A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae.

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.

A genome scan for quantitative trait loci affecting resistance to Trichostrongylus colubriformis in sheep.

A genome linkage scan was carried out using a resource flock of 1029 sheep in six half-sib families. The families were offspring of sires derived by crossing divergent lines of sheep selected for response to challenge with the intestinal parasitic nematode Trichostrongylus colubriformis. All animals in the resource flock were phenotypically assessed for worm resistance soon after weaning using a vaccination/challenge regime. After correcting for fixed effects using a least squares linear model the faecal egg count data obtained following the first challenge and the faecal egg count data obtained after the second challenge were designated Trait 1 and Trait 2, respectively. A total of 472 lambs drawn from the phenotypic extremes of the Trait 2 faecal egg count distribution were genotyped with a panel of 133 microsatellite markers covering all 26 sheep autosomes. Detection of quantitative trait loci (QTL) for each of the faecal egg count traits was determined using interval analysis with the Animap program with recombination rates between markers derived from an existing marker map. No chromosomal regions attained genome-wide significance for QTL influencing either of the traits. However, one region attained chromosome-wide significance and five other regions attained point-wise significance for the presence of QTL affecting parasite resistance.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Molecular analysis and nematode resistance association of a polymorphism at the 5' end of the sheep IgE gene.

Previous work using Southern analysis of genomic DNA detected a polymorphism at the 5' end of the sheep and cattle IgE gene. Identical length differences found between fragments following digestion with restriction enzymes indicated that the basis for the polymorphism was an insertion/deletion event. To characterise the polymorphism, the entire cattle and sheep Cvarepsilon genes were sequenced including 668bp of 5' untranslated DNA. Sequence comparison revealed a high degree of similarity between the ovine and bovine genes at both the nucleotide and amino acid level. A feature of the 5' untranslated DNA was the presence of an 87bp repeat starting at -365 upstream of the Cvarepsilon start site. PCR primers were designed to span most of the 5' untranslated sequence, including the repeat unit, and used to amplify genomic DNA from a panel of 40 sheep. Three alleles were found with frequencies of 0.7, 0.29, 0.01 which were identical to the Southern analysis results. Sequencing of the two commonest alleles revealed the basis for the polymorphism was a 36bp deletion from the 87bp repeat. Association studies in a sheep selection flock phenotypically assessed for parasite resistance found a highly significant association between one of the IgE alleles and resistance to the intestinal nematode parasite Trichostrongylus colubriformis (P=0.005). Attempts to confirm this finding in two other flocks using linkage analysis and genotype association failed to find any significant associations between the IgE polymorphism and resistance to either T. colubriformis or Haemonchus contortus.

Production and verification of an anti-ovine IgE monoclonal antibody.

Recombinant mouse/sheep IgE was used in the production of an anti-IgE monoclonal using conventional hybridoma techniques. The specificity of hybridomas secreting anti sheep IgE monoclonal antibodies was verified using a number of assays including competitive ELISAs, ability to induce mediator release from mast cells, and IgE binding using western blotting. Immunohistochemical staining demonstrated the binding of putative anti-IgE monoclonals to the sheep mast cell surface. The isotype of the antibody was IgG1:K.

A tandemly repetitive DNA sequence is present at diverse locations in the genome of Ostertagia circumcincta.

A novel repetitive DNA sequence in the sheep parasitic nematode Ostertagia circumcincta was cloned and sequenced. This 1.2-kb sequence (Oc1B) was not found in the closely related cattle parasite Ostertagia ostertagi, nor in the more distantly related sheep parasites Haemonchus contortus or Trichostronylus colubriformis. Sequences similar to Oc1B were found at various genomic locations and contained a pair of 33-bp direct repeats. Oc1B also contained a single copy of a 218-bp sequence (designated OcREP) which was present in 100 to 200 copies in the O. circumcincta genome and mostly organized in distinctive tandem arrays. The dual organizational pattern of OcREP as both a satellite-like sequence as well as interspersed as single copies amongst dissimilar sequences adds to the growing evidence for the fluidity of the parasitic nematode genome, and of eukaryotic genomes in general.

