A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Notes on the type, synonyms, and other specimens of the balansioid fungus, Nigrocornus scleroticus

The type, and other specimens of the balansioid fungus, Nigrocornus scleroticus, its synonyms, and similar fungi studied during extensive research on the taxonomy and biology of the fungus, are described. Two hypocrealean fungi found parasitising the ascostromata of N. scleroticus are also discussed.

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.

The performance of irrigated mixtures of tall fescue, ryegrass and white clover in subtropical Australia. 2. The effects on yield and botanical composition of managing for quality.

The effects on yield, botanical composition and persistence, of using a variable defoliation schedule as a means of optimising the quality of the tall fescue component of simple and complex temperate pasture mixtures in a subtropical environment was studied in a small plot cutting experiment at Gatton Research Station in south-east Queensland. A management schedule of 2-, 3- and 4-weekly defoliations in summer, autumn and spring and winter, respectively, was imposed on 5 temperate pasture mixtures: 2 simple mixtures including tall fescue (Festuca arundinacea) and white clover (Trifolium repens); 2 mixtures including perennial ryegrass (Lolium perenne), tall fescue and white clover; and a complex mixture, which included perennial ryegrass, tall fescue, white, red (T. pratense) and Persian (T. resupinatum) clovers and chicory (Cichorium intybus). Yield from the variable cutting schedule was 9% less than with a standard 4-weekly defoliation. This loss resulted from reductions in both the clover component (13%) and cumulative grass yield (6%). There was no interaction between cutting schedule and sowing mixture, with simple and complex sowing mixtures reacting in a similar manner to both cutting schedules. The experiment also demonstrated that, in complex mixtures, the cutting schedules used failed to give balanced production from all sown components. This was especially true of the grass and white clover components of the complex mixture, as chicory and Persian clover components dominated the mixtures, particularly in the first year. Quality measurements (made only in the final summer) suggested that variable management had achieved a quality improvement with increases in yields of digestible crude protein (19%) and digestible dry matter (9%) of the total forage produced in early summer. The improvements in the yields of digestible crude protein and digestible dry matter of the tall fescue component in late summer were even greater (28 and 19%, respectively). While advantages at other times of the year were expected to be smaller, the data suggested that the small loss in total yield was likely to be offset by increases in digestibility of available forage for grazing stock, especially in the critical summer period.

The performance of irrigated mixtures of tall fescue, ryegrass and white clover in subtropical Australia. 1. The effects of sowing mixture combinations, nitrogen and oversowing on establishment, productivity, botanical composition and persistence.

In the subtropics of Australia, the ryegrass component of irrigated perennial ryegrass (Lolium perenne) - white clover (Trifolium repens) pastures declines by approximately 40% in the summer following establishment, being replaced by summer-active C4 grasses. Tall fescue (Festuca arundinacea) is more persistent than perennial ryegrass and might resist this invasion, although tall fescue does not compete vigorously as a seedling. This series of experiments investigated the influence of ryegrass and tall fescue genotype, sowing time and sowing mixture as a means of improving tall fescue establishment and the productivity and persistence of tall fescue, ryegrass and white clover-based mixtures in a subtropical environment. Tall fescue frequency at the end of the establishment year decreased as the number of companion species sown in the mixture increased. Neither sowing mixture combinations nor sowing rates influenced overall pasture yield (of around 14 t/ha) in the establishment year but had a significant effect on botanical composition and component yields. Perennial ryegrass was less competitive than short-rotation ryegrass, increasing first-year yields of tall fescue by 40% in one experiment and by 10% in another but total yield was unaffected. The higher establishment-year yield (3.5 t/ha) allowed Dovey tall fescue to compete more successfully with the remaining pasture components than Vulcan (1.4 t/ha). Sowing 2 ryegrass cultivars in the mixture reduced tall fescue yields by 30% compared with a single ryegrass (1.6 t/ha), although tall fescue alone achieved higher yields (7.1 t/ha). Component sowing rate had little influence on composition or yield. Oversowing the ryegrass component into a 6-week-old sward of tall fescue and white clover improved tall fescue, white clover and overall yields in the establishment year by 83, 17 and 11%, respectively, but reduced ryegrass yields by 40%. The inclusion of red (T. pratense) and Persian (T. resupinatum) clovers and chicory (Cichorium intybus) increased first-year yields by 25% but suppressed perennial grass and clover components. Yields were generally maintained at around 12 t/ha/yr in the second and third years, with tall fescue becoming dominant in all 3 experiments. The lower tall fescue seeding rate used in the first experiment resulted in tall fescue dominance in the second year following establishment, whereas in Experiments 2 and 3 dominance occurred by the end of the first year. Invasion by the C4 grasses was relatively minor (<10%) even in the third year. As ryegrass plants died, tall fescue and, to a lesser extent, white clover increased as a proportion of the total sward. Treatment effects continued into the second, but rarely the third, year and mostly affected the yield of one of the components rather than total cumulative yield. Once tall fescue became dominant, it was difficult to re-introduce other pasture components, even following removal of foliage and moderate renovation. Severe renovation (reducing the tall fescue population by at least 30%) seems a possible option for redressing this situation.

