Analysis of pooled genome sequences from Djallonke and Sahelian sheep of Ghana reveals co-localisation of regions of reduced heterozygosity with candidate genes for disease resistance and adaptation to a tropical environment.

BACKGROUND: The Djallonke sheep is well adapted to harsh environmental conditions, and is relatively resistant to Haemonchosis and resilient to animal trypanosomiasis. The larger Sahelian sheep, which cohabit the same region, is less well adapted to these disease challenges. Haemonchosis and Trypanosomiasis collectively cost the worldwide animal industry billions of dollars in production losses annually. RESULTS: Here, we separately sequenced and then pooled according to breed the genomes from five unrelated individuals from each of the Djallonke and Sahelian sheep breeds (sourced from Ghana), at greater than 22-fold combined coverage for each breed. A total of approximately 404 million (97%) and 343 million (97%) sequence reads from the Djallonke and Sahelian breeds respectively, were successfully mapped to the sheep reference genome Oar v3.1. We identified approximately 11.1 million and 10.9 million single nucleotide polymorphisms (SNPs) in the Djallonke and Sahelian breeds, with approximately 15 and 16% respectively of these not previously reported in sheep. Multiple regions of reduced heterozygosity were also found; 70 co-localised within genomic regions harbouring genes that mediate disease resistance, immune response and adaptation in sheep or cattle. Thirty- three of the regions of reduced heterozygosity co-localised with previously reported genes for resistance to haemonchosis and trypanosomiasis. CONCLUSIONS: Our analyses suggest that these regions of reduced heterozygosity may be signatures of selection for these economically important diseases.

Insights on the relationship between complement component C4 serum concentrations and C4 gene copy numbers in a Western Australian systemic lupus erythematosus cohort.

The relationship between serum concentration of complement C4 ([C4]) and C4 gene copy number (GCN) was investigated in 56 systemic lupus erythematosus (SLE) patients and 33 age and sex-matched controls in a Western Australian population. C4A and C4B gene copy numbers (C4A & B GCN) together with the presence or absence of the ≈6.4-kb human endogenous retroviral element type K (hereafter HERV-K) in intron 9 were estimated by two TaqMan™ real-time PCR (RT-PCR) assays that measured total C4 and HERV-K GCNs, respectively. There was good correlation between the two methods; however, the HERV-K GCN method showed a positive bias (≈6%) relative to the C4A & B total GCN. Despite individual variation, excellent correlation between total C4 GCN and mean [C4] per GCN was observed for both the SLE and control cohorts ( R2 = 88% and R2 = 99%, respectively). It was noted that serum [C4] was significantly lower in the SLE patients than the controls ( p = 0.006) despite there being no difference between C4A and C4B GCN in both cohorts. The data therefore confirm previous reports that the C4A genes are preferentially associated with the presence of the HERV-K insertion relative to C4B genes and does not support the hypothesis that low [C4] in SLE is explained by low C4A GCNs. There was no evidence also that the presence of the HERV-K insertion in C4 genes influenced [C4]. This study supports the view that low [C4] in SLE patients is due to consumption rather than deficient synthesis related to lower C4A & B GCN.

Combatting African Animal Trypanosomiasis (AAT) in livestock: The potential role of trypanotolerance.

African Animal Trypanosomiasis (AAT) is endemic in at least 37 of the 54 countries in Africa. It is estimated to cause direct and indirect losses to the livestock production industry in excess of US$ 4.5 billion per annum. A century of intervention has yielded limited success, owing largely to the extraordinary complexity of the host-parasite interaction. Trypanotolerance, which refers to the inherent ability of some African livestock breeds, notably Djallonke sheep, N'Dama cattle and West African Dwarf goats, to withstand a trypanosomiasis challenge and still remain productive without any form of therapy, is an economically sustainable option for combatting this disease. Yet trypanotolerance has not been adequately exploited in the fight against AAT. In this review, we describe new insights into the genetic basis of trypanotolerance and discuss the potential of exploring this phenomenon as an integral part of the solution for AAT, particularly, in the context of African animal production systems.

