Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.

Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice.

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-[gamma] 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-[gamma] 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-[gamma] 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.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Achievements in forest tree improvement in Australia and New Zealand 6: Genetic improvement and conservation of Araucaria cunninghamii in Queensland

Araucaria cunninghamii (hoop pine) typically occurs as an emergent tree over subtropical and tropical rainforests, in a discontinuous distribution that extends from West Irian Jaya at about 0°30'S, through the highlands of Indonesian New Guinea and Papua New Guinea, along the east coast of Australia from 11°39'S in Queensland to 30°35'S in northern New South Wales. Plantations established in Queensland since the 1920s now total about 44000 ha, and constitute the primary source for the continuing supply of hoop pine quality timber and pulpwood, with a sustainable harvest exceeding 440 000 m3 y-1. Establishment of these managed plantations allowed logging of all native forests of Araucaria species (hoop pine and bunya pine, A. bidwillii) on state-owned lands to cease in the late 1980s, and the preservation of large areas of araucarian forest types within a system of state-owned and managed reserves. The successful plantation program with this species has been strongly supported by genetic improvement activities since the late 1940s - through knowledge of provenance variation and reproductive biology, the provision of reliable sources of improved seed, and the capture of substantial genetic gains in traits of economic importance (for example growth, stem straightness, internode length and spiral grain). As such, hoop pine is one of the few tropical tree species that, for more than half a century, has been the subject of continuous genetic improvement. The history of commercialisation and genetic improvement of hoop pine provides an excellent example of the dual economic and conservation benefits that may be obtained in tropical tree species through the integration of gene conservation and genetic improvement with commercial plantation development. This paper outlines the natural distribution and reproductive biology of hoop pine, describes the major achievements of the genetic improvement program in Queensland over the past 50+ y, summarises current understanding of the genetic variation and control of key selection traits, and outlines the means by which genetic diversity in the species is being conserved.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Assessment and rationalization of genetic diversity of Papua New Guinea taro (Colocasia esculenta) using SSR DNA fingerprinting

Taro (Colocasia esculenta) accessions were collected from 15 provinces of Papua New Guinea (PNG). The collection, totalling 859 accessions was collated for characterization and a core collection of 81 accessions (10%) was established on the basis of characterization data generated on 30 agro-morphological descriptors, and DNA fingerprinting using seven SSR primers. The selection of accessions was based on cluster analysis of the morphological data enabling initial selection of 20% accessions. The 20% sample was then reduced and rationalized to 10% based on molecular data generated by SSR primers. This represents the first national core collection of any species established in PNG based on molecular markers. The core has been integrated with core from other Pacific Island countries, contributing to a Pacific regional core collection, which is conserved in vitro in the South Pacific Regional Germplasm Centre at Fiji. The core collection is a valuable resource for food security of the South Pacific region and is currently being utilized by the breeding programmes of small Pacific Island countries to broaden the genetic base of the crop.

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.

The effects of high stocking rates on milk production from dryland and irrigated Mediterranean pastures

An experiment using herds of similar to 20 cows (farmlets) assessed the effects of high stocking rates on production and profitability of feeding systems based on dryland and irrigated perennial ryegrass-based pastures in a Mediterranean environment in South Australia over 4 years. A target level of milk production of 7000 L/cow.year was set, based on predicted intakes of 2.7 t DM/cow.year as concentrates, pasture intakes from 1.5 to 2.7 t/cow.year and purchased fodder. In years 1 and 2, up to 1.5 t DM/cow.year of purchased fodder was used and in years 3 and 4 the amounts were increased if necessary to enable levels of milk production per cow to be maintained at target levels. Cows in dryland farmlets calved in March to May inclusive and were stocked at 2.5, 2.9, 3.3, 3.6 and 4.1 cows/ha, while those in irrigated farmlets calved in August to October inclusive and were stocked at 4.1, 5.2, 6.3 and 7.4 cows/ha. In the first 2 years, when inputs of purchased fodder were limited, milk production per cow was reduced with higher stocking rates (P < 0.01), but in years 3 and 4 there were no differences. Mean production was 7149 kg/cow.year in years 1 and 2, and 8162 kg/cow.year in years 3 and 4. Production per hectare was very closely related to stocking rate in all years (P < 0.01), increasing from 18 to 34 t milk/ha.year for dryland farmlets (1300 to 2200 kg milk solids/ha) and from 30 to 60 t milk/ha.year for irrigated farmlets (2200 to 4100 kg milk solids/ha). Almost all of these increases were attributed to the increases in grain and purchased fodder inputs associated with the increases in stocking rate. Net pasture accumulation rates and pasture harvest were generally not altered with stocking rate, though as stocking rate increased there was a change to more of the pasture being grazed and less conserved in both dryland and irrigated farmlets. Total pasture harvest averaged similar to 8 and 14 t DM/ha.year for dryland and irrigated pastures, respectively. An exception was at the highest stocking rate under irrigation, where pugging during winter was associated with a 14% reduction in annual pasture growth. There were several indications that these high stocking rates may not be sustainable without substantial changes in management practice. There were large and positive nutrient balances and associated increases in soil mineral content (P < 0.01), especially for phosphorus and nitrate nitrogen, with both stocking rate and succeeding years. Levels under irrigation were considerably higher (up to 90 and 240 mg/kg of soil for nitrate nitrogen and phosphorus, respectively) than under dryland pastures (60 and 140 mg/kg, respectively). Soil organic carbon levels did not change with stocking rate, indicating a high level of utilisation of forage grown. Weed ingress was also high (to 22% DM) in all treatments and especially in heavily stocked irrigated pastures during winter. It was concluded the higher stocking rates used exceeded those that are feasible for Mediterranean pastures in this environment and upper levels of stocking are suggested to be 2.5 cows/ha for dryland pastures and 5.2 cows/ha for irrigated pastures. To sustain these suggested stocking rates will require further development of management practices to avoid large increases in soil minerals and weed invasion of pastures.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes - adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

