Unique Genetic Markers Research Articles

Variable regions of the glyS, infB and rplB genes usable as novel genetic markers for identification and phylogenetic purposes of genera belonging to the family Propionibacteriaceae.

No common, unique genetic markers applicable to classification and phylogenetics for significant genera within the Propionibacteriaceae family have been suggested yet. Therefore, the aim of the study was to propose those genes in the genera Acidipropionibacterium, Cutibacterium, Propionibacterium and Pseudopropionibacterium. These genera were recently elicited from the genus Propionibacterium through whole genomic analyses. Three housekeeping genes, glyS, infB and rplB, were selected from many others according to the requirements for appropriate classification/phylogenetic markers. Concrete fragments of the genes were amplified using specific primers in most of the type (14) and 11 wild strains (originating from dairy products, human skin and the crop of a laying hen) recently classified into the genus Propionibacterium. Sequences obtained from amplicons were used to perform gene statistics and phylogenetic analyses with respect to applicability in classification, typing and phylogeny. The 16S rRNA gene sequences, still considered relevant in spite of its proven shortcomings as a basic tool for evaluation of bacterial phylogeny, were used as a baseline for comparative analyses. The statistics of the gene sequences revealed that the variable regions of all three genes have higher resolution capabilities among strains examined compared to the 16S rRNA gene analysis. Phylogenetic analyses based on individual gene sequences and their concatenate enabled to distinguish clusters of species belonging to the genera Acidipropionibacterium, Cutibacterium and Propionibacterium, which corresponds with a recently reported genomic study. Thus, the crucial importance of this study is the economically advantageous classification and typing of propionibacterial isolates and strains through the three gene regions in contrast to the requirement for whole genomic assays.

Amplified Detection of a Unique Genomic Viral Marker Using Fluorescently Labeled Liposomes

AbstractSensitive and enzyme‐free detection of specific oligonucleotide sequences is a pressing need in the field of precision and personalized medicine. In this work the detection of a unique genetic marker for hepatitis B virus with a low detection limit of 5 × 10−12m and a fast assay time is reported. In the assay presented here, fluorescently labeled liposomes are captured by gold‐decorated polystyrene beads using the target sequence as the building block. This is achieved by modifying the microbeads and the liposomes each with half of the complementary sequence. After a short incubation time with the target of interest, the fluorescent signal of the bridged bead–liposome assembly is detected via flow cytometry. Each liposome is labeled with ≈48 lipid soluble dyes resulting in an amplified fluorescent signal for every sensing event. The detection of the viral marker is even possible in an artificial serum‐spiked sample.

Genetic profile of polyps and risk of advanced metachronous lesions.

555 Background: The role of genetic profile of polyps to predict advanced metachronous lesions (AML) remains unknown. The aim is to study the relation between genetic profile of polyps and both risk of AMLs and time to develop them in surveillance. Methods: 308 patients with colonic polyps were consecutively enrolled between 2007 and 2009 for this cohort study, and followed up to 2014 (median 26 months, range 63). Variables as age, sex, smoking, weight, number of colonoscopies and number and characteristics of polyps were collected. 995 polyps were analyzed for somatic mutations on BRAF and KRAS genes using allelic discrimination by real-time PCR and direct DNA sequenciation, respectively. High level of methylation on CpG islands (CIMP-H) was also tested using MS-MLPA. AML was defined by a size higher than 9mm, high grade dysplasia or villous component. Risk of developing AML for individual genetic markers was studied using Chi-square tests and logistic regression. Log-rank test with Kaplan Meier survival curves and Cox-regression model were also performed. Multivariate analysis were adjusted by sex, age, familial colorectal cancer, smoking and features of AML in first colonoscopy. Results: 21% of polyps in first colonoscopy were CIMP-H. KRAS and BRAF mutations accounted for 25% and 17% of polyps, respectively. In univariate analysis, KRAS-mutated polyps were related to higher risk of AML in surveillance (52% KRAS-mutated polyps vs 31% non-mutated; p = 0.01). Similar results were obtained regarding CIMP-H (77% CIMP-H polyps vs 38% non-CIMP; p = 0.005). Logistic regression showed CIMP-H as the unique genetic marker of risk for AML (OR 11.41, 95% CI 2.04-63.70; p = 0.006). Regarding time to develop AML, shorter intervals were found related to CIMP-H (median of 31 vs 48 months in non-CIMP-H; p = 0.002) and KRAS-mutations (median of 36 vs 49 months in non-mutated; p = 0.029) in univariate analysis. Multivariate analysis highlighted CIMP-H as the unique independent marker associated to shorter time to develop AML (HR 4.01, 95% CI 1.36-10.46; p = 0.01). Conclusions: Presence of CIMP-H in polyps associates higher risk of subsequent AML and shorter interval to their development. Genetic profile of polyps emerges as useful tool for colonoscopy surveillance.

