David Waddington

Analysis of telomere lengths in cloned sheep

The development of nuclear-transfer techniques using cultured somatic cells allows animals to be produced without involving germline cells. This enables us to examine the importance of the repair of chromosome ends (telomeres) in the germ line and to test the telomere hypothesis of ageing.

The dynamics of chromosome evolution in birds and mammals.

Comparative mapping, which compares the location of homologous genes in different species, is a powerful tool for studying genome evolution. Comparative maps suggest that rates of chromosomal change in mammals can vary from one to ten rearrangements per million years. On the basis of these rates we would expect 84 to 600 conserved segments in a chicken comparison with human or mouse. Here we build comparative maps between these species and estimate that numbers of conserved segments are in the lower part of this range. We conclude that the organization of the human genome is closer to that of the chicken than the mouse and by adding comparative mapping results from a range of vertebrates, we identify three possible phases of chromosome evolution. The relative stability of genomes such as those of the chicken and human will enable the reconstruction of maps of ancestral vertebrates.

Analysis of Telomere Length in Dolly, a Sheep Derived by Nuclear Transfer

We have used a (TTAGGG) oligonucleotide probe to demonstrate that ovine telomeres are composed of (TTAGGG) repeat arrays and to compare the terminal restriction fragment lengths of sheep derived by natural mating and nuclear transfer. Here we show that ovine somatic telomeres decrease in length with age, and that Dolly, derived by the transfer of 6-year-old adult somatic nucleus, exhibits diminished terminal restriction fragment lengths. The decrease is consistent with the age of the donor tissue and telomere erosion during in vitro culture. Nuclear transfer does not restore telomere lengths. Dolly otherwise appears physiologically and phenotypically normal for her breed and age. We further report on apparent telomere lengthening in sheep, occurring during the first year in naturally derived lambs.

Development and validation of a bovine macrophage specific cDNA microarray.

BackgroundThe response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited.ResultsHere we report the development of a bovine macrophage specific (BoMP) cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate) and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-γ and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4.ConclusionA 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo.

Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.

Analysis of the real EADGENE data set: Comparison of methods and guidelines for data normalisation and selection of differentially expressed genes (Open Access publication)

A large variety of methods has been proposed in the literature for microarray data analysis. The aim of this paper was to present techniques used by the EADGENE (European Animal Disease Genomics Network of Excellence) WP1.4 participants for data quality control, normalisation and statistical methods for the detection of differentially expressed genes in order to provide some more general data analysis guidelines. All the workshop participants were given a real data set obtained in an EADGENE funded microarray study looking at the gene expression changes following artificial infection with two different mastitis causing bacteria: Escherichia coli and Staphylococcus aureus. It was reassuring to see that most of the teams found the same main biological results. In fact, most of the differentially expressed genes were found for infection by E. coli between uninfected and 24 h challenged udder quarters. Very little transcriptional variation was observed for the bacteria S. aureus. Lists of differentially expressed genes found by the different research teams were, however, quite dependent on the method used, especially concerning the data quality control step. These analyses also emphasised a biological problem of cross-talk between infected and uninfected quarters which will have to be dealt with for further microarray studies.

Allograft responses can interfere with the development of immunity against Theileria annulata following vaccination with parasite infected cell lines

Theileria annulata macroschizont‐infected cell lines are successfully used as vaccines in several countries. The inoculated animals produce a strong allogeneic response against the MHC antigens of the immunizing cell line followed by an anti‐parasite response. Immunity against the parasite wanes in the absence of challenge and re‐immunization is sometimes recommended. However, it is not known if allogeneic responses generated by the first immunization with a T. annulata infected cell line will interfere with the boosting of immunity against the parasite at the time of re‐immunization with the same cell line. Animals were primed against MHC antigens by skin grafting, followed by immunization with a T. annulata infected cell line prepared from the skin donor. A strong anti‐MHC response was produced. This interfered with parasite transfer and the development of an anti‐parasite immune response; the effect was more marked when a low vaccine cell dose was used. There was a negative correlation between the ease of isolating infected cells from the animals after cell line immunization, and the subsequent response to challenge. Where no cell lines could be isolated, the animals were fully susceptible to sporozoite challenge. These observations are of immediate importance in endemic areas where cell lines of T. annulata schizonts are being used as vaccines to control the disease.

