Ecology of uncultivated oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

A sampling technique for two-spotted mite in papaya

Two-spotted mite, Tetranychus urticae Koch, was until recently regarded as a minor and infrequent pest of papaya in Queensland through the dry late winter/early summer months. The situation has changed over the past 4-5 years, so that now some growers consider spider mites significant pests all year round. This altered pest status corresponded with a substantial increase in the use of fungicides to control black spot (Asperisporium caricae). A project was initiated in 1998 to examine the potential reasons for escalating mite problems in commercially-grown papaya, which included regular sampling over a 2 year period for mites, mite damage and beneficial arthropods on a number of farms on the wet tropical coast and drier Atherton Tableland. Differences in soil type, papaya variety, chemical use and some agronomic practices were included in this assessment. Monthly visits were made to each site where 20 randomly-selected plants from each of 2 papaya lines (yellow and red types) were surveyed. Three leaves were selected from each plant, one from each of the bottom, middle and top strata of leaves. The numbers of mobile predators were recorded, along with visual estimates of the percentage and age of mite damage on each leaf. Leaves were then sprayed with hairspray to fix the mites and immature predators to the leaf surface. Four leaf disks, 25 mm in diameter, were then punched from each leaf into a 50 ml storage container with a purpose-built disk-cutting tool. Disks from each leaf position were separated by tissue paper, within the container. On return to the laboratory, each leaf disk was scrutinised under a binocular microscope to determine the numbers of two-spotted mites and eggs, predatory mites and eggs, and the immature stages of predatory insects (mainly Stethorus, Halmus and lacewings). A total of 2160 leaf disks have been examined each month. All data have been entered into an Access database to facilitate comparisons between sites.

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.

Chlorophyll fluorescence measures of seagrasses Halophila ovalis and Zostera capricorni reveal differences in response to experimental shading

In coastal waters and estuaries, seagrass meadows are often subject to light deprivation over short time scales (days to weeks) in response to increased turbidity from anthropogenic disturbances. Seagrasses may exhibit negative physiological responses to light deprivation and suffer stress, or tolerate such stresses through photo-adaptation of physiological processes allowing more efficient use of low light. Pulse Amplitude Modulated (PAM) fluorometery has been used to rapidly assess changes in photosynthetic responses along in situ gradients in light. In this study, however, light is experimentally manipulated in the field to examine the photosynthesis of Halophila ovalis and Zostera capricorni. We aimed to evaluate the tolerance of these seagrasses to short-term light reductions. The seagrasses were subject to four light treatments, 0, 5, 60, and 90% shading, for a period of 14 days. In both species, as shading increased the photosynthetic variables significantly (P < 0.05) decreased by up to 40% for maximum electron transport rates (ETRmax) and 70% for saturating irradiances (Ek). Photosynthetic efficiencies (a) and effective quantum yields (ΔF/Fm′ ) increased significantly (P < 0.05), in both species, for 90% shaded plants compared with 0% shaded plants. H. ovalis was more sensitive to 90% shading than Z. capricorni, showing greater reductions in ETR max, indicative of a reduced photosynthetic capacity. An increase in Ek, Fm′ and ΔF/Fm′ for H. ovalis and Z. capricorni under 90% shading suggested an increase in photochemical efficiency and a more efficient use of low-photon flux, consistent with photo-acclimation to shading. Similar responses were found along a depth gradient from 0 to10 m, where depth related changes in ETRmax and Ek in H. ovalis implied a strong difference of irradiance history between depths of 0 and 5-10 m. The results suggest that H. ovalis is more vulnerable to light deprivation than Z. capricorni and that H. ovalis, at depths of 5-10 m, would be more vulnerable to light deprivation than intertidal populations. Both species showed a strong degree of photo-adaptation to light manipulation that may enable them to tolerate and adapt to short-term reductions in light. These consistent responses to changes in light suggest that photosynthetic variables can be used to rapidly assess the status of seagrasses when subjected to sudden and prolonged periods of reduced light

Ultraviolet radiation effects on fruit surface respiration and chlorophyll fluorescence

