Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance.

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10-7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana.

Hydrophobins are small amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signalling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathogenic fungus used as an arthropod biological control agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, biopanning experiments were performed using a variety of affinity resins, including N,N'-diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads. After five rounds of iterative biopanning, cDNAs corresponding to two B. bassiana (class I) hydrophobins were selectively enriched using melibiose- or lactose-coupled agarose beads. Expression analysis revealed that the hyd1 gene was expressed in all samples tested, including aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle, with hyd1 expression peaking in growing mycelia. In contrast, the hyd2 gene was not appreciably expressed in any of the single-cell types (aerial conidia, blastospores and submerged conidia), but was constitutively expressed in growing mycelia and when cells were sporulating on chitin and insect cuticle. MS fingerprinting of an approximately 10 kDa protein found in boiling SDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.

Surface characteristics of the entomopathogenic fungus Beauveria (Cordyceps) bassiana.

Marked differences in surface characteristics were observed among three types of single-cell propagules produced by the entomopathogenic fungus Beauveria bassiana. Atomic force microscopy (AFM) revealed the presence of bundles or fascicles in aerial conidia absent from in vitro blastospores and submerged conidia. Contact angle measurements using polar and apolar test liquids placed on cell layers were used to calculate surface tension values and the free energies of interaction of the cell types with surfaces. These analyses indicated that the cell surfaces of aerial conidia were hydrophobic, whereas those of blastospores and submerged conidia were hydrophilic. Zeta potential determinations of the electrostatic charge distribution across the surface of the cells varied from +22 to -30 mV for 16-day aerial conidia at pH values ranging from 3 to 9, while the net surface charge ranged from +10 to -13 mV for submerged conidia, with much less variation observed for blastospores, +4 to -4 mV, over the same pH range. Measurements of hydrophobicity using microbial adhesion to hydrocarbons (MATH) indicated that the surfaces of aerial conidia were hydrophobic, and those of blastospores hydrophilic, whereas submerged conidia displayed cell surface characteristics on the borderline between hydrophobic and hydrophilic. Insect pathology assays using tobacco budworm (Heliothis virescens) larvae revealed some variation in virulence among aerial conidia, in vitro blastospores and submerged conidia, using both topical application and haemocoel injection of the fungal cells.

Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata.

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.