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Sample records for lato mediante pcr

  1. Detection of tick blood parasites in Egypt using PCR assay II- Borrelia burgdorferi sensu lato.

    PubMed

    Adham, Fatma K; El-Samie-Abd, Emtithal M; Gabre, Refaat M; El Hussein, Hala

    2010-12-01

    The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent of Lyme borrelosis (LB), was determined for the first time in Egypt by using polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28% of examined soft and hard ticks. High infection rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft ticks Ornithodoros savignyi. Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt, where the infection rate was between 0.0-50.0%. Sequence analysis of PCR product of Borrelia burgdorferi sensu lato shares high degree of similarity in sequence compared to similar species in GenBank.

  2. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

    PubMed

    Tavares, Eliandro Reis; Azevedo, Caroline Souza; Panagio, Luciano Aparecido; Pelisson, Marsileni; Pinge-Filho, Phileno; Venancio, Emerson José; Barros, Tânia Fraga; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2016-01-01

    In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

  3. Real-time PCR-based identification of Borrelia burgdorferi sensu lato species in ticks collected from humans in Romania.

    PubMed

    Briciu, Violeta T; Meyer, Fabian; Sebah, Daniela; Tăţulescu, Doina F; Coroiu, Georgiana; Lupşe, Mihaela; Carstina, Dumitru; Mihalca, Andrei D; Hizo-Teufel, Cecilia; Klier, Christiane; Huber, Ingrid; Fingerle, Volker

    2014-09-01

    The aims of our study were to determine (i) which tick species bite humans in Romania and (ii) the prevalence of Borrelia (B.) burgdorferi genospecies in these ticks. All ticks collected from patients who presented to the Clinic of Infectious Diseases Cluj Napoca in spring/summer 2010 were morphologically identified by an entomologist and tested for B. burgdorferi genospecies prevalence by a real-time PCR assay targeting the hbb gene and melting curve analysis. Out of 532 ticks, 518 were Ixodes ricinus, 10 Dermacentor marginatus, and 3 Haemaphysalis spp. ticks, and one unidentified tick due to destruction. Since evaluation of the hbb PCR revealed that it was not possible to differentiate between B. spielmanii/B. valaisiana and B. garinii/B. bavariensis, sequencing of an 800-bp fragment of the ospA gene was performed in these cases. Out of 389 investigated ticks, 43 were positive by hbb PCR for B. burgdorferi sensu lato. The positive samples were 42 Ixodes ricinus (11.1% B. burgdorferi sensu lato prevalence) and the one unidentified tick. Species identification revealed the presence of mainly B. afzelii, but also of B. garinii, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae. In 4 samples, differentiation between B. spielmanii/B. valaisiana was impossible. Our study shows that the most relevant human pathogenic B. burgdorferi genospecies - predominantly B. afzelii - are present in ticks collected from Romanian patients.

  4. Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of patients with clinical symptoms of Lyme borreliosis.

    PubMed

    Santino, Iolanda; Berlutti, Francesca; Pantanella, Fabrizio; Sessa, Rosa; del Piano, Massimo

    2008-06-01

    Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies complex Borrelia burgdorferi sensu lato (s.l.) transmitted by Ixodes ticks. At present, serology remains the main diagnostic tool for laboratory diagnosis of Lyme borreliosis. Recently, the PCR technique has been applied for diagnosis of B. burgdorferi s.l., but, until now, a reliable, easy-to-perform and sensitive method has not been described. Here we present a new PCR-based method for the detection of both B. burgdorferi s.l. and Borrelia genospecies DNAs in serum samples collected from patients showing Lyme disease symptoms. Of 265 serum samples of patients included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were negative for antibodies against B. burgdorferi by enzyme-linked immunosorbent assay and Western blotting. The B. burgdorferi s.l. 16S rRNA gene was detected by PCR in all serum-positive and in two borderline samples. None of the serum-negative samples nor serum samples collected from healthy subjects gave positive PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for Borrelia afzelii, 18% for Borrelia garinii and 23% for two Borrelia species. Two samples (9%) were not identified to species level. The new protocol could be considered to be reliable as neither false-positive nor false-negative reactions were recorded, and to be sensitive as it detects DNA from one bacterial cell.

  5. PCR Detection of Borrelia burgdorferi Sensu Lato, Tick-Borne Encephalitis Virus, and the Human Granulocytic Ehrlichiosis Agent in Ixodes persulcatus Ticks from Western Siberia, Russia

    PubMed Central

    Morozova, Olga V.; Dobrotvorsky, Andrey K.; Livanova, Natalya N.; Tkachev, Sergey E.; Bakhvalova, Valentina N.; Beklemishev, Anatoly B.; Cabello, Felipe C.

    2002-01-01

    PCR assays were used to test adult Ixodes persulcatus ticks from Western Siberia, Russia, for Borrelia burgdorferi sensu lato, tick-borne encephalitis virus (TBEV), and the human granulocytic ehrlichiosis (HGE) agent. Of the 150 ticks that were studied, 38% were infected with B. burgdorferi, 46% were infected with TBEV, and 8% were infected with the HGE agent. These three pathogens were distributed in the ticks independently of one another. PMID:12354885

  6. Immunohistochemistry and real-time PCR as diagnostic tools for detection of Borrelia burgdorferi sensu lato in ticks collected from humans.

    PubMed

    Briciu, Violeta T; Sebah, Daniela; Coroiu, Georgiana; Lupşe, Mihaela; Cârstina, Dumitru; Ţăţulescu, Doina F; Mihalca, Andrei D; Gherman, Călin M; Leucuţa, Daniel; Meyer, Fabian; Hizo-Teufel, Cecilia; Fingerle, Volker; Huber, Ingrid

    2016-05-01

    The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent.

  7. First Detection of Borrelia burgdorferi sensu lato DNA in Serum of the Wild Boar (Sus scrofa) in Northern Portugal by Nested-PCR.

    PubMed

    Faria, Ana S; Paiva-Cardoso, Maria das Neves; Nunes, Mónica; Carreira, Teresa; Vale-Gonçalves, Hélia M; Veloso, Octávia; Coelho, Catarina; Cabral, João A; Vieira-Pinto, Madalena; Vieira, Maria L

    2015-03-01

    Lyme borreliosis is the most common tick-borne zoonosis in the northern hemisphere. Several vertebrates are crucial in the epidemiological cycle of Borrelia burgdorferi sensu lato, but the role of wild boar as a reservoir is still unknown. Sera were collected from 90 wild boars shot in the Trás-os-Montes region, Northern Portugal (hunting season 2011/2012). In this study, Borrelia DNA was detected for the first time by nested-PCR in three different sera, suggesting that the wild boar may be a potential reservoir for this spirochete. Sequencing results show 100% similarity with Borrelia afzelii. Further studies are needed to evaluate the public health risks associated with boar hunting.

  8. Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

    PubMed

    Krak, Karol; Janoušková, Martina; Caklová, Petra; Vosátka, Miroslav; Štorchová, Helena

    2012-05-01

    Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

  9. Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR

    PubMed Central

    2010-01-01

    Background Borrelia burgdorferi sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan™ minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs. Results Quantitative real time PCR (qPCR) identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii. Conclusions The evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick

  10. Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

    PubMed Central

    Souza, Ana Carolina R.; Ferreira, Renata C.; Gonçalves, Sarah S.; Quindós, Guillermo; Eraso, Elena; Bizerra, Fernando C.; Briones, Marcelo R. S.

    2012-01-01

    Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species. PMID:22535986

  11. Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic.

    PubMed

    Derdáková, Markéta; Beati, Lorenza; Pet'ko, Branislav; Stanko, Michal; Fish, Durland

    2003-01-01

    In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

  12. Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic

    PubMed Central

    Derdáková, Markéta; Beati, Lorenza; Pet'ko, Branislav; Stanko, Michal; Fish, Durland

    2003-01-01

    In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ∼230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used. PMID:12514035

  13. Development and evaluation of a two-step multiplex TaqMan real-time PCR assay for detection/quantification of different genospecies of Borrelia burgdorferi sensu lato.

    PubMed

    Nunes, Mónica; Parreira, Ricardo; Carreira, Teresa; Inácio, João; Vieira, Maria Luísa

    2017-09-11

    Nowadays, at least four clinically important B. burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. garinii, B. burgdorferi sensu stricto (s.s.) and B. lusitaniae) circulate in Portugal. Each genospecies has a different tropism that resuls in a diverse array of clinical manifestations. The standard diagnostic procedure used is normally simple, nevertheless, during the "window-period" phase, in which specific antibodies cannot yet be detected, diagnosis becomes difficult, and calls for reliable, sensitive and specific laboratory methods, such as molecular tests. The aim of this study was to develop and evaluate a multiplex TaqMan real-time PCR assay to infer the presence of B. burgdorferi s.l. genospecies in clinical and vector-derived samples. The assay consists of two steps: (i) a first duplex real-time PCR targeting both flaB of B. burgdorferi s.l., and an internal control (18S rDNA for tick samples or the mammal β-actin gene for clinical samples); and (ii) a second tetraplex real-time PCR targeting the flaB gene of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae. The first step revealed a high specificity and sensitivity, allowing the detection of as low as 20 genome equivalents (GE) of B. burgdorferi s.l. from isolated cultures, clinical samples and ticks. The second step revealed high specificity, but a slightly lower sensitivity (2×10(2) GE) for detection of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae in purified DNA extracts, and particularly when testing cerebrospinal fluid (CSF) samples. Nonetheless, both real-time PCR protocols were developed to be applied at the beginning of the infection, to improve early diagnosis of Lyme borreliosis (LB), where detection of Borrelia should not rely on the use of CSF samples. The assay here described is of special interest for the analysis of both environmental and clinical samples, being advantageous in the former phase screening of Lyme borreliosis, when the efficiency of

  14. Molecular detection of Borrelia burgdorferi sensu lato - An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories.

    PubMed

    Lager, Malin; Faller, Maximilian; Wilhelmsson, Peter; Kjelland, Vivian; Andreassen, Åshild; Dargis, Rimtas; Quarsten, Hanne; Dessau, Ram; Fingerle, Volker; Margos, Gabriele; Noraas, Sølvi; Ornstein, Katharina; Petersson, Ann-Cathrine; Matussek, Andreas; Lindgren, Per-Eric; Henningsson, Anna J

    2017-01-01

    Lyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient´s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement. The purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark. Each participating laboratory was asked to analyse three different sets of samples (reference panels; all blinded) i) cDNA extracted and transcribed from water spiked with cultured Borrelia strains, ii) cerebrospinal fluid spiked with cultured Borrelia strains, and iii) DNA dilution series extracted from cultured Borrelia and relapsing fever strains. The results and the method descriptions of each laboratory were systematically evaluated. The analytical sensitivities and the concordance between the eight protocols were in general high. The concordance was especially high between the protocols using 16S rRNA as the target gene, however, this concordance was mainly related to cDNA as the type of template. When comparing cDNA and DNA as the type of template the analytical sensitivity was in general higher for the protocols using DNA as template regardless of the use of target gene. The analytical specificity for all eight protocols was high. However, some protocols were not able to detect Borrelia spielmanii, Borrelia lusitaniae or Borrelia japonica.

  15. Molecular Identification and Analysis of Borrelia burgdorferi Sensu Lato in Lizards in the Southeastern United States

    PubMed Central

    Clark, Kerry; Hendricks, Amanda; Burge, David

    2005-01-01

    Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States. PMID:15870353

  16. Detection of Lyme disease spirochetes in the skin of naturally infected wild sika deer (Cervus nippon yesoensis) by PCR.

    PubMed Central

    Kimura, K; Isogai, E; Isogai, H; Kamewaka, Y; Nishikawa, T; Ishii, N; Fujii, N

    1995-01-01

    We demonstrated the presence of Borrelia burgdorferi sensu lato DNA in the skin tissues of naturally infected wild sika deer, using PCR. The risk of transmission of B. burgdorferi sensu lato is recognized in sika deer. PMID:7747979

  17. Distinct Combinations of Borrelia burgdorferi Sensu Lato Genospecies Found in Individual Questing Ticks from Europe

    PubMed Central

    Kurtenbach, Klaus; De Michelis, Simona; Sewell, Henna-Sisko; Etti, Susanne; Schäfer, Stefanie M.; Hails, Rosie; Collares-Pereira, Margarida; Santos-Reis, Margarida; Haninçová, Klára; Labuda, Milan; Bormane, Antra; Donaghy, Michael

    2001-01-01

    The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis. PMID:11571205

  18. Analysis of the bmp Gene Family in Borrelia burgdorferi Sensu Lato

    PubMed Central

    Gorbacheva, Victoria Y.; Godfrey, Henry P.; Cabello, Felipe C.

