David Berryman

The genus term Isospora is now applied specifically to parasites of birds, with the term Cystoisospora preferred for parasites which infect mammals. Isospora is a common parasitic coccidian in birds worldwide, especially in passerine birds, in which it can cause systemic coccidiosis. The complete mitochondrial genome sequences from two recently identified Isospora species; Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner, were sequenced and compared with those of other closely related coccidian species. The complete mitochondrial genome sequence for Isospora serinuse is 6260. bp in size and 6223. bp for Isospora manorinae. The mitochondrial genomes of Isospora serinuse and Isospora manorinae include three protein-coding genes (COI, COIII and CytB), 19 LSU and 14 SSU rDNA fragments, including one newly identified putative LSU fragment in Isospora sp. The arrangement of coding regions in these two Isospora species were identical to that of available Isospora sp. and Eimeria spp. mitochondrial genomes and the start codon usage for protein coding genes was conservative. Phylogenetic analysis of the mt genome of the two Isospora species based on the three coding regions further support that the monophyletic nature of avian Isospora.

Glucocorticoids induce lung fibroblasts to produce fibroblast-pneumocyte factor, a peptide that stimulates type II cells to synthesize pulmonary surfactant. This effect is known to be more apparent in cells derived from female fetuses, a characteristic that has been attributed to sex-linked differences in the fibroblasts. In the current study, it has been shown that dexamethasone enhances both β-adrenergic receptor (β-AR) activity (1.3- to 1.6-fold increase) and (-)-isoproterenol-induced secretion of surfactant (1.8- to 1.9-fold increase) in type II cells. However, fibroblast-conditioned media (FCM), prepared in the presence of dexamethasone, generates a much greater response to (-)-isoproterenol (3.1- to 3.8-fold increase). Furthermore, each of these effects is more pronounced if both cell types are female-derived. It is hypothesized that the enhanced response to glucocorticoids is the result of a synergistic effect between the steroid and a component of FCM. Neuregulin-1β (NRG1β), which is elevated in FCM generated in the presence of dexamethasone, influences not only the rate of surfactant secretion and the β-AR activity in type II cells, but also enhances in both sexes the cellular response to (-)-isoproterenol. These results suggest that NRG1β might be more effective than glucocorticoids in treating prematurely born male infants, which are known to respond poorly to glucocorticoids. Given that glucocorticoids are known to induce higher levels of β-AR mRNA, the effect of NRG1β, alone and in combination with dexamethasone, on β-AR gene expression was measured using qRTPCR. Whereas NRG1β had no effect alone, in combination with dexamethasone it produced up to a 4.2-fold elevation in the level of β-AR mRNA.

It is well established that glucocorticoids elevate the production of fibroblast-pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin-1β (NRG1β) in conditioned medium is elevated 2-fold (P < 0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3-fold the rate of phospholipid synthesis (P < 0.05), suggests that NRG1β is a component of FPF that promotes lung development.

Surfactant production is known to involve a cellular communication between lung fibroblasts and the type II pneumocytes. Glucocorticoids induce the production of a peptide by lung fibroblasts, fibroblast-pneumocyte factor (FPF), which sequentially acts on type II cells to enhance the synthesis of surfactant phospholipid. Our findings show that fibroblast-conditioned medium (FCM), generated in the presence of dexamethasone, not only enhanced surfactant phospholipid synthesis in type II cells but also contained an elevated concentration of neuregulin-1β (NRG1β). Even though it has been earlier proposed that leptin has many of the characteristics of FPF, recent research has revealed that NRG1β also has many similar attributes. In the current study, exposure of the type II cells to a commercially available form of NRG1β (heregulin-1β) directly stimulated by more than three-fold the rate of phospholipid synthesis (p <0.05). Although similar in magnitude, the effect of heregulin-1β appeared to require a longer time of exposure to that reported for leptin. There was no increase in the gene expression of NRG1β when lung fibroblasts were exposed to dexamethasone, irrespective of the concentration of dexamethasone used, or the time of contact of the cells to the steroid. Thus the glucocorticoid-induced increase in the level of NRG1β in FCM was not the result of enhanced expression of the NRG1β gene. The inability of dexamethasone to induce a significant increase in NRG1β gene expression in lung fibroblasts suggests that the elevated concentration of NRG1β might be the result of enhanced cleavage of the membrane-bound neuregulin precursor. In summary, these findings not only support but significantly extend the concept previously promoted that NRG1β plays an essential role in the differentiation and maturation of the lung in the later stages of gestation. Moreover, together these studies suggest that FPF may be a complex mixture of agents capable of motivating surfactant synthesis.

