A candidate-SNP retrospective cohort study for fracture risk in Japanese Thoroughbred racehorses.

Fractures are medical conditions that compromise the athletic potential of horses and/or the safety of jockeys. Therefore, the reduction of fracture risk is an important horse and human welfare issue. The present study used molecular genetic approaches to determine the effect of genetic risk for fracture at four candidate SNPs spanning the myostatin (MSTN) gene on horse chromosome 18. Among the 3706 Japanese Thoroughbred racehorses, 1089 (29.4%) had experienced fractures in their athletic life, indicating the common occurrence of this injury in Thoroughbreds. In the case/control association study, fractures of the carpus (carpal bones and distal radius) were statistically associated with g.65809482T/C (P = 1.17 x 10-8 ), g.65868604G/T (P = 2.66 x 10-9 ), and g.66493737C/T (P = 6.41 x 10-8 ). In the retrospective cohort study using 1710 racehorses born in 2000, the relative risk (RR) was highest for male horses at g.65868604G/T, based on the dominant allele risk model (RR = 2.251, 95% confidence interval 1.407-3.604, P = 0.00041), and for female horses at g.65868604G/T, based on the recessive allele risk model (RR = 2.313, 95% confidence interval 1.380-3.877, P = 0.00163). Considering the association of these SNPs with racing performance traits such as speed, these genotypes may affect the occurrence of carpus fractures in Japanese Thoroughbred racehorses as a consequence of the non-genetic influence of the genotype on the distance and/or intensity of racing and training. The genetic information presented here may contribute to the development of strategic training programs and racing plans for racehorses that improve their health and welfare.

Comparison of alfaxalone, ketamine and thiopental for anaesthetic induction and recovery in Thoroughbred horses premedicated with medetomidine and midazolam.

REASONS FOR PERFORMING STUDY: There is limited information on clinical use of the new injectable anaesthetic agent alfaxalone in Thoroughbred horses. OBJECTIVES: To compare anaesthetic induction and recovery characteristics and cardiopulmonary responses between alfaxalone, ketamine and thiopental in Thoroughbred horses premedicated with medetomidine and midazolam. STUDY DESIGN: Randomised blinded experimental cross-over study. METHODS: Six Thoroughbred horses were anaesthetised 3 times with alfaxalone 1 mg/kg bwt, ketamine 2.5 mg/kg bwt or thiopental 4 mg/kg bwt after premedication with medetomidine 6 µg/kg bwt and midazolam 20 µg/kg bwt. Qualities of anaesthetic induction and recovery were scored on a scale of 1 (poor) to 5 (excellent). Induction time and recovery time were recorded. Cardiopulmonary values (heart rate, respiratory rate, arterial blood pressures, and arterial blood gases) were recorded throughout anaesthesia. Data were analysed with nonparametric methods. RESULTS: The anaesthetic induction (P = 0.2) and recovery (P = 0.1) quality scores (median, range) were not different amongst protocols and were 4.0, 3-5; 5.0, 4-5; 4.5, 3-5; and 4.5, 3-5; 3.5, 2-5; 4.0, 2-5 for alfaxalone, ketamine and thiopental, respectively. Induction time for ketamine (67, 53-89 s) was significantly longer than that for alfaxalone (49, 40-51 s, P = 0.01) and thiopental (48, 43-50 s, P = 0.01). Time to standing for alfaxalone (44, 40-63 min, P = 0.01) and thiopental (39, 30-58 min, P = 0.01) was significantly longer than that for ketamine (25, 18-26 min). Cardiovascular values were maintained within the clinically acceptable level throughout anaesthesia. Respiratory rate significantly decreased during anaesthesia for all 3 drugs; however, spontaneous breathing did not disappear, and PaCO2 values were maintained at approximately 50 mmHg. CONCLUSIONS: All 3 drugs showed similar effects in relation to anaesthetic induction and recovery qualities and cardiopulmonary responses. However, alfaxalone and thiopental prolonged recovery time compared with ketamine.

Habitual intake of fermented milk products containing Lactobacillus casei strain Shirota and a reduced risk of hypertension in older people.

