Microbial ecology of the equine hindgut during oligofructose-induced laminitis

Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3-7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.

Estimation in a multiplicative mixed model involving a genetic relationship matrix.

Genetic models partitioning additive and non-additive genetic effects for populations tested in replicated multi-environment trials (METs) in a plant breeding program have recently been presented in the literature. For these data, the variance model involves the direct product of a large numerator relationship matrix A, and a complex structure for the genotype by environment interaction effects, generally of a factor analytic (FA) form. With MET data, we expect a high correlation in genotype rankings between environments, leading to non-positive definite covariance matrices. Estimation methods for reduced rank models have been derived for the FA formulation with independent genotypes, and we employ these estimation methods for the more complex case involving the numerator relationship matrix. We examine the performance of differing genetic models for MET data with an embedded pedigree structure, and consider the magnitude of the non-additive variance. The capacity of existing software packages to fit these complex models is largely due to the use of the sparse matrix methodology and the average information algorithm. Here, we present an extension to the standard formulation necessary for estimation with a factor analytic structure across multiple environments.

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

Prediction of breeding values for average fruit weight in mango using a multivariate individual mixed model

Mango is an important horticultural fruit crop and breeding is a key strategy to improve ongoing sustainability. Knowledge of breeding values of potential parents is important for maximising progress from breeding. This study successfully employed a mixed linear model methods incorporating a pedigree to predict breeding values for average fruit weight from highly unbalanced data for genotypes planted over three field trials and assessed over several harvest seasons. Average fruit weight was found to be under strong additive genetic control. There was high correlation between hybrids propagated as seedlings and hybrids propagated as scions grafted onto rootstocks. Estimates of additive genetic correlation among trials ranged from 0.69 to 0.88 with correlations among harvest seasons within trials greater than 0.96. These results suggest that progress from selection for broad adaptation can be achieved, particularly as no repeatable environmental factor that could be used to predict G x E could be identified. Predicted breeding values for 35 known cultivars are presented for use in ongoing breeding programs.

Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing

Kangaroos ferment forage material in an enlarged forestomach analogous to the rumen, but in contrast to ruminants, they produce little or no methane. The objective of this study was to identify the dominant organisms and pathways involved in hydrogenotrophy in the kangaroo forestomach, with the broader aim of understanding how these processes are able to predominate over methanogenesis. Stable isotope analysis of fermentation end products and RNA stable isotope probing (RNA-SIP) were used to investigate the organisms and biochemical pathways involved in the metabolism of hydrogen and carbon dioxide in the kangaroo forestomach. Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos. In in vitro fermentations, the microbial community of the kangaroo foregut produced very little methane, but produced a significantly greater proportion of acetate derived from carbon dioxide than the microbial community of the bovine rumen. A bacterial operational taxonomic unit closely related to the known reductive acetogen Blautia coccoides was found to be associated with carbon dioxide and hydrogen metabolism in the kangaroo foregut. Other bacterial taxa including members of the genera Prevotella, Oscillibacter and Streptococcus that have not previously been reported as containing hydrogenotrophic organisms were also significantly associated with metabolism of hydrogen and carbon dioxide in the kangaroo forestomach.The ISME Journal advance online publication, 13 March 2014; doi:10.1038/ismej.2014.25.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 × 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ~50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 x 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ∼50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Shelf life of minimally processed honeydew, kiwifruit, papaya, pineapple and cantaloupe

Shelf life of minimally processed (peeled, deseeded, and diced) honeydew melon, kiwifruit, papaya, pineapple, and cantaloupe stored at 4°C was studied. Sensory assessments were carried out at 3-day intervals by highly trained panels until the end of shelf life. Microbiological counts were made immediately after dicing fruit and at the end of shelf life. Results indicated that both the length of shelf life and type of spoilage were related to fruit species. Minimally processed fruit had longer shelf life at 4°C than at temp. recommended for whole fruit when these were >4°C. Spoilage of 4°C-stored kiwifruit, papaya, and pineapple pieces was found to be not as a consequence of microbial growth

Aseptic Packaging of Tropical Fruit Purees

The object of this investigation was to develop high quality aseptically packaged mango and passionfruit puree products. Kensington mango puree (acidified to ph 3.5) and deseeded passionfruit pulp (ph 3.0) were sterilised in a scraped-surface heat exchanger, cooled to 20°C in a tubular heat-exchanger, aseptically packaged in sterile laminate bags. Six sterilising time/temperature combinations were compared - 85°C/15 secs, 85°C/60 secs, 90° C/15 secs, 90°/60 secs, 95°C/15 secs, 95°C/60 secs. Products were assessed immediately after processing, and after eight months ambient storage, for microbial, physical, chemical, and sensory quality. All treatments were microbiologically sound and showed no enzyme activity. Sensory quality was very acceptable, and there was no evidence of heat damage. Quality (especially colour and flavour) decreased during storage in all heat treatments.

