Description of serum symmetric dimethylarginine concentration and of urinary SDS-AGE pattern in dogs with ACTH dependent hyperadrenocorticism.

Serum symmetric dimethylarginine (SDMA) and patterns of urinary protein separated by sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE) have not been investigated as biomarkers in dogs with ACTH-dependent hyperadrenocorticism (ADHAC). This exploratory prospective study aimed to evaluate SDMA, serum creatinine (sCR), and SDS-AGE in dogs with ADHAC with and without proteinuria (ADHAC-P and ADHAC-nP, respectively). Thirty-five pet dogs classified as ADHAC-P (n=16), ADHAC-nP (n=6) and healthy (n=13) were included. Renal biomarkers were evaluated in all dogs at diagnosis. Baseline concentration of SDMA was not significantly different between the three groups (P = 0.15) whereas sCr was significantly lower in dogs in ADHAC dogs compared to healthy dogs (88.0 µmol/L [70.4-132.6; 79.2-114.4]) whether they had proteinuria or not (P = 0.014 and 0.002, respectively). However, baseline concentrations of sCr and SDMA were not significantly different between dogs with ADHAC-P dogs (SDMA, 8 µg/dL [5-12; 7-9]; sCr, 57.2 µmol/L [35.2-212.2; 52.8-92.4]) and ADHAC-nP dogs (SDMA, 8.5 µg/dL [7-13; 8-10]; sCr, 70.4 µmol/L [61.6-79.2; 61.6-70.4]) (P = 0.35 and P = 0.41, respectively). Proteinuria in dogs with ADHAC-P was mainly of glomerular origin (SDS-AGE pattern: glomerular in 10/16 dogs; mixed glomerular/tubular in four dogs). In our study, SDMA was neither significantly different in dogs with ADHAC whether they were proteinuric or not, nor between ADHAC and healthy dogs. Urinary electrophoresis provides additional information to the UPC and further investigations are needed to determine whether it may help identify dogs with ADHAC-P requiring specific antiproteinuric treatment.

Short course of immune-suppressive doses of prednisolone, evaluated through a prospective double-masked placebo-controlled clinical trial in healthy Beagles, is associated with sustained modifications in renal, hydration, and electrolytic status.

OBJECTIVE: To investigate the effects and duration of orally administered prednisolone on renal function evaluated by glomerular filtration rate (GFR) determination and creatinine (Cr) and symmetric dimethylarginine (SDMA) concentrations as well as on urinalysis, electrolytes, and hydric status in healthy dogs. ANIMALS: 14 healthy Beagles. PROCEDURES: In this prospective double-masked placebo-controlled study, dogs were randomized after baseline evaluation to receive a 7-day course of either prednisolone (1.5 to 2.0 mg/kg, PO, q 12 h) or a placebo. A repeated-measure design was performed, each dog participating in 4 successive sampling sessions. Clinical data, systolic blood pressure, CBC, and biochemical analyses including serum SDMA concentration, GFR determination, urine output quantification, and complete urinalysis were performed for all dogs the day before (D0) and at the end of steroid administration (D7) as well as 2 weeks (D21) and 4 weeks (D35) after the end of treatment. RESULTS: At D7, when compared with baseline, GFR increased significantly in treated dogs, whereas creatinine and SDMA concentrations decreased significantly. GFR and Cr but not SDMA modifications persisted significantly at D21. None of the variables differed significantly from baseline at D35. The OR of presenting an albumin band on urine electrophoresis was 2.4 times as high in treated versus control dogs (OR, 36; 95% CI, 1.8 to 719.4; P = 0.02). CLINICAL RELEVANCE: A short-term course of immune-suppressive prednisolone treatment in healthy dogs leads to a sustained but reversible renal hyperfiltration state. Modification in electrolytic variables can affect the clinical interpretation of blood work in such patients.

Asymptomatic carriage of canine infectious respiratory disease complex pathogens among healthy dogs.

