Effect of selected feed meals and starches on diet digestibility in the mud crab, Scylla serrata

The present study examined the capacity of the mud crab, Scylla serrata to digest experimental diets that contained different animal and plant-based feed meals or different levels or types of starch. The apparent dry matter digestibility (ADMD) coefficients for all feed meals tested in the first part of this study, except meat meal, were similar (78–88%). Crude protein digestibility (ACPD) coefficients for all feed meals were relatively high, with values ranging from 86% to 96%. Cotton seed meal, poultry meal, canola meal, fishmeal, soybean meal and lupin meal had similar gross energy digestibility (AGED) values (P>0.05) ranging from 84% to 89%. In the second part of this study, the impact of selected starches on the digestibility of fishmeal-based formulated diets was assessed. The apparent starch digestibility (ASD) of wheat starch decreased significantly as the inclusion level was increased from 15% to 60%, however, there was no significant effect on ACPD values. At a 30% inclusion level, the ASD of diets containing different starches decreased in the order corn>wheat>potato=rice. Moreover, ACPD values were significantly higher (P<0.05) in the diets containing corn or rice starch than in those containing wheat or potato starches.

Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

Influence of biochars on flux of N2O and CO2 from Ferrosol

Biochars produced by slow pyrolysis of greenwaste (GW), poultry litter (PL), papermill waste (PS), and biosolids (BS) were shown to reduce N₂O emissions from an acidic Ferrosol. Similar reductions were observed for the untreated GW feedstock. Soil was amended with biochar or feedstock giving application rates of 1 and 5%. Following an initial incubation, nitrogen (N) was added at 165 kg/ha as urea. Microcosms were again incubated before being brought to 100% water-filled porosity and held at this water content for a further 47 days. The flooding phase accounted for the majority (<80%) of total N₂O emissions. The control soil released 3165 mg N₂O-N/m², or 15.1% of the available N as N₂O. Amendment with 1 and 5% GW feedstock significantly reduced emissions to 1470 and 636 mg N₂O-N/m², respectively. This was equivalent to 8.6 and 3.8% of applied N. The GW biochar produced at 350°C was least effective in reducing emissions, resulting in 1625 and 1705 mg N₂O-N/m² for 1 and 5% amendments. Amendment with BS biochar at 5% had the greatest impact, reducing emissions to 518 mg N₂O-N/m², or 2.2% of the applied N over the incubation period. Metabolic activity as measured by CO₂ production could not explain the differences in N₂O emissions between controls and amendments, nor could NH₄⁺ or NO₃^– concentrations in biochar-amended soils. A decrease in NH₄⁺ and NO₃^– following GW feedstock application is likely to have been responsible for reducing N₂O emissions from this amendment. Reduction in N₂O emissions from the biochar-amended soils was attributed to increased adsorption of NO₃^–. Small reductions are possible due to improved aeration and porosity leading to lower levels of denitrification and N₂O emissions. Alternatively, increased pH was observed, which can drive denitrification through to dinitrogen during soil flooding.

An examination of the factorial invariance and refinement of the multidimensional school anger inventory for five Pacific Rim countries

The validity of the Multidimensional School Anger Inventory (MSAI) was examined with adolescents from 5 Pacific Rim countries (N ¼ 3,181 adolescents; age, M ¼ 14.8 years; 52% females). Confirmatory factor analyses examined configural invariance for the MSAI’s anger experience, hostility, destructive expression, and anger coping subscales. The model did not converge for Peruvian students. Using the top 4 loaded items for anger experience, hostility, and destructive expression configural invariance and partial metric and scalar invariances were found. Latent means analysis compared mean responses on each subscale to the U.S. sample. Students from other countries showed higher mean responses on the anger experience subscale (ds ¼ .37–.73). Australian (d ¼ .40) and Japanese students (d ¼ .21) had significantly higher mean hostility subscale scores. Australian students had higher mean scores on the destructive expression subscale (d ¼ .30), whereas Japanese students had lower mean scores (d ¼ 2.17). The largest latent mean gender differences (females lower than males) were for destructive expression among Australian (d ¼ 2.67), Guatemalan (d ¼ 2.42), and U.S. (d ¼ 2.66) students. This study supported an abbreviated, 12-item MSAI with partial invariance. Implications for the use of the MSAI in comparative research are discussed.

