Animal Olympics 2014 Poster Abstracts
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Animal Olympics 2014
Poster Abstracts
QAAFI – Animal Science Olympics 2014Page 2
Awawdeh, Leann – Zoetis (STUDENT)
Avian colibacillosis has received considerable attention worldwide in the poultry industry
due to its direct association with significant economic losses. Economic losses are a result
of reduced production due to high mortality and morbidity. Avian pathogenic E. coli (APEC)
is the causative agent of avian colibacillosis and is capable of causing localised or systemic
extraintestinal infections. The mechanism by which APEC causes disease is not yet fully
understood. Recent overseas studies have sought to define the APEC pathotype and have
indicated that the prevalence of various virulence genes among isolates from chickens
with colibacillosis were useful markers for the detection and characterisation of APEC.
In Australia, the review of the literature shows few studies on the molecular and general
epidemiology of APEC in poultry. The initial research aims to a) evaluate methodology for
DNA extraction from the cloacal swabs of broiler chickens, b) to investigate the assumption
that in healthy birds E. coli is only present in the intestinal tract and not in the internal
organs, and c) to determine if birds with colibacillosis have APEC present on cloacal swabs
that have similar VGs to those cultured from internal organs. APEC was defined as an E. coli
that possesses two out of the following five virulence genes: iroN, iutA, iss, hlyF and ompT
using an existing and validated pentaplex PCR. These VGs were selected because they have
been previously identified as marker genes for APEC.
QAAFI – Animal Science Olympics 2014Page 3
Bishop-Hurley, Greg – Virbac and Zoetis
The viability and public acceptance of the Australian beef industry is dependent on
mitigating methane emissions from grazing cattle. However, to assess the success of
newly developed and emerging mitigation strategies it is necessary to be able to readily
measure enteric gas emissions from livestock. The measurement of gas emissions in an
autonomous fashion has the capacity of capturing and recording data in a manner that
does not significantly disturb or impede animals in their natural grazing environment.
The livestock industries require a technology to rapidly and accurately measure enteric
methane emissions from large numbers of free-grazing individual animals. An intra-
rumen capsule incorporating gas sensors (methane, carbon dioxide and hydrogen) and
a wireless sensor network platform (WSN) have been developed to measure rumen
methane concentrations. Membranes have been developed that offer stable, passive
and economical hydrogen sulphide separation from other gasses, in particular methane,
carbon dioxide and hydrogen. The gas sensing device also monitors the pressure and
temperature of the rumen. Gas concentrations obtained by the intra-rumen device
can be corrected for changes in pressure. Data are relayed through nodes outside the
rumen and displayed on the web in near real-time. Further work is required to validate
the utility of the device for determining methane yield by the animal under a range of
feeding systems. This work involves collaboration between researchers at CSIRO Animal
QAAFI – Animal Science Olympics 2014Page 4
Food and Health Sciences, CSIRO Communication Information and the Royal Melbourne
Institute of Technology (RMIT).A purpose built intra-rumen gas sensing device has been
developed and fabricated and has undergone testing to quantify both the lifespan in
the rumen environment and longevity of operation as it relates to energy consumption.
This device includes a number of improvements over the previous prototype including
measuring temperature and pressure near the sensor, more energy dense batteries,
better isolation of electronics and battery from harmful gasses and more sophisticated
software. Ruminant metabolism trials were conducted in sheep under controlled animal-
house conditions and in open-circuit respiration chambers at the CSIRO Armidale
Laboratories. Animals were fed a conventional (commercial) feedlot diet once a day.
Quantities of expired gases (methane, carbon dioxide and hydrogen) per unit feed intake
were compared with the concentration of in-rumen gases obtained by the intra-rumen gas
sensing device.
QAAFI – Animal Science Olympics 2014Page 5
Blackall, Pat – Zoetis and Virbac
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a
disease that causes significant economic losses where ever pigs are raised. There are
currently 15 serovars of A. pleuropneumoniae, with serovar 1, 5, 7 and 15 being the
dominant serovars in Australia. As the current vaccines provide protection only against
the serovars in the vaccine, the ability to recognize serovars is a key support for effective
prevention and control programs. Conventional serotyping is laborious and limited to a few
laboratories around the world with access to the necessary unique antisera. A multiplex
PCR was developed to serotype the dominant Australian serovars – 1, 5, 7, 12 and 15.
All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia,
five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with
the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A.
pleuropneumoniae species. The new multiplex PCR gave a species-specific band with all
411 A. pleuropneumoniae isolates and agreed with conventional serotyping for all serovar
1 (46 field isolates), 5 (81 field isolates), 7 (80 field isolates), 12 (16 field isolates) and
15 (117 field isolates). Out of the 25 non-typable field isolates examined, only two did not
yield a serovar band in the multiplex PCR. This multiplex PCR for serovar 1, 5, 7, 12 and 15
is species specific and capable of recognizing the serovar of diverse field isolates.
QAAFI – Animal Science Olympics 2014Page 6
Busch, Greta – MLA, Virbac, Elanco and Zoetis (STUDENT)
There are more than 890 pecies of ticks (Acari: Ixodida) found worldwide# with several
species of medical and agricultural importance. Distributed along the eastern coast of
Australia, the paralysis tick, Ixodes holocyclus, is one of at least 55 tick species capable of
causing toxicoses e.g. paralysis#. Paralysis can result in death and affects a large number
of Australian companion animals and livestock each year. The cause of paralysis is at least
one toxin produced in the salivary glands and secreted into the host while feeding. Several
toxic fractions have been isolated# with the sequence and structure of one (HT-1) published#.
