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Vietnamese smallholder dairy cows (VDC) are the result of crossbreeding between different zebu (ZEB) and taurine dairy breeds through many undefined generations. Thus, the predominant breed composition of VDC is currently unknown. This study aimed to evaluate the level of genetic diversity and breed composition of VDC. The SNP data of 344 animals from 32 farms located across four dairy regions of Vietnam were collected and merged with genomic reference data, which included three ZEB breeds: Red Sindhi, Sahiwal and Brahman, three taurine breeds: Holstein (HOL), Jersey (JER) and Brown Swiss (BSW), and a composite breed: Chinese Yellow cattle. Diversity and admixture analyses were applied to the merged data set. The VDC were not excessively inbred, as indicated by very low inbreeding coefficients (Wright's F ranged from -0.017 to 0.003). The genetic fractions in the test herds suggested that the VDC are primarily composed of HOL (85.0%); however, JER (6.0%), BSW 5.3%) and ZEB (4.5%) had also contributed. Furthermore, major genotype groupings in the test herds were pure HOL (48%), B3:15/16HOL_1/16ZEB (22%) and B2:7/8HOL_1/8ZEB (12%). The genetic makeup of the VDC is mainly components of various dairy breeds but also has a small percentage of ZEB; thus, the VDC could be a good genetic base for selecting high milk-producing cows with some degree of adaptation to tropical conditions.

This study aimed to rank potential drivers of cow productivity and welfare in tropical smallholder dairy farms (SDFs) in Vietnam. Forty-one variables were collected from 32 SDFs located in four geographically diverse dairy regions, with eight SDFs per region. Twelve variables, including milk yield (MILK), percentages of milk fat (mFA), protein (mPR), dry matter (mDM), energy-corrected milk yield (ECM), heart girth (HG), body weight (BW), ECM per 100 kg BW (ECMbw), body condition score (BCS), panting score (PS), inseminations per conception (tAI), and milk electrical resistance (mRE) of cows, were fitted as outcome variables in the models. Twenty-one other variables describing farm altitude, housing condition, and diet for the cows, cow genotypes, and cow physiological stage were fitted as explanatory variables. Increased farm altitude was associated with increases in ECM and mRE and with decreases in PS and tAI (P < 0.05). Increases in roof heights and percentage of shed side open were associated with increases in ECM, mFA, and mDM (P < 0.05). Increased dry matter intake and dietary densities of dry matter and fat were associated with increased MILK, ECM, and ECMbw and decreased tAI (P < 0.05). Increased dietary lignin density was associated with increased PS. Increased genetic proportion of Brown Swiss in the herd was associated with increased MILK, ECM, and ECMbw (P < 0.05). Thus, to improve cow productivity and welfare in Vietnamese SDFs, the following interventions were identified for testing in future cause-effect experiments: increasing floor area per cow, roof heights, shed sides open, dry matter intake, dietary fat density, and the genetic proportion of Brown Swiss and decreasing dietary lignin density.

The Wagyu breed of taurine cattle possess favourable genetics for intramuscular fat (IMF) but genomic loci associated with the trait remain under characterised. Here, we report the identification of a previously unidentified genomic region possessing a particular haplotype structure in Wagyu. Through deployment of a genome-wide haplotype detection analysis that captures regions conserved in a target population but not other populations we screened 100 individual Wagyu and contrasted them with 100 individuals from two independent comparison breeds, Charolais and Angus, using high-density SNPs. An extreme level of Wagyu conservation was assigned to a single genomic window (spanning genomic coordinates BTA28:41 088-300 265 bp). In fact, a five-SNP region spanning 27 096 bp is almost perfectly conserved among the 100 Wagyu individuals assayed and partially overlaps RAB4A. Focussing in, two consecutive SNPs (genomic coordinates 236 949 and 239 950) are apparently fixed within the Wagyu (BB and AA respectively), but at mixed frequencies in the other two breeds. These SNPs are located in the two introns straddling exon 7. In a separate analysis using the 1000 Bulls database, we found that, coincident with exon 7 of RAB4A first allele frequencies were highest in the high IMF Japanese Native (Wagyu) breeds (0.78) and lowest in the low IMF indicine breeds (Nelore and Brahman), with intermediate marbling breeds (Angus and Charolais) assigned intermediate rankings (0.42). RAB4A is known to encode a protein that regulates intracellular trafficking of the insulin-regulated glucose transporter GLUT4. RAB4A can be considered an attractive new positional candidate for IMF development.

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species.