OBJECTIVES: The objectives of this study were to determine the prevalence of canine infectious respiratory disease pathogens among asymptomatic client-owned dogs, and to compare the risks of asymptomatic pathogen carriage between client-owned dogs and dogs in an animal shelter. MATERIALS AND METHODS: Pooled tonsillar, conjunctival and nasal cavity swabs from asymptomatic client-owned dogs (n=133) were tested using a real-time polymerase chain reaction canine respiratory panel. Identical samples from asymptomatic dogs in an animal shelter (n=295) were similarly tested for selected pathogens. Risk differences were calculated between client-owned dogs and shelter dogs for each of the respiratory pathogens included in the analyses. RESULTS: A total of 15 of 133 (11.3%) asymptomatic client-owned dogs were positive for at least one pathogen in the complex. Seven dogs (6.1%) were positive for M. cynos, six (5.2%) were positive for B. bronchiseptica, two (1.7%) were positive for canine herpesvirus type 1 and two (1.7%) were positive for canine respiratory coronavirus. For all eight pathogens tested in both groups, the proportion of positive cases was higher among shelter dogs than among client-owned dogs. Shelter dogs had a higher risk for M. cynos (0.18, 95% confidence interval: 0.12 to 0.25), canine respiratory coronavirus (0.15, 95% confidence interval: 0.10 to 0.19), canine distemper virus (0.06, 95% confidence interval: 0.03 to 0.09), and canine pneumovirus (0.05, 95% confidence interval: 0.03 to 0.08) than client-owned dogs. Odds ratios for M. cynos (0.31, 95% confidence interval: 0.08 to 0.92) and canine respiratory coronavirus (0.05, 95% confidence interval: 0.01 to 0.18) were significantly different between client-owned and shelter dogs. In all cases except for canine herpesvirus type 1, dogs within the shelter population were observed to be at higher risk of exhibiting asymptomatic carriage of a respiratory pathogen as compared to client-owned dogs. The strength of this association was strongest for M. cynos and canine respiratory coronavirus. CLINICAL SIGNIFICANCE: The risk of canine infectious respiratory disease pathogen exposure posed by asymptomatic client-owned dogs is poorly defined. This study also corroborates previous reports of high canine infectious respiratory disease prevalence among clinically healthy shelter dogs, and further determined that the overall prevalence of canine infectious respiratory disease pathogen carriage among clinically healthy client-owned dogs is low but is highest for the traditional pathogen B. bronchiseptica and the emerging pathogen M. cynos.

Reactions of nitrite with goethite and surface Fe(II)-goethite complexes.

Chemodenitrification-the abiotic (chemical) reduction of nitrite (NO2-) by iron (II)-plays an important role in nitrogen cycling due in part to this process serving as a source of nitrous oxide (N2O). Questions remain about the fate of NO2- in the presence of mineral surfaces formed during chemodenitrification, such as iron(III) (hydr) oxides, particularly relative to dissolved iron(II). In this study, stirred-batch kinetic experiments were conducted under anoxic conditions (to mimic iron(III)-reducing conditions) from pH 5.5-8 to investigate NO2- reactivity with goethite (FeOOH(s)) and Fe(II)-treated goethite using wet chemical and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Nitrite removal from solution by goethite was more rapid at pH 5.5 than at pH 7 and 8. Spectral changes upon nitrite adsorption imply an inner-sphere surface interaction (monodentate and bidentate) at pH 5.5 based on ATR-FTIR spectra of the nitrite-goethite interface over time. In iron(II)-amended experiments at pH 5.5 with high aqueous Fe(II) in equilibrium with goethite, nitrous oxide was generated, indicating that nitrite removal involved a combination of sorption and reduction processes. The presence of a surface complex resembling protonated nitrite (HONO) with an IR peak near ~1258 cm-1 was observed in goethite-only and iron(II)-goethite experiments, with a greater abundance of this species observed in the latter treatment. These results might help explain gaseous losses of nitrogen where nitrite and iron(II)/goethite coexist, with implications for nutrient cycling and release of atmospheric air pollutants.

Urinary clusterin and cystatin B as biomarkers of tubular injury in dogs following envenomation by the European adder.

Diagnosing acute kidney injury remains a challenge since the established renal biomarkers, serum creatinine (sCr) and symmetric dimethylarginine (SDMA) reflect glomerular function and not tubular injury. Sensitive tubular markers such as urinary clusterin (uClust) and cystatin B (uCysB) have been proposed to detect AKI at an earlier stage. Since envenomation by the European adder (Vipera berus berus) could serve as a spontaneous disease model of AKI we investigated these new biomarkers in affected dogs. Concentrations of uClust and uCysB as well as sCr and SDMA were analyzed retrospectively in stored samples from 26 dogs with snake envenomation and 13 healthy controls. Higher concentrations of uClust (P < 0.012) and uCysB (P < 0.001) were observed in the snake-envenomed group. Normalization of uClust and uCysB to urinary creatinine did not alter the results. No differences were observed in sCr and SDMA between the snake-envenomed group and the healthy control group. Spearman rank correlation analysis revealed a strong association of uClust with uCysB in the snake-envenomed dogs (r = 0.75 P < 0.001) but not in the healthy controls. The high percentage of snake-envenomed dogs with increased uClust and uCysB concentrations in the absence of increased sCr and SDMA suggests renal tubular injury in the affected dogs. Larger prospective case-controlled studies are warranted to evaluate the clinical utility and prognostic value of these biomarkers.