There is currently no in vitro test available for the mandatory screening of putative toxins
prior to testing using in vivo assays. Aims : Develop and test an in vitro assay suitable for
screening the activity of putative toxins. Screen putative toxins and determine their in vitro
activity. Synaptosomes were isolated from rat brain homogenate using a discontinuous Percoll
gradient as previously described by Dunkley et al. 2008. All proteins were labelled using a
commercially available fluorescent dye (Alexa 488, Life Technologies). Taipan (Oxyuranus
scutellatus scutellatus) venom (Venom Supplies) was used as a binding positive control.
BSA (Sigma-Aldrich) was used as a negative non-binding control. Water was used as a blank
control. As a proof of concept, synaptosomes (50 µg and 75 µg) were incubated with varying
amounts of protein (0.5 µg, 1 µg, 5 µg) for 90 minutes at 37 °C. Binding was terminated by
centrifugation and the total relative fluorescence measured. Total relative fluorescence units
were used to indicate the level of binding activity.
QAAFI – Animal Science Olympics 2014Page 7
Castells Domingos, Yuri – Virbac, Elanco and Zoetis (presented by co-author)
In phosphorus (P) deficient rangelands in northern Australia it is recommended that P
supplements should be fed during the wet season when requirements for growth and milk
synthesis are highest. However, on commercial properties P supplements are fed only during
the dry season or not at all. The following experiment examined the capacity of pregnant heifers
to store P fed during the dry season into body reserves, and to subsequently mobilise this P
during lactation when diet P intake was inadequate. Forty heifers were fed diets either deficient
or adequate in P (0.08g and 0.28g P/kg DM, respectively) during the last 4 months of pregnancy
and the first 3 months of lactation in a 2 x 2 factorial experiment design. Thus there were 4
treatments: high-P in pregnancy/high-P in lactation (HH); high-P in pregnancy/low-P in lactation
(HL); low-P in pregnancy/high-P in lactation (LH); low-P in pregnancy/low-P in lactation (LL).
During pregnancy the HL heifers grew more and used these body nutrient stores to produce
about 33% more milk during lactation than the LL heifers. However, the LH heifers produced
60% more milk than the LL heifers. Provision of P in late pregnancy increased feed intake and
allowed some storage of P into body P reserves which could be utilized by the cow in early
lactation. Thus feeding P supplements during the late dry season when the heifer or cow is in
late pregnancy has some subsequent benefit during the wet season when the cow is lactating.
QAAFI – Animal Science Olympics 2014Page 8 Castro-Olivera, Matias – Elanco and Zoetis (STUDENT) Laminitis, a crippling foot disease of horses, is characterised by failure of the attachment between the distal phalanx and the inner hoof wall. Although several conditions can lead to laminitis, insulin dysregulation appears to be involved in the majority of cases. Hyperinsulinemia can trigger laminitis experimentally, possibly modulating lamellar epidermal cell proliferation, however the pathophysiology remains unclear. Objectives: To determine the in vivo effects of local delivery of excess insulin on lamellar perfusion, energy metabolism and histomorphometry. Methods: Both forefeet of 6 horses were instrumented with microdialysis probes perfused with a polyionic isotonic solution containing urea for 25 h. One randomly assigned foot from each horse (treatment) had human insulin 100µg/ml added to the perfusate. Dialysate samples were collected hourly for glucose, lactate, pyruvate and urea determinations. After euthanasia, lamellar tissue sections were analysed for epidermal cell proliferation (EdU stain) and histomorphometry. Wilcoxon signed ranks tests or paired t tests were used for comparisons where appropriate. Results: Median [interquartile range] glucose concentrations over the 25h period were significantly lower in treatment (0.57mmol/L[0.52 - 0.68] compared with control (0.84mmol/L[0.72 - 0.98]; p
Page 9 in other metabolites or urea clearance. Insulin treatment cause a significant change in the number of proliferating (EdU+) cells and primary epidermal lamellae surrounding the probe were significantly longer in the treatment feet (p
Page 10
Chemonges, Saul – MLA, Virbac, Elanco, Zoetis (STUDENT)
Sheep are invaluable production animals and not only contribute to the human food chain, but
a source of natural wool, for diverse cultural use including fulfilling sacrificial requirements and
their burgeoning contribution to translational research. Whilst there are many parallels in the
pathophysiology of ailments or production needs and translational relevance between sheep
and other livestock animals such as cattle and pigs, most research has only been performed
with respect to the latter two species. A problem however arises in that not enough is known
about the responses of sheep to a range of physiological and pathological events. This problem
is confounded further by potential breed differences in response. It is therefore essential that
a proteogenomic model is developed to accurately define sheep’s response to stimuli, such as
minimally invasive non-fatal experimental acute injury, before sheep can successfully be used
as a model for other species. These observations taken together, led to the research question:
How can learning from sheep help in the development of proteogenomic assays? It is believed
that every injury is associated with characteristic changes in protein expression. This work aims
to develop and optimise methods for understanding injury through proteogenomic approaches
using mass spectrometry (MS). Understanding strategies for surviving acute injury down to
molecular mechanisms involving protein expression could help in developing novel therapies
for the benefit of sheep themselves and better production outcomes for sheep growers. Most
crucial is that proteogenomic information could be used to improve gene annotations for the
recently sequenced sheep genome.
QAAFI – Animal Science Olympics 2014Page 11
Dayao, Denise – Elanco and Zoetis (STUDENT)
Antimicrobial resistance in bacterial porcine respiratory pathogens has been shown to
exist in many countries. However, little is known about the variability in antimicrobial
susceptibility within a population of a single bacterial respiratory pathogen on a pig farm.
