Duration of thermal support for preventing hypothermia induced by anesthesia with medetomidine-midazolam-butorphanol in mice.

Hypothermia during anesthetic events is a common adverse effect of anesthesia in laboratory animals. In particular, small rodents such as mice is susceptible to hypothermia during anesthetic events. Therefore, the animals will need additional thermal support by external heating devices during and after anesthesia. In general, the time of recovery from anesthesia is typically longer in case of injectable anesthesia rather than inhalant anesthesia. However, the durations of thermal support have been almost limited to 1 hr from administration of anesthesia in general. Our study objectives are two-fold: 1) to compare the levels of hypothermia induced by injectable anesthesia with medetomidine-midazolam-butorphanol (MMB) and inhalant anesthesia with isoflurane (ISO); 2) to find the adequate durations of thermal support for preventing hypothermia induced by their anesthesia in mice. Adult male ICR mice were anesthetized during 40 min without and with the thermal support for 1 (both anesthetic groups), 2, 3, and 5 hr (in MMB group). Without thermal support, the decrease of body temperature in MMB group were more severe than that in ISO group. The durations of thermal support completely prevented hypothermia at 5 hr-support in MMB group and that at 1 hr-support in ISO group. However, the other short durations did not prevent hypothermia at 1, 2 and 3 hr-support in MMB group. These results suggest that the mice should be received thermal support over 5 hr after injection of MMB anesthesia to prevent hypothermia.

Evaluation of response to restraint stress by salivary corticosterone levels in adult male mice.

Saliva as a sampling method is a low invasive technique for the detection of physiologically active substances, as opposed to sampling the plasma or serum. In this study, we obtained glucocorticoids transferred from the blood to the saliva from mice treated with 2.0 mg/kg via an intraperitoneal injection of cortisol. Next, to evaluate the effect of restraint stress using mouse saliva-collected under anesthesia by mixed anesthetic agents-we measured plasma and salivary corticosterone levels at 60 min after restraint stress. Moreover, to evaluate salivary corticosterone response to stress in the same individual mouse, an adequate recovery period (1, 3 and 7 days) after anesthesia was examined. The results demonstrate that exogenous cortisol was detected in the saliva and the plasma, in mice treated with cortisol. Restraint stress significantly increased corticosterone levels in both the plasma and saliva (P<0.001). Monitoring the results of individual mice showed that restraint stress significantly increased salivary corticosterone levels in all three groups (1-, 3- and 7-day recovery). However, the statistical evidence of corticosterone increase is stronger in the 7-day recovery group (P<0.001) than in the others (P<0.05). These results suggest that the corticosterone levels in saliva reflect its levels in the plasma, and salivary corticosterone is a useful, less-invasive biomarker of physical stress in mice. The present study may contribute to concepts of Reduction and Refinement of the three Rs in small animal experiments.

A novel experimental platform for toxigenic and non-toxigenic Corynebacterium ulcerans infection in mice.

Corynebacterium ulcerans is a zoonotic pathogen that can produce diphtheria toxin and causes an illness categorized as diphtheria in the European Union because its clinical appearance is similar to that of diphtheria caused by Corynebacterium diphtheriae. Despite the importance of the pathogen in public health, the organism's mechanism of infection has not been extensively studied, especially in experimental animal models. Therefore in the present study we constructed an intranasal infection system for mice. Mice are insensitive to diphtheria toxin and this has the advantage of excluding the cytotoxic effect of the toxin that might interfere with the analysis of the early stage of infection. Both the toxigenic and non-toxigenic C. ulcerans strains were capable of killing mice within 3 days after inoculation at 10(7) colony-forming units per mouse. In experimentally infected animals, C. ulcerans was detected in the respiratory tract but not in the intestinal tract. The bacterium was also detected in peripheral blood and it disseminated into the lung, kidney and spleen to produce a systemic infection. This experimental infection system provides a platform for analyzing the virulence of C. ulcerans in future studies.

Screening for intestinal microflora influencing superoxide dismutase activity in mouse cecal mucosa.

We have suggested that intestinal microflora reduces the activity of the antioxidant enzyme superoxide dismutase (SOD) in the mouse cecal mucosa. In this study, gnotobiotic mice were used to examine the species of intestinal microflora influencing SOD activity in the cecal mucosa. The total SOD activity in the cecal mucosa of each germ-free (GF), gnotobiotic mouse with Escherichia coli, Lactobacillus and Bacteroides was significantly higher than that in the cecal mucosa of gnotobiotic mice with chloroform-treated feces (CHF), conventionalized (CVz) mice and conventional (CV) mice (P<0.05). In addition, CuZnSOD mRNA expression showed similar tendencies. Our results suggest that the antioxidant defense status in the cecal mucosa is influenced by CHF inoculation.

