Estimation of the outcrossing rate for annual Asian wild rice under field conditions

The Asian wild rice, Oryza rufipogon, has partial outcrossing behavior and shows high levels of genetic variation. To estimate an accurate outcrossing rate of annual form of O. rufipogon, two backcross lines (Lines R1 and R2) between Oryza sativa Nipponbare and O. rufipogon W630 were examined under field conditions. A chromosome survey confirmed that these lines had wild chromosomal segments at more than 92% of marker loci. As for the traits of glume, stamen and pistil, Line R1 showed similar floral morphology as that of O. rufipogon W630, whereas Line R2 had larger glumes. In 2005, 2006 and 2008, a total of 22 backcross plants were planted in the middle of wild rice plots. The successive progenies of each plant were examined using microsatellite markers that could clearly detect self-pollination and outcrossing. The outcrossing rates of Line R1 plants ranged from 4.04% to 25.50% with an average of 10.20%. This indicates that cross-pollination of wild rice is a chance event affected by many environmental factors. The outcrossing rates of Line R2 plants also varied, however, no significant difference was observed between the averages of Lines R1 and R2, suggesting that the glume sizes are not critical for outcrossing ability.     

Variation in floral scent compounds recognized by honeybees in Brassicaceae crop species

Floral scent attracts pollinators. We investigated the floral scent compounds recognized by pollinators in six Brassica crop species, including allogamous species with different genomes and autogamous species with two parental genomes and radish (Raphanus sativus). Biologically active compounds recognized by honeybees were screened from all floral compounds by combined gas chromatography–electroantennogram analysis and their profiles were determined by gas chromatography–mass spectrometry. Fourteen of the 52 compounds were active. All accessions had more than two active compounds, but the compounds greatly differed between the two genera. On the basis of similarities in whether active compounds were presence or absence, their amount and their composition ratio, we divided the Brassica accessions into three to five groups by cluster analyses. Most groups were composed of a mixture of allogamous and autogamous species sharing same genome, indicating that the variation depended on genome, not species. These results suggest that all species require pollinator visits for reproduction, despite their different reproductive systems. However, the inter-genus and intra-specific variations shown by the multiple groups within a species might cause different visitation frequencies by pollinators between genera and among accessions within a species, resulting in insufficient seed production in some accessions or species.     

Association of the amino acid motifs of BoLA-DRB3 alleles with mastitis pathogens in Japanese Holstein cows

The association of the polymorphism of bovine leukocyte antigen ( BoLA-DRB3 ) genes, identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method, with resistance and susceptibility to mastitis caused by Streptococci , coagulase-negative Staphylococci, Escherichia coli and Staphylococcus aureus was investigated. Blood samples for DNA extraction were collected from 170 Holstein cows (129 mastitis and 41 healthy cows) from 5 districts in Chiba prefecture, Japan. Susceptibility or resistance to the mastitis-causing pathogens was thought to vary by the presence of amino acid substitutions at the 9, 11, 13, and 30 positions. DRB3*0101 and DRB3*1501 had amino acid motifs of Glu⁹, Ser¹¹, Ser¹³, and Tyr³⁰, and they were considered to have susceptibility to all 4 mastitis pathogens. In contrast, DRB3*1101 and DRB3*1401 had amino acid motifs of Gln⁹, His¹¹, Gly¹³, and His³⁰ in these positions, and they also had Val⁸⁶, so these alleles were considered to have resistance to Streptococcal and coagulase-negative Staphylococcal mastitis. However, in the case of Escherichia coli mastitis, amino acid substitutions at the 9, 11, 13, and 30 positions had little effect, but rather substitutions at the 47, 67 positions of pocket 7, and at the 71, 74 positions of pocket 4, Tyr⁴⁷, Ile⁶⁷, Ala⁷¹, and Ala⁷⁴, were associated with resistance. This motif was present in DRB3*1201 .     

Clinicopathological survey of 101 canine mammary gland tumors: differences between small-breed dogs and others

Clinicopathological features of mammary gland tumors (MGTs) in 101 dogs were evaluated retrospectively. The incidence of histological malignancy in 60 small- and 41 other-breed dogs were 25% and 58.5%, respectively. In 82 epithelial MGTs, small-sized tumors (< 3 cm) or non-invasive tumors were predominant in small breeds. In multivariate survival analysis, small breed (p=0.048) and lower stage of tumor cell invasion (p=0.006) were significantly associated with longer survival time. These results suggest that the incidence of histological or biological malignancy in MGTs is lower in small-breed dogs than in others.     