Prospects for development of genetic markers for resistance to gastrointestinal parasite infection in sheep.

Selection of sheep for resistance to internal parasites represents a viable option for future parasite control. Many phenotypic measures are available for determining the level of infection in individual sheep, although no phenotypic markers are available which allow prediction of an individual's resistance status. Genetic markers are therefore the best way to incorporate parasite resistance into selection programmes. With the recent development of genetic maps, several experiments are underway to search for markers linked to parasite-resistance genes in sheep. It can be predicted confidently that markers associated with resistance will be discovered within 12 months. Markers useful as selection criteria will be available within 5 years, although considerable quantitative genetic analysis needs to be done to find the best way to utilise marker information in selection programmes. In future, methods for differential DNA analysis or mRNA expression will lead to isolation of the genes involved.

A middle-repetitive DNA sequence element in the sheep parasitic nematode, Trichostrongylus colubriformis.

A novel repetitive DNA sequence in the sheep parasitic nematode Trichostrongylus colubriformis was cloned and sequenced. A 1.1 kb repetitive sequence (Tc15) which hybridized with DNA from T. colubriformis but not with DNA from two other parasitic nematodes, Haemonchus contortus and Ostertagia circumcincta, or sheep was further characterized. Southern blot analysis showed that the repeat hybridized to a range of fragments in restriction digested T. colubriformis DNA and existed in multiple copy number tandem arrays. However, to define clearly the repetitive monomeric unit further screening of phagemid libraries containing BamH I restriction fragments using a subclone of Tc15 as a probe was carried out. Restriction map and sequence data were compiled for 3 clones containing a 145 bp highly repetitive sequence (designated TcREP) which shared homology with the original pTc15 clone. TcREP hybridized to a tandemly repeating sequence monomer of 145 bp in T. colubriformis DNA which was cloned from various genetic environments in the T. colubriformis genome. TcREP homologous sequences were also found in the genomes of two other species of the same genus (Trichostrongylus axei and Trichostrongylus vitrinus) but not in a fourth species (Trichostrongylus rugatus).

Cellular inflammatory responses in skin of sheep selected for resistance or susceptibility to fleece rot and fly strike.

Leucocyte populations were examined in normal and inflamed skin of sheep bred for resistance (R) or susceptibility (S) to bacterial fleece rot and the common sequela, body strike caused by the dipteran parasite Lucilia cuprina. No differences between R and S lines were found in numbers of neutrophils accumulating in acute inflammatory lesions induced by activated complement, leukotriene B4, interleukin (IL)-1 beta, tumour necrosis factor-alpha, IL-8 or endotoxin from Pseudomonas aeruginosa. T19+ (alpha gamma delta T cell subset) lymphocytes and eosinophils were more prevalent in skin of sheep from the S line whereas IgE+ cells were more prevalent in skin of sheep from the R line. In an unrelated population of sheep, animals with low fleece rot scores had more intense neutrophil migration into inflammatory lesions induced by all the mediators examined than did animals with high fleece rot scores. IgE+ cells were more prevalent in animals with low fleece rot scores, although in contrast to R and S lines, T19+ cells tended to be elevated in this group of animals. The results suggest that defence mechanisms associated with IgE+ cells in skin may play an important role in resistance to fleece rot and fly strike.

Characterization of a tandemly repetitive DNA sequence from Haemonchus contortus.

Genomic DNA from the sheep parasitic nematode Haemonchus contortus was shotgun cloned in the plasmid vector pUC18. Recombinants which gave the strongest hybridization signals to 32P-radiolabelled genomic DNA were selected as representatives of the repetitive component of the parasite DNA. One repetitive sequence which hybridized only with DNA from H. contortus and not with DNA from two other sheep nematodes (Trichostrongylus colubriformis and Ostertagia circumcincta) was further characterized by sequencing and dot blot analysis. A related repeat was found in the closely related species Haemonchus placei. Experiments to determine the genomic organization of the repeat showed that it existed in a multi-copy number tandem array. This is the first report on the characterization of repetitive DNA in sheep parasite nematodes.