Plant species identification using fecal DNAs from red-eared slider and Reeves’ pond turtle in agricultural canals for rural ecosystem conservation

Fecal DNA samples from the red-eared slider and Reeves’ pond turtle, suspected pests of lotus root paddies, were used to identify the plant species eaten by these turtles in order to develop a strategy for rural ecosystem conservation. The fecal samples were obtained from young and adult individuals (mostly female) of both species living in agricultural canals surrounding lotus root paddies in Tokushima Prefecture, Japan. The samples were screened for the presence or absence of DNA from nine plant species using PCR and plant species-specific primers for the rbcL gene of chloroplast DNA. In the red-eared slider, our analysis identified seven plant species in the fecal DNA samples of adults and three plant species in those of young individuals. In Reeves’ pond turtle, our analysis identified two plant species from adult fecal samples and one species from those of young individuals. Thus, adult red-eared sliders consume a greater range of plants than young red-eared sliders or Reeves’ pond turtles. Both turtle species, independently of age, consumed lotus plants and were likely to cause feeding damage to lotus roots. Considering the plant species detected in adult red-eared sliders and these plant habitats, we suggest that this adult turtle is likely to travel between the agricultural canals and the lotus root paddies. These findings will help the development of strategies for preventing damage to lotus roots by these turtles; furthermore, they indicate that fecal DNA analysis will be applicable to investigation of the feeding habits of other animal species.     

Examination of the pathogenesis of diabetic nephropathy in OLETF rats

We conducted protein loading to examine the progression and pathogenesis of diabetic nephropathy. For this experiment, male OLETF, LETO, F344 and BN rats were used. This experiment was performed on rats between 5 and 30 weeks of age. Examination parameters included body weight, food intake, oral glucose tolerance test (OGTT), urinary protein level (UP), urinary albumin level (UA), glomerular filtration rate (GFR), kidney weights, light microscopy (LM) and electron microscopy (EM). In the protein-loaded OLETF group, the UP level was markedly increased 20 weeks or more after birth. In OLETF control group, GFR were higher than those in other strains. Glomerular hypertrophy and kidney weights were markedly increased in protein-loaded groups in OLETF rats. Thirty weeks after birth, EM showed that the number of polyethyleneimine (PEI) of the glomerular basement membrane (GBM) in protein-loaded OLETF group was significantly decreased compared to that in control group. These changes in OLETF rats were more marked in the protein-loaded group than those in the control group. LM showed that the number of exudative lesions with fibrin-cap in the protein-loaded OLETF group was significantly increased than those in control group. In OLETF rats, protein loading caused deterioration of nephropathy at 30 weeks of age. Therefore, it was demonstrated that not only blood sugar control but also protein intake factors play important roles in the deterioration of nephropathy in OLETF rats.     

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor on bovine peripheral blood neutrophil functions in vitro and in vivo

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) on bactericidal activity of bovine peripheral blood neutrophils in vitro and in vivo were studied. In in vitro experiment, bovine blood neutrophils were cultured for 9 hr in media containing 0.005, 0.05 or 0.5 microg/ml of rboGM-CSF. Neutrophils treated with rboGM-CSF showed significantly higher luminol-dependent chemiluminescence (LDCL) than control cells. In in vivo experiment, neutrophils isolated from cows injected 5.0 microg/kg of rboGM-CSF showed significantly higher Nitrobluetetrazolium (NBT) reduction value than that from control cows 24 hr post injection. Total leukocyte counts of cows injected rboGM-CSF sharply decreased 6 hr post injection and recovered to normal level 2 days post injection. Body temperature of these cows rose 6 hr post injection and back to normal level at 24 hr post injection. It was suggested that rboGM-CSF enhanced bactericidal activity of bovine neutrophils both in vitro and in vivo.     

Hog Cholera: I. Investigation of the Agar Double-Diffusion Precipitation Test for The Detection of the Virus in Swine Tissue

The agar double-diffusion precipitation test was investigated to detect hog cholera virus in splenic and pancreatic tissues of swine. Special attention was given to the conditions influencing the sensitivity and specificity of the test. These studies emphasize the strict techniques and methods required in the test in order to detect specific antigen — antibody reaction. Absorption studies performed on serum fractions prepared by DEAE cellulose chromatography and studied electrophoretically, indicated the specific reaction was given by fractions, migrating in the gamma-globulin region and was absorbed by infected but not by normal spleens. The sensitivity of the test is dependent to a great extent on the successful liberation of the viral antigen from the tissue.     

