Influence of Escherichia coli infection on intestinal mucosal barrier integrity of germ-free piglets.

AIMS: We focused on investigating the influence of Escherichia coli (E. coli) on the intestinal barrier. MATERIAL AND METHODS: We studied changes in the distribution and secretory activities of goblet cells and enteroendocrine cells (EECs), as well as changes in the population of mast cells (MCs) in the jejunal and colonic mucosa of germ-free (GF) piglets as a healthy control group and GF piglets whose intestines were colonised with E. coli bacteria on day 5. KEY FINDINGS: The results suggest that the colon of GF piglets is more resistant and less prone to coliform bacterial infection compared to the jejunum. This can be confirmed by a lower degree of histopathological injury index as well as an improvement of the morphometric parameters of the colonic mucosa, together with a significantly increased (p < 0.05) expression of MUC1/EMA, and ZO-3. We also observed a significant decrease in the population of activated MCs (p < 0.001) and EECs (p < 0.001). These findings may indicate a rapid response and better preparation of the intestinal barrier for possible pathological attacks and the subsequent development of mucosal lesions during the development and progression of the intestinal diseases. SIGNIFICANCE: To date, gut-targeted therapeutic approaches that can modulate bacterial translocation and chronic inflammation are still in their infancy but represent one of the most promising areas of research for the development of new effective treatments or clinical strategies in the future. Therefore, a better understanding of these processes can significantly contribute to the development of these targeted strategies for disease prevention and treatment.

Antibiofilm Activity of Weissella spp. and Bacillus coagulans Isolated from Equine Skin against Staphylococcus aureus.

The aim of this study was to evaluate the antimicrobial and antibiofilm activity of Weissella cibaria, Weissella hellenica and Bacillus coagulans, isolated from equine skin, against biofilm-forming Staphylococcus aureus CCM 4223 and clinical isolate methicillin-resistant S. aureus (MRSA). Non-neutralized cell-free supernatants (nnCFS) of tested skin isolates completely inhibited the growth and biofilm formation of S. aureus strains and caused dispersion of the 24 h preformed biofilm in the range of 21-90%. The majority of the pH-neutralized cell-free supernatants (nCFS) of skin isolates inhibited the biofilm formation of both S. aureus strains in the range of 20-100%. The dispersion activity of B. coagulans nCFS ranged from 17 to 77% and was significantly lower than that of nnCFS, except for B. coagulans 3T27 against S. aureus CCM 4223. Changes in the growth of S. aureus CCM 4223 in the presence of catalase- or trypsin-treated W. hellenica 4/2D23 and W. cibaria 4/8D37 nCFS indicated the role of peroxides and/or bacteriocin in their antimicrobial activities. For the first time, the presence of the fenD gene, associated with biosurfactants production, was detected in B. coagulans. The results of this study showed that selected isolates may have the potential for the prevention and treatment of biofilm-forming S. aureus infections.

Immunohistochemical visualisation of the enteric nervous system architecture in the germ-free piglets.

The enteric nervous system (ENS), considered as separate branch of the autonomic nervous system, is located throughout the length of the gastrointestinal tract as a series of interconnected ganglionic plexuses. Recently, the ENS is getting more in the focus of gastrointestinal research. For years, the main interest and research was aimed to the enteric neurons and their functional properties in normal conditions, less attention has been paid to the germ-free animals. Germ-free (GF) piglets have clear microbiological background and are reared in sterile environment. GF piglets are regarded as clinically relevant models for studying of human diseases, as these piglets' manifest similar clinical symptoms to humans. In this study we briefly summarised the main characteristics in immunohistochemical distribution of ENS elements in the wall of jejunum and colon of germ-free piglets.

Detection of Periodontal Pathogens from Dental Plaques of Dogs with and without Periodontal Disease.

