The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer

Background  

Although mice have long served as an animal model for periodontitis, information on the composition of their indigenous oral microbiota is limited. The aim of the current study was to characterize mouse oral bacterial flora by applying extensive parallel pyrosequencing using the latest model pyrosequencer, a Roche/454 Genome Sequencer FLX Titanium. In addition, the effect of Toll-like receptor (TLR) 2 deficiency on oral microbiota was evaluated.     

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6 · 10³ colony forming units per rotifer^–1) and free-living (0.6–25 10⁷ cells·ml^–1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.After enrichment of the cultures with squid meal, the similarity in the composition of the bacterial flora between the rotifers and water was reduced. However, acclimation of the culture at 6 °C resulted in better agreement of the rotifer associated flora and that in water. Enrichment of the cultures induced a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. The bacterial flora of the rotifer cultures are dominated by presumably opportunistic species after enrichment, which may have detrimental effects when rotifers are fed as live food to marine fish larvae.     

Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment

The mammalian gastrointestinal (GI) tract is inhabited by over a hundred species of symbiotic bacteria. Differences among individuals in the composition of the GI flora may contribute to variation in in vivo experimental analyses and disease susceptibility. To investigate potential interindividual differences in GI flora composition, we developed real-time quantitative PCR-based assays for the detection of the eight members of the Altered Schaedler Flora (ASF) as representative members of different bacterial niches within the mammalian GI tract. Quantitative and reproducible strain-specific variations in the numbers of the ASF members were observed across 23 different barrier-housed inbred mouse strains, suggesting that the ASF assays can be used as sentinels for changes in GI flora composition. A significant cage effect was also detected. Isogenic mice that cohabited at weaning, whether from the same or different litters, showed little variation in ASF profiles. Conversely, litters split among different cages at weaning showed divergence in ASF profiles after three weeks. Individual ASF profiles, once established, were highly stable over time in the absence of environmental perturbation. Furthermore, cohabitation of different inbred strains maintained most of the interstrain variation in the GI flora, supporting a role of host genetics in determining GI flora composition.     

Study of murine faecal microflora by cellular fatty acid analysis; effect of age and mouse strain

Analysis of bacteria-derived cellular fatty acids was applied to study differences in faecal floras of inbred mice. The bacterial composition of the faecal flora clearly changes with age, whereas the sex does not affect it. Most interestingly, different mouse strains were found to have different faecal floras. This was particularly observed at the age of 17–19 weeks for stool samples of four different mouse strains; the mice were handled identically in identical environments, and the two congenic strains used were different from each other only by the major histocompatibility complex (MHC). These results suggest that composition of the faecal flora is genetically regulated.     

Candidal vaginitis in hormone-treated mice: Prevention by a chitin extract

In view of findings from previous studies that a chitin soluble extract (CSE) blocked adhesion of Candida albicans in vitro and in vivo and prevented thereby a short lived candidal infection in naive mice, we attempted in the present study to block by CSE the development of a persistent infection, induced in hormone-treated animals. Continuous oestrus phase was obtained in mice by repeated weekly subcutaneous injections with estradiol benzoate. Intravaginal inoculation of the hormone-treated mice with 10⁷–10¹⁰ C. albicans cells induced a persistent candidal infection. Fifty three mice were pretreated intravaginally prior to inoculation of C, albicans with 2.5, 5.0 or 10 mg/mouse of a CSE cream and followed up for development of infection in comparison to 30 untreated animals. Twenty four hrs post fungus inoculation the infection rate among the CSE treated mice was 11–23% VS 84% among the controls; the rate increased a week later to 97% among the controls VS 41–50% among the CSE treated. Administering the CSE to the mice prior — and post-yeast inoculation (37 mice), led to increased efficacy of the treatment. The data, indicating that CSE is an effective measure for preventing persistent candidal vaginitis, may open the way to consider a similar approach for prophylaxis of vaginitis in human susceptible populations.     

Influence of hydrological seasonality on bacterioplankton in two neotropical floodplain lakes

Seasonal fluctuation in river stage strongly affects the ecological functioning of tropical floodplain lakes. This study was conducted to assess the influence of hydrological seasonality on bacterial production and abundance in two floodplain lakes of the Autana River, a blackwater river in the Middle Orinoco basin, Venezuela. Water samples for nutrient chemistry, chlorophyll a, and microbiological determinations were collected in two floodplain lakes and in the mainstem of the river during 1997–98. DOC and chlorophyll a concentrations were similar between mainstem and lake sites during high water when river and lakes were well connected but became different during the period of low water when the interaction was minimal. Higher values of bacterial production were observed in the floodplain lakes (0.62–1.03 g C l^–1 h^–1) compared to the mainstem sites (0.17–0.19 g C l^–1 h^–1) during the period of low water, while during the period of high water river and lake sites showed similar levels (0.04 g C l^–1 h^–1). Bacterial numbers followed bacterial production in the floodplain lakes, reaching higher numbers during the period of low water (1.41–2.40 × 10⁶ cells ml^–1). Availability of substrate and inorganic nutrients, pH, and inputs and losses of bacterial cells could be determining the observed seasonal patterns in bacterial production and abundance. The Autana lakes exhibited a strong seasonal pattern in the chemical and biological conditions, showing higher productivity during the lentic phase that lasted between 5 and 6 months.     

Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Ecology of bacterial communities in the schistosomiasis vector snailBiomphalaria glabrata

The internal colony-forming bacterial flora of the schistosome intermediate host snailBiomphalaria glabrata (Say) has been characterized in ca. 500 individual snails from Puerto Rico, Guadeloupe, and St. Lucia, and from laboratory aquaria. Freshly captured wild snails harbor 2–40×10⁶ CFU·g^–1, and healthy aquarium snails harbor 4–16×10⁷ CFU·g^–1, whereas moribund individuals have 4–10 times as many bacteria as healthy individuals from the same habitats.Pseudomonas spp. are the most common predominant bacteria in normal snails, whereasAcinetobacter, Aeromonas, andMoraxella spp. predominate in moribund snails. External bacterial populations in water appear to have little effect on the composition and size of the flora in any snail. In addition to normal (healthy) and moribund snails, a third group of snails has been distinguished on the basis of internal bacterial density and predominating genera. These high-density snails may have undergone stresses and may harbor opportunistic pathogens. The microfloras of wild and laboratory-reared snails can be altered and stimulated to increase in density by crowding the snails or treating them with antibiotics.     

An anaerobic continuous-flow culture model of interactions between intestinal microflora and Candida albicans

Summary The finding by earlier workers that Escherichia coli suppressed the growth of Candida albicans in vitro or in gnotobiotic mice has led to numerous, erroneous conclusions regarding the identity of the organisms and mechanisms responsible for the suppression of Candida in the gut. This is due, in part, to the fact that nearly all studies to date have not reflected interactions as they occur in the intestinal tract. This paper describes a series of experiments that establish that an anaerobic continuous-flow (CF) culture model, of the ecology of the large intestinal flora reproduces interactions between bacteria and Candida as they occur in the large intestine. This was determined in the following ways. (i) Bacterial counts in CF cultures of conventional mouse cecal flora or human fecal flora closely resembled that found in the mouse intestine and human feces. (ii) Dense layers of bacterial growth that formed on the glass walls of the CF culture vessels resembled bacterial populations that colonize intestinal mucosa. (iii) Total and individual levels of certain metabolic end-products of the predominant anaerobic bacterial flora present in CF cultures coincided with those found in the large intestine of conventional mice or human feces used to establish the CF cultures. (iv) C. albicans was eliminated from CF cultures of mouse cecal flora at a rate similar to that of untreated experimental animals. (v) Contents of CF cultures fed to antibiotic-treated mice redressed several cecal abnormalities, and suppressed Candida populations to levels found in conventional animals. Thus, a number of complex ecological mechanisms were maintained in CF cultures which normally control Candida populations in the large intestine. It is suggested, therefore, that the CF culture model should help to further define the mechanisms which control C. albicans and other fungi in the intestinal tract, as well as define which components of the indigenous microflora are responsible for suppression of Candida in the gut.     

The Dynamics of Physiological and Biochemical Parameters of Mouse Status as a Function of Concentration of Chemical Toxicants in Drinking Water Extracts

The effect of single oral administration of oil solution of hexane–ether extracts with different contents of benzo(a)pyrene and polychlorinated biphenyls on the dynamics of physiological and biochemical parameters in the liver and blood of mice proved to significantly depend on chemical composition and concentration of the extract as well as on the time period after administration. No linear dose–effect relationships have been revealed for both physiological and biochemical parameters. Administration of low doses of the extracts changed the scale and the degree of the relationship between mouse body weight and liver weight. The most substantial changes in most biochemical parameters were observed in the groups of mice administered with the lowest concentration of the extracts (5.3 × 10^–5 mg/kg).     