A comprehensive mapping of the structure and gene organisation in the sheep MHC class I region.

BACKGROUND: The major histocompatibility complex (MHC) is a chromosomal region that regulates immune responsiveness in vertebrates. This region is one of the most important for disease resistance because it has been associated with resistance or susceptibility to a wide variety of diseases and because the MHC often accounts for more of the variance than other loci. Selective breeding for disease resistance is becoming increasingly common in livestock industries, and it is important to determine how this will influence MHC polymorphism and resistance to diseases that are not targeted for selection. However, in sheep the order and sequence of the protein coding genes is controversial. Yet this information is needed to determine precisely how the MHC influences resistance and susceptibility to disease. METHODS: CHORI bacterial artificial chromosomes (BACs) known to contain sequences from the sheep MHC class I region were sub-cloned, and the clones partially sequenced. The resulting sequences were analysed and re-assembled to identify gene content and organisation within each BAC. The low resolution MHC class I physical map was then compared to the cattle reference genome, the Chinese Merino sheep MHC map published by Gao, et al. (2010) and the recently available sheep reference genome. RESULTS: Immune related class I genes are clustered into 3 blocks; beta, kappa and a novel block not previously identified in other organisms. The revised map is more similar to Bovidae maps than the previous sheep maps and also includes several genes previously not annotated in the Chinese Merino BAC assembly and others not currently annotated in the sheep reference chromosome 20. In particular, the organisation of nonclassical MHC class I genes is similar to that present in the cattle MHC. Sequence analysis and prediction of amino acid sequences of MHC class I classical and nonclassical genes was performed and it was observed that the map contained one classical and eight nonclassical genes together with three possible pseudogenes. CONCLUSIONS: The comprehensive physical map of the sheep MHC class I region enhances our understanding of the genetic architecture of the class I MHC region in sheep and will facilitate future studies of MHC function.

Characterisation of tetra-nucleotide microsatellite loci in the ostrich (Struthio camelus).

1. Eleven polymorphic tetra-nucleotide microsatellite loci were identified in the ostrich (Struthio camelus) using a selective enrichment protocol. 2. The average number of alleles observed was 6·6 with an average heterozygosity of 0·4. 3. The population was found to be in Hardy-Weinberg equilibrium and two of the loci had a greater than 95% probability of having null alleles. 4. These microsatellite loci will add to the existing pool of markers available for the ostrich and help to facilitate analysis of population structure and pedigree determination.

Conserved haplotype blocks within the sheep MHC and low SNP heterozygosity in the Class IIa subregion.

This report describes single-nucleotide polymorphisms (SNPs) in the sheep major histocompatibility complex (MHC) class II and class III regions and provides insights into the internal structure of this important genomic complex. MHC haplotypes were deduced from sheep family trios based on genotypes from 20 novel SNPs representative of the class II region and 10 previously described SNPs spanning the class III region. All 30 SNPs exhibited Hardy-Weinberg proportions in the sheep population studied. Recombination within an extended sire haplotype was observed within the class II region for 4 of 20 sheep chromosomes, thereby supporting the presence of separated IIa and IIb subregions similar to those present in cattle. SNP heterozygosity varied across the class II and III regions. One segment of the class IIa subregion manifested very low heterozygosity for several SNPs spanning approximately 120 Kbp. This feature corresponds to a subregion within the human MHC class II region previously described as a 'SNP desert' because of its paucity of SNPs. Linkage disequilibrium (LD) was reduced at the junction separating the putative class IIb and IIa subregions and also between the class IIa and the class III subregions. The latter observation is consistent with either an unmapped physical separation at this location or more likely a boundary characterized by more frequent recombination between two conserved subregions, each manifesting high within-block LD. These results identify internal blocks of loci in the sheep MHC, within which recombination is relatively rare.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone.

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

The influence of MHC and immunoglobulins a and e on host resistance to gastrointestinal nematodes in sheep.