Bacteria-assisted removal of fluoroquinolones from wheat rhizospheres in an agricultural soil.

Extensive fluoroquinolone antibiotics use results in their widespread occurrence in various environments including soil, which threatens the soil ecology and public health. The fate of fluoroquinolones in agricultural soil and the efficacy of enhanced degradation in the presence of an agricultural crops and antibiotic degrading bacteria could be better understood. The current study examined ciprofloxacin (CIP), enrofloxacin (ENR), and levofloxacin (LEV) biodegradation in a Maury Silt Loam soil in greenhouse conditions by bacterial-assisted removal of individual and mixed antibiotics in wheat rhizospheres. Fluoroquinolones were added at rates of 5, 50, and 100 mg kg-1. Three bacterial isolates were applied at 106 CFU g-1 soil individually and in consortium. Antibiotics appeared in wheat tissue, with more accumulation in roots than shoots. Low recoveries (<50%) of CIP, ENR, and LEV were observed at all levels and treatments in a bacteria and wheat-free control compared to the initial concentrations applied Contaminated soil with wheat had greater antibiotic recovery than the wheat-free control. Antibiotic recovery with bacterial inoculum was less than that of the indigenous bacteria. The least antibiotic recovery occurred with wheat and bacterial inoculum together. At concentrations of 5 and 50 mg kg-1, but not at 100 mg kg-1, CIP, ENR, and LEV were below detection limits in soil after 30 days through the combination of wheat and bacteria compared to the control. This synergistic removal of the fluoroquinolone antibiotics is proposed to be due to enhanced antibiotic bioavailability, which suggests it as an environment-friendly approach to biodegradation.

Molecular diversity of Scottish Cryptosporidium hominis isolates.

Cryptosporidium hominis is one of the most prevalent protozoan parasites to infect humans where transmission is via the consumption of infective oocysts. This study describes sporadic cases in addition to the molecular diversity of outbreak cases in Scotland using the glycoprotein-60 subtyping tool. From a total of 187 C. hominis isolates, 65 were subjected to further molecular analysis and 46 were found to be the common IbA10G2 subtype. Unusual subtypes included four isolates belonging to the Ia family (IaA14R3, n = 12; IaA14R2, n = 1; IaA9G3, n = 1; IaA25R3, n = 2), two from the Id family (IdA24, n = 1; IdA17, n = 1) and one belonging to the Ie family, namely IeA11G3T3. These data contribute significantly to our knowledge and understanding of the molecular diversity of C. hominis isolates from outbreak investigations involving Scottish residents which will be beneficial for the management of future outbreaks.

Pan-atlantic analysis of the overlap of a highly migratory species, the leatherback turtle, with pelagic longline fisheries.

Large oceanic migrants play important roles in ecosystems, yet many species are of conservation concern as a result of anthropogenic threats, of which incidental capture by fisheries is frequently identified. The last large populations of the leatherback turtle, Dermochelys coriacea, occur in the Atlantic Ocean, but interactions with industrial fisheries could jeopardize recent positive population trends, making bycatch mitigation a priority. Here, we perform the first pan-Atlantic analysis of spatio-temporal distribution of the leatherback turtle and ascertain overlap with longline fishing effort. Data suggest that the Atlantic probably consists of two regional management units: northern and southern (the latter including turtles breeding in South Africa). Although turtles and fisheries show highly diverse distributions, we highlight nine areas of high susceptibility to potential bycatch (four in the northern Atlantic and five in the southern/equatorial Atlantic) that are worthy of further targeted investigation and mitigation. These are reinforced by reports of leatherback bycatch at eight of these sites. International collaborative efforts are needed, especially from nations hosting regions where susceptibility to bycatch is likely to be high within their exclusive economic zone (northern Atlantic: Cape Verde, Gambia, Guinea Bissau, Mauritania, Senegal, Spain, USA and Western Sahara; southern Atlantic: Angola, Brazil, Namibia and UK) and from nations fishing in these high-susceptibility areas, including those located in international waters.