High-value fruit crops are exposed to a range of environmental conditions that can reduce fruit quality. Solar injury (SI) or sunburn is a common disorder in tropical, sub-tropical, and temperate climates and is related to: 1) high fruit surface temperature; 2) high visible light intensity; and, 3) ultraviolet radiation (UV). Positional changes in fruit that are caused by increased weight or abrupt changes that result from summer pruning, limb breakage, or other damage to the canopy can expose fruit to high solar radiation levels, increased fruit surface temperatures, and increased UV exposure that are higher than the conditions to which they are adapted. In our studies, we examined the effects of high fruit surface temperature, saturating photosynthetically-active radiation (PAR), and short-term UV exposure on chlorophyll fluorescence, respiration, and photosynthesis of fruit peel tissues from tropical and temperate fruit in a simulation of these acute environmental changes. All tropical fruits (citrus, macadamia, avocado, pineapple, and custard apple) and the apple cultivars 'Gala', 'Gold Rush', and 'Granny Smith' increased dark respiration (A0) when exposed to UV, suggesting that UV repair mechanisms were induced. The maximum quantum efficiency of photosystem II (Fv/Fm) and the quantum efficiency of photosystem II (ΦII) were unaffected, indicating no adverse effects on photosystem II (PSII). In contrast, 'Braeburn' apple had a reduced Fv/Fm with no increase in A0 on all sampling dates. There was a consistent pattern in all studies. When Fv/Fm was unaffected by UV treatment, A0 increased significantly. Conversely, when Fv/Fm was reduced by UV treatment, then A0 was unaffected. The pattern suggests that when UV repair mechanisms are effective, PSII is adequately protected, and that this protection occurs at the cost of higher respiration. However, when the UV repair mechanisms are ineffective, not only is PSII damaged, but there is additional short-term damage to the repair mechanisms, indicated by a lack of respiration to provide energy.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

In the article 'Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis' (Milinovich et al., 2007), it is found that with reference to Horse 1, the histological signs of laminitis were first observed at 12 h post-oligofructose administration, and not 30 h as was indicated in the Results section under the subheading 'Induction of Laminitis' and in Fig. 1.

A digital imaging technique to quantify the incidence of internal browning in potato (Solanum tuberosum) tubers

Internal browning disorders, including brown fleck (BF), in potato (Solanum tuberosum) tubers greatly reduce tuber quality, but the causes are not well understood. This is due, in part, to the highly variable data provided by visual value-based rating systems. A digital imaging technique was developed to quantify accurately the incidence of internal browning in potato tubers. Images of tuber sections were scanned using a flatbed scanner and digitally enhanced to highlight tuber BF lesions, and the area of affected tissue calculated using pixel quantification software. Digital imaging allowed for the determination of previously unused indices of the incidence and severity of internal browning in potato tubers. Statistical analysis of the comparison between digitally derived and visual-rating BF data from a glasshouse experiment showed that digital data greatly improved the delineation of treatment effects. The F-test probability was further improved through square root or logarithmic data transformations of the digital data, but not of the visual-rating data. Data from a field experiment showed that the area of tuber affected by BF and the number of small BF lesions increased with time and with increase in tuber size. The results from this study indicate that digital imaging of internal browning disorders of potato tubers holds much promise in determining their causes that heretofore have proved elusive.

An improved technique for estimating short-term survival of released line-caught fish, and an application comparing barotrauma-relief methods in red emperor (Lutjanus sebae Cuvier 1816).

Promotion of better procedures for releasing undersize fish, advocacy of catch-and-release angling, and changing minimum legal sizes are increasingly being used as tools for sustainable management of fish stocks. However without knowing the proportion of released fish that survive, the conservation value of any of these measures is uncertain. We developed a floating vertical enclosure to estimate short-term survival of released line-caught tropical and subtropical reef-associated species, and used it to compare the effectiveness of two barotrauma-relief procedures (venting and shotline releasing) on red emperor (Lutjanus sebae). Barotrauma signs varied with capture depth, but not with the size of the fish. Fish from the greatest depths (40-52 m) exhibited extreme signs less frequently than did those from intermediate depths (30-40 m), possibly as a result of swim bladder gas being vented externally through a rupture in the body wall. All but two fish survived the experiment, and as neither release technique significantly improved short-term survival of the red emperor over non-treatment we see little benefit in promoting either venting or shotline releasing for this comparatively resilient species. Floating vertical enclosures can improve short-term post-release mortality estimates as they overcome many problems encountered when constraining fish in submerged cages.

Raw data for project "Development of a DNA based aging technique for use in fisheries assessments" Fisheries and Development Corporation project 2007/033