Biobanking in Precision Medicine

Precise treatment of a disease, with a concerted analysis at the genomic and phenotypic level, is a paradigm shift from the one-size-fits-all treatment practiced for centuries. As part of the Precision Medicine Initiative, a million cohort samples will be collected for research and development of unique genetic markers in the study of the relationship of genomic markers to cancer. The million cohort samples are only as good as the conditions under which the samples are collected and stored. The purpose of this review is to discuss an economically viable biobanking solution for tissue, blood, and nucleic acid storage for such an endeavor! Tissue biopsy and whole blood are two common types of tissues collected for the Precision Medicine Initiative. Tissue samples can be stored as formalin-fixed paraffin-embedded (FFPE) blocks at ambient for decades, but fresh tissue samples although limited in sample size are better suited for downstream application. Postmortem collection of tissue is a good alternative to fresh tissue samples if the samples can be acquired in a timely manner before cold ischemia sets in. Blood is the preferred tissue sample for the Precision Medicine Initiative as it is easy to collect compared to other tissue types. Energy and space limitations are going to be crucial for storing a million samples for decades. Dry storage at ambient temperature is an alternative to the ultra-low-temperature storage of samples. Dry storage of whole blood samples as dried blood spots (DBS) or of the isolated components such as nucleic acids at ambient is ideal. In this review, we discuss the ambient temperature storage of blood samples and of nucleic acid. The million cohort biobanked blood and tissue samples will be crucial references for decades to come as new discoveries are made and new markers identified. Collection of blood samples at ambient as DBS and storage of the associated nucleic acid at ambient will be the key to the long-term success of biobanking of this large cohort.

Attenuation and protective efficacy of Rift Valley fever phlebovirus rMP12-GM50 strain.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the differentiation from wild-type RVFV strains. In this study, we aimed to strengthen the attenuation profile of the MP-12 vaccine strain via the introduction of 584 silent mutations. To minimize the impact on protective efficacy, codon usage and codon pair bias were not de-optimized. The resulting rMP12-GM50 strain showed 100% protective efficacy with a single intramuscular dose, raising a 1:853 mean titer of plaque reduction neutralization test. Moreover, outbred mice infected with one of three pathogenic reassortant ZH501 strains, which encoded rMP12-GM50 L-, M-, or S-segments, showed 90%, 50%, or 30% survival, respectively. These results indicate that attenuation of the rMP12-GM50 strain is significantly attenuated via the L-, M-, and S-segments. Recombinant RVFV vaccine strains encoding similar silent mutations will be also useful for the surveillance of reassortant strains derived from vaccine strains in endemic countries.

A specific indel marker for the Philippines Schistosoma japonicum revealed by analysis of mitochondrial genome sequences

In the present study, near-complete mitochondrial (mt) genome sequences for Schistosoma japonicum from different regions in the Philippines and Japan were amplified and sequenced. Comparisons among S. japonicum from the Philippines, Japan, and China revealed a geographically based length difference in mt genomes, but the mt genomic organization and gene arrangement were the same. Sequence differences among samples from the Philippines and all samples from the three endemic areas were 0.57-2.12 and 0.76-3.85%, respectively. The most variable part of the mt genome was the non-coding region. In the coding portion of the genome, protein-coding genes varied more than rRNA genes and tRNAs. The near-complete mt genome sequences for Philippine specimens were identical in length (14,091bp) which was 4bp longer than those of S. japonicum samples from Japan and China. This indel provides a unique genetic marker for S. japonicum samples from the Philippines. Phylogenetic analyses based on the concatenated amino acids of 12 protein-coding genes showed that samples of S. japonicum clustered according to their geographical origins. The identified mitochondrial indel marker will be useful for tracing the source of S. japonicum infection in humans and animals in Southeast Asia.

