Evaluation of the characteristics of venous occlusion after placement of an ameroid constrictor in dogs

OBJECTIVES: To evaluate the mechanism of vascular occlusion after placement of an ameroid constrictor (AC) on a large intra-abdominal vein and document changes in blood flow. STUDY DESIGN: Experimental study. ANIMALS: Six adult dogs. METHODS: Six 6.5 mm ACs were digitally scanned to measure area and circumference of the ameroid and inner lumen before surgery. ACs were surgically positioned around the left common iliac vein (CIV) in each dog. Peri-vascular ultrasonic flow probes were positioned on the left CIV cranial to the AC, and on the right CIV as an internal control. Blood flow measurements were recorded daily until there was no, or prolonged minimal, blood flow, at which time the dogs were euthanatized. Left and right CIVs were removed for histologic evaluation, and the ACs re-scanned to evaluate degree and direction of expansion of the ameroid. RESULTS: Reduction in blood flow occurred within 10 days in all dogs. Three dogs had complete occlusion by day 10 from thrombus formation. One dog damaged the flow probes at 13 days when blood flow was approximately 50% of intra-operative values; the dog was euthanatized at 37 days; there was partial occlusion from thrombosis. Two dogs had persistent low blood flow and occlusion from thrombus formation when euthanatized at 59 and 98 days. There was a significant increase in total AC area and outer circumference, and decrease in luminal area and inner circumference when presurgical and postocclusion measurements were compared. CONCLUSIONS: Vascular occlusion after placement of an AC on a large intra-abdominal vein occurred from thrombus formation. CLINICAL RELEVANCE: A similar pattern of venous occlusion may occur after placement of an AC on a portosystemic shunt, which may lead to both short- and long-term complications associated with portal hypertension.     

Low biodegradability of fluoxetine HCl,diazepam and their human metabolites in sewage sludge-amended soil

Background, aim, and scope

The European Union banned disposal of sewage sludge (SS) at sea in Europe in 1998. Since that time, the application rate of SS to land has risen significantly and is set to rise further. Fifty-two percent of SS was disposed to land in the UK in 2000. Land application is, thus, possibly an important transport route for SS-associated organic chemicals into the environment. There are now over 3,000 different pharmaceutical ingredients in use in the EU and many enter sewage systems. Possibly as a result, the last decade has seen an increase in reports of pharmacologically active compounds in the environment (e.g. in watercourses, open ocean and soils). Surprisingly, there is still a significant lack of knowledge of the transport and fate of pharmaceuticals in the environment, particularly in soils. The present project, therefore, investigated the susceptibility to microbial degradation of the selective serotonin re-uptake inhibitor, Prozac® (fluoxetine HCl), and the 1,4-benzodiazepine, Valium® (Diazepam) and their major human metabolites (norfluoxetine HCl, temazepam and oxazepam) in short-term (60 day) bacterial liquid cultures derived from UK SS-amended soil and of fluoxetine HCl in a longer-term (270 day) SS-amended soil culture.

Methodology

Recently developed extraction techniques, including solid phase extraction, allowed all analytes to be isolated from the biodegradation cultures (aqueous and soil matrices), and subsequently analysed using novel high performance liquid chromatography–electrospray ionization-multistage mass spectrometry (HPLC-ESI-MSⁿ) techniques. Ratio calibration using deuterated internal standards allowed the generation of quantitative data. A simple basified tautomerism experiment was also performed to aid in the identification of a bacterial transformation product. Alongside the biodegradation studies, HPLC-APCI-MSⁿ profiling of bacteriohopanepolyols (BHPs; bacterial membrane marker chemicals) allowed assessment of the microbial community structure in the SS-amended soils.

Results

The pharmaceuticals were found to be resistant to biodegradation in liquid culture studies (60 days), and even after prolonged exposure in SS-amended soil (>200 days; fluoxetine HCl only). Oxazepam was the only 1,4-benzodiazepine studied which underwent biotic transformation (~40%) in liquid culture studies. Evidence to support the theory that the transformation product was a 1,4-benzodiazepine tautomer, is presented. BHP profiles of eight different SS-amended soils suggested that the restricted bacterial community of the soil used as a culture source in these biodegradation studies was typical of SS-amended soils.