The primary aim of this study was to determine the relationship between telomere length and age in a range of marine invertebrates including abalone (Haliotis spp) oysters (Saccostrea glomerata), spiny lobsters (Sagmariasus verreauxi formerly Jasus verreauxi and Jasus edwardsii) and school prawns (Metapenaeus macleayi). Additionally, this relationship was studied in a vertebrate organism using the freshwater fish Silver perch (Bidyanus bidyanus). Telomere length differences between tissues were also examined in some species such as Saccostrea glomerata, Sagmariasus verreauxi and Bidyanus bidyanus. In some cases cultured specimens of known age were used and this is quoted in the spreadsheets. For other wild-caught specimens where age was not known, size was used as a proxy for age. This may be a broad size class, or be determined by shell size or carapace length depending on the organism. Each spreadsheet contains raw data of telomere length estimates from Terminal Restriction Fragment Assays (TRF) for various individuals of each species including appropriate details such as age or size and tissue. Telomere length estimates are given in base pairs (bp). In most cases replicate experiments were conducted on groups of samples three times but on a small number of occasions only two replicate experiments were conducted. Further description of the samples can be found in final report of FRDC 2007/033. The arithmetic average for each individual (sample ID) across the two or three replicate experiments is also given. Bidyanus bidyanus (SilverPerch) Two sheets are contained within. a) Comparison of telomere length between different tissues (heart, liver and muscle) within the three year old age class - two replicate experiments were conducted. b) Comparison of telomere length between fish of different but known ages (0.25, 1, 2, and 3 years old) in each of three tissues, heart, liver and muscle – three replicate experiments were conducted per tissue. Haliotis spp (Abalone species) Three species were tested. H. asinina Telomere length was compared in two age classes-11 month and 18 month old abalone using muscle tissue from the foot. Within gel-variation was also estimated using a single sample run three times on one gel (replicate experiment). H. laevigata x H. rubra hybrids Telomere length was compared in three known age classes – two, three and four years old using muscle tissue from the foot. H. rubra Telomere length was compared in a range of different sized abalone using muscle tissue from the foot. Shell size is also given for each abalone Saccostrea glomerata Three sheets are contained within the file. a) Samples came from Moreton Bay Queensland in 2007. Telomere length was compared in two tissues (gill and mantle) of oysters in three age groups (1, 3 and 4 years) b) Samples came from Moreton Bay Queensland in 2009. Telomere length was compared in three age classes using DNA from gill tissue only c) Samples came from Wallis Lake, New South Wales. Telomere length was estimated from whole body minus the shell from 1 year old oysters, gill tissue of 3 age classes (1.5 years, 3 and 4 years), mantle tissue of two age classes (3 and 4 years). Sagmariasus verreauxi (formerly Jasus verreauxi) Telomere length was estimated from abdomen tissue of puerulus, gill and muscle tissue of 3 year old, large and very large size classes of lobsters. Jasus edwardsii Telomere length was measured in two size classes of lobsters- adults of varying sizes using muscle tissue and puerulus using tissues from the abdomen minus the exoskeleton. Metapenaeus macleayi Telomere length was measured in three size classes of school prawns adults. Muscle tissue was used, minus the exoskeleton.

Development of a DNA based aging technique for use in fisheries assessments. Final report, Australian Fisheries Research and Development Corporation Project 2007/033.

The productivity of a fisheries resource can be quantified from estimates of recruitment, individual growth and natural and fisheries-related mortality, assuming the spatial extent of the resource has been quantified and there is minimal immigration or emigration. The sustainability of a fisheries resource is facilitated by management controls such as minimum and maximum size limits and total allowable catch. Minimum size limits are often set to allow individuals the opportunity to reproduce at least once before the chance of capture. Total allowable catches are a proportion of the population biomass, which is estimated based on known reproduction, recruitment, mortality and growth rates. In some fisheries, however, management actions are put in place without quantification of the resource through the stock assessment process. This occurs because species-specific information, for example individual growth, may not be available. In these circumstances, management actions need to be precautionary to protect against future resource collapse, but this often means that the resource is lightly exploited. Consequently, the productivity of the resource is not fully realised. Australia’s most valuable fisheries are invertebrate fisheries (Australian Department of Agriculture Fisheries and Forestry, 2008). For example, Australian fisheries (i.e. excluding aquaculture) production of crustaceans (largely prawns, rock lobster and crab) was 41,000 tonnes in 2006/7, worth $778 million. Production from mollusc (largely abalone, scallops, oysters and squid) fisheries was 39,000 tonnes, worth $502 million. Together, in 2006/7 crustacean and mollusc fisheries represented 58% of the total value of Australian wild fisheries production. Sustainable management of Australia’s invertebrate fisheries is frustrated by the lack of data on species-specific growth rates. This project investigated a new method to estimate age, and hence individual growth rates, in invertebrate fisheries species. The principle behind the new aging method was that telomeres (i.e. DNA end-caps of chromosomes) get shorter as an individual gets older. We studied commercial crustacean and molluscan species. A vertebrate fish species (silver perch, Bidyanus bidyanus) was used as a control to standardise our work against the literature. We found a clear relationship between telomere length and shell size for temperate abalone (Haliotis rubra). Further research is recommended before the method can be implemented to assist management of wildharvested abalone populations. Age needs to be substituted for shell size in the relationship and it needs to be studied for abalone from several regions. This project showed that telomere length declined with increasing age in Sydney rock oysters (Saccostrea glomerata) and was affected by regional variation. A relationship was not apparent between telomere length and age (or size as a surrogate for age) for crustacean species (school prawns, Metapenaeus macleayi; eastern rock lobster, Sagmariasus verreauxi; southern rock lobster, Jasus edwardsii; and spanner crabs, Ranina ranina). For school prawns, there was no difference between telomere length in males and females. Further research is recommended, however, as telomeric DNA from crustaceans was difficult to analyse using the terminal restriction fragment (TRF) assay. Telomere lengths of spanner crabs and lobsters were at the upper limit of resolution of the assay used and results were affected by degradation and possible contamination of telomeric DNA. It is possible that telomere length is an indicator of remaining lifespan in molluscan and crustacean individuals, as suggested for some vertebrate species (e.g. Monaghan, 2010). Among abalone of similar shell size and among lobster pueruli, there was evidence of individuals having significantly longer or shorter telomeres than the group average. At a population level, this may be a surrogate for estimates of future natural mortality, which may have usefulness in the management of those populations. The method used to assay telomere length (terminal restriction fragment assay) performed adequately for most species, but it was too expensive and time-consuming to be considered a useful tool for gathering information for fisheries management. Research on alternative methods is strongly recommended.

