Isolation of multiple novel paramyxoviruses from pteropid bat urine

Bats have been found to harbor a number of new emerging viruses with zoonotic potential and there has been a great deal of interest in identifying novel bat pathogens to determine risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid, however virus isolation is still a challenge and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports we have described the isolation of Hendra virus, Menangle virus and Cedar virus, in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.

Novel paramyxoviruses in Australian flying-fox populations support host–virus co-evolution

Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses (UPVs) in Australian flying-fox species (Pteropus alecto, Pteropus scapulatus, Pteropus poliocephalus and Pteropus conspicillatus) and in the Christmas Island species Pteropus melanotus natalis. Using a degenerative reverse transcription-PCR specific for the L gene of known species of the genus Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously UPVs, with a representative 31 UPVs clustering in eight distinct groups (100 UPVs/495 samples). No new henipaviruses were identified. The findings were consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Review of bats and SARS

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horse-shoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses.

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.

Suppression of populations of Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), with a novel blowfly trap

The Australian sheep blowfly, Lucilia cuprina initiates more than 85% of fly strikes on sheep in Australia with an estimated average annual cost of A$280 million to the Australian wool industry. LuciTrap® is a commercially available, selective trap for L. cuprina consisting of a plastic bucket with multiple fly entry cones and a synthetic attractant. The impact of LuciTrap on populations of L. cuprina on sheep properties in five Australian states was evaluated by comparing L. cuprina populations on paired properties with and without LuciTraps over seasons when significant fly populations could be expected. Twenty-four comparisons (trials) were conducted over four years. During times of ‘higher fly density’ (when the 48 h geometric mean of trap catches on the control property was greater than five L. cuprina), the overall geometric mean trap catches for control and trapped properties differed significantly (P<0.001) with mean trap catches of 19.4 and 7.74 L. cuprina respectively. The selectivity of the LuciTrap was confirmed with 59% of all trapped flies being L. cuprina. Chrysomya spp. and Calliphora spp. constituted 9.3% and 1.1% of the catches with a variety of other flies (mainly Sarcophagidae and Muscidae) providing the remainder (31%). L. sericata was only trapped in Tasmania and made up 7.7% of the Lucilia spp. catch in this State. Seventy-two percent of the trapped L. cuprina were female. The deployment of LuciTrap on sheep properties at one trap per 100 sheep from the beginning of the anticipated fly season suppressed the populations of L. cuprina by 60% compared to matched control properties. The LuciTrap is a selective and easy to use fly trap and constitutes an effective, non-insecticidal tool for use in integrated management programs for L. cuprina.

Tadpole mouthpart depigmentation as an accurate indicator of chytridiomycosis, an emerging disease of amphibians

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis, and its role in causing population declines and species extinctions worldwide has created an urgent need for methods to detect it. Several reports indicate that in anurans chytridiomycosis can cause the depigmentation of tadpole tnouthparts, but the accuracy of using depigmentation to determine disease status remains uncertain. Our objective was to determine for the Mountain Yellow-legged Frog (Rana muscosa) whether visual inspections of the extent of tadpole mouthpart depigmentation could be used to accurately categorize individual tadpoles or R. muscosa populations as B. dendrobatidis-positive or negative. This was accomplished by assessing the degree of mouthpart depigmentation in tadpoles of known disease status (based on PCR assays). The depigmentation of R. muscosa tadpole mouthparts was associated with the presence of B. dendrobatidis, and this association was particularly strong for upper jaw sheaths. Using a rule that classifies tadpoles with upper jaw sheaths that are 100% pigmented as uninfected and those with jaw sheaths that are <100% pigmented as infected resulted in the infection status of 86% of the tadpoles being correctly classified. By applying this rule to jaw sheath pigmentation scores averaged across all tadpoles inspected per site, we were able to correctly categorize the infection status of 92% of the study populations. Similar research on additional anurans is critically needed to determine how broadly applicable our results for R. muscosa are to other species.