Turtle groups or turtle soup: dispersal patterns of hawksbill turtles in the Caribbean.

Despite intense interest in conservation of marine turtles, spatial ecology during the oceanic juvenile phase remains relatively unknown. Here, we used mixed stock analysis and examination of oceanic drift to elucidate movements of hawksbill turtles (Eretmochelys imbricata) and address management implications within the Caribbean. Among samples collected from 92 neritic juvenile hawksbills in the Cayman Islands we detected 11 mtDNA control region haplotypes. To estimate contributions to the aggregation, we performed 'many-to-many' mixed stock analysis, incorporating published hawksbill genetic and population data. The Cayman Islands aggregation represents a diverse mixed stock: potentially contributing source rookeries spanned the Caribbean basin, delineating a scale of recruitment of 200-2500 km. As hawksbills undergo an extended phase of oceanic dispersal, ocean currents may drive patterns of genetic diversity observed on foraging aggregations. Therefore, using high-resolution Aviso ocean current data, we modelled movement of particles representing passively drifting oceanic juvenile hawksbills. Putative distribution patterns varied markedly by origin: particles from many rookeries were broadly distributed across the region, while others would appear to become entrained in local gyres. Overall, we detected a significant correlation between genetic profiles of foraging aggregations and patterns of particle distribution produced by a hatchling drift model (Mantel test, r = 0.77, P < 0.001; linear regression, r = 0.83, P < 0.001). Our results indicate that although there is a high degree of mixing across the Caribbean (a 'turtle soup'), current patterns play a substantial role in determining genetic structure of foraging aggregations (forming turtle groups). Thus, for marine turtles and other widely distributed marine species, integration of genetic and oceanographic data may enhance understanding of population connectivity and management requirements.

Potential nitrification in alum-treated soil slurries amended with poultry manure.

Alum is used to reduce environmental pollutants in poultry production. Alum decreases NH3 volatilization and increases total N and NH4+-N compared to untreated poultry manure. Nitrification in poultry wastes could therefore be stimulated due to higher NH4+ concentrations or could be inhibited because the soil environment is acidified. A 10-day laboratory study was conducted to study potential nitrification rates in soil slurries (20 g soil in 150 ml water) amended with 2.0 g alum-treated poultry manure. Fecal bacteria, NH4+, NO2-, NO3-, orthophosphate, pH, and NH3 were measured at 2-day intervals. Alum significantly reduced fecal bacteria concentrations through day 6. Water-soluble P was reduced 82% by day 10. Alum-treated manure had significantly increased NH4+ concentrations by day 8 and 10, and significantly decreased NO2- and NO3- concentrations by days 6-10. Alum's effect on potential nitrification was inhibitory in the soil environment. Slurries with alum-treated poultry manure had reduced nitrification rates, fecal bacteria, and soluble P. Therefore, in addition to reducing P loss, alum could temporarily reduce the risk for environmental pollution from land-applied manures in terms of both NO3- and fecal bacteria loss.

Antibiotic use is associated with resistance of environmental organisms in a teaching hospital.

An intensive care unit (ICU), acute stroke unit (ASU) and medical day bed unit (MDBU) underwent a standardized four-month environmental screening programme. The aim was to examine environmental organisms from these wards and compare bacterial resistances in association with antimicrobial usage. Hand-touch and other sites were screened using commercial dip-slides, and staff were asked to provide fingertip cultures. Patient blood isolates were retained throughout the study. Organisms were quantitatively and qualitatively assessed including antimicrobial susceptibility testing. Antibiotic consumption data in defined daily doses/1000 patient-days were obtained for each unit for the previous year. Two hundred and seventy-six staphylococci and 67 Gram-negative bacilli were recovered. Antibiotic resistance was significantly associated with individual wards for staphylococci (P<0.0001) and coliforms (P=0.04), and trends were also demonstrated for other Gram-negative organisms (P=0.06) despite fewer numbers. Antibiotic consumption on the ICU was six-fold higher than on the ASU and MDBU. Associations were found between consumption of selected antibiotic groups and corresponding resistances among staphylococci and Gram-negative bacilli. Antibacterial resistance was the only significant difference between environmental bacteria from different wards, and appeared to reflect prescribing pressure. Visual inspection of a ward may not provide a reliable guide regarding the presence of multi-resistant organisms in the hospital environment or the potential risk of infection. These findings have implications for local antibiotic policies, infection control and cleaning schedules.

