Lysozyme mRNA Expression Research Articles

Coated sodium butyrate and vitamin D3 supplementation improve gut health through influencing intestinal immunity, barrier, and microflora in early-stage broilers.

Intestinal development and function are critical to maintaining sustained broiler growth. The present study aimed to evaluate the effects of coated sodium butyrate (CSB) and vitamin D3 (VD3) on the intestinal immunity, barrier, oxidative stress and microflora in early-stage broilers. In total, 192 one-day-old broilers were assigned to a 2 × 2 factorial design including two dietary supplements at two different levels, in which the main effects were VD3 (3000 or 5000 IU kg-1) and CSB (0 or 1 g kg-1). The results showed that CSB supplementation increased ileal goblet cells (GCs) numbers, villus height and decreased crypt depth in broilers. CSB increased ileal proliferating cell nuclear antigen expression and high-level VD3 decreased cluster of differentiation 3 expression. CSB reduced serum d-lactate, endotoxin (ET), adrenocorticotropic hormone, corticosterone and malondialdehyde (MDA) concentrations and increased total antioxidant capacity (T-AOC) level. Meanwhile, high-level VD3 decreased serum ET concentration. Furthermore, CSB increased ileal T-AOC, lysozyme (LYZ) and transforming growth factor (TGF)-β and decreased MDA, whereas high-level VD3 decreased ileal MDA and increased secretory immunoglobulin A. CSB up-regulated ileal claudin1, superoxide dismutase 1, TGF-β and LYZ mRNA expression and down-regulated interleukin-1β mRNA expression. CSB combined with high-level VD3 increased ileal Faecalibaculum abundance. Spearman correlation analysis showed that Faecalibaculum was related to the immune and barrier function. Dietary supplementation with CSB and high-level VD3 improved early gut health in broilers by promoting intestinal development, enhancing antioxidant capacity, strengthening barrier function and enhancing the favorable composition of the gut bacterial flora. © 2024 Society of Chemical Industry.

Illumination of different light wavelengths on growth performance and physiological response of juvenile sweetfish, Plecoglossus altivelis

Light-emitting diodes (LEDs) have gained increasing popularity in fish farming because certain wavelengths of light may promote the growth and innate immune system of fish. In this study, we reported the effects of different LED wavelengths on growth performance, antioxidant defense, and innate immune responses in sweetfish. According to the results, the blue-light group showed significantly increased body weight and length after 12 weeks. In contrast, the red-light group showed significantly inhibited growth performance and decreased feeding motivation. There was no difference in survival rate among the groups during the 12-week experiment. The antioxidant defense analysis results showed that red light may cause increased splenic oxidative stress. Splenic tnf-α mRNA expression increased in the blue-light group at weeks 4, 8, and 12; however, in the red-light group, this increase was significant only at week 12. il-1β mRNA level increased at week 4 in both groups. The lysozyme mRNA expression was downregulated in the blue-light group after 4 weeks, but it decreased only at week 12 in the red-light group. Histological sections of the ovary in the blue-light group showed a higher synchronization of oocyte development compared with the red-light group. In summary, light wavelength in a captivity environment can influence the physiological response of sweetfish. This study thus demonstrated that blue LED has a positive effect on fish growth performance, antioxidant ability, innate immunity, and reproduction.

Vitamin A- and D-Deficient Diets Disrupt Intestinal Antimicrobial Peptide Defense Involving Wnt and STAT5 Signaling Pathways in Mice.

Vitamin A and D deficiencies are associated with immune modulatory effects and intestinal barrier impairment. However, the underlying mechanisms remain unclear. C57BL/6J mice were fed either a diet lacking in vitamin A (VAd), vitamin D (VDd) or a control diet (CD) for 12 weeks. Gut barrier function, antimicrobial peptide (AMP) defense and regulatory pathways were assessed. VAd mice compared to CD mice showed a reduced villus length in the ileum (p < 0.01) and decreased crypt depth in the colon (p < 0.05). In both VAd- and VDd-fed mice, ileal α-defensin 5 (p < 0.05/p < 0.0001 for VAd/VDd) and lysozyme protein levels (p < 0.001/p < 0.0001) were decreased. Moreover, mRNA expression of lysozyme (p < 0.05/p < 0.05) and total cryptdins (p < 0.001/p < 0.01) were reduced compared to controls. Furthermore, matrix metalloproteinase-7 (Mmp7) mRNA (p < 0.0001/p < 0.001) as well as components of the Wnt signaling pathway were decreased. VAd- and VDd-fed mice, compared to control mice, exhibited increased expression of pro-inflammatory markers and β-defensins in the colon. Organoid cell culture confirmed that vitamins A and D regulate AMP expression, likely through the Jak/STAT5 signaling pathway. In conclusion, our data show that vitamin A and D regulate intestinal antimicrobial peptide defense through Wnt and STAT5 signaling pathways.

