The aim of this study was to determine the effects of probiotics-feeding on the gene expression and protein localization of avian β-defensins (AvBDs) in the proventriculus of broiler chicks. Male broiler chicks were arranged in 3 groups: control group, probiotics group I and probiotics group II, which were fed with starter rations containing 0%, 0.2% or 0.4% probiotics, respectively, from day 0 (D0; at one day old) to D14. Proventriculi in all groups were collected at D0, D7 and D14 for analysis of AvBDs expression and AvBD12 protein localization. The expression of AvBDs genes was examined by reverse transcription-PCR and changes in the expression upon probiotics-feeding were examined by real-time PCR. The AvBD12 localization was examined by immunohistochemistry, and density of immunoreaction products was examined by image analysis under a microscope. Out of 14 AvBDs genes, seven AvBDs were detected in the proventriculus of chicks, namely, AvBD1, 2, 4, 6, 7, 10 and 12. The expression of the 7 detected genes did not show any significant differences between control and probiotics groups at D7 and D14. The immunoreactive (ir) -AvBD12 was localized in surface epithelium and cells in the connective tissues of proventricular glands. The ir-AvBD12 density in surface epithelium was significantly higher at D7 than at D0 or D14 in control group. At D7 and D14, the ir-AvBD12 density was significantly lower in probiotics groups than in control group. The ir-AvBD12 cells in proventricular gland increased in number with age; however, there were no significant differences between control and probiotics groups at D7 and D14. These results suggest that, although probioticsfeeding does not affect the gene expression of AvBDs, it may induce AvBD12 secretion from the surface epithelium of the proventriculus in broiler chicks.

The aim of this study was to determine the effects of probiotics on T cell subsets induction in the intestine of broiler chicks. Day-old male broiler chicks were fed with or without probiotics consisting of Streptococcus faecalis, Clostridium buthricum, Bacillus mesentericus (probiotics group and control group, respectively). Cryostat sections of their ileum, cecum and rectum at day 0, 7 and 14 of feeding were immunostained for CD4, CD8 and TCRγδ, and the frequencies of positive cells in the mucosal tissue were analyzed. The CD4+, CD8+, TCRγδ+ T cells were localized in the lamina propria of intestinal mucosa in all birds. At day 7 and 14, CD8+ T cells were localized also in the mucosal epithelium of all segments in the probiotics group and of cecum in control group, and TCRγδ+ T cells were observed in the mucosal epithelium of all birds. The frequencies of CD4+, CD8+, TCRγδ+ T cells were increased with age from day 0 to day 14 in both control and probiotics groups. The frequency of CD8+ T cells was significantly greater in probiotics group than control group in the ileum and rectum (P＜0.05) and the cecum (P＜0.01) at day 7. There were no significant differences in the frequency of CD4+ and TCRγδ+ T cells between control and probiotics groups in all intestinal segments at day 7 and 14. The ratio of CD8+/CD4+ T cells was greater than 1.0 in all tissues. The ratio in the ileum at day 7 was significantly greater in the probiotics group than control group (P＜0.05). These results further suggest that probiotics cause an influx of the CD8+ T cells into the intestinal mucosa, which may enhance the intestinal immunity by CD8+ T cells in young chicks.