Management of plant invasions mediated by frugivore interactions

1. Some of the most damaging invasive plants are dispersed by frugivores and this is an area of emerging importance in weed management. It highlights the need for practical information on how frugivores affect weed population dynamics and spread, how frugivore populations are affected by weeds and what management recommendations are available. 2. Fruit traits influence frugivore choice. Fruit size, the presence of an inedible peel, defensive chemistry, crop size and phenology may all be useful traits for consideration in screening and eradication programmes. By considering the effect of these traits on the probability, quality and quantity of seed dispersal, it may be possible to rank invasive species by their desirability to frugivores. Fruit traits can also be manipulated with biocontrol agents. 3. Functional groups of frugivores can be assembled according to broad species groupings, and further refined according to size, gape size, pre- and post-ingestion processing techniques and movement patterns, to predict dispersal and establishment patterns for plant introductions. 4. Landscape fragmentation can increase frugivore dispersal of invasives, as many invasive plants and dispersers readily use disturbed matrix environments and fragment edges. Dispersal to particular landscape features, such as perches and edges, can be manipulated to function as seed sinks if control measures are concentrated in these areas. 5. Where invasive plants comprise part of the diet of native frugivores, there may be a conservation conflict between control of the invasive and maintaining populations of the native frugivore, especially where other threats such as habitat destruction have reduced populations of native fruit species. 6. Synthesis and applications. Development of functional groups of frugivore-dispersed invasive plants and dispersers will enable us to develop predictions for novel dispersal interactions at both population and community scales. Increasingly sophisticated mechanistic seed dispersal models combined with spatially explicit simulations show much promise for providing weed managers with the information they need to develop strategies for surveying, eradicating and managing plant invasions. Possible conservation conflicts mean that understanding the nature of the invasive plant-frugivore interaction is essential for determining appropriate management.

Action learning in partnership with Landcare and catchment management groups to support increased pasture sowings in southern inland Queensland

The incorporation of sown pastures as short-term rotations into the cropping systems of northern Australia has been slow. The inherent chemical fertility and physical stability of the predominant vertisol soils across the region enabled farmers to grow crops for decades without nitrogen fertiliser, and precluded the evolution of a crop–pasture rotation culture. However, as less fertile and less physically stable soils were cropped for extended periods, farmers began to use contemporary farming and sown pasture technologies to rebuild and maintain their soils. This has typically involved sowing long-term grass and grass–legume pastures on the more marginal cropping soils of the region. In partnership with the catchment management authority, the Queensland Murray–Darling Committee (QMDC) and Landcare, a pasture extension process using the LeyGrain™ package was implemented in 2006 within two Grain & Graze projects in the Maranoa-Balonne and Border Rivers catchments in southern inland Queensland. The specific objectives were to increase the area sown to high quality pasture and to gain production and environmental benefits (particularly groundcover) through improving the skills of producers in pasture species selection, their understanding and management of risk during pasture establishment, and in managing pastures and the feed base better. The catalyst for increasing pasture sowings was a QMDC subsidy scheme for increasing groundcover on old cropping land. In recognising a need to enhance pasture knowledge and skills to implement this scheme, the QMDC and Landcare producer groups sought the involvement of, and set specific targets for, the LeyGrain workshop process. This is a highly interactive action learning process that built on the existing knowledge and skills of the producers. Thirty-four workshops were held with more than 200 producers in 26 existing groups and with private agronomists. An evaluation process assessed the impact of the workshops on the learning and skill development by participants, their commitment to practice change, and their future intent to sow pastures. The results across both project catchments were highly correlated. There was strong agreement by producers (>90%) that the workshops had improved knowledge and skills regarding the adaptation of pasture species to soils and climates, enabling a better selection at the paddock level. Additional strong impacts were in changing the attitudes of producers to all aspects of pasture establishment, and the relative species composition of mixtures. Producers made a strong commitment to practice change, particularly in managing pasture as a specialist crop at establishment to minimise risk, and in the better selection and management of improved pasture species (particularly legumes and the use of fertiliser). Producers have made a commitment to increase pasture sowings by 80% in the next 5 years, with fourteen producers in one group alone having committed to sow an additional 4893 ha of pasture in 2007–08 under the QMDC subsidy scheme. The success of the project was attributed to the partnership between QMDC and Landcare groups who set individual workshop targets with LeyGrain presenters, the interactive engagement processes within the workshops themselves, and the follow-up provided by the LeyGrain team for on-farm activities.