Discussion

The lack of substantial degradation of all target analytes except oxazepam under simulated, but realistic, SS-amended soil conditions indicates their likely persistent nature. Although oxazepam did undergo significant biotic (and abiotic) losses, the metabolite formed under biotic influences was hypothesised to be another bioactive 1,4-benzodiazepine (either 2-enol or 3-enol nordiazepam) which is likely to be resistant to further change. The bacterial communities in SS-amended soils may be unable to degrade such compounds.

Recommendations and perspectives

With such pharmaceuticals as tested here, there is a potential for accumulation within environments such as field soil to which SS is regularly added both as a disposal mechanism and as a fertiliser. When compounds undergo accumulation, the risk of transport to other environmental components becomes more likely. From field soils, these may include potential exposure to flora and fauna and possible bioaccumulation or effects on terrestrial organisms and accumulation in plants, including crops grown on the SS-treated soils. The fate and subsequent impact of many pharmaceuticals within the terrestrial environment is still largely unknown and further research is required before the risks, if any, of SS-associated pharmaceuticals can be fully assessed.

     

Taxon-specific responses of soil bacteria to the addition of low level C inputs

The addition of small or trace amounts of carbon to soils can result in the release of 2-5 times more C as CO₂ than was added in the original solution. The identity of the microorganisms responsible for these so-called trigger effects remains largely unknown. This paper reports on the response of individual bacterial taxa to the addition of a range of ¹⁴C-glucose concentrations (150, 50 and 15 and 0 μg C g⁻¹ soil) similar to the low levels of labile C found in soil. Taxon-specific responses were identified using a modification of the stable isotope probing (SIP) protocol and the recovery of [¹⁴C] labelled ribosomal RNA using equilibrium density gradient centrifugation. This provided good resolution of the ‘heavy’ fractions ([¹⁴C] labelled RNA) from the ‘light’ fractions ([¹²C] unlabelled RNA). The extent of the separation was verified using autoradiography. The addition of [¹⁴C] glucose at all concentrations was characterised by changes in the relative intensity of particular bands. Canonical correspondence analysis (CCA) showed that the rRNA response in both the ‘heavy’ and ‘light’ fractions differed according to the concentration of glucose added but was most pronounced in soils amended with 150 μg C g⁻¹ soil. In the ‘heavy RNA’ fractions there was a clear separation between soils amended with 150 μg C g⁻¹ soil and those receiving 50 and 15 μg C g⁻¹ soil indicating that at low C inputs the microbial community response is quite distinct from that seen at higher concentrations. To investigate these differences further, bands that changed in relative intensity following amendment were excised from the DGGE gels, reamplified and sequenced. Sequence analysis identified 8 taxa that responded to glucose amendment (Bacillus, Pseudomonas, Burkholderia, Bradyrhizobium, Actinobacteria, Nitrosomonas, Acidobacteria and an uncultured β-proteobacteria). These results show that radioisotope probing (RNA-RIP) can be used successfully to study the fate of labile C substrates, such as glucose, in soil.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Reliability of indicators of sheep welfare assessed by a group observation method

Assessments of animal-based outcomes form the core of routine veterinary clinical examinations and are being increasingly used as indicators of animal welfare. A method of group observation that did not require gathering and handling of individual sheep, was used to assess eight animal-based indicators of sheep welfare: demeanour, skin irritation, wool loss, excessive panting, coughing, lameness, and cleanliness of the ventral abdominal and 'breech' (perineum/gluteal/caudal hindlimb) areas. The inter-observer reliability of two or three observers who independently assessed these indicators was tested on 2406 adult sheep and growing lambs across 36 farms and the intra-observer reliability of an experienced, veterinary assessor--the 'test standard observer'--was assessed on 88 adult sheep during four on-farm assessments. Observer reliability was evaluated using Cronbach's alpha (α) comparison of the recorded proportion (%) of sheep affected by each welfare condition and by binomial logistic regression modelling. High levels of inter-observer reliability were identified for the assessment of group lameness (α 0.76-1.00) and cleanliness of the breech area (α 0.97-1.00). Excellent intra-observer reliability was determined for lameness (α 0.99), cleanliness of the breech area (α 0.97), demeanour (α 1.00) and wool loss (α 1.00). In addition, proportion data and logistic regression models identified few between-observer differences. The results suggest that welfare outcomes based on observations of the behaviour and physical appearance of individual animals within a group may offer a reliable and feasible measurement tool for the on-farm assessment of sheep welfare.     