Parabacteroides chinchillae sp. nov., isolated from chinchilla (Chincilla lanigera) faeces.

Strains of Gram-stain-negative, anaerobic, rod-shaped bacteria were isolated from chinchilla (Chinchilla lanigera) faeces, and strain ST166(T) was investigated taxonomically. Phylogenetic analyses of 16S rRNA gene sequences revealed that strain ST166(T) belonged to the genus Parabacteroides. Strain ST166(T) formed a distinct line of descent, and the highest sequence similarity to ST166(T) was found with Parabacteroides merdae JCM 9497(T) (95.6%) and Parabacteroides johnsonii JCM 13406(T) (95.0%). Analysis of hsp60 gene sequences also supported these relationships. Based on the phenotypic and phylogenetic characteristics, the novel species Parabacteroides chinchillae sp. nov. is proposed. The type strain of P. chinchillae sp. nov. is ST166(T) ( = JCM 17104(T) =CCUG 62154(T)).

Upregulation of superoxide dismutase activity in the intestinal tract mucosa of germ-free mice.

Superoxide dismutase (SOD) catalyzes the breakdown of superoxide into hydrogen peroxide and oxygen in the antioxidant defense system. We had reported that the SOD activities in the ceca of germ-free (GF) mice were significantly higher than those in conventional (CV) mice. In this study, we confirmed the location where SOD activity and protein expression increased in the ceca of GF mice. An immunohistochemical analysis and total SOD activity assay were conducted using the mucosa and other remaining tissues in the ceca. In addition to SOD activity in the ceca, 4 sites of intestinal (duodenal, jejunal, ileal and colonic) mucosae in GF mice were compared with those of CV mice. Total SOD activity in the cecal mucosa of GF mice was significantly higher than that in CV mice (P<0.01), and the intensity of CuZnSOD-positive cells in cecal mucosa was increased in all GF mice. Total and CuZnSOD activities in the duodenal, jejunal, ileal, cecal and colonic mucosae of GF mice were significantly higher than those in CV mice (P<0.05, or P<0.01). Furthermore, CuZnSOD mRNA showed similar tendencies with respect to these activities. Our results suggest for the first time that upregulation of SOD activity occurs in the entire intestinal mucosa of GF mice.

Bacteroides stercorirosoris sp. nov. and Bacteroides faecichinchillae sp. nov., isolated from chinchilla (Chinchilla lanigera) faeces.

Strains of gram-negative anaerobic rods were isolated from chinchilla (Chinchilla lanigera) faeces, and three strains, ST161(T), ST33 and ST37(T), were investigated taxonomically. Based on phylogenetic analyses and specific phenotypic characteristics, the three strains were allocated to the genus Bacteroides. Phylogenetic analyses of their 16S rRNA gene sequences revealed that strain ST161(T) formed a distinct line of descent, with highest sequence similarity to strain ST33 (98.7 %) and Bacteroides oleiciplenus JCM 16102(T) (97.7 %). High levels of DNA-DNA relatedness (79-89 %) were found between strains ST161(T) and ST33, but low levels were found between strain ST161(T) and B. oleiciplenus JCM 16102(T) (33-37 %) and between strain ST33 and B. oleiciplenus JCM 16102(T) (33-37 %). These data clearly indicated that strains ST161(T) and ST33 represent a single novel species. 16S rRNA gene sequence analyses showed that strain ST37(T) also formed a distinct line of descent, with highest sequence similarity to Bacteroides acidifaciens JCM 10556(T) (96.5 %) and Bacteroides caccae JCM 9498(T) (95.6 %). Analysis of hsp60 gene sequences also supported these relationships. Based on phenotypic and phylogenetic characteristics, two novel species, Bacteroides stercorirosoris sp. nov. and Bacteroides faecichinchillae sp. nov., are thus proposed. The type strains of B. stercorirosoris and B. faecichinchillae are ST161(T) ( = JCM 17103(T) = CCUG 60872(T)) and ST37(T) ( = JCM 17102(T) = CCUG 60873(T)), respectively. The DNA G+C contents of strains ST161(T) and ST37(T) were 45.7 and 41.0 mol%, respectively.