Effects of commercial diazinon and imidacloprid on microbial urease activity in soil and sod.

Diazinon [O,O-diethyl O-2-isopropyl-6-methyl(pyrimidine-4-yl) phosphorothioate] and imidacloprid [1-(1-[6-chloro-3-pyridinyl]methyl)-N-nitro-2-imidazolidinimine] are applied to lawns for insect control simultaneously with nitrogenous fertilizers such as urea, but their potential effect on urease activity and nitrogen availability in turfgrass management has not been evaluated. Urease activity in enzyme assays, washed cell assays, and soil slurries was examined as a function of insecticide concentration. Intact cores from field sites were used to assess the effect of insecticide application on urease activity in creeping bentgrass (Agrostis palustris Huds.) and bluegrass (Poa pratensis L.) sod. Bacterial urease from Bacillus pasteurii and plant urease from jack bean [Canavalia ensiformis (L.) DC.] were unaffected by the insecticides. Both insecticides inhibited the growth of Proteus vulgaris, a urease-producing bacterium, but only diazinon significantly reduced urease activity in washed cells; neither insecticide inhibited urease activity in sonicated cells. Neither diazinon nor imidacloprid inhibited urease activity in Woolper soil (fine, mixed, mesic Typic Argiudoll) slurries, but diazinon slightly inhibited urease activity in Maury soil (fine, mixed, semiactive, mesic Typic Paleudalf) slurries. Imidacloprid had no effect on urease activity in creeping bentgrass or bluegrass sod at up to 10 times the commercial application rate. Diazinon briefly, but significantly, reduced urease activity in bluegrass sod. Co-application of imidacloprid and urea appears to be benign with respect to urease activity in soil and sod. Diazinon, in contrast, appears to have a significant, short-term, inhibitory effect on the microbial urease-producing community, but that effect depends on soil type.

Fertilizer, tillage, and dairy manure contributions to nitrate and herbicide leaching.

Few studies have examined the water quality impact of manure use in no-tillage systems. A lysimeter study in continuous corn (Zea mays L.) was performed on Maury silt loam (fine, mixed, semiactive, mesic Typic Paleudalf) to evaluate the effect(s) of tillage (no-till [NT] and chisel-disk [CD]), nitrogen fertilizer rate (0 and 168 kg N ha(-1)), and dairy manure application timing (none, spring, fall, or fall plus spring) on NO3-N, atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), and alachlor [2-chloro-2'-6'-diethyl-N-(methoxymethyl)acetanilide] concentrations in leachate collected at a 90-cm depth. Herbicides were highest immediately after application, declining to less than 4 mug L(-1) in about two months. Manure and manure timing by tillage interactions had little effect on leachate herbicides; rather, the data suggest that macropores rapidly transmitted atrazine and alachlor through the soil. Tillage usually did not significantly affect leachate NO3-N, but no-tillage tended to cause higher NO(3)-N. Manuring caused higher NO3-N concentrations; spring manuring had more impact than fall, but fall manure contained about 78% of the N found in spring manure. Nitrate under spring "only fertilizer" treatment exceeded 10 mg L(-1) 38% of the time, compared with 15% for spring only manure treatment. After three years, manured soil leachate NO3-N exceeded that for soil receiving only N fertilizer. Soil profile (90 cm) NO3-N after corn harvest exceeding 22 kg N ha(-1) was associated with winter leachate NO3-N greater than 10 mg N L(-1). Manure can be used effectively in conservation tillage systems on this and similar soils. Accounting for all N inputs, including previous manure applications, will be important.

Determining nitrogen fractions in swine slurry.

Nutrient management plans are used to reduce non-point source pollution from animal operations. These plans require manure analysis and use indices to determine nutrient availability. This study evaluated a modified method for determining nitrogen fractions in swine slurry stored under slatted floors, calculating plant available nitrogen, and characterizing nitrogen fractions by holding pit depth. Manure samples were collected from gestating sow and finishing pig holding pits at discrete depths. Ammonium, amino acid, and amino sugar concentrations were significantly different for gestation holding pits by depth, but finishing pig holding pit values were not significantly different by depth. Plant available nitrogen was approximately 74% for gestation and 67% for finishing barn manures. Nitrogen fraction analysis suggests land application of swine manure for crop growth should be managed based on type of nitrogen present in the manure, which can be highly correlated to animal growth stage.