    2000-01-01

    BmpA, BmpB, BmpC, and BmpD are homologous Borrelia burgdorferi lipoproteins of unknown functions, encoded by the bmp genes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of the bmp region has been characterized for a variety of B. burgdorferi sensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All four bmp genes were present in the same relative order in all B. burgdorferi sensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of the bmp genes in these species, variations in DNA sequence in the bmpD-bmpC and bmpC-bmpA intergenic regions were significantly more common than in the corresponding 3′ bmpD and bmpC coding regions. The genetic structure of the chromosomal region containing the bmp genes thus appears to be well conserved across different species of B. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest that bmp gene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced by B. burgdorferi sensu lato. PMID:10715014

  19. The Phylogeny of Bacillus cereus sensu lato.

    PubMed

    Okinaka, Richard T; Keim, Paul

    2016-02-01

    The three main species of the Bacillus cereus sensu lato, B. cereus, B. thuringiensis, and B. anthracis, were recognized and established by the early 1900 s because they each exhibited distinct phenotypic traits. B. thuringiensis isolates and their parasporal crystal proteins have long been established as a natural pesticide and insect pathogen. B. anthracis, the etiological agent for anthrax, was used by Robert Koch in the 19th century as a model to develop the germ theory of disease, and B. cereus, a common soil organism, is also an occasional opportunistic pathogen of humans. In addition to these three historical species designations, are three less-recognized and -understood species: B. mycoides, B. weihenstephanensis, and B. pseudomycoides. All of these "species" combined comprise the Bacillus cereus sensu lato group. Despite these apparently clear phenotypic definitions, early molecular approaches to separate the first three by various DNA hybridization and 16S/23S ribosomal sequence analyses led to some "confusion" because there were limited differences to differentiate between these species. These and other results have led to frequent suggestions that a taxonomic change was warranted to reclassify this group to a single species. But the pathogenic properties of B. anthracis and the biopesticide applications of B. thuringiensis appear to "have outweighed pure taxonomic considerations" and the separate species categories are still being maintained. B. cereus sensu lato represents a classic example of a now common bacterial species taxonomic quandary.

  20. Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil.

    PubMed

    Monteiro, Danieli Urach; Botton, Sônia de Avila; Tonin, Alexandre Alberto; Haag, Karen Luisa; Musskopf, Germano; Azevedo, Maria Isabel; Weiblen, Carla; Ribeiro, Tatiana Correa; de la Rue, Mário Luiz

    2015-01-01

    This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

  1. Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications

    PubMed Central

    Wang, Guiqing; van Dam, Alje P.; Schwartz, Ira; Dankert, Jacob

    1999-01-01

    Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especiallly correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail. PMID:10515907

  2. Genetic Diversity of Borrelia burgdorferi Sensu Lato in Ticks from Mainland Portugal

    PubMed Central

    De Michelis, Simona; Sewell, Henna-Sisko; Collares-Pereira, Margarida; Santos-Reis, Margarida; Schouls, Leo M.; Benes, Vladimir; Holmes, Edward C.; Kurtenbach, Klaus

    2000-01-01

    To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin. PMID:10834965

  3. Molecular Evidence of Coinfection of Ticks with Borrelia burgdorferi Sensu Lato and the Human Granulocytic Ehrlichiosis Agent in Switzerland

    PubMed Central

    Leutenegger, Christian M.; Pusterla, Nicola; Mislin, Caroline N.; Weber, Rainer; Lutz, Hans

    1999-01-01

    Adult Ixodes ricinus ticks were collected in Switzerland and tested for the presence of coinfection with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for B. burgdorferi and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for B. burgdorferi. PMID:10488215

  4. Assessment of two new molecular methods for identification of Candida parapsilosis sensu lato species.

    PubMed

    Garcia-Effron, Guillermo; Canton, Emilia; Pemán, Javier; Dilger, Amanda; Romá, Eva; Perlin, David S

    2011-09-01

    Candida parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis replaced C. parapsilosis groups I, II, and III in 2005. Since then, an increased interest in studying their epidemiology has arisen based on the observed differences in antifungal susceptibilities and virulence the three species. A strict differentiation of these species cannot be achieved by phenotypic methods. We evaluate two new molecular methodologies to differentiate among these species by the use of a collection of 293 bloodstream infection isolates of C. parapsilosis sensu lato. For the first method, the isolates were studied using PCR amplification of a fragment of the C. parapsilosis sensu lato FKS1 gene and a universal primer pair followed by EcoRI enzyme digestion. The other method used the allele discrimination ability of molecular beacons in a multiplex real-time PCR format. Both methods of identification showed 100% concordance with internal transcribed spacer 1 (ITS1)/ITS2 sequencing and proved to be effective for clinical applications, even with mixed-species DNAs.

  5. An alternative method for the analysis of melanin production in Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato.

    PubMed

    Brilhante, Raimunda S N; España, Jaime D A; de Alencar, Lucas P; Pereira, Vandbergue S; Castelo-Branco, Débora de S C M; Pereira-Neto, Waldemiro de A; Cordeiro, Rossana de A; Sidrim, José J C; Rocha, Marcos F G

    2017-10-01

    Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC 22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L-DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using grayscale images. All Cryptococcus spp. strains produced melanin after growth on minimal medium agar containing L-DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L-DOPA, while H. werneckii presented the strongest pigmentation. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production. © 2017 Blackwell Verlag GmbH.

  6. Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA.

    PubMed

    Clark, Kerry L; Leydet, Brian F; Threlkeld, Clifford

    2014-05-01

    The present study investigated the cause of illness in human patients primarily in the southern USA with suspected Lyme disease based on erythema migrans-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. The study also included some patients from other states throughout the USA. Several PCR assays specific for either members of the genus Borrelia or only for Lyme group Borrelia spp. (Borrelia burgdorferi sensu lato), and DNA sequence analysis, were used to identify Borrelia spp. DNA in blood and skin biopsy samples from human patients. B. burgdorferi sensu lato DNA was found in both blood and skin biopsy samples from patients residing in the southern states and elsewhere in the USA, but no evidence of DNA from other Borrelia spp. was detected. Based on phylogenetic analysis of partial flagellin (flaB) gene sequences, strains that clustered separately with B. burgdorferi sensu stricto, Borrelia americana or Borrelia andersonii were associated with Lyme disease-like signs and symptoms in patients from the southern states, as well as from some other areas of the country. Strains most similar to B. burgdorferi sensu stricto and B. americana were found most commonly and appeared to be widely distributed among patients residing throughout the USA. The study findings suggest that human cases of Lyme disease in the southern USA may be more common than previously recognized and may also be caused by more than one species of B. burgdorferi sensu lato. This study provides further evidence that B. burgdorferi sensu stricto is not the only species associated with signs and/or symptoms consistent with Lyme borreliosis in the USA.

  7. Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds.

    PubMed Central

    Olsén, B; Jaenson, T G; Bergström, S

    1995-01-01

    The prevalence of Lyme disease Borrelia-infected ticks on migrating birds was studied in Scandinavia. A total of 22,998 birds were caught at eight different bird observatories and examined for ticks. Five different species of ticks were found infesting the birds. The dominant species, Ixodesricinus, constituted 98.3% of the ticks collected. The presence of spirochetes was determined by an immunofluorescence assay of tick larvae and DNA amplification by PCR on all ticks. To determine which Borrelia burgdorferi sensu lato species were present, a species classification was performed by DNA amplification with species-specific 16S rDNA primers and by DNA sequencing (rDNA is DNA coding for rRNA). Flagellin gene sequences of all species of B. burgdorferi sensu lato previously recorded in Europe were observed. Borrelia garinii was the most prevalent Lyme disease Borrelia species in ticks collected from birds arriving from the South or Southeast in the spring, whereas the distribution was more heterogeneous in ticks from birds migrating from the Southwest. These data support the notion that birds are partly responsible for the heterogeneous distribution of Lyme disease Borrelia spirochetes in Europe. PMID:7487041

  8. First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri.

    PubMed

    Oliver, J H; Kollars, T M; Chandler, F W; James, A M; Masters, E J; Lane, R S; Huey, L O

    1998-01-01

    Five Borrelia burgdorferi sensu lato isolates from Missouri are described. This represents the first report and characterization of such isolates from that state. The isolates were obtained from either Ixodes dentatus or Amblyomma americanum ticks that had been feeding on cottontail rabbits (Sylvilagus floridanus) from a farm in Bollinger County, Mo., where a human case of Lyme disease had been reported. All isolates were screened immunologically by indirect immunofluorescence by using monoclonal antibodies to B. burgdorferi-specific outer surface protein A (OspA) (antibodies H3TS and H5332), B. burgdorferi-specific OspB (antibody H6831), Borrelia (genus)-specific antiflagellin (antibody H9724), and Borrelia hermsii-specific antibody (antibody H9826). Analysis of the isolates also involved a comparison of their protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the isolates were analyzed by PCR with six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain B. burgdorferi B-31. Although some genetic variability was detected among the five isolates as well as between them and the B-31 strain, enough similarities were found to classify them as B. burgdorferi sensu lato.

  9. Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark.

    PubMed

    Hagen, Ferry; Hare Jensen, Rasmus; Meis, Jacques F; Arendrup, Maiken Cavling

    2016-09-01

    Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC50 of 4-8 mg l(-1) except for C. curvatus (MICs >32 mg l(-1) ). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l(-1) ), voriconazole (≥0.5 mg l(-1) ) and isavuconazole (0.06 and 0.25 mg l(-1) respectively) were elevated compared to the wild-type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l(-1) ). Antifungal susceptibility revealed species-specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.

  10. Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

    PubMed

    Krause, Peter J; Narasimhan, Sukanya; Wormser, Gary P; Barbour, Alan G; Platonov, Alexander E; Brancato, Janna; Lepore, Timothy; Dardick, Kenneth; Mamula, Mark; Rollend, Lindsay; Steeves, Tanner K; Diuk-Wasser, Maria; Usmani-Brown, Sahar; Williamson, Phillip; Sarksyan, Denis S; Fikrig, Erol; Fish, Durland

    2014-07-01

    Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease-endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato-infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991-2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from ≈10% of B. miyamotoi sensu lato-seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our results suggest that human B. miyamotoi sensu lato infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.

  11. A new taxonomic classification for species in Gomphus sensu lato

    Treesearch

    Admir J. Giachini; Michael A. Castellano

    2011-01-01

    Taxonomy of the Gomphales has been revisited by combining morphology and molecular data (DNA sequences) to provide a natural classification for the species of Gomphus sensu lato. Results indicate Gomphus s.l. to be non-monophyletic, leading to new combinations and the placement of its species into four genera: Gomphus...

  12. Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from southern Poland.

    PubMed

    Strzelczyk, Joanna K; Gaździcka, Jadwiga; Cuber, Piotr; Asman, Marek; Trapp, Gizela; Gołąbek, Karolina; Zalewska-Ziob, Marzena; Nowak-Chmura, Magdalena; Siuda, Krzysztof; Wiczkowski, Andrzej; Solarz, Krzysztof

    2015-12-01

    In 2008-2011 ticks were collected from southern Poland. Out of 6336 individuals collected and identified as Ixodes ricinus, 768 (2 larvae, 84 nymphs, 417 females, 265 males) were included in molecular study. The aim of this study was to investigate the prevalence and types of genospecies of Borrelia burgdorferi sensu lato in ticks. The polymerase chain reaction (PCR) was applied to detect the presence of pathogens in ticks. Subsequently the amplified DNA was digested with TasI enzyme. The infection rate was 15% (116) of examined ticks. PCR-RFLP analysis allowed distinguishing three genospecies of B. burgdorferi s.l.: B. burgdorferi sensu stricto, B. afzelii, and B. garinii. RFLP analyses of 116 positive samples revealed 96 (83%) monoinfections and 13 (11%) coinfections, whereas unidentified genospecies were present in 7 (6%) of positive samples. In the case of monoinfections, B. burgdorferi s.s. was the predominant species of pathogen in infected ticks - 61.4%. Other genospecies: B. garinii and B. afzelii were detected in 22.9% and 15.6% of the samples, respectively. To sum up, 15 % of ticks were infected by B. burgdorferi s.l which increases the risk of human infections in the recreational areas of southern Poland. Furthermore, there is a need to increase public awareness and implement more preventive measures concerning Lyme disease.