Povidone iodine exposure alters the immune response and microbiota of the gill and skin in koi carp, Cyprinus carpio

Povidone‑iodine (PVP-I) is a disinfectant widely used to prevent and treat bacterial diseases in fish. The use of PVP-I may disturb the microbial community on the fish body surface and thereby facilitate pathogen invasion. We aimed to assess the effects of PVP-I on the immune system and bacterial communities of skin and gill in Cyprinus carpio. Fish were disinfected with 7.5 mL/m3 of PVP-I (1%) for 30 min, and then skin and gill tissues were sampled on days 0, 1, 3, 7, and 14 for the determination of immunity parameters and characterization of surface bacterial communities. Control fish were not disinfected. The results indicated that exposure to PVP-I decreased the activities of gill lysozyme on day 1, and significantly increased the activities of gill SOD and Na-ATPase on day 7. The activity of skin SOD was enhanced on day 3. The PVP-I bath enhanced the mRNA expression of lysozyme, sod, and cat in the gill but decreased lysozyme on days 1–7 and sod on days 7–14 in the skin. The mRNA expression of tight junction proteins was enhanced in both gill and skin tissue. PVP-I decreased gill bacterial richness on days 3 and 7, and reduced bacterial diversity on day 7. The gill microbiota on day 7 was remarkably separate from the control. The skin microbiota on days 1 and 3 was significantly separated from that of the control. In summary, PVP-I elicited shifts in the immune response and changes in the surface microbiota of fish, and the sensitive time windows of microbial fluctuation after PVP-I treatment were three days in the skin and seven days in the gill.

Combined application of glycerol monolaurate and tributyrin improves the growth performance, antioxidant capacity, nonspecific immunity, gene expression and intestinal health of juvenile Litopenaeus vannamei

This study aimed to investigate the effects of the glycerol monolaurate (GML) and tributyrin (TB) and their interaction on the growth performance, body composition, nonspecific immunity, gene expression and intestinal microbiota of Litopenaeus vannamei (initial weight 0.24 ± 0.00 g). The feeding trial comprised a completely randomized 3 × 2 factorial design and six formulated diets with two different GML levels (0.10% and 0.15%) and three different TB levels (0.10%, 0.15%, and 0.20%) named G10T10, G10T15, G10T20, G15T10, G15T15, and G15T20, respectively. Results showed that three groups (G10T15, G15T10, and G15T15) had significantly higher final mean weight, weight gain rate, specific growth rate (P < 0.05) and lower feed conversion ratio than the other groups (P < 0.05). Catalase at low and medium addition levels of TB (G10T10, G10T15, G15T10, and G15T15) was promoted by GML (P < 0.05), and malondialdehyde was inhibited by GML (P < 0.05). Glutathione in G10T15 and G15T10 had the highest values among the groups (P < 0.05). Acid phosphatase was greatly influenced by GML, TB, and their interaction, and its maximum value was found in G15T10 (P < 0.05). Alkaline phosphatase was influenced by TB and was significantly higher in G10T15 and G15T15 than in the other groups (P < 0.05). The mRNA expression of lysozyme (LZM), heat shock protein 70 (HSP70), and phenol oxidase (PO) were significantly affected by GML, TB, and their interaction (P < 0.05). At the phylum level, Firmicutes and Firmicutes/Bacteroidetes ratio increased and then decreased at low level of GML (0.10%) and gradually decreased at high level of GML (0.15%), with minimum values in the G10T15 and G15T10 groups (P < 0.05). In conclusion, 0.15% GML and 0.10% TB were the optimal dietary addition on the basis of growth performance, nonspecific immunity, and intestinal health of the juvenile shrimp (Litopenaeus vannamei).