This study examined the antimicrobial susceptibility of Actinobacillus pleuropneumoniae
using multiple isolates within a pig and across the pigs in three different slaughter
batches. Initially, the isolates from the three batches were identified, serotyped and
subsample genotyped. All isolates were identified as A. pleuropneumoniae serovar 1,
and only a single genetic profile was detected in the examined isolates. The susceptibility
of 367 isolates of A. pleuropneumoniae to ampicillin, tetracycline and tilmicosin was
determined by a disc diffusion technique. For tilmicosin, the three batches were found
to consist of a mix of susceptible and resistant isolates. The zone diameters of the three
antimicrobials varied considerably among isolates in the second sampling. Additionally,
the second sampling provided statistically significant evidence of binmodal populations
in terms of zone diameters for both tilmicosin and ampicillin. The results support the
hypothesis that the antimicrobial susceptibility of one population of a porcine respiratory
pathogen can vary within a batch of pigs on a farm.
QAAFI – Animal Science Olympics 2014Page 12 Fu, Minghai – Elanco ECR (STUDENT) Pigs start using major part of the net energy in depositing fat than muscle which results in excess fat in carcasses and losses in feed efficiency during the finishing period. Recent findings of the physiological effects of bitter taste receptors in the tongue (e.g. aversion and rejection responses) and gastrointestinal tract (e.g. secreting satiety hormones and regulating gut motility) might facilitate the potential of regulating feed intake. The current study aims to reduce feed intake and fat deposition in pigs using non-toxic bitter compounds. Two experiments were conducted. In Exp.1, 175 male pigs (72.0±5.7kg) were selected to test six bitter compounds mixed in the standard feed, i.e. caffeine (0.05%) and distillation extracts (0.1%) of rhubarb, semen brassicae, gentiana, quassia and artemisia absinthian. In Exp.2, 180 male pigs (62.7±5.2kg) were used to study dose-response effect of two effective compounds from Exp.1, i.e. gentiana and quassia (0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%). The results in Exp.1 showed that caffeine significantly decreased (p
Page 13
Gilbert, Ros – Virbac
Viruses of prokaryotes (phages) are known to naturally occur in dense populations in the
intestinal tract of herbivores, infecting and actively replicating within the enteric microbial
populations. Developments in high through-put sequencing technology have enabled these
viral populations to be characterised on an unprecedented scale, revealing extensive viral
genetic diversity. In this study viral metagenomes were prepared from the rumen fluid of
Bos taurus cattle maintained on a high grain feedlot ration, Bos indicus x Bos taurus cattle
maintained on a forage diet and from forestomach material of native Eastern Grey kangaroos
(Macropus giganteus) grazing native and improved pasture. All of the viral metagenomes
were dominated by extensive populations of tailed phages (Caudovirales) including phages
known to infect bacterial species normally found in faecal and other gut-associated microbial
ecosystems, such as Lactobacillus, Lactococcus, Clostridium and Bacteroides. The viral
metagenomes of forage-fed cattle had the most sequence similarity, indicating that the phage
types present had similar structural and functional gene requirements. In contrast, sequences
related to phages infecting Proteobacteria such as Salmonella and Aeromonas, were more
abundant in cattle fed the high grain diet. Sequences related to phages of Clostridium and
Bacillus were highly abundant in the kangaroo fore-stomach material, as were phages of
bacteria not normally found within the bovine rumen, such as Riemerella and Tetrasphaera.
This study has provided new insights into the nature of the enteric phage populations of
Queensland cattle breeds and native Australian macropods.
QAAFI – Animal Science Olympics 2014Page 14
Godwin, Rosamond, – Virbac, Elanco and Zoetis
The House fly (Musca domestica) is a cosmopolitan and ubiquitous pest of intensive animal
facilities such as cattle feedlots. An abundant mix of moisture, feed and manure provide
the perfect breeding conditions for flies and consequently vast populations can arise.
Uncontrolled fly populations are of agricultural and public health concern through their
nuisance value and the fact that they act as vectors for a wide variety of pathogens. The
challenge to control flies is great as this pest has developed resistance to almost every
class of insecticide. Non-chemical methods are preferred because of public concerns over
the build-up of chemical residues in our food and the environment. Our group has isolated
strains of a naturally occurring fungal pathogen Metarhizium anisopliae which infects
and kills housefly. We’ve demonstrated proof of concept that this fungus, as part of an
Integrated Pest Management, program offers a potential alternative to synthetic chemicals
for house fly control. The goal of our current project is to develop fungal formulations that
are commercially acceptable. Successful formulations must be economical, practical and
effective for use in the field. We have successfully identified our best isolate, optimised
production systems and have tested a range of components in lab-based assays. In the next
12 months we aim to test our best formulations in the field. Demonstrated field efficacy of
our formulations is the next step towards commercialization and more efficient fly control will
save intensive animal industries billions of dollars globally each year.
QAAFI – Animal Science Olympics 2014Page 15
Grant, Lucas – Elanco, Virbac, Zoetis (STUDENT)
Highly fermentable dietary fibre can make a direct contribution to gastrointestinal health in
pigs. It is known that modifications to the diet can lead to beneficial shifts in the microbiota,
both in terms of profile and activity. However, the relationships between the gut microbial
population and specific fibres are poorly understood. Research that links community profiling
of microbes, from animals with high and low dietary fibre is imperative to understanding
this symbiotic environment, as the production of short chain fatty acids by gut microbes is
essential for increased nutritional and health value to the animal. A controlled in vivo study,
using pigs, was undertaken to examine the effect of fibre substrates on the community
profile of bacteria, within sites of the gastrointestinal tract. Samples were collected for
analysis from animals fed a low fibre or a high fibre diet. The diets were fed for two weeks
(after a gradual change-over period), and amplicon sequencing of bacterial 16S ribosomal
RNA genes was conducted using digesta from the caecum and distal colon. Preliminary
results show differences at the Phylum and OTU level (~species). This study is novel in its
characterisation of the in vivo response of the gut-associated microbial community to a fibre-
rich food (a complex prebiotic source) within pigs.