Further developments in fingermark detection rely heavily on improving our knowledge of latent fingermark composition. Investigations into the factors responsible for compositional variation , such as traits including age, gender and lifestyle habits , as well as a better understanding of fingermark degradation processes, are vital to overcome the limitations of current development methods. In recent years, there has been much interest in employing mass spectrometry imaging (MSI) for these purposes due to its ability to simultaneously identify and map the substances present on surfaces [1] . A number of investigations have applied SIMS, MALDI and DESI MSI techniques to the examination of both endogenous and exogenous fingermark components. Results so far indicate the potential of MSI to be applied to in - depth studies into the factors that influence finger mark composition. This presentation gives an overview of current developments in the field and reports on our progress so far in applying it to the study of latent fingermark chemistry

Many of the EPPs that pose the biggest threat to the biosecurity of Australia’s plant industries are bacterial, but difficulties in identification to the subspecific or ‘ pathovar ’ level can seriously delay incursion management and affect market access. Pathovars are defined by host specificity so bio assays remain the definitive means of identification, but these require high level physical containment and can be slow and subjective , delaying diagnosis . Some pathovar - specific serological and molecular tests are available but better diagnostic methods are often required. This project used proteomics and metabolomics, platforms that identify functional molecules potentially associated with plant - pathogen interactions, to identify biomarkers that differentiate pathovars in species of Xanthomonas . Membrane - associated proteins from a collection of bacterial isolates were compared on 2Dimensional gels. Proteins that were found to be differentially expressed between distinct pathovars may be important modulators of host specificity so they were identified and the genes that encode them located by reference to genomic sequences . DNA - based assays targeting these genes were designed and validated for their specificity to the pathovar level . We have developed two new assays that provide levels of specificity not reported elsewhere in the literature. These assays specifically target the bacteria causing the different forms of citrus canker, but without cross - reaction to the closely - related organisms causing bacterial blight on cotton and Citrus Bacterial Spot. The molecular assays will be incorporated into the National Diagnostic Protocol for citrus canker through the SPHDS process. The metabolomics component has analysed metabolite expression in selected bacterial pathovars. Results showed separation between the different pathovars based on differential levels of expression of particular metabolites. These metabolites may be important determinants of pathogenicity. Neither proteomics nor metabolomics had been implemented before in the study of phytopathogenic bacteria and whilst both proved to be technically demanding, each delivered new biomarkers that differentiate phytopathogenic bacteria to a subspecific level . This confirmed the viability of these approaches as platforms to discover novel diagnostic targets. The new methods developed will be implemented into the national incursion response capability , improving the specificity of diagnostic testing available and reducing the possibility of false positive diagnosis . The project has fostered new collaborative partnerships both nationally (NSW, Victoria, WA) and internationally (to Thailand and the USA). The next phase of this work will provide a strong start - up project to the Plant Biosecurity Cooperative Research Centre ( PBCRC ) . This project has directly enhanced the plant bacteriology capacity of NSW and Australia trough the recruitment and training of science professionals and an undergraduate student , and supported the specialist training of a Thai scientist through allied project CRC20093.

Many of the biggest threats to the biosecurity of Australia’s plant industries are bacterial, but difficulties in identification to pathovar level could seriously delay incursion management and affect market access. Pathovars are defined by host specificity so bioassays remain the definitive means of identification, but require physical containment and can be slow and subjective. Some pathovar‐specific serological and molecular tests are available but better diagnostic methods are often required. We have evaluated the use of proteomics and metabolomics, platforms that identify functional molecules potentially associated with plant‐pathogen interactions, to identify biomarkers that differentiate pathovars in Xanthomonas species. The proteomics component has focused on profiling membrane‐associated proteins extracted from selected bacterial isolates. Profiles show isolates of the same pathovar cluster together and proteins are differentially expressed between distinct pathovars. Differentially expressed proteins have been analysed by digestions and mass spectrometry and the genes that encode them identified by reference to genomic sequences. Based on this information, molecular tests to differentiate the pathovars are being designed and validated. The metabolomics component has analysed metabolite expression in selected bacterial pathovars. Results show separation between the different pathovars and differentially expressed metabolites are evident.

Sowing seed stocks with minimal virus content provides a key control measure in preventing damaging epidemics of Cucumber mosaic virus (CMV) in crops of narrow-leafed lupin (Lupinus angustifolius). A seed testing service provides an estimate of percent CMV infection based on a dry seed test in which bulked subsamples of ungerminated seed are ground to a fine powder for testing. When enzyme-linked immunosorbent assay (ELISA) was used, CMV antiserum that gave low background optical density (A405) values with extracts of powder from subsamples of healthy seed provided greatest accuracy, readily detecting one infected seed in subsamples of 100 seeds. In comparative ELISAs on duplicate subsamples from eight different seed stocks, germination and dry seed tests always gave similar percent infection values. When seed coats were separated from the embryos of CMV-infected and healthy lupin seeds before testing by ELISA, the virus was only detected in embryos from infected seeds and never in their seed coats. Treatment with trisodium phosphate did not alter the low ELISA optical density (A405) values obtained with seed coats separated from infected seeds. Therefore, seed coat contamination with CMV is lacking in lupin, justifying large-scale routine use of a dry seed test to estimate percent virus infection in commercial seed samples.