Revision of a Australoxenella Howden & Storey in Australia (Coleoptera: Scarabaeidae: Aphodiinae)

Australoxeneffa Howden & Storey, the only Australian member of the aphodiine tribe Stereomerini, is revised. Eleven species are described of which the following are new: concinna, kalpara, midgee, mirreen, moogoon, peckonim, teeta, wurrook, zborowskii. Relationships between species are discussed. Most new specimens were taken in flight interception traps. No information is available on the biology of these suspected termitophiles.

Nocturnal Perching of Scarabaeine Dung Beetles (Coleoptera, Scarabaeidae) in an Australian Tropical Rain Forest

Wongabel, a northeastern Queensland tropical, wet, evergreen forest, contains 22 species of Scarabaeinae dung beetles. Five of these species were observed to perch commonly on leaves at night. Length of the beetle and the height of its perch were recorded for each of 56 1 specimens. Unlike the New World tropical dung beetle perchers, no clear evidence was found that small species perched closer to the ground than larger species. The evidence gathered, at least for the four most common perchers, supports the hypothesis that perching is one type of foraging strategy. The similarities and differences between the Australian and New World perchers are discussed.

The Biosynthesis of Sesquiterpene Isocyanides and Isothiocyanates in the Marine Sponge Acanthella cavernosa (Dendy); Evidence for Dietary Transfer to the Dorid Nudibranch Phyllidiella pustulosa

The tropical marine sponge Acanthella cavernosa (Dendy) converts potassium [14C] cyanide to axisonitrile-3 (1); this precursor is also used for the synthesis of axisothiocyanate-3 (2) suggesting that isocyanides are precursors to isothiocyanates in A. cavernosa. Likewise, potassium [14C] thiocyanate is used for the synthesis of axisothiocyanate-3; unexpectedly this precursor also labelled axisonitrile-3. These results demonstrate either an interconversion between cyanide and thiocyanate prior to secondary metabolite formation or that the secondary metabolites can themselves be interconverted. Specimens of the dorid nudibranch Phyllidiellu pustulosa, preadapted to a diet of A. cavernosa, fed on 14C-labelled sponges and were subsequently found to contain the radioactive terpenes (1) and (2). Specimens of P. pustulosa, which had not expressed a dietary preference for A. cavernosa in the field, did not generally feed in aquarium tests with 14C-labelled sponges and, therefore, provided non-radioactive extracts. Since control experiments demonstrated the inability of P. pustulosa to synthesise the metabolites de novo, we therefore conclude that P. pustulosa acquires secondary metabolites by dietary transfer from A. cavernosa.

