Establishing a reproducible method for the culture of primary equine corneal cells

Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.     

Reducing insecticide and fungicide loads in runoff from plastic mulch with vegetative-covered furrows

A common management practice for the production of fresh-market vegetables utilizes polyethylene (plastic) mulch because it increases soil temperature, decreases weed pressure, maintains soil moisture, and minimizes soil contact with the product. However, rain events afford much more erosion and runoff because 50-75% of the field is covered with an impervious surface. A plot study was conducted to compare and to quantify the off-site movement of soil, insecticides, and fungicides associated with runoff from plots planted with Sunbeam tomatoes (Lycopersicon esculentum Mill) using the conventional polyethylene mulch management practice vs an alternative management practice-polyethylene mulch-covered beds with cereal rye (Secale cereale) planted in the furrows between the beds. The use of cereal rye-covered furrows with the conventional polyethylene system decreased runoff volume by more than 40%, soil erosion by more than 80%, and pesticide loads by 48-74%. Results indicate that vegetative furrows are critical to minimizing the negative aspects of this management practice.     

Scope for growth of Texas Bay scallop Argopecten irradians amplicostatus

Bivalves have exceptional potential as sentinel organisms because their sedentary benthic lifestyle and ability to pump large volumes of water in short periods of time results in bioaccumulation of pollutants and toxins present in the water column. Scope for Growth (SFG) is an instantaneous measure of production that ranges from maximum positive values under optimal conditions to negative valves when the organism is stressed and utilizing its body reserves for maintenance. We determined baseline SFG for the Texas bay scallop Argopecten irradians amplicostatus so that this species might be used to determine sublethal effects of toxins, contaminants, or other environmental stressors in light of recent harmful algal bloom events in the Gulf of Mexico. We also quantified the effects of different diets (phytoplankton, zooplankton, mixed phytoplankton/zooplankton) on Texas bay scallop SFG. SFG of starved animals was negative, indicating that body reserves were used for maintenance. SFG varied significantly (P < 0.001) by diet, with rotifers yielding the highest value (mean = 14.88 Joules hr^?1g dry weight, SE = 0.72). We determined that the Texas bay scallop has presently unrecognized potential as sentinel organisms to define effects of various environmental and anthropogenic perturbations.     

Computed tomographic contrast tenography of the digital flexor tendon sheath of the equine hindlimb

Pre‐surgical investigation of digital flexor tendon sheath pathology remains challenging with current standard imaging techniques. The aim of this prospective, anatomical, pilot study was to describe the anatomy of the equine hind limb digital flexor tendon sheath using a combination of computed tomography (CT) and computed tomographic contrast tenography in clinically normal cadaver limbs. Ten pairs of hind limbs with no external abnormalities were examined from the level of the tarsometatarsal joint distally. Limbs initially underwent non‐contrast CT examination using 120 kVp, 300 mAs, and 1.5 mm slice thickness. Sixty millilitres of ioversol iodinated contrast media and saline (final concentration 100 mg/ml) were injected using a basilar sesamoidean approach. The computed tomographic contrast tenography examination was then repeated, before dissection of the specimens to compare gross and imaging findings. The combined CT and computed tomographic contrast tenography examinations provided excellent anatomical detail of intra‐thecal structures. The borders of the superficial and deep digital flexor tendons, and the manica flexoria were consistently identifiable in all limbs. Detailed anatomy including that of the mesotenons, two of which are previously undescribed, and the plantar annular ligament were also consistently identifiable. Dissection of all 10 pairs of limbs revealed there to be no pathology, in accordance with the imaging findings. In conclusion, the combination of CT and computed tomographic contrast tenography may be useful adjunctive diagnostic techniques to define digital flexor tendon sheath pathology prior to surgical exploration in horses.     

Exogenous polyunsaturated fatty acids (PUFAs) influence permeability,antimicrobial peptide resistance,biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.