QAAFI – Animal Science Olympics 2014Page 16
Guo, Bing – MLA AND Zoetis (STUDENT) Abstract 1
Intramuscular fat percentage (IMF%) is a commercially important phenotype for beef
cattle. A test that could predict an animal’s potential for IMF generation could be used
to better match elite performing animals to high cost grain diets. This study aimed to
develop a novel method to predict the future IMF% status of cattle using fewer animals
and more quickly than the conventional approaches. A cattle correlation dataset (gene
expression vs. IMF% in 48 Brahman steers), a sheep correlation dataset (gene expression
vs. IMF% in 20 sheep) and a cattle DE dataset (differential gene expression of Wagyu
x Hereford and Piedmontese x Hereford) were used to identify IMF% gene expression
estimators. Five genes (CIDEA, THRSP, ACSM1, DGAT2 and FABP4) most correlated
with IMF% were identified as the IMF 5 gene set by integrated analysis. Compared with
Ultrasound and spectrophotometry measurements, the IMF 5 gene set is the best choice
to make an accurate estimation on IMF% in cattle with IMF% around 2%. 31 cattle from
3 breeds across 5 developmental stages were used to develop a qPCR-based assay.
Excluding a small number of outliers, the expression of the IMF 5 gene set showed a very
high correlation (>0.8) with IMF%, demonstrating the wide utility of the IMF 5 gene set
between 2% and 20% IMF across breeds and developmental stages. Based on the current
measured HSCW and IMF%, using gene expression level of the IMF 5 gene set may be a
good predictor of the future marbling status of live animals.
QAAFI – Animal Science Olympics 2014Page 17
Guo, Bing – MLA AND Zoetis (STUDENT) Abstract 2
Enhancing growth rate is crucial for the beef cattle industry because it provides more meat
for human consumption. However, there is a paucity of tools for fundamental research on the
cellular and molecular mechanisms of cattle muscle growth which will be important for the
manipulation of the commercially important phenotype, average daily gain (ADG) of muscle.
This study aimed to identify markers for the different cellular contributors to cattle muscle
growth, and to use them to interpret differences in gene expression profiles. The expression
of two groups of genes, significantly enriched for “cell cycle” and “ECM (extracellular matrix)
organization” Gene Ontology terms was correlated with average daily gain/kg liveweight (ADG/
kg) in the longissimus muscle (LM) of 48 Brahman steers of similar age; but not across a time
course of postnatal muscle development of cattle, particularly once rates of intramuscular
fat (IMF) deposition increased. The clustering analysis based on 5,596 genes with similar
expression profiles to the ECM related genes indicated that the “cell cycle” and “ECM
organization” signals appeared to be generated by the fibro/adipogenic progenitors (FAPs).
The extent of differential expression between genotypes and the timing of the postnatal
increase in expression of the “cell cycle” and “ECM organization” gene sets and the expression
profiles of WISP2, ZNF423 and the IMF deposition genes suggests that FAPs division, whilst
responding to adipogenesis was not proportional to IMF deposition or adipocyte number;
although FAPs activity appeared to be influenced by differences in adipogenesis and IMF
deposition between genotypes.
QAAFI – Animal Science Olympics 2014Page 18
Hall, Robyn – Elanco (STUDENT) presented by co-author
The capacity to create infectious bacterial artificial chromosomes (iBAC) of the genomes
of avian herpesviruses has revolutionised genomic studies if these important viruses.
The methodologies used for the creation of iBAC clones are strongly dependent on the
capacity to grow the virus of interest in an in vitro system with high enough efficiency to
increase the likelihood of sufficient recombination events to occur to transfer the BAC
vector into the viral genome. This requirement has either hindered or prevented the
cloning of some virus strains, particularly those of Gallid herpesvirus 2. However even a
high replication capacity does not guarantee the successful characterisation of an iBAC,
which is exemplified by Gallid herpesvirus 1. To enable the cloning of herpesvirus genomes
that is not dependent on replication capacity a novel strategy for iBAC construction has
been developed which is independent of virus replication. The strategy has been reduced
to practice using Meleagrid herpesvirus 1 (MeHV-1) as an experimental model. The
concepts and strategies behind this methodology will be illustrated. There is considerable
potential to expand this approach to those herpesviruses of avian and other herpesviruses
for which iBAC construction has proven problematic will also be addressed.
QAAFI – Animal Science Olympics 2014Page 19
Harriot, Lana – Zoetis (STUDENT)
New research has recently revealed that wild dogs utilise populated areas such as school
grounds, parklands and public backyards. Close proximity to these locations increases the
opportunity for humans, and particularly children, to be exposed to wild dogs, their faecal
material and vector-borne diseases. There are numerous microorganisms that wild dogs
can carry and transmit either directly or indirectly, through environmental contamination or
vectors. These can include bacterial, parasitic and viral pathogens, of which may have serious
implications for human health. Currently the prevalence and associated risk factors of many
pathogens remains largely unknown and unexplored. The role of wild dogs in the maintenance
and transmission of pathogens with public health significance needs to be investigated to
determine the scale of the problem and if management action is required. Prevalence data on
targeted zoonotic diseases amongst peri-urban wild dogs is currently being collected utilising
faecal samples, blood samples and cadavers. Necropsy, microbiological and molecular
methods are being utilised for detection and identification of pathogens. Information will be
further integrated with geographical information to assist in the quantification of risk factors
and the public health effect of diseases carried. Preliminary results suggest that parasitic
pathogens are common in peri-urban wild dogs and the zoonotic tapeworm Echinococcus
granulosus is present in a high proportion of dogs across the landscape. Further diagnostic
testing is currently being conducted. Results from these investigations will lead to more
informed management programs for wild dogs in peri-urban areas.
