Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2', 3'-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.

A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen

BACKGROUND: The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti-KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti-KEL1 reagents. STUDY DESIGN AND METHODS: Kell genotyping methods, including KEL exon 6 direct sequencing, were applied. KEL cDNA from D1 was sequenced. Flow cytometry was used to assess KEL1 and KEL2 RBC expression. RESULTS: RBCs from the donor, her mother, and an unrelated donor gave weak or negative reactions with some anti-KEL1 reagents. Other Kell-system antigens appeared normal. The three individuals were homozygous for KEL C578 (KEL*2) but heterozygous for a 577A>T transversion, encoding Ser193. They appeared to be KEL*2 homozygotes by routine genotyping methods. Flow cytometry revealed weak KEL1 expression and normal KEL2, similar to that of KEL*2 homozygotes. CONCLUSION: Ser193 in the Kell glycoprotein appears to result in expression of abnormal KEL1, in addition to KEL2. The mutation is not detected by routine Kell genotyping methods and, because of unpredicted KEL1 expression, could lead to a misdiagnosis.

Demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats

Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the env gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.

Dysphagia due to triple A syndrome: successful treatment of achalasia by balloon dilatation

Triple A syndrome is a rare autosomal recessive inherited disorder which is characterized by alacrima, adrenal insufficiency, and achalasia. We report on a 14-year old girl with dysphagia, regurgitation, and vomiting since 5 years. At the age of five years an Addison crisis was diagnosed and cortisone substitution was initiated. In addition, the patient had episodes of conjunctivitis. Severe esophagitis and candida infection were diagnosed by esophago-gastro-duodenoscopy and treated with omeprazole and fluconazole. The esophageal barium swallow was typical for achalasia. Medical treatment of achalasia with oral nifedipine resulted only in a partial and temporal improvement. But after seven balloon dilatations dysphagia and nocturnal coughing improved clearly and a remarkable gain of weight could be seen. Direct sequencing showed a homozygous nonsense mutation in exon 11 of the AAAS gene leading to truncation at position 342 of the 546 amino acid protein. CONCLUSION: Triple A syndrome has to be considered in patients with dysphagia. In our patient, the absence of tears since birth followed by adrenal insufficiency were early signs of the triple A syndrome. Balloon dilatation of the esophago-gastric junction is an effective treatment, which can avoid surgical interventions.

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

The Mycoplasma conjunctivae genome sequencing, annotation and analysis

BACKGROUND: The mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra). METHODS: The genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database. RESULTS: The annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen http://cbi.labri.fr/outils/molligen/ allowing for comparative genomics. CONCLUSION: We show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

Intestinal Tritrichomonas foetus infection in cats in Switzerland detected by in vitro cultivation and PCR

Tritrichomonas foetus, a parasite well known for its significance as venereally transmitted pathogen in cattle, has recently been identified as a cause of chronic large-bowel diarrhea in domestic cats in the US, UK, and, more recently, also in Norway. In a period of 3 months (October to December 2007), 45 cats of Switzerland suffering from chronic diarrhea were investigated for intestinal infections, including a search for trichomonads. A commercially available in vitro culture system was used to screen for infection, complemented with a PCR and subsequent amplicon sequencing to support speciation. The PCR is based upon amplification of a sequence derived from the internal transcribed spacer region 1 (ITS1) on the ribosomal RNA gene (rRNA) using primers designed to detect a broad range of genera and species belonging to the family of Trichomonadidae. The method was furthermore adapted to the uracil DNA glycosylase (UDG) system in order to prevent carry-over contamination and it included a recombinant internal control to track for inhibitory reactions. Eleven out of the 45 cats were culture-positive, as revealed by microscopic identification of trichomonadid organisms. One of the isolates was subjected to scanning electron microscopy and findings revealed the presence of three flagella, thus placing the isolate into the gender Tritrichomonas sp. PCR and subsequent amplicon sequencing were carried out with ten of the 11 isolates. A total homology with published T. foetus sequences was confirmed in all of the cases. T. foetus therefore appears to range among those organisms that can cause chronic diarrhea in cats in Switzerland.

Multiplex strategy for multilocus sequence typing, fla typing, and genetic determination of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.

Occurrence, quantification, and genotyping of Mycoplasma conjunctivae in wild Caprinae with and without infectious keratoconjunctivitis

