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.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Availability of soil potassium and diagnostic soil tests

Thirty-seven surface (0-0.10 or 0-0.20 m) soils covering a wide range of soil types (16 Vertosols, 6 Ferrosols, 6 Dermosols, 4 Hydrosols, 2 Kandosols, 1 Sodosol, 1 Rudosol, and 1 Chromosol) were exhaustively cropped in 2 glasshouse experiments. The test species were Panicum maximum cv. Green Panic in Experiment A and Avena sativa cv. Barcoo in Experiment B. Successive forage harvests were taken until the plants could no longer grow in most soils because of severe potassium (K) deficiency. Soil samples were taken prior to cropping and after the final harvest in both experiments, and also after the initial harvest in Experiment B. Samples were analysed for solution K, exchangeable K (Exch K), tetraphenyl borate extractable K for extraction periods of 15 min (TBK15) and 60 min (TBK60), and boiling nitric acid extractable K (Nitric K). Inter-correlations between the initial levels of the various soil K parameters indicated that the following pools were in sequential equilibrium: solution K, Exch K, fast release fixed K [estimated as (TBK15-Exch K)], and slow release fixed K [estimated as (TBK60-TBK15)]. Structural K [estimated as (Nitric K-TBK60)] was not correlated with any of the other pools. However, following exhaustive drawdown of soil K by cropping, structural K became correlated with solution K, suggesting dissolution of K minerals when solution K was low. The change in the various K pools following cropping was correlated with K uptake at Harvest 1 ( Experiment B only) and cumulative K uptake ( both experiments). The change in Exch K for 30 soils was linearly related to cumulative K uptake (r = 0.98), although on average, K uptake was 35% higher than the change in Exch K. For the remaining 7 soils, K uptake considerably exceeded the change in Exch K. However, the changes in TBK15 and TBK60 were both highly linearly correlated with K uptake across all soils (r = 0.95 and 0.98, respectively). The slopes of the regression lines were not significantly different from unity, and the y-axis intercepts were very small. These results indicate that the plant is removing K from the TBK pool. Although the change in Exch K did not consistently equate with K uptake across all soils, initial Exch K was highly correlated with K uptake (r = 0.99) if one Vertosol was omitted. Exchangeable K is therefore a satisfactory diagnostic indicator of soil K status for the current crop. However, the change in Exch K following K uptake is soil-dependent, and many soils with large amounts of TBK relative to Exch K were able to buffer changes in Exch K. These soils tended to be Vertosols occurring on floodplains. In contrast, 5 soils (a Dermosol, a Rudosol, a Kandosol, and 2 Hydrosols) with large amounts of TBK did not buffer decreases in Exch K caused by K uptake, indicating that the TBK pool in these soils was unavailable to plants under the conditions of these experiments. It is likely that K fertiliser recommendations will need to take account of whether the soil has TBK reserves, and the availability of these reserves, when deciding rates required to raise exchangeable K status to adequate levels.

Cyanide poisoning in cattle from Dysphania glomulifera (red crumbweed): Using the internet for rapid plant identification and diagnostic advice: Case Report

A 300-strong Angus-Brahman cattle herd near Springsure, central Queensland, was being fed Acacia shirleyi (lancewood) browse during drought and crossed a 5-hectare, previously burnt area with an almost pure growth of Dysphania glomulifera subspecies glomulifera (red crumbweed) on their way to drinking water. Forty cows died of cyanide poisoning over 2 days before further access to the plant was prevented. A digital image of a plant specimen made on a flat-bed scanner and transmitted by email was used to identify D glomulifera. Specific advice on the plant's poisonous properties and management of the case was then provided by email within 2 hours of an initial telephone call by the field veterinarian to the laboratory some 600 km away. The conventional method using physical transport of a pressed dried plant specimen to confirm the identification took 5 days. D glomulifera was identified in the rumen of one of two cows necropsied. The cyanogenic potential of D glomulifera measured 4 days after collection from the site of cattle deaths was 18,600 mg HCN/kg in dry matter. The lethal dose of D glomulifera for a 420 kg cow was estimated as 150 to 190 g wet weight. The plant also contained 4.8% KNO3 equivalent in dry matter, but nitrate-nitrite poisoning was not involved in the deaths.

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.

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.

Enhanced diagnostic platforms for Post Entry Quarantine (PEQ)

Enhanced diagnostic platforms for Post Entry Quarantine (PEQ) and market access (Phase 1).

Determining Optimum Irrigation Scheduling Techniques for Key Wildflower Crops

Determining Optimum Irrigation Scheduling Techniques for Key Wildflower Crops.

CRC27012 A National Diagnostic Database for Emergency Plant Pests

Development of a national diagnostic database for Emergency Plant Pests which will be web-accessible.

Diagnostic Agronomy

Develop and implement a diagnostic framework designed to help advisors and farmers identify the causes of poor crop performance and implement appropriate remedial measures.

CRC20093 Increasing Diagnostic Capacity in Thailand

Accurate identification of pests is essential for practically all aspects of agricultural development and is critical to the operations of biosecurity that safeguard agricultural integrity and facilitate trade. Diagnostic capability is at the forefront of and complementary to, activities such as border protection, incursion management, surveillance and pest and disease certification. The efficiency of a biosecurity system therefore depends largely on the feedback between these activities and diagnostics. Australian scientists will train Thai scientists in diagnostics and surveillance to provide the Thai DOA with skills that will aid in the development of a Thai Diagnostic Network. The skills will be taught using a range of pests, including some which have particular biosecurity importance for both Australia and Thailand such as citrus canker, potato viruses and fruit flies.

CRC20080 Diagnostic technologies for phosphine resistance management

Development of molecular markers for rapid diagnosis of phosphine resistance in insects.

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.