Analysis of unique and specific genetic markers for diagnosis of antibiotic resistant, pathogenic escherichia coli (E. coli) encoding resistance to the third generation antibiotic, cefotaxime

Among the wide range of antibiotic available, beta-lactam antibiotics are used widely in bacterial pathogenic infections. The most common cause of bacterial resistance against beta-lactam antibiotics is the production of beta-lactamases by many members of Enterobacteriaceae family, including especially, E. col. (Harwalkar, Sataraddi et al. 2013). Extended-spectrum β-lactamases (ESBLs) are resistant to the third generation antibiotics, cephaloosporins and monobactum, additionally, these ESBL producing organisms™ exibit co-resistant to many other classes of antibiotics resulting limited treatment alternatives (Paterson, Bonomo 2005, Lartigue, Zinsius et al. 2007, Lewis, Herrera et al. 2007). Over the past decade, it has been observed dramatic increase of these ESBL producing E. coli strains, especially, CTX-M family of ESBL producers, to the third-generation cephalosporin antibiotic, Cefotaxime (Bonnet 2004, Lartigue, Zinsius et al. 2007, Paterson, Hujer et al. 2003, Paterson, Bonomo 2005, Ruppe, Lixandru et al. 2013). Although studies have published the diagnosis of various ESBL producers in worldwide, only few reports of molecular identification of CTX-M family of genes have been published(Al-Mayahie 2013, Harwalkar, Sataraddi et al. 2013, Literacka, Bedenic et al. 2009). This study aims to identify the DNA segments of CTX-M genes unique to E. coli, and analyse the surrounding genetic makeup of CTX-M genes. Collectively, understanding the genetic makeup of these resistance genes will shed a light on their mechanistic of antimicrobial resistance. Unlike other conventional clinical diagnostic methods, molecular diagnostics seeks evidence of a disease at the very basic causative level by detecting the nucleic acid identity. The main advantage of this diagnostic method is that the diagnosis carried out in the molecular level, and the method will identify the microbial pathogens at the early stages of the infectious diseases (Versalovic, Lupski 2002). Different types of rapid detection systems such as Real-Time PCR have been used to identify the different CTX-M family target genes (Birkett, Ludlam et al. 2007, Li, Chen 2012, Versalovic, Lupski 2002, Woodford, Fagan et al. 2006, Woodford, Sundsfjord 2005). The identified genes so far, has some degree of potency for identification, but the majority of these genes lacks the characteristics of a strong and unique genetic marker that can used as a specific target solely for identification of any CTX-M family of ESBL producing E. coli strains. Therefore this study analyse short DNA segments and develop a sensitive, specific and reliable method that can be used for rapid selective detection of ESBL producers resistant to Cefotaxime in antibiotic resistant E. coli strains.

The 80-kb DNA duplication on BTA1 is the only remaining candidate mutation for the polled phenotype of Friesian origin.

BackgroundThe absence of horns, called polled phenotype, is the favored trait in modern cattle husbandry. To date, polled cattle are obtained primarily by dehorning calves. Dehorning is a practice that raises animal welfare issues, which can be addressed by selecting for genetically hornless cattle. In the past 20 years, there have been many studies worldwide to identify unique genetic markers in complete association with the polled trait in cattle and recently, two different alleles at the POLLED locus, both resulting in the absence of horns, were reported: (1) the Celtic allele, which is responsible for the polled phenotype in most breeds and for which a single candidate mutation was detected and (2) the Friesian allele, which is responsible for the polled phenotype predominantly in the Holstein-Friesian breed and in a few other breeds, but for which five candidate mutations were identified in a 260-kb haplotype. Further studies based on genome-wide sequencing and high-density SNP (single nucleotide polymorphism) genotyping confirmed the existence of the Celtic and Friesian variants and narrowed down the causal Friesian haplotype to an interval of 145 kb.ResultsAlmost 6000 animals were genetically tested for the polled trait and we detected a recombinant animal which enabled us to reduce the Friesian POLLED haplotype to a single causal mutation, namely a 80-kb duplication. Moreover, our results clearly disagree with the recently reported perfect co-segregation of the POLLED mutation and a SNP at position 1 390 292 bp on bovine chromosome 1 in the Holstein-Friesian population.ConclusionWe conclude that the 80-kb duplication, as the only remaining variant within the shortened Friesian haplotype, represents the most likely causal mutation for the polled phenotype of Friesian origin.

