Comparison of Culture and a Novel 5' Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Detection of Helicobacter pullorum in meat chickens in Australia

The bacterial genus Helicobacter is a member of the Campylobacteriales in the Epsilonproteobacteria subphylum, and is comprised of organisms that are morphologically similar to Campylobacter. The term ‘campylobacteria’ is used to encompass the genera Campylobacter, Arcobacter, Helicobacter and Anaerobiospirillum. Helicobacter was separated from the genus Campylobacter in 1989. Helicobacter spp have been isolated from gastric tissue or intestinal contents of humans and a wide range of animal species, with some associated with disease...

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Detection and characterisation of strong resistance to phosphine in Brazilian Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

As failure to control Rhyzopertha dominica (F.) with phosphine is a common problem in the grain-growing regions of Brazil, a study was undertaken to investigate the frequency, distribution and strength of phosphine resistance in R. dominica in Brazil. Nineteen samples of R. dominica were collected between 1991 and 2003 from central storages where phosphine fumigation had failed to control this species. Insects were cultured without selection until testing in 2005. Each sample was tested for resistance to phosphine on the basis of the response of adults to discriminating concentrations of phosphine (20 and 48 h exposures) and full dose-response assays (48 h exposure). Responses of the Brazilian R. dominica samples were compared with reference susceptible, weak-resistance and strong-resistance strains from Australia in parallel assays. All Brazilian population samples showed resistance to phosphine: five were diagnosed with weak resistance and 14 with strong resistance. Five samples showed levels of resistance similar to the reference strong-resistance strain. A representative highly resistant sample was characterised by exposing mixed-age cultures to a range of constant concentrations of phosphine for various exposure periods. Time to population extinction (TPE) and time to 99.9% suppression of population (LT99.9) values of this sample were generally similar to those of the reference strong-resistance strain. For example, at 0.1, 0.5 and 1.0 mg L-1, LT99.9 values for BR33 and the reference strong-resistance strain were respectively 21, 6.4 and 3.7 days and 17, 6.2 and 3.8 days. With both strains, doubling phosphine concentrations to 2 mg L -1 resulted in increased LT99.9 and TPE. High level and frequency of resistance in all population samples, some of which had been cultured without selection for up to 12 years, suggest little or no fitness deficit associated with phosphine resistance. The present research indicates that widespread phosphine resistance may be developing in Brazil. Fumigation practices should be monitored and resistance management plans implemented to alleviate further resistance development.

The assessment of TGGE for the detection of interspecific and intergeneric DNA-marker polymorphism within Solanaceae

RFLP markers are currently the most appropriate marker system for the identification of uncharacterised polymorphism at the interspecific and intergeneric level. Given the benefits of a PCR-based marker system and the availability of sequence information for many Solanaceous cDNA clones, it is now possible to target conserved fragments, for primer development, that flank sequences possessing interspecific polymorphism. The potential outcome is the development of a suite of markers that amplify widely in Solanaceae. Temperature gradient gel electrophoresis (TGGE) is a relatively inexpensive gel-based system that is suitable for the detection of most single-base changes. TGGE can be used to screen for both known and unknown polymorphisms, and has been assessed here, for the development of PCR-based markers that are useful for the detection of interspecific variation within Solanaceae. Fifteen markers are presented where differences between Lycopersicon esculentum and L. pennellii have been detected by TGGE. The markers were assessed on a wider selection of plant species and found to be potentially useful for the identification of interspecific and intergeneric polymorphism in Solanaceous plants.

A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.

Processes leading to the detection of tropical weed infestations during an eradication program.

The authors identify and track processes that have resulted in the detection of six tropical weeds targeted for eradication. The habitats and distributions of these species make detection by field officers and members of the public more likely than targeted searches. The eradication program is increasing the scope of detection processes by conducting and documenting activities to improve weed recognition amongst public, government and industry stakeholders.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

Detection and use of QTL for complex traits in multiple environments.

