Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

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.

Large cryptic internal sequence repeats in protein structures from Homo sapiens

Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats - be it structure, function or evolution - would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.

Thermally Induced Acyl Migration in Salicylamides. 1. Structures of the Reactants, O-Acetylsalicylamide and O-Benzoylsalicylamide

O-Acetylsalicylamide (Ia), C9H9NO3, M r =179.18, monoclinic, P2Jc, a=8.155(5), b=8.571 (2), c= 13.092 (3)A, fl=99.54 (5) ° , V= 902.4(6)A 3, Z=4, Dm=l.31, Dx=l.319gcm -3, 2(Mo Ka) = 0.71069 A,/~ = 1.08 cm -1, F(000) = 376, T = 295 K, R = 0.076 for 1604 reflections. O-Benzoylsalicylamide (Ib), C14HtlNO 3, M,=241.2, monoclinic, P2t/e, a=9.423(1), b=5.116(1), e= 26.424 (2) A, fl= 103.97 (1)% V= 1236.2 (3)/~3, Z= 4, D~ = 1.28, D x = 1.296 gcm -3, ,;L(Cu Ks) = 1.5418 A, p = 7.71 cm-', F(000) = 504, T= 295 K, R =0.050 for 2115 reflections. The dihedral angles between the amide group and the benzene ring are 39.9 ° (Ia) and 37.9 ° (Ib), whereas between the acyl group and the benzene ring they are 78.1 ° (Ia) and 93.4 ° (Ib). The differences in the packing of the two structures are brought out in terms of the observed hydrogen-bonding patterns. Based on the crystallographic results, an intramolecular mechanism for the migration of the acyl group from the O to the N position is suggested in both compounds.

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.

The effect of crack closure on the grain size dependence of fatigue crack growth threshold

Recent studies (I-7) clearly indicate a strong dependence of fatigue threshold parameter, A K on grain size in several alloy systems. Attempts to explain these observations on the basis of crat~tortuosity (1,8), fracture surface roughness (5,9) and crack closure (6) appear to present a fairly clear picture of the mechanisms that cause a reduction in crack growth rates at threshold. In general, it has been shown that coarse grained microstructures exhibit higher fatigue threshold in low carbon steels (1,5) aluminium alloys (7) and titanium alloys (6). In spite of these observations, there exists (10-1#) considerable uncertainity about the manner in which the AK~L depends on prior austenitic grain size in quenched and tempered steels. Studies in quenched and tempered steels demonstrating both an increase (3,12,14) as well as a decrease (11,12) in AKth with an increase in prior austenitic grain size can be sought to illustrate this point. Occasionally , the absence of any sensitivity of AKth to the variations in prior austenitJc grain size has also been reported (11,13). While a few investigators (5-7) comfortably rationalised the grain size effects on AK~L on the basis of crack closure by a comparison in terms of the closure-free component of the thresho~Ifc~, AK -f such an approach has yet to be extended to high strength steels, An attempt has been made in t~et ,pthrg sent study to explai. n the effect of pri, or austeniti.c grain size on &Kth on the basis of crack closure measurements in a high strength steel.

Spatial and temporal variation and effects of changes in management in discard rates from the commercial reef line fishery of the Great Barrier Reef, Australia

Discarding in commercially exploited fisheries has received considerable attention in the last decade, though only more recently in Australia. The Reef Line fishery (RLF) of the Great Barrier Reef (GBR) in Australia is a large-scale multi-sector, multi-species, highly regulated hook and line fishery with the potential for high levels of discarding. We used a range of data sources to estimate discard rates and discard quantities for the two main target groups of the RLF, the coral trout, Plectropomus spp, and the red throat emperor, Lethrinus miniatus, and investigated possible effects on discarding of recent changes in management of the fishery. Fleet-wide estimates of total annual quantities discarded from 1989 to 2003 were 292-622 t and 33-95 t for coral trout and red throat emperor, respectively. Hypothetical scenarios of high-grading after the introduction of a total allowable commercial catch for coral trout resulted in increases in discard quantities up to 3895 t, while no high-grading still meant 421 t were discarded. Increasing the minimum size limit of red throat emperor from 35 to 38 cm also increased discards to an estimated 103 t. We provide spatially and temporally explicit estimates of discarding for the two most important species in the GBR RLF of Australia to demonstrate the importance of accounting for regional variation in quantification of discarding. Effects of management changes on discarding are also highlighted. This study provides a template for exploring discarding levels for other species in the RLF and elsewhere.

