Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice.

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-[gamma] level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-[gamma] was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-[gamma] production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.

The determination of essential oils in Sandalwood via NIR Spectroscopy

Sandalwood oil is widely used in the medicinal, cosmetic and aromatherapy industries. The oil is distilled from the heartwood of the sandalwood tree Santalum - a genus of hemi-parasitic tree species occurring throughout South and Southeast Asia, Australia and the Pacific. With international concern on the sustainability Sandalwood oil (Fox, 2000), the quality of oil entering the market is being compromised either through extraction from underdeveloped heartwoods or through adulteration with lower grade Sandalwood oils or synthetic substitutes (Howes et al. 2004). Although no standard method exists to assess the quality of Sandalwood oil, the International Organisation for Standardisation recommends GCMS analysis of santalol oil content. NIR spectroscopy has had a demonstrated success for other essential oils (Schulz et al. 2004, Steur et al. 2001). In addition, NIR spectroscopy has also been applied as both a qualitative and quantitative analytical tool in the forestry industry (Steur et al. 2001). This project aimed to assess the ability of NIR spectroscopy as a non-invasive, rapid and cheap analytical alternative to GCMS for Santalol determination.

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.

Non-destructive prediction of 'Hass' avocado dry matter via FT-NIR spectroscopy.

BACKGROUND: The inability to consistently guarantee internal quality of horticulture produce is of major importance to the primary producer, marketers and ultimately the consumer. Currently, commercial avocado maturity estimation is based on the destructive assessment of percentage dry matter (%DM), and sometimes percentage oil, both of which are highly correlated with maturity. In this study the utility of Fourier transform (FT) near-infrared spectroscopy (NIRS) was investigated for the first time as a non-invasive technique for estimating %DM of whole intact 'Hass' avocado fruit. Partial least squares regression models were developed from the diffuse reflectance spectra to predict %DM, taking into account effects of intra-seasonal variation and orchard conditions. RESULTS: It was found that combining three harvests (early, mid and late) from a single farm in the major production district of central Queensland yielded a predictive model for %DM with a coefficient of determination for the validation set of 0.76 and a root mean square error of prediction of 1.53% for DM in the range 19.4-34.2%. CONCLUSION: The results of the study indicate the potential of FT-NIRS in diffuse reflectance mode to non-invasively predict %DM of whole 'Hass' avocado fruit. When the FT-NIRS system was assessed on whole avocados, the results compared favourably against data from other NIRS systems identified in the literature that have been used in research applications on avocados.

Improving information for the macadamia industry via an information "bank"

Macadamia growers are under increasing pressure to remain viable in an increasingly competitive global market. A key need is quick access to high quality information. Current industry information is poorly integrated, poorly updated, and because it is largely in hard-copy, is difficult to access efficiently. With the dramatic growth in the use of the internet by growers, as evidenced in a recent industry communications survey, an opportunity exists to address this problem through the development of a high quality, internet-based information “bank”. The bank would bring together the macadamia information resources and collective knowledge of R&D and other relevant agencies into a one-stop information shop, aligned more effectively with grower needs.

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.

Mixture Effects of Benzene, Toluene, Ethylbenzene and Xylenes (BTEX) to Lung Carcinoma Cells via a Hanging Drop Air Exposure System

A recently developed hanging drop air exposure system for toxicity studies of volatile chemicals was applied to evaluate the cell viability of lung carcinoma A549 cells after 1 h and 24 h of exposure to benzene, toluene, ethylbenzene and xylenes (BTEX) as individual compounds and mixtures of 4 or 6 components. The cellular chemical concentrations causing 50% reduction of cell viability (EC50) were calculated use a mass balance model and came to 17, 12, 11, 9, 4 and 4 mmol/kg cell dry weight for benzene, toluene, ethylbenzene, m-xylene, o-xylene and p-xylene respectively after 1 h of exposure. The EC50 decreased by a factor of four after 24 h of exposure. All mixture effects were best described by the mixture toxicity model of concentration addition, which is valid for chemicals with the same mode of action. Good agreement with the model predictions were found for benzene, toluene, ethylbenzene and m-xylene at four different representative fixed concentration ratios after 1 h of exposure but lower agreement to mixture prediction was obtained after 24 h of exposure. A recreated car exhaust mixture, which involved the contribution of the more toxic p-xylene and o-xylene, yielded an acceptable but lower quality prediction as well.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.