Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk

A novel real-time quantitative PCR assay for detecting the pathogenic and contagious Staphylococcus aureus genotype B (GTB) in bulk tank milk was developed and evaluated. The detection of this pathogen in bulk tank milk would greatly facilitate its control, as it is responsible for great economic loss in Swiss dairy herds. The assay is based on the simultaneous detection of 3 GTB-typical target sequences, including 2 enterotoxin genes and a polymorphism within the leucotoxin E gene. A variety of mastitis-associated bacteria was used to validate the assays, resulting in an analytical specificity of 100% and high repeatability. The analytical sensitivity in milk was 40 cfu/mL. An exponential association between simulated cow prevalence and quantitative PCR result was observed. An initial field study revealed 1 GTB-positive herd among the 33 studied herds. This novel assay for bulk tank milk analysis is suitable for routine purposes and is expected to be an effective tool for minimizing Staph. aureus GTB in Swiss dairy herds.

Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.

Huntington's disease: new aspects on phenotype and genotype

Huntington's disease typically presents with involuntary movements, cognitive decline and behavioural abnormalities; however, new data show a greater spectrum and more complexity in the mode of presentation than previously appreciated. On one hand efforts are under way to better assess all aspects of the evolving phenotype over the course of the disease, on the other hand large cohorts have been prospectively followed-up and similar efforts are now being started in China. In this communication, we briefly review the most salient findings from the last couple of years. The recently established large cohorts allow the performance of accurate studies examining correlation of genetic polymorphisms with specific aspects of the phenotype thus allowing for some mechanistic insight into the causes of phenotypic variation. While Huntington's disease is the most frequent hereditary cause of chorea, other disorders with similar clinical phenotypes, including neuroacanthocytosis, are now better known, including a better understanding of the primary cause as well as the pathophysiology at the molecular level. Studies on the mechanisms of disease in these different disorders may shed light on the respective pathomechanisms and may open new approaches to a better understanding and additional treatment options for choreatiform neurodegenerative disorders.

Associations between prion protein genotype and type traits in East Friesian milk sheep

Linear models were used to analyse the relationships between the prion protein genotypes and the height at the withers and rump, the heart girth and the length of the trunk of 440 East Friesian milk sheep. Significant associations were found between the ARR allele and the height of the withers and rump, and heart girth. The average height at the withers of the homozygous ARR/ARR sheep was 1.9 cm less than that of ARQ/ARQ sheep and 1.6 cm less than in sheep heterozygous for ARR; the height at the rump, length of the trunk and heart girth were similarly smaller. In the ARR/ARR ewes, the average height at the withers was 2.6 cm less and the height at the rump was 1.9 cm less than in the ARQ/ARQ ewes.

Miotic action of tramadol is determined by CYP2D6 genotype

Polymorphic CYP2D6 is the enzyme that activates the opioid analgesic tramadol by O-demethylation to M1. Our objective was to determine the opioid effects measured by pupillary response to tramadol of CYP2D6 genotyped volunteers in relation to the disposition of tramadol and M1 in plasma. Tramadol displayed phenotypic pharmacokinetics and it was possible to identify PM subjects with >99% confidence from the metabolic ratio (MR) in a single blood sample taken between 2.5 and 24 h post-dose. Homozygous extensive metabolizers (EM) differed from poor metabolizers (PM), with an almost three-fold greater (P=0.0014) mean maximal pupillary constriction (Emax). Significant correlations between the AUC and Cmax values of M1 versus pupillary constriction were found. The corresponding correlations of pharmacokinetic parameters for tramadol itself were weaker and negative. The strongest correlations were for the single-point metabolic ratios at all sampling intervals versus the effects, with rs ranging from 0.85 to 0.89 (p0.01). It is concluded that the concept of dual opioid/non-opioid action of the drug, though considerably stronger in EMs, is valid for both EM and PM subjects. This is the theoretical basis for the frequent use and satisfactory efficacy of tramadol in clinical practice when given to genetically non-selected population.

CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis

OBJECTIVE: To determine whether pharmacogenetic tests such as N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) genotyping are useful in identifying patients prone to antituberculosis drug-induced hepatotoxicity in a cosmopolite population. METHODS: In a prospective study we genotyped 89 patients treated with isoniazid (INH) for latent tuberculosis. INH-induced hepatitis (INH-H) or elevated liver enzymes including hepatitis (INH-ELE) was diagnosed based on the clinical diagnostic scale (CDS) designed for routine clinical practice. NAT2 genotypes were assessed by fluorescence resonance energy transfer probe after PCR analysis, and CYP2E1 genotypes were determined by PCR with restriction fragment length polymorphism analysis. RESULTS: Twenty-six patients (29%) had INH-ELE, while eight (9%) presented with INH-H leading to INH treatment interruption. We report no significant influence of NAT2 polymorphism, but we did find a significant association between the CYP2E1 *1A/*1A genotype and INH-ELE (OR: 3.4; 95% CI:1.1-12; p = 0.02) and a non significant trend for INH-H (OR: 5.9; 95% CI: 0.69-270; p = 0.13) compared with other CYP2E1 genotypes. This test for predicting INH-ELE had a positive predictive value (PPV) of 39% (95% CI: 26-54%) and a negative predictive value (NPV) of 84% (95% CI: 69-94%). CONCLUSION: The genotyping of CYP2E1 polymorphisms may be a useful predictive tool in the common setting of a highly heterogeneous population for predicting isoniazid-induced hepatic toxicity. Larger prospective randomized trials are needed to confirm these results.

QUANTIFYING UNCERTAINTY IN GENOTYPE CALLS

Genome-wide association studies (GWAS) are used to discover genes underlying complex, heritable disorders for which less powerful study designs have failed in the past. The number of GWAS has skyrocketed recently with findings reported in top journals and the mainstream media. Mircorarrays are the genotype calling technology of choice in GWAS as they permit exploration of more than a million single nucleotide polymorphisms (SNPs)simultaneously. The starting point for the statistical analyses used by GWAS, to determine association between loci and disease, are genotype calls (AA, AB, or BB). However, the raw data, microarray probe intensities, are heavily processed before arriving at these calls. Various sophisticated statistical procedures have been proposed for transforming raw data into genotype calls. We find that variability in microarray output quality across different SNPs, different arrays, and different sample batches has substantial inuence on the accuracy of genotype calls made by existing algorithms. Failure to account for these sources of variability, GWAS run the risk of adversely affecting the quality of reported findings. In this paper we present solutions based on a multi-level mixed model. Software implementation of the method described in this paper is available as free and open source code in the crlmm R/BioConductor.

EXPLORATION, NORMALIZATION, AND GENOTYPE CALLS OF HIGH DENSITY OLIGONUCLEOTIDE SNP ARRAY DATA

In most microarray technologies, a number of critical steps are required to convert raw intensity measurements into the data relied upon by data analysts, biologists and clinicians. These data manipulations, referred to as preprocessing, can influence the quality of the ultimate measurements. In the last few years, the high-throughput measurement of gene expression is the most popular application of microarray technology. For this application, various groups have demonstrated that the use of modern statistical methodology can substantially improve accuracy and precision of gene expression measurements, relative to ad-hoc procedures introduced by designers and manufacturers of the technology. Currently, other applications of microarrays are becoming more and more popular. In this paper we describe a preprocessing methodology for a technology designed for the identification of DNA sequence variants in specific genes or regions of the human genome that are associated with phenotypes of interest such as disease. In particular we describe methodology useful for preprocessing Affymetrix SNP chips and obtaining genotype calls with the preprocessed data. We demonstrate how our procedure improves existing approaches using data from three relatively large studies including one in which large number independent calls are available. Software implementing these ideas are avialble from the Bioconductor oligo package.

A HIDDEN MARKOV MODEL FOR JOINT ESTIMATION OF GENOTYPE AND COPY NUMBER IN HIGH-THROUGHPUT SNP CHIPS

Amplifications and deletions of chromosomal DNA, as well as copy-neutral loss of heterozygosity have been associated with diseases processes. High-throughput single nucleotide polymorphism (SNP) arrays are useful for making genome-wide estimates of copy number and genotype calls. Because neighboring SNPs in high throughput SNP arrays are likely to have dependent copy number and genotype due to the underlying haplotype structure and linkage disequilibrium, hidden Markov models (HMM) may be useful for improving genotype calls and copy number estimates that do not incorporate information from nearby SNPs. We improve previous approaches that utilize a HMM framework for inference in high throughput SNP arrays by integrating copy number, genotype calls, and the corresponding confidence scores when available. Using simulated data, we demonstrate how confidence scores control smoothing in a probabilistic framework. Software for fitting HMMs to SNP array data is available in the R package ICE.