Mycoplasma conjunctivae, the causative agent of infectious keratoconjunctivitis (IKC), was recently detected in asymptomatic Alpine ibex (Capra ibex ibex). This suggested that an external source of infection may not be required for an IKC outbreak in wildlife but might be initiated by healthy carriers, which contradicted previous serologic investigations in chamois. Our aims were to 1) assess the prevalence of M. conjunctivae among asymptomatic ibex and Alpine chamois (Rupicapra rupicapra rupicapra) and its frequency in IKC-affected animals, 2) determine mycoplasma loads in different disease stages, and 3) characterize the M. conjunctivae strains involved. Eye swabs from 654 asymptomatic and 204 symptomatic animals were collected in diverse Swiss regions between 2008 and 2010, and tested by TaqMan real-time PCR. Data analysis was performed considering various patterns of IKC occurrence in the respective sampling regions. Strains from 24 animals were compared by cluster analysis. Prevalence of M. conjunctivae was 5.6% (95% confidence interval [CI]: 3.7-8.1%) in asymptomatic ibex and 5.8% (CI: 3.0-9.9%) in asymptomatic chamois, with significant differences between years and regions in both species. Detection frequency in symptomatic animals was significantly higher during IKC outbreaks than in nonepidemic situations (i.e., regular but low incidence or sporadic occurrence). Mycoplasma load was significantly lower in eyes from healthy carriers and animals with mild signs than from animals with moderate and severe signs. Although some strains were found in both asymptomatic and diseased animals of the same species, others apparently differed in their pathogenic potential depending on the infected species. Overall, we found a widespread occurrence of M. conjunctivae in wild Caprinae with and without IKC signs. Our results confirm the central role of M. conjunctivae in outbreaks but suggest that other infectious agents may be involved in IKC cases in nonepidemic situations. Additionally, presence and severity of signs are related to the quantity of M. conjunctivae in the eyes rather than to the strain. We propose that individual or environmental factors influence the clinical expression of the disease and that persistence of M. conjunctivae in populations of wild Caprinae cannot be excluded.

Unique mixed phenotype and unexpected functional effect revealed by novel compound heterozygosity mutations involving SCN5A.

BACKGROUND Functional characterization of mutations involving the SCN5A-encoded cardiac sodium channel has established the pathogenic mechanisms for type 3 long QT syndrome and type 1 Brugada syndrome and has provided key insights into the physiological importance of essential structure-function domains. OBJECTIVE This study sought to present the clinical and biophysical phenotypes discerned from compound heterozygosity mutations in SCN5A on different alleles in a toddler diagnosed with QT prolongation and fever-induced ventricular arrhythmias. METHODS A 22-month-old boy presented emergently with fever and refractory ventricular tachycardia. Despite restoration of sinus rhythm, the infant sustained profound neurological injury and died. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open-reading frame/splice mutational analysis of the 12 known long QT syndrome susceptibility genes was performed. RESULTS The infant had 2 SCN5A mutations: a maternally inherited N-terminal frame shift/deletion (R34fs/60) and a paternally inherited missense mutation, R1195H. The mutations were engineered by site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. As expected, the frame-shifted and prematurely truncated peptide, SCN5A-R34fs/60, showed no current. SCN5A-R1195H had normal peak and late current but abnormal voltage-dependent gating parameters. Surprisingly, co-expression of SCN5A-R34fs/60 with SCN5A-R1195H elicited a significant increase in late sodium current, whereas co-expression of SCN5A-WT with SCN5A-R34fs/60 did not. CONCLUSIONS A severe clinical phenotype characterized by fever-induced monomorphic ventricular tachycardia and QT interval prolongation emerged in a toddler with compound heterozygosity involving SCN5A: R34fs/60, and R1195H. Unexpectedly, the 94-amino-acid fusion peptide derived from the R34fs/60 mutation accentuated the late sodium current of R1195H-containing Na(V)1.5 channels in vitro.

Genome-wide analysis in German shepherd dogs reveals association of a locus on CFA 27 with atopic dermatitis

Humans and dogs are both affected by the allergic skin disease atopic dermatitis (AD), caused by an interaction between genetic and environmental factors. The German shepherd dog (GSD) is a high-risk breed for canine AD (CAD). In this study, we used a Swedish cohort of GSDs as a model for human AD. Serum IgA levels are known to be lower in GSDs compared to other breeds. We detected significantly lower IgA levels in the CAD cases compared to controls (p = 1.1 × 10(-5)) in our study population. We also detected a separation within the GSD cohort, where dogs could be grouped into two different subpopulations. Disease prevalence differed significantly between the subpopulations contributing to population stratification (λ = 1.3), which was successfully corrected for using a mixed model approach. A genome-wide association analysis of CAD was performed (n cases = 91, n controls = 88). IgA levels were included in the model, due to the high correlation between CAD and low IgA levels. In addition, we detected a correlation between IgA levels and the age at the time of sampling (corr = 0.42, p = 3.0 × 10(-9)), thus age was included in the model. A genome-wide significant association was detected on chromosome 27 (praw = 3.1 × 10(-7), pgenome = 0.03). The total associated region was defined as a ~1.5-Mb-long haplotype including eight genes. Through targeted re-sequencing and additional genotyping of a subset of identified SNPs, we defined 11 smaller haplotype blocks within the associated region. Two blocks showed the strongest association to CAD. The ~209-kb region, defined by the two blocks, harbors only the PKP2 gene, encoding Plakophilin 2 expressed in the desmosomes and important for skin structure. Our results may yield further insight into the genetics behind both canine and human AD.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.