Effects of alpha-pinene odor in different concentrations on stress-induced hyperthermia in rats

Stress-induced hyperthermia is observed in animals exposed to stressful conditions. In our previous study, plant-derived fragrances such as green odor and alpha-pinene were shown to suppress this stress response in rats. In the present study, we examined the concentration-dependence of the alpha-pinene effects on stress-induced hyperthermia. Male rats carrying telemetry transmitters were transferred individually to a new cage containing bedding that had been sprayed with 0.3, 0.03, and 0.003% concentrations of alpha-pinene or control solvent. Following transfer to the novel environment, the body temperature increased significantly, and this response was clearly suppressed when the cage was scattered with 0.03% alpha-pinene only. These results suggest that the effect of alpha-pinene on stress-induced hyperthermia can be observed only at a certain concentration.     

Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords

To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.     

Novel species specific antigens of trypanosoma congolense and their different localization among life-cycle stages

Seven monoclonal antibodies (mAbs) were raised against Trypanosoma congolense procyclic form (PCF). Localization of the antigens recognized by the mAbs was determined in bloodstream form (BSF), PCF, epimastigote form (EMF) and metacyclic form (MCF) by confocal laser scanning microscopy (CLSM). Two mAbs (10F9 and 20H12) showed different fluorescent patterns among different life-cycle stages of the parasite. The 10F9 recognized a 76 kDa antigen of all life-cycle stages of the parasite and the antigen localization corresponded with that of a mitochondrion. While the 20H12 recognized 119 and 122 kDa antigens of all the life-cycle stages and the antigen localization corresponded with a flagellum in BSF and MCF, tip of a flagellum in PCF, and part of cytoplasm in EMF. Moreover, the 20H12 did not react to T. brucei gambiense, T. b. rhodesiense and T. evansi antigens in both CLSM and immunoblotting. Therefore, the antigens recognized by the 20H12 seem to be T. congolense specific. Although, further studies will be required for a full characterization of the T. congolense specific 119 and 122 kDa antigens, the mAb 20H12 and the specific antigens may be useful in not only establishment of T. congolense specific diagnosis methods but also studies on molecular mechanisms regulating differentiation of the parasite during life-cycle.     

Regional differences of pheromone production in the sebaceous glands of castrated goats treated with testosterone

The primer pheromone is responsible for the "male effects" in goats and produced in the sebaceous glands testosterone-dependently. In the present study, the responses of sebaceous glands obtained from the head and rump regions of castrated goats were examined by our bioassay system after testosterone treatment to demonstrate the presence of regional differences in the pheromone production in male goats. The testosterone treatment resulted in the marked development of sebaceous glands and the induction of pheromone bioactivity in the head region of the goats. On the contrary, this treatment brought neither development of the sebaceous glands nor induction of pheromone bioactivity in the rump region. The treatment increased immunoreactivities to androgen receptors (AR) and 5alpha-reductase in the sebaceous glands of both regions, although the activities were more apparent in the head region than the rump region. These findings suggest that the primer pheromone of male goats is produced specifically in the sebaceous glands of the head region due partly to regional differences in the expression of AR and 5alpha-reductase mediating testosterone bioactivities.     

Effect of liquid paraffin on antibody responses and local adverse reactions of bivalent oil adjuvanted vaccines containing newcastle disease virus and infectious bronchitis virus

Effects of liquid paraffin on antibody responses and local adverse reactions after intramuscular injection of oil adjuvanted vaccines containing Newcastle disease (ND) and infectious bronchitis (IB) virus were investigated in chickens. Each vaccine was prepared with a liquid paraffin such as Carnation, Crystol 52 and Lytol. These vaccines induced sustained antibody responses against ND and IB. Among local adverse reactions, Lytol induced granulomatous reactions and abscesses, but Carnation and Crystol 52 did not. The residual weight of liquid paraffin at the injection site decreased in the order Carnation, Crystol 52, Lytol. Crystol 52 was composed of relatively few short-chain hydrocarbons (i.e., n-C20H42). The vaccine with liquid paraffin mainly composed of n-C16H34-n-C20H42 was suggested to induce fewer adverse reactions.