Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection

The objectives of these studies were to assess the diagnostic performance (sensitivity and specificity) of the IgM capture enzyme-linked immunosorbent assay (ELISA; MAC) for diagnosis of West Nile (WN) virus in horses and to examine the performance of this test by using different criteria for seropositivity. A total of 36 horses classified as WN virus infected (group 1) and 383 horses from 4 subpopulations of hoses classified as noninfected (groups 2, 3, 4, and 5) were used in the study. The sensitivity (proportion of infected horses that tested positive for WN virus IgM antibodies) and specificity (proportion of noninfected horses that tested negative) were calculated at different cutoff points by using receiver operating curve (ROC) analysis. Using a selected cutoff point = 2.0, the sensitivity and specificity of the MAC were 91.7 and 99.2%, respectively. The area under the ROC curve = 0.95 (95% confidence interval [CI], 0.89 to 1.0), suggesting that the MAC is a useful tool for diagnosis of recent WN virus exposure in horses. In fulfillment of the 2nd objective, 2 other indices were developed and these indices approached 1.0 for the AUC with smaller 95% CIs. These indices were then used to test 602 additional diagnostic samples submitted from suspect horses between 2002 and 2004. Using the standard cutoff, 194 (32%) of the horses were interpreted as positive. Utilizing newly predicted cutoff criteria from each index, additional horses were identified as positive. In conclusion, the MAC as used for identification of WN virus-diseased horses undergoing recent exposure performs reliably at the standard cutoff for seropositivity. A negative test might not completely rule out WN virus disease, but horses that test negative were most likely not exposed to WNV. Performance of the test can be further improved by investigation of other indexes of seropositivity.     

Biological and Chemical Approaches for the Detection and Identification of Illegal Estrogens in Water-based Solutions

The continuous introduction of new products used as growth promoters in animal husbandry, for sports doping and as products for body-building requires residue laboratories to initiate research on developing a strategy for the identification of ’unknown’ components. In this study, a strategy is presented for elucidating the identity, the structure and the possible effects of illegal estrogenic compounds in an unidentified water-based solution. To obtain complete information on the composition and activity of the unidentified product, a multidisciplinary approach was needed. A case-study is described with a ’solution X’ found during a raid. First, in vivo techniques (animal trials with mice, anatomical and histological research) were combined with in vitro techniques (the yeast estrogenic screen (YES)). In a later stage of the investigation, HPLC-fractionation, liquid chromatography–multiple mass spectrometry (LC-MS ⁿ and gas chromatography-multiple mass spectrometry (GC-MS ⁿ were used. Finally, the identity of ’solution X’ was confirmed in a very low concentration range (10 ng/L estrone and 400 ng/l ethinyloestradiol).     

Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post-thaw Incubation in Capacitating Conditions

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca²⁺ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca²⁺ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca²⁺ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca²⁺ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.     

Orthotropic elastic constants of wood

Wood tracheids are essentially tubular structures but wood cross sections are characterized by large numbers of triple points or junctures of wall segments from three adjacent cells. A symmetric triple point is taken as an approximation to the basic unit of wood structure. This element is analysed as a linearly elastic, isotropic body. It is shown that bending effects enhance the deformations arising from simple strains so that the overall response of the element is anisotropic. The resulting stiffnesses are ordered $$E_L \user2{ > }E_R \user2{ > }G_{LR} \sim G_{LT} \user2{ > }E_T \user2{ > }G_{RT} $$ for what are considered to be fairly typical element geometries. It is shown that for all geometries the longitudinal Youngs modulus is proportional to the volume fraction of cell wall material.     

Gold nanoelectrodes of varied size: transition to molecule-like charging

A transition from metal-like double-layer capacitive charging to redox-like charging was observed in electrochemical ensemble Coulomb staircase experiments on solutions of gold nanoparticles of varied core size. The monodisperse gold nanoparticles are stabilized by short-chain alkanethiolate monolayers and have 8 to 38 kilodaltons core mass (1.1 to 1.9 nanometers in diameter). Larger cores display Coulomb staircase responses consistent with double-layer charging of metal-electrolyte interfaces, whereas smaller core nanoparticles exhibit redox chemical character, including a large central gap. The change in behavior is consistent with new near-infrared spectroscopic data showing an emerging gap between the highest occupied and lowest unoccupied orbitals of 0.4 to 0.9 electron volt.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.     

Serratia marcescens associated with bell pepper (Capsicum annuum L.) soft-rot disease under greenhouse conditions

Soft-rotting bacteria affecting bell peppers crops represent an economically destructive disease of growing importance worldwide. In Venezuela since 2006, soft-rot symptoms have been occasionally observed in bell pepper fruits grown under greenhouse conditions. Affected fruits presented water-soaked lesions that progressed to complete fruit maceration. Bacteria were isolated from water-soaked lesions in order to identify the causal agent of this disease. Of 13 bacterial isolates recovered from affected fruits, only isolate AGPim1A was able to produce a hypersensitive reaction in tobacco plants and to reproduce soft-rot symptoms in bell peppers fruits. Several methods, including classical bacteriological tests and carbon utilization profiling, alongside with sequence analysis of 16S rRNA and housekeeping genes gyrB and groES-groEL, allowed identification of the soft-rotting bacterium as Serratia marcescens. To our knowledge, this is the first report showing a S. marcescens strain associated with soft-rot disease in bell pepper fruits.     

Forest structure,habitat and carbon benefits from thinning floodplain forests: Managing early stand density makes a difference

Forest ecosystems are increasingly expected to produce multiple goods and services, such as timber, biodiversity, water flows, and sequestered carbon. While many of these are not mutually exclusive, they cannot all be simultaneously maximised so that management compromise is inevitable. We used a 42-year dataset from a naturally regenerating floodplain forest of the river red gum (Eucalyptus camaldulensis) to investigate the effects of pre-commercial thinning on long-term patterns in habitat quality, forest structure and rates of carbon storage (i.e. standing aboveground carbon). Estimates of habitat quality were based on the density of hollow-bearing trees because hollows are ecologically important to many species of vertebrates and invertebrates in these forests. Thinning improved habitat value by producing 20 (±8) hollow-bearing trees per ha after 42 years, while the unthinned treatment produced none. Unthinned (highest density) stands were dominated by many slender trees, mostly <25 cm in diameter, whereas thinned stands produced negatively skewed size distributions with higher median and maximum stem diameters. Moderately thinned stands (560 trees ha⁻¹) had the highest aboveground carbon storage rate (4.1 t C year⁻¹) and the highest aboveground carbon stocks (200.2 ± 9.6 t C ha⁻¹) after 42 years, while the unthinned treatment had the lowest carbon storage rate (1.6 t C year⁻¹) and an intermediate level of aboveground standing carbon (165.1 ± 31.1 t C ha⁻¹). Our results highlight the importance of early stand density as a determinant of long-term forest structure, habitat quality and carbon storage rates. We recommend that thinning be considered as one component of a broader strategy for enhancing the structure, habitat value and aboveground carbon storage of developing floodplain forests.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.