Dental plaque bacteria are one of the main factors responsible for the development of a periodontal disease, which is the most common infectious disease in dogs. The aim of this study was to identify the presence of periodontal disease-related bacteria in the dental plaque of dogs. Plaque samples were taken from dogs with and without periodontal disease. Samples were analyzed for the presence of Porphyromonas gulae, Tannerella forsythia and Treponema denticola using a PCR technique amplifying 16S rRNA genes of P. gulae and T. forsythia and flaB2 genes of Treponema species, including T. denticola. The presence of T. forsythia was confirmed in all samples. P. gulae was detected in all dogs with periodontal disease and in 71.43% of dogs without periodontal disease. Treponema spp. were detected in 64.29% of the samples. Based on Sanger sequencing and Basic Local Alignment Search Tool algorithm, Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was present in 28.57% of dogs with periodontal disease, while T. putidum was present in 42.86% of dogs with periodontal disease and in 57.14% of dogs without periodontal disease. T. putidum was positively correlated with both P. gulae and T. forsythia, suggesting that it may be involved in the development of periodontal disease.

Antiadhesive, antibiofilm and dispersion activity of biosurfactants isolated from Bacillus amyloliquefaciens 3/22.

The aim of this work was to monitor the potential antibiofilm properties of biosurfactants (BS) isolated from Bacillus amyloliquefaciens 3/22 against biofilm formation of the indicator strain Staphylococcus aureus CCM 4223. In this work, the effect of BS 3/22 on biofilm growth during co-incubation, inhibition of biofilm-forming cell adhesion and biofilm dispersion was studied. BS 3/22 inhibited biofilm formation, with its formation decreasing significantly (p < 0.05; p < 0.01; p < 0.001) with increasing BS 3/22 concentration. BS 3/22 also showed antiadhesive activity, which correlated with the concentration used. The dispersing effect of isolated BS 3/22 on a 24-hour biofilm was also detected. BS 3/22 were effective in biofilm dispersion even at lower concentrations compared to antiadhesive activity and inhibition of biofilm formation.

Study of microbiocenosis of canine dental biofilms.

Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

Bacillus amyloliquefaciens-Derived Lipopeptide Biosurfactants Inhibit Biofilm Formation and Expression of Biofilm-Related Genes of Staphylococcus aureus.

Biosurfactants (BSs) are surface-active compounds produced by diverse microorganisms, including the genus Bacillus. These bioactive compounds possess biological activities such as antiadhesive, antimicrobial and antibiofilm effects that can lead to important applications in combating many infections. Based on these findings, we decided to investigate the antibiofilm activity of BSs from the marine Bacillus amyloliquefaciens against Staphylococcus aureus CCM 4223. Expression of biofilm-related genes was also evaluated using qRT-PCR. Isolated and partially purified BSs were identified and characterized by molecular tools and by UHPLC-DAD and MALDI-TOF/MS. Bacillus amyloliquefaciens 3/22, that exhibited surfactant activity evaluated by oil spreading assay, was characterized using the 16S rRNA sequencing method. Screening by PCR detected the presence of the sfp, srfAA, fenD and ituD genes, suggesting production of the lipopeptides (LPs) surfactin, fengycin and iturin. The above findings were further supported by the results of UHPLC-DAD and MALDI-TOF/MS. As quantified by the crystal violet method, the LPs significantly (p < 0.001) reduced biofilm formation of S. aureus in a dose-dependent manner and decreased expression of biofilm-related genes fnbA, fnbB, sortaseA and icaADBC operon. Data from our investigation indicate a promising therapeutic application for LPs isolated from B. amyloliquefaciens toward prevention of S. aureus biofilm infections.

Antimicrobial and Antibiofilm Activity of the Probiotic Strain Streptococcus salivarius K12 against Oral Potential Pathogens.

Oral probiotics are increasingly used in the harmonization of the oral microbiota in the prevention or therapy of various oral diseases. Investigation of the antimicrobial activity of the bacteriocinogenic strain Streptococcus salivarius K12 against oral pathogens shows promising results, not only in suppressing growth, but also in eliminating biofilm formation. Based on these findings, we decided to investigate the antimicrobial and antibiofilm activity of the neutralized cell-free supernatant (nCFS) of S. salivarius K12 at various concentrations against selected potential oral pathogens under in vitro conditions on polystyrene microtiter plates. The nCFS of S. salivarius K12 significantly reduced growth (p < 0.01) in Streptococcus mutans Clarke with increasing concentration from 15 to 60 mg/mL and also in Staphylococcus hominis 41/6 at a concentration of 60 mg/mL (p < 0.001). Biofilm formation significantly decreased (p < 0.001) in Schaalia odontolytica P10 at nCFS concentrations of 60 and 30 mg/mL. Biofilm inhibition (p < 0.001) was also observed in Enterobacter cloacae 4/2 at a concentration of 60 mg/mL. In Schaalia odontolytica P10 and Enterobacter cloacae 4/2, the nCFS had no effect on their growth.

