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.

Using Alkanes to Predict Voluntary Intake, Faecal Output and Digestibility of Buffel Grass Hay Fed to Steers

The cuticular waxes of forage plants contain long chain n-alkanes with odd carbon chain lengths in the range C25-C37 which are quantitatively recovered in faeces. When these concentrations are used with the concentrations of administered synthetic even chain length alkanes, the voluntary intake (VI), faecal output (FO) and digestibility (DMD) of forages can be estimated (Dove and Mayes 1991, 1996).

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.

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.

In vitro propagation of Corymbia torelliana x C. citriodora (Myrtaceae) via cytokinin-free node culture

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively to establish forestry plantations in subtropical Australia. Methods were developed for in vitro seed germination, shoot multiplication and plantlet formation that could be used to establish in vitro and ex vitro clone banks of juvenile Corymbia hybrids. Effects of sodium hypochlorite concentration and exposure time on seed contamination and germination, and effects of cytokinin and auxin concentrations on shoot multiplication and subsequent rooting, were assessed. A two-step surface sterilisation procedure, involving 70% ethanol followed by 1% sodium hypochlorite, provided almost no contamination and at least 88% germination. A novel method of cytokinin-free node culture proved most effective for in vitro propagation. Lateral bud break of primary shoots was difficult to induce by using cytokinin, but primary shoots rooted prolifically, elongated rapidly and produced multiple nodes in the absence of exogenous cytokinin. Further multiplication was obtained either by elongating lateral shoots of nodal explants in cytokinin-free medium or by inducing organogenic callus and axillary shoot proliferation with 2.2 µm benzyladenine. Plantlets were produced using an in vitro soil-less method that provided extensive rooting in sterile propagation mixture. These methods provide a means for simultaneous laboratory storage and field-testing of clones before selection and multiplication of desired genotypes.

A new culture method for lesser mealworm, Alphitobius diaperinus

A new culture method for lesser mealworm, Alphitobius diaperinus (Panzer), was developed to provide large numbers of adult lesser mealworms of approximately the same age for insecticide resistance testing. Culturing entailed allowing 100 adults to reproduce for 4 days in a wheat-based culture medium contained inside a plastic culture box, removing the adults from the medium, and then rearing their progeny to adulthood therein, in approximately 56 days at 32 degrees C and 55% RH. During their development, progeny were supplied water via apple slices at 0, 21 and 35 days, and a foam substrate in which to pupate, also at 35 days. During 2004-2005, adult lesser mealworms were collected from six broiler-house populations and then cultured with this method. Each population produced 4500 adults required to complete resistance testing with one insecticide within ten culture boxes, at an average of 798 adults per culture box.

Advances in the culture of crabs

Farmed crab production in 2005 reached 660,000 tonnes globally of which virtually all was produced in Asia. The freshwater Chinese mitten crab Eriocheir japonica sinensis accounts for two thirds of global crab production with the remainder, estuarine portunid crabs such as Scylla species. Initially reliant upon harvest of wild juveniles, the adoption of hatchery methods to supply “seed” makes a significant increase in aquaculture production possible. Many fundamental husbandry issues such as feeding and reproduction are only now receiving research attention.

Advances in the culture of lobsters.

Commercial aquaculture of marine lobsters is an attractive proposition, as most species are high value with established market demand, and fishery production is static or diminishing. Nevertheless, achievement of commercial success will necessitate resolution of technical difficulties associated with on-growing of aggressive species (clawed lobsters) or with rearing the larvae, which for spiny and slipper lobsters is generally a painstaking and protracted process. Notwithstanding these technical challenges, increasing market demand for the product is driving a substantial research and development effort around the world to develop commercial lobster farming technology. This chapter reports on the status of that effort, the successes and obstacles.