Smallholder dairy farms (SDFs) are distributed widely across lowland and highland regions in Vietnam, but data on the productivity and welfare status of these cows remains limited. This cross-sectional study was conducted to describe and compare the productivity and welfare status of SDF cows across contrasting regions. It was conducted in autumn 2017 on 32 SDFs randomly selected from four typical but contrasting dairy regions (eight SDFs per region), a south lowland, a south highland, a north lowland, and a north highland region. Each farm was visited over a 24-h period (an afternoon followed by a morning milking and adjacent husbandry activities) to collect data of individual lactating cows (n = 345) and dry cows (n = 123), which included: milk yield and concentrations, body weight (BW), body condition score (BCS, 5-point scale, 5 = very fat), inseminations per conception, and level of heat stress experienced (panting score, 4.5-point scale, 0 = no stress). The high level of heat stress (96% of lactating cows were moderate to highly heat-stressed in the afternoon), low energy corrected milk yield (15.7 kg/cow/d), low percentage of lactating cows (37.3% herd), low BW (498 and 521 kg in lactating and dry cows, respectively), and low BCS of lactating cows (2.8) were the most important productivity and welfare concerns determined and these were most serious in the south lowland. By contrast, cows in the north lowland, a relatively hot but new dairying region, performed similarly to those in the south highland, a region historically considered to be one of the most suitable for dairy cows in Vietnam due to its cool environment. This indicates the potential to mitigate heat stress through new husbandry strategies. Cows in the north highland had the highest BW (535 and 569 kg in lactating and dry cows, respectively) and the highest energy corrected milk yield (19.2 kg/cow/d). Cows in all regions were heat-stressed during the daytime, although less so in the highlands compared to the lowlands. Opportunities for research into improving the productivity and welfare of Vietnamese SDF cows are discussed.

In smallholder dairy farms (SDFs), farmers often build cowsheds using local materials and based on self-accumulated experience without due consideration to reducing the risk of heat stress. This study aimed to characterise the heat stress abatement strategies and microclimate within SDF cowsheds from four typical dairy regions of Vietnam (south lowland, south highland, north lowland and north highland) and identify the housing parameters most associated with the microclimate. The study was conducted on 32 SDFs (eight SDFs per region) in autumn 2017. Twelve housing management variables, illustrating cowshed design and heat stress abatement methods of each SDF, were collected. Six microclimate parameters, collected within the cowshed, were temperature (AT), humidity, air speed (AS), heat load index (HLI), Temperature-humidity index (THI) and accumulated heat load units (AHLU) during a day (06:00 h to 18:00 h). Factor analysis and cluster analysis was applied to group cowsheds of SDFs into clusters where SDFs in the cluster had the same cowshed characteristics. Multivariable linear models were applied to define the parameters most likely to inform future research into heat stress mitigation on SDF. Averaged from 08:00 h to 18:00 h, microclimate inside the cowsheds was considered hot (HLI &gt, 79) in the highland and very hot (HLI &gt, 86) in the lowland regions. Cows in the lowland regions accumulated high heat load (AHLU &gt, 50) by 18:00 h. Cowsheds of SDFs varied widely and grouped into seven cowshed types, but no type was more effective than others in reducing heat stress conditions within cowsheds. Using roof soakers together with fans decreased AT and HLI by 1.3 °C and 3.2 units, respectively, at 14:00 h compared to 11:00 h. Each 100 m increase in altitude was associated with decreases of 0.4 °C in AT, 1.3 units in HLI and 0.8 units in THI (p &lt, 0.001). Each meter increase in the eave height of the cowshed roof was associated with decreases of 0.87 °C in AT, 3.31 units in HLI and 1.42 units in THI, and an increase of 0.14 m/s in AS (p &lt, 0.05). The cowshed parameters that should be prioritised for future research into the amelioration of heat stress in SDF cows include using the roof soakers together with fans, increasing altitude, eave roof height and floor area per cow.

Brahman cattle (Bos indicus) are well adapted to thrive in tropical environments. Since their introduction to Australia in 1933, Brahman’s ability to grow and reproduce on marginal lands has proven their value in the tropical beef industry. The poll phenotype, which describes the absence of horns, has become desirable in the cattle industry for animal welfare and handler safety concerns. The poll locus has been mapped to chromosome one. Four alleles, each a copy number variant, have been reported across this locus in B. indicus and Bos taurus. However, the causative mutation in Brahman cattle has not been fully characterized. Oxford Nanopore Technologies’ minION sequencer was used to sequence four homozygous poll (PcPc), four homozygous horned (pp), and three heterozygous (Pcp) Brahmans to characterize the poll allele in Brahman cattle. A total of 98 Gb were sequenced and an average coverage of 3.33X was achieved. Read N50 scores ranged from 9.9 to 19 kb. Examination of the mapped reads across the poll locus revealed insertions approximately 200 bp in length in the poll animals that were absent in the horned animals. These results are consistent with the Celtic poll allele, a 212-bp duplication that replaces 10 bp. This provides direct evidence that the Celtic poll allele is segregating in the Australian Brahman population.

Heifers that have an earlier age at puberty often have greater lifetime productivity. Age at puberty is moderately heritable so selection should effectively reduce the number of days to puberty, and improve heifer productivity and profitability as a result. However, recording age at puberty is intensive, requiring repeat ovarian scanning to determine age at first corpus luteum (AGECL). Genomic selection has been proposed as a strategy to select for earlier age at puberty; however, large reference populations of cows with AGECL records and genotypes would be required to generate accurate GEBV for this trait. Reproductive maturity score (RMS) is a proxy trait for age at puberty for implementation in northern Australia beef herds, where large scale recording of AGECL is not feasible. RMS assigns a score of 0 to 5 from a single ovarian scan to describe ovarian maturity at ~600 d. Here we use multivariate genomic prediction to evaluate the value of a large RMS data set to improve accuracy of GEBV for age at puberty (AGECL). There were 882 Brahman and 990 Tropical Composite heifers with AGECL phenotypes, and an independent set of 974 Brahman, 1,798 Santa Gertrudis, and 910 Droughtmaster heifers with RMS phenotypes. All animals had 728,785 real or imputed SNP genotypes. The correlation of AGECL and RMS (h2 = 0.23) was estimated as -0.83 using the genomic information. This result also demonstrates that using genomic information it is possible to estimate genetic correlations between traits collected on different animals in different herds, with minimal or unknown pedigree linkage between them. Inclusion of heifers with RMS in the multi-trait model improved the accuracy of genomic evaluations for AGECL. Accuracy of RMS GEBV generally did not improve by adding heifers with AGECL phenotypes into the reference population. These results suggest that RMS and AGECL may be used together in a multi-trait prediction model to increase the accuracy of prediction for age at puberty in tropically adapted beef cattle.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