Gastrointestinal nematode parasites in farmed animals are of particular importance due to their effects on production. In Australia, it is estimated that the direct and indirect effects of parasite infestation cost the animal production industries hundreds of millions of dollars each year. The main factors considered by immunologists when studying gastrointestinal nematode infections are the effects the host's response has on the parasite, which immunological components are responsible for these effects, genetic factors involved in controlling immunological responses, and the interactions between these forming an interconnecting multilevel relationship. In this paper, we describe the roles of immunoglobulins, in particular IgA and IgE, and the major histocompatibility complex in resistance to gastrointestinal parasites in sheep. We also draw evidence from other animal models to support the involvement of these immune components. Finally, we examine how IgA and IgE exert their influence and how methods may be developed to manage susceptible animals.

Multiple incursions and putative species revealed using a mitochondrial and nuclear phylogenetic approach to the Trogoderma variabile (Coleoptera: Dermestidae) trapping program in Australia.

The Warehouse beetle, Trogoderma variabile (Coleoptera: Dermestidae), is an internationally significant invasive pest of packed goods and stored grain. When it was first documented in Australia at Griffith, New South Wales, in 1977, an eradication campaign was initiated. After several years and considerable effort, the eradication campaign was abandoned. To monitor the presence and spread of T. variabile, surveys were carried out by government agencies in 1992 and 2002. When survey data was compared, it was concluded that the distribution of morphologically identified T. variabile had doubled in most Australian states. Here, we used samples from the 2002 survey to conduct a phylogenetic study using partial sequences of mitochondrial genes Cytochrome oxidase I and Cytochrome B, and the nuclear gene 18S, to examine the distribution and dispersal of T. variabile and detect the presence of misidentified species. Based on our molecular results, we show that only 47% of the samples analysed were T. variabile, and the remaining were a mixture of six putative species. In addition, T. variabile was found in only 78% of the trapping sites. We discuss the importance of correct diagnosis in relation to the eradication campaign.

Polymorphism of sheep MHC Class IIb gene TAPASIN.

The Major Histocompatibility Complex (MHC) is one of the most gene dense regions in the genome and studies in several species have shown significant associations between the MHC and disease. The endoplasmic reticular glycoprotein, tapasin, is involved in the MHC class I antigen presentation pathway. Sheep TAPASIN is located in the class IIb region of the MHC. Sheep TAPASIN was subcloned from BAC and cosmid genomic clones and DNA sequenced. TAPASIN is 9549bp in length and encodes a protein of 447 amino acids. The structure of sheep TAPASIN was similar to other mammals and consisted of eight exons with a distinctively larger intron between exon three and four. Sheep TAPASIN gene had high sequence identity with other mammalian TAPASINs. The TAPASIN gene sequence is conserved across many mammalian species and is possibly maintained through purifying selection with the average ratio of ds/dn of 3.9. Twenty-six SNPs in sheep TAPASIN were identified.

A map of the class III region of the sheep major histocompatibilty complex.

BACKGROUND: The central, or class III, region of the major histocompatibility complex (MHC) is an important gene rich sub-region of the MHC of mammals and contains many loci implicated in disease processes and potential productivity traits. As a prelude to identifying MHC loci associated with productivity traits in sheep, we have used BAC and cosmid libraries of genomic DNA to generate a physical map of the sheep MHC class III region. This map will facilitate association studies and provide insights into the distribution of recombination events in this chromosomal segment. RESULTS: Twenty eight sheep genes were identified in 10 BAC clones which spanned approximately 700 kbp of a chromosomal region adjacent to the class I region of the sheep MHC and which therefore covers most, if not all, of the class III of the sheep MHC. The relative positions of 17 of these genes was established as well as two additional groups of genes for which the intragroup order was not known. Cosmid mapping permitted a more detailed mapping of the complement genes present in the class III and showed a local inversion (relative to humans) of one pair of the duplicated complement C4 and CYP21 loci. A panel of 26 single nucleotide polymorphisms (SNPs) was identified in 10 loci, covering approximately 600 kbp of the mapped region. CONCLUSION: This report provides a physical map covering approximately 700 kbp of the class III of the sheep MHC together with a SNP panel which will facilitate disease and productivity association studies. The presence of a local inversion (relative to humans) of one pair of the duplicated C4 and CYP21 loci and a previously described dinucleotide tandem repeat locus (BfMs) has been located within an intron of the SK12VL gene.

