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.

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.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

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.

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.

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.

Microsatellite analysis of genetic diversity in wild and farmed Emus (Dromaius novaehollandiae).

The emu (Dromaius novaehollandiae) occupies most regions of the Australian continent and in recent times has been farmed for meat, oil, and leather. Very little is known about the genetic structure of natural or farmed populations of these birds. We report a preliminary study of genetic variation in emus undertaken by typing birds from five farms and two natural populations at five polymorphic microsatellite loci. Genetic diversity was high for all populations and there was little evidence of inbreeding, with most populations conforming to Hardy-Weinberg equilibrium for most loci. Significant heterozygote deficiencies at one locus in a number of populations were detected and may indicate the presence of null alleles. Comparisons of allele frequencies showed little evidence of genetic differentiation either among farmed populations or between farmed and natural populations.

PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer.

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA fingerprint Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS-PCR size groups (1.35-1.6 kb) were observed, which correlated closely with the major genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS-PCR size groups A (1.42 kb) C (1.4 kb) and D (1.35 kb) corresponded to isoenzyme assemblage A, and IGS-PCR group B (1.6 kb) to isoenzyme assemblage B. Amplified products from IGS-PCR size groups A and B, which contained 50/52 isolates, were subsequently digested with 8 different restriction enzymes and their profiles compared. Analysis separated isolates within each IGS-PCR size group into 2 distinct clusters which correlated almost exactly with the same genetic groups established previously using isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they established different genetic relationships between individual isolates. Much of the variability associated with the IGS was attributed to isolates harbouring multiple IGS-sequence types. Restriction analysis of IGS-PCR products amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are homogeneous with respect to the types of IGS-variants they maintain. Furthermore, in vitro culture of the cloned isolate BAH39c9 over a 6-year period also failed to reveal variation in IGS-PCR digestion profiles. These results suggest that IGS-PCR RFLP profiles are inherently stable. IGS-PCR analysis was successfully applied to 11 Giardia cyst samples highlighting the potential for this approach to genotype Giardia isolates without the need for in vitro culture.

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.

Particle-enhanced turbidimetric immunoassay of sex-hormone-binding globulin in serum.

A particle-enhanced turbidimetric immunoassay (PETIA) for human sex-hormone-binding globulin (SHBG) is described. The method involves use of antibody covalently coupled to latex particles and is almost fully automated, with sample processing being complete in less than 20 min. The working reagents are stable for at least three months, and full calibration of the assay each day is not essential. A particular advantage is that pretreatment of samples is rarely required because the working range of the assay is from 2.0 to 320 nmol/L for nondiluted serum. Intra- and interassay CVs were less than 4.5% and 8.5%, respectively, and mean analytical recovery was 101.5%. SHBG concentrations of 129 serum samples determined by this method and by a commercially available immunoradiometric assay correlated highly.

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.

Sex hormone-binding globulin secretion by human hepatocarcinoma cells is increased by both estrogens and androgens.

The secretion of sex hormone-binding globulin (SHBG) by the human hepatocarcinoma cell line Hep G2 was increased significantly not only by estradiol (E2) but also by testosterone (T), dihydrotestosterone, and the synthetic androgen danazol in the presence, but not the absence, of fetal calf serum. The secretion of SHBG also was stimulated by the antiestrogen tamoxifen, although this required the use of longer incubation periods and higher concentrations than were required for the steroids. The antiandrogen cyproterone acetate and cortisol (5 microM) decreased SHBG secretion. Pregnanediol (5 microM) had no discernible effect. These changes were considered specific as none of the compounds altered either the secretion of total protein by the cells or their total protein content. Cells incubated with a mixture of E2 (0.5 microM) and T (0.5 microM) secreted significantly more SHBG than did cells incubated with E2 (1.0 microM), indicating these steroids exert their effects through different mechanisms. The increases with E2 and T were reduced significantly by tamoxifen and cyproterone acetate, respectively, suggesting receptor mediation of the steroid effects. The E2-related increase was abolished by cycloheximide, indicating that the changes were due to alterations in the synthesis of SHBG rather than to alterations in the release of previously synthesized protein. These findings suggest the T-related decrease in plasma SHBG levels may be due to causes other than a decrease in the hepatic synthesis of the protein. Additionally, they indicate that Hep G2 cells may prove suitable for examining the regulation of the SHBG gene by a variety of compounds.

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.