[Show abstract][Hide abstract] ABSTRACT: 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 probiotics-feeding does not affect the gene expression of AvBDs, it may induce AvBD12 secretion from the surface epithelium of the proventriculus in broiler chicks.
The Journal of Poultry Science 01/2015; 52(1):57-67. DOI:10.2141/jpsa.0140114 · 0.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Psoriasin (S100A7) is a member of the S100 protein family of calcium-binding proteins and plays a crucial role in local host defences. The present study aimed to identify the expression of S100A7 in the goat mammary gland and in milk. The goat S100A7 coding DNA sequence was identified using direct sequencing. An S100A7 antibody was raised in rabbits by immunization with a synthetic S100A7 peptide consisting of 13 amino acids. Messenger RNA expression and protein localization in different regions of a healthy mammary gland were detected by reverse transcription-polymerase chain reaction and immunohistochemistry. Changes in the concentration of S100A7 in the milk after an intramammary infusion of Escherichia coli lipopolysaccharide (LPS) were examined by an enzyme immunoassay.
The goat S100A7 peptide had 98% and 86% sequence similarity to that of sheep and bovines, respectively. The S100A7 mRNA expression was higher in the teat and udder skin than in the cistern and parenchyma of the mammary gland. Immunoreactive S100A7 was localized in the epithelial cells of the alveolus and gland cistern, and stratified squamous epithelium of the teat. Psoriasin as a secreted protein was detectable in healthy milk, and an intramammary LPS infusion increased the concentration of S100A7 in the milk. The results suggest that S100A7 is produced in the epithelial cells of the mammary gland and is secreted into the milk.
The Veterinary Journal 10/2014; 202(1). DOI:10.1016/j.tvjl.2014.06.013 · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to examine the effect of the somatic cell count (SCC) in milk on reproductive performance, such as pregnancy status in the prepartum period and ovarian function in the postpartum period, in dairy cows. Blood samples were collected every week from one month prepartum to parturition in order to measure the concentrations of 13,14-dihydro-15-keto-PGF2α (PGFM), estrone sulfate (E1S) and progesterone. Milk samples were collected three times per week in both the prepartum (for one month before the dry period) and postpartum periods (for 3 months immediately after parturition) to measure the SCC. Progesterone was also determined in the whole milk of postpartum cows to define the day of the first ovulation. In the prepartum period, the maximum SCC negatively correlated with the pregnancy period (r = –0.77), but not the calf birth weight. Positive and negative correlations were observed between the average SCC and PGFM or progesterone
concentrations in plasma, respectively (r = 0.84 or –0.92, respectively), at 39 weeks of pregnancy. In the postpartum period, a correlation was observed between the day of the first ovulation and both the average and maximum SCC (r = –0.74 and –0.75, respectively), whereas days open was not related to the SCC. These results suggest that a high SCC in the prepartum period may advance parturition by increasing PGF2α and decreasing progesterone and that the first ovulation in the postpartum period was affected by a high SCC.
