Using simulation techniques to investigate methods to determine resistance of helminths to anthelmintic treatment

The widespread and increasing resistance of internal parasites to anthelmintic control is a serious problem for the Australian sheep and wool industry. As part of control programmes, laboratories use the Faecal Egg Count Reduction Test (FECRT) to determine resistance to anthelmintics. It is important to have confidence in the measure of resistance, not only for the producer planning a drenching programme but also for companies investigating the efficacy of their products. The determination of resistance and corresponding confidence limits as given in anthelmintic efficacy guidelines of the Standing Committee on Agriculture (SCA) is based on a number of assumptions. This study evaluated the appropriateness of these assumptions for typical data and compared the effectiveness of the standard FECRT procedure with the effectiveness of alternative procedures. Several sets of historical experimental data from sheep and goats were analysed to determine that a negative binomial distribution was a more appropriate distribution to describe pre-treatment helminth egg counts in faeces than a normal distribution. Simulated egg counts for control animals were generated stochastically from negative binomial distributions and those for treated animals from negative binomial and binomial distributions. Three methods for determining resistance when percent reduction is based on arithmetic means were applied. The first was that advocated in the SCA guidelines, the second similar to the first but basing the variance estimates on negative binomial distributions, and the third using Wadley’s method with the distribution of the response variate assumed negative binomial and a logit link transformation. These were also compared with a fourth method recommended by the International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) programme, in which percent reduction is based on the geometric means. A wide selection of parameters was investigated and for each set 1000 simulations run. Percent reduction and confidence limits were then calculated for the methods, together with the number of times in each set of 1000 simulations the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been said to occur. These simulations provide the basis for setting conditions under which the methods could be recommended. The authors show that given the distribution of helminth egg counts found in Queensland flocks, the method based on arithmetic not geometric means should be used and suggest that resistance be redefined as occurring when the upper level of percent reduction is less than 95%. At least ten animals per group are required in most circumstances, though even 20 may be insufficient where effectiveness of the product is close to the cut off point for defining resistance.

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae (Erysiphaceae)

Some whole leaf-clearing and staining techniques are described for the microscopic observation of the origin of powdery mildew conidiophores, whether from external mycelium or internal mycelium, emerging through stomata. These techniques enable separation of the two genera, Oidiopsis and Streptopodium, in the Erysiphaceae.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species: Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warned, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warned were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body. E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

Control techniques and management strategies for the problematic Navua sedge (Cyperus aromaticus)

Navua sedge, a member of the Cyperaceae family, is an aggressive weed of pastures in Fiji, Sri Lanka, Malay Peninsula, Vanuatu, Samoa, Solomons, and Tahiti and is now a weed of pastures and roadsides in north Queensland, Australia. Primarily restricted to areas with an annual rainfall exceeding 2500 mm, Navua sedge is capable of forming dense stands smothering many tropical pasture species. Seventeen herbicides were field tested at three sites in north Queensland, with glyphosate, halosulfuron, hexazinone, imazapic, imazapyr, or MSMA the most effective for Navua sedge control. Environmental problems such as persistence in soil, lack of selectivity and movement off-site may occur using some herbicides at the predicted LC90 control level rates. A seasonality trial using halosulfuron (97.5 g ai/ha) gave better Navua sedge control (84%) spraying March to September than spraying at other times (50%). In a frequency trial, sequential glyphosate applications (2,160 g ae/ha) every two months was more effective for continued Navua sedge control (67%) than a single application of glyphosate (36%), though loss of ground cover would occur. In a management trial, single applications of glyphosate (2,160 to 3,570 g ae/ha) using either a rope wick, ground foliar spraying or a rotary rope wick gave 59 to 73% control, while other treatments (rotary hoe (3%), slashing (-13%) or crushing (-30%)) were less effective. In a second management trial, four monthly rotary wick applications were much more effective (98%) than four monthly crushing applications (42%). An effective management plan must include the application of regular herbicide treatments to eliminate Navua sedge seed being added to the soil seed bank. Treatments that result in seed burial, for example, discing are likely to prolong seed persistence and should be avoided. The sprouting activity of vegetative propagules and root fragmentation needs to also be considered when selecting control options.

Regional development, commercialisation and target adoption of new integrated management techniques for Sheep industry

This Sheep CRC2 project is directed towards regional development of, support for, commercialisation and target adoption of, new integrated management techniques, which will lift productivity and better match product with market specification. The outputs and benefits will help support and improve the productivity and profitability of producers remaining in the industry, and support industry in the current move towards one in which both meat and wool will be joint drivers of industry productivity.

Determining Optimum Irrigation Scheduling Techniques for Key Wildflower Crops

Determining Optimum Irrigation Scheduling Techniques for Key Wildflower Crops.

