Moira Menzies

The sheep genome illuminates biology of the rumen and lipid metabolism

A genome for ewe and ewe Sheep-specific genetic changes underlie differences in lipid metabolism between sheep and other mammals, and may have contributed to the production of wool. Jiang et al. sequenced the genome of two Texel sheep, a breed that produces high-value meat, milk, and wool. The genome information will provide an important resource for livestock production and aid in the understanding of mammalian evolution. Science, this issue p. 1168 A genomic analysis of sheep explains specializations in digestive system physiology and wool production. Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.

Nematode challenge induces differential expression of oxidant, antioxidant and mucous genes down the longitudinal axis of the sheep gut

To characterize the role of a range of oxidant, antioxidant and mucous‐related genes in the primary response to gastrointestinal nematodes, groups of genetically resistant sheep were challenged with either Haemonchus contortus or Trichostrongylus colubriformis and necropsied for retrieval of tissue at days 0, 3, 7, 14 and 21. To determine if the response was localized to the site of parasite infection, four different gut tissues were sampled: the abomasum, proximal and distal jejunum and ileum. Basal expression patterns of all candidate genes were determined using the day 0 (pre‐challenge) samples. A conserved innate response involving elevated expression of dual oxidase, glutathione peroxidase and trefoil factor was initiated within 3 days of challenge and extended out to 21 days. An increase in host gene expression levels at the preferred site of infection (the abomasum for H. contortus and the proximal jejunum for T. colubriformis) was also common to both nematodes. However, these increases were concomitant with reduced expression in other areas of the gut suggesting a compartmentalized response. Other aspects of the response were parasite‐specific, with T. colubriformis challenge inducing expression peaks at times corresponding to nematode life‐stage transitions.

Identification of a dieldrin resistance-associated mutation in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

ABSTRACT The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), is a major vector of tick fever organisms affecting cattle in many parts of the world, including Australia, Africa, and South America. Control of the southern cattle tick through acaricide use is an important approach in disease management, Resistance has emerged to many of the acaricides currently and previously used, including the cyclodienes. Although cyclodiene resistance mechanisms have been characterized in many insect species, this report is the first to identify mutations associated with dieldrin resistance in the cattle tick. A novel two base pair mutation in the GABA-gated chloride channel gene has been identified at position 868–9 and causes a codon change from threonine to leucine. Analysis of a small number of field-collected samples resistant to dieldrin shows this mutation has been maintained without selection pressure since the withdrawal of dieldrin in Australia >20 yr ago. The mutation is not found in other laboratory-maintained strains of R. microplus that were subject to selection pressure with various acaricides.

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.

Potential for recombinant Babesia bovis antigens to protect against a highly virulent isolate

Two antigens from Babesia bovis,12D3 and 11C5, were expressed and purified as recombinant proteins in Escherichia coli and used to vaccinate groups of six Babesia‐susceptible cattle. These were subsequently challenged with a highly virulent strain of B. bovis. All cattle showed symptoms of disease and most required treatment. Cattle vaccination groups receiving either 12D3 or 11C5 or a combination of both, reduced parasitaemia by approximately fourfold and a number of individual animals appeared to control the parasite infection. Control of parasites correlated with high monocyte numbers late in infection. The results thus confirm the potential usefulness of both antigens but also demonstrate the limitations of current formulations.

Divergent ghrelin expression patterns in sheep genetically resistant or susceptible to gastrointestinal nematodes