Journal of Reproduction and Development 09/2014; 60(6). DOI:10.1262/jrd.2014-065 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day −6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days −6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 106 cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the mechanism by which the avian infectious bronchitis virus (IBV) affects eggshell formation. Attenuated IBV (aIBV group) or vehicle (control group) was injected into the oviductal magnum lumen of White Leghorn laying hens. The changes in the expression of genes related to eggshell formation (collagen types I and V, and CaBP-D28 K), densities of cytotoxic cells (CD8+ cells and TCR-γδ+ T cells) as well as gene expression of molecules related to cytotoxic immunoreaction (B-NK, perforin, granzyme and IL-2) and proinflammatory cytokines (IL-1β, IL-6 and IFN-γ) were examined by quantitative RT-PCR or immunohistochemistry in the isthmus and uterus. Gene expression of IL-1β and IL-6 receptors in the tubular gland cells of the isthmus and uterus was analyzed by RT-PCR. Gene expression of collagen type I, but not collagen type V, in the isthmus and CaBP-D28 K in the uterus was decreased in the aIBV group compared with that in the control. The frequencies of CD8+ cells and TCR-γδ+ T cells in the isthmus and uterus were significantly higher in the aIBV group than in the control group. The expression of cytotoxic molecular and proinflammatory cytokines was also higher in the aIBV group than in the control. The expression of IL-6 receptor, but not IL-1β receptor, was identified in the tubular gland cells in the isthmus and uterus. These results suggest that IBV infection causes disorder of eggshell formation by disturbing gene expression of collagen type I in the isthmus and CaBP-D28 K in the uterus, probably via the effects of substances from cytotoxic cells and proinflammatory cytokines.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the presence and localisation of gonadotropin releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR) and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR and PGRMCI, mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles, and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all of the receptors were primarily localised in the granulosa and theca cells. In addition, LHR was also localised in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors except GnRHRI in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR and PGRMCI decreased from the preantral follicles (primordial, primary and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, while FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that bacterial lipopolysaccharide (LPS), a ligand of Toll-like receptor 4 (TLR4), induced mucin mRNA to enhance the mucosal barrier in the hen vagina. The aim of this study was to determine the intracellular signaling molecules for that mucin induction, and the effect of molting and estrogen on their expression. The expression of TLR4, its adaptor molecules, and transcriptional factors in the vaginal mucosa of laying and molting hens treated with or without estradiol was examined by reverse-transcription PCR. The expression of mucin in the cultured mucosal tissue stimulated by LPS together with inhibitors of transcriptional factors was analyzed by quantitative reverse-transcription PCR. The expression of TLR4, its adaptor molecule, namely, myeloid differentiation factor 88 (MyD88) or Toll-interleukin 1 receptor domain-containing adaptor-inducing IFN-β (TRIF), and transcriptional factors, namely, cFos and cJun, declined in molting hens compared with that in laying hens, and were upregulated by estradiol. In vagina of laying hens, mucin expression was upregulated by LPS, whereas it was suppressed by inhibitors of transcriptional factors, namely, ALLN (an inhibitor of IκB proteolysis), BAY-117085 (an NFκB inhibitor), U0126 [a mitogen-activated protein kinase (MAPK) inhibitor], and transhinone IIA [an activated protein 1 (AP-1) inhibitor]. These results suggest that a MyD88-dependent pathway downstream of TLR4 and transcriptional factors of NFκB and AP-1 participate in the induction of mucin expression by LPS in the vaginal mucosa. These signaling functions may decline during molting owing to the decline in the level of circulating estrogen. Such mucin expression system may play a role in the mucosal barrier against infection in the vaginal mucosa.
[Show abstract][Hide abstract] ABSTRACT: Cathelicidins are a family of antimicrobial peptides found in neutrophils and the epithelium that have broad-spectrum activity against bacteria. This study aimed to investigative the mRNA expression of cathelicidins and protein localization of cathelicidin-2 in the goat mammary gland and its secretion into milk. The mRNA expression of cathelicidins was examined in different regions of the mammary gland by reverse transcription PCR. A cathelicidin-2 antibody was raised in rabbits by immunization with a synthetic cathelicidin-2 peptide consisting of 17 amino acids. The protein localization of cathelicidin-2 was investigated in the mammary gland by immunohistochemistry. Skim milk was collected before (0h) and 2, 4, 8, 12, and 24h after the intramammary infusion of lipopolysaccharide and saline, and the concentration of cathelicidin-2 was examined by an enzyme immunoassay. The mRNAs of cathelicidin-1, 2, 3, 6, and 7 were expressed in both the teat and deep region of the mammary gland from healthy and mastitic goats. Immunoreactive cathelicidin-2 was not localized in the epithelial cells of the teat skin, teat cistern, or mammary alveoli, whereas it was localized in polymorphonuclear cells in the mammary gland and those collected from the blood and milk. Cathelicidin-2 was detected in skim milk by Western blotting. The concentration of cathelicidin-2 in milk increased 4h after the intramammary infusion of lipopolysaccharide. These results suggest that cathelicidin-2 is expressed in polymorphonuclear cells and is secreted into milk in goat.