We introduce an innovative approach to lowering the overall cost of obtaining genomic EBV (GEBV) and encourage their use in commercial extensive herds of Brahman beef cattle. In our approach, the DNA genotyping of cow herds from 2 independent properties was performed using a high-density bovine SNP chip on DNA from pooled blood samples, grouped according to the result of a pregnancy test following their first and second joining opportunities. For the DNA pooling strategy, 15 to 28 blood samples from the same phenotype and contemporary group were allocated to pools. Across the 2 properties, a total of 183 pools were created representing 4,164 cows. In addition, blood samples from 309 bulls from the same properties were also taken. After genotyping and quality control, 74,584 remaining SNP were used for analyses. Pools and individual DNA samples were related by means of a "hybrid" genomic relationship matrix. The pooled genotyping analysis of 2 large and independent commercial populations of tropical beef cattle was able to recover significant and plausible associations between SNP and pregnancy test outcome. We discuss 24 SNP with significant association ( < 1.0 × 10) and mapped within 40 kb of an annotated gene. We have established a method to estimate the GEBV in young herd bulls for a trait that is currently unable to be predicted at all. In summary, our novel approach allowed us to conduct genomic analyses of fertility in 2 large commercial Brahman herds managed under extensive pastoral conditions.

An endometrial biopsy allows for a comprehensive assessment of the uterine environment of a breeding female. Although routine in mares, devices used for endometrial biopsies are impracticable in heifers due to the size and structure of the cervix. This report describes the use of a human bronchoscopy biopsy device (Karl Storz® 10366L) for collection of endometrial biopsies in Bos indicus beef heifers. The Storz® device is smaller and thinner and enabled the collection of an endometrial biopsy in 86% of heifers (n = 44/51). The biopsied tissue was of good quality and suitable for transcriptomic assessment of the endometrium, with total RNA yield and RNA integrity number (RIN) averaging 1.3 μg (range 0.4-5.3 μg) and 7.4 (range 5.7-8.4), respectively.

Brahman cattle have a Bos indicus and Bos taurus mosaic genome, as a result of the process used to create the breed (repeat backcrossing of Bos taurus females to Bos indicus bulls). With the aim of identifying Bos taurus segments in the Brahman genome at sequence level resolution, we sequenced the genomes of 46 influential Brahman bulls. Using 36 million variants identified in the sequences, we searched for regions close to fixation for Bos indicus or Bos taurus segments that were longer than expected by chance (from simulation of the breed formation history of Brahman cattle). Regions close to fixation for Bos indicus content were enriched for protein synthesis genes, while regions of higher Bos taurus content included genes of the G-protein coupled receptor family (including genes implicated in puberty, such as THRS). The region with the most extreme Bos taurus enrichment was on chromosome 14 surrounding PLAG1. The introgressed Bos taurus allele at PLAG1 increases stature and the high frequency of the allele likely reflects strong selection for the trait. Finally, we provide evidence that the polled mutation in Brahmans, a desirable trait under very strong recent selection, is of Celtic origin and is introgressed from Bos taurus.

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

Global aquaculture has expanded rapidly to address the increasing demand for aquatic protein needs and an uncertain future for wild fisheries. To date, however, most farmed aquatic stocks are essentially wild and little is known about their genomes or the genes that affect important economic traits in culture. Biologists have recognized that recent technological advances including next generation sequencing (NGS) have opened up the possibility of generating genome wide sequence data sets rapidly from non-model organisms at a reasonable cost. In an era when virtually any study organism can ‘go genomic’, understanding gene function and genetic effects on expressed quantitative trait locus phenotypes will be fundamental to future knowledge development. Many factors can influence the individual growth rate in target species but of particular importance in agriculture and aquaculture will be the identification and characterization of the specific gene loci that contribute important phenotypic variation to growth because the information can be applied to speed up genetic improvement programmes and to increase productivity via marker-assisted selection (MAS). While currently there is only limited genomic information available for any crustacean species, a number of putative candidate genes have been identified or implicated in growth and muscle development in some species. In an effort to stimulate increased research on the identification of growth-related genes in crustacean species, here we review the available information on: (i) associations between genes and growth reported in crustaceans, (ii) growth-related genes involved with moulting, (iii) muscle development and degradation genes involved in moulting and (iv) correlations between DNA sequences that have confirmed growth trait effects in farmed animal species used in terrestrial agriculture and related sequences in crustacean species. The information in concert can provide a foundation for increasing the rate at which knowledge about key genes affecting growth traits in crustacean species is gained.

