomplished until now and remains to be further optimized.