Development of an assay to determine single nucleotide polymorphisms in the prion gene for the genetic diagnosis of relative susceptibility to classical scrapie in sheep.

The objective of this study was to develop a reliable Taqman 5' Nuclease Assay for genotyping sheep for scrapie susceptibility. The sheep prion gene contains 2 single nucleotide polymorphisms (SNPs) that may mediate resistance to classical scrapie, one at codon 136, alanine (A) or valine (V), and another at codon 171, arginine (R) or glutamine (Q). The R allele appears to confer resistance to classical scrapie, with the AA(136) RR(171) genotype the most resistant to scrapie and QR(171) only rarely infected in the US sheep population. The Assays by Design protocol was used for development of probes and primers for codon 136 and Primer Express for codon 171. Commercially available kits were used to isolate genomic DNA from blood or muscle. For validation, 70 SNP determinations for each codon were compared to commercial testing with an error rate of less than 1%. Then, 935 samples from blood (n = 818) and muscle (n = 117) were tested for both codons with 928 successful determinations and only 7 samples (<1% of total samples) that needed repeating. Genotypes were AA QQ (n = 102; 11.0%), AV QQ (n = 28; 3.0%), AA QR (n = 396; 42.7%), AV QR (n = 54; 5.8%), and AA RR (n = 348; 37.5%). Thus, 86% of the sheep tested (n = 798) contained R at codon 171 and were expected to be scrapie-resistant. This new Taqman 5' Nuclease SNP genotyping assay is accurate, easy to perform, and useful in the study of classical scrapie in sheep and its prevention through selective breeding programs to eliminate highly susceptible animals.     

Microchip-associated leiomyosarcoma in an Egyptian fruit bat (Rousettus aegyptiacus).

Microchips are commonly used in companion, research, and zoo animal medicine for easy, reliable, and cost-effective identification with relatively low risk of side effects. This report describes development of a metastatic leiomyosarcoma associated with a microchip in an Egyptian fruit bat (Rousettus aegyptiacus).     

The Impact of Egg Ozonation on Hatching Success,Larval Growth,and Survival of Atlantic Cod,Atlantic Salmon,and Rainbow Trout

The direct exposure of fish eggs to ozonated water has generated interest as a means of ensuring pathogen-free eggs without the use of harsh chemicals. However, there are numerous knowledge gaps, including safe contact times, exposure levels, and potential long-term effects on aquaculture species in both freshwater and seawater. The effect of different ozone (O₃) doses (0.5–1.0, 1.5–2.0, and 2.5–3.0 mg of O₃/L for 90 s) on recently fertilized eggs of Atlantic Cod Gadus morhua and eyed eggs of Atlantic Salmon Salmo salar and Rainbow Trout Oncorhynchus mykiss was evaluated in comparison with the effects of two commercial disinfectants: Perosan (0.004 mg/L) and Ovadine (100 mg/L). The impact of ozone application was evaluated based on hatching success, larval nucleic acid concentration, larval growth, and survival. Overall, results indicated that ozonation of Atlantic Cod eggs at a dose less than 3.0 mg/L for 90 s produced no negative effect on the larvae up to 30 d posthatch. Furthermore, ozonation of Atlantic Salmon and Rainbow Trout eggs generated no negative effect on the larvae, based on monitoring until 85% yolk sac re-absorption (16 d posthatch).

Received May 6, 2014; accepted October 24, 2014     

Evolution of antioxidant capacity during storage of selected fruits and vegetables

Interest in the consumption of fresh fruits and vegetables is, to a large extent, due to its content of bioactive nutrients and their importance as dietary antioxidants. Among all of the selected fruits and vegetables, strawberries and black grapes have relatively high antioxidant capacities associated with high contents of total phenolic compounds, ascorbic acid, and flavonols. More interesting, the results of this study indicated that in most fruits and vegetables storage did not affect negatively the antioxidant capacity. Better, in some cases, an increase of the antioxidant capacity was observed in the days following their purchase, accompanied by an increase in phenolic compounds. In general, fruits and vegetables visually spoil before any significant antioxidant capacity loss occurs except in banana and broccoli. When ascorbic acid or flavonoids (aglycons of flavonols and anthocyanins) were concerned, the conclusions were similar. Their content was generally stable during storage.     

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49 μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.     