QAAFI – Animal Science Olympics 2014Page 20
Hay, Karen – MLA and Zoetis (STUDENT)
The National Bovine Respiratory Disease Initiative was a nationwide cohort study
which aimed to identify and quantify risk factors for bovine respiratory disease (BRD)
in Australian feedlot cattle. Population-level effects depend on both the strength of
association with BRD and the proportion of animals in the population that are exposed
to the risk factor. This study describes the estimation of the population-level effects of
management-related risk factors and hence the identification of priority preventative
strategies or interventions most likely to reduce the impact of BRD at the population level.
Shared pen water was a common risk factor that was strongly associated with BRD
incidence. The population-level effects estimated from this study indicated that ensuring
water is not shared between feedlot pens could result in a major reduction in BRD
incidence (from 17.6% to 5.1%) in the study population assuming this exposure is causal.
Other management-related risk factors with a large to moderate population-level effect
included lifetime mixing history, timing of the move to the feedlot and group size before
entry to the feedlot. The population-level effect of the presence of bovine viral diarrhoea
virus (BVDV) in the pen indicated that eradication of BVDV would be expected to result in a
reduction in BRD incidence from 17.6% to 12.0%. Factors with a more modest population-
level effect included yard weaning and prior vaccination.
QAAFI – Animal Science Olympics 2014Page 21
James, Peter – Virbac and Zoetis
Buffalo fly (BF), Haematobia exigua and horn fly (HF), Haematobia irritans infestations are
consistently listed by northern Australian and USA beef and dairy producers as amongst
their main cattle health problems. Since the 1980s HF has spread through most of South
America and the area in Australia infested with BF is projected to extend significantly
southward with climate change. Research into new methods of BF control has been
limited by the need to maintain live cattle hosts. This is expensive, severely restricts the
type of research that can be conducted and is increasingly problematic from an animal
ethics standpoint. We report development of an in vitro rearing technique for buffalo fly
whereby all stages can be grown in the laboratory without the need for a live animal host.
The current laboratory colony has now been maintained for 47 generations. Availability
of this colony will help maintain or improve the efficiency of currently available control
methods by providing a characterised and consistent strain of BF for use in detecting
and monitoring resistance and for testing new candidate control compounds. It will also
facilitate the development of new control techniques such as methods incorporating
the use of Wolbachia and the release of genetically modified strains. We have also
established the first ever H. irritans and H. exigua cell lines. The H irritans lines have
undergone multiple passages and have now been cryopreserved for future use. The H.
exigua line was lost during the Brisbane floods but we expect to re-establish a BF line
QAAFI – Animal Science Olympics 2014Page 22
with the methods utilised successfully for HF. These cell lines will be critical to attempts
to transfect Wolbachia into buffalo fly. They will also provide an important resource for
research on BF and HF in areas such as early screening and clarification of modes of
action of new biocidal molecules, the use of viruses and baculoviruses for biocontrol,
vaccine design, clarification of gene action and elaboration of intracellular biochemical
pathways that could provide future insecticide or vaccine targets.
QAAFI – Animal Science Olympics 2014Page 23 Karbanowicz, Thomas – Elanco (STUDENT) Cell surface display libraries (CSDLs) are a powerful tool for screening peptides, expressed single-chain variables or fragment antigens exposed upon the cell surface of Escherichia coli or yeast. This methodology allows for high throughput screening and selection for antibodies based on affinity, utilizing fluorescence activated cell sorting (FACS). Current technologies rely upon phage display libraries however significant limitations in affinity maturation and unpredictable expression bias are difficult to overcome. Previous attempts to express holocyclotoxin-1 (HT-1) a toxin from the Australian paralysis tick, Ixodes holocyclus, using prokaryotic expression systems have been unsuccessful. Eukaryotic display libraries utilizing Saccharomyces cerevisiae possess protein folding and secretory machines homologous to mammalian cells. In this research, a novel S. cerevisiae cell line, IMTV014, was developed to extracellularly display HT-1. Holocyclotoxin-1 was efficiently displayed (90%, P
Page 24 Lees, Angela – MLA (STUDENT) Body temperature (BT) is considered to be the most reliable indicator of thermal balance. Continuous measurement of BT is often difficult and data collection is often restricted to short intervals (≤ 10 d). Technological advancement with remote sensing technology, through rumen boluses, has the potential to obtain BT over long periods of time. However studies on the impact of environmental heat load on rumen temperature (TRUM) are limited. The objective of this study was to investigate the diurnal rhythm TRUM of shaded and un-shaded steers over 130 days. The study described herein was part of a larger 180 day feedlot study involving 36 steers (12 Angus, 12 Charolais and 12 Brahman). Only the results from Brahman and Angus will be discussed here. Cattle were allocated to two treatments; un-shaded and shaded (3 m2 shade/animal); treatments were replicated with 3 pens per treatment. Rumen temperature was recorded at 10 min intervals using active RFID rumen boluses. Individual TRUM data were converted to an hourly average and then mean hourly TRUM within breed x treatment were calculated. Rumen temperature was analysed using a repeated measures model for the effect of treatment (P=0.60); breed×treatment (P=0.57); breed (P=0.0002); time of day (hour; P