This study investigated relationships between the frequent intake of fermented milk products containing Lactobacillus casei strain Shirota (LcS) and the onset of hypertension (resting systemic pressure ≥140 mmHg [systolic]/≥90 mmHg [diastolic], a doctor's diagnosis and/or antihypertensive medicine use) during a 5-year period in 352 communityliving Japanese aged 65 to 93 years (125 men and 227 women). Initially normotensive subjects were divided into two groups (n=254 and n=98) on the basis of their intake of fermented milk products (<3 or ≥3 times/week, respectively), as estimated during an interview by a certified nutritionist. The incidence of hypertension over the 5-year interval was significantly lower in those who took fermented milk products ≥3 rather than <3 times/week (6.1 vs 14.2%, P=0.037). A multivariate-adjusted proportional hazards model predicted that blood pressures were significantly more likely to remain normal over 5 years in subjects who took ≥3 fermented milk products rather than <3 times/ week (relative risk 0.398 [95% confidence interval 0.167-0.948], P=0.037). These results suggest that after adjustment for potential confounders, the risk of developing hypertension is substantially lower in elderly people who take fermented milk products containing LcS at least 3 times a week.

Fermented milk containing Lactobacillus casei strain Shirota prevents the onset of physical symptoms in medical students under academic examination stress.

This pilot study investigated the effects of the probiotic Lactobacillus casei strain Shirota (LcS) on psychological, physiological, and physical stress responses in medical students undertaking an authorised nationwide examination for promotion. In a double-blind, placebo-controlled trial, 24 and 23 healthy medical students consumed a fermented milk containing LcS and a placebo milk, respectively, once a day for 8 weeks until the day before the examination. Psychophysical state, salivary cortisol, faecal serotonin, and plasma L-tryptophan were analysed on 5 different sampling days (8 weeks before, 2 weeks before, 1 day before, immediately after, and 2 weeks after the examination). Physical symptoms were also recorded in a diary by subjects during the intervention period for 8 weeks. In association with a significant elevation of anxiety at 1 day before the examination, salivary cortisol and plasma L-tryptophan levels were significantly increased in only the placebo group (P<0.05). Two weeks after the examination, the LcS group had significantly higher faecal serotonin levels (P<0.05) than the placebo group. Moreover, the rate of subjects experiencing common abdominal and cold symptoms and total number of days experiencing these physical symptoms per subject were significantly lower in the LcS group than in the placebo group during the pre-examination period at 5-6 weeks (each P<0.05) and 7-8 weeks (each P<0.01) during the intervention period. Our results suggest that the daily consumption of fermented milk containing LcS may exert beneficial effects preventing the onset of physical symptoms in healthy subjects exposed to stressful situations.

Multilocus sequence typing of bifidobacterial strains from infant's faeces and human milk: are bifidobacteria being sustainably shared during breastfeeding?

Bifidobacteria are considered to be one of the most important beneficial intestinal bacteria for infants, contributing to the priming of the mucosal immune system. These microbes can also be detected in mother's milk, suggesting a potential role of human milk in the colonisation of infant's gut. However, little is known about the timing of bacteria appearance in human milk, and whether human milk is the first source of inoculation. Here, we investigated whether specific strains are shared sustainably between maternal milk and infant's gut. Faecal samples and human milk were collected from 102 healthy mother-infant pairs (infant's faeces: meconium, 7, 30 days of age; mother's milk: once before delivery, colostrum, 7, 30 days after delivery). Bifidobacterial strains were isolated from these samples, and were discriminated by means of multilocus sequencing typing. No bifidobacteria were detected from human milk collected before delivery, or colostrum. Strains were isolated only from human milk samples obtained 7 days after birth or later. On the other hand, bifidobacterial strains were obtained from infant's faeces throughout the study period, sometimes as early as the first day of life (meconium). We have found that bifidobacterial species belonging to Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum subsp. longum could be identified as monophyletic between infant's faeces and their mother's milk. These strains were confirmed to be sustainably shared between maternal milk and infant's gut. Moreover, monophyletic strains were isolated at the same time point or earlier from infant's faeces than from human milk, and none were isolated earlier from human milk than from infant's faeces. Although it remains unclear whether human milk is the first source of microbes for infants, our results confirm that human milk is a reservoir of bifidobacteria, and specific strains are shared between infant's intestine and human milk during breastfeeding.

