Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Dynamics of plant populations in Heteropogon contortus(black speargrass) pastures on a granite landscape in southern Queensland. 1. Dynamics of H. contortus populations

The dynamics of Heteropogon contortus (black speargrass) populations were measured in a subset of treatments contained within an extensive grazing study conducted between 1990 and 1996 in H. contortus pasture in southern Queensland. This subset included 2 landscape positions and 3 stocking rates in both native pasture and legume-oversown native pasture. Severe drought conditions throughout much of the study necessitated ongoing adjustments to the original stocking rates and, as a result, drought was the major influence on the dynamics of H. contortus populations. Plant density and basal area in the silver-leaved ironbark landscape were consistently higher than those in the narrow-leaved ironbark landscape. There was limited evidence of any impact by either light or moderate stocking rate but there was evidence of an impact at the heaviest stocking rate. There was minimal impact of legume oversowing. Relatively large fluctuations in plant density occurred during this study resulting from the death of existing plants, due mainly to drought, and seedling recruitment. Similarly, there were relatively large fluctuations in basal area caused mainly by changes in plant size. Rates for turnover of plant numbers were relatively high whereas plant turnover rates of basal areas were relatively low. Regular seedling recruitment appeared necessary to ensure the persistence of this species. Despite the high turnover, populations were maintained at reasonable levels indicating the overall resilience of H. contortus.

1-Methylcyclopropene Delays Softening in Tomato Slices

I-Methylcyclopropene (1-MCP) has the potential in tomato to reduce ethylene-associated changes in texture. Tomato cv. 'Revolution' was harvested at the 'pink' maturity stage and whole fruit treated with 0, 0.1, 1.0 or 10.0 µL.L-' 1-MCP at 20 "C for 12 h. Slices of 7-mm thickness were cut using a commercial slicer, and the slices stored in vertical stacks in plastic containers at 5°C for 7 days. The application of 1-MCP reduced both ethylene production and respiration rate of slices and resulted in firmer pericarp firmness. Ethylene production was 24%, 40%, and 62% lower following 0.1, 1.0, 10.0 µL L-' 1-MCP, respectively, compared with controls. In addition, respiration rate was reduced 6%, 10% and 20% by those 1-MCP treatments. 1-MCP treatments produced 20%, 34%, and 24% higher pericarp firmness, respectively, than in fruit not treated with 1-MCP.

Exposure of Tomato Fruit to 1-MCP Improves Quality of Stored Slices

Maintenance of quality, such as firmness, is important during storage of fresh cut produce. This study compared the effects of I-MCP on the quality of tomato slices when intact tomatoes were treated with 1-MCP and then sliced, or tomatoes were sliced and the slices treated with I-MCP. In both instances the MCP treatment was 1 µL Lˉٰ at 20 ºC for 12 h. Tomato cv. 'Revolution' was harvested at the 'pink' stage of maturity, cut into 7-mm slices, and stored as vertical stacks in closed plastic containers at 5ºC for up to 7 days after the 1-MCP treatment. Exposure of intact tomatoes to I-MCP resulted in reduced ethylene production (31%) and firmer (22%) slices than when tomatoes were not I-MCP treated. The application of I-MCP prior to slicing of tomatoes appears a useful strategy to retain quality of stored tomato slices.

Vaccination with an Insulin-like Growth Factor Binding Protein-1 (IGFBP-1)-bBased Vaccine Improves Growth in Feed-Restricted Brahman Steers

Dry-season weight loss in grazing cattle in northern Australia has been attenuated using a number of strategies (Hunter and Vercoe, 1987, Sillence et al. 1993, Gazzola and Hunter, 1999). Furthermore, the potential to improve efficiency of feed utilisation (and thus, dry-season performance) in ruminants through conventional modulation of the insulin-like growth factor (IGF) axis (Oddy and Owens, 1997, Hill et al., 1999) and through immunomodulation of the IGF axis (Hill et al., 1998a,b) has been demonstrated. The present study investigated the use of a vaccine directed against IGFBP-1 in Brahman steers which underwent a period of nutritional restriction followed by a return to wet-season grazing.

Piggery pond sludge as a nitrogen source for crops. 1. Mineral N supply estimated from laboratory incubations and field application of stockpiled and wet sludge

Piggery pond sludge (PPS) was applied, as-collected (Wet PPS) and following stockpiling for 12 months (Stockpiled PPS), to a sandy Sodosol and clay Vertosol at sites on the Darling Downs of Queensland. Laboratory measures of N availability were carried out on unamended and PPS-amended soils to investigate their value in estimating supplementary N needs of crops in Australia's northern grains region. Cumulative net N mineralised from the long-term (30 weeks) leached aerobic incubation was described by a first-order single exponential model. The mineralisation rate constant (0.057/week) was not significantly different between Control and PPS treatments or across soil types, when the amounts of initial mineral N applied in PPS treatments were excluded. Potentially mineralisable N (No) was significantly increased by the application of Wet PPS, and increased with increasing rate of application. Application of Wet PPS significantly increased the total amount of inorganic N leached compared with the Control treatments. Mineral N applied in Wet PPS contributed as much to the total mineral N status of the soil as did that which mineralised over time from organic N. Rates of C02 evolution during 30 weeks of aerobic leached incubation indicated that the Stockpiled PPS was more stabilised (19-28% of applied organic C mineralised) than the WetPPS (35-58% of applied organic C mineralised), due to higher lignin content in the former. Net nitrate-N produced following 12 weeks of aerobic non-leached incubation was highly correlated with net nitrate-N leached during 12 weeks of aerobic incubation (R^2 = 0.96), although it was <60% of the latter in both sandy and clayey soils. Anaerobically mineralisable N determined by waterlogged incubation of laboratory PPS-amended soil samples increased with increasing application rate of Wet PPS. Anaerobically minemlisable N from field-moist soil was well correlated with net N mineralised during 30 weeks of aerobic leached incubation (R^2 =0.90 sandy soil; R^2=0.93 clay soil). In the clay soil, the amount of mineral N produced from all the laboratory incubations was significantly correlated with field-measured nitrate-N in the soil profile (0-1.5 m depth) after 9 months of weed-free fallow following PPS application. In contrast, only anaerobic mineralisable N was significantly correlated with field nitrate-N in the sandy soil. Anaerobic incubation would, therefore, be suitable as a rapid practical test to estimate potentially mineralisable N following applications of different PPS materials in the field.

Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov.

This paper describes a phenotypic and genotypic investigation of the taxonomy of [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium, a major subcluster within the avian 16S rRNA cluster 18 of the family Pasteurellaceae. An extended phenotypic characterization was performed of the type strain of [Haemophilus] paragallinarum, which is NAD-dependent, and eight NAD-independent strains of [Haemophilus] paragallinarum. Complete 16S rRNA gene sequences were obtained for one NAD-independent and four NAD-dependent [Haemophilus] paragallinarum strains. These five sequences along with existing 16S rRNA gene sequences for 11 other taxa within avian 16S rRNA cluster 18 as well as seven other taxa from the Pasteurellaceae were subjected to phylogenetic analysis. The analysis demonstrated that [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium formed a monophyletic group with a minimum of 96·8% sequence similarity. This group can also be separated by phenotypic testing from all other recognized and named taxa within the Pasteurellaceae. As both genotypic and phenotypic testing support the separate and distinct nature of this subcluster, the transfer is proposed of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium to a new genus Avibacterium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. The type strains are NCTC 1118T (Avibacterium gallinarum), NCTC 11296T (Avibacterium paragallinarum), NCTC 11297T (Avibacterium avium) and NCTC 3438T (Avibacterium volantium). Key characteristics that separate these four species are catalase activity (absent only in Avibacterium paragallinarum) and production of acid from galactose (negative only in Avibacterium paragallinarum), maltose (negative only in Avibacterium avium) and mannitol (negative in Avibacterium gallinarum and Avibacterium avium).

Assessment of internal quality attributes of mandarin fruit. 1. NIR calibration model development

The utility of near infrared spectroscopy as a non-invasive technique for the assessment of internal eating quality parameters of mandarin fruit (Citrus reticulata cv. Imperial) was assessed. The calibration procedure for the attributes of TSS (total soluble solids) and DM (dry matter) was optimised with respect to a reference sampling technique, scan averaging, spectral window, data pre-treatment (in terms of derivative treatment and scatter correction routine) and regression procedure. The recommended procedure involved sampling of an equatorial position on the fruit with 1 scan per spectrum, and modified partial least squares model development on a 720–950-nm window, pre-treated as first derivative absorbance data (gap size of 4 data points) with standard normal variance and detrend scatter correction. Calibration model performance for the attributes of TSS and DM content was encouraging (typical Rc2 of >0.75 and 0.90, respectively; typical root mean squared standard error of calibration of <0.4 and 0.6%, respectively), whereas that for juiciness and total acidity was unacceptable. The robustness of the TSS and DM calibrations across new populations of fruit is documented in a companion study.

Inferential, non-parametric statistics to assess quality of probabilistic forecast systems

Many statistical forecast systems are available to interested users. In order to be useful for decision-making, these systems must be based on evidence of underlying mechanisms. Once causal connections between the mechanism and their statistical manifestation have been firmly established, the forecasts must also provide some quantitative evidence of `quality’. However, the quality of statistical climate forecast systems (forecast quality) is an ill-defined and frequently misunderstood property. Often, providers and users of such forecast systems are unclear about what ‘quality’ entails and how to measure it, leading to confusion and misinformation. Here we present a generic framework to quantify aspects of forecast quality using an inferential approach to calculate nominal significance levels (p-values) that can be obtained either by directly applying non-parametric statistical tests such as Kruskal-Wallis (KW) or Kolmogorov-Smirnov (KS) or by using Monte-Carlo methods (in the case of forecast skill scores). Once converted to p-values, these forecast quality measures provide a means to objectively evaluate and compare temporal and spatial patterns of forecast quality across datasets and forecast systems. Our analysis demonstrates the importance of providing p-values rather than adopting some arbitrarily chosen significance levels such as p < 0.05 or p < 0.01, which is still common practice. This is illustrated by applying non-parametric tests (such as KW and KS) and skill scoring methods (LEPS and RPSS) to the 5-phase Southern Oscillation Index classification system using historical rainfall data from Australia, The Republic of South Africa and India. The selection of quality measures is solely based on their common use and does not constitute endorsement. We found that non-parametric statistical tests can be adequate proxies for skill measures such as LEPS or RPSS. The framework can be implemented anywhere, regardless of dataset, forecast system or quality measure. Eventually such inferential evidence should be complimented by descriptive statistical methods in order to fully assist in operational risk management.

Chemical synthesis and structure elucidation of bovine K-casein (1-44)

The caseins (αs1, αs2, β, and κ) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1–44) of bovine κ-casein, the protein which maintains the micellar structure of the caseins. κ-Casein (1–44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro8 to Arg34. This is the first report which demonstrates extensive secondary structure within the casein class of proteins.

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.

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV-1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50-60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.