Chlamydia pneumoniae infection significantly exacerbates aortic atherosclerosis in an LDLR–/– mouse model within six months

We have previously shown that infection with the C. pneumoniae AR39 strain once monthly for 9 consecutive months significantly exacerbated atherosclerosis in mice with LDL receptor deficiency (LDLR–/–) in the presence of a high cholesterol diet. To further optimize the LDLR–/– mouse model for studying the mechanisms of C. pneumoniae atherogenesis, we have tested a different infection protocol with intranasal inoculation twice monthly for 6 consecutive months in the present study. We found that C. pneumoniae infection for 6 months was sufficient to produce a 130%, significantly greater exacerbation of aortic atherosclerosis in LDLR–/– mice in the presence of a high cholesterol diet. Mice receiving a high cholesterol diet alone displayed a lesion area index of 18.2 ± 6.1 (S.D.) while mice treated with both the high cholesterol diet and C. pneumoniae infection had a lesion area index of 41.8 ± 15.2 (S.D.). However, the chlamydial infection did not significantly alter the mouse serum total cholesterol or the LDL levels induced by the high cholesterol diet. This study not only confirms our previous findings that C. pneumoniae infection can exacerbate aortic atherosclerosis lesion in the LDLR–/– mice, but also further optimizes the LDLR–/– mouse model for future mechanism studies.     

l-arginine supplementation of milk liquid or dry diets fed to pigs after weaning has a positive effect on production in the first three weeks after weaning at 21 days of age

A total of 48, 21-day-old weaned pigs, was used in a 2 × 2 factorial arrangement of treatments with the factors being diet type (milk liquid replacer vs. dry feed) and l-arginine (ARG) supplementation (0 vs. 6 g ARG/kg) to test the hypothesis that dietary supplementation with ARG would increase performance of pigs after weaning. Pigs were fed the experimental diets for 10 d (days) after weaning and then transitioned over a 3-d period to a dry Phase II diet fed in meal form devoid of supplemental ARG. The study ended at d 21. There were five replicates (pens) per treatment (a total of 12 pigs per treatment). Blood samples were collected from two pigs per replicate on d 7 and 16 of the experiment, and free amino acids (AA) and plasma urea nitrogen (PUN) levels analysed. Milk-fed pigs outperformed (P<0.001) dry-fed pigs for the first 10 d of the experiment as well as for the total 21-d period. At d 7, milk-fed pigs had higher (P<0.05) levels of most free indispensable and dispensable amino acids in their plasma. In both the milk-fed and dry-fed pigs supplemented with ARG, average daily feed intake (ADFI, P<0.05) and average daily gain (ADG, P<0.05) were increased during the dietary transition period (d 11–14), when pigs were being changed to the Phase II diet. The difference in production in the transition period caused a tendency for ARG-supplemented pigs to eat more feed (P<0.1) and grow faster (P<0.5) over the 21-d experimental period. Pigs supplemented with ARG had higher plasma ARG levels (P<0.05) at d 7 after weaning and lower plasma urea levels (P<0.05) at both d 7 and 16 after weaning. These data show the benefits of feeding a milk liquid diet as well as of ARG supplementation after weaning on production indices.     

Evolution of resident oral bacterial biota in BALB/c mice during pregnancy and lactation

To assess the influence of pregnancy and lactation on the oral microbial ecology of BALB/c mice, we followed the distribution of the predominant oral bacteria of four groups of these mice during these two periods. Compared with nonpregnant control female mice of the same age maintained under the same conditions, the distribution of the resident oral bacterial species differed significantly only during the lactation period (8–16 days after parturition). This difference could possibly be attributed to hormonal influences and/or grooming habits.     

Bacteriology of the oral cavity of BALB/c mice

To be used as a model in dental research, an animal must fulfil experimental needs and information on the composition and variation of its oral flora must be available. Only limited data are available on the indigenous oral bacterial flora of BALB/c mice. In this work, a total of 671 isolates from different sites (saliva, tongue, teeth, and mucosa) of the oral cavity of BALB/c mice were identified. Only 18 different species were isolated, which indicates the relative simplicity of the flora. The predominant species of the total cultivable flora were "Lactobacillus murinus" (38%), Staphylococcus aureus (37%), Streptococcus faecalis (8%), Staphylococcus sciuri (4%), and Escherichia coli (3%). The other species each represent less than 2% of the flora. "Lactobacillus murinus" is found in greater proportion on mucosa than in the other sites, Staph. aureus predominates in saliva, and Strep. faecalis was found in greater proportion in tooth samples. Statistical analyses, using the minimum percentage of similarity, indicate that there is some variation among the microflora of different mice but that this difference is smaller for mice from the same lot. These results set the basis for the study of the variations of the indigenous oral microflora of BALB/c mice under different conditions.     

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

An adequate assessment of scorpion and snake venom LD₅₀is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD₅₀variation of Tunisian scorpion (Androctonus australis garzonii: Aag andButhus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57Bl/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990–1996). Aag venom is 3–4 times more toxic than Bot venom. However for both venoms, the LD₅₀determined in C57Bl/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD₅₀variations (25–50%) were also seen from one electrically prepared batch to another. A good correlation (r=0·982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD₅₀values when assessedin vivo.The LD₅₀variation of Tunisian viper (Cerastes cerastes: Cc andVipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990–1992). No significant LD₅₀variations were observed with the mouse strain, the sex or the season of venom milking. However, LD₅₀varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS–PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD₅₀evaluation or the venom quality used for the development of an efficient antivenom.     