  13. Isolation of Borrelia burgdorferi sensu lato from the skin of the European badger (Meles meles) in Switzerland.

    PubMed

    Gern, Lise; Sell, Katy

    2009-04-01

    No data are available on the role of badgers in the ecology of Lyme borreliosis spirochetes in Europe. In a recent study describing validation of a molecular method allowing host DNA identification and Borrelia burgdorferi sensu lato detection in Ixodes ricinus, the simultaneous presence of B. afzelii DNA and of European badger (Meles meles) DNA was detected in I. ricinus ticks in Switzerland. This suggested that badgers might be reservoir hosts for B. afzelii. Here, we present results obtained in a study on badgers conducted in 1996-1997. Thirty-one tissue samples (ear biopsy: n = 25, aspiration fluid: n = 6) from 8 badgers were placed in BSK medium to isolate B. burgdorferi sensu lato and were then examined by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Globally, six Borrelia isolates (6/31, 19.4%) were obtained from 3/8 (37.5%) badgers. These isolates were identified as B. afzelii (n = 3) and B. valaisiana (n = 3).

  14. Bacterial tick-borne diseases caused by Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, and Rickettsia spp. among patients with cataract surgery

    PubMed Central

    Chmielewski, Tomasz; Brydak-Godowska, Joanna; Fiecek, Beata; Rorot, Urszula; Sędrowicz, Elżbieta; Werenowska, Małgorzata; Kopacz, Dorota; Hevelke, Agata; Michniewicz, Magdalena; Kęcik, Dariusz; Tylewska-Wierzbanowska, Stanisława

    2014-01-01

    Background Clinical data have shown that tick-borne diseases caused by Borrelia burgdorferi sensu lato, Bartonella spp., Coxiella burnetii, and Rickettsia spp. can affect the central nervous system, including the eye. The aim of this study was to establish a relationship between the incidence of cataract and evidence of bacterial infections transmitted by ticks. Material/Methods Fluid with lenticular masses from inside of the eye and blood from 109 patients were tested by PCR and sequencing. Sera from patients and the control group were subjected to serological tests to search specific antibodies to the bacteria. Results Microbiological analysis revealed the presence of Bartonella sp. DNA in intraoperative specimens from the eye in 1.8% of patients. Serological studies have shown that infections caused by B. burgdorferi sensu lato and Bartonella sp. were detected in 34.8% and 4.6% of patients with cataract surgery, respectively. Conclusions Presence of DNA of yet uncultured and undescribed species of Bartonella in eye liquid indicates past infection with this pathogen. Specific antibodies to B. burgdorferi sensu lato and Bartonella sp. are detected more frequently in patients with cataract compared to the control group. This could indicate a possible role of these organisms in the pathological processes within the eyeball, leading to changes in the lens. Further studies are needed to identify Bartonella species, as well as to recognize the infectious mechanisms involved in cataract development. PMID:24902636

  15. Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in Southern Norway

    PubMed Central

    2010-01-01

    Background Borrelia burgdorferi sensu lato (s.l.) are the causative agent for Lyme borreliosis (LB), the most common tick-borne disease in the northern hemisphere. Birds are considered important in the global dispersal of ticks and tick-borne pathogens through their migration. The present study is the first description of B. burgdorferi prevalence and genotypes in Ixodes ricinus ticks feeding on birds during spring and autumn migration in Norway. Methods 6538 migratory birds were captured and examined for ticks at Lista Bird Observatory during the spring and the autumn migration in 2008. 822 immature I. ricinus ticks were collected from 215 infested birds. Ticks were investigated for infection with B. burgdorferi s.l. by real-time PCR amplification of the 16S rRNA gene, and B. burgdorferi s.l. were thereafter genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, or by direct sequencing of the PCR amplicon generated from the rrs (16S)-rrl (23S) intergenetic spacer. Results B. burgdorferi s.l. were detected in 4.4% of the ticks. The most prevalent B. burgdorferi genospecies identified were B. garinii (77.8%), followed by B.valaisiana (11.1%), B. afzelii (8.3%) and B. burgdorferi sensu stricto (2.8%). Conclusion Infection rate in ticks and genospecies composition were similar in spring and autumn migration, however, the prevalence of ticks on birds was higher during spring migration. The study supports the notion that birds are important in the dispersal of ticks, and that they may be partly responsible for the heterogeneous distribution of B. burgdorferi s.l. in Europe. PMID:21054890

  16. First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the Marbled polecat, Vormela peregusna and the European mink, Mustela lutreola (Mammalia: Mustelidae)

    PubMed Central

    2012-01-01

    Background Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease. Case presentation Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto. Conclusions This is the first report of the Lyme disease spirochetes in the two mentioned hosts. PMID:22901862

  17. Detection of Lyme Disease Bacterium, Borrelia burgdorferi sensu lato, in Blacklegged Ticks Collected in the Grand River Valley, Ontario, Canada.

    PubMed

    Scott, John D; Foley, Janet E; Anderson, John F; Clark, Kerry L; Durden, Lance A

    2017-01-01

    We document the presence of blacklegged ticks, Ixodes scapularis, in the Grand River valley, Centre Wellington, Ontario. Overall, 15 (36%) of 42 I. scapularis adults collected from 41 mammalian hosts (dogs, cats, humans) were positive for the Lyme disease bacterium, Borrelia burgdorferi sensu lato (s.l.). Using real-time PCR testing and DNA sequencing of the flagellin (fla) gene, we determined that Borrelia amplicons extracted from I. scapularis adults belonged to B. burgdorferi sensu stricto (s.s.), which is pathogenic to humans and certain domestic animals. Based on the distribution of I. scapularis adults within the river basin, it appears likely that migratory birds provide an annual influx of I. scapularis immatures during northward spring migration. Health-care providers need to be aware that local residents can present with Lyme disease symptoms anytime during the year.

  18. Detection of Lyme Disease Bacterium, Borrelia burgdorferi sensu lato, in Blacklegged Ticks Collected in the Grand River Valley, Ontario, Canada

    PubMed Central

    Scott, John D.; Foley, Janet E.; Anderson, John F.; Clark, Kerry L.; Durden, Lance A.

    2017-01-01

    We document the presence of blacklegged ticks, Ixodes scapularis, in the Grand River valley, Centre Wellington, Ontario. Overall, 15 (36%) of 42 I. scapularis adults collected from 41 mammalian hosts (dogs, cats, humans) were positive for the Lyme disease bacterium, Borrelia burgdorferi sensu lato (s.l.). Using real-time PCR testing and DNA sequencing of the flagellin (fla) gene, we determined that Borrelia amplicons extracted from I. scapularis adults belonged to B. burgdorferi sensu stricto (s.s.), which is pathogenic to humans and certain domestic animals. Based on the distribution of I. scapularis adults within the river basin, it appears likely that migratory birds provide an annual influx of I. scapularis immatures during northward spring migration. Health-care providers need to be aware that local residents can present with Lyme disease symptoms anytime during the year. PMID:28260991

  19. Tick vectors of Cercopithifilaria bainae in dogs: Rhipicephalus sanguineus sensu lato versus Ixodes ricinus.

    PubMed

    Ramos, Rafael Antonio Nascimento; Giannelli, Alessio; Brianti, Emanuele; Annoscia, Giada; Cantacessi, Cinzia; Dantas-Torres, Filipe; Otranto, Domenico

    2013-08-01

    Recently, dermal microfilariae of a Cercopithifilaria species (Spirurida, Onchocercidae), namely Cercopithifilaria bainae , were detected in dogs from several geographical areas of the Mediterranean basin. Evidence from both laboratory and field studies support the role of the brown dog tick, Rhipicephalus sanguineus sensu lato, as an intermediate host of this nematode. In the present study, we investigated the competence of Ixodes ricinus nymphs as vectors of C. bainae. On November 2012, fully engorged nymphs of I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) were collected from a dog infected by C. bainae. The presence of C. bainae in I. ricinus was assessed by both microscopic dissection of specimens and detection of nematode DNA (PCR), at days 3, 10, 20 and 30 (T1-T4) post-collection; due to the small number of specimens available, R. sanguineus s.l. were examined using the same methods at T4 only. No developing larva of C. bainae was detected in I. ricinus specimens at different time points (T1-T4), even if four of these specimens were PCR-positive at T1. Seven out of ten R. sanguineus s.l. were positive for C. bainae third-stage larvae (L3) at both microscopical and molecular analysis at T4. This study indicates that C. bainae does not develop in I. ricinus nymphs, which preclude the role of this tick as an intermediate host of this parasite. Data presented herein provide new insights into the biology of this filarioid species and will lead to a better understanding of the role of different tick species as vectors of nematodes.

  20. Borrelia burgdorferi sensu lato in Ixodes cf. neuquenensis and Ixodes sigelos ticks from the Patagonian region of Argentina.

    PubMed

    Sebastian, Patrick S; Bottero, Maria Noelia Saracho; Carvalho, Luis; Mackenstedt, Ute; Lareschi, Marcela; Venzal, José M; Nava, Santiago

    2016-10-01

    This study was conducted to detect Borrelia burgdorferi sensu lato infection in ixodid ticks from the Patagonia region in the south of Argentina. Therefore, ticks were collected on rodents in the provinces of Chubut, Río Negro and Santa Cruz. These ticks were identified as nymphs of Ixodes cf. neuquenensis and Ixodes sigelos. The B. burgdorferi s.l. infection was tested by a battery of PCR methods targeting the gene flagellin (fla) and the rrfA-rrlB intergenic spacer region (IGS). Three pools of I. sigelos nymphs from Chubut and Santa Cruz provinces as well as one pool of I. cf. neuquenensis nymphs from Río Negro province were tested positive in the fla-PCR. The samples of I. sigelos were also positive for the IGS-PCR. Phylogenetically, the haplotypes found in the positive ticks belong to the B. burgdorferi s.l. complex, and they were closely related to Borrelia chilensis, a genospecies isolated from Ixodes stilesi in Chile. The pathogenic relevance of the Borrelia genospecies detected in both I. neuquenensis and I. sigelos is unknown.

  1. A proposal for the classification of biological weapons sensu lato.

    PubMed

    Rozsa, Lajos

    2014-12-01

    Due to historical and legislation reasons, the category of bioweapons is rather poorly defined. Authors often disagree on involving or excluding agents like hormones, psychochemicals, certain plants and animals (such as weeds or pests) or synthetic organisms. Applying a wide definition apparently threatens by eroding the regime of international legislation, while narrow definitions abandon several important issues. Therefore, I propose a category of 'biological weapons sensu lato' (BWsl) that is defined here as any tool of human aggression whose acting principle is based on disciplines of biology including particularly microbiology, epidemiology, medical biology, physiology, psychology, pharmacology and ecology, but excluding those based on inorganic agents. Synthetically produced equivalents (not necessarily exact copies) and mock weapons are also included. This definition does not involve any claim to subject all these weapons to international legislation but serves a purely scholarly purpose. BWsl may be properly categorized on the base of the magnitude of the human population potentially targeted (4 levels: individuals, towns, countries, global) and the biological nature of the weapons' intended effects (4 levels: agricultural-ecological agents, and non-pathogenic, pathogenic, or lethal agents against humans).

  2. Lyme borreliosis caused by diverse genospecies of Borrelia burgdorferi sensu lato in northeastern China.

    PubMed

    Ni, X-B; Jia, N; Jiang, B-G; Sun, T; Zheng, Y-C; Huo, Q-B; Liu, K; Ma, L; Zhao, Q-M; Yang, H; Wang, X; Jiang, J-F; Cao, W-C

    2014-08-01

    The variety of Borrelia burgdorferi sensu lato (B. burgdorferi) genospecies leads to distinction in clinical manifestations of Lyme borreliosis (LB). There are reports of LB clinical characteristics in China, where the B. burgdorferi genospecies in ticks and animal hosts are different from those in Europe and North America. During May to September in 2010 and 2011, all patients who had erythema migrans (EM, more than 5 cm in diameter) after a recent tick-bite, and sought medical care at Mudanjiang Forestry Central Hospital, Heilongjiang Province of northeastern China, were enrolled in the study. Specific PCR was used to determine the B. burgdorferi genospecies in the disseminated patients. Of 265 EM patients, B. burgdorferi DNA was detected in blood specimens from 15 of 55 disseminated patients. Sequence analyses of 5S-23S rRNA, flagellin, ospC, 16S rRNA and ospA genes revealed that 11 patients were infected with Borrelia garinii, three with Borrelia afzelii and one with Borrelia valaisiana-related genospecies. Among 15 patients, 40%, 13.3% and 13.3% manifested pruritus, pain and ulceration, respectively. Systemic symptoms, arthralgia or a swollen joint and lymphadenopathy were observed in 26.7%, 13.3% and 6.7% patients, respectively. In northeastern China, three genospecies of LB patients were detected. The B. burgdorferi genospecies identified in this study was predominantly B. garinii. A case infected with B. valaisiana-related genospecies was reported for the first time.