Extranodal lymphoblastic lymphoma of suspected B-cell lineage in the gingiva of a racehorse, accompanied by mandibular osteolysis.

A mass developed in the mandibular gingiva of a thoroughbred racehorse. When the horse could no longer eat unassisted, it was killed and immediately autopsied. Macroscopically, the mandible exhibited extensive osteolysis, with only a small amount of bone remaining around the tooth roots. The cut surface of the mass around the mandible consisted of neoplastic medullary tissue, in which osteogenesis was observed. The medullary tissue was composed of pleomorphic medium-sized to large cells, interlaced by collagen bundles. These cells had large, pale, round or ovoid, sometimes cleaved nuclei, with one or two prominent nucleoli. Mitoses were numerous. Electron microscopy showed that the cells in the medullary tissues were similar in shape to undifferentiated lymphocytes. Immunohistochemically, these cells were positive for B-cell associated antigen in the pre-B-cell stage. Our findings suggest that the horse had extranodal lymphoblastic lymphoma of suspected B-cell lineage, possibly originating from the lymphatic system of the gingiva. We consider that the osteolysis resulted from activation of osteoclasts by proliferation of the tumour cells.

Antitumor effects of the intravesical instillation of heat killed cells of the Lactobacillus casei strain Shirota on the murine orthotopic bladder tumor MBT-2.

PURPOSE: To characterize the potential of heat killed Lactobacillus casei, Shirota strain (LC9018), as an alternative to bacillus Calmette-Guerin (BCG) for treating patients with bladder cancer we investigated the antitumor effects of intravesical instillation of LC9018 in the MBT-2 orthotopic bladder tumor implantation model in C3H/He mice. MATERIALS AND METHODS: LC9018 or BCG, Tokyo 172 strain, was instilled once daily for 10 days starting on the day after orthotopic implantation of MBT-2. Tumor appearance and mean bladder weight on day 21 after tumor implantation were evaluated. Moreover, we investigated the augmentation of local cellular immunity in bladder mucosa by immunohistochemical staining and reverse transcription polymerase chain reaction. RESULTS: Intravesical LC9018 instillation significantly reduced the rate of tumor appearance in 8 of 38 subjects (p <0.001) and mean tumor growth plus or minus standard deviation with a bladder weight of 37 +/- 49 mg. (p <0.001) compared with tumor appearance in 41 of 58 subjects and mean bladder weight 146 +/- 183 mg. in controls. BCG had no significant antitumor activity in the orthotopic implantation model. Intravesical instillation of LC9018 augmented the local expression of antitumor cytokine messenger RNA (interferon-gamma and tumor necrosis factor-alpha) and induced the infiltration of neutrophils surrounded by macrophages that phagocytosed LC9018 cells at the bladder mucosa. CONCLUSIONS: These results suggest that LC9018 is potentially more potent and safer as a therapeutic agent than BCG for superficial bladder tumors. Furthermore, the antitumor effect of LC9018 is exerted via the augmentation of local cell mediated antitumor immunity.

Lactobacillus casei acquires the binding activity to fibronectin by the expression of the fibronectin binding domain of Streptococcus pyogenes on the cell surface.

Fibronectin binding domain was expressed on the cell surface of Lactobacillus casei strain Shirota which hardly adheres to fibronectin. DNA for the fibronectin binding domain of the sfbl gene, which encodes a fibronectin binding protein of Streptococcus pyogenes ATCC 21059, was amplified with polymerase chain reaction, cloned into a surface display vector pSAK332, and introduced into L. casei. The fibronectin binding domain was expressed as a fusion protein consisting of staphylokinase of Staphylococcus aureus and the anchor sequence of cell wall-associated 763 proteinase of Lactococcus lactis NCDO 763. The fibronectin binding ability of the resulting L. casei was confirmed with Western blot analysis, immunoelectron microscopic analysis, and adherence to fibroblast cells. These results indicate that L. casei has acquired a new phenotype to bind fibronectin upon the expression of the fibronectin binding domain on the cell surface. This L. casei also shows binding affinity to fibrinogen, indicating that fibronectin binding domain is involved in the binding to fibrinogen as well.

Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli.

Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L. crispatus, L. reuteri, and L. agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniques. These lactobacilli associated with the horse, except for L. reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats. A symbiotic relationship of these lactobacilli with the horse is suggested.