Laboratory-rearing Techniques for Tephritid Fruit Flies in the South Pacific

Laboratory colonies of 15 economically important species of multi-host fruit flies (Diptera:Tephritidae) have been established in eight South Pacific island countries for the purpose of undertaking biological studies, particularly host status testing and research on quarantine treatments. Laboratory rearing techniques are based on the development of artificial diets for larvae consisting predominately of the pulp of locally available fruits including pawpaw, breadfruit and banana. The pawpaw diet is the standard diet and is used in seven countries for rearing 11 species. Diet ingredients are standard proportions of fruit pulp, hydrolysed protein and a bacterial and fungal inhibitor. The diet is particularly suitable for post-harvest treatment studies when larvae of known age are required. Another major development in the laboratory rearing system is the use of pure strains of Enterobacteriaceae bacterial cultures as important adult-feeding supplements. These bacterial cultures are dissected out of the crop of wild females, isolated by sub-culturing, and identified before supply to adults on peptone yeast extract agar plates. Most species are egged using thin, plastic receptacles perforated with 1 mm oviposition holes, with fruit juice or larval diet smeared internally as an oviposition stimulant. Laboratory rearing techniques have been standardised for all of the Pacific countries. Quality control monitoring is based on acceptable ranges in per cent egg hatch, pupal weight and pupal mortality. Colonies are rejuvenated every 6 to 12 months by crossing wild males with laboratory-reared females and vice versa. The standard rearing techniques, equipment and ingredients used in collecting, establishment, maintenance and quality control of these fruit fly species are detailed in this paper.

Effect of Packaging Materials and Storage on Major Volatile Compounds in Three Australian Native Herbs

Lemon myrtle, anise myrtle, and Tasmanian pepper leaf are commercial Australian native herbs with a high volatile or essential oil content. Packaging of the herbs in high- or low-density polyethylene (HDPE and LDPE) has proven to be ineffective in preventing a significant loss of volatile components on storage. This study investigates and compares the effectiveness of alternate high-barrier property packaging materials, namely, polyvinylidene chloride coated polyethylene terephthalate/casted polypropylene (PVDC coated PET/CPP) and polyethylene terephthalate/polyethylene terephthalate/aluminum foil/linear low-density polyethylene (PET/PET/Foil/LLDPE), in prevention of volatile compound loss from the three native herbs stored at ambient temperature for 6 months. Concentrations of major volatiles were monitored using gas chromatography?mass spectrometry (GC-MS) techniques. After 6 months of storage, the greatest loss of volatiles from lemon myrtle was observed in traditional LDPE packaging (87% loss) followed by storage in PVDC coated PET/CPP (58% loss) and PET/PET/Foil/LLDPE (loss of 23%). The volatile loss from anise myrtle and Tasmanian pepper leaf stored in PVDC coated PET/CPP and PET/PET/Foil/LLDPE packaging was <30%. This study clearly indicates the importance of selecting the correct packaging material to retain the quality of herbs with high volatile content.

Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions

Amino functionalised mesoporous silica nanoparticles (AM-41) have been identified as a promising vaccine delivery material. The capacity of AM-41 to stabilise vaccine components at ambient temperature (23–27 °C) was determined by adsorbing the model antigen ovalbumin (OVA) to AM-41 particles (OVA-41). The OVA-41 was successfully freeze-dried using the excipients 5% trehalose and 1% PEG8000. Both the immunological activity of OVA and the nanoparticle structure were maintained following two months storage at ambient temperature. The results of immunisation studies in mice with reconstituted OVA-41 demonstrated the induction of humoral and cell-meditated immune responses. The capacity of AM-41 particles to facilitate ambient storage of vaccine components without loss of immunological potency will underpin the further development of this promising vaccine delivery platform.

Perennial pastures for marginal farming country in southern Queensland. 1. Grass establishment techniques

Efficient ways to re-establish pastures are needed on land that requires a rotation between pastures and crops. We conducted trials in southern inland Queensland with a range of tropical perennial grasses sown into wheat stubble that was modified in various ways. Differing seedbed preparations involved cultivation or herbicide sprays, with or without fertilizer at sowing. Seed was broadcast and sowing time ranged from spring through to autumn on 3 different soil types. Seed quality and post-sowing rainfall were major determinants of the density of sown grass plants in the first year. Light cultivation sometimes enhanced establishment compared with herbicide spraying of standing stubble, most often on harder-setting soils. A nitrogen + phosphorus mixed fertilizer rarely produced any improvement in sown grass establishment and sometimes increased weed competition. The effects were similar for all types of grass seed from hairy fascicles to large, smooth panicoid seeds and minute Eragrostis seeds. There was a strong inverse relationship between the initial density of sown grass established and the level of weed competition.