We outline an in vivo cellular program of bovine longissimus muscle development inferred from expression data from 60 days post conception to 3months postnatal. Analytic challenges included changes in cellular composition, ambiguous 'diagnostic' markers of cell type and contrasts between cattle human and mouse myogenesis. Nevertheless, the expression profiles of the myosin isoforms support slow and fast muscle fibres emanating from primary and secondary myogenesis respectively, while expression of the prenatal myosin subunits is down regulated prior to birth. Of the canonical pro-myogenic transcription factors (TF), MYF6 and MYF5 are negatively co-expressed, with MYF6 displaying higher expression in the post-natal samples and MYF5, MYOG, HES6 and PAX7 displaying higher expression in early development. A set of TFs (SIX1, EYA2 and DACH2) considered important in undifferentiated murine cells were equally abundant in differentiated bovine cells. An examination of mammalian regulators of fibre composition, muscle mass and muscle metabolism, underscored the roles of PPARGC1A, TGFβ signalling and the NHR4 Nuclear Hormone Receptors on bovine muscle development. Enriched among the most variably expressed genes from the entire data set were molecules regulating mitochondrial metabolism of carbohydrate (PDK4), fat (UCP3), protein (AGXT2L1) and high energy phosphate (CKMT2). The dramatic increase in the expression of these transcripts, which may enable the peri-natal transition to metabolic independence critical for new-born herbivores, provides surprising evidence for substantial developmental remodelling of muscle mitochondria and reflects changes in nutrient availability. Overall, despite differences in size, metabolism and physiology, the muscle structural subunit expression program appears very similar in ruminants, rodents and humans.

This chapter is intended to provide an overview of previous studies into heat stress and markers that are associated with thermo-tolerance in sheep, as well as other ruminant species that may be considered models for biological processes in sheep. The chapter is divided into two major parts. The first part examines the roles of well-documented heat stress-related biological markers such as heat shock proteins and genes associated with favourable phenotypes such as coat colour and texture. The second part looks at research using methodologies such as microarray, transcriptomics and genomics that have been employed for the identification of novel genes or markers associated with traits of interest. Finally, the chapter concludes with a summary of the observed and expected impacts that climate change will have upon disease. Advances in our understanding of the physiological and biochemical challenges associated with heat stress in sheep and other ruminants, and utilizing this information to deliver increased thermo-tolerance, will be critical to the continued productivity of dairy, meat and fibre sectors in livestock globally.

The giant freshwater prawn, Macrobrachium rosenbergii, a sexually dimorphic decapod crustacean is currently the world’s most economically important cultured freshwater crustacean species. Despite its economic importance, there is currently a lack of genomic resources available for this species, and this has limited exploration of the molecular mechanisms that control the M. rosenbergii sex-differentiation system more widely in freshwater prawns. Here, we present the first hybrid transcriptome from M. rosenbergii applying RNA-Seq technologies directed at identifying genes that have potential functional roles in reproductive-related traits. A total of 13,733,210 combined raw reads (1720 Mbp) were obtained from Ion-Torrent PGM and 454 FLX. Bioinformatic analyses based on three state-of-the-art assemblers, the CLC Genomic Workbench, Trans-ABySS, and Trinity, that use single and multiple k-mer methods respectively, were used to analyse the data. The influence of multiple k-mers on assembly performance was assessed to gain insight into transcriptome assembly from short reads. After optimisation, de novo assembly resulted in 44,407 contigs with a mean length of 437 bp, and the assembled transcripts were further functionally annotated to detect single nucleotide polymorphisms and simple sequence repeat motifs. Gene expression analysis was also used to compare expression patterns from ovary and testis tissue libraries to identify genes with potential roles in reproduction and sex differentiation. The large transcript set assembled here represents the most comprehensive set of transcriptomic resources ever developed for reproduction traits in M. rosenbergii, and the large number of genetic markers predicted should constitute an invaluable resource for future genetic research studies on M. rosenbergii and can be applied more widely on other freshwater prawn species in the genus Macrobrachium.

We describe the development of a highly elastic and adhesive surgical tissue sealant, based on photochemically crosslinked gelatin, for sealing sutured incisions in the gastrointestinal (GI) tract in a rabbit surgical model and in a canine colon anastomosis study. The study included in vitro assessment of mechanical parameters of the tissue sealant and in vivo analysis of burst strength and histology at 24 h, 3 days and 7 days post surgery, in a rabbit model, to assess progress of wound healing at the suture sites. Utility of this sealant to repair and seal a lower colonic resection and anastomosis procedure in a canine model was also investigated. We show that a photopolymerised gelatin tissue sealant provides effective sealing of GI incisions and facilitates wound healing with no evidence of inflammation up to 28 days post-surgery. Blending of derivatised gelatin with underivatised gelatin allowed tuning of elasticity and elastic modulus of the photopolymerised sealant to suit surgical applications. High tissue adhesive strength was maintained at all blend ratios and exceeded 100 kPa. This highly elastic and adhesive photopolymerised gelatin tissue sealant offers a number of advantages over currently available sealants suitable for GI surgical procedures.