Stem injection: a control technique often overlooked for exotic woody weeds

In the rangelands of northern Australia, basal bark, cut stump, hand applied residual herbicides and foliar spraying have traditionally been the main herbicide techniques for control of individual exotic woody weeds growing within scattered to medium density infestations. In this paper we report on the preliminary results of stem injection as an alternate technique for the control of yellow oleander ( Cascabela thevetia (L.) Lippold), a woody weed that is difficult to kill. A randomised complete block experiment comprising 12 herbicide treatments (including a control) and three replicates was undertaken. Two rates of triclopyr + picloram, hexazinone, glyphosate, 2,4- D + picloram and metsufuron methyl and one rate of imazapyr were tested. At 15 months after application, triclopyr + picloram, glyphosate, 2,4-D + picloram and imazapyr all recorded high mortality (>90%) for at least one application rate. These results suggest that stem injection warrants further investigation as a control technique for other exotic woody weeds growing in rangelands.

Evaluation of the impacts of spaying by either the dropped ovary technique or ovariectomy via flank laparotomy on the welfare of Bos indicus beef heifers and cows

The welfare outcomes for Bos indicus cattle (100 heifers and 50 cows) spayed by either the dropped ovary technique (DOT) or ovariectomy via flank laparotomy (FL) were compared with cattle subjected to physical restraint (PR), restraint by electroimmobilization in conjunction with PR (EIM), and PR and mock AI (MAI). Welfare assessment used measures of morbidity, mortality, BW change, and behavior and physiology indicative of pain and stress. One FL heifer died at d 5 from peritonitis. In the 8-h period postprocedures, plasma bound cortisol concentrations of FL, DOT, and EIM cows were not different and were greater (P < 0.05) than PR and MAI. Similarly, FL and DOT heifers had greater (P < 0.05) concentrations than PR and MAI, with EIM intermediate. Creatine kinase and aspartate aminotransferase concentrations were greater (P < 0.05) in FL and EIM heifers compared with the other treatments, with a similar pattern seen in the cows. Haptoglobin concentrations were significantly (P < 0.05) increased in the FL heifers compared with other treatments in the 8- to 24-h and 24- to 96-h periods postprocedures, and in cows were significantly (P < 0.05) increased in the FL and DOT compared with PR in the 24- to 96-h period. Behavioral responses complemented the physiological responses; standing head down was shown by more (P < 0.05) FL cows and heifers to 3 d postprocedures compared with other treatments, although there was no difference between FL and DOT heifers at the end of the day of procedures. At this same time, fewer (P < 0.05) FL and DOT heifers and cows were observed feeding compared with other treatments, although in cows there was no difference between FL, DOT, and EIM. There were no significant differences (P > 0.05) between treatments in BW changes. For both heifers and cows, FL and DOT spaying caused similar levels of acute pain, but FL had longer-lasting adverse impacts on welfare. Electroimmobilization during FL contributed to the pain and stress of the procedure. We conclude that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization causes pain and stress and should not be routinely used as a method of restraint.

Immuno-fluorescence staining patterns of leukocyte subsets in the skin of taurine and indicine cattle

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein–Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein–Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25+ cells labelled regularly shaped cells that showed a wide range of intensities of staining.