We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin- 12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cellmediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.

Black point (BP) can cause severe losses to the barley industry through downgrading and discounting of malting barley. The genetic improvement in BP resistance of barley is complex, requiring reliable screening tools, an understanding of genotype by environment interactions and an understanding of the biochemical mechanisms of melanisation involved in BP development. Thus the application of molecular markers for resistance to BP may be a useful tool for plant breeders. We have investigated the genetic regions associated with BP resistance in the barley F2 population, Valier/Binalong. Quantitative trait loci (QTLs) contributed by the resistant parent Valier, were detected on chromosomes 2HS, 2HC, 3HL, 4HL and a QTL contributed by the susceptible parent, Binalong was detected on 5HL. Three of the four QTLs were detected in two distinctly different environments. The differences observed in BP resistance between these two environments and the implications for accelerated screening are discussed. Identified SSR markers in these regions may be useful for selecting black point resistance in related breeding materials.

Ensemble classification methods have shown promise for achieving higher classification accuracy for microarray data classification analysis. As noise values do exist in all microarray data even after microarray data preprocessing stage, robustness is therefore another very important criteria in addition to accuracy for evaluating reliable microarray classification algorithms. In this paper, we conduct experimental comparison of our newly developed MDMT with C4.5, BaggingC4.5, Ad-aBoostingC4.5, Random Forest and CS4 on four microarray cancer data sets. We test and evaluate how well a given single or ensemble classifier can tolerate noise data in unseen test datasets, particularly with increasing levels of noise. The experimental results show that MDMT tolerates the noise values in unseen test data sets better than other compared methods do, particularly with increasing levels of noise data. We observe that a random forests is comparable to MDMT in term of resistance to noise. The experimental results also show that ensemble decision tree methods tolerate the noise values better than single tree C4.5 does. We conclude that avoiding overlapping genes exist among the ensemble trees is an intuitive, simple and effective way to achieve higher degree of diversity for ensemble decision tree methods. The algorithm based on this principal is more reliable to deal with microarray data sets with certain level of noise data.

The immunogenicity and protective efficacy of a DNA vaccine encoding a genetically inactivated S1 domain of pertussis toxin was evaluated using a murine respiratory challenge model of Bordetella pertussis infection. It was found that mice immunized via the intramuscular route elicited a purely cell-mediated immune response to the DNA vaccine, with high levels of gamma interferon (IFN-gamma) and interleukin (IL)-2 detected in the S1-stimulated splenocyte supernatants and no serum IgG. Despite the lack of an antibody response, the lungs of DNA-immunized mice were cleared of B. pertussis at a significantly faster rate compared with mock-immunized mice following an aerosol challenge. To gauge the true potential of this S1 DNA vaccine, the immune response and protective efficacy of the commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccine were included as the gold standard. Immunization with DTaP elicited a typically strong T-helper (Th)2-polarized immune response with significantly higher titres of serum IgG than in the DNA vaccine group, but a relatively weak Th1 response with low levels of IFN-gamma and IL-2 detected in the supernatants of antigen-stimulated splenocytes. DTaP-immunized mice cleared the aerosol challenge more efficiently than DNA-immunized mice, with no detectable pathogen after day 7 post-challenge.

Black Point (BP) is a dark discolouration of the embryo end of wheat and barley grains. It can result in reduced grain quality and value and is a significant problem in most Australian wheat growing areas. Estimated losses through downgrading have been as high as $50 million annually. Quantitative trait loci (QTL) for BP resistance have previously been identified in Sunco and Cascades. The aim of this study was to use a full diallele to validate these QTL and also to determine if two other varieties with increased resistance mirror existing sources of resistance or are potentially novel sources of resistance for BP. The BP resistant lines Lang, Cascades, SW95-50213, and Genaro, and one BP susceptible line, Cunningham were used as parental lines in the full diallele. As both Lang and Sunco are Cook derivatives, Lang was used to validate the Sunco QTL. Marker regression analysis confirmed the QTL on chromosome 2BS in Lang crosses. Genaro and SW95-50213, which are more resistant to BP than Sunco or Cascades, indicated an association between BP resistance and chromosomes 2B and 2A, respectively. The identification of markers for BP resistance in a number of different resistant sources will facilitate the pyramiding of genes for BP resistance in wheat.

Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

Flour yield quantitative trait loci (QTLs) were identified in 3 Australian doubled haploid populations, Sunco × Tasman, CD87 × Katepwa, and Cranbrook × Halberd. Trial data from 3 to 4 sites or years were available for each population. QTLs were identified on chromosomes 2BS, 4B, 5AL, and 6BL in the Sunco × Tasman population, on chromosomes 4B, 5AS, and 6DL in the CD87 × Katepwa population, and on chromosomes 4DS, 5DS, and 7AS in the Cranbrook × Halberd population. In the Sunco × Tasman cross the highest genetic variance was detected with the QTL on chromosome 2B (31.3%), in the CD87 × Katepwa cross with the QTL on chromosome 4B (23.8%), and in the Cranbrook × Halberd cross with the QTL on chromosome 5D (18%). Only one QTL occurred in a similar location in more than one population, indicating the complexity of the flour yield character across different backgrounds.

We investigate the idea of using diversified multiple trees for Microarray data classification. We propose an algorithm of Maximally Diversified Multiple Trees (MDMT), which makes use of a set of unique trees in the decision committee. We compare MDMT with some well-known ensemble methods, namely AdaBoost, Bagging, and Random Forests. We also compare MDMT with a diversified decision tree algorithm, Cascading and Sharing trees (CS4), which forms the decision committee by using a set of trees with distinct roots. Based on seven Microarray data sets, both MDMT and CS4 are more accurate on average than AdaBoost, Bagging, and Random Forests. Based on a sign test of 95% confidence, both MDMT and CS4 perform better than majority traditional ensemble methods tested. We discuss differences between MDMT and CS4.