Resilin is an important elastomeric protein of insects, with roles in the storage and release of energy during a variety of different functional categories including flight and jumping. To date, resilin genes and protein function have been characterised only in a small number of flying insects, despite their importance in fleas and other jumping insects. Microscopy and immunostaining studies of resilin in flea demonstrate the presence of resilin pads in the pleural arch at the top of the hind legs, a region responsible for the flea's jumping ability. A degenerate primer approach was used to amplify resilin gene transcripts from total RNA isolated from flea (Ctenocephalides felis), buffalo fly (Haematobia irritans exigua) and dragonfly (Aeshna sp.) pharate adults, and full-length transcripts were successfully isolated. Two isoforms (A and B) were amplified from each of flea and buffalo fly, and isoform B only in dragonfly. Flea and buffalo fly isoform B transcripts were expressed in an Escherichia coli expression system, yielding soluble recombinant proteins Cf-resB and Hi-resB respectively. Protein structure and mechanical properties of each protein before and after crosslinking were assessed. This study shows that resilin gene and protein sequences are broadly conserved and that crosslinked recombinant resilin proteins share similar mechanical properties from flying to jumping insects. A combined use of degenerate primers and polyclonal sera will likely facilitate characterisation of resilin genes from other insect and invertebrate orders.

Feather keratin has been widely studied for use as a bio-based material. In this paper, we dissolve feather keratin using industrial sodium sulfide to investigate the yield, dissolved keratin characteristics, and properties of regenerated products to assess the potential of using sodium sulfide as a means of converting waste feathers into a bio-polymer. Optimal conditions appeared to require short incubation times in order to give maximum strength in the regenerated product. This limits the yield to approximately 55%. Air-dried films and acid-precipitated samples are all readily re-crosslinked, suggesting the re-crosslinking process is robust. Minimizing exposure to the highly alkaline conditions appears favorable to final product strength through minimizing alkaline chain damage. The β-sheet structure of the parent keratin is largely maintained. The regenerated keratin was shown to have potentially attractive physical properties for use as a bio-polymer.

Gelatin is widely used as a medical biomaterial because it is readily available, cheap, biodegradable and demonstrates favourable biocompatibility. Many applications require stabilisation of the biomaterial by chemical crosslinking, and this often involves derivatisation of the protein or treatment with cytotoxic crosslinking agents. We have previously shown that a facile photochemical method, using blue light, a ruthenium catalyst and a persulphate oxidant, produces covalent di-tyrosine crosslinks in resilin and fibrinogen to form stable hydrogel biomaterials. Here we show that various gelatins can also be rapidly crosslinked to form highly elastic (extension to break >650%) and adhesive (stress at break >100 kPa) biomaterials. Although the method does not require derivatisation of the protein, we show that when the phenolic (tyrosine-like) content of gelatin is increased, the crosslinked material becomes resistant to swelling, yet retains considerable elasticity and high adhesive strength. The reagents are not cytotoxic at the concentration used in the photopolymerisation reaction. When tested in vivo in sheep lung, the photopolymerised gelatin effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. The elastic properties, thermal stability, speed of curing and high tissue adhesive strength of this photopolymerised gelatin, offer considerable improvement over current surgical tissue sealants.

We assessed correlations between single nucleotide polymorphisms (SNPs) in the actin and crustacean hyperglycemic hormone (CHH) genes with individual growth performance in giant freshwater prawn Macrobrachium rosenbergii. Thirty SNPs were detected in the actin and CHH genes in broodstock of three M. rosenbergii strains (Dong Nai, Mekong, and Hawaiian) and their offspring. A preliminary study that evaluated all SNPs suggested an association between SNPs in intron 3 of the CHH gene and individual growth performance. A larger number of offspring (n = 243) then were genotyped and tested for the associations between SNPs in intron 3 and individual growth performance. Four intronic SNPs were associated significantly with three growth traits (body weight, carapace length, and standard length). Of these, CH3-2402 and CH3-2561 were highly correlated with all three traits, while CH3-2407 and CH3-2409 were correlated significantly only with body weight. A further haplotype-trait association analysis confirmed that these four SNP markers were in linkage disequilibrium, and the specific haplotype TGAA had significant associations with high growth (P

Two novel recombinant proteins An16 and Dros16 have recently been generated. These recombinant proteins contain, respectively, sixteen copies of an 11 amino acid repetitive domain (AQTPSSQYGAP) observed in a resilin-like gene from Anopheles gambiae and sixteen copies of a 15 amino acid repetitive domain (GGRPSDSYGAPGGGN) observed in the first exon of the Drosophila melanogaster CG15920 gene. We compare structural characteristics of the proteins and material properties of resulting biopolymers relative to Rec1-resilin, a previously characterized resilin-like protein encoded by the first exon of the Drosophila melanogaster CG15920 gene. While the repetitive domains of natural resilins display significant variation both in terms of amino acid sequence and length, our synthetic polypeptides have been designed as perfect repeats. Using techniques including circular dichroism, atomic force microscopy, and tensile testing, we demonstrate that both An16 and Dros16 have similar material properties to those previously observed in insect and recombinant resilins. Modulus, elasticity, resilience, and dityrosine content in the cross-linked biomaterials were assessed. Despite the reduced complexity of the An16 and Dros16 proteins compared to natural resilins, we have been able to produce elastic and resilient biomaterials with similar properties to resilin.