APOE-epsilon4 and APOE -491A polymorphisms in individuals with subjective memory loss.

The accurate clinical diagnosis of Alzheimer's disease can only be made with a high degree of certainty in specialized centres. The identification of predictive or diagnostic genetic factors may improve accuracy of disease prediction or diagnosis. One major genetic risk factor, the epsilon4 allele of the apolipoprotein E gene, is universally recognised. We have recently shown that the A allele of the apolipoprotein E, -491A/T promoter polymorphism is also an important risk factor for Alzheimer's disease in an Australian population. We designed the present study to investigate the association between apolipoprotein E genotype, -491A/T polymorphism, plasma apoE levels and the subjective experience of memory decline among 98 subjects and 49 age, gender and education-matched normal controls. An increased frequency of the epsilon4 allele of apolipoprotein E was significantly associated with the 'memory complainers' group (OR = 2.35, P = 0.02) as was the A allele of the -491A/T polymorphism (OR = 2, P = 0.02). Among all subjects, only seven individuals were homozygous for both of these alleles, and six of these seven individuals belonged to the 'memory complainers' group. This sub-group also had relatively elevated plasma apolipoprotein E levels (P < 0.01) and tended to score lower on the Mini-Mental State Examination (MMSE) and Cambridge Cognition Test. These data suggest that the epsilon4 allele of apolipoprotein E and the -491A allele are over-represented among individuals who complain of memory difficulties. Follow-up studies should clarify whether these genotypes and phenotypes are useful in the prediction and/or diagnosis of Alzheimer's disease.

Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality.

A polymerase chain reaction-based method for genotyping Giardia duodenalis isolates using a polymorphic region near the 5' end of the small subunit ribosomal (SSU) RNA gene is described. Analysis was performed using Giardia cysts purified directly from feces. Isolates were collected from humans and dogs living in isolated Aboriginal communities where Giardia infections are highly endemic. This is the first report of the genetic characterization of Giardia from dogs and humans living in the same locality. Comparison of the SSU-rRNA sequences from 13 human and 9 dog isolates revealed 4 different genetic groups. Groups 1 and 2 contained all of the human isolates, whereas groups 3 and 4 consisted entirely of Giardia samples recovered from dogs. One dog sample contained templates from both groups 2 and 3. These results suggest that zoonotic transmission of Giardia infections between humans and dogs does not occur frequently in these communities. The dog-associated SSU-rRNA sequences have not been reported before, suggesting a new G. duodenalis subgroup. A genetic basis for the differences observed between the groups was supported by sequence analysis of 9 in vitro cultured isolates that were placed into the same genetic groups established by enzyme electrophoresis.

Typing of methicillin-resistant Staphylococcus aureus with an M13 repeat probe.

A bacteriophage M13 tandem repeat has been used to probe EcoRI digested genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA). The patterns generated were found to be useful in typing MRSA and generally confirmed the relationships that had previously been recognized in other studies based on antimicrobial resistance and plasmid profiles. The epidemic MRSA of London hospitals (EMRSA) and the majority of the epidemic MRSA of eastern Australian hospitals (EA MRSA) gave the same pattern. However, two isolates previously classified as EA MRSA gave a different pattern and a third another pattern. One isolate from Dublin, two isolates from Nuneaton and two isolates from Singapore gave the same pattern as the two EA MRSA. With the exception of the early or classic MRSA all the other isolates examined gave their own distinctive patterns. With one exception the classic MRSA belonged to a separate group. The exception was of particular interest because it gave the same pattern as the majority of the EA MRSA. This suggests that there may be an evolutionary relationship between some of the classic MRSA and the EMRSA of London and the EA MRSA of Australia.

Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma.

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.