Differences in Immune Response and Biochemical Parameters of Mice Fed by Kefir Milk and Lacticaseibacillus paracasei Isolated from the Kefir Grains.

The health benefits of kefir consumption have been well-known for hundreds of years. The objective of this study was to investigate the effect of kefir milk and the probiotic strain Lacticaseibacillus paracasei Z2 isolated from kefir grains on the immune response and selected parameters of the lipid and liver enzymatic profiles of mice. Mice fed with kefir milk showed significantly increased phagocytic activity and percentages of B cells in the blood and increased gene expression for mucins and percentages of CD8+ lymphocytes in the gut. By applying kefir, we achieved a significant reduction in serum LDL cholesterol and an LDL/HDL ratio that favored an increase in HDL cholesterol. Regarding the hepatic enzymes, in particular a significant reduction in ALT activity was observed. L. paracasei Z2 alone stimulated the immune response more markedly compared with kefir milk. Regarding the systemic level, we observed increases in the proportion of all T cells (CD3+), CD4+ lymphocytes and the ratio of CD4+:CD8+ cells, and regarding the local intestinal level we noted a significant increase in gene expression for mucins (MUC-1 and MUC-2) and IgA. Moreover, we confirmed the formation of a biofilm on the surface of the forestomach only after the application of L. paracasei Z2 alone, but not after kefir administration. The results confirmed the hypothesis that the final effect of the probiotic does not correspond with the effect of the individual strain but is the result of mutual interactions of the microorganisms presented in a preparation, and therefore in the case of multi-strain probiotics, in vivo testing of the complex preparation is necessary.

Biofilm-forming lactic acid bacteria of honey bee origin intended for potential probiotic use.

Scientists around the world are focusing their interest on the use of probiotics in honey bees as an alternative method of prophylaxis against causative agents of both American and European foulbrood. In our study we tested inhibitory activity against Paenibacillus larvae and the biofilm formation activity by various lactic acid bacteria isolated from honey bee guts or fresh pollen samples in the presence of different sugars added to the cultivation media. In addition, we tested the probiotic effect of a newly selected Apilactobacillus kunkeei V18 in an in situ experiment in bee colonies. We found antibacterial activity against P. larvae in four isolates. Biofilm formation activity of varying intensity was noted in six of the seven isolates in the presence of different sugars. The strongest biofilm formation (OD570 ≥ 1) was noted in A. kunkeei V18 in the presence of fructose; moreover, this isolate strongly inhibited the growth of P. larvae under laboratory conditions. Inhibition of P. larvae and Melissococcus plutonius by A. kunkeei V18 in situ was confirmed in a pilot study.

Antiadhesive, antibiofilm and dispersion activity of biosurfactants isolated from Bacillus amyloliquefaciens 3/22.

The aim of this work was to monitor the potential antibiofilm properties of biosurfactants (BS) isolated from Bacillus amyloliquefaciens 3/22 against biofilm formation of the indicator strain Staphylococcus aureus CCM 4223. In this work, the effect of BS 3/22 on biofilm growth during co-incubation, inhibition of biofilm-forming cell adhesion and biofilm dispersion was studied. BS 3/22 inhibited biofilm formation, with its formation decreasing significantly (p < 0.05; p < 0.01; p < 0.001) with increasing BS 3/22 concentration. BS 3/22 also showed antiadhesive activity, which correlated with the concentration used. The dispersing effect of isolated BS 3/22 on a 24-hour biofilm was also detected. BS 3/22 were effective in biofilm dispersion even at lower concentrations compared to antiadhesive activity and inhibition of biofilm formation.

Innovative Animal Model of DSS-Induced Ulcerative Colitis in Pseudo Germ-Free Mice.

