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This thesis was designed under the umbrella of the collaborative research centre (German: Sonderforschungsbereich) “SFB 852 Nutrition and intestinal microbiota - host interactions in the pig”, where different feeding strategies for pigs were examined in vivo, including probiotic treatment. There are many literature reports on the efficacy of the frequently-used probiotic Enterococcus faecium (E. faecium). The described effects range from no effect at all to a positive influence on performance (e.g. weight gain or feed conversion ratio) or protection from pathogens or reduced resistance to pathogens. Since the intestine is the interface where nutrition, microbiota, pathogens and host meet, the objective of the thesis was to further our knowledge on the effect of E. faecium in a Salmonella challenge situation, with particular focus on critically involved components of the intestinal mucosal network.
Chapter 1 gives a general introduction on the pig as one of the most important farm animals for meat production as well as for biomedical research. For a long time, antibiotic growth promotors (in-feed-antibiotics) were used to compensate for the negative effects of intensive pig husbandry. It is known that the overly intensive and uncontrolled use of antibiotics affects human as well as animal health due to a rise in multiple resistances of bacterial infections. Consequently, research efforts of the SFB were focused on feed additives, particularly on mechanistic studies on the effects of probiotics.
The field of work for this thesis was the “Histology Platform”, whose purpose was to apply and refine a broad spectrum of histological methodologies to the porcine organism in the course of several feeding trials. The effects of different nutritional strategies on gastrointestinal histological parameters under either normal housing conditions or in challenge situations were to be examined.
Chapter 2 gives a literature overview on the pig as a model organism in biomedical sciences; porcine intestinal morphology, physiology and immunology; the intestinal mucosal network; nutritional influences on microbiota, pathogens and host and histological techniques applied to investigate the gastrointestinal tract.
The objective of this thesis as well as the hypotheses are summarized in chapter 3 and are also found in the following descriptions.
Chapters 4, 5 and 6 cover the three main publications for this thesis.
Chapter 4 reports on intraepithelial lymphocyte numbers and histomorphological parameters in the porcine gut after Enterococcus faecium NCIMB 10415 feeding in a Salmonella Typhimurium challenge. Morphological parameters and the number of intraepithelial lymphocytes (IEL) were evaluated for the effect of the factors “time post infection/age” and “probiotic treatment” by light microscopy. The time post-infection had significant effects (P < 0.05) on the treated animals as well as in the control animals. Older animals showed longer and wider villi, deeper and wider crypts, a higher villus enlargement factor, a higher ratio between villus and crypt enlargement factors as well as more intraepithelial lymphocytes. Probiotic treatment resulted in a non-significant tendency for longer villi (P = 0.037), a slightly, but non-significant, higher ratio of villus surface/crypt circumference enlargement factors (P = 0.046) and significantly more IEL (P < 0.025). As the most important result, the population of intraepithelial lymphocytes situated at the nuclear level of the epithelium was identified to be strongly influenced by probiotic treatment (P = 0.004 at villus tip and P < 0.001 at villus base). It was concluded that the probiotic may have an immune modulatory effect by increasing the number of intraepithelial lymphocytes. These results confirmed the hypothesis that favourable effects of E. faecium treatment under a Salmonella challenge would involve beneficial changes in performance and immunological related parameters of the intestinal mucosal network. As an indicator for enhanced performance, the mucosal surface available for nutrient absorption was slightly enlarged (1.14-1.31-fold increase in absorptive surface related parameters “villus length”, “enlargement factor villi” and “ratio of villus surface/crypt circumference enlargement factors”). The epithelial barrier defending or regenerating intraepithelial lymphocytes were increased (1.09-2.32-fold), wich is a sign for improved immune protection. As an additional indicator for improved immune protection, the number of immunohistochemically detectable bacteria (Salmonella) invading the mucosa was expected to be significantly lower. Due to technical issues (see chapter 5), this hypothesis could not be tested.
Chapter 5 reports on the enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs. Samples were obtained from a challenge trial in which piglets were infected with Salmonella enterica serovar Typhimurium DT104. Tissue samples were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues, and detergents like Triton X-100 or Saponin were found to enhance the sensitivity of the detection method. Additionally, a detection limit for Salmonella in immunohistochemical preparations was estimated (102-103 CFU per g tissue). It was concluded that the use of detergents could result in a higher sensitivity in the immunohistochemical detection of salmonellae. To the best of my knowledge, this is the first report on this issue. The results confirmed the hypothesis that (species-) specific histological protocols improve the detection of pathogens within porcine tissues.
Since mast cells were identified as a central cell population with a multitude of physiological and pathological functions, including regulation of intestinal barrier function and host defence within the intestinal mucosal network, it was hypothesised that these cells are influenced by probiotic and other treatments in the SFB 852 trials. A species-specific protocol was to be established to reliably quantify mast cells in porcine tissues and find a basal cell number for further investigations in other SFB 852 trials. This aim was accomplished and in Chapter 6 the evaluation of different fixatives for histochemical staining techniques for porcine intestinal mast cells is described, including the effect of tissue shrinkage during fixation and embedding. Different tissue fixation and staining methods were evaluated in the porcine intestine. Metachromatic staining of mast cells was found to be critically dependent on the fixation and staining technique. The study revealed that zinc salt fixation preserved metachromatic staining in mast cells, which is the first report on this topic. Polychromatic methylene blue was deemed the optimal staining and in order to compare mast cell counting results between different fixation methods, tissue shrinkage has to be taken into account. These results also confirmed the hypothesis that (species-) specific histological protocols improve the detection and identification of immune cells within porcine tissues.
In a general discussion (chapter 7), 3 topics concerning the intestinal mucosal network are discussed that turned out to be important issues in histological studies on nutrition-microbiota-pathogen-host-interactions: developmental changes, nutrition-related changes and methodological aspects.