Motorcycle riders' self-reported aggression when riding compared with car driving

Aggressive driving has been shown to be related to increased crash risk for car driving. However, less is known about aggressive behaviour and motorcycle riding and whether there are differences in on-road aggression as a function of vehicle type. If such differences exist, these could relate to differences in perceptions of relative vulnerability associated with characteristics of the type of vehicle such as level of protection and performance. Specifically, the relative lack of protection offered by motorcycles may cause riders to feel more vulnerable and therefore to be less aggressive when they are riding compared to when they are driving. This study examined differences in self-reported aggression as a function of two vehicle types: passenger cars and motorcycles. Respondents (n = 247) were all motorcyclists who also drove a car. Results were that scores for the composite driving aggression scale were significantly higher than on the composite riding aggression scale. Regression analyses identified different patterns of predictors for driving aggression from those for riding aggression. Safety attitudes followed by thrill seeking tendencies were the strongest predictors for driving aggression, with more positive safety attitudes being protective while greater thrill seeking was associated with greater self-reported aggressive driving behaviour. For riding aggression, thrill seeking was the strongest predictor (positive relationship), followed by self-rated skill, such that higher self rated skill was protective against riding aggression. Participants who scored at the 85th percentile or above for the aggressive driving and aggressive riding indices had significantly higher scores on thrill seeking, greater intentions to engage in future risk taking, and lower safety attitude scores than other participants. In addition participants with the highest aggressive driving scores also had higher levels of self-reported past traffic offences than other participants. Collectively, these findings suggest that people are less likely to act aggressively when riding a motorcycle than when driving a car, and that those who are the most aggressive drivers are different from those who are the most aggressive riders. However, aggressive riders and drivers appear to present a risk to themselves and others on road. Importantly, the underlying influences for aggressive riding or driving that were identified in this study may be amenable to education and training interventions.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

from "The Roving Party"

They walked down through a dry creek bed lined with swamp gums grown so close together they appeared as one living whole. The men passed around these trees in single file, among sun shafts which pierced the canopy but threw no light upon their faces nor warmed their bones. In the gloom the air was thick with flies and the mushrooms grew like the sightless larvae of some queer and unnamed vermin. Before long they found themselves among a stand of trees which had been stripped of their bark for windbreaks. The naked trunks were carved over with bisected circles, detailings of the moon and sun, images of snakes and roo. The Parramatta men gazed at the finely wrought icons but John Batman found more to hold his attention. Pressed onto the flesh of the tree was a bloody handprint. Batman removed his hat and crouched to examine the ground and Black Bill joined him. One injured man had passed this way...

Children’s personal exposure to air pollution in rural villages in Bhutan

Exposure assessment studies conducted in developing countries have been based on fixed-site monitoring to date. This is a major deficiency, leading to errors in estimating the actual exposures, which are a function of time spent and pollutant concentrations in different microenvironments. This study quantified school children’s daily personal exposure to ultrafine particles (UFP) using real-time monitoring, as well as volatile organic compounds (VOCs) and NO2 using passive sampling in rural Bhutan in order to determine the factors driving the exposures. An activity diary was used to track children’s time activity patterns, and difference in mean exposure levels across sex and indoor/outdoor were investigated with ANOVA. 82 children, attending three primary schools participated in this study; S1 and S2 during the wet season and S3 during the dry season. Mean daily UFP exposure (cm-3) was 1.08 × 104 for children attending S1, 9.81 × 103 for S2, and 4.19 × 104 for S3. The mean daily NO2 exposure (µg m-3) was 4.27 for S1, 3.33 for S2 and 5.38 for S3 children. Likewise, children attending S3 also experienced higher daily exposure to a majority of the VOCs than those attending S1 and S2. Time-series of UFP personal exposures provided detailed information on identifying sources of these particles and quantifying their contributions to the total daily exposures for each microenvironment. The highest UFP exposure resulted from cooking/eating, contributing to 64% of the daily exposure, due to firewood combustion in houses using traditional mud cookstoves. The lowest UFP exposures were during the hours that children spent outdoors at school. The outcomes of this study highlight the significant contributions of lifestyle and socio-economic factors in personal exposures and have applications in environmental risk assessment and household air pollution mitigation in Bhutan.

Managing for rainfall variability: effect of grazing strategy on cattle production in a dry tropical savanna