Crown gall of tobacco caused by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> biovar 1 in tobacco fields

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.     

Effect of amino acid substitutions in the epitope regions of pyolysin from Arcanobacterium pyogenes

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY, cholesterol-dependent cytolysin) family of bacterial toxins. Recently, we demonstrated that the epitopes of monoclonal antibodies (mAbs) S, H, C, and G lie in the regions of amino acids regions 55-73, 123-166, 482-506, and 482-506 of PLO, respectively, by the reaction of mAbs with truncated PLOs. In this study, we substituted the amino acids in these epitope regions of PLO by site-directed mutagenesis and examined the effect of these amino acid substitutions. Mutants I70S/R71A/L73S, Y131S/P132S, and L163S/P164S for mAbs H or S completely lost the hemolytic activity of the proteins, but these mutants still bound to erythrocyte membranes. Mutants L495S/W497S and W500S/W501S for mAbs C and G also completely lost their hemolytic activity, but still bound to erythrocyte membranes. In the undecapeptide region of PLO, the cysteine residue required for thiol activation is replaced with alanine. Therefore, we substituted Ala-492 of the undecapeptide region for Cys. The hemolytic activity of this mutant A492C decreased by adding hydrogen peroxide or storing at 4 degrees C, and the decreased hemolytic activity was restored by adding L-cysteine.     

Effects of supplementary corn silage on the feed intake and milk production of time-restricted grazing dairy cows

The effects of supplementary corn silage (CS) of either 2 or 4 kg of dry matter (DM; S + 2 and S + 4, respectively) above the energy requirement for milk production and maintenance for grazing dairy cows (S) were determined. Time‐restricted grazing was used to compare the feed intake, milk production, and nitrogen and energy use of lactating cows. The experiment was carried out on two different pastures using a 3 × 3 Latin square design for each pasture. Cows were grazed for 5 h on a rotational grazing system and were fed concentrate (1 kg per 5 kg of milk yield). Herbage intake was measured using a weighing technique. To calculate the energy and nitrogen use, whole feces and urine were collected. There was no statistical effect of the pastures. Herbage intake decreased by the addition of CS (P = 0.02). The reduction of herbage DM intake per unit consumption of supplementary CS towards the S group were 0.80 and 0.45 kg for the S + 2 and S + 4 groups, respectively. The total DM intake for the S + 4 group was higher than that for the S and S + 2 groups (P = 0.02). Milk yield did not differ among treatments, even though the total DM intake for the S + 4 group was higher than that of the S and S + 2 groups. Nitrogen and energy use did not differ with the addition of CS.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Structural gene and strain specificity of a novel cysteine protease produced by Staphylococcus aureus isolated from a diseased chicken

Recently whole genome sequencing of Staphylococcus aureus has revealed the genes encoding cysteine proteases such as staphopain and SspB. In this study, we cloned and sequenced the structural gene (ScpA) encoding a cysteine (thiol) protease of S. aureus strain CH-91 from a chicken with dermatitis using polymerase chain reaction (PCR) and inverse PCR methods. The sequence information revealed a coding sequence (CDS) of 1200 nucleotides encoding the ScpA preproenzyme of 399 amino acids with a molecular mass of 45,071 Da. The deduced amino acid sequence of the ScpA differed at many positions from those of staphopain and SspB with identities of 64 and 42%, respectively. In the Southern blot analysis with a total DNA of S. aureus strain CH-91, the ScpA probe hybridized with a single 7.7 kb XbaI fragment or 2.8 and 0.8 kb EcoRI fragments, whereas the staphopain and SspB probes did not hybridize with these DNA fragments. These results suggest that this ScpA gene is a single-copy gene and is a novel gene, which is not found in the published whole genome sequences of S. aureus. In immunoblot, PCR, and Southern blot assays, the ScpA or its gene was detected in high protease-producing strains from chickens, but was not recognized in bovine and porcine strains or low protease-producing avian strains. These results indicate that the ScpA of CH-91 type may be specific to the high protease-producing strains of S. aureus from chickens, namely, there is a strain specificity of the ScpA.