The aim of this study was to investigate the use of a standardized animal model subjected to antibiotic treatment, and the effects of this treatment on the course of dextran sodium sulphate (DSS)-induced colitis in mice. By decontamination with selective antibiotics and observation of pathogenesis of ulcerative colitis (UC) induced chemically by exposure of mice to various concentrations of DSS, we obtained an optimum animal PGF model of acute UC manifested by mucin depletion, epithelial degeneration and necrosis, leading to the disappearance of epithelial cells, infiltration of lamina propria and submucosa with neutrophils, cryptitis, and accompanied by decreased viability of intestinal microbiota, loss of body weight, dehydration, moderate rectal bleeding, and a decrease in the selected markers of cellular proliferation and apoptosis. The obtained PGF model did not exhibit changes that could contribute to inflammation by means of alteration of the metabolic status and the induced dysbiosis did not serve as a bearer of pathogenic microorganisms participating in development of ulcerative colitis. The inflammatory process was induced particularly by exposure to DSS and its toxic action on compactness and integrity of mucosal barrier in the large intestine. This offers new possibilities of the use of this animal model in studies with or without participation of pathogenic microbiota in IBD pathogenesis.

Effects of Lactobacillus Fermentum Supplementation on Body Weight and Pro-Inflammatory Cytokine Expression in Campylobacter Jejuni-Challenged Chickens.

Due to the interest in using probiotic bacteria in poultry production, this research was focused on evaluating the effects of Lactobacillus fermentum Biocenol CCM 7514 administration on body weight gain and cytokine gene expression in chickens challenged with Campylobacter jejuni. One-hundred and eight 1-day old COBB 500 broiler chickens were equally assigned to four experimental groups at random. In the control group (C) chicks were left untreated, whereas in groups LB and LBCj a suspension of L. fermentum was administered. A suspension of C. jejuni was subsequently applied to groups Cj and LBCj. Body weight was registered, and the individuals were later slaughtered; cecum samples were collected at 12, 36 and 48 h post-infection (hpi). The entire experiment lasted seven days. Reverse transcription quantitative PCR (RT-qPCR) was used to determine expression levels of IL-1ß, IL-15, IL-17, and IL-18 at each time point. Pathogen-infected individuals were observed to weigh significantly less than those fed with the probiotic. Significant differences were also found in transcript abundance; expression of IL-15 was downregulated by the probiotic and upregulated by C. jejuni. The effects of bacterial treatments were time-dependent, as the expression profiles differed at later stages. The present outcomes demonstrate that L. fermentum both reduces the impact of C. jejuni infection on chicken body weight and regulates positively pro-inflammatory cytokine expression, which ultimately increase bird well-being and improves production.

The Influence of Feed-Supplementation with Probiotic Strain Lactobacillus reuteri CCM 8617 and Alginite on Intestinal Microenvironment of SPF Mice Infected with Salmonella Typhimurium CCM 7205.

Alginite is a non-ore raw material arising by fossilization of accumulated organic (algae) and inorganic material, particularly clay, carbonates, quartz, and amorphous modification of silicic acid in the aqueous environment. Humic acids as a component of organic portion of alginite are known for very good buffering ability which allows them to stabilise pH throughout the digestion system of animals, stimulate receptors of the immune system in intestinal villi against pathogenic bacteria, and support proliferation and activity of beneficial bacteria (lactobacilli, bifidobacteria, and similar). Our investigations focused on the influence of a probiotic strain in combination with alginite on intestinal microenvironment of SPF mice infected with Salmonella Typhimurium. The 66 female mice (BALB/c) used in our study were divided to four experimental groups, control NC1, control NC2 (alginite), IC (alginite + Salmonella Typhimurium CCM 7205NAL), LAB (Lact. reuteri CCM 8617 + alginite + Salm. Typhimurium CCM 7205NAL). The group supplemented with Lact.reuteri CCM 8617 and alginite showed significant reduction in growth of Salm. Typhimurium in mice faeces at 24 and 72 h (P < 0.001) post infection. The supplementation of additives affected positively also nitrogen, enzymatic, hepatic and energy metabolism of mice. The demonstrable positive influence of additives alleviated the negative impact of Salm. Typhimurium infection on the morphology investigated in the jejunum and ileum of LAB group of mice. The livers of mice treated with both alginite and Lact.reuteri CCM 8617 showed marked reduction of overall inflammation, hepatocyte necrosis and size of typhoid nodules.