Page 25 Lees, Jarrod – Virbac and Elanco (STUDENT) The aim was to determine the effect of shade on vaginal temperature (TVAG) of dairy cows housed outside in a subtropical environment. Holstein Friesian cows (n=40) were paired by milk yield and live weight, and allocated to a treatment (shade/no shade). Pairs were randomly allocated to a TVAG group (5 pairs/group). Data were collected on one group per week, over eight weeks (1 replication/4 weeks). A temperature logger was used to collect TVAG over 5 d. Temperature Humidity Index (THI) was calculated using data from an onsite weather station. Cows were under treatment conditions from 0700-1445 h daily and turned out to pasture overnight. TVAG and THI were averaged for each h over each 5 d period. These data were split into three periods Day (D), Night (N), and Evening (E); TVAG_D and THI_D (0900-1500 h); TVAG_N and THI_N (2200-0600 h); and TVAG_E and THI_E (1700-2100 h). Difference between D and N (TVAG_D-N and THI_D-N) were also calculated. Pearsons correlations were performed on TVAG against THI to determine any if relationships existed for shade, no shade and all cows. Relationships were found for N (r = -0.26; P = 0.041); D (r = 0.34; P=0.006); and D-N (r=0.61; P
Page 26 Othman, Azalea Hani – MLA, Virbac, Elanco and Zoetis (STUDENT) Noxious experiences of surgical husbandry procedures are an increasingly controversial aspect of animal production systems. Various types of objective parameters (cortisol, behaviour) have been postulated to quantify noxiousness, however they do not specifically reflect pain. This study aims to refine a multi-parameter ‘toolkit’ in assessment of painful husbandry procedure, using a sheep surgical castration model. Twelve juvenile Merino cross sheep were assigned to 2 groups of 6 : surgically castrated (CAST) or sham-castrated (CON). Castration was an open surgical technique without the provision of anaesthesia or analgesia. Blood samples were collected immediately prior to castration (0), and at 10, 20, 30, 45 min, 1, 2, 8, 12 hr and daily until day 7 post-castration. Plasma concentrations of the biomarkers were determined : cortisol (CORT), haptoglobin (Hp), interleukin-6 (IL-6), substance P (SP), beta-endorphin (β-EP) and prostaglandin E2 (PGE2). CORT in CAST was significantly (P
Page 27 Ong, Chian Teng – Zoetis (STUDENT) Ixodes holocyclus (Australian paralysis tick) occurs along the eastern coast of the Australian continent. The primary host is the indigenous marsupial (bandicoot) and secondary host toxicoses annually include ~100,000 domestic animals and up to 10,000 companion animals with high levels of fatality. Holocyclotoxin, the causative agent of tick paralysisis produced in saliva as the adult female tick engorges. Treatment of affected animals involves the timely delivery of antiserum prepared from hyperimmune dogs. Limited I. holocyclus sequence data is available and thus next generation sequencing was undertaken for the first time. Roche 454 sequence analysis was conducted on cDNA from salivary glands (SG) of bandicoot-engorged female ticks and also on whole nymphs. Subsequent Illumina HiSeq analysis examined the transcriptome of SG and the viscera of fully-engorged I. holocyclus ticks collected from dogs and cats with paralysissymptoms. Transcriptome assemblies resulted in 87,493 transcripts with a minimum length of 200 bp with 2877, 4247, 41669 and 38700 attributed to 454 SG bandicoot- engorged, 454 nymph, Illumina SG dog/cat-engorged, and Illumina viscera dog/cat-engorged, respectively. Transcripts can be categorised into 6 functional groups - Housekeeping, Pathogen, Secreted proteins, Transposable elements, Previously Identified and Novel. Each library is found to have different compositions of the 6 functionally groups. Interestingly, different holocyclotoxin transcripts were obtained from cat/dog-engorged ticks compared with transcripts obtained from bandicoot-engorged I. holocyclus. Data showed that holocyclotoxins are members of a multivariable protein family with a highly conserved structure. Research is underway to develop novel anti-toxin treatments and or vaccines. QAAFI – Animal Science Olympics 2014
Page 28
Martinez Fernandez, Gonzalo – Elanco ECR (STUDENT)
The aim of this trial was study the effect of chloroform on methanogenesis and hydrogen
production at different doses in cattle. Eight fistulated steers were allocated in two
experimental groups fed with one of the experimental diets, a forage diet consisted of low
quality Rhodes grass hay or a forage diet supplemented with a concentrate at a ratio of 60:40.
Three chloroform- cyclodextrin (CD) doses were used throughout the experiment at different
effect levels (low, medium and high). Following the control period animals received each dose
of chloroform-CD (increasing progressively up to the final dose) for 10 d with the last two days
being confined in open-circuit respiration chambers for direct measurement of methane and
hydrogen production and collection of rumen samples.
Dry matter intakes were not dramatically decreased on both diets when animals were
treated with chloroform-CD. Methane production (g) per day and per kg of DMI was reduced
progressively when doses of chloroform were increased on both diets. Conversely hydrogen
expelled by treated animals increased progressively as methane production was reduced,
with the greatest amounts of hydrogen loss occurring in animals supplemented with the
grass/concentrate diet. Rumen fermentation parameters showed a shift in the fermentation
pathways towards a more propionic acid production. The observed results are in accordance
with previous studies (Mitsumori et al., 2012), which suggest that ruminants could adapt
to a high hydrogen rumen concentration. Therefore, dietary strategies would be required to
facilitate the consumption of this hydrogen excess for improving energy supply to the animal.