  3. Borrelia burgdorferi sensu lato in Ixodes longiscutatus ticks from Brazilian Pampa.

    PubMed

    Dall'Agnol, Bruno; Michel, Thaís; Weck, Bárbara; Souza, Ugo Araújo; Webster, Anelise; Leal, Bruna Ferreira; Klafke, Guilherme Marcondes; Martins, João Ricardo; Ott, Ricardo; Venzal, José Manuel; Ferreira, Carlos Alexandre Sanchez; Reck, José

    2017-10-01

    Borrelia burgdorferi sensu lato (s.l.) complex includes the agents of Lyme disease/borreliosis in North America, Europe, and Asia, such Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, Borrelia bissettiae, and Borrelia mayonii. In 2013 B. burgdorferi s.l. was reported for the first time in the Neotropical region, from Ixodes aragaoi ticks in Uruguayan Pampa. In addition, from 2011 to 2016, 17 suspected human cases of borreliosis-like syndrome were reported in Rio Grande do Sul (RS) state, Brazil, which contains only part of country in the Pampa biome. The goal of this work is to report the results of a state surveillance program conducted in order to investigate the presence of B. burgdorferi s.l. in its classic vector, Ixodes spp. ticks, from the Brazilian Pampa. For this, we searched for Ixodes spp. ticks in 307 rodents from 11 municipalities of RS state. We then tested the ticks for the presence of B. burgdorferi s.l. DNA using PCR analysis. Of 35 Ixodes spp. ticks tested, one larva and one nymph of Ixodes longiscutatus ticks tested positive for Borrelia sp. DNA. The phylogenetic analysis of the flaB fragment grouped our samples (referred as Borrelia sp. haplotype Pampa) into B. burgdorferi s.l. group in a particular branch with other South American haplotypes, and this group was close to Borrelia carolinensis, B. bissettiae, and Borrelia californiensis. This is the first evidence of B. burgdorferi s.l. circulation in ticks of the genus Ixodes in Brazil. These results highlight the need for the implementation of public health policies for the diagnosis and prevention of potential cases of human borreliosis in Brazil. Further studies are needed to fill the gaps in our knowledge of the distribution, pathogenicity, reservoirs, and vectors of these emerging South American B. burgdorferi s.l. haplotypes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  4. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisher™ magnetic particle processor prior to genome sequencing

    NASA Astrophysics Data System (ADS)

    Mäkinen, Johanna; Marttila, Harri; Viljanen, Matti K.

    2001-01-01

    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher™ magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher™ magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification.

  5. Borrelia miyamotoi sensu lato Seroreactivity and Seroprevalence in the Northeastern United States

    PubMed Central

    Narasimhan, Sukanya; Wormser, Gary P.; Barbour, Alan G.; Platonov, Alexander E.; Brancato, Janna; Lepore, Timothy; Dardick, Kenneth; Mamula, Mark; Rollend, Lindsay; Steeves, Tanner K.; Diuk-Wasser, Maria; Usmani-Brown, Sahar; Williamson, Phillip; Sarksyan, Denis S.; Fikrig, Erol; Fish, Durland

    2014-01-01

    Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease�?"endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato�?"infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991�?"2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from �%^10% of B. miyamotoi sensu lato�?"seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our resultsA suggest thatA human B. miyamotoiA sensu latoA infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection. PMID:24960072

  6. Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

    USGS Publications Warehouse

    Zhioua, E.; Bouattour, A.; Hu, C.M.; Gharbi, M.; Aeschliman, A.; Ginsberg, H.S.; Gern, L.

    1999-01-01

    Free-living adult Ixodes ricinus L. were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.

  7. The Western progression of lyme disease: infectious and Nonclonal Borrelia burgdorferi Sensu Lato populations in Grand Forks County, North Dakota.

    PubMed

    Stone, Brandee L; Russart, Nathan M; Gaultney, Robert A; Floden, Angela M; Vaughan, Jefferson A; Brissette, Catherine A

    2015-01-01

    Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota.

  8. The Western Progression of Lyme Disease: Infectious and Nonclonal Borrelia burgdorferi Sensu Lato Populations in Grand Forks County, North Dakota

    PubMed Central

    Stone, Brandee L.; Russart, Nathan M.; Gaultney, Robert A.; Floden, Angela M.; Vaughan, Jefferson A.

    2014-01-01

    Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota. PMID:25304515

  9. Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy.

    PubMed

    Mannelli, Alessandro; Nebbia, Patrizia; Tramuta, Clara; Grego, Elena; Tomassone, Laura; Ainardi, Romina; Venturini, Lucia; De Meneghi, Daniele; Meneguz, Pier Giuseppe

    2005-03-01

    Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2-55.5) and mean number of ticks per host was 3.3 (1.6-7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8-85.8) and mean number of ticks per host was 6.9 (4.4-10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40-0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9-78.5) of larvae with engorgement ratio > or = 3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.

  10. First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae)

    USDA-ARS?s Scientific Manuscript database

    The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The indiscriminate use of chemicals for tick control exerts a strong selective pressure...

  11. A new species of Chaenusa Haliday sensu lato (Hymenoptera: Braconidae) from the Nearctic Region

    USDA-ARS?s Scientific Manuscript database

    Chaenusa psillosae Kula, new species from the Nearctic Region is described. Specimens upon which the new species is described were reared from an undetermined species of Hydrellia Robineau-Desvoidy infesting Sagittaria latifolia Willd. A key to the New World species of Chaenusa sensu lato is amended...

  12. Habitat-Specific Diversity of Borrelia burgdorferi Sensu Lato in Europe, Exemplified by Data from Latvia

    PubMed Central

    Etti, Susanne; Hails, Rosie; Schäfer, Stefanie M.; De Michelis, Simona; Sewell, Henna-Sisko; Bormane, Antra; Donaghy, Michael; Kurtenbach, Klaus

    2003-01-01

    The distribution of Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks from ecologically distinct habitats in Latvia was analyzed. A significant variation in the frequency of the genospecies across sites was observed, pointing to the importance of the host community in the ecology of Lyme borreliosis. PMID:12732580

  13. Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification.

    USDA-ARS?s Scientific Manuscript database

    Since 2002, severe leaf spotting on parsley (Petroselinum crispum L.) has occurred in Monterey County, California. One of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from seven distinct outbreaks and twice from the same outbreak (2002 and 2009). Frag...

  14. In vitro repellency of DEET against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum

    USDA-ARS?s Scientific Manuscript database

    Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repell...

  15. Evaluation of Borrelia real time PCR DNA targeting OspA, FlaB and 5S-23S IGS and Borrelia 16S rRNA RT-qPCR.

    PubMed

    de Leeuw, Bertie H C G M; Maraha, Boulos; Hollemans, Leonie; Sprong, Hein; Brandenburg, Afke H; Westenend, Pieter J; Kusters, Johannes G

    2014-12-01

    Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

  16. Use of FTA(®) card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

    PubMed

    Boué, Franck; El Berbri, Ikhlass; Hormaz, Vanessa; Boucher, Jean-Marc; El Mamy, Ahmed Bezeid; Traore, Abdallah; Fihri, Ouafaa Fassi; Petavy, Anne-Françoise; Dakkak, Allal; Umhang, Gérald

    2017-02-01

    Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA(®) card technology. The DNA samples were taken by applying the FTA(®) paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA(®) samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. First molecular evidence of [i]Borrelia burgdorferi[/i] sensu lato in goats, sheep, cattle and camels in Tunisia.

    PubMed

    Ben Said, Mourad; Belkahia, Hanène; Alberti, Alberto; Abdi, Khaoula; Zhioua, Manel; Daaloul-Jedidi, Monia; Messadi, Lilia

    2016-09-01

    Borrelia burgdorferi sensu lato (s.l.) are tick-transmitted spirochaetes of veterinary and human importance. Molecular epidemiology data on ruminants are still lacking in most countries of the world. Therefore, the aim of this study was to estimate the rate of B. burgdorferi s.l. infection in ruminants from Tunisia. A total of 1,021 ruminants (303 goats, 260 sheep, 232 cattle and 226 camels) from different bioclimatic areas in Tunisia were investigated for the presence of B. burgdorferi s.l. DNA in blood by real time PCR. Prevalence rates were 30.4% (92/303) in goats, 6.2% (16/260) in sheep, 1.3% (3/232) in cattle, and 1.8% (4/226) in camels. Only tick species belonging to Rhipicephalus and Hyalomma genera were found on the investigated animals. In small ruminants, the prevalence of B. burgdorferi s.l. varied significantly according to localities and farms. Goats located in humid areas were statistically more infected than those located in sub-humid areas. Prevalence rates varied significantly according to age and breed in sheep, and age and tick infestation in goats. This study provides the first insight into the presence of B. burgdorferi s.l. DNA in ruminants in Tunisia, and demonstrates that host species such as goats and sheep may play an important role in natural Lyme disease cycles in this country.

  18. Pan-genome and phylogeny of Bacillus cereus sensu lato.

    PubMed

    Bazinet, Adam L

    2017-08-02

    Bacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group of medical and agricultural significance. In this study, I use publicly available genomes and novel bioinformatic workflows to characterize the B. cereus s. l. pan-genome and perform the largest phylogenetic and population genetic analyses of this group to date in terms of the number of genes and taxa included. With these fundamental data in hand, I identify genes associated with particular phenotypic traits (i.e., "pan-GWAS" analysis), and quantify the degree to which taxa sharing common attributes are phylogenetically clustered. A rapid k-mer based approach (Mash) was used to create reduced representations of selected Bacillus genomes, and a fast distance-based phylogenetic analysis of this data (FastME) was performed to determine which species should be included in B. cereus s. l. The complete genomes of eight B. cereus s. l. species were annotated de novo with Prokka, and these annotations were used by Roary to produce the B. cereus s. l. pan-genome. Scoary was used to associate gene presence and absence patterns with various phenotypes. The orthologous protein sequence clusters produced by Roary were filtered and used to build HaMStR databases of gene models that were used in turn to construct phylogenetic data matrices. Phylogenetic analyses used RAxML, DendroPy, ClonalFrameML, PAUP*, and SplitsTree. Bayesian model-based population genetic analysis assigned taxa to clusters using hierBAPS. The genealogical sorting index was used to quantify the phylogenetic clustering of taxa sharing common attributes. The B. cereus s. l. pan-genome currently consists of ≈60,000 genes, ≈600 of which are "core" (common to at least 99% of taxa sampled). Pan-GWAS analysis revealed genes associated with phenotypes such as isolation source, oxygen requirement, and ability to cause diseases such as anthrax or food poisoning. Extensive phylogenetic analyses using an unprecedented amount of data

  19. Environmental Contamination by Echinococcus granulosus sensu lato Eggs in Relation to Slaughterhouses in Urban and Rural Areas in Tunisia

    PubMed Central

    Chaâbane-Banaoues, Raja; Oudni-M’rad, Myriam; M’rad, Selim; Mezhoud, Habib; Babba, Hamouda

    2016-01-01

    Hydatidosis has become a real concern for health care institutions and animal rearers in Tunisia. The Tunisian endemicity is aggravated by the growing number of dogs and the difficulty of getting rid of contaminated viscera because of the lack of equipment in most slaughterhouses. Therefore, microscopic and molecular tools were applied to evaluate the role of slaughterhouses in canine infection and Echinococcus granulosus sensu lato (s. l.) egg dissemination. Exposure risk to E. granulosus s. l. eggs in urban and rural areas was explored in order to implant preventive and adapted control strategies. Microscopic examinations detected taeniid eggs in 152 amongst 553 fecal samples. The copro-PCR demonstrated that 138 of 152 taeniid samples analyzed were positive for E. granulosus s. l. DNA. PCR-RFLP demonstrated that all isolated samples belonged to E. granulosus sensu stricto (s. s.). An important environmental contamination index (25.0%) by E. granulosus s. l. eggs was demonstrated. The average contamination index from the regions around slaughterhouses (23.3%; 95% CI: 17.7-28.9%) was in the same range as detected in areas located far from slaughterhouses (26.0%, 95% CI: 21.3-30.8%). Echinococcosis endemic areas were extended in both rural (29.9%, 95% CI: 24.8-34.9%) and urban locations (18.1%, 95% CI: 13.0-22.9%). The pathogen dissemination is related neither to the presence/absence of slaughterhouses nor to the location in urban or rural areas, but is probably influenced by human activities (home slaughtering) and behavior towards the infected viscera. PMID:26951990

