Detection of castor contamination by real-time polymerase chain reaction

Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay. The detection limit for castor contamination in ground beef samples was <0.001% (<10 microg of castor acetone powder per gram of beef, corresponding to 0.5 microg of ricin), indicating excellent sensitivity for the assay, well below the threshold for oral toxicity.     

Insoluble fraction of buckwheat (Fagopyrum esculentum Moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by Caco-2 cells

Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.     

An investigation of avoidance by Antarctic krill of RRS James Clark Ross using the Autosub-2 autonomous underwater vehicle

The autonomous underwater vehicle (AUV) Autosub-2 was deployed on eight missions ahead of RRS James Clark Ross in the northern Weddell Sea and in the Bransfield Strait, Southern Ocean, to assess avoidance of the research vessel by Antarctic krill Euphausia superba. The AUV was equipped with the same type of scientific echosounder as the research vessel (Simrad EK500 operating at 38 and 120 kHz) and measured the density of krill along transect acoustically (g m⁻² wet mass) prior to the ship’s arrival. We hypothesised that if krill avoided the ship, perhaps in response to radiated noise, then the ship should detect less krill than the AUV which is known to have much lower noise levels than the ship. We were unable to detect any significant difference between the density of krill detected by the ship or the AUV, either at the transect level or at finer scales within transects. We conclude, therefore, that avoidance by krill of RRS James Clark Ross will not significantly bias acoustic estimates of krill abundance by this vessel.     

Primary Production in Antarctic Sea Ice

A numerical model shows that in Antarctic sea ice, increased flooding in regions with thick snow cover enhances primary production in the infiltration (surface) layer. Productivity in the freeboard (sea level) layer is also determined by sea ice porosity, which varies with temperature. Spatial and temporal variation in snow thickness and the proportion of first-year ice thus determine regional differences in sea ice primary production. Model results show that of the 40 teragrams of carbon produced annually in the Antarctic ice pack, 75 percent was associated with first-year ice and nearly 50 percent was produced in the Weddell Sea.     

Analysis of fenbendazole residues in bovine milk by ELISA

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.     

Implications of intraguild predation for sea turtle nest protection

In the United States, raccoons Procyon lotor are often removed from sea turtle nesting beaches to decrease egg mortality. However, raccoons also consume ghost crabs Ocypode quadrata, another common egg predator. Reducing predator populations can benefit secondary predators, inflating total predation pressure and leading to a decline in prey species. We used track and burrow counts to compare raccoon and ghost crab abundance at four beaches in Florida, USA, that differ in management activity and determined predation rates on loggerhead Caretta caretta nests by each predator. Mean raccoon abundance (range 0.12-0.46 tracks plot⁻¹ night⁻¹) and ghost crab density (0.09-0.19 burrows m⁻²) were inversely correlated. Ghost crabs were largest at the site with the fewest raccoons. The stable nitrogen isotope ratios of ghost crabs (mean 9.8‰) were positively correlated with body mass, indicating larger ghost crabs feed at a higher trophic level and suggesting large ghost crabs may consume more loggerhead eggs. The highest rates of egg predation by both predators (31%) occurred where raccoon abundance was lowest and ghost crab abundance was highest, suggesting ghost crab burrows may facilitate predation by raccoons. Our data suggest that predation by raccoons limits ghost crabs and that removing raccoons can increase ghost crab abundance and sea turtle egg mortality. Although predator removal can be effective when nest predation rates are quite high, maintaining moderate raccoon densities may be important for controlling ghost crabs. These results highlight the importance of understanding food web connectivity in developing management strategies to achieve conservation goals, especially when the species of concern are threatened or facing extinction.     

A functional and biochemical analysis of bovine class II MHC antigens using monoclonal antibodies

Monoclonal antibodies (MAbs) reacting with bovine (2) ovine (3), murine (1) or human (1) Class II MHC antigens were examined for reactivity with bovine peripheral blood leucocytes (PBL) and lymph node cells (LNC) by immunofluorescence, immunoprecipitation and the capacity to inhibit mixed lymphocyte responses (MLR), lectin- and antigen-induced blastogenesis. The 6 MAbs identified comparable percentages of Class II positive lymphocytes in PBL (40.8 to 54.2%) and LNC (6 to 11.5%) regardless of BoLA-A phenotype. Immunohistological staining of Class II MAb was localized principally to the lymphoid follicles in lymph nodes and to isolated epithelial reticular cells in the thymus. The anti-Class II MAb immunoprecipitated alpha- and beta- chains of 26-29K and 32-34K, respectively. These MAb inhibited proliferative responses in the MLR by between 25 and 74%, and diminished blastogenesis induced by specific antigens (purified protein derivative + PPD and ovalbumin) and B-lymphocyte mitogens (PPD, lipopolysaccharide and dextran sulphate) by between 45 and 75%, regardless of BoLA-A phenotype. In contrast, proliferation in response to concanavalin A and phytohaemagglutinin were unaffected by the anti- Class II MAb. Similarly these MAb did not affect lysis by cytotoxic T-lymphocytes, the activity of which was depressed by anti-Class I MAbs and monospecific alloantisera.     

Dexamethasone and prednisolone in the horse: pharmacokinetics and action on the adrenal gland

Pharmacokinetics of dexamethasone and prednisolone were studied in 6 horses given dexamethasone alcohol (IV or IM) or dexamethasone 21-isonicotinate as a solution IV or IM (50 micrograms/kg of body weight), prednisolone 21-sodium succinate IV or IM (0.6 mg/kg of body weight), or prednisolone acetate IM (0.6 mg/kg of body weight). Plasma concentrations were determined using a high-performance liquid chromatographic method. After dexamethasone alcohol (IV) or dexamethasone 21-isonicotinate (IV), the half-life of elimination was similar (53 minutes) for both formulations. After dexamethasone (alcohol and isonicotinate, IM), concentrations were low or nondetected. After prednisolone 21-sodium succinate (IV), the half-life of elimination (99.5 minutes) was significantly (P less than 0.01) longer than that for dexamethasone. After prednisolone 21-sodium succinate (IM), absorption was rapid and bioavailability was high. After prednisolone acetate (IM), absorption was slow and prednisolone was present in plasma for about 7 days. Due to the nonlinearity of prednisolone kinetics, a bioavailability higher than 100% was obtained. The basal plasma hydrocortisone concentration was approximately 70 ng/ml. After dexamethasone (IV or IM), plasma hydrocortisone values decreased after a 2-hour delay and returned to base line after a 3 to 4 day delay. After prednisolone 21-sodium succinate (IV or IM), plasma hydrocortisone decreased immediately (IV) or rapidly (IM) and returned to base line after a 24-hour delay. After prednisolone acetate (IM), plasma hydrocortisone decreased for up to 21 days.