Optimising ketocarotenoid production in potato tubers : effect of genetic background, transgene combinations and environment

Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits,as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring.

Dietary intake in Australian children aged 4–24 months: Consumption of meat and meat alternatives

Meat/meat alternatives (M/MA) are key sources of Fe, Zn and protein, but intake tends to be low in young children. Australian recommendations state that Fe-rich foods, including M/MA, should be the first complementary foods offered to infants. The present paper reports M/MA consumption of Australian infants and toddlers, compares intake with guidelines, and suggests strategies to enhance adherence to those guidelines. Mother–infant dyads recruited as part of the NOURISH and South Australian Infants Dietary Intake studies provided 3 d of intake data at three time points: Time 1 (T1) (n 482, mean age 5·5 (SD 1·1) months), Time 2 (T2) (n 600, mean age 14·0 (SD 1·2) months) and Time 3 (T3) (n 533, mean age 24 (SD 0·7) months). Of 170 infants consuming solids and aged greater than 6 months at T1, 50 (29 %) consumed beef, lamb, veal (BLV) or pork on at least one of 3 d. Commercial infant foods containing BLV or poultry were the most common form of M/MA consumed at T1, whilst by T2 BLV mixed dishes (including pasta bolognaise) became more popular and remained so at T3. The processed M/MA increased in popularity over time, led by pork (including ham). The present study shows that M/MA are not being eaten by Australian infants or toddlers regularly enough; or in adequate quantities to meet recommendations; and that the form in which these foods are eaten can lead to smaller M/MA serve sizes and greater Na intake. Parents should be encouraged to offer M/MA in a recognisable form, as one of the first complementary foods, in order to increase acceptance at a later age.

La música orquestal peruana de 1945 a 2005 : Identidades en la diversidad

Peruvian orchestral music 1945–2005. Identities in diversity Peruvian music for orchestra has not been studied as a whole before, and is hardly known by Peruvian musicians and public. The aim of the thesis is to give a panoramic view of Peruvian orchestral music after 1945, study the particular historical context in which these works were created and how they reflect the search for a musical identity of its own, be it individual, local, national or Latin American. Identity is a construction that changes permanently, and individuals can share many identities at the same time. This is a central issue in multicultural societies as the Peruvian, and music is an important mean for constructing cultural identity. The hypothesis of this research is that orchestral work is a medium for Peruvian composers to express their relationship with traditional and popular musics of the country in different ways, from quotation of melodies to a more abstract appropiation of concepts or suggestive title references. Representative works by selected composers, of different techniques, styles or special reception are chosen and analyzed. Research methodology includes analysis of works with various methods according to their stylistic and technical features, in order to find the particular ways in which composers have approached or expressed diverse identities. The investigation shows that Peruvian orchestral music includes works in the main stylistic trends and using the main compositional techniques of the modernist and postmodern periods. It also shows that the construction and expression of particular identities through the study and use of other Peruvian musical traditions is a constant interest shared by composers of different age and esthetic. In a multicultural society as the Peruvian, characterized by its diversity, different forms of transcultural composition are an important mean of dealing with identity issues in music. This thesis also includes for the first time a list of all orchestral works composed in the country or by Peruvian composers in the period, their composers and genres. KEYWORDS: Peruvian music, contemporary music for orchestra, identity

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia

Objective To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. Design Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. Results Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h)and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. Conclusion Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