  20. Molecular phylogeny of the megadiverse insect infraorder Bibionomorpha sensu lato (Diptera)

    PubMed Central

    Kaspřák, David; Mantič, Michal; Fitzgerald, Scott; Ševčíková, Tereza; Tóthová, Andrea; Jaschhof, Mathias

    2016-01-01

    The phylogeny of the insect infraorder Bibionomorpha (Diptera) is reconstructed based on the combined analysis of three nuclear (18S, 28S, CAD) and three mitochondrial (12S, 16S, COI) gene markers. All the analyses strongly support the monophyly of Bibionomorpha in both the narrow (sensu stricto) and the broader (sensu lato) concepts. The major lineages of Bibionomorpha sensu lato (Sciaroidea, Bibionoidea, Anisopodoidea, and Scatopsoidea) and most of the included families are supported as monophyletic groups. Axymyiidae was not found to be part of Bibionomorpha nor was it found to be its sister group. Bibionidae was paraphyletic with respect to Hesperinidae and Keroplatidae was paraphyletic with respect to Lygistorrhinidae. The included Sciaroidea incertae sedis (except Ohakunea Edwards) were found to belong to one clade, but the relationships within this group and its position within Sciaroidea require further study. PMID:27781163

  1. Rhipicephalus sanguineus sensu lato (Ixodidae) in synantropic rodents in Rio Grande do Sul, Brazil.

    PubMed

    Winkel, Kathleen Tavares; Ribeiro, Paulo Bretanha; Antunes, Lidiane Oliveira; Cárcamo, Marcial Corrêa; Vianna, Elvia Elena Silveira

    2014-01-01

    Rhipicephalus sanguineus, the brown dog tick, is responsible for maintaining and transmitting various pathogens, both in animals and human beings, and it is of great sanitary importance. This communication reports the first occurrence of Rhipicephalus sanguineus sensu lato parasitizing Rattus norvegicus in the state of Rio Grande do Sul, Brazil, and it is also the first record of this tick species parasitizing Rattus rattus in Brazil. The rodents were captured from the port area, located in the city of Pelotas, Rio Grande do Sul, Brazil. We collected 6 larvae of this tick species from 2 male R. rattus individuals, and 3 larvae from 2 female R. norvegicus individuals; parasitized specimens of both rodent species were captured from different sites within the experimental area. This record broadens the number of Rhipicephalus sanguineus sensu lato hosts in urban areas, indicating the need for continued monitoring on population density for both R. sanguineus and synanthropic rodents.

  2. Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group

    PubMed Central

    2012-01-01

    Background The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species. Results During the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence. Conclusions Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool. PMID:23088190

  3. Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

    SciTech Connect

    Casjens, S.R.; Dunn, J.; Fraser-Liggett, C. M.; Mongodin, E. F.; Qiu, W. G.; Luft, B. J.; Schutzer, S. E.

    2011-03-01

    Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named 'Borrelia finlandensis.'

  4. Molecular and Pathogenic Characterization of Borrelia burgdorferi Sensu Lato Isolates from Spain

    PubMed Central

    Escudero, Raquel; Barral, Marta; Pérez, Azucena; Vitutia, M. Mar; García-Pérez, Ana L.; Jiménez, Santos; Sellek, Ricela E.; Anda, Pedro

    2000-01-01

    Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic. PMID:11060064

  5. Fourier transform infrared spectroscopy of DNA from Borrelia burgdorferi sensu lato and Ixodes ricinus ticks

    NASA Astrophysics Data System (ADS)

    Muntean, Cristina M.; Stefan, Razvan; Bindea, Maria; Cozma, Vasile

    2013-06-01

    In this work we present a method for detection of motile and immotile Borrelia burgdorferi genomic DNA, in relation with infectious and noninfectious spirochetes. An FT-IR study of DNA isolated from B. burgdorferi sensu lato strains and from positive and negative Ixodes ricinus ticks, respectively, is reported. Motile bacterial cells from the species B. burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii were of interest. Also, FT-IR absorbance spectra of DNA from immotile spirochetes of B. burgdorferi sensu stricto, in the absence and presence of different antibiotics (doxycycline, erythromycin, gentamicin, penicillin V or phenoxymethylpenicillin, tetracycline, respectively) were investigated. FT-IR spectra, providing a high molecular structural information, have been analyzed in the wavenumber range 400-1800 cm-1. FT-IR signatures, spectroscopic band assignments and structural interpretations of these DNAs are reported. Spectral differences between FT-IR absorbances of DNAs from motile bacterial cells and immotile spirochetes, respectively, have been found. Particularly, alterations of the sugar-phosphate B-form chain in the case of DNA from Borrelia immotile cells, as compared with DNA from B. burgdorferi sensu lato motile cells have been observed. Based on this work, specific B. burgdorferi sensu lato and I. ricinus DNA-ligand interactions, respectively, might be further investigated using Fourier transform infrared spectroscopy.

  6. Incidence, Antibiotic Susceptibility, and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products.

    PubMed

    Yim, Jin-Hyeok; Kim, Kwang-Yeop; Chon, Jung-Whan; Kim, Dong-Hyeon; Kim, Hong-Seok; Choi, Da-Som; Choi, In-Soo; Seo, Kun-Ho

    2015-06-01

    Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10(4) CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases. © 2015 Institute of Food Technologists®

  7. Expression Profiles of Toll-Like Receptors in the Differentiation of an Infection with Borrelia burgdorferi Sensu Lato Spirochetes.

    PubMed

    Dudek, Slawomir; Ziółko, Ewa; Kimsa-Dudek, Magdalena; Solarz, Krzysztof; Mazurek, Urszula; Wierzgoń, Aleksander; Kokot, Teresa; Muc-Wierzgoń, Małgorzata

    2017-04-01

    The similarity of Lyme borreliosis to other diseases and its complex pathogenesis present diagnostic and therapeutic difficulties. The changes that occur at the cellular and molecular levels after a Borrelia sp. infection still remain poorly understood. Therefore, the present study focused on the expression of TLR and TLR-signaling genes in human dermal fibroblasts in the differentiation of an infection with Borrelia burgdorferi sensu lato spirochetes. Normal human dermal fibroblasts were cultured with the spirochetes of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Total RNA was extracted from the cells using TRIzol reagent. The analysis of the expression profiles of TLRs and TLR-related genes was performed using commercially available oligonucleotide microarrays of HG-U133A. The GeneSpring 12.0 platform and significance analysis of microarrays were used for the statistical analysis of microarray data. The analyses using the oligonucleotide microarray and QRT-PCR techniques permitted to identify the genes encoding TLR4 and TLR6 as specific for infection with B. afzelii and B. burgdorferi sensu stricto. In turn, TLR3 was only characteristic for an infection with B. burgdorferi sensu stricto. There were no changes in the TLR gene expression after infection with B. garinii. Our findings confirm that Borrelia has a major effect on fibroblast gene expression. Further characterization of changes in gene expression may lead to valuable insights into the role of the toll-like receptor in the pathogenesis of Lyme disease and may provide guidelines for the development of diagnostic markers for an infection with a particular Borrelia genospecies. Moreover, this will help to identify better treatment strategies for Lyme disease.

  8. Molecular detection of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato genospecies in red foxes (Vulpes vulpes) from Romania.

    PubMed

    Dumitrache, Mirabela Oana; Matei, Ioana Adriana; Ionică, Angela Monica; Kalmár, Zsuzsa; D'Amico, Gianluca; Sikó-Barabási, Sándor; Ionescu, Dan Traian; Gherman, Călin Mircea; Mihalca, Andrei Daniel

    2015-10-08

    Red foxes (Vulpes vulpes) are one of the most widespread wild carnivores in the world, being recognized to harbor and transmit a wide range of vector-borne diseases. Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato are zoonotic tick-borne pathogens causing emerging diseases. Wild animals play an essential role in the transmission of diseases and pathogens maintenance in nature. Epidemiological studies regarding the prevalence of tick-borne pathogens in red foxes are of public health importance, as they may successfully act as a pathogen transmission interface between wildlife, domestic animals and humans. This study included 14 counties from Romania. A total number of 353 red foxes (Vulpes vulpes) were examined. Heart tissue samples were collected during necropsy and stored at -20 °C. Genomic DNA extraction was performed and all samples were examined by polymerase chain reaction (PCR). Specific primers for A. phagocytophilum, A. platys, E. canis and Borrelia burgdorferi s.l. were used. Sequence analysis was performed (Macrogen Europe, Amsterdam) and obtained sequences are available at GenBank™. Out of the 353 samples, 9 (2.55 %; 95 % CI: 1.25-4.96 %) were positive for A. phagocytophilum. Positive animals originated from 5 counties. In total, 5 out of 353 heart tissue samples (1.42 %; 95 % CI: 0.52-3.47 %) collected from red foxes were positive for B. burgdorferi s.l. Red foxes originated from 4 counties. None of the samples were positive for A. platys or E. canis. No co-infection with A. phagocytophilum and B. burgdorferi s.l. was found. This first report of A. phagocytophilum and B. burgdorferi s.l. in red foxes from Romania suggests a limited role of foxes in the maintenance of the two related pathogens, but may represent a potential risk from a public health perspective.

  9. Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands.

    PubMed

    Koetsveld, Joris; Tijsse-Klasen, Ellen; Herremans, Tineke; Hovius, Joppe W R; Sprong, Hein

    2016-03-01

    Only a few reported cases indicate that Rickettsia helvetica and Rickettsia monacensis can cause disease in humans. Exposure to these two spotted fever group (SFG) rickettsiae occurs through bites of Ixodes ricinus, also the primary vector of Lyme borreliosis in Europe. To date, it is unclear how often exposure to these two microorganisms results in infection or disease. We show that of all the Borrelia burgdorferi s.l.-positive ticks, 25% were co-infected with rickettsiae. Predominantly R. helvetica was detected while R. monacensis was only found in approximately 2% of the ticks. In addition, exposure to tick-borne pathogens was compared by serology in healthy blood donors, erythema migrans (EM)-patients, and patients suspected of Lyme neuroborreliosis (LNB). As could be expected, seroreactivity against B. burgdorferi sensu lato was lower in blood donors (6%) compared to EM patients (34%) and suspected LNB cases (64%). Interestingly, seroreactivity against SFG Rickettsia antigens was not detected in serum samples from blood donors (0%), but 6% of the EM patients and 21% of the LNB suspects showed anti-rickettsial antibodies. Finally, the presence of B. burgdorferi s.l. and Rickettsia spp. in cerebrospinal fluid samples of a large cohort of patients suspected of LNB (n=208) was investigated by PCR. DNA of B. burgdorferi s.l., R. helvetica and R. monacensis was detected in seventeen, four and one patient, respectively. In conclusion, our data show that B. burgdorferi s.l. and SFG rickettsiae co-infection occurs in Dutch I. ricinus and that Lyme borreliosis patients, or patients suspected of Lyme borreliosis, are indeed exposed to both tick-borne pathogens. Whether SFG rickettsiae actually cause disease, and whether co-infections alter the clinical course of Lyme borreliosis, is not clear from our data, and warrants further investigation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  10. Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: relevance for Lyme vaccine design.

