Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10⁶ cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10⁶ cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10‐fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.     

Effects of warming basal ends of Carolina poplar (Populus × canadensis Moench.) softwood cuttings at controlled low-air-temperature on their root growth and leaf damage after planting

We investigated the effects of warming the basal ends of Carolina poplar (Populus × canadensis Moench.) softwood cuttings at controlled low-air-temperature on their root growth and leaf damage after planting. The warming treatment was applied to the cuttings by soaking 10 mm beyond the cut end in warmed water maintained at 30 °C in a cold chamber maintained at an air temperature of 10 °C and a photosynthetic photon flux density (PPFD) of 10 μmol m^?2 s^?1 (near the light compensation point at 10 °C) until rooting was observed. The warmed cuttings were then grown in a growth chamber at an air temperature of 30 °C, relative humidity 85–90 %, and a PPFD of 100 μmol m^?2 s^?1. Control cuttings were grown in the growth chamber throughout the experiment. Rooting occurred simultaneously for both warmed and control cuttings, irrespective of air temperature. Root development was greater and leaf damage, evaluated on the basis of extent of necrosis, was less for warmed cuttings than for control cuttings. The reduction of leaf damage for warmed cuttings probably resulted from reduced post-planting water stress and leaf senescence, because of improved root development as a result of the pre-planting warming treatment. This technique could improve the propagation of cuttings of woody plants, because it would ensure that the cuttings are ready to develop roots with minimum loss of carbohydrates, irrespective of weather conditions.     

Time dependence of Poisson’s effect in wood III: asymmetry of three-dimensional viscoelastic compliance matrix of Japanese cypress

To understand the viscoelasticity of wood three dimensionally, matched samples of Japanese cypress were loaded in uniaxial tensile creep in the longitudinal (L), radial (R), and tangential (T) directions at approximately 9.7 % equilibrium moisture content. Longitudinal and transverse strains were measured for the determination of viscoelastic Poisson’s ratios and three-dimensional viscoelastic compliance tensors concerning the normal strain. The changes in the transverse strains showed the same tendencies as those in the longitudinal strains, in all directions of loading. That is, during creep, the absolute value of transverse strain continued to increase with the gradual reduction in the increase rate; immediately after the removal of the load, it recovered rapidly, after which it continued to recover slowly. The transverse strain increased most easily in the T direction, followed by R and L, during creep. All the viscoelastic Poisson’s ratios and the absolute values of all elements of the viscoelastic compliance increased logarithmically with creep time. The three-dimensional viscoelastic compliance matrix for Japanese cypress is concluded to be asymmetric.     

Glycosylated Porphyra-334 and Palythine-Threonine from the Terrestrial Cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.     

Transition of Historical Control Data for High Incidence Tumors in F344 Rats

Historical control data of tumor incidence were collected from the control groups (215 animals of each sex) in four recent carcinogenicity studies that were started between 2005 to 2009 (terminally sacrificed between 2007 and 2011) at BoZo Research Center Inc. (Gotemba, Shizuoka, Japan) using Fischer 344 rats (F344/DuCrlCrlj). These data were compared to the previous historical control data (from 1990 to 2004, previously reported) in the same facility. In the results, the incidence of C-cell adenoma in the thyroid tended to increase in both sexes in recent years (30.8% for males and 24.4% for females in 2005-2009) as compared with the previous data (17.4% and 20.1% for males and 11.5% and 11.8% for females in 1990–1999 and 2000–2004, respectively). In addition, the incidences of pancreatic islet cell adenoma in males and uterine adenocarcinoma tended to increase from around 2000 and remained high in recent years (incidences of islet cell adenoma in males of 10.5%, 17.1% and 20.5% in 1990–1999, 2000–2004 and 2005–2009; incidences of uterine adenocarcinoma of 3.3%, 12.0% and 13.5% in 1990–1999, 2000–2004 and 2005–2009, respectively). There was no apparent difference in the incidence of other tumors.     

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.     

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.     

Construction of an expression vector for improved secretion of canine IL-18

To construct a vector for caspase-1 independent expression of canine IL-18, the signal sequence of canine IL-12p40 was fused to the sequence of mature IL-18 on the NdeI restriction site which is located at the 3' end of the signal sequence. The resulting vector expressed coding protein from transfected mammalian cells. The expressed protein was shown to have IL-18 bioactivity in a INF-gamma-inducing assay. These results suggest that the expression vector is the desired tool for advancement of dendritic cell (DC)-based cancer therapy, provided that the vector can successfully be transfected into dendritic cells. We propose a simple and widely applicable method for providing the signal sequence.     

Bovine colostral CD8-positive cells are potent IFN-gamma-producing cells

IFN-gamma plays an important role in cellular immunity contributing to microorganism elimination. We have previously reported that bovine colostrum contains high levels of IFN-gamma as well as immunoglobulin. Lymphocytes are potent producers of IFN-gamma, so establishing the lymphocyte population in colostrum will help to identify the source of colostral cytokines. In this study, we used flow cytometric analysis to quantify the population of three types of lymphocytes found in colostrum; namely, CD4 (Th) cells, CD8 (cytotoxic T) cells, and gammadelta-T cells. We also quantified the concentration of colostral IFN-gamma using ELISA. IFN-gamma concentrations were measured in colostrum obtained from 96 healthy Holstein cows; the levels tended to decrease on the first day post-parturition. Flow cytometric analysis showed that many gammadelta-T- and CD8-positive cells were present in the colostrum, and that the CD4/CD8 ratio was low. The ratios of CD8- and gammadelta-T-positive cells to cells of other types decreased during the 5 days after parturition, but that of CD4-positive cells showed no change during the observation period. To identify IFN-gamma-expressing colostral lymphocytes, we used magnetic separation technology to separate the lymphocytes (CD4, CD8 and gammadelta-T) from colostral cells, then examined them for IFN-gamma mRNA expression with real-time PCR (RT-PCR). RT-PCR analysis revealed potent expression of the IFN-gamma gene in CD8-positive cells, reaching higher levels than in CD4- or gammadelta-T-positive cells. These results suggest that the CD8-positive T cells in colostrum play a role as producers of IFN-gamma.