Dietary supplementation of curcumin-loaded chitosan nanoparticles stimulates immune response in the white leg shrimp Litopenaeus vannamei challenged with Vibrio harveyi

Pathogen infections in shrimps trigger the release of reactive oxygen species (ROS) as a part of immune response. The excessive accumulation of ROS causes the production of oxidative stress, which leads to oxidative damage of the biomolecules in the host cells. The inclusion of dietary antioxidants is known to mitigate oxidative stress and stimulate immunity. Curcumin, a potential antioxidant was encapsulated in chitosan nanoparticles to surge its bioavailability and was administered orally to Vibrio harveyi challenged and non-challenged Litopenaeus vannamei. The non-challenged shrimps fed with curcumin-loaded chitosan nanoparticles (Cur-CSNPs) showed a significant increase (p ≤ 0.05) in the specific growth rate, daily growth coefficient and survival rate. A significant increase (p ≤ 0.05) in the phenoloxidase activity, total hemocyte count and superoxide dismutase activity was observed in both the challenged and non-challenged shrimps fed with Cur-CSNPs. Additionally, a significant increase (p ≤ 0.05) in the relative mRNA expression of lysozyme, cMnSOD and lectin was observed in the Cur-CSNPs fed shrimps. The findings of this research suggest that Cur-CSNPs reinforce the immune system of L. vannamei against V. harveyi infection. Moreover, the non-challenged shrimps showed improvement in the growth parameters in addition to immunostimulation. Thereby a routine inclusion of dietary Cur-CSNPs could mitigate the oxidative damage caused by the incidence of environmental or pathogen-mediated oxidative stress.

Retinal Pigment Epithelial Cells Express Antimicrobial Peptide Lysozyme - A Novel Mechanism of Innate Immune Defense of the Blood-Retina Barrier.

PurposeFor this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens.MethodsThe expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology.ResultsThe mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation.ConclusionsRPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.

Mucosal expression of defensin-5, soluble phospholipase A2 and lysozyme in the intestine in a rat model of acute liver failure and its relationship to intestinal bacterial translocation

IntroductionTo study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). Patients and methodsSprague–Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8h, 16h, 24h, 48h, and 72h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d-galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. ResultsNo bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. DiscussionThe immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation.

Effect of antioxidants on lysozyme and complement activities in heat-stressed rabbits

The negative influence of heat stress (HS) on rabbit farms cannot be ignored. The current study aims to detect the effect of Moringa oleifera leaf extract (MOE), Vitamin C (Vit C) and sodium bicarbonate (NaHCO3) on HS-induced changes in the complement, lysozyme, and antioxidant activities. A total of 45 growing male NZW rabbits were divided into five groups: Negative control rabbits were not exposed to HS (group 1), while HS groups (2-5) were exposed to high temperatures (39?). Group 2 was exposed to HS but non-treated while the rest of groups were treated with MOE, Vit C and NaHCO3, respectively. Expression of lysozyme mRNA, as well as the activities of lysozyme, complement and antioxidants, were assayed. Serum IL-1β was also assayed using ELISA. Furthermore, the histopathological changes of liver and kidney, blood urea nitrogen (BUN), body weight gain (BWG), and mortality rate of experimental animals, were investigated. Our data showed a decrease in lysozyme, antioxidant activities, and BWG versus increases in complement activity, IL-1β level, and BUN in HS. Histological investigation showed an increase in inflammatory reactions. MOE, Vit C and NaHCO3 could alleviate HS effects through increased activities of lysozyme and antioxidants versus decreased complement activity leading to improved health status.

Dietary Glycyrrhiza uralensis extracts supplementation elevated growth performance, immune responses and disease resistance against Flavobacterium columnare in yellow catfish (Pelteobagrus fulvidraco)

The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1β and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation.

Lactobacillus reuteri Stimulates Intestinal Epithelial Proliferation and Induces Differentiation into Goblet Cells in Young Chickens.