    PubMed

    Schnell, Gilles; Boeuf, Amandine; Jaulhac, Benoît; Boulanger, Nathalie; Collin, Elody; Barthel, Cathy; De Martino, Sylvie; Ehret-Sabatier, Laurence

    2015-04-01

    Lyme borreliosis is the most important vector-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC-MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild-type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT-PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 (http://proteomecentral.proteomexchange.org/dataset/PXD000876). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  12. Comparative histology of floral elaiophores in the orchids Rudolfiella picta (Schltr.) Hoehne (Maxillariinae sensu lato) and Oncidium ornithorhynchum H.B.K. (Oncidiinae sensu lato)

    PubMed Central

    Davies, Kevin L.; Stpiczyńska, Malgorzata

    2009-01-01

    Background and Aims Floral elaiophores, although widespread amongst orchids, have not previously been described for Maxillariinae sensu lato. Here, two claims that epithelial, floral elaiophores occur in the genus Rudolfiella Hoehne (Bifrenaria clade) are investigated. Presumed elaiophores were compared with those of Oncidiinae Benth. and the floral, resin-secreting tissues of Rhetinantha M.A. Blanco and Heterotaxis Lindl., both genera formerly assigned to Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto). Methods Putative, floral elaiophore tissue of Rudolfiella picta (Schltr.) Hoehne and floral elaiophores of Oncidium ornithorhynchum H.B.K. were examined by means of light microscopy, histochemistry, scanning electron microscopy and transmission electron microscopy. Key Results and Conclusions Floral, epithelial elaiophores are present in Rudolfiella picta, indicating, for the first time, that oil secretion occurs amongst members of the Bifrenaria clade (Maxillariinae sensu lato). However, whereas the elaiophore of R. picta is borne upon the labellar callus, the elaiophores of O. ornithorhynchum occur on the lateral lobes of the labellum. In both species, the elaiophore comprises a single layer of palisade secretory cells and parenchymatous, subsecretory tissue. Cell wall cavities are absent from both and there is no evidence of cuticular distension in response to oil accumulation between the outer tangential wall and the overlying cuticle in R. picta. Distension of the cuticle, however, occurs in O. ornithorhynchum. Secretory cells of R. picta contain characteristic, spherical or oval plastids with abundant plastoglobuli and these more closely resemble plastids found in labellar, secretory cells of representatives of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) than elaiophore plastids of Oncidiinae. In Rhetinantha, such plastids are involved in the synthesis of resin-like material or wax. Despite these differences, the elaiophore anatomy of

  13. Revisiting phylogenetic diversity and cryptic species of Cenococcum geophilum sensu lato.

    PubMed

    Obase, Keisuke; Douhan, Greg W; Matsuda, Yosuke; Smith, Matthew E

    2016-08-01

    The fungus Cenococcum geophilum Fr. (Dothideomycetes, Ascomycota) is one of the most common ectomycorrhizal fungi in boreal to temperate regions. A series of molecular studies has demonstrated that C. geophilum is monophyletic but a heterogeneous species or a species complex. Here, we revisit the phylogenetic diversity of C. geophilum sensu lato from a regional to intercontinental scale by using new data from Florida (USA) along with existing data in GenBank from Japan, Europe, and North America. The combination of internal transcribed spacer (ITS) ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene resolved six well-supported lineages (87-100 % bootstrap values) that are closely related to each other and a seventh lineage that is phylogenetically distinct. A multi-locus analysis (small subunit (SSU), large subunit (LSU), translational elongation factor (TEF), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2)) revealed that the divergent lineage is the sister group to all other known Cenococcum isolates. Isolates of the divergent lineage grow fast on nutrient media and do not form ectomycorrhizas on seedlings of several pine and oak species. Our results indicate that C. geophilum sensu lato includes more phylogenetically distinct cryptic species than have previously been reported. Furthermore, the divergent lineage appears to be a non-mycorrhizal sister group. We discuss the phylogenetic diversity of C. geophilum sensu lato and argue in favor of species recognition based on phylogenetic and ecological information in addition to morphological characteristics. A new genus and species (Pseudocenococcum floridanum gen. et sp. nov.) is proposed to accommodate a divergent and putatively non-mycorrhizal lineage.

  14. Prevalence of Borrelia burgdorferi Sensu Lato in Ticks Collected from Migratory Birds in Switzerland

    PubMed Central

    Poupon, Marie-Angèle; Lommano, Elena; Humair, Pierre-François; Douet, Véronique; Rais, Olivier; Schaad, Michael; Jenni, Lukas; Gern, Lise

    2006-01-01

    The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae. PMID:16391149

  15. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  16. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  17. Milk-originated Bacillus cereus sensu lato strains harbouring Bacillus anthracis-like plasmids are genetically and phenotypically diverse.

    PubMed

    Bartoszewicz, Marek; Marjańska, Paulina Sylwia

    2017-10-01

    Bacillus cereus sensu lato is widely distributed in food products, including raw and processed milk. Plasmids often determine bacterial virulence and toxicity, but their role in the evolution of B. cereus sensu lato is only partly known. Here, we observed that nearly 8% of B. cereus sensu lato isolates were positive for pXO1-like plasmids and 12% for pXO2-like plasmids in raw and ultra-heat-treated (UHT) milk from one dairy plant. However, pXO1-like plasmids were significantly more frequent in raw milk, while pXO2-like plasmids were more frequent in processed milk. Strains from raw and UHT milk were enterotoxigenic, with up to one-fifth of the isolates being psychrotolerant. Phylogenetic assessment using multi-locus sequence typing revealed a polyphyletic structure for these bacilli, with distinct groups of cold-adapted isolates and pathogenic strains (including emetic B. cereus). Populations corresponding to both sampling sites exhibited significant linkage disequilibrium and the presence of purifying selection. The far-from-clonal population structure indicated the presence of sequence types or ecotypes adapted to specific conditions in the dairy industry. A high recombination-to-mutation ratio suggested an important role for horizontal gene transfer among B. cereus sensu lato isolates in milk. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Mutation in the sodium channel gene corresponds with phenotypic resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to pyrethroids

    USDA-ARS?s Scientific Manuscript database

    The brown dog tick, Rhipicephalus sanguineus sensu lato, is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne d...

  19. Draft Genome Sequences of Human Pathogenic Fungus Geomyces pannorum Sensu Lato and Bat White Nose Syndrome Pathogen Geomyces (Pseudogymnoascus) destructans.

    PubMed

    Chibucos, Marcus C; Crabtree, Jonathan; Nagaraj, Sushma; Chaturvedi, Sudha; Chaturvedi, Vishnu

    2013-12-19

    We report the draft genome sequences of Geomyces pannorum sensu lato and Geomyces (Pseudogymnoascus) destructans. G. pannorum has a larger proteome than G. destructans, containing more proteins with ascribed enzymatic functions. This dichotomy in the genomes of related psychrophilic fungi is a valuable target for defining their distinct saprobic and pathogenic attributes.

  20. Sporothrix schenckii Sensu Lato identification in fragments of skin lesion cultured in NNN medium for differential diagnosis of cutaneous leishmaniasis.

    PubMed

    Antonio, Liliane de Fátima; Pimentel, Maria Inês Fernandes; Lyra, Marcelo Rosandiski; Madeira, Maria de Fátima; Miranda, Luciana de Freitas Campos; Paes, Rodrigo Almeida; Brito-Santos, Fábio; Carvalho, Maria Helena Galdino Figueredo; Schubach, Armando de Oliveira

    2017-02-01

    Eighty-nine patients with clinical suspicion of leishmaniasis were referred for differential diagnosis. Sporothrix schenckii sensu lato was isolated in Novy-MacNeal-Nicolle + Schneider media in 98% of 64 patients with final diagnosis of sporotrichosis. This medium may be suitable for diagnosis of sporotrichosis in areas where cutaneous leishmaniasis is also endemic. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. An Invasive Mammal (the Gray Squirrel, Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato

    PubMed Central

    Magierecka, Agnieszka; Gilbert, Lucy; Edoff, Alissa; Brereton, Amelia; Kilbride, Elizabeth; Denwood, Matt; Birtles, Richard; Biek, Roman

    2015-01-01

    Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n = 679), an invasive species in the United Kingdom, as B. burgdorferi sensu lato hosts. We found that gray squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi sensu lato in the United Kingdom, and 11.9% were infected with B. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospecies Borrelia garinii was most common. The second most frequent infection was with Borrelia afzelii. Genotyping of B. garinii and B. afzelii produced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi sensu lato infection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains of B. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species in B. burgdorferi sensu lato epidemiology can be highly variable and thus difficult to predict. PMID:25888168

  2. Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina.

    PubMed

    Cicuttin, Gabriel L; Tarragona, Evelina L; De Salvo, M Nazarena; Mangold, Atilio J; Nava, Santiago

    2015-09-01

    Natural infection with Ehrlichia canis and Anaplasma platys in ticks belonging to the tropical and temperate lineages of Rhipicephalus sanguineus sensu lato from Argentina was evaluated. Samples were tested for Ehrlichia canis infection by PCR assays using 16S rRNA, dsb and p28 gene, while detection of A. platys was performed with 16S rRNA and groESL gene. The assignment of the ticks to each lineage was corroborated with 16S rDNA sequences. All ticks infected with E. canis and A. platys belonged to the tropical lineage. These results constitute the first record of E. canis infection in R. sanguineus s.l ticks from Argentina. No ticks from the temperate lineage were found to be infected with E. canis, coinciding with previous studies performed in Argentina and Uruguay where E. canis infection was not detected in R. sanguineus s.l from the temperate lineage. Because the presence of the tropical lineage of R. sanguineus s.l has been documented in tropical areas of northern Argentina between 22° and 24° of south latitude, the findings of this work indicate that transmission of E. canis and A. platys to dogs by R. sanguineus s.l probably occurs along this region.

  3. Human seroprevalence against Borrelia burgdorferi sensu lato in two comparable regions of the eastern Alps is not correlated to vector infection rates.

    PubMed

    Sonnleitner, S T; Margos, G; Wex, F; Simeoni, J; Zelger, R; Schmutzhard, E; Lass-Flörl, C; Walder, G

    2015-04-01

    Seroprevalences were determined by testing sera of 1607 blood donors from North, East, and South Tyrol. In the Tyrols, the continental divide delimitates areas with high seroprevalences of IgG antibodies against Borrelia burgdorferi sensu lato in the North (7.2%) from areas with low seroprevalences in the South (1.5%). To determine Borrelia prevalences in unfed Ixodes ricinus ticks, 755 questing ticks were tested by PCR. Prevalences in nymphal and adult ticks were found to be 19.7% (n=132) and 21.5% (n=205) in North Tyrol and 23% (n=43) and 23.7% (n=376) in South Tyrol, respectively. Sequencing of 46 Borrelia-positive ticks yielded 74% Borrelia (B.) afzelii, 11% B. garinii, 7% B. lusitaniae, 7% B. burgdorferi sensu stricto, and 2% B. valaisiana infections. Distinct genetic clusters could not be delimitated on either side of the continental divide. This study describes occurrence and geographic dispersion of Borrelia spp. in the Tyrols, discusses possible reasons for significant differences in human seroprevalence, and indicates that prevalence of Borrelia in vector ticks is not a direct predictive factor for the local seroprevalence in humans.

  4. Borrelia mayonii sp. nov., a member of the Borrelia burgdorferi sensu lato complex, detected in patients and ticks in the upper midwestern United States.

    PubMed

    Pritt, Bobbi S; Respicio-Kingry, Laurel B; Sloan, Lynne M; Schriefer, Martin E; Replogle, Adam J; Bjork, Jenna; Liu, Gongping; Kingry, Luke C; Mead, Paul S; Neitzel, David F; Schiffman, Elizabeth; Hoang Johnson, Diep K; Davis, Jeffrey P; Paskewitz, Susan M; Boxrud, David; Deedon, Alecia; Lee, Xia; Miller, Tracy K; Feist, Michelle A; Steward, Christopher R; Theel, Elitza S; Patel, Robin; Irish, Cole L; Petersen, Jeannine M

    2016-11-01

    Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

  5. Emergence of tick-borne pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ricketsia raoultii and Babesia microti) in the Kyiv urban parks, Ukraine.

    PubMed

    Didyk, Yuliya M; Blaňárová, Lucia; Pogrebnyak, Svyatoslav; Akimov, Igor; Peťko, Branislav; Víchová, Bronislava

    2017-02-01

    To date, only limited data about the presence of ticks and circulation of tick-borne pathogens in urban parks of Kyiv in northern Ukraine are available. In total, 767 ticks (696 Ixodes ricinus and 69 Dermacentor reticulatus) collected in seven urban parks and one suburban oak wood park in Kyiv were individually analyzed by the PCR assays. Tick-borne pathogens, namely spirochetes from Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum, and Babesia microti, were detected in 11.1% of tested I. ricinus ticks. In total, 4% of I. ricinus ticks tested positive for the presence of B. burdorferi s.l. (Borrelia afzelii and Borrelia garinii), 5.2% for A. phagocytophilum, and Ba. microti was confirmed in 1.9% of examined ticks. Mixed infections were recorded in four DNA samples, representing the prevalence of 0.6%. One female and two I. ricinus nymphs were simultaneously infected with B. afzelii and A. phagocytophilum, and one female carried B. afzelii and Ba. microti. In addition, 10.1% of D. reticulatus ticks tested positive for Rickettsia raoultii. Identification of infectious agents and their diversity, assessment of the relative epidemiological importance and determination of the prevalence in questing ticks from central parts of the cities are crucial steps towards the tick-borne diseases surveillance in urban environment.