Effects of intestinal microflora on superoxide dismutase activity in the mouse cecum.

In the antioxidant defense system, superoxide dismutase (SOD) catalyzes the breakdown of superoxide into hydrogen peroxide and oxygen. In the cecum, the influence of intestinal microflora on SOD activity is unknown. In this study, we used germ-free (GF) mice to examine the effect of intestinal microflora on SOD activity in the cecum, and SOD activity was compared between GF and conventional (CV) mice. The activity of CuZnSOD and MnSOD was determined using the SOD Assay Kit-WST. Expressions of CuZnSOD mRNA and protein were determined by real-time PCR and western blot analyses, respectively. The activities of CuZnSOD and MnSOD were significantly higher in the ceca of GF IQI and FVB/N strain mice than in CV mice (P<0.01-0.05). The gene expressions of CuZnSOD mRNA in the ceca of GF mice were significantly higher than those in CV mice (P<0.05), and CuZnSOD protein expression showed similar tendencies. Consistent with the abovementioned results, the total SOD activity in conventionalized mice decreased to the level of total SOD activity observed in the ceca of CV mice. Furthermore, no differences between GF and CV mice were observed in the SOD activities in the liver and thymus. Our results suggest that the antioxidant defense system in the mouse cecum is influenced by the intestinal microflora that downregulate SOD activity.

Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica.

BACKGROUND: Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system. RESULTS: The RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA. CONCLUSIONS: The results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

Bacteroides chinchillae sp. nov. and Bacteroides rodentium sp. nov., isolated from chinchilla (Chinchilla lanigera) faeces.

Gram-negative anaerobic rods were isolated from chinchilla (Chinchilla lanigera) faeces and three strains, ST170(T), ST180 and ST28(T), were investigated taxonomically. On the basis of phylogenetic analyses and specific phenotypic characteristics, the three strains belonged to the genus Bacteroides. Phylogenetic analysis of their 16S rRNA gene sequences revealed that strains ST170(T) and ST180 formed a single cluster and a distinct line of descent. Strain ST170(T) exhibited 99.7 % 16S rRNA gene sequence similarity with strain ST180 and 95.1, 94.6 and 94.4 % 16S rRNA gene sequence similarity with Bacteroides massiliensis JCM 13223(T), Bacteroides dorei JCM 13471(T) and Bacteroides vulgatus JCM 5826(T), respectively. Strain ST28(T) also formed a distinct line of descent and exhibited the highest 16S rRNA gene sequence similarity with Bacteroides uniformis JCM 5828(T) (98.1 %). Low DNA-DNA relatedness (1 %) between strain ST28(T) and B. uniformis JCM 5828(T) clearly indicated that they belonged to different species. Analysis of hsp60 sequences also supported these relationships. The DNA G+C contents of strains ST170(T) and ST28(T) were 45.2 and 41.0 mol%, respectively. On the basis of phenotypic characteristics and phylogenetic data, two novel species, Bacteroides chinchillae sp. nov. (type strain ST170(T)  = JCM 16497(T)  = CCUG 59335(T)) and Bacteroides rodentium sp. nov. (type strain ST28(T)  = JCM 16496(T)  = CCUG 59334(T)), are proposed.

Prevalence and analysis of Pseudomonas aeruginosa in chinchillas.

BACKGROUND: Chinchillas (Chinchilla laniger) are popular as pets and are often used as laboratory animals for various studies. Pseudomonas aeruginosa is a major infectious agent that causes otitis media, pneumonia, septicaemia enteritis, and sudden death in chinchillas. This bacterium is also a leading cause of nosocomial infections in humans. To prevent propagation of P. aeruginosa infection among humans and animals, detailed characteristics of the isolates, including antibiotic susceptibility and genetic features, are needed. In this study, we surveyed P. aeruginosa distribution in chinchillas bred as pets or laboratory animals. We also characterized the isolates from these chinchillas by testing for antibiotic susceptibility and by gene analysis. RESULTS: P. aeruginosa was isolated from 41.8% of the 67 chinchillas included in the study. Slide agglutination and pulsed-field gel electrophoresis discriminated 5 serotypes and 7 unique patterns, respectively. For the antibiotic susceptibility test, 40.9% of isolates were susceptible to gentamicin, 77.3% to ciprofloxacin, 77.3% to imipenem, and 72.7% to ceftazidime. DNA analyses confirmed that none of the isolates contained the gene encoding extended-spectrum ß-lactamases; however, 2 of the total 23 isolates were found to have a gene similar to the pilL gene that has been identified in the pathogenicity island of a clinical isolate of P. aeruginosa. CONCLUSIONS: P. aeruginosa is widely spread in chinchillas, including strains with reduced susceptibility to the antibiotics and highly virulent strains. The periodic monitoring should be performed to help prevent the propagation of this pathogen and reduce the risk of infection from chinchillas to humans.

