A mutation confers Monochoria vaginalis resistance to sulfonylureas that target acetolactate synthase

Acetolactate synthase (ALS) is the target enzyme for four distinct families of compounds: sulfonylureas (SUs), imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We cloned and sequenced the fragments encoding ALS genes from biotypes of Monochoria vaginalis susceptible (S) and resistant (R) to SU-herbicides. The nucleotide sequences of the 39 bp Domain A region for R M. vaginalis biotype differed from that of the S biotype by a single nucleotide substitution at variable Pro codon of Domain A (CCT to TCT), predicting a Pro in the S but a Ser in the R biotype. No nucleotide differences between S and R M. vaginalis were observed in Domain D. We suggest that the amino acid substitution at Domain A region is responsible for resistance to SU-herbicides in M. vaginalis collected from Ushiku City, Ibaraki Prefecture, Japan.     

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.     

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.     

Comparison of the blood coagulation profiles of ferrets and rats

The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.     

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet [DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.     

Narthecium asiaticum Maxim. Poisoning of grazing cattle: observations on spontaneous and experimental cases

A total of 39 Holstein cattle were grazed in tracts of wild grassland on account of shortage in pasture grass. Twenty-nine cattle were affected and 26 of them died during a 21-day period. The main signs were depression, anorexia, ascites, and oliguria. There was elevated serum urea nitrogen and sugar and protein in the urine. Pathological examination revealed turbid swelling of the kidney, an increase in the amount of fluid in the body cavity, edema in the perirenal adipose tissue and hemorrhage in various visceral organs and tissues. Histologically, acute tubular necrosis in the kidney, hypoplasia of the erythroblast series in the bone marrow, atrophy and degeneration of the lymphatic tissue and focal necrosis of the liver were observed in many of the cattle. Among cows experimentally fed Narthecium asiaticum Maxim., Polygonum sachalinense Fr. Schum., and Vitis coignetiae Pulliat which were presumed to have been ingested in large amounts by grazing cattle in the field, the cows fed N. asiaticum revealed the clinical, biochemical and pathological changes similar to those noticed in naturally affected cattle. Cows fed P. sachalinense and V. coignetiae showed no distinct systemic symptoms except transient anorexia and hypothermia.     

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Magnetic resonance imaging of alveolar echinococcosis experimentally induced in the rat lung

Pulmonary alveolar echinococcosis (AE) caused by the metacestode of Echinococcus multilocularis is a lethal zoonosis and is a lesion secondarily induced by hematogenous dissemination from hepatic AE lesions. In the present study, a hematogenous pulmonary AE model was experimentally induced in rats by the injection of echinococcal larval tissue homogenate to the tail vein, and then the pathological and diagnostic aspects of pulmonary AE were examined by magnetic resonance imaging (MRI). Histological primary, mature and degenerated AE lesions were observed 5, 18 and 50 weeks after injection, respectively. These lesions were discriminated as signal-void, hypointense and hyperintense regions in T1-weighted MRI (T1WI), respectively. The change in signal intensity in T1WI might reflect the content of proteinaceous fluid as a result of AE cyst degeneration. Western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis provided evidence for AE infection in the early stage. T1WI in combination with Western blot analysis could possibility become definitive and early signs of hematogenous pulmonary AE infection.