Probiotics, such as Lactobacillus, have been proven to be effective in maintaining intestinal homeostasis. The modulatory effect of Lactobacillus on intestinal epithelial development in early life is still unclear. In this study, Lactobacillus isolates with good probiotic abilities were screened and orally administered to detect their regulatory effect on intestinal development in chickens. L. reuteri 22 was isolated from chickens and chosen for subsequent chicken experiments due to its strong acid and bile salt resistance and ability to adhere to epithelial cells. The 3-day-old chickens were orally administrated with 108 CFU L. reuteri 22 for consecutive 7 days. L. reuteri 22 increased Lgr5 mRNA expression (3.23 ± 0.40, P = 0.001) and activated the Wnt/β-catenin signaling pathway, with increasing expression of proliferating cell nuclear antigen (PCNA) (49.27 ± 9.81, P = 0.021) to support the proliferation of chicken intestinal epithelial cells. Moreover, L. reuteri 22 also inhibited the Notch signaling pathway to induce intestinal stem cell differentiation into goblet cells with increased mucin 2 (Muc-2) expression (1.72 ± 0.34, P = 0.047). L. reuteri 22 significantly enhanced lysozyme mRNA expression (2.32 ± 0.55, P = 0.019) to improve intestinal innate mucosal immunity. This study demonstrated that L. reuteri administration could regulate chicken intestinal epithelium development to ensure the function of the intestinal mucosal barrier, which is beneficial for newborn animals.

Melanoma Explorer: a web application to allow easy reanalysis of publicly available and clinically annotated melanoma omics data sets.

Validating newly discovered biomarkers in large, publicly available data sets is often difficult and requires specialized computer programming skills. Melanoma Explorer is a web application that enables easy interrogation of melanoma omics data sets that are freely available in online data repositories with a point-and-click interface. Two use cases are demonstrated. First, the relationship of lysozyme mRNA expression is shown to be prognostic in two independent gene expression microarray data sets. Second, a figure from a journal article showing the relationship of tumour thickness and miR-382 abundance is reproduced. Melanoma Explorer is demonstrated to be a useful tool for reproducing results of published studies and providing additional evidence for biomarkers in independent data sets.

Can dietary ginger (Zingiber officinale) alter biochemical and immunological parameters and gene expression related to growth, immunity and antioxidant system in zebrafish (Danio rerio)?

Effects of the dietary ginger (Zingiber officinale) on growth, some immunological and biochemical parameters and gene expression related to growth, immunity and antioxidant systems in zebrafish were studied. Immune parameters such as immunoglobulin level, alternative complement activity, and lysozyme activity were significantly higher in treatment groups fed with 2% and 3% ginger than the control fish. Amylase activity also increased significantly in the same pattern as the immunological parameters. Catalase and lysozyme mRNA expression levels representatives of antioxidant and immune genes respectively, were up-regulated in the fish fed with 3% ginger. Conversely, expression of gene encoding ghrelin was down-regulated in all treatment groups compared with the control group. Present study suggests that powdered ginger especially at 3% might augment some immunological and biochemical responses as well as expression of genes relevant to antioxidant and immune systems in zebrafish.

Dysbiosis of Intestinal Microbiota and Decreased Antimicrobial Peptide Level in Paneth Cells during Hypertriglyceridemia-Related Acute Necrotizing Pancreatitis in Rats.

Hypertriglyceridemia (HTG) aggravates the course of acute pancreatitis (AP). Intestinal barrier dysfunction is implicated in the pathogenesis of AP during which dysbiosis of intestinal microbiota contributes to the dysfunction in intestinal barrier. However, few studies focus on the changes in intestine during HTG-related acute necrotizing pancreatitis (ANP). Here, we investigated the changes in intestinal microbiota and Paneth cell antimicrobial peptides (AMPs) in HTG-related ANP (HANP) in rats. Rats fed a high-fat diet to induce HTG and ANP was induced by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct. Rats were sacrificed at 24 and 48 h, respectively. Pancreatic and ileal injuries were evaluated by histological scores. Intestinal barrier function was assessed by plasma diamine oxidase activity and D-lactate level. Systemic and intestinal inflammation was evaluated by tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-17A expression. 16S rRNA high throughput sequencing was used to investigate changes in intestinal microbiota diversity and structure. AMPs (α-defensin5 and lysozyme) expression was measured by real-time polymerase chain reaction (PCR) and immunofluorescence. The results showed that compared with those of normal-lipid ANP (NANP) groups, the HANP groups had more severe histopathological injuries in pancreas and distal ileum, aggravated intestinal barrier dysfunction and increased TNFα, IL-1β, and IL-17A expression in plasma and distal ileum. Principal component analysis showed structural segregation between the HANP and NANP group. α-Diversity estimators in the HANP group revealed decreased microbiota diversity compared with that in NANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. In the HANP group, at phyla level, Candidatus_Saccharibacteria and Tenericutes decreased significantly, whereas Actinobacteria increased. At genus level, Allobaculum, Bifidobacterium, and Parasutterella increased significantly, while Alloprevotella, Anaerotruncus, Candidatus_Saccharimonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Ruminiclostridium_5, Ruminococcaceae_UCG-005, and Ruminococcaceae_UCG-014 decreased. Compared with those in the NANP rats, mRNA expression of lysozyme and α-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats. Moreover, in the NANP rats and the HANP rats, Allobaculum abundance was inversely correlated with lysozyme expression, while Anaerotruncus abundance was positively correlated with it by Spearman test. In conclusion, intestinal microbiota dysbiosis and decreased AMPs of Paneth cells might participate in the pathogenesis of intestinal barrier dysfunction in HANP.