Ptesculentoside, a novel norsesquiterpene glucoside from the Australian bracken fern Pteridium esculentum.

A novel norsesquiterpene glucoside ptesculentoside has been isolated from the Australian bracken Pteridium esculentum, together with the known bracken carcinogen ptaquiloside and lesser amounts of caudatoside. The structure of ptesculentoside is determined by analysis of 1D and 2D NMR spectra, and via its conversion into previously known pterosin G.

WHIPPET: a novel tool for prioritizing invasive plant populations for regional eradication

Large geographic areas can have numerous incipient invasive plant populations that necessitate eradication. However, resources are often deficient to address every infestation. Within the United States, weed lists (either state-level or smaller unit) generally guide the prioritization of eradication of each listed species uniformly across the focus region. This strategy has several limitations that can compromise overall effectiveness, which include spending limited resources on 1) low impact populations, 2) difficult to access populations, or 3) missing high impact populations of low priority species. Therefore, we developed a novel science-based, transparent, analytical ranking tool to prioritize weed populations, instead of species, for eradication and tested it on a group of noxious weeds in California. For outreach purposes, we named the tool WHIPPET (Weed Heuristics: Invasive Population Prioritization for Eradication Tool). Using the Analytic Hierarchy Process that included expert opinion, we developed three major criteria, four sub-criteria, and four sub-sub-criteria, taking into account both species and population characteristics. Subject matter experts weighted and scored these criteria to assess the relative impact, potential spread, and feasibility of eradication (major criteria) for 100 total populations of 19 species. Species-wide population scores indicated that conspecific populations do not necessarily group together in the final ranked output. Thus, priority lists based solely on species-level characteristics are less effective compared to a blended prioritization based on both species attributes and individual population and site parameters. WHIPPET should facilitate a more efficacious decision-making process allocating limited resources to target invasive plant infestations with the greatest predicted impacts to the region under consideration.

A novel species of mastrevirus (family Geminiviridae) isolated from Digitaria didactyla grass from Australia.

Mastreviruses (family Geminiviridae) that infect monocotyledonous plants occur throughout the temperate and tropical regions of Asia, Africa, Europe and Australia. Despite the identification of a very diverse array of mastrevirus species whose members infect African monocots, few such species have been discovered in other parts of the world. For example, the sequence of only a single monocot-infecting mastrevirus, Chloris striate mosaic virus (CSMV), has been reported so far from Australia, even though earlier biological and serological studies suggested that other distinct mastreviruses were present. Here, we have obtained the complete nucleotide sequence of a virus from the grass Digitaria didactyla originating from Australia. Analysis of the sequence shows the virus to be a typical mastrevirus, with four open reading frames, two in each orientation, separated by two non-coding intergenic regions. Although it showed the highest levels of sequence identity to CSMV (68.7%), their sequences are sufficiently diverse for the virus to be considered a member of a new species in the genus Mastrevirus, based on the present species demarcation criteria. We propose that the name first used during the 1980s be used for this species, Digitaria didactyla striate mosaic virus (DDSMV).

Two novel mastreviruses from chickpea (Cicer arietinum) in Australia

Two novel mastreviruses (genus Mastrevirus; family Geminiviridae), with proposed names chickpea chlorosis virus (CpCV) and chickpea redleaf virus, are described from chickpea (Cicer arietinum) from eastern Australia. The viruses have genomes of 2,582 and 2,605 nucleotides, respectively, and share similar features and organisation with typical dicot-infecting mastreviruses. Two distinct strains of CpCV were suggested by phylogenetic analysis. Additionally, a partial mastrevirus Rep sequence from turnip weed (Rapistrum rugosum) indicated the presence of a distinct strain of Tobacco yellow dwarf virus (TYDV). In phylogenetic analyses, isolates of Bean yellow dwarf virus, Chickpea chlorotic dwarf Pakistan virus and Chickpea chlorotic dwarf Sudan virus from southern and northern Africa and south-central and western Asia clustered separately from these three viruses from Australia. An Australian, eastern Asian, or south-eastern Asian origin for the novel mastreviruses and TYDV is discussed.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.