Biosurfactants and their role in the inhibition of the biofilmforming pathogens.

Resistance of pathogenic bacteria is currently one of the major medical problems. Most microbial infections are based on the formation of biofilms, which are a significant reservoir of pathogens. The aim of this study is to determine the antibiofilm and antimicrobial activity of biosurfactants isolated from intestinal lactobacilli and marine bacteria. Biosurfactants (BS) isolated from the strains L. fermentum 2I3, L. fermentum B2/6, L. reuteri SL16, L. reuteri B6/1, S. luteola 3/22, Brevibacillus sp. 4/9, Brevibacillus sp. 2/30 and B. amyloliquefaciens 1/6K significantly (p < 0.001) inhibited the biofilm formation of S. aureus CCM 3953 and P. mirabilis CCM 7188, with higher inhibition detected in BS of marine bacteria when compared to BS isolated from lactobacilli. The results suggest that the mechanism of the antibiofilm effect of BS isolated from lactobacilli against both the reference strains is the same and it is not the result of their antimicrobial action. In contrast, the mechanism of the antibiotic effect of BS isolated from marine bacteria probably depends on the properties of the indicator strain. Key words: biosurfactants biofilm pathogens inhibition.

Amoxicillin-clavulanic acid and ciprofloxacin-treated SPF mice as gnotobiotic model.

The experiment was carried out on 24 SPF BALB/c female mice and lasted for 15 days with a 5-day antibiotic (ATB) treatment and then 10 days without ATB treatment. The aim of our study was to acquire an animal model with reduced and controlled microflora and, at the same time, to ensure that the good health of these animals is maintained. Per oral administration of amoxicillin and clavulanate potassium in Amoksiklav (Sandoz, Slovenia) at a dose of 387.11 mg/kg body weight (0.2 ml of dilution per mouse) and subcutaneous administration of ciprofloxacin in Ciloxan (Alcon, Spain) at a dose of 18.87 mg/kg body weight (0.1 ml of dilution per mouse) were performed every 12 h during first 5 days of experiment. Five-day treatment with ATB led to a reduced survivability of microorganisms in faeces (28.33 ± 0.43 % on day 2) and caecum content (28.10 ± 1.56 %), where no cultivable microorganisms in faeces were present. Ten-day convalescence of decontaminated animals under gnotobiotic conditions prevented recovery of species diversity in mice gut microflora. This was reduced to two detectable cultivable species, namely Escherichia coli (GenBank KX086704) and Enterococcus sp. (GenBank KX086705) which were capable to restore its metabolic (CRL 2012) and morphological potential (Baratta et al. Histochem Cell Biol 131:713-726, 2009) within physiological range. Animals obtained under this procedure can be used in further studies. As a result, we created a mouse gnoto model with reduced and controlled microflora without alteration of the overall health status of the respective animals.

Bovine vaginal strain Kocuria kristinae and its characterization.

Kocuria spp. are widely distributed in nature. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria. In general, limited knowledge exists concerning the properties associated with the representants of the genus Kocuria, Kocuria kristinae as well. Following our previous results, K. kristinae Kk2014 Biocenol(™) (CCM 8628) was isolated from vagina of a healthy cow. Its taxonomical allottation was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system and phenotypic characteristics. Kk2014 strain showed strong adherence capability to the vaginal mucus, produced organic acids which can play a role in prevention of unsuitable contamination, and showed in vitro antagonistic/antimicrobial activity against strains Arcanobacterium pyogenes CCM 5753, Fusobacterium necrophorum CCM 5982, Streptococcus equi subsp. zooepidemicus CCM 7316, and Gardnerella vaginalis CCM 6221. Antimicrobial activity ranged from 100 to 200 AU/mL, up to 32 mm in size, respectively.

Analysis of biofilm formation by intestinal lactobacilli.