Effects of melatonin and green-wavelength LED light on the physiological stress and immunity of goldfish, Carassius auratus, exposed to high water temperature.

This study investigated the effects of increasing water temperature (22-30°C) on the physiological stress response and immunity of goldfish, Carassius auratus, and the ability of green light-emitting diode (LED) irradiation or melatonin injections to mitigate this temperature-induced stress. To evaluate the effects of either green-wavelength LED light or melatonin on stress in goldfish, we measured plasma triiodothyronine (T3), thyroxine (T4), and thyroid hormone receptor (TR) mRNA expression; plasma cortisol and glucose; and immunoglobulin M (IgM) and lysozyme mRNA expression. The thyroid hormone activities, TR mRNA expression, and plasma cortisol and glucose were higher in goldfish exposed to high-temperature water, but were lower after exposure to melatonin or green-wavelength LED light. Lysozyme mRNA expression and plasma IgM activity and protein expression were lower after exposure to high water temperatures and higher after melatonin or green-wavelength LED light treatments. Therefore, high water temperature induced stress and decreased immunity; however, green-wavelength LED light and melatonin treatments mitigated the effects of stress and enhanced immunity. The benefits of melatonin decreased with time, whereas those of green-wavelength LED treatment did not.

The activity parameters, cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia, Veneridae) responding to the pathogenic bacterium Vibrio anguillarum

ABSTRACTLysozyme is an important component of the innate immune response against pathogen infection. The functional forms have been well-studied in the c-type lysozymes of vertebrates but less so in i-type lysozymes prevalent in most invertebrate animals. Cyclina sinensis is a commercially important marine venerid bivalve that is abundant and widely distributed around the maritime coasts of Asia. We obtained an i-type lysozyme (i-lyz) gene in C. sinensis by large scale EST sequencing of a SMART-cDNA library. In C. sinensis, the i-lyz gene encodes 181 amino acids. The lysozyme activities and the mRNA levels of the i-lyz gene in haemocytes were upregulated during the infection by a bacterium, Vibrio anguillarum. The lysozyme activity in haemocytes was increased after 12 h infection by V. anguillarum, and reached the highest level at 24 h post-infection, which was significantly higher than that of the control group (P < 0.01). The expression level of the i-lyz gene in haemocytes was increased after 3 h infection of Vibrio, and reached the highest level at 6 h post-infection. A recombinant i-lyz was obtained through prokaryotic expression which, when isolated and purified, had a molecular weight of approximately 19 kDa. Western blotting was used to validate the expression of the fusion protein. Furthermore, we demonstrate that the recombinant i-lyz protein has obvious bacteriostatic activity upon Escherichia coli whereby the K value of the curve was significantly lower than control and blank groups. Our study shows that the C. sinensis i-type lysozyme plays an important role in the immune defence against V . anguillarum and provides a theoretical reference for clarifying the mechanism of immune response against pathogen invasion in this bivalve.

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure

To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation. Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance. ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining. The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.

Optimization of culture conditions for larval GIFT tilapia Oreochromis niloticus using response surface methodology and effects of HAMP-1 and c-type lysozyme mRNA expression in liver

Response surface methodology (RSM) in concert with central composite experimental design was firstly applied to optimize the culture condition for larval Genetically Improved Farmed Tilapia (GIFT) tilapia. Larvae were reared at different water temperature (16–37 °C) and salinity (0–20 ‰) for 35 days. Results showed that the linear and quadratic effects of temperature and salinity on specific growth rate (SGR), survival and hepatic HAMP-1 mRNA levels were statistically significant (P 0.05). A significant increase in the levels of c-type lysozyme mRNA was observed as salinity increases; the quadratic effects of salinity were insignificant (P > 0.05). The regression equations of SGR, survival, the levels of hepatic HAMP-1 and c-type lysozyme mRNA toward the two factors of interest were established using multiple regression analysis, with the coefficients of determination being 0.980, 0.982, 0.968, and 0.949, respectively (P < 0.01). Based on RSM, the optimal temperature/salinity combination was obtained at 28.2 °C/6.8 ‰ of which the greatest SGR (10.08 % day−1) and survival (91.34 %) were simultaneously attained. Adequately increasing salinity would improve growth, survival, and innate immune function in larval GIFT tilapia.