Estimating the Number of Conserved Segments Between Species Using a Chromosome Based Model

Comparative genetic maps between two species which position genes on chromosomes allow syntenic blocks of one or more genes to be identified. These may then be used, with appropriate models, to estimate the number of chromosomal segments with conserved content between species. We outline a model for the distribution of the lengths of unobserved segments on each chromosome which allows for widely differing chromosome lengths. The model is motivated by, and applied to data from two comparative maps for the chicken, one with human and one with mouse. The performance of two genome based models is also compared for these maps, and for simulations based upon them.

The deposition of the cuticle on laying hens eggs is a moderately heritable trait

Various varieties of Mucuna pruriens are widely grown in the tropics. It is an important legume used for soil improvement, as a green manure or cover crop. Besides, its prolific production of beans is an essential attribute for use as human food or animal feed. The use of seeds of Mucuna species as non-conventional protein source in poultry feed could be a significant relief in developing countries where conventional protein ingredients such as soybean meal, fish meal and protein concentrates have become scarce and expensive. Mucuna beans are a rich source of crude protein (230 to 384 g/kg) with high protein digestibility (Adebowale et al., 2005). However, the beans contain anti-nutritive factors such as tannins, protease inhibitors, saponins, phytic acid and L-dopa (Adebowale et al., 2005) which limit their nutritive value for poultry (Carmen et al., 1999). It has been observed that hydrothermal treatments of the Mucuna seeds were most effective among the processing methods for the reduction of various anti-nutritive factors and improved protein digestibility (Siddhuraju and Becker, 2005). Preliminary studies (Carmen et al., 1999) have shown that a single processing method is not adequate in eliminating the adverse effects of the antinutritive factors. Hence there is a need to use combined processing methods such as soaking, dehulling and heat treatment. The objectives of this study were to determine dietary effects of two varieties of Ghanaian Mucuna pruriens beans processed by combined methods of soaking, dehulling and cooking on the growth performance of broiler chickens during the finishing phase as well as economics of production. Two experiments were conducted concurrently to evaluate the nutritive value of two Ghanaian Mucuna pruriens varieties (white and mottle beans) in concentrate-based diets for broiler chickens. Both types of beans were soaked in water for 48 h, dehulled by hand and cooked for 2 h (white beans) or one hour (mottle beans). A total of 270 (4-week-old) broiler chicks (Lohmann strain) were randomly allotted in groups of 15 birds (9 males, 6 females) in triplicate to the dietary treatments using a completely randomised design. In each experiment (Table 1), three diets that comprised the control (no Mucuna beans) and the processed Mucuna beans (150 and 200 g/kg) were formulated to be isonitrogenous (200 g/kg CP) with slight variation in ME (12 1 to 13 0 MJ/kg). Diets were formulated using maize, broiler— finisher concentrate and wheat bran. The birds were fed the diets from 4 to 8 weeks of age. Feed and water were supplied ad libitum and light was provided for 24 h. Data collected included feed intake, weight gain, feed/gain, mortality, feed cost and economy of gain. The data were subjected to ANOVA using the ‘Genstat’. In both experiments, there were no significant differences (P > 0 05) in the growth parameters and economic indicators among the treatments (Table 2). This could be attributed to the fact that the adverse effects of antinutritive factors such as tannins, L-dopa and protease inhibitors might have been minimised substantially by the combined processing methods. Other studies (Udedibie et al., 2001; Dei et al., 2006) showed significant improvement in the performance of broilers fed processed Mucuna beans. The incorporation of both processed Mucuna beans at 200 g/kg, also, spared some of the maize (4 to 6%), wheat bran (50%) and broiler—finisher concentrate (23 to 27%). Therefore, a processed Mucuna bean has a potential to be used as poultry feed.

Analysis of the real EADGENE data set: Multivariate approaches and post analysis (Open Access publication)

The aim of this paper was to describe, and when possible compare, the multivariate methods used by the participants in the EADGENE WP1.4 workshop. The first approach was for class discovery and class prediction using evidence from the data at hand. Several teams used hierarchical clustering (HC) or principal component analysis (PCA) to identify groups of differentially expressed genes with a similar expression pattern over time points and infective agent (E. coli or S. aureus). The main result from these analyses was that HC and PCA were able to separate tissue samples taken at 24 h following E. coli infection from the other samples. The second approach identified groups of differentially co-expressed genes, by identifying clusters of genes highly correlated when animals were infected with E. coli but not correlated more than expected by chance when the infective pathogen was S. aureus. The third approach looked at differential expression of predefined gene sets. Gene sets were defined based on information retrieved from biological databases such as Gene Ontology. Based on these annotation sources the teams used either the GlobalTest or the Fisher exact test to identify differentially expressed gene sets. The main result from these analyses was that gene sets involved in immune defence responses were differentially expressed.