Proteomic Characterization of Equine Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a biofluid that is reflective of overall health. Although proteomic profiling of human CSF has been performed in the context of a variety of disease states, this report represents the first comprehensive proteomic analysis of equine CSF. A total of 320 proteins were confidently identified across six healthy horses, and these proteins were further characterized by gene ontology terms mapped in UniProt, and normalized spectral abundance factors were calculated as a measure of relative abundance. Theses results provide an optimized protocol for analysis of equine CSF and lay the groundwork for future studies involving the study of equine CSF in the context of pathogenic disease states.     

Microbial Composition before and after Conservation of Grass-Dominated Haylage Harvested Early,Middle, and Late in the Season

Haylage for horses is often harvested in late plant maturity, which could be associated with an increased risk of impaired hygienic quality in the forage and short aerobic storage stability after bale opening, but knowledge in this area is scant. An experiment was conducted in which the microbial composition was analyzed before and after conservation of primary growth haylage harvested early (June), middle (July), and late (August) in the season during 1 year. The counts of yeast, enterobacteria, and lactic acid bacteria (LAB) in preconserved herbage increased with the advancing harvest time (P ≤ .02). After conservation, the August haylage had increased counts of enterobacteria (log 4.3 colony-forming unit [CFU]/g) and LAB (log 6.5 CFU/g), compared with the June and July haylage (log ≤1.7 CFU/g for enterobacteria and ≤5.7 CFU/g for LAB, P < .001). The yeast counts were the lowest in the June haylage (log 5.0 CFU/g) compared with the July and August haylage (log ≥6.3 CFU/g, P < .001). After conservation, the mold counts were lower in the June haylage and greater in the August haylage (P = .01). In the preconserved herbage, Cladosporium cladosporioides was the most common mold species in June but Fusarium poae was in July, and Mucor fragilis in August. After conservation, Penicillium carneum was the only species found in the June haylage, with M. circinelloides most frequently found in the July haylage and M. hiemalis and M. circinelloides found at similar frequencies in the August haylage. An advanced harvest time resulted in greater counts of enterobacteria, yeast, and LAB and an increased number of mold species in the conserved haylage. The aerobic storage stability of the opened haylage bales measured by temperature was similar among the harvests.     

Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells

Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer.     

US strategy for forest management adaptation to climate change: building a framework for decision making

Context

Recent policy changes in the USA direct agencies managing federal forests to analyze the potential effects of climate change on forest productivity, water resource protection, wildlife habitat, biodiversity, and other values.

Aims

This paper describes methods developed to (1) assess current risks, vulnerabilities, and gaps in knowledge; (2) engage internal agency resources and external partners in the development of options and solutions; and (3) manage forest resources for resilience, not just in terms of natural ecosystems but in affected human communities as well.

Methods

We describe an approach designed to characterize certain climate change effects on forests, and estimate the effectiveness of response options ranging from resistance to a realignment of management objectives.

Results

Field testing on a 6,300 km² area of conifer forest in the northwestern USA shows this decision model to be useful and cost-effective in identifying the highest sensitivities relating to vegetation management, biological diversity, water resources and forest transportation systems, and building consensus for adaptive strategies and actions.

Conclusions

Results suggest that this approach is an effective means for guiding management decisions to adapt to the effects of climate change, and provides an empirical basis for setting budgetary and management priorities.     

Feasibility and repeatability of cold and mechanical quantitative sensory testing in normal dogs

Feasibility and inter-session repeatability of cold and mechanical quantitative sensory testing (QST) were assessed in 24 normal dogs. Cold thermal latencies were evaluated using a thermal probe (0 °C) applied to three pelvic limb sites. Mechanical thresholds were measured using an electronic von Frey anesthesiometer (EVF) and a blunt-probed pressure algometer (PA) applied to the dorsal aspect of the metatarsus. All QST trials were performed with dogs in lateral recumbency. Collection of cold QST data was easy (feasible) in 19/24 (79%) dogs. However, only 18.4%, 18.9% and 13.2% of cold QST trials elicited a response at the medial tibia, third digital pad and plantar metatarsal regions, respectively. Collection of mechanical QST data was easy (feasible) in 20/24 (83%) dogs for both EVF and PA.At consecutive sampling times, approximately 2 weeks apart, the average EVF sensory thresholds were 414 ± 186 g and 379 ± 166 g, respectively, and the average PA sensory thresholds were 1089 ± 414 g and 1028 ± 331 g, respectively. There was no significant difference in inter-session or inter-limb threshold values for either mechanical QST device. The cold QST protocol in this study was achievable, but did not provide consistently quantifiable results. Both mechanical QST devices tested provided repeatable, reliable sensory threshold measurements in normal, client-owned dogs. These findings contribute to the validation of the EVF and PA as tools to obtain repeated QST data over time in dogs to assess somatosensory processing changes.