Intestinal integrity, endotoxin transport and detoxification in pigs divergently selected for residual feed intake1

ABSTRACTMicrobes and microbial components potentially impact the performance of pigs through immune stimulation and altered metabolism. These immune modulating factors can include endotoxin from gram negative bacterial outer membrane component, commonly referred to as lipopolysaccharide (LPS). In this study, our objective was to examine the relationship between intestinal barrier integrity, endotoxin and inflammation with feed efficiency (FE), using pig lines divergently selected for residual feed intake (RFI) as a model. Twelve gilts (62 ± 3 kg BW) from the low RFI (LRFI, more efficient) and 12 from the high RFI (HRFI, less efficient) were used. Individual performance data was recorded for 5 wk. At the end of the experimental period, ADFI of LRFI pigs was less (P < 0.001), ADG not different between the 2 lines (P = 0.72) but the G:F of LRFI pigs was greater than for HRFI pigs (P = 0.019). Serum endotoxin concentration (P < 0.01) and the acute phase protein haptoglobin (P < 0.05) were greater in HRFI pigs. Transepithelial resistance of the ileum, transport of fluorescein isothiocyanate labeled-Dextran and-LPS in ileum and colon, as well as tight junction protein mRNA expression in ileum, did not differ between the lines, indicating the 2 lines did not differ in transport characteristics at the intestinal level. Ileum inflammatory markers, myeloperoxidase (P < 0.05) and IL-8 (P < 0.10), were found to be greater in HRFI pigs. Alkaline phosphatase (ALP) activity was significantly increased in the LRFI pigs in ileum and liver tissues and negatively correlated with blood endotoxin (P < 0.05). Lysozyme activity in the liver was not different between the lines; however, the LRFI pigs had a twofold greater lysozyme activity in ileum (P < 0.05). Despite the difference in their activity, ALP or lysozyme mRNA expression was not different between the lines in either tissue. Decreased endotoxin and inflammatory markers and the enhanced activities of antimicrobial enzymes in the LRFI line may not fully explain the difference in the FE between the lines, but they have the potential to prevent the growth potential in HRFI pigs. Further studies are needed to identify the other mechanisms that may contribute to the greater endotoxin and acute phase proteins in the HRFI pigs and the greater FE in the LRFI pigs.

Molecular cloning and characterization of a novel c-type lysozyme gene in swimming crab Portunus trituberculatus

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. In this study, the full-length cDNA of a novel c-type lysozyme was cloned from the haemocytes of swimming crab Portunus trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of lysozyme was of 984 bp, containing a 5′ untranslated region (UTR) of 90 bp, a 3′ UTR of 222 bp with a poly (A) tail, and an open reading frame (ORF) of 672 bp encoding an 18 amino acids signal peptide and a mature polypeptide of 205 amino acids with the predicted molecular weight of 23.76 kDa. The conserved catalytic sites Glu51 and Asp69, and the eight conserved cysteine residues identified in lysozyme suggesting it belonged to c-type lysozyme. Also, the swimming crab c-type lysozyme may belong to calcium-binding c-type lysozymes because of containing the three Asp residues (Asp98, Asp103 and Asp104), which are conserved in calcium-binding c-type lysozymes. Quantitative real-time RT-PCR analysis revealed that the mRNA transcripts of lysozyme were expressed at the highest level in haemocytes, with higher level in gill, moderately in hepatopancreas and muscle while low in stomach and heart. The mRNA expression of lysozyme in haemocytes was down-regulated significantly after challenge with Vibrio alginolyticus at 3 h, and then kept at the low expression level until 24 h. As time progressed, the mRNA expression of c-type lysozyme was recovered to the control level after 24 h injection. The results indicate swimming crab c-type lysozyme may be an inducible acute-phase protein and bacterial challenge could decline its mRNA expression.