Effect of intraperitoneal needling on pancreatic beta-cell cytotoxicity mediated via alloxan in mice with an FVB/N genetic background.

The present study investigated whether pre-stimulation with intraperitoneal (i.p.) needling protects against development of diabetes in alloxan-treated transgenic (Tg) mice overexpressing the human Cu/Zn superoxide dismutase gene or non-Tg littermates of the FVB/N strain. Twenty minutes before the alloxan treatment (60 mg/kg) the mice were injected intraperitoneally with 0.05 ml saline while control mice received only the alloxan treatment. Hyperglycemic responses of the saline-injected mice to alloxan were significantly suppressed in the Tg mice (P<0.05). A similar reduction of response was also observed in non-Tg littermates, but the effect was less than that in the Tg mice. This protective effect on the diabetogenic action of alloxan was also demonstrated by an analysis of the number of days positive for urinary glucose, and by immunohistochemical analysis of pancreatic insulin-positive cells. A similar suppressive effect on the hyperglycemic response of alloxan was observed in the mice stimulated by i.p. needling alone. However, suppression of the hyperglycemic response was not observed in ICR mice receiving an i.p. injection. These results suggest that the diabetogenic action of alloxan can be suppressed by i.p. needling-mediated stimulation in mice that have a genetic background of the FVB/N strain. Since a slight protective effects of alloxan-induced diabetes was also observed in the Tg mice compared to FVB/N mice treated with only alloxan, this phenomenon could be more clearly seen in the Tg mice than in non-Tg littermates with an FVB/N background.

Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence.

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis.

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens.

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.

Age-related changes on marking, marking-like behavior and the scent gland in adult Mongolian gerbils (Meriones unguiculatus).

Marking behavior, marking-like behavior [3], and changes of the scent glands were observed in aged Mongolian gerbils. In Experiment 1, changes in the marking and marking-like behavior with aging were evaluated in adult male and female Mongolian gerbils of an inbred strain aged 6 to 36 months. The frequency of marking behavior in males was significantly higher than females throughout the observation period except at 36 months of age. On the other hand, frequency of marking-like behavior in males, but not in females decreased with aging, significantly. In Experiment 2, changes of the scent gland in adult males and females aged 6 to 36 months were morphologically evaluated. Macroscopic examination revealed an increase in the size length and width of the glands of males aged 12 months and females aged 6 months. Histologically the glands of all the males and females aged 6 months developed moderately or well. Some of the 12-month-old males and females showed acinar atrophy of the glands, and all the females aged 18 months or more had highly atrophied scent glands. From these results, we concluded that there is no relationship between the changes of marking behavior and those of the scent glands in aged male Mongolian gerbils, and assume that marking behavior in aged animals does not have an important meaning as marking. In Experiment 3, marking and marking-like behavior in castrated adult Mongolian gerbils aged 16 weeks were observed. The result showed that marking behavior, not marking-like behavior was inhibited after castration. From these findings, we consider that generally marking behavior in Mongolian gerbils consists of androgen-dependent marking behavior and androgen-independent marking behavior (marking-like behavior).

Detection of Corynebacterium kutscheri from the oral cavity of rats.

A simple and useful method for the detection of C. kutscheri from the oral cavity of living rats was devised. In 10 sacrificed rats from two naturally and subclinically infected conventional colonies, 10(4.28) or 10(3.84) CFU/ml C. kutscheri were isolated from upper incisor swab extractions, while 10(1.38) or 10(1.58) and < 10 or 10(1.56) CFU/ml from the upper soft palate and pharynx, respectively. In another survey with 26 living animals, which were reared on the same rack, organisms were detected from the upper incisor and gingival swabs in 15 of 26 rats (57.7%). The results were reproducible at a second survey 10 days later. No organisms were isolated from any sites of the orally negative rats. These results indicated that culture of swab specimens from the upper incisors and gingivae of incisors is useful for the detection of C. kutscheri infection in living rats.