Rainfall variability is a challenge to sustainable and pro. table cattle production in northern Australia. Strategies recommended to manage for rainfall variability, like light or variable stocking, are not widely adopted. This is due partly to the perception that sustainability and profitability are incompatible. A large, long-term grazing trial was initiated in 1997 in north Queensland, Australia, to test the effect of different grazing strategies on cattle production. These strategies are: (i) constant light stocking (LSR) at long-term carrying capacity (LTCC); (ii) constant heavy stocking (HSR) at twice LTCC; (iii) rotational wet-season spelling (R/Spell) at 1.5 LTCC; (iv) variable stocking (VAR), with stocking rates adjusted in May based on available pasture; and (v) a Southern Oscillation Index (SOI) variable strategy, with stocking rates adjusted in November, based on available pasture and SOI seasonal forecasts. Animal performance varied markedly over the 10 years for which data is presented, due to pronounced differences in rainfall and pasture availability. Nonetheless, lighter stocking at or about LTCC consistently gave the best individual liveweight gain (LWG), condition score and skeletal growth; mean LWG per annum was thus highest in the LSR (113 kg), intermediate in the R/Spell (104 kg) and lowest in the HSR(86 kg). MeanLWGwas 106 kg in the VAR and 103 kg in the SOI but, in all years, the relative performance of these strategies was dependent upon the stocking rate applied. After 2 years on the trial, steers from lightly stocked strategies were 60-100 kg heavier and received appreciable carcass price premiums at the meatworks compared to those under heavy stocking. In contrast, LWG per unit area was greatest at stocking rates of about twice LTCC; mean LWG/ha was thus greatest in the HSR (21 kg/ha), but this strategy required drought feeding in four of the 10 years and was unsustainable. Although LWG/ha was lower in the LSR (mean 14 kg/ha), or in strategies that reduced stocking rates in dry years like the VAR(mean 18 kg/ha) and SOI (mean 17 kg/ha), these strategies did not require drought feeding and appeared sustainable. The R/Spell strategy (mean 16 kg/ha) was compromised by an ill-timed fire, but also performed satisfactorily. The present results provide important evidence challenging the assumption that sustainable management in a variable environment is unprofitable. Further research is required to fully quantify the long-term effects of these strategies on land condition and profitability and to extrapolate the results to breeder performance at the property level.

Development and testing of an evaluation procedure for commercial manure additive products.

Manure additive products can be used to reduce odour emissions (OE) from livestock farms. The standardised evaluation of these manure additive products under specific farm conditions is important. In this study, the efficacy of a manure additive (WonderTreat(TM), CKLS, Inc., Hong-Kong) was assessed under Australian conditions utilising a combination of laboratory and field-scale evaluation techniques. As a first step, the efficacy of the manure additive was assessed in a laboratory-scale trial using a series of uniformly managed digesters and standard odour, liquor ammonia and hydrogen sulphide concentration measurement procedures. This showed that the addition of WonderTreat(TM) at the 'low dose rate' (LDR) (102.6 g m-2) used during the trial significantly, but only marginally (30%; P = 0.02) reduced the OE rate (mean 13.9 OU m-2 s-1) of anaerobic pig liquor relative to an untreated control (UC) (19.9 OU m-2 s-1). However, the 'high dose rate' (HDR) (205.3 g m-2) also assessed during the trial preformed similarly (19.7 OU m-2 s-1) to the UC. No statistically significant difference in the concentrations of a range of measured water quality variables at the 5% level was observed between the treatments or controls digesters. As a second step, a field-scale assessment of the manure additive was undertaken at a commercial piggery. Two piggery manure lagoons (each with approximately 2500 m2 surface area) were included in the study; one was treated with WonderTreat(TM) while the other was used as control. The efficacy of the treatment was assessed using olfactometric evaluation of odour samples collected from the surface of the pond using a dynamic wind tunnel and ancillary equipment. No statistically significant reduction in OE rate could be demonstrated (P = 0.35), partially due to the limited number of samples taken during the assessment. However, there was a numerical reduction in the average OE rate of the treatment pond (29 OU m-2 s-1 at 1 m s-1) compared to the control lagoon (38 OU m-2 s-1 at 1 m s-1).

Is the distribution of Fiji leaf gall in Australian sugarcane explained by variation in the vector Perkinsiella saccharicida?

Fiji leaf gall (FLG) is an important virally induced disease in Australian sugarcane. It is confined to southern canegrowing areas, despite its vector, the delphacid planthopper Perkinsiella saccharicida, occurring in all canegrowing areas of Queensland and New South Wales. This disparity between distributions could be a result of successful containment of the disease through quarantine and/or geographical barriers, or because northern Queensland populations of Perkinsiella may be poorer vectors of the disease. These hypotheses were first tested by investigating variation in the ITS2 region of the rDNA fragment among eastern Australian and overseas populations of Perkinsiella. The ITS2 sequences of the Western Australian P. thompsoni and the Fijian P. vitiensis were distinguishable from those of P. saccharicida and there was no significant variation among the 26 P. saccharicida populations. Reciprocal crosses of a northern Queensland and a southern Queensland population of P. saccharicida were fertile, so they may well be conspecific. Single vector transmission experiments showed that a population of P. saccharicida from northern Queensland had a higher vector competency than either of two southern Queensland populations. The frequency of virus acquisition in the vector populations was demonstrated to be important in the vector competency of the planthopper. The proportion of infected vectors that transmitted the virus to plants was not significantly different among the populations tested. This study shows that the absence of FLG from northern Queensland is not due to a lack of vector competency of the northern population of P. saccharicida.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.