In response to the rapid development of DNA Microarray technology, many classification methods have been used for Microarray classification. SVMs, decision trees, Bagging, Boosting and Random Forest are commonly used methods. In this paper, we conduct experimental comparison of LibSVMs, C4.5, BaggingC4.5, AdaBoostingC4.5, and Random Forest on seven Microarray cancer data sets. The experimental results show that all ensemble methods outperform C4.5. The experimental results also show that all five methods benefit from data preprocessing, including gene selection and discretization, in classification accuracy. In addition to comparing the average accuracies of ten-fold cross validation tests on seven data sets, we use two statistical tests to validate findings. We observe that Wilcoxon signed rank test is better than sign test for such purpose.

The immunogenicity of P97 adhesin repeat region R1 (P97R1) ofMycoplasma hyopneumoniae, an important pathogenesis-associated region of P97, was evaluated in mice as a mucosal vaccine. Mice were immunized orally with attenuatedSalmonella typhimurium aroAstrain CS332 harbouring a eukaryotic or prokaryotic expression vector encoding P97R1. Local and systemic immune responses were analysed by ELISA on mouse sera, lung washes and splenocyte supernatants following splenocyte stimulation with specific antigensin vitro. Although no P97R1-specific antibody responses were detected in serum and lung washes, significant gamma interferon was produced by P97R1-stimulated splenocytes from mice immunized orally withS. typhimurium aroAharbouring either expression system, indicating induction of a cell-mediated immune response. These results suggested that live bacterial vectors carrying DNA vaccines or expressing heterologous antigens preferentially induce a Th1 response. Surprisingly, however, mice immunized with the vaccine carrierS. typhimurium aroACS332 induced serum IgG, but not mucosal IgA, against P97R1 orS. typhimurium aroACS332 whole-cell lysate, emphasizing the importance of assessing the suitability of attenuatedS. typhimuriumantigen-carrier delivery vectors in the mouse model prior to their evaluation as potential vaccines in the target species, which in this instance was pigs.

A wheat×maize induced doubled haploid population that segregates at the Awned locus for awned and awnless phenotypes were studied at two field sites using a genetic linkage map. Interval QTL analysis indicated that significant QTLs for wheat flour water absorption and protein content were located on a linkage group associated with the morphological marker, awns. The QTL peak for flour water absorption was located at the Awned locus (B1, 5AL), whilst the QTL peak for protein content was located nearby, 10.1cm away from the Awned locus. The locations of those QTL were confirmed by analysing data from two independent field trials conducted under different environment conditions. The QTL identified for water absorption controlled 12% and 11% of the observed variance at the two field trials, whilst for flour protein content the QTL explained 7% and 19% of the variance respectively. Variance component analysis indicated that the QTL for water absorption controlled approximately 14.8–25.0% and 13.6–23% of the genetic variance at the two sites studied (Roma and Jimbour) whilst the QTL for protein content explained between 12.8% and 30.4% of the genetic variance at Roma and 34.7–82.6% at Jimbour. Cross-site analysis with composite interval mapping approach resulted in significant LOD values of 6.12 and 9.94 for water absorption and protein content, respectively. The QTL for water absorption was independent from the hardness locus.

Spring frosts in wheat growing areas of Australia can result in significant damage during the heading stage, resulting in losses millions of dollars each year due to decreased yields. To address this issue, wheat has been co-transformed via micro-projectile bombardment with an antifreeze protein (AFP) gene targeted to the apoplast or endoplasmic reticulum. T0 transgenic plants expressed AFP and showed the potential for frost tolerance in in vitro tests on plant extracts (Khanna and Daggard 2005). Additional transgenic wheat plants carrying the AFP gene targeted to both apoplast and ER have subsequently been produced. DNA extracted from plants surviving selection on phosphinothricin revealed 63.8% were PCR positive for presence of the AFP gene, indicating a wheat transformation frequency of 3.97%. Co-transformation of AFP and bar genes occurred in 53% of samples tested. PCR and gene expression analysis (via RT-PCR) have been carried out on plants that were subsequently tested in a controlled frost environment at the Australian Genome Research Facility (AGRF Adelaide). Analysis of frost induced sterility (FIS) showed that frosted control plants were significantly higher in FIS compared to unfrosted controls, and that FIS of plants transformed with one of the constructs was not significantly different to that of the unfrosted controls. This result suggests that the introduced AFP may protect the transgenic wheat plants against frost.

In recent years, the rapid development of DNA Microarray technology has made it possible for scientists to monitor the expression level of thousands of genes in a single experiment. As a new technology, Microarray data presents some fresh challenges to scientists since Microarray data contains a large number of genes (around tens thousands) with a small number of samples (around hundreds). Both filter and wrapper gene selection methods aim to select the most informative genes among the massive data in order to reduce the size of the expression database. Gene selection methods are used in both data preprocessing and classification stages. We have conducted some experiments on different existing gene selection methods to preprocess Microarray data for classification by benchmark algorithms SVMs and C4.5. The study suggests that the combination of filter and wrapper methods in general improve the accuracy performance of gene expression Microarray data classification. The study also indicates that not all filter gene selection methods help improve the performance of classification. The experimental results show that among tested gene selection methods, Correlation Coefficient is the best gene selection method for improving the classification accuracy on both SVMs and C4.5 classification algorithms.

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-γ level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-γ was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-γ production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.