QAAFI – Animal Science Olympics 2014Page 29
Ramirez, Orlando – MLA, Virbac, Elanco and Zoetis (STUDENT)
Endometrial biopsies have been used to evaluate uterine function. However, its application
in the embryo industry is limited since biopsy may affect embryo development and
implantation. The objective is to evaluate the effect of endometrial biopsy on (1) histological
changes in the bovine uteri (2) expression of pro-inflammatory markers (3) corpus luteum
(CL) function (4) pregnancy success. Biopsies were collected from the uterine horn ipsilateral
to the CL using circular biopsy forceps. In Experiment 1, 10 Bos indicus/Droughmaster
heifers/cows were biopsied (n=6) at day 7th of the cycle before embryo transfer. In
Experiment 2, 9 Droughmaster heifers/cows were biopsied (n=5) at day 4th of the same
cycle of embryo transfer. The uterus were collected at day 7 for histopathological analysis
and molecular expression (IL-1β, TNFα). In Experiment 3 (in progress), 15 Charbrays heifers
are either biopsied at day 4th (n=8) or at day 7th (n=7). In both groups at day 7th, an
embryo is transferred to the same biopsied-horn. Progesterone and PGFM concentration
are measured and pregnancy is diagnosed at day 35th. In Experiment 1, the macroscopic
evaluation revealed a focal-well demarcated, dark-red discolouration of the endometrium
in four uterus. However, the histopathological examination did not show evidence of
inflammatory infiltrate in the biopsied horn and the adjacent segments.The histopathological
examination, gene expression and corpus luteum evaluation will allow us to understand
whether the uterine function is compromised by the biopsy procedure.
QAAFI – Animal Science Olympics 2014Page 30
Raza, Ali – Future farming Virbac (STUDENT)
Anthelmintic resistance poses a threat to our ability to control parasitic intestinal worms in
livestock production systems worldwide. There is also an increasing possibility that resistance
will affect our ability to control intestinal worms in humans in coming years. Certain specific
and non-specific mechanisms of drug resistance have been implicated in nematodes. Our
knowledge of non-specific mechanisms is, however, less advanced as most attention to date
has focused on specific drug target–based resistance mechanisms. We aim to explore the
role of drug efflux systems (P-glycoproteins, P-gps) in anthelmintic resistance in Haemonchus
contortus by testing the following hypotheses: (i) multidrug-resistant inhibitors (MDRIs) can
reverse anthelmintic resistance either partially or completely, (ii) expression of P-gps varies
between resistant and susceptible isolates of H. contortus, and (iii) anthelmintics induce
P-gps in nematodes, hence providing drug-exposed worms with a mechanism to tolerate the
presence of the drug. Our initial results suggest that some modern MDRIs are able to reverse
the anthelmintic resistance shown by the Wallangra (HcW) isolate of H. contortus in larval
development and migration assays. We have established a rhodamine123 efflux assay in
order to study efflux activities in nematode eggs and larvae, and will be utilising this assay
to examine induction of P-gp activity in resistant and susceptible isolates. Future studies will
include the analysis of expression levels of P-gps in resistant and susceptible isolates of H.
contortus and induction of P-gps following exposure to anthelmintics.
QAAFI – Animal Science Olympics 2014Page 31
Schofield, Benjamin – Elanco and Zoetis (STUDENT)
In ruminants such as sheep and cattle the digestion of plant material is primarily
driven by the rumen microbiological community. Within this community key organisms
play a specific role in digestion, ranging from fibre degradation, VFA production, to the
regeneration of essential coenzymes such as those involved with methanogenesis.
It is therefore important to maintain a healthy rumen microbial community to ensure
maximum production efficiency. With the use of probiotic organisms a healthy rumen
microbial ecosystem can be achieved. Bacillus amyloliquefaciens H57 is a probiotic
isolated specifically for its antimicrobial activity and its ability to form spores. In this
study B. amyloliquefaciens H57 was fed to pregnant Dorper ewes to monitor its effect on
the rumen microbial community. Rumen fluid samples were collected and the microbial
community established through Illumina paired-end sequencing of the 16S rRNA
amplicon. After comparing the microbial populations of control animals to that of animals
fed the probiotic, it was evident that there was a significant difference between the two
groups. In the probiotic treated group the dominant organism was classified as part of the
S24-7 family whilst the dominant organisms within the control group were members of the
Prevotella genus. As not a lot is known about the S24-7 family it is hoped that with further
genomic analysis that its role in the rumen can be established as well as its interaction
with the probiotic B. amyloliquefaciens H57.
QAAFI – Animal Science Olympics 2014Page 32
Tan, Eddie – Virbac, Elanco, Zoetis (STUDENT) Abstract 1
Indospicine is a natural hepatotoxic amino acid found in Indigofera plant species and numerous
reports of mild to severe liver disease reports in animals ingesting these plants have been
published. Dogs are highly susceptible to indospicine, and dietary exposure to indospicine residues
present in camel meat has recently caused secondary hepatotoxic indospicine poisoning of dogs.
Several native Indigofera species are known to be palatable to feral camels in central Australia,
and questions have been raised about the prevalence of indospicine residues in their tissues.
Indospicine quantitation has previously been done by amino acid analysis, HPLC and more
recently by LC-MS/MS with phenylisothiocyanate derivitization. The analysis methods were lengthy
(nearly 2 h) with poor resolution from other amino acids. These factors restrict rapid advance
in indospicine related research. In this study, an analytical method with no derivatization was
developed and validated, based on heptafluorobutyric acid ion-pairing reagent, with the use of
ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine
the level of indospicine in camel muscle samples using Stable Isotope Dilution Assay (SIDA). This
is the first high throughout non-derivatized UPLC-MS/MS method with high recovery efficiency and
repeatability for the quantitation of indospicine in camel meat samples. This method has no amino
acids resolution problem with short 10 minutes analysis time. Camel meat samples collected from
the Simpson Desert region of central Australia were largely contaminated with indospicine (≈50%)
and the highest indospicine residue is 3.73 mg/kg (fresh weight). However, the majority of samples
(95%) contained less than 1 mg/kg indospicine.
