Utilization of Sake lees as Broiler Feedstuff and its Effects on Growth Performance and Intestinal Immunity

Increasing food loss and waste (FLW) is a global problem, and efforts are being made to use waste food as potential livestock feed material. The amount of self-supplied feed is lower in Japan than in other countries, and the government recommends FLW use for animal feed. Sake (Japanese rice wine) is a traditional alcoholic beverage. During the sake manufacturing process, large amounts of squeezed solids or “lees” (sake lees) are generated. Sake lees are nutritious and functional, but are prone to spoilage. In this study, we investigated whether sake lees should be mixed with animal feed immediately or after drying. To assess the usefulness of sake lees as a poultry feed ingredient and determine the effect of sake lees on intestinal immunity, we performed a feeding trial with three treatments: a raw sake lees (RSL) diet, dried sake lees (DSL) diet, and control diet. Three-week-old broilers were fed these diets (n=8 per group) for two weeks. We then calculated feed efficiency and performed RT-qPCR to assess the effects of diet on intestinal immunity. The growth performance in the RSL diet group was equivalent to that in the control diet group. The DSL diet became difficult for broilers to eat, resulting in decreased growth performance. In the ileum of RSL-diet broilers, the mRNA expression levels of TGF-β1 and avian β-defensin (AvBD)12 were significantly increased compared to those of control diet broilers (p<0.05), and a significant correlation was observed between the two genes (p<0.05). Our results indicated that sake lees should not be dried and should be mixed immediately with feed, and this sake lees when fed to chicken activates the intestinal immunity. However, sake lees have a lower fat content than corn, and it is thus important to combine sake lees with high-energy feed.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.     

Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

We previously reported that the release of O₂ ⁻ from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response. The nucleotide sequence data of PsCu/Zn-SOD1 reported are available in the DDBJ/EMBL/GenBank databases under accession number AB189165.     

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana.     

Effects of polyline simplification of dynamic GPS data under forest canopy on area and perimeter estimations

The objectives of this study were to determine Global Positioning System (GPS) positional errors while moving under the forest canopy and to clarify the effects of polyline simplification on area and perimeter estimations. We used the Pathfinder Pro XR and GPSMAP 76S, which are categorized as “high-end mapping” and “general navigation” GPS receivers, respectively. The field tests were conducted in both natural and plantation forests. The results showed that the Pathfinder Pro XR, which has better multipath rejection technology, worked well, especially in the plantation forest under unfavorable conditions of higher stand density. We used analysis of variance to clarify the effects of the receiver type, positioning mode, stand type, and polyline simplification method on area and perimeter estimations. The receiver type and positioning mode were found to be significant factors that affected area estimation. The Pathfinder Pro XR estimated the area more accurately than the GPSMAP 76S, and differential GPS estimated the area more accurately than autonomous GPS. With respect to the perimeter, the receiver type, positioning mode, and polyline simplification method were found to be significant factors. The results showed that perimeter estimation was improved by using the velocity filter, and further improved by using the velocity filter and Douglas-Peucker algorithm, especially when the Pathfinder Pro XR was used. The GPSMAP 76S estimated the perimeter accurately without any filtering because its default speed filter worked well, even though the GPSMAP 76S is a general navigation GPS receiver.