Fruit maturity and soundness relevant to feeding choice by fruit-piercing moths (Lepidoptera: Noctuidae) in citrus crops in northeast Australia

Fruit-piercing moths are significant pests of a range of fruit crops throughout much of the world's tropics and subtropics. Feeding damage by the adult moths is most widely reported in varieties of citrus. In the years 2003 and 2004, fruit-piercing moth activity was observed regularly at night in citrus crops in northeast Australia, to determine the level of maturity (based on rind colour) and soundness of fruit attacked. 'Navelina' navel and 'Washington' navel orange, grapefruit and mixed citrus crops were assessed, and fruit was rated and placed into five categories: green, colouring, ripe, overripe and damaged. There were no statistical differences in the percentage of fruit attacked in each category across crops. However, within the individual crops significant proportions of green 'Navelina' fruit (58.7%) and green mixed citrus (57.1%) were attacked in 2004. Among all the crops assessed, 25.1% of moth feeding occurred on overripe or damaged fruit. Crops started to be attacked at least 8 weeks before picking, but in two crops there were large influxes of moths (reaching 27 and 35 moths/100 trees, respectively) immediately before harvest. Moth activity was most intense between late February and late March. Eudocima fullonia (Clerck) represented 79.1% of all moths recorded on fruit, with Eudocima materna (L.), Eudocima salaminia (Cramer) and Serrodes campana (Guen.) the only other species observed capable of inflicting primary damage. Our results suggest that growers should monitor moth activity from 8 weeks before harvest and consider remedial action if moth numbers increase substantially as the crop matures or there is a history of moth problems. The number of fruit pickings could be increased to progressively remove ripe fruit or early harvest of the entire crop contemplated if late influxes of moths are known.

Growth and species interactions of Eucalyptus pellita in a mixed and monoculture plantation in the humid tropics of north Queensland

This study investigated whether mixed-species designs can increase the growth of a tropical eucalypt when compared to monocultures. Monocultures of Eucalyptus pellita (E) and Acacia peregrina (A) and mixtures in various proportions (75E:25A, 50E:50A, 25E:75A) were planted in a replacement series design on the Atherton Tablelands of north Queensland, Australia. High mortality in the establishment phase due to repeated damage by tropical cyclones altered the trial design. Effects of experimental designs on tree growth were estimated using a linear mixed-effects model with restricted maximum likelihood analysis (REML). Volume growth of individual eucalypt trees were positively affected by the presence of acacia trees at age 5 years and this effect generally increased with time up to age 10 years. However, the stand volume and basal area increased with increasing proportions of E. pellita, due to its larger individual tree size. Conventional analysis did not offer convincing support for mixed-species designs. Preliminary individual-based modelling using a modified Hegyi competition index offered a solution and an equation that indicates acacias have positive ecological interactions (facilitation or competitive reduction) and definitely do not cause competition like a eucalypt. These results suggest that significantly increased in growth rates could be achieved with mixed-species designs. This statistical methodology could enable a better understanding of species interactions in similarly altered experiments, or undesigned mixed-species plantations.

Limitations of soil microbial biomass carbon as an indicator of soil pollution in the field

The size of the soil microbial biomass carbon (SMBC) has been proposed as a sensitive indicator for measuring the adverse effects of contaminants on the soil microbial community. In this study of Australian agricultural systems, we demonstrated that field variability of SMBC measured using the fumigation-extraction procedure limited its use as a robust ecotoxicological endpoint. The SMBC varied up to 4-fold across control samples collected from a single field site, due to small-scale spatial heterogeneity in the soil physicochemical environment. Power analysis revealed that large numbers of replicates (3-93) were required to identify 20% or 50% decreases in the size of the SMBC of contaminated soil samples relative to their uncontaminated control samples at the 0.05% level of statistical significance. We question the value of the routine measurement of SMBC as an ecotoxicological endpoint at the field scale, and suggest more robust and predictive microbiological indicators.