[Show abstract][Hide abstract] ABSTRACT: The ovary and oviduct of the hen are susceptible to various pathogenic microorganisms, and infections in these organs may not only contaminate the eggs, but also disorder the egg formation. The immune function of the ovary and oviduct is essential to protect these tissues from infection as well as for the production of hygienic eggs. This paper reviews recent studies on the host defence system in the reproductive organs with reference to their innate immune functions, with emphasis on the important role of Toll-like receptors and avian β-defensins in this defence system.
Avian biology research 02/2014; 7(1). DOI:10.3184/175815514X13902927945697 · 0.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucins play an essential role as mucosal barrier to prevent invasion of pathogens in the oviductal tissue of hens. The aim of this study was to determine the effect of estradiol and lipopolysaccharide (LPS) on the mucin expression in the lower oviductal segments (vagina and uterus) of hens. The mucosal tissues of the vagina and uterus were collected from White Leghorn laying and molting hens, and molting hens with or without intramuscular injection with 1 mg of estradiol-benzoate (EB) daily for 7 d. Part of these tissues was cultured in TCM-199 culture medium with or without LPS (10, 100, or 1,000 ng/mL) for 1.5 or 3 h. Mucin expression in the mucosa of laying, molting, and EB-treated molting hens (EB-group) and in those tissues cultured with or without LPS was analyzed by quantitative reverse-transcription PCR. Cultured tissues were also processed for paraffin sections and stained with Alcian blue (AB). In both the vagina and uterus, mucin expression and density of AB-positive mucopolysaccharide were reduced in molting hens compared with laying hens, and upregulated by EB. Mucin expression in the cultured vagina and uterus tissues of laying and molting hens was upregulated by LPS in a dose- and time-dependent manner. However, there was no response to LPS for induction of mucin in the tissues of EB-group hens. The mucin expression level in the vagina and uterus tissues stimulated by LPS was lower in the EB-group hens than in laying and molting hens, and that in the uterus was lower in the molting hens than in laying hens. These results suggest that mucin expression is stimulated by LPS in the vagina and uterus of laying and molting hens. Estrogen may upregulate mucin expression in those tissues in association with epithelial development, whereas it may suppress the response to LPS for mucin induction. The mucin expression caused by LPS may enhance mucosal barrier function and play a role in preventing infections by bacteria in the vagina and uterus.
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase.
[Show abstract][Hide abstract] ABSTRACT: Mastitis is most critical disease in dairy cows and causes huge cost in the dairy industry. To prevent and treat it, it is important to understand the mechanisms of immune function in the mammary gland. Innate immunity is non-specific acute-response immune function. Some components of innate immunity in the mammary gland are found, e.g. Lingual Antimicrobial Peptide (LAP), Lactoferrin (LF). These components are found to be localized in the alveolar epithelium of mammary gland. LAP belongs to the beta-defensin family, and plays a crucial role in killing a large variety of microorganisms. LF belongs to an iron-binding glycoprotein and has antibacterial activity. It is reported that LF has been localized immunohistochemically in mammary epithelial cells of lactating cows. Our previous study revealed that secretion of LAP into milk proceeded to that of LF after lipopolysaccharide (LPS) injection into the mammary gland. From this result, it is hypothesized that immunohistochemistry probably shows positive to either LF or LAP but not both in the alveolus vs epithelium in the mammary gland. Therefore, the aim of the present study is to investigate the immunolocalization of LAP and LF in the same bovine mammary tissue. Bovine mammary tissues were collected in the slaughterhouse and were fixed with neutralized formalin immediately. Paraffin sections (2-um thickness) were processed with antigen retrieval treatment followed by blocking with casein milk. Sections were cultured with LF antibody or LAP antibody. Immunoreaction products were visualized by incubation with a DAB. LAP and LF were localized in the cytoplasm of epithelial cell of alveolus. In some cases, LAP and LF were seen clearly in the same alveoli of section. In other cases, some epithelial cells were stained only LAP, but not LF, and other epithelial cells of alveolus were stained only LF, but not LAP. These results suggest the possibility that LAP and LF are differentially synthesized in the alveolar epithelium and may support our previous findings that their secretion occurs at the different time course.
