Lactobacillus equicursoris sp. nov., isolated from the faeces of a thoroughbred racehorse.

We previously isolated five strains of putative lactobacilli from the faeces of a thoroughbred horse (a 4-year-old male). Of the five strains, four were identified as members of existing Lactobacillus species; however, sequence analysis of the 16S rRNA gene revealed that the fifth isolate, DI70(T), showed approximately 97 % identity (1325/1366 bp) with the type strain of Lactobacillus delbrueckii. Therefore, we considered the possibility that DI70(T) represents a novel species of the genus Lactobacillus. Cells of strain DI70(T) were Gram-stain-positive, catalase-negative, non-spore-forming, non-motile rods. In phylogenetic trees constructed on the basis of 16S rRNA gene sequences, strain DI70(T) formed a subcluster in the L. delbrueckii phylogenetic group and was closely related to L. delbrueckii, Lactobacillus crispatus and Lactobacillus jensenii. However, analysis of DNA-DNA relatedness showed that DI70(T) was genetically distinct from its phylogenetic relatives. The isolate also exhibited distinct biochemical and physiological characteristics when compared with its phylogenetic relatives. It required anaerobic conditions for growth on agar medium. The results indicate that isolate DI70(T) indeed represents a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus equicursoris sp. nov. The type strain is DI70(T) (=JCM 14600(T) =DSM 19284(T)).

Lactobacillus hayakitensis, L. equigenerosi and L. equi, predominant lactobacilli in the intestinal flora of healthy thoroughbreds.

To detect the predominant lactobacilli in the intestinal flora of healthy thoroughbreds, we isolated lactobacilli from the feces of nine thoroughbreds (five males and four females; 0-15-year-old). The isolated lactobacilli comprise 17 species (37 strains), and they were classified into five groups: Lactobacillus salivarius (6 species), L. reuteri (6 species), Lactobacillus delbrueckii (3 species), L. buchneri (1 species) and L. vitulinus (1 species). On the basis of 16S rRNA gene sequences, we identified 3 other phylogenetic relatives belonging to the genus Lactobacillus. These results suggest that the intestinal flora of thoroughbreds may comprise many species of the genus Lactobacillus. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the 340-bp fragments of the 16S rRNA genes from the same nine fecal samples showed that L. hayakitensis, L. equigenerosi and L. equi are contained in all the samples, suggesting that these species are predominant lactobacilli in the intestinal flora of thoroughbreds.

Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses.

Four bacterial strains, designated ST18(T), HM244, HM250 and DI49, were isolated from the fresh faeces of four thoroughbred horses in Japan. Cells were Gram-positive, strictly anaerobic, catalase-negative, non-spore-forming, non-motile rods that occurred in chains. They were placed in the same subcluster based on 16S rRNA gene sequence analysis, phenotypic characteristics and levels of DNA-DNA relatedness. Their DNA G+C content ranged from 36 to 38 mol%. Lactobacillus catenaformis, Lactobacillus vitulinus and Catenibacterium mitsuokai belong to cluster XVII of the Clostridium subphylum. Strain ST18(T) was most closely related to L. catenaformis ATCC 25536(T) in the phylogenetic tree, but these strains shared only 89.9 % (1336/1486 bp) 16S rRNA gene sequence similarity. L. catenaformis, L. vitulinus and C. mitsuokai are homofermentative bacteria, whereas ST18(T) produced CO(2) from glucose. Whereas the cell-wall peptidoglycan type of L. catenaformis and L. vitulinus wasL-Lys-L-Ala(3), that of C. mitsuokai and the subgroup represented by ST18(T) was A1gamma (L-Ala-D-Glu-meso-diaminopimelic acid). On the basis of 16S rRNA gene sequence divergence of more than 10 % from L. catenaformis as well as phenotypic characteristics, strains ST18(T), HM244, HM250 and DI49 are considered to represent a novel species of a new genus belonging to the Clostridium subphylum cluster XVII, for which the name Sharpea azabuensis gen. nov., sp. nov. is proposed. The type strain of Sharpea azabuensis is ST18(T) (=JCM 14210(T) =DSM 18934(T)).

Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Lactobacillus hayakitensis sp. nov., isolated from intestines of healthy thoroughbreds.

Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species. In the representative strain, KBL13(T), the DNA G+C content was 34.3 mol%. Lactobacillus salivarius JCM 1231(T) (=ATCC 11741(T); AF089108) is the type strain most closely related to the strain KBL13(T) as shown in the phylogenetic tree, and the 16S rRNA gene sequence identity showed 96.0 % (1425/1484 bp). Comparative 16S rRNA gene sequence analysis of this strain indicated that the two isolated strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives, L. salivarius, Lactobacillus aviarius, Lactobacillus saerimneri and Lactobacillus acidipiscis. DNA-DNA reassociation experiments with L. salivarius and L. aviarius confirmed that KBL13(T) represents a novel species, for which the name Lactobacillus hayakitensis sp. nov. is proposed. The type strain is KBL13(T) (=JCM 14209(T)=DSM 18933(T)).

Fertility of spermatozoa cryopreserved with 2% acetamide or glycerol through artificial insemination in the Japanese white rabbit.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P < 0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P < 0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa.

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.

Neonatal cerebral cortical lesion abolishes the anxiolytic action of diazepam in adult rats: effects of location and extent of cortical lesion.

In our previous study, diazepam (DZP), a benzodiazepine receptor agonist, failed to suppress foot-shock-elicited ultrasonic vocalizations (USVs) in adult rats that had been neonatally lesioned in the neocortex. Because neonatal lesion of the neocortex did not influence the production of USVs, the presence of an anxiolytic mechanism of DZP is suggested apart from any anxiogenic mechanism in the brain. However, the previous study did not indicate any specific cortical regional lesions that impaired the normal development of the anxiolytic mechanism in the brain. The present study was undertaken in order to examine whether neonatal lesion of the neocortex, smaller and more localized than that in the previous study, abolishes the anxiolytic effect of DZP on foot-shock-elicited and air-puff-elicited USVs. A neonatal lesion about 2 mm diameter was made in the unilateral frontal cortex frontal to the hindlimb area or in the occipital cortex caudal to the hindlimb area. The attenuating effect of DZP on the USVs elicited by both aversive stimuli was found to be abolished only in the frontal cortex-lesioned rats. This finding indicates that the frontal cortex is likely to be specifically involved in the normal development of the benzodiazepine-anxiolytic mechanism in the brain.

Mammary gland differentiation in female rats after prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl.

Recently we reported finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) increases the rate of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreases it. One of the most important factors determining sensitivity of the mammary gland to neoplastic stimuli is its stage of differentiation at the time of exposure to the carcinogenic agent. Hence, to verify a biphasic dose-response relationship (enhancement of carcinogenesis at low dose, and inhibition at high dose), we investigated the effects of prenatal exposure to PCB126 on mammary gland differentiation. Female SD rats were injected (i.g.) with 25 pg, 2.5 ng, 250 ng, 7.5 microg of PCB126/kg, or the vehicle, on days 13-19 postconception. In 50-day-old offspring, regardless of the day of exposure to DMBA, only the 7.5 microg group showed statistically significant high levels of PCB126 in the fatty tissue of their mammary glands. Fifty-day-old female offspring of the 250 ng group showed apparent inhibition of the normal differentiation of terminal end buds (TEB) to alveolar buds and lobules (ABL), while those of the 7.5 microg group showed mammary gland hypoplasia. Expression levels of the estrogen receptor-alpha (ER) in TEBs and the ER mRNA in mammary glands were higher in the 7.5 microg, 250 ng, 2.5 ng groups. Proliferating cell nuclear antigen (PCNA) expression in TEBs of 50-day-old rats was statistically significantly higher in the 250 ng group and lower in the 7.5 microg group. In the developing mammary gland, TEBs are considered the most susceptible to mammary carcinogenesis, while ABLs are relatively protected from mammary carcinogenesis. Thus, prenatal exposure to a relatively low dose of PCB126 induced an alteration of mammary gland differentiation that might potentially increase the risk of DMBA-induced mammary carcinoma.