Phylogenetic considerations for predicting the host range of Ustilago sporoboli-indici, a potential biological control agent for Sporobolus species in Australia

The ribosomal DNA internal transcribed spacer region was amplified and sequenced from a selection of specimens of the Sporobolus smut Ustilago sporoboli-indici. Phylogenetic comparison with other Ustilago and Sporisorium species revealed strong support for an evolutionary radiation of Ustilago species infecting the Chloridoideae and Pooideae, of which U. sporoboli-indici forms a major lineage. Comparisons are made with other groups of plant pathogenic fungi, and it is concluded that phylogenetic analyses of potential biocontrol agents are useful for identifying pathogens that are derived from evolutionary lineages that parasitize a wide range of unrelated plants. Such pathogens are less desirable as biocontrol agents as they may have a greater likelihood of infecting plants outside their normal host ranges.

Borate protection of softwood from Coptotermes acinaciformis (Isoptera: Rhinotermitidae) damage: Variation in protection thresholds explained

Laboratory and field data reported in the literature are confusing with regard to “adequate” protection thresholds for borate timber preservatives. The confusion is compounded by differences in termite species, timber species and test methodology. Laboratory data indicate a borate retention of 0.5% mass/mass (m/m) boric acid equivalent (BAE) would cause >90% termite mortality and restrict mass loss in test specimens to ≤5%. Field data generally suggest that borate retentions appreciably >0.5% m/m BAE are required. We report two field experiments with varying amounts of untreated feeder material in which Coptotermes acinaciformis (Froggatt) (Isoptera: Rhinotermitidae) responses to borate-treated radiata (Monterey) pine, Pinus radiata D. Don, were measured. The apparently conflicting results between laboratory and field data are explained by the presence or absence of untreated feeder material in the test environment. In the absence of untreated feeder material, wood containing 0.5% BAE provided adequate protection from Coptotermes sp., whereas in the presence of untreated feeder material, increased retentions were required. Furthermore, the retentions required increased with increased amounts of susceptible material present. Some termites, Nasutitermes sp. and Mastotermes darwiniensis Froggatt, for example, are borate-tolerant and borate timber preservatives are not a viable management option with these species. The lack of uniform standards for termite test methodology and assessment criteria for efficacy across the world is recognized as a difficulty with research into the performance of timber preservatives with termites. The many variables in laboratory and field assays make “prescriptive” standards difficult to recommend. The use of “performance” standards to define efficacy criteria (“adequate” protection) is discussed.

Field assessment of the resistance of timber products to termite (Isoptera) damage: Results can change with the amount of bait wood

Whether or not termites initiate damage to timber via the end grain may determine the need for spot-treating the exposed untreated cut ends of envelope-treated softwood framing material. Australian Coptotermes acinaciformis (Froggatt) were field-tested for their ability to initiate feeding via the end grain of timber (35 × 90 mm) treated with a repellent Tanalith® T envelope. Specimens of commercial radiata pine Pinus radiata D.Don framing timber (untreated) and slash pine Pinus elliottii Englem. (untreated and envelope-treated) were partially clad in fine stainless steel mesh. Clad and unclad specimens were exposed to C. acinaciformis near Townsville, North Queensland, Australia, for four months. Results showed that this species of termite can indeed damage timber via the end grain, including exposed untreated cut ends of envelope-treated material as demonstrated earlier for different populations of C. acinaciformis. Differences between the test conditions in field trials carried out at different times (where C. acinaciformis either did or did not damage timber via the end grain) are discussed. Clearly, outcomes from field studies with preservative-treated materials are dependent upon experimental conditions. Notably, the amount of bait wood (highly termite-susceptible timber substrate) offered in a given method can strongly influence the termite response. Further investigation is required to standardise this aspect of conditions in protocols for the assessment of wood preservatives.

