Rapid enumeration of bacteria grown on surimi-based products by flow cytometry

ABSTRACT: A rapid method to enumerate bacteria adhered on a surimi-based product (kamaboko) by flow cytometry (FCM) is described. To remove Escherichia coli cells from the surface of kamaboko, ultrasonic energy was used. Almost all cells can be removed from kamaboko in 3 min with ultrasonic treatment. Because the sample might contain various non-bacterial particles such as food additives and debris of products, propidium iodide was used to discriminate bacterial cells from non-bacterial particles. Fluorescence scattergrams could distinguish bacteria from the particles, and the FCM method could be used to enumerate bacteria adhered on the surface of kamaboko during storage. Cell numbers determined by FCM paralleled well with those measured using a traditional colony counting method in the range of 10⁴–10⁸ cells/g. The FCM assay could enumerate cells within 1 min and the total assay time, including sample preparation, was less than 30 min.     

Molecular cloning and sequencing of the cDNA encoding the bat CD4

Chiroptera is thought to be a vector or a natural reservoir of various pathogenic microbes. However, there are few basic studies on the subject of chiroptera immune systems. This is the first report to determine the sequence of bat CD4 cDNA. Comparison with other animals' CD4 and phylogenetic analysis have shown that bat CD4 had a higher homology to cat and dog CD4 than to human and mouse CD4. Moreover, from the analysis of the structure of the CD4 Ig-like C-type 1 region, in bat CD4 there was an insertion of 18 extra amino acids. In addition, bat CD4 lacked cystein, which suggested that the disulfide bond could not be formed. Human, monkey and mouse CD4 have the cystein and the disulfide bond, but pig, cat, whale and dog CD4, like that of the bat, lacked the cystein. We conducted the present study in order to help elucidate the infectious diseases derived from the bat as well as bat immune systems.     

Association of a single nucleotide polymorphism in ribosomal protein L27a gene with marbling in Japanese Black beef cattle

Marbling, defined by the amount and distribution of intramuscular fat, is an economically important trait of beef cattle in Japan. The c2‐11#2 expressed sequence tag (EST) has been previously shown to possess expression difference in musculus longissimus muscle between low‐marbled and high‐marbled steer groups, and to be located within genomic region of a quantitative trait locus for marbling. Thus, the ribosomal protein L27a (RPL27A) gene containing the c2‐11#2 EST sequence was considered as a positional candidate for the gene responsible for marbling. In the present study, a single nucleotide polymorphism (SNP) in the promoter region of the RPL27A, referred to as g.3109537C>T, was detected between the 2 steer groups. The SNP was associated with the predicted breeding value for beef marbling standard number by the analyses using Japanese Black beef cattle population. The effect of genotypes of the SNP on the predicted breeding value for subcutaneous fat thickness was not statistically significant. These findings suggest that the RPL27A SNP may be useful for effective marker‐assisted selection to increase the levels of marbling in Japanese Black beef cattle.     

Binding capacity of a barley beta-D-glucan to the beta-glucan recognition molecule dectin-1

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.     

Biochemical,physiological, and molecular characterization of <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> (formerly named <Emphasis Type="Italic"> Erwinia chrysanthemi</Emphasis>) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.     

Difference in age at first conception between early and late litters of feral raccoon (Procyon lotor) in Kanagawa prefecture

Due to the long parturition period of raccoons, we assumed that age at first conception of late born females was later than that of early born females. From March 2005 to September 2008, 201 females estimated to be younger than 24 months were separated into early- and late-born groups on the basis of their estimated birth month (to the nearest 2 months), and parous status and body mass index (BMI) were examined. Age at first conception of late-born females (over 18 months old) was estimated to be later than that of early-born females (over 12 months old). The average BMI in early-born individuals at 12 months old might have affected their ability to conceive because of body fat deposition.     

Diagnosis of subacute ruminal acidosis (SARA) by continuous reticular pH measurements in cows

The objective of this study was to determine whether subacute ruminal acidosis (SARA) could be diagnosed by continuous measurements of the reticular pH, as compared with the ruminal pH, using healthy cows fed a control diet and SARA cows fed a rumen acidosis-inducing diet. The reticular and ruminal pH were measured simultaneously by a radio transmission pH measurement system. The mean reticular pH at 1-h intervals decreased gradually from the morning feeding to the next feeding time in both healthy and SARA cows, though the decrease in the ruminal pH was observed to be more drastic as compared with that observed in the reticular pH. The threshold of the 1-h mean pH in the reticulum for a diagnosis of SARA was considered to be 6.3, and a significant positive correlation was observed between the reticular and ruminal pH. No differences in the concentrations of lactic acid, ammonia nitrogen, and volatile fatty acids were noted between the reticular and ruminal fluids in SARA cows. These results demonstrate that the reticular pH can be used to detect SARA in cows, as opposed to using the ruminal pH.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.