Developments in controlled green-water larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere

Since 1989, researchers with the Department of Primary Industries and Fisheries (DPI&F) in Queensland, Australia, have successfully used controlled low-water exchange green-water cultures to rear the larvae of estuarine fishes and crustaceans through to metamorphosis. High survivals and excellent fry condition have been achieved for several commercially important endemic species produced for various projects. They include barramundi or sea bass, Lates calcarifer, Australian bass, Macquaria novemaculeata, dusky flathead, Platycephalus fuscus, sand whiting, Sillago ciliata, red sea bream or snapper, Pagrus auratus, banana prawn, Fenneropenaeus merguiensis, and others. The consistent success of our standardised and relatively simple approach at different localities has led to it being incorporated into general fingerling production practices at several establishments in Australia. Although post-metamorphosis rearing methods have differed for each species investigated, due to various biological and behavioural traits and project requirements, these larval rearing methods have been successful with few species-specific modifications. Initially modelled on the Taiwanese approach to rearing Penaeids in aerated low-water exchange cultures, the approach similarly appears to rely on a beneficial assemblage of micro-organisms. Conceptually, these micro-organisms may include a mixture of the air-borne primary invaders of pure phytoplankton cultures when exposed to outdoor conditions. Whilst this would vary with different sites, our experiences with these methods have consistently been favourable. Mass microalgal cultures with eco-physiological youth are used to regularly augment larval fish cultures so that rearing conditions simulate an exponential growth-phase microalgal bloom. Moderate to heavy aeration prevents settlement of particulate matter and encourages aerobic bacterial decomposition of wastes. The green-water larval rearing approach described herein has demonstrated high practical utility in research and commercial applications, and has greatly simplified marine finfish hatchery operations whilst generally lifting production capacities for metamorphosed fry in Australia. Its potential uses in areas of aquaculture other than larviculture are also discussed.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Light environment and tree development of young Acacia melanoxylon in mixed-species regrowth forest, Tasmania, Australia

Blackwood (Acacia melanoxylon R. Br.) is a valuable leguminous cabinetwood species which is commonly found as a canopy or subcanopy tree in a broad range of mixed-species moist forests on tablelands and coastal escarpments in eastern Australia. This paper reports on the competitive light environment of a commercially valuable multi-species regrowth forest in NW Tasmania, in order to define some of the functional interactions and competitive dynamics of these stands. Comparative observations were made of the internal forest light environment in response to small-gap silvicultural treatments, in a young regenerative mix of three codominant tree species. Light measurements were made during periods of maximum external irradiance of the regrowth Eucalyptus obliqua/A. melanoxylon forest canopy at age 10.5 years. This was at a time of vigourous stand development, 4.5 years following the application of three experimental silvicultural treatments whose effects were observed in comparison with an untreated canopy sample designed as a control. Minimal irradiance was observed within and beneath the dense subcanopy of the native nurse species (Pomaderris apetala) which closely surrounds young blackwood regeneration. Unlike current plantation nurse systems, the dense foliage of the native broadleaved Pomaderris all but eliminated direct side-light and low-angle illumination of the young blackwood, from the beginning of tree establishment. The results demonstrated that retention of these densely stocked native codominants effectively suppressed both size and frequency of blackwood branches on the lower bole, through effective and persistent interception of sunlight. Vigorous young blackwood crowns later overtopped the codominant nurse species, achieving a predictable height of branch-free bole. This competitive outcome offers a valuable tool for management of blackwood crown dynamics, stem form and branch habit through manipulation of light environment in young native regrowth systems. Results demonstrate that effective self-pruning in the lower bole of blackwood is achieved through a marked reduction in direct and diffuse sunlight incident on the lower crown, notably to less than 10-15% of full sunlight intensity during conditions of maximum insolation. The results also contain insights for the improved design of mixed-species plantation nurse systems using these or functionally similar species' combinations. Based on evidence presented here for native regrowth forest, plantation nurse systems for blackwood will need to achieve 85-90% interception of external side-light during early years of tree development if self-pruning is to emulate the results achieved in the native nurse system.