Differences in genetic diversity between wild and domesticated Black Tiger shrimp (Penaeus monodon) were assessed in five stocks using molecular markers. These stocks represent an Australian wild population from a single location, and four populations of domesticated animals originating from the same location. Two established high-throughput genotyping systems with a total of 12 microsatellite markers were used to assess diversity of farmed stocks in comparison to wild-caught animals. Genetic diversity analyses showed a large decline in diversity from the wild-caught stocks to the domesticated stocks. Diversity shifts were noted in both allele numbers per locus corrected for sample size (allelic richness) and heterozygosity, declining from 14.2 to 4.6, and 0.77 to 0.61, respectively. The effective population size (Ne) of a commercial fourth generation domesticated strain, following multiple introductions of wild stocks was estimated at 10, highlighting the bottleneck effects possible within a few generations. Significant differences were noted between all pairs of stocks using FST analysis (0.042 to 0.160, P < 0.05), with the greatest difference observed between wild animals and a fourth generation domesticated stock sampled from a commercial farm. This study illustrates the rapid loss of genetic diversity and reduction in effective population size that can occur during domestication when rigid genetic management of the stocks is not possible. This study also demonstrates the value of using molecular (microsatellite) marker systems in shrimp breeding programs to monitor genetic diversity and the effective population size.

Growth rate of the Kuruma prawn, Marsupenaeus japonicus is an important economic trait, with larger animals commanding higher market prices. To identify gene markers associated with growth, a genetic map of a full-sib F(2) intercross family of M. japonicus has previously been generated and quantitative trait loci (QTL) influencing weight, total length, and carapace length were identified. In this study, amplified fragment length polymorphism (AFLP) markers associated with the major QTL region, contributing 16% to phenotypic variation, were characterized. Flanking sequence has been obtained and allelic variants responsible for segregation patterns of these markers have been identified. The genomic sequence surrounding the AFLP band 7.21a, residing under the QTL peak, contains a gene sequence homologous to the elongation of very long chain fatty acids-like (ELOVL) protein family. A full-length mRNA (ELOVL-MJ) encoding this protein was isolated from M. japonicus, representing both the first ELOVL gene in crustacea and the first candidate gene identified via QTL studies in crustacea.

Despite large numbers of putative microsatellites currently listed in databases for Penaeus monodon , there are no publications on assessing these markers for multiplexed high throughput systems either for fingerprinting or population genetics study purposes in P. monodon . Accordingly, we started our investigation on the development of high throughput systems for P. monodon . Ninety publicly-available P. monodon microsatellite sequences were initially screened for suitability. They were assessed for the presence of tri- or tetra-nucleotide repeats, repeat number and type, suitability of flanking sequences for primer design and estimated size of product (100 to 350 bp). Nineteen sequences were chosen for preliminary assessment on a panel of 15 animals. Of the 19 tested, only 12 were suitable for further investigation. Therefore a 2-step enrichment library approach was adopted to develop additional microsatellites. Of 42 new unique microsatellite sequences obtained, eight sequences were assessed and seven showed polymorphism. Together, these 19 markers were examined further for their ease of amplification and reliability of allele calling for inclusion in high throughput systems. Thirteen polymorphic markers were incorporated into two multiplex systems (six and seven markers, respectively). These multiplexed systems were then used to evaluate the genetic diversity between two populations of P. monodon , one from the East Coast of Australia and a single pond containing farmed animals from Thailand. There were significant differences between the two populations. Three markers in system 1 showed Hardy–Weinberg disequilibrium in both populations, indicating their unsuitability as high throughput system markers. Using two systems and the UPGMA clustering methods revealed the existence of sub-populations within the Australian wild population. The results indicate the usefulness of the two multiplexed microsatellite systems in genetic diversity studies.

Resilin is an elastic protein with outstanding material properties: high resilience and a very high fatigue lifetime. We are interested in the production of resilin-like proteins which can be photo-chemically cross-linked to form rubbery biomaterials to be used in a variety of industrial and medicinal applications. A method has been developed for producing soluble recombinant proteins in small scale fermentation equipment using glycerol batch for initial growth and primary induction by IPTG at carbon source depletion, followed by new growth in lactose-induced culture. Recombinant rec1-resilin has been over-expressed in the host strain Escherichia coli BL21(DE3)pLysS at a level of up to 300 mg/l, a greater than 20-fold increase in volumetric productivity, relative to that obtained from conventional IPTG induction in LB medium. The primary induction step before lactose induction in fresh medium resulted in a 2.5- to 3-fold increase of both volumetric productivity and cell specific yield compared to that without primary induction under the same conditions. This method is amenable and suitable for large scale production of soluble resilin-like proteins at a low operating cost. In addition, a simple 'salt precipitation and heat purification' method allowed rapid and efficient downstream processing of a large quantity of soluble recombinant resilin-like proteins. These methods will enable investigation of the structural and functional properties of resilin-like proteins, and the development of highly resilient biomaterials.

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.