The reliability of supply of feed grains in the northern region

Reliability of supply of feed grain has become a high priority issue for industry in the northern region. Expansion by major intensive livestock and industrial users of grain, combined with high inter-annual variability in seasonal conditions, has generated concern in the industry about reliability of supply. This paper reports on a modelling study undertaken to analyse the reliability of supply of feed grain in the northern region. Feed grain demand was calculated for major industries (cattle feedlots, pigs, poultry, dairy) based on their current size and rate of grain usage. Current demand was estimated to be 2.8Mt. With the development of new industrial users (ethanol) and by projecting the current growth rate of the various intensive livestock industries, it was estimated that demand would grow to 3.6Mt in three years time. Feed grain supply was estimated using shire scale yield prediction models for wheat and sorghum that had been calibrated against recent ABS production data. Other crops that contribute to a lesser extent to the total feed grain pool (barley, maize) were included by considering their production relative to the major winter and summer grains, with estimates based on available production records. This modelling approach allowed simulation of a 101-year time series of yield that showed the extent of the impact of inter-annual climate variability on yield levels. Production estimates were developed from this yield time series by including planted crop area. Area planted data were obtained from ABS and ABARE records. Total production amounts were adjusted to allow for any export and end uses that were not feed grain (flour, malt etc). The median feed grain supply for an average area planted was about 3.1Mt, but this varied greatly from year to year depending on seasonal conditions and area planted. These estimates indicated that supply would not meet current demand in about 30% of years if a median area crop were planted. Two thirds of the years with a supply shortfall were El Nino years. This proportion of years was halved (i.e. 15%) if the area planted increased to that associated with the best 10% of years. Should demand grow as projected in this study, there would be few years where it could be met if a median crop area was planted. With area planted similar to the best 10% of years, there would still be a shortfall in nearly 50% of all years (and 80% of El Nino years). The implications of these results on supply/demand and risk management and investment in research and development are briefly discussed.

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera

A 5′ Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5′ Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5′ Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Purification of immunoglobulins from chicken sera by thiophilic gel chromatography

Immunoglobulin Y is different from most of the other immunoglobulins because it does not bind protein A or protein G. Thiophilic gel chromatography has been successfully used to purify IgY from chicken egg yolk, but the technology has not previously been used to purify IgY from serum. In this research note, we describe the optimization of T-gel chromatography for purification of IgY from serum. Data are provided on the recovery and purity of IgY obtained using potassium sulfate buffers of different concentrations. Decreasing the strength of potassium sulfate buffer from 0.5 to 0.3 M did not alter the amount of IgY recovered but increased the purity. Using 0.3 M potassium sulphate, we recovered approximately 63.7% of the serum Ig as almost pure IgY.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Quantifying Transmission of Campylobacter jejuni in Commercial Broiler Flocks

Since meat from poultry colonized with Campylobacter spp. is a major cause of bacterial gastroenteritis, human exposure should be reduced by, among other things, prevention of colonization of broiler flocks. To obtain more insight into possible sources of introduction of Campylobacter into broiler flocks, it is essential to estimate the moment that the first bird in a flock is colonized. If the rate of transmission within a flock were known, such an estimate could be determined from the change in the prevalence of colonized birds in a flock over time. The aim of this study was to determine the rate of transmission of Campylobacter using field data gathered for 5 years for Australian broiler flocks. We used unique sampling data for 42 Campylobacter jejuni-colonized flocks and estimated the transmission rate, which is defined as the number of secondary infections caused by one colonized bird per day. The estimate was 2.37 +/- 0.295 infections per infectious bird per day, which implies that in our study population colonized flocks consisting of 20,000 broilers would have an increase in within-flock prevalence to 95% within 4.4 to 7.2 days after colonization of the first broiler. Using Bayesian analysis, the moment of colonization of the first bird in a flock was estimated to be from 21 days of age onward in all flocks in the study. This study provides an important quantitative estimate of the rate of transmission of Campylobacter in broiler flocks, which could be helpful in future studies on the epidemiology of Campylobacter in the field.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes - adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

Mechanically Ventilated Broiler Sheds: a Possible Source of Aerosolized Salmonella, Campylobacter, and Escherichia coli.

This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were similar to 10(8) CFU g(-1) and, as a consequence, were in the range of 10(2) to 10(4) CFU m(-3) in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (10(3) to 10(5) most probable number [MPN] g(-1)) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m(-3)) and once outside (2.3 MPN m(-3)). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g(-1). Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m(-3). Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.