Growth and carcass characteristics of cast-for-age Merino ewes fed sorghum-based feedlot diets

Grain feeding low bodyweight, cast-for-age (CFA) sheep from pastoral areas of eastern Australia at the end of the growing season can enable critical carcass weight grades to be achieved and thus yield better economic returns. The aim of this work was to compare growth and carcass characteristics for CFA Merino ewes consuming either simple diets based on whole sorghum grain or commercial feed pellets. The experiment also compared various sources of additional nitrogen (N) for inclusion in sorghum diets and evaluated several introductory regimes. Seventeen ewes were killed initially to provide baseline carcass data and the remaining 301 ewes were gradually introduced to the concentrate diets over 14 days before being fed concentrates and wheaten hay ad libitum for 33 or 68 days. Concentrate treatments were: (i) commercial feed pellets, (ii) sorghum mix (SM; whole sorghum grain, limestone, salt and molasses) + urea and ammonium sulfate (SMU), (iii) SMU + whole cottonseed at 286 g/kg of concentrate dry matter (DM), (iv) SM + cottonseed meal at 139 g/kg of concentrate DM, (v) SMU + virginiamycin (20 mg/kg of concentrate) for the first 21 days of feeding, and (vi) whole cottonseed gradually replaced by SMU over the first 14 days of feeding. The target carcass weight of 18 kg was achieved after only 33 days on feed for the pellets and the SM + cottonseed meal diet. All other whole grain sorghum diets required between 33 and 68 days on feed to achieve the target carcass weight. Concentrates based on whole sorghum grain generally produced significantly (P < 0.05) lower carcass weight and fat score than pellets and this may have been linked to the significantly (P < 0.05) higher faecal starch concentrations for ewes consuming sorghum-based diets (270 v. 72 g/kg DM on day 51 of feeding for sorghum-based diets and pellets, respectively). Source of N in whole grain sorghum rations and special introductory regimes had no significant (P > 0.05) effects on carcass weight or fat score of ewes with the exception of carcass weight for SMU + whole cottonseed being significantly lower than SM + cottonseed meal at day 33. Ewes finished on all diets produced acceptable carcasses although muscle pH was high in all ewe carcasses (average 5.8 and 5.7 at 33 and 68 days, respectively). There were no significant (P > 0.05) differences between diets in concentrate DM intake, rumen fluid pH, meat colour score, fat colour score, eye muscle area, meat pH or meat temperature.

Integrated pond culture for improved production and environmental performance

This presentation given at the World Aquaculture conference in 2008 describes research undertaken at the Bribie Island Research Centre involving zero water exchange co-culture of whiting and banana prawns.

In vitro culture of buffalo fly and infections with Wolbachia

"Develop and optimise reliable in vitro culture methods for buffalo fly "Use the in vitro system to determine whether experimental Wolbachia infection can be established in buffalo fly. "Prepare further applications for related work towards better control of buffalo fly, exploiting the in vitro culture system.

Strategic banana tissue culture industry development and biosecurity act

Application and development of activities based on in vitro technologies delivering research, industry development and biosecurity activities to sustain and improve the Australian banana industry.

Residue Potential of Norsesquiterpene Glycosides in Tissues of Cattle Fed Austral Bracken (Pteridium esculentum).

Austral bracken, Pteridium esculentum, occurs widely in Australian grazing lands and contains both the known carcinogen ptaquiloside and its hydroxy analogue, ptesculentoside, with untested carcinogenic potential. Calves were fed a diet containing 19% P. esculentum that delivered 1.8 mg of ptaquiloside and 4.0 mg of ptesculentoside per kilogram of body weight (bw) per day to explore the carcass residue potential of these compounds. Concentrations of ptaquiloside and ptesculentoside in the liver, kidney, skeletal muscle, heart, and blood of these calves were determined as their respective elimination products, pterosin B and pterosin G, by HPLC-UV analysis. Plasma concentrations of up to 0.97 mu g/mL ptaquiloside and 1.30 mu g/mL ptesculentoside were found, but were shown to deplete to <10% of these values within 24 h of bracken consumption. Both glycosides were also detected in all tissues assayed, with ptesculentoside appearing to be more residual than ptaquiloside. Up to 0.42 and 0.32 mu g/g ptesculentoside was present in skeletal muscle and liver, respectively, 15 days after bracken consumption ended. This detection of residual glycosides in tissues of cattle feeding on Austral bracken raises health concerns for consumers and warrants further investigation.