QTL mapping methods for complex traits are challenged by new developments in marker technology, phenotyping platforms, and breeding methods. In meeting these challenges, QTL mapping approaches will need to also acknowledge the central roles of QTL by environment interactions (QEI) and QTL by trait interactions in the expression of complex traits like yield. This paper presents an overview of mixed model QTL methodology that is suitable for many types of populations and that allows predictive modeling of QEI, both for environmental and developmental gradients. Attention is also given to multi-trait QTL models which are essential to interpret the genetic basis of trait correlations. Biophysical (crop growth) model simulations are proposed as a complement to statistical QTL mapping for the interpretation of the nature of QEI and to investigate better methods for the dissection of complex traits into component traits and their genetic controls.

An innovative microplate assay to facilitate the detection of antimicrobial activity in plant extracts.

A microplate assay was modified for the detection of antimicrobial activity in plant extracts. The aim was to develop an in vitro assay that could rapidly screen plant extracts to provide quantitative data on inhibition of microbial growth. A spectrophotometric assay using a microplate with serial dilutions of the plant extract and the bacteria was developed. Two bacteria, Staphylococcus aureus and Escherichia coli, were used for this study. Essential oils, oregano (Origanum vulgare) and lemon myrtle (Backhousia citriodora), and three active components carvacrol, thymol and citral were evaluated. The reproducibility of the assay was high, with correlation coefficients (r aureus and E. coli between 0.9321 and 0.9816. Similarly, r and 0.9814. This assay could also be used to measure antimicrobial activity in plant extracts which vary in pH and color.

Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

Resin defect impacts on the value of graded recovery and evaluation of technologies for internal defect detection in slash pine logs.

The key outcome will be to identify a technology that is practical to use to scan logs identified by the modelling as suspect or marginal for sawing and to confirm their unsuitability for value adding sawing by internal scanning.

Detection of Polymyxa graminis in a barley crop in Australia.

Polymyxa graminis was detected in the roots of barley plants from a field near Wondai, Queensland, in 2009. P. graminis was identified by characteristic sporosori in roots stained with trypan blue. The presence of P. graminis f. sp. tepida (which is hosted by wheat and oats as well as barley) in the roots was confirmed by specific PCR tests based on nuclear ribosomal DNA. P. graminis is the vector of several damaging soil-borne virus diseases of cereals in the genera Furovirus, Bymovirus and Pecluvirus. No virus particles were detected in sap extracts from leaves of stunted barley plants with leaf chlorosis and increased tillering. Further work is required to determine the distribution of P. graminis in Australian grain crops and the potential for establishment and spread of the exotic soil-borne viruses that it vectors.