Synergism of natural selection and introgression in the origin of a new species.

This article explores how introgressive hybridization enhances the evolutionary effects of natural selection and how, reciprocally, natural selection can enhance the evolutionary effects of introgression. Both types of interaction were observed during a 40-year study of Darwin's finches (Geospiza) on the small Galápagos island of Daphne Major. Hybrids, produced rarely by Geospiza fortis (medium ground finch) breeding with Geospiza scandens (cactus finch) and Geospiza fuliginosa (small ground finch), survived and bred as well as the parental species in the past 3 decades. By backcrossing, they increased the standing genetic variation and thereby the evolutionary responsiveness of the populations to natural selection. Natural selection occurred in droughts and oscillated in direction as a result of climatically induced fluctuations in food composition. Introgressive hybridization has led to the formation of a new lineage. It was initiated by a large, introgressed, hybrid male with a unique song and genetic marker that immigrated from the nearby island of Santa Cruz and bred with local hybrids and with G. fortis. All members of the lineage died in the 2003-2005 drought except a brother and a sister, who then bred with each other. Subsequent increase in the lineage was facilitated by selective mortality of the largest G. fortis. Breeding endogamously, the lineage is behaving as a biological species.

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Evidence of Associations between Cytokine Genes and Subjective Reports of Sleep Disturbance in Oncology Patients and Their Family Caregivers

The purposes of this study were to identify distinct latent classes of individuals based on subjective reports of sleep disturbance; to examine differences in demographic, clinical, and symptom characteristics between the latent classes; and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. Among 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their FCs, growth mixture modeling (GMM) was used to identify latent classes of individuals based on General Sleep Disturbance Scale (GSDS) obtained prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in candidate cytokine genes were interrogated for differences between the two latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on GSDS group membership. Two latent classes were identified: lower sleep disturbance (88.5%) and higher sleep disturbance (11.5%). Participants who were younger and had a lower Karnofsky Performance status score were more likely to be in the higher sleep disturbance class. Variation in two cytokine genes (i.e., IL6, NFKB) predicted latent class membership. Evidence was found for latent classes with distinct sleep disturbance trajectories. Unique genetic markers in cytokine genes may partially explain the interindividual heterogeneity characterizing these trajectories.

Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages

Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged as a leading cause of antibiotic associated diarrheal disease in humans. The genetic basis of the pathogenicity of C. difficile remains poorly understood. In this study we aimed at characterizing the genetic diversity of C. difficile strains by three different methods (PCR ribotyping, multilocus sequence typing and genetic markers) to improve the typing of C. difficile. Our study was performed on a reference collection (Leeds–Leiden/ECDC) of C. difficile PCR ribotype (RT) strains (n=70) expanded with six PCR RT strains highly related to the emerging PCR RTs 027 and 078. Besides PCR ribotyping we used multilocus sequence typing (MLST) using seven housekeeping genes (MLST 7HG) that has recently been developed for characterizing C. difficile isolates as well as analysis of unique genetic markers. Evolutionary relatedness of the sequences determined by MLST 7HG was analyzed in phylogenetic analysis. In total 56 MLST 7HG sequence types (STs) were identified, nine of which were new. Phylogeny reconstruction of the reference set of strains supplemented with the online available C. difficile MLST reference database, revealed six monophyletic lineages of closely related STs. ST-122 (PCR RT131) formed a well-separated branch in the tree and was thus designated as a novel lineage. Furthermore, we confirmed that several PCR RTs are highly related to the emerging PCR RTs 027 and 078 since these types display the same STs (ST-1 and ST-11, respectively). Based on the observed results, we conclude that MLST 7HG is a valuable method to study C. difficile phylogeny.