Extensive surface diversity of a commensal microorganism by multiple DNA inversions.

The dynamic interactions between a host and its intestinal microflora that lead to commensalism are unclear. Bacteria that colonize the intestinal tract do so despite the development of a specific immune response by the host. The mechanisms used by commensal organisms to circumvent this immune response have yet to be established. Here we demonstrate that the human colonic microorganism, Bacteroides fragilis, is able to modulate its surface antigenicity by producing at least eight distinct capsular polysaccharides-a number greater than any previously reported for a bacterium-and is able to regulate their expression in an on-off manner by the reversible inversion of DNA segments containing the promoters for their expression. This means of generating surface diversity allows the organism to exhibit a wide array of distinct surface polysaccharide combinations, and may have broad implications for how the predominant human colonic microorganisms, the Bacteroides species, maintain an ecological niche in the intestinal tract.

Duration of immunity in dogs after vaccination or naturally acquired infection.

This paper reviews current scientific information about the duration of immunity induced in dogs by infection or vaccination. It describes the shortcomings of the methods used to measure the immune responses of dogs, and explains the need for basic studies on the nature of protective humoral and cellular responses, and standardised assays for the long-term duration of immunity to pathogens other than rabies. The information is inadequate to warrant uniform recommendations on the ideal intervals for vaccination; each vaccine must be evaluated on the basis of its own merits and the characteristics of the disease it is intended to guard against.

Duration of immunity in cats after vaccination or naturally acquired infection.

The necessity for cats to be vaccinated annually against common pathogens has been questioned because sarcomas have infrequently been reported at the injection site. However, with few exceptions, the duration of immunity induced by vaccination or infection is uncertain, and there may therefore be a risk associated with a decision not to revaccinate. This article reviews the information available about the duration of immunity induced by vaccination or infection in cats, and reveals many shortcomings that make blanket recommendations impossible. Each vaccine must be considered individually.

Evaluation of the clinical efficacy of marbofloxacin (Zeniquin) tablets for the treatment of canine pyoderma: an open clinical trial.

The efficacy and field safety of marbofloxacin (Zeniquin) for the treatment of superficial and deep bacterial pyoderma were evaluated. Seventy-two dogs were treated with 2.75 mg kg-1 of marbofloxacin orally once daily for 21 or 28 days. Sixty-two dogs (86%) had superficial pyoderma and 10 (14%) had deep pyoderma. A history of prior pyoderma was reported in 39/72 dogs. Pretreatment aerobic bacteriologic cultures of skin lesions were performed in 47 cases and the predominant pathogen isolated was Staphylococcus intermedius. Treatment was successful in 62/72 (86.1%) dogs, improvement was noted in 6/72 (8.3%) dogs and treatment failed in 4/72 (5.6%) dogs. Adverse effects associated with treatment included listlessness, anorexia, vomiting, soft stool, flatulence and polydipsia; these adverse effects were seen in only 6/81 dogs. Marbofloxacin was safe and effective for the treatment of superficial and deep pyoderma in dogs at the dosage used in this study.

Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis.

Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides-polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.

Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina.

Viral delivery of neurotrophins or other therapeutic genes is an attractive option for treating retinal degeneration. Regulated expression of these genes in the retina is needed to aid in dose delivery and to promote safety. To evaluate whether tetracycline (tet)-inducible transgenes encapsidated in recombinant adeno-associated viruses (rAAV) can provide controlled gene expression in vitro and in the rat retina, two viruses were constructed: a silencer/activator vector and an inducible doxycycline (dox)-responsive GFP vector. Combinations of these two viruses were subretinally injected into wild-type rats and dox was orally administered through the drinking water. Retinal GFP expression was monitored in vivo with a noninvasive fluorescence imaging method. Eyes were also examined by histology, Western analysis, and electroretinography. Subretinal injection of rAAV efficiently delivers inducible genes to both photoreceptors and retinal pigment epithelial cells. GFP expression was initially observed 1 week postinduction, and GFP protein was undetectable after removal of dox. In uninduced animals, GFP expression was negligible. The dox dosage was varied in vivo and showed a correlation to the level of GFP expression. Thus, transduction of retinal cells with tet-inducible vectors allows for tight regulation of gene expression.