In this study, the biofilm-forming potential of intestinal Lactobacillus reuteri strains under different culture conditions was characterized by microtiter plate biofilm assays. Moreover, the spatial organization of exogenously applied L. reuteri L2/6 (a pig isolate) at specific locations in gastrointestinal tract of monoassociated mice was investigated by fluorescence in situ hybridization. We did not detect biofilm formation by tested strains in nutrient-rich de Man-Rogosa-Sharpe (MRS) medium. On the contrary, a highly positive biofilm formation was observed in medium with lower accessibility to the carbon sources and lack of salts. The results obtained confirmed the significant role of Tween 80 and the quantity and nature of the sugars in the growth medium in biofilm formation. The omission of Tween 80 in MRS medium favored the formation of biofilm. Abundant biofilm formation was detected in the presence of lactose, galactose, and glucose. However, a gradual increase in sugar concentration triggered a significant decrease in biofilm formation. In addition, conditions related to the gastrointestinal environment, such as low pH and the presence of bile and mucins, highly modulated biofilm production. This effect seems to be dependent on the specificity and properties of the medium used for cultivation. From the evidence provided by this study we conclude that the biofilm formation capacity of L. reuteri is strongly dependent on the environmental factors and culture medium used.

Influence of dietary supplementation with flaxseed and lactobacilli on the mucosal morphology and proliferative cell rate in the jejunal mucosa of piglets after weaning.

The aim of the study was to investigate the influence of flaxseed and lactobacilli supplementation to the diet of piglets during the time period between 10 days before and 21 days after weaning. The morphometry of the jejunal mucosa and proliferative ratio of both epithelial and lamina propria cells were compared with those found in a group of piglets fed with the usual diet added with sunflower oil during the same time period. The addition of flaxseed oil to the diet significantly increased the crypt depth in comparison with both groups supplemented with sunflower (P < 0.05 and 0.001 respectively) on the weaning day. Moreover, the flaxseed addition caused a significant decrease in villus height (P < 0.01) and crypt depth (P < 0.01) 21 days postweaning in comparison with the sunflower group. The proliferative ratio of the epithelial cells in the sunflower group on the weaning day was significantly higher than in both flaxseed groups (P < 0.01). Paradoxically, significantly higher proliferative activity in the mucosal connective tissue in the group with flaxseed supplementation in comparison with the sunflower group was observed on the day of weaning, as well as 3 days later (P < 0.05 both). A combination of flaxseed with lactobacilli showed significantly lower proliferative activity in the connective tissue cells from weaning up to 7 days after weaning (P < 0.05 all) in comparison with the flaxseed group.

Influence of dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning.

A histological study was designed to determine the influence of flaxseed and/or lactobacilli inclusion in the diet of piglets from 10 days before to 21 days after weaning. The selected inflammatory cell population incidence in the piglet jejunal mucosa was investigated. Significantly higher numbers of myeloperoxidase-positive (P<0.01) and CD163-positive (P<0.001) cells in the jejunal mucosa were recorded on the weaning day and for 7 days after (P<0.001 and P<0.01, respectively) in the flaxseed group compared with the basal diet. The number of intraepithelial lymphocytes was also significantly increased until 3 days after weaning (P<0.001). A prolonged significant increase in the myeloperoxidase-positive cells and intraepithelial lymphocyte numbers in the flaxseed+lactobacilli group was detected. In contrast, the number of CD163-positive cells in the flaxseed+lactobacilli group was significantly lower on the day of weaning (P<0.05) and 3 days after (P<0.01). The same effect was observed in the group with lactobacilli alone during the first 3 days after weaning (P<0.05 and P<0.01, respectively) and these findings indicate down-regulation of CD163 expression in the jejunal mucosa by lactobacilli. The presence of lactobacilli in the diet had a stimulatory effect on goblet cell quantity in the epithelium (P<0.001) and a distinct 50% reduction in the flaxseed group (P<0.01) compared with the basal diet was observed on the weaning day. A significant increase in myeloperoxidase-positive cell number in the jejunal mucosa in the flaxseed+lactobacilli group was the only significant difference (P<0.05 and P<0.01, respectively) found 21 days after weaning in comparison with all the other groups, indicating the pro-inflammatory effect of this feed additive combination. We conclude that dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning might be linked with significant anti-inflammatory effects in the jejunal mucosa.