The objective of this study was to use Amplified Fragment Length Polymorphism (AFLP) markers to determine the genetic location and effects of genomic regions controlling weight (WT), total length (TL) and carapace length (CL) in one full-sib Penaeus japonicus family. One hundred and two extreme progeny from an intermediate F2 cross of high (HH) and low (LL) growth parents were used to identify these regions. Two analytical methods were applied in this study. They were interval mapping (IM) and composite interval mapping (CIM). There was no significant marker-trait association in the female map. One region controlling part of the variation for CL and TL was identified in linkage group 1 of the male map by IM, and the same region and one other suggestive QTL marker in linkage group 25 of the male map were detected by CIM. Both regions were located on the end of linkage groups. In order to derive additional markers, the Bulk Segregant Analysis (BSA) technique was applied to these regions to generate further AFLP markers. From 45 markers scored using 11 AFLP primer combinations, one AFLP marker was mapped to the QTL region in linkage group 1 and one in the middle of group 25. The new results supported the findings from the early analyses prior to using BSA. The magnitude of phenotypic variation explained by the joint action of the QTL markers indicated that genetic factors of large effect were involved in the control of the studied characters. This study is the first to identify QTLs for growth traits in a penaeid species.

Studies using antibodies to immunolocalize theToxoplasma gondiidense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules ofT. gondiiand is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa inT. gondiiexcretory–secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified inNeospora caninum. Finally, GRA3 possesses a dilysine ‘KKXX’ endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

Forty-three microsatellites were isolated from grey mullet (Mugil cephalus) of Mediterranean origin using the standard enrichment methods followed by enhanced chemiluminescence for the rapid identification of clones containing microsatellites. Eleven microsatellites were characterized to assess genetic diversity of different populations surrounding the island of Sardinia in Italy. The versatility of these markers was also assessed using an Australian population. All 11 microsatellites were highly polymorphic with a mean heterozygosity of 0.834. The number of alleles ranged from 14 to 38 in the Sardinian populations and from 11 to 25 in the Australian samples.

The role of interleukin-4 (IL-4) during the course of Toxoplasma gondii infection was studied using IL-4-/- mice and their wild-type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL-4 was demonstrated and IL-4-/- mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL-4-/- mice although conversely liver pathology was greater than in wild-type mice. Significantly, plasma IL-12 and IFN-gamma levels, which peaked at days 6 and 8, respectively, were higher in IL-4-/- mice. The exacerbatory role of IL-4 in the intestine was found by competitive RT-PCR not to be associated with increased parasite burdens but was related to comparative expression of IL-10.

We have previously identified that Leishmania mexicana cysteine proteases (CPs) are virulence factors. We have now produced a recombinant L. mexicana CP, CPB2.8, which has similar enzymatic activity to native enzyme. Inoculation of CPB2.8 (≤5 μg) into the footpads of BALB/c mice not only up-regulated mRNA transcripts for IL-4 and IL-4 production in the draining popliteal lymph nodes, but also polarized splenocyte anti-CD3 stimulated responses toward a Th2 bias as measured by increased IL-5 production compared with controls. In agreement with promoting a Th2 response, CPB2.8 also induced strong specific IgE responses in treated mice as well as increasing whole IgE levels. Inhibition of the enzyme activity of CPB2.8 by treatment with E-64 ablated the enzyme’s ability to induce IgE. Significantly, infection of mice with CPB-deficient parasites failed to stimulate production of IgE, unlike infection with wild-type parasites. Furthermore, enzymatically active (

During infection in the intermediate host, Toxoplasma gondii undergoes stage conversion between the rapidly dividing tachyzoite that is responsible for acute toxoplasmosis and the slowly replicating, encysted bradyzoite stage. This process of tachyzoite-bradyzoite interconversion is central to the pathogenesis and longevity of infection. Recent research has identified several stage-specific genes and proteins. However, despite recent advances in the understanding of Toxoplasma cell biology, more research is necessary to elucidate the complex events occurring during tachyzoite-bradyzoite interconversion. Here, a brief summary of this process is provided and a new method to characterize gene expression during interconversion is introduced.

The shikimate pathway is essential for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential conversion of erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then used as a substrate for other pathways that culminate in production of folates, ubiquinone, napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The shikimate pathway is absent from animals and present in the apicomplexan parasites Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway by glyphosate is effective in controlling growth of these parasites. These findings emphasize the potential benefits of developing additional effective inhibitors of the shikimate pathway. Such inhibitors may function as broad-spectrum antimicrobial agents that are effective against bacterial and fungal pathogens and apicomplexan parasites.

Engineered protein polymers that display responsiveness to multiple stimuli are emerging as a promising class of soft material with unprecedented functionality. The remarkable advancement in genetic engineering and biosynthesis has created the opportunity for precise control over the amino acid sequence, size, structure and resulting functions of such biomimetic proteins. Herein, we describe the multi-stimuli-responsive characteristics of a resilin-mimetic protein, An16-resilin (An16), derived from the consensus sequence of resilin gene in the mosquito Anopheles gambiae. We demonstrate that An16 is an intrinsically disordered protein that displays unusual dual-phase thermal transition behavior along with responsiveness to pH, ion, light and humidity. Identifying the molecular mechanisms that allow An16 to sense and switch in response to varying environments furthers the ability to design intelligent biomacromolecules. Refereed/Peer-reviewed