A re-consideration of the taxonomic status of Nebria lacustris Casey (Coleoptera, Carabidae, Nebriini) based on multiple datasets – a single species or a species complex?

This study gathered evidence from principal component analysis (PCA) of morphometric data and molecular analyses of nucleotide sequence data for four nuclear genes (28S, TpI, CAD1, and Wg) and two mitochondrial genes (COI and 16S), using parsimony, maximum likelihood, and Bayesian methods. This evidence was combined with morphological and chorological data to re-evaluate the taxonomic status of Nebria lacustris Casey sensu lato. PCA demonstrated that both body size and one conspicuous aspect of pronotal shape vary simultaneously with elevation, latitude, and longitude and served to distinguish populations from the southern Appalachian highlands, south of the French Broad, from all other populations. Molecular analyses revealed surprisingly low overall genetic diversity within Nebria lacustris sensu lato, with only 0.39% of 4605 bp varied in the concatenated dataset. Evaluation of patterns observed in morphological and genetic variation and distribution led to the following taxonomic conclusions: (1) Nebria lacustris Casey and Nebria bellorum Kavanaugh should be considered distinct species, which is a NEW STATUS for Nebria bellorum. (2) No other distinct taxonomic subunits could be distinguished with the evidence at hand, but samples from northeastern Iowa, in part of the region known as the “Driftless Zone”, have unique genetic markers for two genes that hint at descent from a local population surviving at least the last glacial advance. (3) No morphometric or molecular evidence supports taxonomic distinction between lowland populations on the shores of Lake Champlain and upland populations in the adjacent Green Mountains of Vermont, despite evident size and pronotal shape differences between many of their members.

Diagnostic and therapeutic management in patients with hypereosinophilic syndromes

The hypereosinophilic syndromes (HES) are rare disorders characterized by sustained, nonreactive hypereosinophilia with eosinophilia-associated organ damage/dysfunction. The most frequent clinical manifestations include skin abnormalities, cardiac failure, and neurological deficits, but disease presentations differ between patients and every organ may be affected. HES patients are currently categorized according to 2 classifications: World Health Organization 2008 and Working Classification 2006, but both have several limitations in daily practice. Despite advances in our understanding of HES pathogenesis, more than 50% of patients are still diagnosed with idiopathic disease, while the remaining subset has myeloproliferative (M-HES) or lymphocytic (L-HES) variants. In 10% to 20% of patients with M-HES, a unique genetic marker, fip1-like1/platelet-derived growth factor receptor α (FIP1L1-PDGFRA), was identified. It has dramatically changed disease management since imatinib, a tyrosine kinase inhibitor, appeared to be highly effective in these patients with up to 100% of long-term hematological response. L-HES is associated with abnormal T-cell populations secreting excessive amounts of eosinophilopoietic cytokines, mainly interleukin 5 (IL-5). Recently, encouraging results of treatment with monoclonal antibody neutralizing IL-5, mepolizumab, have been published. Corticosteroids remain the first-line therapeutic option for patients who do not have FIP1L1-PDGFRA fusion transcript, but treatment discontinuation leads to the recurrence of eosinophilia. This review reflects the current state of knowledge on the pathogenesis and therapy of HES. The shortcomings of current definitions and classifications are also discussed.