Generation of polymeric immunoglobulin receptor-deficient mouse with marked reduction of secretory IgA.

We generated mouse lacking exon 2 of polymeric Ig receptor (pIgR) gene by a gene-targeting strategy (pIgR-deficient mouse; pIgR-/- mouse) to define the physiological role of pIgR in the transcytosis of Igs. pIgR-/- mice were born at the expected ratio from a cross between pIgR+/- mice, indicating that disruption of the pIgR gene in mice is not lethal. pIgR and secretory component proteins were not detected in pIgR-/- mice by Western blot analysis. Moreover, immunohistochemical analysis showed that pIgR protein is not expressed in jejunal and colonic epithelial cells of pIgR-/- mice, whereas IgA+ cells are present in the intestinal mucosa of pIgR-/- mice as well as wild-type littermates. Disruption of the pIgR gene caused a remarkable increase in serum IgA concentration and a slight increment of serum IgG and IgE levels, leaving serum IgM level unaltered. In contrast, IgA was much reduced but not negligible in the bile, feces, and intestinal contents of pIgR-/- mice. Additionally, IgA with a molecular mass of 280 kDa preferentially accumulated in the serum of pIgR-/- mice, suggesting that transepithelial transport of dIgA is severely blocked in pIgR-/- mice. These results demonstrate that dIgA is mainly transported by pIgR on the epithelial cells of intestine and hepatocytes, but a small quantity of IgA may be secreted via other pathways.

Polymeric immunoglobulin receptor gene of mouse: sequence, structure and chromosomal location.

We have isolated genomic clones of a mouse gene (pIgR) for polymeric immunoglobulin receptor which mediates transport of polymeric immunoglobulins. The four overlapping clones obtained retain a DNA fragment spanning approx. 32 kb altogether, and the base sequences of these clones were determined. Comparison with cDNA sequence identified 11 exons and 10 introns, as well as a polyadenylation site. We have also identified presumptive regulatory elements on the 5' presumptive untranscribed region and a polyadenylation signal on the 3' untranslated region. Thus, the DNA cloned covers the whole area which is transcribed into mRNA. Also, in situ hybridization locates this gene on the long arm of the first chromosome of mouse.

Enterotoxin-binding glycoproteins in a proteose-peptone fraction of heated bovine milk.

The binding of Escherichia coli heat-labile enterotoxin to caseins, whey proteins, milk fat globule membrane, and proteose-peptone fraction from bovine milk was studied by using the Western blot technique. Two toxin-binding glycoproteins, pp16k and pp20k, with molecular weights of 15,500 and 20,000, respectively, were detected only in a proteose-peptone fraction. These glycoproteins were partially purified by ammonium sulfate precipitation and Toyopearl HW 55 gel filtration chromatography. The binding ability to the toxin was destroyed by periodate treatment or beta-galactosidase treatment, indicating that a carbohydrate moiety, particularly a terminal galactosyl residue, was essential for the binding of the toxin. In contrast, the binding ability was not changed by mild acid treatment, and these glycoproteins did not bind cholera toxin, which can bind to ganglioside GM1, suggesting that the carbohydrate structure of the glycoproteins is different from that of GM1. The N-terminal amino acid sequence and immunoblot analysis indicated that the protein moieties of pp16k and pp20k are identical to alpha-lactalbumin and beta-lactoglobulin, respectively. These toxin-binding glycoproteins were not detected in whey proteins isolated from unheated skim milk, suggesting that they are newly generated during heat treatment of skim milk before the preparation of a proteose-peptone fraction.

Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8.

The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.

Inquiries into the cloning and expression of the tuf gene of Thermus thermophilus HB8.

The tuf gene, which encodes the elongation factor Tu (EF-Tu), of Thermus thermophilus HB8 was cloned and sequenced. The whole G + C content of the tuf gene was 64.9%, and 84.5% of the third base in codon usage was either G or C, in which G was much favorable than C. The tuf gene was expressed in Escherichia coli under the control of the E. coli trp promoter. For the highly efficient expression, the SD sequence of the E. coli trpL was used instead of that of T. thermophilus HB8 and the length between the SD sequence and the initiation codon was controlled to keep 9 nucleotides.