QAAFI – Animal Science Olympics 2014Page 33
Tan, Eddie – Virbac, Elanco, Zoetis (STUDENT) Abstract 2
There are nearly 700 leguminous Indigofera plants in the world with more than 40 species of this
genus recorded in grazing regions of Australia. These plants are generally palatable to livestock
with several species considered as potential feed and fodder legumes due to their high protein
content. However, certain Indigofera species contain the hepatotoxic nonproteinogenic amino
acid indospicine, and primary and secondary animals’ hepatotoxicosis have been reported due
to consumption of Indigofera plants. Indospicine is unusual in that it accumulates in animal
tissues as the free amino acid and such residues persists for several months after cessation of
consumption of Indigofera plant material. Dogs are particularly sensitive to indospicine toxicity
with reported domestic dog deaths in Australia attributed to the consumption of indospicine
contaminated horse and camel meat. The indospicine concentration in many Indigofera taxa
has not previously been established and is of interest to determine the risk posed by these
pasture plant species. This paper determines the indospicine content of 11 of the more
common Indigofera species in Australia by a validated high throughput ultra-performance liquid
chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Only the naturalised
non-native species I. spicata followed by the widespread native species I. linnaei contained high
levels of indospicine and may pose a hepatotoxic threat to animals. Other Indigofera species
analysed (I. colutea, I. linifolia, I. adesmiifolia, I. georgei, I. hirsuta, I. leucotricha, I. oblongifolia, I.
parviflora and I. trita) contain negligible or non-detectable levels of indospicine and do not pose a
similar risk.
QAAFI – Animal Science Olympics 2014Page 34
Xiang, Ruidong – Virbac, Elanco and Zoetis
Parent-of-origin dependent (epi)genetic factors are important determinants of prenatal
development that program adult phenotype. However, data on magnitude and specificity
of parental genome effects on fetal bone are lacking. We used an outbred bovine model
to dissect and quantify effects of parental genomes, fetal sex and non-genetic maternal
effects on the fetal skeleton and analyzed phenotypic and molecular relationships
between fetal muscle and bone. Analysis of 51 bone morphometric and weight
parameters from 72 fetuses recovered at Day153 gestation (54% term) identified six
principal components (PC1-6) that explained 80% of the variation in skeletal parameters.
Parental genomes accounted for most of the variation (72.1%-99.7%) in bone wet weight
(PC1), limb ossification (PC2), flat bone size (PC4) and axial skeletal growth (PC5). Limb
length showed lesser effects of parental genomes (PC3, 40.8%) and a significant non-
genetic maternal effect (gestational weight gain, 29%). Partitioning of variation explained
by parental genomes revealed strong maternal genome effects on bone wet weight
(74.1%, P < 0.0001) and axial skeletal growth (93.5%, P < 0.001), while paternal genome
controlled limb ossification (95.1%, P < 0.0001). Histomorphometric data revealed strong
maternal genome effects on growth plate height (98.6%, P < 0.0001) and trabecular
thickness (85.5%, P < 0.0001) in distal femur. Parental genome effects on fetal bone
were mirrored by maternal genome effects on fetal serum 25-hydroxyvitamin D (96.9%,
QAAFI – Animal Science Olympics 2014P < 0.001) and paternal genome effects on alkaline phosphatase (90.0%, P < 0.001) Page and 35
their correlations with maternally controlled bone wet weight and paternally controlled
limb ossification, respectively. Bone wet weight and flat bone size correlated positively with
muscle weight (r = 0.84 and 0.77, P < 0.0001) and negatively with muscle H19 expression
(r= -0.34 and -0.31, P < 0.01). Since Imprinted H19 regulates growth factors by miRNA
interference, this suggests muscle-bone interaction via epigenetic factors.
QAAFI – Animal Science Olympics 2014Page 36
Xu, Tao, MLA and Elanco (STUDENT)
The cattle tick, Rhipicephalus microplus causes the most significant loss for tropical and sub-
tropical beef industries worldwide estimated at $US2.5b annually. Members of the serine protease
inhibitor (serpin) family play important roles during the interaction between hosts and ticks. Tick
serpins manipulate the hosts’ coagulation system for successful blood feeding, disrupt the hosts’
inflammatory and immune responses, and are also involved in tick physiological regulations.
Studies of R. microplus serpins are limited and thus this study aimed to identify, express and
determine the biological activity of the recombinant R.microplus serpins. CattleTickBase was
mined for serpin sequences and 13 serpins were identified, named RMS-1 to RMS-14. Seven
full length serpins were selected for transcriptional analyses and semi-quantitative RT-PCR
demonstrated differential expression in different female adult organs as well as during different
tick life stages. The biological function was examined using the recombinant R. microplus serpins
(rRMSs) expressed by the yeast Pichia pastoris. rRMS-4 prolonged blood clotting in an RCT
assay and specifically inhibited Thrombin at the inhibition rate of 6.0±0.35x104M-1s-1. rRMS-3
inhibited Chymotrypsin and Elastase. Chymotrypsin was also inhibited by rRMS-9. ELISA screening
demonstrated that rRMS-3 and -4 were recognised by sera collected from tick exposed cattle. To
further understand the mechanism of serpin inhibition, the tertiary structure of cleaved rRMS-9
was resolved by crystallisation and X-ray diffraction confirming that RMS-9 undergoes the serpin
signature conformational change. This preliminary study demonstrates the function of R. microplus
serpins and provides potential avenues in the development of innovative tick control methods.
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