Application of the Enfer chemiluminescent multiplex ELISA system for the detection of Mycobacterium bovis infection in goats

A study was conducted to optimise a multiplex serological immunoassay for use in identification of goats infected with Mycobacterium bovis. To assess assay specificity, 31 goats with a history of being free from M. bovis infection were used. To determine assay sensitivity, 180 Single Intradermal Comparative Tuberculin test (SICTT) positive goats were recruited. Additionally, 286 SICTT negative goats classed as potentially exposed animals present in the same positive herds were also included in the study. The results of the assay demonstrated a specificity of 100%. The multiplex assay detected 57/60 SICTT (95.0%) positive animals in one M. bovis infected herd and 120/120 (100%) in a second herd. In a separate experiment, 28 M. caprae culture confirmed infected goats from Spain were assayed, of which 24 (85.7%) were found positive in the test. The results show that inclusion of an antibody based assay can improve the ability to identify M. bovis and M. caprae infected goats. With further development and validation the multiplex assay may prove to be a useful tool for control of M. bovis and M. caprae infection in goats.     

Efficiently locating conservation boundaries: Searching for the Tasmanian devil facial tumour disease front

Conservation management actions and decisions are often defined by the location of ecological boundaries, for example, the present range of invasive or threatened species. The position of these boundaries can be cryptic, and managers must therefore directly sample sites, an expensive and time-consuming process. While accurate boundary location techniques have been considered by ecological theorists, the issue of cost-effective, or optimal boundary location has not. We propose a general framework for boundary location which incorporates both cost-efficiency and uncertainty. To illustrate its application, we use it to help locate an infectious disease front in the endangered Tasmanian devil population. The method ensures optimal spatial sampling by maximizing the expected information gained from each sample. When resources are limited, our method provides more accurate estimates of the boundary location than traditional sampling protocols. Using a formal decision theory sampling design encourages economically efficient actions, and provides defensible and transparent rationale for management actions.     

Electrodes with high power and high capacity for rechargeable lithium batteries

New applications such as hybrid electric vehicles and power backup require rechargeable batteries that combine high energy density with high charge and discharge rate capability. Using ab initio computational modeling, we identified useful strategies to design higher rate battery electrodes and tested them on lithium nickel manganese oxide [Li(Ni(0.5)Mn(0.5))O2], a safe, inexpensive material that has been thought to have poor intrinsic rate capability. By modifying its crystal structure, we obtained unexpectedly high rate-capability, considerably better than lithium cobalt oxide (LiCoO2), the current battery electrode material of choice.     

In vitro evaluation of the effect of a novel immunosuppressive agent, FTY720, on the function of feline neutrophils

OBJECTIVE: To use in vitro assays to evaluate the effects of a novel immunosuppressive agent, FTY720, on biological functions (migration, phagocytosis, and production of reactive-oxygen species [ROS]) of feline peripheral neutrophils and determine the cytotoxic effects of FTY720 on feline peripheral neutrophils. SAMPLE POPULATION: Peripheral neutrophils obtained from 8 healthy cats. PROCEDURE: Peripheral neutrophils were isolated from blood samples obtained from the 8 cats and exposed to the phosphorylated form of FTY720 (FTY720-P). A fluorescence-based in vitro evaluation of migration was performed. Phagocytosis of microbes and production of ROS were evaluated by use of a 2-color flow cytometry system. Samples of whole blood obtained from the cats were incubated with various concentrations of FTY720-P, fluorescein-labeled Staphylococcus aureus, and dihydroethidium. Cytotoxic effects were evaluated by use of propidium iodide staining. RESULTS: Addition of FTY720-P caused a slight non-significant decrease in phagocytosis and production of ROS by feline peripheral neutrophils. Migration activity of feline peripheral neutrophils was significantly increased by the addition of FTY720-P. Addition of FTY720-P at concentrations considered for clinical use did not increase the death rate of feline peripheral neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: FTY720 does not inhibit critical functions of feline peripheral neutrophils in vitro.