Gastrointestinal nematodes are a major problem for pastoral ruminant production systems. This problem could be reduced by the application of breeding strategies that select for nematode resistant sheep, but no suitable molecular markers are available. Research selection flocks containing lines that are resistant (R) or susceptible (S) to gastrointestinal nematodes provide an excellent resource for discovering selectable markers, and for studying the underlying mechanisms of an effective anti-nematode response. In this study we have used a combination of quantitative real time PCR assays and ELISA to determine if nematode challenge impacts on the expression of the satiety-regulating hormone ghrelin. The expression responses were then compared between the selection flock R and S lines. The results show that the basal levels of ghrelin in plasma were greater than 2-fold higher in nematode naïve S line sheep. Three days after a primary nematode challenge divergent ghrelin expression patterns were observed between the selection lines, with levels increasing in R sheep while decreasing in S sheep. After a secondary challenge this trend was repeated, but following a third challenge ghrelin expression levels rose in both R and S sheep, by which time the S animals had acquired an effective immune response to the nematodes, as measured by a significant reduction in faecal egg output. Importantly, this phenomenon was observed in gene expression studies in gut tissues and also in ELISA measurements of ghrelin peptide levels in plasma. A regression analysis showed that ghrelin transcript expression in the gut accounted for >40% of the variation in faecal egg count measured following Haemonchus or Trichostrongylus infection. We therefore hypothesise that the direction of ghrelin expression (up or down) immediately following nematode exposure may play an important role in regulating the differing anti-nematode responses that occur in the R and S lines. Such differences identify ghrelin as a previously unrecognized factor influencing the acquisition of immunity to nematodes.

Gene expression profiles of BMP4, FGF10 and cognate inhibitors, in the skin of foetal Merino sheep, at the time of secondary follicle branching

Abstract:  The high concentration of secondary branched follicles is a distinctive feature of the Merino sheep. These follicles initiate from 100 days of gestation. Here, we report a transition in abundance of the BMP4 and FGF10 morphogens occurring at this time. At 103 days of gestation, FGF10 gene expression dropped steadily from maximal levels, in a trend that continued until day 143. Conversely, from day 105, BMP4 transcript levels rapidly increased to maximal levels that were maintained until 131 days, before declining. This profile closely matches reported changes in branched follicle numbers, which peak in density at day 134. SPRY4, a known regulator of FGF10, increased to maximal levels concomitant with the fall in FGF10, suggesting a relationship. Levels of the BMP4 inhibitor NOG matched the initial rise of BMP4, with a fivefold spike at 108 days; but consistent with the rise in BMP4, this high level was not sustained.

RNF14 is a regulator of mitochondrial and immune function in muscle.

BackgroundMuscle development and remodelling, mitochondrial physiology and inflammation are thought to be inter-related and to have implications for metabolism in both health and disease. However, our understanding of their molecular control is incomplete.ResultsIn this study we have confirmed that the ring finger 14 protein (RNF14), a poorly understood transcriptional regulator, influences the expression of both mitochondrial and immune-related genes. The prediction was based on a combination of network connectivity and differential connectivity in cattle (a non-model organism) and mice data sets, with a focus on skeletal muscle. They assigned similar probability to mammalian RNF14 playing a regulatory role in mitochondrial and immune gene expression. To try and resolve this apparent ambiguity we performed a genome-wide microarray expression analysis on mouse C2C12 myoblasts transiently transfected with two Rnf14 transcript variants that encode 2 naturally occurring but different RNF14 protein isoforms. The effect of both constructs was significantly different to the control samples (untransfected cells and cells transfected with an empty vector). Cluster analyses revealed that transfection with the two Rnf14 constructs yielded discrete expression signatures from each other, but in both cases a substantial set of genes annotated as encoding proteins related to immune function were perturbed. These included cytokines and interferon regulatory factors. Additionally, transfection of the longer transcript variant 1 coordinately increased the expression of 12 (of the total 13) mitochondrial proteins encoded by the mitochondrial genome, 3 of which were significant in isolated pair-wise comparisons (Mt-coxII, Mt-nd2 and mt-nd4l). This apparent additional mitochondrial function may be attributable to the RWD protein domain that is present only in the longer RNF14 isoform.ConclusionsRNF14 influences the expression of both mitochondrial and immune related genes in a skeletal muscle context, and has likely implications for the inter-relationship between bioenergetic status and inflammation.