Polychaete-assisted sand filters – prawn farm wastewater remediation trial

Medium bedding sand which is commonly available in coastal sedimentary deposits, and a marine polychaete-worm species from Moreton Bay recently classified as Perinereis helleri (Nereididae), were deployed in a simple low-maintenance sand filter design that potentially has application at large scale. Previous work had shown that this physical and biological combination can provide a new option for saline wastewater treatment, since the worms help to prevent sand filter blocking with organic debris and offer a profitable by-product. To test the application of this new concept in a commercial environment, six 1.84 m2 Polychaete-assisted sand filters were experimentally tested for their ability to treat wastewater from a semi-intensive prawn culture pond. Polychaetes produced exclusively on the waste nutrients that collected in these gravity-driven sand filters were assessed for their production levels and nutritional contents. Water parameters studied included temperature, salinity, pH, dissolved oxygen (DO), oxidation/ reduction potential (redox), suspended solids, chlorophyll a, biological oxygen demand (BOD), and common forms of nitrogen and phosphorus. Pond water which had percolated through the sand bed had significantly lower pH, DO and redox levels compared with inflow water. Suspended solids and chlorophyll a levels were consistently more than halved by the process. Reductions in BOD appeared dependant on regular subsurface flows. Only marginal reductions in total nitrogen and phosphorus were documented, but their forms were altered in a potentially useful way: dissolved forms (ammonia and orthophosphate) were generated by the process, and this remineralisation also seemed to be accentuated by intermittent flow patterns. Flow rates of approximately 1,500 L m-2 d-1 were achieved suggesting that a 1 ha polychaete bed of this nature could similarly treat the discharge from a 10 ha semi-intensive prawn farm. Sixteen weeks after stocking sand beds with one-month-old P. helleri, over 3.6 kg of polychaete biomass (wet weight) was recovered from the trial. Production on a sand bed area basis was 328 g m-2. Similar (P>0.05) overall biomass production was found for the two stocking densities tested (2000 and 6000 m-2; n = 3), but survival was lower and more worms were graded as small (<0.6 g) when produced at the higher density (28.2 ± 1.5 % and approx. 88 %, respectively) compared with the lower density (46.8 ± 4.4 % and approx. 76 %, respectively). When considered on a weight for weight basis, about half of the worm biomass produced was generally suitable for use as bait. The nutritional contents of the worms harvested were analysed for different stocking densities and graded sizes. These factors did not significantly affect their percentages of dry matter (DM) (18.23 ± 0.57 %), ash (19.77 ± 0.80 % of DM) or gross energy 19.39 ± 0.29 MJ kg-1 DM) (n = 12). Although stocking density did not affect the worms’ nitrogen and phosphorus contents, small worms had a higher mean proportion of nitrogen and phosphorus (10.57 ± 0.17 % and 0.70 ± 0.01 % of DM, respectively) than large worms (9.99 ± 0.12 % and 0.65 ± 0.01 % of DM, respectively) (n = 6). More lipid was present in large worms grown at the medium density (11.20 ± 0.19 %) compared with the high density (9.50 ± 0.31 %) and less was generally found in small worms (7.1-7.6 % of DM). Mean cholesterol and total phospholipid levels were 5.24 ± 0.15 mg g-1 and 13.66 ± 2.15 mg g-1 DM, respectively (n = 12). Of the specific phospholipids tested, phosphatidyl-serine or sphingomyelin were below detection limits (<0.05 mg g-1), whilst mean levels of phosphatidyl-ethanolamine, phosphatidyl-inositol, phosphatidyl-choline and lysophosphatidyl-choline were 6.89 ± 1.09, 0.89 ± 0.26, 4.04 ± 1.17 and 1.84 ± 0.37 mg g-1, respectively (n = 12). Culture density generally had a more pronounced effect on phospholipid contents than did size of worms. By contrast, worm size had a more pronounced effect on total fatty acid contents, with large worms containing significantly higher (P<0.001) levels on a DM basis (46.88 ± 2.46 mg g-1) than smaller worms (27.76 ± 1.28 mg g-1). A very broad range of fatty acids were detected with palmitic acid being the most heavily represented class (up to 14.23 ± 0.49 mg g-1 DM or 27.28 ± 0.22 % of total fatty acids). Other heavily represented classes included stearic acid (7.4-8.8 %), vaccenic acid (6.8-7.8 %), arachidonic acid (3.5-4.4 %), eicosapentaenoic acid (9.9-13.8 %) and docosenoic acid (5.7-7.0 %). Stocking density did not affect (P>0.05) the levels of amino acids present in polychaete DM, but there was generally less of each amino acid tested on a weight per weight basis in large worms than in small worms. This difference was significant (P<0.05) for the most heavily represented classes being glutamic acid (73-77 mg g-1), aspartic acid (50-54 mg g-1), and glycine (46-53 mg g-1). These results demonstrate how this polychaete species can be planted and sorted at harvest according to various strategies aimed at providing biomass with specific physical and nutritional qualities for different uses.

Establishing forest pest detection systems in South Pacific and Australia

The aim of the project is to reduce the risk of serious damage by exotic pests to the valuable timber resources of Fiji, Vanuatu and Australia by establishing efficient detection systems for target pests in high hazard sites. In particular, the project aims to minimise losses in the valuable plantations of Fiji and the emerging plantation industry of Vanuatu. This is part of a 'neighbourhood watch' approach to incursion management that will benefit all regional countries, including Australia.