The hypereosinophilic syndromes: current concepts and treatments

The hypereosinophilic syndromes (HES) encompass a spectrum of diseases that have increased blood eosinophils and tissue damage in common. The clinical manifestations are protean and may involve any organ system, but especially the skin. Our understanding of these diseases has drastically changed over the past 15 years, along with new classifications that characterize patients with marked eosinophilia. One HES variant, myeloproliferative, is actually chronic eosinophilic leukaemia with a unique genetic marker, FIP1L1-PDGFRA. Such patients are well-controlled by administration of the kinase inhibitor, imatinib, and remissions appear durable with continued imatinib therapy. FIP1L1-PDGFRA is expressed in several cell lineages, thus explaining increases in neutrophils and mast cells in HES. The lymphocytic HES variant is associated with T-cell clones producing interleukin-5 (IL-5) and can evolve into lymphoma. While myeloproliferative and lymphocytic HES are well established and permit elimination of the term, idiopathic, to these varieties, most HES patients do not fall into these categories and are classified as complex (using the 2006 Workshop Report). A recent study showed that a monoclonal antibody to IL-5, mepolizumab, reduced glucocorticoid therapy in HES patients who did not possess the FIP1L1-PDGFRA mutation while controlling eosinophilia and preventing recurrence or progression of tissue damage. These advances augur well for continued progress in the understanding and treatment of HES.

Pollinator visitation patterns strongly influence among-flower variation in selfing rate

Adjacent flowers on Mimulus ringens floral displays often vary markedly in selfing rate. We hypothesized that this fine-scale variation in mating system reflects the tendency of bumble-bee pollinators to probe several flowers consecutively on multiflower displays. When a pollinator approaches a display, the first flower probed is likely to receive substantial outcross pollen. However, since pollen carryover in this species is limited, receipt of self pollen should increase rapidly for later flowers. Here the first direct experimental test of this hypothesis is described. In order to link floral visitation sequences with selfing rates of individual flowers, replicate linear arrays were established, each composed of plants with unique genetic markers. This facilitated unambiguous assignment of paternity to all sampled progeny. A single wild bumble-bee was permitted to forage on each linear array, recording the order of floral visits on each display. Once fruits had matured, 120 fruits were harvested (four flowers from each of five floral displays in each of six arrays). Twenty-five seedlings from each fruit were genotyped and paternity was unambiguously assigned to all 3000 genotyped progeny. The order of pollinator probes on Mimulus floral displays strongly and significantly influenced selfing rates of individual fruits. Mean selfing rates increased from 21 % for initial probes to 78 % for the fourth flower probed on each display. Striking among-flower differences in selfing rate result from increased deposition of geitonogamous (among-flower, within-display) self pollen as bumble-bees probe consecutive flowers on each floral display. The resulting heterogeneity in the genetic composition of sibships may influence seedling competition and the expression of inbreeding depression.

“Escapes” of Eggs from Farmed Cod Spawning in Net Pens: Recruitment to Wild Stocks

Escapes of farmed fish from aquaculture installations, as well as large scale stock enhancement and sea ranching activities, are regarded as risk factors for negative genetic impacts on native gene pools. These aspects have been discussed for several decades, and a number of recent experiments on Atlantic salmon have clearly demonstrated dramatically reduced overall fitness of farmed offspring compared with offspring of wild origin. The Atlantic cod (Gadus morhua L.) is now the most promising new marine species for large scale farming in Norway, and more than 500 commercial farming licenses have been issued, corresponding to a maximum annual production capacity of about 300,000 tonnes. In comparison with the environmental problems caused by escapes from the salmon farming industry, cod farming presents new challenges. As a marine species, cod are able to spawn in the net pens during the ongrowing period and thus release genetic material into the environment. Coastal cod stocks in Norwegian waters are depleted and therefore may be particularly vulnerable to genetic interaction with farmed cod. Thus, there is an urgent need for more detailed knowledge of potential hazards. A pilot experiment performed in Heimarkspollen in Austevoll (a practically landlocked fjord) demonstrated for the first time that farmed cod are capable of producing viable cod larvae that mix with larvae from wild cod. The farmed cod used in the pilot experiment were homozygotic for a rare allele (GPI-1*30), and mature fish were placed in a net pen to allow spawning and egg drift into the surrounding water to take place. Surveys of eggs found the highest densities in the vicinity of the net pen. Later larval surveys found that 20% to 25% of the larvae sampled in the area possessed the unique genetic marker and could therefore be traced to the spawning cod in the net cage. Numerous genetically marked larvae were also found outside Heimarkspollen, indicating a high degree of dispersal of eggs and larvae by tidal advection.

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: Morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8–16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.

Distribution of Epidemic Clonal Genetic Markers among Listeria monocytogenes 4b Isolates

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a −127 G→A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.