3rd International Conference on Chemical Engineering and Advanced; 09/2013
[Show abstract][Hide abstract] ABSTRACT: Dectin-1 plays a critical role in the pathogenesis of intestinal inflammation by recognizing the pathogenic agents and mediating cytokine responses. The objective of this study was to establish the association between dectin-1 polymorphisms and susceptibility to non-specific digestive disorders (NSDD) and cytokine expression in rabbits. A total of seven coding SNPs were detected in dectin-1 gene. The genetic association between SNP (ss707197675A>G) and susceptibility to NSDD was evaluated using a case-control study (178 cases and 174 controls). The results revealed that the A allele was associated with an increased risk of developing NSDD in rabbits. The AA genotype significantly increased the genetic susceptibility to NSDD with odds ratio of 4.76 (95% confidence interval, 1.92-12.50, P = 0.0002) compared with GG and GA genotypes. We also experimentally induced NSDD in another independent growing rabbit population by feeding a low-fiber diet and subsequently investigated the cytokine mRNA expression. Among the four studied cytokines, the expression levels of IFN-γ, IL-17F, and IL-22 were increased 2.8 to 6.0 fold in AA genotype compared with GG genotype (P < 0.01). The greater IL-17F and IL-22 mRNA expressions indicated a positive correlation with severe intestinal inflammation (P < 0.05). The decreased expression of IL-10 was associated with severe intestinal inflammation (P = 0.006), but IL-10 expression was not influenced by dectin-1 genotype. In conclusion, polymorphism ss707197675 of dectin-1 is related with susceptibility to NSDD and increased expression of proinflammatory cytokines, and dectin-1 could be an important candidate gene associated with NSDD in rabbits.
[Show abstract][Hide abstract] ABSTRACT: Immune function in the vagina of hen oviduct is essential to prevent infection by microorganisms colonizing in the cloaca. The aim of this study was to determine whether CpG oligodeoxynucleotide (CpG-ODN) stimulate the expression of avian β-defensins (AvBDs) in hen vaginal cells. Specific questions were whether CpG-ODN affects the expression of AvBDs and proinflammatory cytokines, and whether the cytokines affect AvBDs expression in vaginal cells. The dispersed vaginal cells of White Leghorn laying hens were cultured and stimulated by different doses of lipopolysaccharide (LPS), CpG-ODN, interleukin (IL)1β, or IL6. The cultured cell population contained epithelial cells, fibroblast-like cells and CD45-positive leukocytes. The immunoreactive AvBD 3, 10 and 12 were localized in the mucosal epithelium in the section of the vagina. The expression of AvBDs, IL1β, and IL6 was analyzed by quantitative RT-PCR. RT-PCR analysis showed the expression of AvBD 1, 3, 4, 5, 10, and 12 in the cultured vaginal cells without stimulation. Toll-like receptors (TLRs) 4 and 21, which recognize LPS and CpG-ODN, respectively, and IL1 and IL6 receptors (IL1R and IL6R) were also expressed in them. The expression of IL1β, IL6, and AvBD 10 and 12 was upregulated by LPS, whereas only IL1β and IL6 were upregulated by CpG-ODN. IL1β stimulation upregulated AvBD 1 and 3 expression, whereas IL6 stimulation did not cause changes in AvBDs expression. These results suggest that CpG-ODN derived from microbes upregulates the expression of IL1β and IL6 by interaction with TLR 21, and then IL1β induces AvBD 1 and 3 to prevent infection in the vagina.
[Show abstract][Hide abstract] ABSTRACT: As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell-cell adhesion) and integrin (cell-extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β1 antibodies and their localizations were compared. Cadherin-positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin-positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β1 was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β1 -immuno reaction in the granulosa layers and integrin β1 -positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell-cell adhesion, resulting in the consequent loss of these layers from the cystic follicle.