  6. Central nervous system infection due to Cryptococcus gattii sensu lato in India: Analysis of clinical features, molecular profile and antifungal susceptibility.

    PubMed

    Lahiri Mukhopadhyay, Shayanki; Bahubali, Veenakumari H; Manjunath, Netravathi; Swaminathan, Aarthi; Maji, Sayani; Palaniappan, Marimuthu; Parthasarathy, Satishchandra; Chandrashekar, Nagarathna

    2017-07-23

    Cryptococcus gattii species complex has evolved as a pathogen in the last two decades causing infection among both immunocompetent and immunocompromised hosts. We aimed to analyse the clinical features of CNS infection caused by C. gattii sensu lato, molecular and antifungal susceptibility profile of this pathogen. Cases diagnosed to have CNS cryptococcosis were included in the study. Cryptococcus recovered from patient's specimen was identified by standard protocol. Species confirmation, mating type and molecular type determination were performed by PCR based methods. Antifungal susceptibility was tested in VITEK2C to amphotericin B, 5-flucytosine, fluconazole and voriconazole. Among 199 cases, 20 (10%) were due to C. gattii, comprising of 75% cryptococcal meningitis and 25% cryptococcoma cases. Young adult males were commonly affected. Headache and vomiting were prominent symptoms and 50% were immunocompromised. Among the isolates, 75%, 20% and 5% were C. tetragattii, C. gattii sensu stricto and C. bacillisporus respectively and all had mating type α. Four (20%) isolates of C. tetragattii and the only isolate of C. bacillisporus were resistant to fluconazole. The most common species isolated from south India is C. tetragattii. The study contributes to the epidemiology of C. gattii and reiterates the need for genotyping and antifungal susceptibility testing. © 2017 Blackwell Verlag GmbH.

  7. Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere

    PubMed Central

    Ivanova, Larisa B.; Tomova, Alexandra; González-Acuña, Daniel; Murúa, Roberto; Moreno, Claudia X.; Hernández, Claudio; Cabello, Javier; Cabello, Carlos; Daniels, Thomas J.; Godfrey, Henry P.; Cabello, Felipe C.

    2014-01-01

    Summary Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by PCR and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group Borrelia chilensis VA1, in honor of its country of origin. PMID:24148079

  8. Comparison of isolation rate of Borrelia burgdorferi sensu lato in two different culture media, MKP and BSK-H.

    PubMed

    Ružić-Sabljić, E; Maraspin, V; Cimperman, J; Strle, F; Lotrič-Furlan, S; Stupica, D; Cerar, T

    2014-07-01

    The aim of the study was to evaluate two culture media for Borrelia burgdorferi sensu lato isolation from a 5 × 2 × 2 mm skin biopsy that was dissected into two pieces and inoculated into modified Kelly-Pettenkofer (MKP) and Barbour-Stoenner-Kelly-H (BSK-H) medium. Samples were incubated at 33°C for up to 9 weeks. Borrelia species was determined by MluI-restriction of whole genome or by MseI-restriction of PCR product. We determined the proportion of isolation rate, 'slow-growers', contaminated specimens and Borrelia species in the two media. In each of the two media 235 skin specimens were cultivated. We found 90/470 (19.1%) contaminated cultures (BSK-H 67/235, 28.5%; MKP 23/235, 9.8%; p <0.0001). Borrelia growth was ascertained in 59/235 (25.1%) BSK-H and 102/235 (43.4%) MKP cultures (p <0.0001); the corresponding values for non-contaminated cultures were 59/168 (35.1%) and 102/212 (48.1%); (p 0.003). Fourteen specimens were positive only in BSK-H, 57 solely in MKP, and 43 in both culture media. Slow growth was present in 8/59 (13.6%) BSK-H and in 4/98 (4.1%) MKP positive cultures (p 0.019). Borrelia afzelii was identified in 44/51 (86.3%) BSK-H and in 88/98 (89.8%) MKP culture-positive samples; the corresponding findings for Boreelia garinii and B. burgdorferi sensu stricto were 6/51 (11.8%) and 9/98 (9.2%), and 1/51 (1.9%) and 1/98 (1.0%), for BSK-H and MKP, respectively. Comparison of MKP and BSK-H medium for Borrelia culturing from skin specimens of European patients with erythema migrans revealed the advantage of MKP over BSK-H.

  9. Spatial distribution of the sibling species of Anopheles gambiae sensu lato (Diptera: Culicidae) and malaria prevalence in Bayelsa State, Nigeria

    PubMed Central

    2014-01-01

    Background Much of the confusing ecophenotypic plasticity of Anopheles gambiae sensu lato is attributable to the differential biological traits of the sibling species, with their heterogeneous geographical distribution, behavioral dissimilarities and divergent population dynamics. These differences are critical to their roles in malaria transmission. Studies were, therefore, undertaken on the spatial distribution of these species and malaria prevalence rates in Bayelsa State, September, 2008-August 2010. Methods Mosquito sampling was in 7 towns/villages in 7 Local Government Areas (LGAs) in 3 eco-vegetational zones: Fresh Water Swamp Forest (FWSF): Sagbama, Yenagoa, Kolokuma-Opokuma LGAs; Brackish Water Swamp Forest (BWSF): Ogbia, Ekeremor, Southern Ijaw LGAs; Mangrove Water Forest (MWF): Nembe LGA. Adults were collected twice quarterly by the Pyrethrum Spray Catch (PSC) technique. Anopheles was separated morphologically and the sibling species PCR- identified. Simultaneously, malaria prevalence rates were calculated from data obtained by the examination of blood smears from consenting individuals at hospitals/clinics. Results An. gambiae s.s. was dominant across the 3-eco-vegetational zones. Spatial distribution analyses by cell count and nearest neighbor techniques indicated a tendency to clustering of species. An. gambiae s.s. and An. arabiensis clustered in Ekeremor LGA while these 2 species and An. melas aggregated in Nembe. The gonotrophic (physiological) status examination revealed that 34.3, 23.5, 23.1 and 18.4% of the population were fed, unfed, gravid and half gravid respectively. The highest malaria prevalence rates were obtained at Kolokuma-Opokuma and Nembe LGAs. Variation in prevalence rates among LGAs was significant (t = 5.976, df = 6, p-value = 0.002, p < 0.05). The highest prevalence rate was in the age group, 30-39 yrs, while the lowest prevalence was in the 0-9 yrs group. Conclusion High malaria prevalence rates were associated

  10. Ovarian structure and oogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (teleostei, Cyprinodontiformes).

    PubMed

    Uribe, Mari Carmen; Grier, Harry J; Parenti, Lynne R

    2012-04-01

    The cyprinodontiform family Goodeidae comprises two biogeographically disjunct subfamilies: the viviparous Goodeinae endemic to the Mexican Plateau, and the oviparous Empetrichthyinae, known only from relict taxa in Nevada and California. Ovarian characteristics of two oviparous species of goodeid, Crenichthys baileyi and Empetrichthys latos, studied using museum collections, are compared with those of viviparous species of goodeids. Both subfamilies have a single, cystovarian ovary. The ovary in the viviparous Goodeinae has an internal septum that divides the ovarian lumen into two compartments, and it may possess oogonia. There is no ovarian septum in the oviparous C. baileyi and E. latos. Oogenesis is similar in both subfamilies with regard to the proliferation of oogonia, initiation of meiosis, primary growth and development of an oocyte during secondary growth in which fluid yolk progressively fuses into a single globule. Notably, eggs of C. baileyi and E. latos are approximately double the size of those of the viviparous Goodeinae in which embryos develop inside the ovarian lumen and are nourished, in part, by nutrients transferred from the maternal tissues, a mode of embryo development called matrotrophy. Egg envelopes of the two subfamilies differ in that those of C. baileyi and E. latos have a relatively thick zona pellucida, attachment fibrils or filaments that develop between the follicle cells during oogenesis, and a micropyle observed only in E. latos. In contrast, viviparous goodeid eggs have a relatively thin zona pellucida, but lack adhesive fibrils, and a micropyle was not observed. These reproductive characters are compared with those of species of the eastern North American Fundulus, a representative oviparous cyprinodontiform. One newlyrecognized shared, derived character, a single, median ovoid ovary with no obvious external evidence of fusion, supports monophyly of the Goodeidae. Differences among the goodeid subfamilies and Fundulus are

  11. Prevalence and infection intensity of Rickettsia massiliae in Rhipicephalus sanguineus sensu lato ticks from Mendoza, Argentina.

    PubMed

    Monje, Lucas D; Linares, María Cielo; Beldomenico, Pablo M

    2016-11-01

    Rickettsia massiliae belongs to the spotted fever group and in the New World is commonly associated with the brown dog tick, Rhipicephalus sanguineus. Herein we investigate the presence of R. massiliae in Rh. sanguineus sensu lato ticks in a location near the Andean foothills (Mendoza, Argentina), to provide a prevalence estimate and to assess the infection intensity of this pathogen. Rickettsia massiliae infection was found in 5.1% of the Rh. sanguineus s.l ticks analyzed, all with high infection intensities. Molecular analysis determined that all R. massiliae-infected Rh. sanguineus s.l. ticks belonged to the temperate lineage. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  12. Helicobacter heilmannii sensu lato: an overview of the infection in humans.

    PubMed

    Bento-Miranda, Mario; Figueiredo, Ceu

    2014-12-21

    Helicobacter heilmannii sensu lato (H. heilmannii s.l.) is a group of gastric non-Helicobacter pylori Helicobacter species that are morphologically indistinguishable from each other. H. heilmannii s.l. infect the stomach of several animals and may have zoonotic potential. Although the prevalence of these infections in humans is low, they are associated with gastric pathology, including mucosa-associated lymphoid tissue lymphoma, making them a significant health issue. Here, the taxonomy, epidemiology, microbiology, diagnosis, and treatment of these infections will be reviewed. The gastric pathology associated with H. heilmannii s.l. infections in humans will also be addressed. Finally, the features of the complete bacterial genomes available and studies on species-specific pathogenesis will be reviewed. The understanding of the mechanisms that underlie gastric disease development mediated by the different bacterial species that constitute H. heilmannii s.l. is essential for developing strategies for prevention and treatment of these infections.

  13. Helicobacter heilmannii sensu lato: An overview of the infection in humans

    PubMed Central

    Bento-Miranda, Mario; Figueiredo, Ceu

    2014-01-01

    Helicobacter heilmannii sensu lato (H. heilmannii s.l.) is a group of gastric non-Helicobacter pylori Helicobacter species that are morphologically indistinguishable from each other. H. heilmannii s.l. infect the stomach of several animals and may have zoonotic potential. Although the prevalence of these infections in humans is low, they are associated with gastric pathology, including mucosa-associated lymphoid tissue lymphoma, making them a significant health issue. Here, the taxonomy, epidemiology, microbiology, diagnosis, and treatment of these infections will be reviewed. The gastric pathology associated with H. heilmannii s.l. infections in humans will also be addressed. Finally, the features of the complete bacterial genomes available and studies on species-specific pathogenesis will be reviewed. The understanding of the mechanisms that underlie gastric disease development mediated by the different bacterial species that constitute H. heilmannii s.l. is essential for developing strategies for prevention and treatment of these infections. PMID:25548476

  14. Borrelia burgdorferi sensu lato-infected Ixodes ricinus collected from vegetation near the Arctic Circle.

    PubMed

    Hvidsten, Dag; Stordal, Frode; Lager, Malin; Rognerud, Bjørg; Kristiansen, Bjørn-Erik; Matussek, Andreas; Gray, Jeremy; Stuen, Snorre

    2015-09-01

    This is the first study to determine the density of questing Ixodes ricinus in northern Norway. It was performed at two sites in Brønnøy, which has been known for its tick permissive habitats for decades and is one of the northernmost habitats with an abundant I. ricinus population in the world. From April to November 2011, all stages of host-seeking I. ricinus were collected from the two sites. The overall prevalence of nymphs infected with Borrelia burgdorferi sensu lato was 21% and that of adult ticks 46%. The rates of the genospecies Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana were similar to findings in most other studies in Scandinavia, with B. afzelii by far the most prevalent at 76%. The high Borrelia-infection prevalence in ticks from Brønnøy may explain the high incidence rate of reported Lyme borreliosis in the municipality.