New records of the River Shark Glyphis (Carcharhinidae) reported from Cape York Peninsula, northern Australia

The distribution of the river shark Glyphis in northern Australia is extended with new records of occurrence in the Gulf of Carpentaria and a reassessment of historical survey data from Cape York Peninsula. Nine new specimens of Glyphis sp. A were collected in 2005 from the Weipa region on the Queensland coast of the Gulf of Carpentaria. A re-examination of archival records from 1978-86 marine and estuarine fish surveys in the Gulf of Carpentaria and along the northern Queensland East Coast allowed a further nineteen Glyphis specimens to be identified. Combined this gives twenty-eight new records of Glyphis specimens from the coasts of Cape York Peninsula, Queensland. Common habitat characteristics for all captures were turbid, shallow, fast running tidal water in the upper reaches of coastal rivers. The substrate was generally muddy and the rivers lined with mangrove. In all surveys the representation of Glyphis was low, being less than 1% of the total shark captures historically and 0.002 sharks 50 m net hour-1 in Weipa 2005. The size range captured was 1000-1800 mm total length historically and 705-1200 mm total length from Weipa 2005, with none recorded as sexually mature. Diagnostic characteristics of the Weipa specimens, identified as Glyphis sp. A, were: lower jaw teeth protruding and "spear-like"; second dorsal fin greater than half the height of the first dorsal fin; the snout relatively short and fleshy in the lateral view; pectoral fin ventral surface black in colouration; the precaudal vertebral count between 118 and 123; and the total vertebral count between 204 and 209.

Fishery biology and management of Protonibea diacanthus (Sciaenidae) aggregations in far Northern Cape York Peninsula waters

The sciaenid Protonibea diacanthus is a large, long-lived predatory fish of inshore northern Australian waters, which forms annual aggregations that are fished extensively by traditional (subsistence) and recreational fishers. There are now widespread concerns that the resource is being overexploited. Indigenous fishers of the Cape York Northern Peninsula Area (NPA) relate that large adult fish (up to 1500 mm total length (TL)) made up the bulk of the catch from the sciaenid aggregations until about 1994. In contrast, sexually mature P. diacanthus comprised only a small component (12 fish out of 270=4.4%) examined in a 1999–2000 sampling programme that was biased towards the largest individuals available. At 790 mm TL, the minimum size at first maturity for female P. diacanthus in this study is much smaller than the 920 mm TL reported previously in Queensland waters. Developing ovaries were observed in specimens sampled from sciaenid aggregations which formed in NPA waters between May and September 2000. However, no fish with ripe or spent gonads were found in the study, so the current timing and location of the spawning season for P. diacanthus in the region remain unknown. Food items observed in the analysis of the diet of P. diacanthus from the NPA included a variety of teleosts and invertebrates. The range of animal taxa represented in the prey items support the description of an ‘opportunistic predator’ attributed to the species. In our sampling, the stomach contents of fish caught during the time of the aggregation events did not differ from those observed at other times of the year. A total of 114 P. diacanthus were tagged and released at aggregation sites during the study period, and 3 fish (2.6%) were subsequently recaptured. The low rate of tag returns from the wild stock tagging programme, both in this study (2.6%) and from recreational fisher tag/release programmes for the sciaenid elsewhere in Queensland (6.5%), were not explained by tag loss nor mortality, given the high retention rate of tags and the zero mortality seen in tank trials. In response to the biological findings from this study, indigenous community councils of the NPA imposed a 2-year fishing moratorium for P. diacanthus. Surveys at aggregation sites in 2002 and 2003 established that much larger fish (mean size 103.5 cm TL) were again present on the grounds, albeit in very low numbers. These recent preliminary results highlight the critical need for continued monitoring and management of the P. diacanthus fishery in the NPA, if prospects for resource recovery are to be realised. The NPA initiative has provided a rare opportunity to negotiate a co-management strategy, based on scientific data and traditional knowledge, for the recovery of a cultural and economically significant fished resource.

An annotated checklist of smut fungi (Ustilaginomycetes) from Thailand

An annotated checklist of the smut fungi (Ustilaginomycetes) from Thailand has been compiled after an examination of the scientific literature, previously deposited herbarium specimens and specimens collected by the authors during a survey in December 2005. Fifty-two species of smut fungi are listed, including 11 species which were newly discovered during our survey. Most of these smut fungi are reported for the first time from Thailand. Several species are very rare, being known only from the type material or a limited number of collections.