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.     

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.     

Functional disorder of the retina in manganese-deficient Japanese quail revealed by electroretinography using a contact lens electrode with built-in light source

Manganese deficiency results in neurological and skeletal defects, together with ultrastructural disarrangement of the retina in rats. Wild birds show a range of Mn concentrations in their tissues, including the liver, raising the possibility of Mn-related disorders in the wild. Electroretinography (ERG) provides a useful noninvasive approach to evaluate visual function. This method is especially useful in birds, as objective analysis of them is very difficult, while they have well-developed vision. In this study, we carried out a convenient and reliable ERG recording using a contact lens electrode with a built-in light source (LED electrode) of Japanese quail (Coturnix coturnix japonica) fed a Mn-deficient diet. After 10 min light adaptation, single-flash and flicker cone responses were reproducibly recorded to cause an intensity-dependent increase in amplitude of both a-wave and b-wave in single-flash ERG. Mn-deficient feeding markedly decreased the Mn concentration in the liver by almost half in 3 to 6 weeks, followed by body weight loss in 13 to 15 weeks. Implicit time of a-wave and b-wave cone response by single-flash stimulation was significantly delayed in quail with a Mn depletion from 3 to 6 weeks. Every cone response of the Mn-deprived quail had a tendency to decrease amplitude. The ultrastructure of cone photoreceptor cells was disorganized by Mn deficiency, including changes in outer segment discs of photoreceptor cells. These results suggest the essential role of Mn in the integrity of the retinal function of birds.     

Role of sorbitol in manufacturing dried seafood from heated squid meat

SUMMARY: Japanese common squid meat was heat-treated at 80°C for 1 min, cured with 1.0 M sorbitol solution (pH 7.0) at 4°C for 18 h, and dried further at 30°C (60% relative humidity) for 16 h. Osmotic dehydration during the sorbitol curing process and slow moisture vaporization at the initial drying period were observed regardless of the heat denaturation of muscle protein. Simultaneously, lowering the amount of moisture vaporized in the falling rate of the drying period caused a shortening of the total drying time. Furthermore, the shear force of the dried product from heat-treated meat was kept at a lower value by sorbitol curing, although the suppression effect of sorbitol on the hardening of dried meat was lost by protein denaturation. These results are useful for understanding the role of sorbitol in reducing drying time and in eliminating excess hardening of dried squid products.