A murine model was used to characterize the local immune and inflammatory response during ocular toxoplasmosis. Major histocompatibility complex (MHC) class I, normally expressed at low levels in immune-privileged sites such as the eye, was up-regulated during infection as determined by competitive reverse transcriptase (RT)-PCR and immunocytochemistry for both β2-microglobulin and the MHC class I heavy chain. However, the eyes of chronically infected mice also had increased levels of mRNA transcripts for transforming growth factor β, a cytokine associated with immune privilege and constitutively expressed in normal eyes. Transcripts for a number of inflammatory mediators, including interleukin-6 (IL-6), were increased during chronic infection. The role of IL-6 was further investigated by comparing disease progression and the development of the local immune response in wild-type (WT) and IL-6-deficient mice (IL-6−/−mice). Following infection, IL-6−/−mice developed more severe inflammation in the retina and vitreous humor compared with WT mice. This increased severity of disease was associated with reduced ocular IL-1α and increased tumor necrosis factor α mRNA production compared with WT mice. Moreover, the increased severity of disease in IL-6−/−mice correlated with increased eye parasite burden as determined by RT-PCR for theToxoplasma gondiibradyzoite-specificLDH2gene. These results demonstrate alterations to components of immune privilege as a result of ocular toxoplasmosis and a role for IL-6 in controlling parasite numbers and inflammation in the eye.

This study compared the genes encoding cytoplasmic 70 kDa heat shock protein in virulent and avirulent strains of Toxoplasma gondii, to determine whether differences may contribute to the variation in protein expression levels previously reported for this protozoan parasite. A T. gondii PCR probe with homology to Eimeria acervulina cytoplasmic 70 kDa heat shock protein was used to screen a genomic DNA mini-library and isolate the gene from the virulent RH strain. The entire coding region was subsequently amplified from the avirulent ME49 strain by PCR. Alignment of the gene sequences revealed that the virulent RH strain had four copies of a seven-aa repeat unit (GGMPGGM) at the 3'- end of the gene compared with five copies in the avirulent ME49 strain. Comparison of this region among other virulent and avirulent strains revealed that this difference was consistent with virulence. Copy number estimation revealed that this gene is single-copy in both the RH and the ME49 strains. Analysis of mRNA expression revealed a 1.5- to 2-fold increase in transcription of this gene in virulent strains when compared with avirulent strains. For each strain, mRNA was observed at similar levels whether grown in vivo or in vitro. Also, heat-shock treatment of tachyzoites prior to harvest did increase mRNA levels in vitro. This suggests that post-transcriptional regulation of cytoplasmic 70 kDa heat shock protein may occur in T. gondii. Differences in cytoplasmic 70 kDa heat shock protein have been demonstrated at genomic and transcriptional levels in virulent strains compared with avirulent strains, suggesting that this 70 kDa heat shock protein may play an important role in the virulence of T. gondii.

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Background Previous genome-wide association studies have identified significant regions of the X chromosome associated with reproductive traits in two Bos indicus-influenced breeds: Brahman cattle and Tropical Composites. Two QTL regions on this chromosome were identified in both breeds as strongly associated with scrotal circumference measurements, a reproductive trait previously shown to be useful for selection of young bulls. Scrotal circumference is genetically correlated with early age at puberty in both male and female offspring. These QTL were located at positions 69–77 and 81–92 Mb respectively, large areas each to which a significant number of potential candidate genes were mapped. Results To further characterise these regions, a bioinformatic approach was undertaken to identify novel non-synonymous SNP within the QTL regions of interest in Brahman cattle. After SNP discovery, we used conventional molecular assay technologies to perform studies of two candidate genes in both breeds. Non-synonymous SNP mapped to Testis-expressed gene 11 (Tex11) were associated (P

In cattle, endometrial gene expression during the first 2 weeks after fertilization is different in cattle that conceive compared with those that fail to conceive. The potential selection of animals based on endometrial marker expression before the day of embryo transfer may improve the efficiency of bovine embryo transfer. This study aimed to evaluate the morphological and molecular changes induced by endometrial biopsy in cycling tropically adapted beef cattle. In both experiments, a single biopsy was performed on the horn ipsilateral to the corpus luteum using a circular cup biopsy forceps. The uterus was collected after slaughter, and sections were taken adjacent to the biopsy site. Five heifers were not biopsied and acted as controls. In Experiment 1, presynchronised heifers (n = 10) were biopsied on Day 7 post-oestrus (B7) and then resynchronised with cloprostenol (Estromil®, 250 μg IM) to induce oestrus (O), 2 to 3 days after injection. At Day 7 post-oestrus (D7), the reproductive tracts were collected, 10 days after biopsy (B7-O-D7). In Experiment 2, presynchronised heifers (n = 7) were biopsied on Day 4 post-oestrus (B4), and reproductive tracts were collected 3 days later (B4-D7). A quantitative scoring system was used to define histological appearance of the endometrium (1 to 25; above 16 indicates endometritis). The RNA expressions of interleukin-1-β (IL-1β) and transforming growth factor-β (TGF-β) were measured by quantitative real-time PCR (qRT-PCR). Macroscopic examination showed the uterus collected from B7-O-D7 heifers exhibited a focal, well demarcated, dark red discoloration of the endometrium (4/10) with no evidence of the biopsy site found in the remaining heifers (6/10). The biopsied uteri from B4-D7 heifers revealed no macroscopic evidence of the biopsied site (4/7), serosa congestion (2/7), and one case where a severe endometrial haemorrhage was evident (1/7). Histologically, the overall mean score was not indicative of endometritis. The RNA expression showed up-regulation of both IL-1β and TGF-β in both B7-O-D7 and B4-D7 heifers compared with non-biopsied heifers and significantly higher expression of IL-1β in B4-D7 than B7-O-D7 (P = 0.031, P