Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia.

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.     

Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle

The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.     

Soil risk maps – Interpreting soils data for policy makers,agencies and industry

Surface runoff, erosion, compaction and the leaching of potential pollutants from land can degrade the soil resource and damage the water environment, reducing crop yields, causing loss of valuable nutrients and organic matter, together with increasing flood risk. Increasingly, it is recognized that scientific information must be translated into practical tools to change practices and protect the soil and water resource. Working alongside agencies in Scotland, we applied a suite of simple, transparent, rule-based models to identify areas most at risk of exporting sediment and pollutants that may degrade water quality based on field-scale (1:25,000) soil maps. The maps have been used by Scottish Environment Protection Agency, Scottish Water and Scottish Government to assess soil risks to waters from field to regional scale. The work brought scientists together with policy makers, agencies and the water industry to pool their knowledge to apply these practical tools for decision-making. It highlights the need to apply existing knowledge to answer salient questions. All three examples described show that providing the right type of information, which is based on fundamental soils data, can directly influence the implementation of policies, investment and monitoring decisions and provide evidence in support of government. However, this requires both researchers and agency scientists to develop skills as knowledge brokers and to normalize the use of soil data in everyday agency settings.     

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.     

Consistent responses of yield and resistance of wheat cultivars to the root-lesion nematode,Pratylenchus thornei,in the Australian northern subtropical region,but not in the temperate southern region

To understand the yield response of cereal cultivars to Pratylenchus thornei, eight experiments were conducted within the subtropical northern, and temperate southern grain-producing regions of Australia. Wheat cultivars (Triticum aestivum) ranging from susceptible to moderately resistant to P. thornei were grown in Year 1 to establish a range of population densities. In Year 2 before sowing, P. thornei was quantified in each plot and six cereal cultivars were each grown on a similar range of population densities (average minimum to maximum of 3.4–60.6 P. thornei/g soil); P. thornei was quantified again at harvest. In the four experiments in the northern region there was a significant, negative logarithmic response of yield of the three most intolerant/susceptible cultivars as P. thornei population densities increased (yield decreased 172–479 kg/ha per unit increase in log_e-transformed P. thornei/g soil). The responsiveness of yield to increasing P. thornei population densities diminished as the tolerance and resistance of the cultivars improved. In the southern region, there was no relationship between yield and P. thornei in three experiments and minor, positive increases in one experiment (1.6 kg/ha per unit increase in P. thornei/g soil). Across both regions, the change in P. thornei population densities from sowing to harvest was logarithmic and positive, and generally greatest in the northern region. The contrast of responses of cereal cultivars between the regions, despite similar population densities of P. thornei, is indicative of the influence of the environment particularly on tolerance, therefore management with a regional focus is essential.     

Plasma-activated water inhibits in vitro conidial germination of Colletotrichum alienum,a postharvest pathogen of avocado

Cold plasma, an ionized gas produced by applying an electrical current to air, can be used to produce plasma-activated water (PAW), which has excellent antimicrobial properties. In this study PAW was applied to conidia of Colletotrichum alienum to investigate its impact on conidial germination in vitro. PAW was produced by treating tap, deionized, or distilled water with cold plasma for 30 or 60 min to produce PAW30 or PAW60, each of which was then incubated for up to 24 hr with a conidial suspension of C. alienum in a ratio of 1:1, 1:2, or 1:3 (conidial suspension:PAW), and the percentage germination measured. The greatest reduction in germination occurred when conidia were incubated with PAW60 produced from deionized water or distilled water, for all ratios. For PAW30, deionized water was the most effective for all three ratios, and on this basis, deionized water was selected for all further experiments. PAW produced from smaller volumes of water and at shorter distances from the cold plasma source was more effective at reducing germination. Treatment of conidia with acidified water was not as effective as PAW at inhibiting germination. Nitrates and nitrites were present in the PAW in varying concentrations and may have contributed to the inhibition of germination. PAW retained activity and reduced germination even after storage for 15 days. These findings demonstrate the potential of PAW as a novel treatment for postharvest fungal pathogens.