Development of profitable milk production systems for northern Australia: a field assessment of the productivity of five potential farming systems using farmlets.

Farmlets, each of 20 cows, were established to field test five milk production systems and provide a learning platform for farmers and researchers in a subtropical environment. The systems were developed through desktop modelling and industry consultation in response to the need for substantial increases in farm milk production following deregulation of the industry. Four of the systems were based on grazing and the continued use of existing farmland resource bases, whereas the fifth comprised a feedlot and associated forage base developed as a greenfield site. The field evaluation was conducted over 4 years under more adverse environmental conditions than anticipated with below average rainfall and restrictions on irrigation. For the grazed systems, mean annual milk yield per cow ranged from 6330 kg/year (1.9 cows/ha) for a herd based on rain-grown tropical pastures to 7617 kg/year (3.0 cows/ha) where animals were based on temperate and tropical irrigated forages. For the feedlot herd, production of 9460 kg/cow.year (4.3 cows/ha of forage base) was achieved. For all herds, the level of production achieved required annual inputs of concentrates of similar to 3 t DM/animal and purchased conserved fodder from 0.3 to 1.5 t DM/animal. This level of supplementary feeding made a major contribution to total farm nutrient inputs, contributing 50% or more of the nitrogen, phosphorus and potassium entering the farming system, and presents challenges to the management of manure and urine that results from the higher stocking rates enabled. Mean annual milk production for the five systems ranged from 88 to 105% of that predicted by the desktop modelling. This level of agreement for the grazed systems was achieved with minimal overall change in predicted feed inputs; however, the feedlot system required a substantial increase in inputs over those predicted. Reproductive performance for all systems was poorer than anticipated, particularly over the summer mating period. We conclude that the desktop model, developed as a rapid response to assist farmers modify their current farming systems, provided a reasonable prediction of inputs required and milk production. Further model development would need to consider more closely climate variability, the limitations summer temperatures place on reproductive success and the feed requirements of feedlot herds.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.

Analysis of the barley bract suppression gene Trd1

A typical barley (Hordeum vulgare) floret consists of reproductive organs three stamens and a pistil, and non-reproductive organs-lodicules and two floral bracts, abaxial called 'lemma' and adaxial 'palea'. The floret is subtended by two additional bracts called outer or empty glumes. Together these organs form the basic structural unit of the grass inflorescence, a spikelet. There are commonly three spikelets at each rachis (floral stem of the barley spike) node, one central and two lateral spikelets. Rare naturally occurring or induced phenotypic variants that contain a third bract subtending the central spikelets have been described in barley. The gene responsible for this phenotype was called the THIRD OUTER GLUME1 (Trd1). The Trd1 mutants fail to suppress bract growth and as a result produce leaf-like structures that subtend each rachis node in the basal portion of the spike. Also, floral development at the collar is not always suppressed. In rice and maize, recessive mutations in NECK LEAF1 (Nl1) and TASSEL SHEATH1 (Tsh1) genes, respectively, have been shown to be responsible for orthologous phenotypes. Fine mapping of the trd1 phenotype in an F-3 recombinant population enabled us to position on the long arm of chromosome 1H to a 10 cM region. We anchored this to a conserved syntenic region on rice chromosome Os05 and selected a set of candidate genes for validation by resequencing PCR amplicons from a series of independent mutant alleles. This analysis revealed that a GATA transcription factor, recently proposed to be Trd1, contained mutations in 10 out of 14 independent trd1 mutant alleles that would generate non-functional TRD1 proteins. Together with genetic linkage data, we confirm the identity of Trd1 as the GATA transcription factor ortholog of rice Nl1 and maize Tsh1 genes.