Seasonal influence on the reproductive performance of swine in the humid zone of Ghana

Relationships between month and climatic factors (ambient temperature, relative humidity, RH, and rainfall) with litter size, conception rate, days from weaning to conception and mortalities at birth and preweaning were studied in large white pigs under research station (RS) and commercial farm (CF) conditions. In RS and CF 868 and 572 farrowing records, respectively, were involved. Litter size was almost evenly distributed in all months of the year, at both farms, despite significant (P<0.05) negative but low correlations between litter size and maximum (r=–0.272, RS) and minimum (r=–0.233, CR) temperatures in the month after conception. There were no significant differences in conception rate between months. However under RS conditions there were significant (P<0.05) correlations between minimum temperature and conception rate in the months prior to (r=–0.362) and at conception (r=–0.221). Days from weaning to conception were bimodally distributed with peak values occurring between January and June and October and December. Significant mortality at birth (P<0.05) and preweaning was generally highest in the cold rainy months of May to October. However, only under CF conditions were there significant correlations between minimum temperature in the month of birth and percentage mortalities at birth (r=–0.217) and preweaning (r=–0.250). Rainfall and RH had no significant (P>0.05) correlation with birth and preweaning mortalities.     

Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD_531–545 epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5 mg of CTB–GAD_(531–545)3 per liter of culture with greater than 90% purity by a Ni–NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB–GAD_(531–545)3 fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.     

Bacterial productivity and microbial biomass in tropical mangrove sediments

Bacterial productivity (³H-thymidine incorporation into DNA) and intertidal microbenthic communities were examined within five mangrove estuaries along the tropical northeastern coast of Australia. Bacteria in mangrove surface sediments (0–2 cm depth) were enumerated by epifluorescence microscopy and were more abundant (mean and range: 1.1(0.02–3.6)×10¹¹ cells·g DW^–1) and productive (mean: 1.6 gC·m^–2· d^–1) compared to bacterial populations in most other benthic environments. Specific growth rates (¯x=1.1) ranged from 0.2–5.5 d^–1, with highest rates of growth in austral spring and summer. Highest bacterial numbers occurred in winter (June–August) in estuaries along the Cape York peninsula north of Hinchinbrook Island and were significantly different among intertidal zones and estuaries. Protozoa (10⁵–10⁶·m^–2, pheopigments (0.0–24.1g·gDW^–1) and bacterial productivity (0.2–5.1 gC·m^–2·d^–1) exhibited significant seasonality with maximum densities and production in austral spring and summer. Algal biomass (chlorophylla) was low (mean: 1.6g·gDW^–1) compared to other intertidal sediments because of low light intensity under the dense forest canopy, especially in the mid-intertidal zone. Partial correlation analysis and a study of possible tidal effects suggest that microbial biomass and bacterial growth in tropical intertidal sediments are regulated primarily by physicochemical factors and by tidal flushing and exposure. High microbial biomass and very high rates of bacterial productivity coupled with low densities of meiofaunal and macroinfaunal consumers observed in earlier studies suggest that microbes may be a sink for carbon in intertidal sediments of tropical mangrove estuaries.     

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.     

Maturation of the intestinal digestion and of microbial activity in the young rabbit: impact of the dietary fibre:starch ratio

The developmental changes of intestinal digestive potential and caecal microbial activity were described in suckling and weaned rabbits according to two feeding programmes. Two groups of thirteen litters were fed from 18 to 42 days old a “High” or a “Medium” NDF:starch ratio diet (resp. 2.7 vs 2.0, groups HL and ML) with similar protein and lipid levels, and from 42 to 70 days old the two groups were fed a “Low” NDF:starch ratio diet (1.7). From 25 to 32 days (weaning), the milk and solid feed intake were 22% and 41% higher in ML group (P < 0.05), and the mortality by diarrhoea was 4 units lower (P < 0.01). The whole tract digestive efficiency increased by 10% before weaning, and remained steady (organic matter) or decreased (lipids, protein) after weaning. Energy digestibility was 0.623 and 0.686 for High and Medium diets respectively. From 25 to 42 days, total enzymatic activity in intestinal content increased for chymotrypsin (5-fold, P < 0.001), lipase (10-fold, P < 0.001), amylase (17-fold, P < 0.01) and maltase (11-fold, P < 0.001), while trypsin doubled after weaning. The feeding programme only affected the amylase and maltase activities, that were higher in HL group (P < 0.05). The volatile fatty acids concentration in the caecum was not significantly different among the groups, but it increased by 44% 10 days after weaning. The bacterial fibrolytic enzymes, increased by 30% after weaning and were similar among the two groups. The study revealed that the intestinal digestive maturation and the caecal microbial activity of the rabbit evolved markedly between 3 and 5 weeks of age, and was weakly affected when the NDF:starch ratio decreased from 2.7 to 2.0.