  15. Advances in antitumor polysaccharides from phellinus sensu lato: Production, isolation, structure, antitumor activity, and mechanisms.

    PubMed

    Yan, Jing-Kun; Pei, Juan-Juan; Ma, Hai-Le; Wang, Zhen-Bin; Liu, Yuan-Shuai

    2017-04-13

    Edible and medicinal fungi (mushrooms) are widely applied to functional foods and nutraceutical products because of their proven nutritive and medicinal properties. Phellinus sensu lato is a well-known medicinal mushroom that has long been used in preventing ailments, including gastroenteric dysfunction, diarrhea, hemorrhage, and cancers, in oriental countries, particularly in China, Japan, and Korea. Polysaccharides represent a major class of bioactive molecules in Phellinus s. l., which have notable antitumor, immunomodulatory, and medicinal properties. Polysaccharides that were isolated from fruiting bodies, cultured mycelia, and filtrates of Phellinus s. l. have not only activated different immune responses of the host organism but have also directly suppressed tumor growth and metastasis. Studies suggest that polysaccharides from Phellinus s. l. are promising alternative anticancer agents or synergizers for existing antitumor drugs. This review summarizes the recent development of polysaccharides from Phellinus s. l., including polysaccharide production, extraction and isolation methods, chemical structure, antitumor activities, and mechanisms of action.

  16. [Genetic divergence and allozymic variability in mice of the genus Apodemus s. lato (Muridae, Rodentia)].

    PubMed

    Mezhzherin, S V; Zykov, A E

    1991-01-01

    Genetic variability of 36 presumed loci was examined in 5 species of subgenus Sylvaemus (sylvaticus, flavicollis, microps, falzfeini, ponticus) and in 3 species of the subgenus Apodemus s. str. (agrarius, peninsulae, speciosus) from different geographic regions of the USSR. Taxonomic status and species affiliation of A. s. chorassanicus from Turkmenia and A. s. tscherga from Altay have been established: the former is identical to A. falzfeini from the Ukraine, the latter is identical to A. microps. Genus Apodemus s. lato can be divided into two different geni (Apodemus s. str. and Sylvaemus) on the basis of genetic distance between them (D = 1,518). Genetic differentiation within subgenus Sylvaemus is 0.311, within subgenus Apodemus s. str. is 1,011. The observed differences in genetic heterozygosity between species (H varies from 0 to 0.067) are, probably, due to the historical events which take place in the formation of areas of these species.

  17. The dynamic history of plastid genomes in the Campanulaceae sensu lato is unique among angiosperms

    PubMed Central

    Knox, Eric B.

    2014-01-01

    Why have some plants lost the organizational stability in plastid genomes (plastomes) that evolved in their algal ancestors? During the endosymbiotic transformation of a cyanobacterium into the eukaryotic plastid, most cyanobacterial genes were transferred to the nucleus or otherwise lost from the plastome, and the resulting plastome architecture in land plants confers organizational stability, as evidenced by the conserved gene order among bryophytes and lycophytes, whereas ferns, gymnosperms, and angiosperms share a single, 30-kb inversion. Although some additional gene losses have occurred, gene additions to angiosperm plastomes were previously unknown. Plastomes in the Campanulaceae sensu lato have incorporated dozens of large ORFs (putative protein-coding genes). These insertions apparently caused many of the 125+ large inversions now known in this small eudicot clade. This phylogenetically restricted phenomenon is not biogeographically localized, which indicates that these ORFs came from the nucleus or (less likely) a cryptic endosymbiont. PMID:25024223

  18. Molecular, biological, and morphometric comparisons between different geographical populations of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

    PubMed

    Sanches, Gustavo S; Évora, Patrícia M; Mangold, Atílio J; Jittapalapong, Sattaporn; Rodriguez-Mallon, Alina; Guzmán, Pedro E E; Bechara, Gervásio H; Camargo-Mathias, Maria I

    2016-01-15

    In this study, different geographical populations of Rhipicephalus sanguineus sensu lato were compared by molecular, biological, and morphometric methods. Phylogenetic trees were constructed using 12S and 16S rDNA sequences and showed two distinct clades: one composed of ticks from Brazil (Jaboticabal, SP), Cuba (Havana) Thailand (Bangkok) and the so-called "tropical strain" ticks. The second clade was composed of ticks from Spain (Zaragoza), Argentina (Rafaela, Santa Fe) and the so-called "temperate strain" ticks. Morphometric analysis showed good separation between females of the two clades and within the temperate clade. Males also exhibited separation between the two clades, but with some overlap. Multiple biological parameters revealed differences between the two clades, especially the weight of the engorged female. These results confirm the existence of at least two species under the name "R. sanguineus".

  19. Larvae of Ixodiphagus wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae) from Brazil.

    PubMed

    Bezerra Santos, Marcos Antônio; de Macedo, Lucia Oliveira; de Souza, Islanne Barbosa; do Nascimento Ramos, Carlos Alberto; Alves, Leucio Câmara; Ramos, Rafael Antonio Nascimento; de Carvalho, Gílcia Aparecida

    2017-03-21

    The biological control of ticks represents an alternative method to the chemical control, given its ecological-friendly approach. Amongst the alternatives, the use of parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) has been largely investigated. The aim of this study was to document and molecularly characterize Ixodiphagus wasps in ticks from a tropical region of Brazil. From October 2015 to March 2016, Rhipicephalus sanguineus sensu lato ticks (n=1814) were collected from naturally infested dogs and Ixodiphagus larvae were detected by microscopic examination. In addition, adult wasps were obtained in the laboratory. Larvae and adults were molecularly identified as Ixodiphagus hookeri. These findings suggest that this type of parasitism deserves to be studied in local tick populations, in order to elucidate the role of these wasps as a potential alternative to chemical tick control.

  20. A taxonomic review of the Neoserica (sensu lato) septemlamellata group (Coleoptera, Scarabaeidae, Sericini)

    PubMed Central

    Ahrens, Dirk; Liu, Wan-Gang; Fabrizi, Silvia; Bai, Ming; Yang, Xing-Ke

    2014-01-01

    Abstract In the present paper the species belonging to the Neoserica (sensu lato) septemlamellata group, that included so far only four known species, are revised. Here we describe eleven new species originating mainly from Indochina and Southern China: N. daweishanica sp. n., N. gaoligongshanica sp. n., N. guangpingensis sp. n., N. igori sp. n., N. jiulongensis sp. n., N. plurilamellata sp. n., N. weishanica sp. n., N. yanzigouensis sp. n. (China) N. sapaensis sp. n. (China, Vietnam), N. bansongchana sp. n., N. takakuwai sp. n. (Laos). The lectotypes of Neoserica septemlamellata Brenske, 1898 and N. septemfoliata Moser, 1915 are designated. Keys to the species and species groups are given, the genitalia of all species and their habitus are illustrated and distribution maps are included. PMID:24843263

  1. Association of urine protein excretion and infection with Borrelia burgdorferi sensu lato in Bernese Mountain dogs.

    PubMed

    Gerber, Bernhard; Eichenberger, Simone; Haug, Katharinan; Wittenbrink, Max M; Reusch, Claudia E

    2009-12-01

    Bernese Mountain dogs (BMDs) are prone to develop a familial glomerulonephropathy and a pathogenic role of Borrelia burgdorferi sensu lato in this disease has been suspected. Glomerular disease in many affected dogs is clinically inapparent and proteinuria is found incidentally. In this study, urine protein excretion was evaluated in 122 clinically healthy BMDs and 55 controls. The seroprevalence of B. burgdorferi in BMDs was 57%, compared to 16% in controls. There were no significant differences in the occurrence of positive dipstick results, microalbuminuria, urine protein-to-urine creatinine ratio or abnormal urine protein pattern (determined by sodium dodecyl sulphate agarose gel electrophoresis) between BMDs and controls and BMDs with and without antibodies against B. burgdorferi. It was concluded that antibodies against B. burgdorferi are not associated with proteinuria as an early sign of renal disease in BMDs.

  2. Divergence of protein-coding capacity and regulation in the Bacillus cereus sensu lato group

    PubMed Central

    2014-01-01

    Background The Bacillus cereus sensu lato group contains ubiquitous facultative anaerobic soil-borne Gram-positive spore-forming bacilli. Molecular phylogeny and comparative genome sequencing have suggested that these organisms should be classified as a single species. While clonal in nature, there do not appear to be species-specific clonal lineages, excepting B. anthracis, in spite of the wide array of phenotypes displayed by these organisms. Results We compared the protein-coding content of 201 B. cereus sensu lato genomes to characterize differences and understand the consequences of these differences on biological function. From this larger group we selected a subset consisting of 25 whole genomes for deeper analysis. Cluster analysis of orthologous proteins grouped these genomes into five distinct clades. Each clade could be characterized by unique genes shared among the group, with consequences for the phenotype of each clade. Surprisingly, this population structure recapitulates our recent observations on the divergence of the generalized stress response (SigB) regulons in these organisms. Divergence of the SigB regulon among these organisms is primarily due to the placement of SigB-dependent promoters that bring genes from a common gene pool into/out of the SigB regulon. Conclusions Collectively, our observations suggest the hypothesis that the evolution of these closely related bacteria is a consequence of two distinct processes. Horizontal gene transfer, gene duplication/divergence and deletion dictate the underlying coding capacity in these genomes. Regulatory divergence overlays this protein coding reservoir and shapes the expression of both the unique and shared coding capacity of these organisms, resulting in phenotypic divergence. Data from other organisms suggests that this is likely a common pattern in prokaryotic evolution. PMID:25350501

  3. Molecular screening for Bartonella henselae and Borrelia burgdorferi sensu lato co-existence within Ixodes ricinus populations in central and eastern parts of Poland.

    PubMed

    Sytykiewicz, Hubert; Karbowiak, Grzegorz; Werszko, Joanna; Czerniewicz, Paweł; Sprawka, Iwona; Mitrus, Joanna

    2012-01-01

    The presented study aimed at establishing the prevalence and co-infection rates of Bartonella henselae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from the central and eastern parts of Poland. The common tick individuals were gathered in the years 2008-2009. Questing ticks were sampled by dragging a white woollen flag over lower vegetation at 17 localities within diverse types of habitats: urban recreational green areas (city parks and squares), suburban forests and rural woodlands throughout the investigated regions of Poland. Detection of B. henselae in tested tick specimens was based on PCR amplification of the citrate synthase (gltA) gene, while screening for the presence of B. burgdorferi s.l. DNA was carried out by analyzing fragments of two genes: the flagellin (fla) and outer surface protein A (ospA). A total number of 1,571 I. ricinus ticks were sampled: 865 (55.1%) nymphs, 377 females (24.0%) and 329 males (20.9%). The application of PCR assays revealed that 76 (4.8%) tick samples were B. henselae-positive, B. burgdorferi s.l. DNA was detected in 194 specimens (12.3%), whereas the co-existence of these pathogens was evidenced in 22 tested ticks (1.4%). Furthermore, the occurrence of bartonellae and co-circulation of analysed microorganisms in I. ricinus was affirmed only within adult individuals, while presence of the screened spirochetes was ascertained in both nymphal and adult ticks. It should be stressed that the suburban woods of Warsaw and rural forests in Warsaw County characterized the highest prevalence levels of dual infection with investigated tick-borne pathogens, whereas the lowest co-infection rates were recorded in tick populations inhabiting rural forests in Płock County and forested areas in Korczew-Mogielnica (within the Nadbużański Landscape Park).

  4. Occurrence of Bartonella henselae and Borrelia burgdorferi sensu lato co-infections in ticks collected from humans in Germany.

    PubMed

    Mietze, A; Strube, C; Beyerbach, M; Schnieder, T; Goethe, R

    2011-06-01

    Bartonella (B.) henselae is the zoonotic agent of cat scratch disease. B. henselae has been associated with therapy-resistant Lyme disease in humans suggesting that B. henselae and Borrelia burgdorferi sensu lato might be transmitted concurrently by ticks. In the present study we found that 16 (6.9%) of 230 Ixodes ricinus collected from humans harboured DNA of Bartonella spp. Fifteen positive ticks were infected with B. henselae and one tick with B. clarridgeiae. Twenty-five percent of the 16 Bartonella positive ticks were co-infected with Borrelia burgdorferi sensu lato. Our data show that B. henselae is present in Ixodes ricinus and that ticks may serve as source of infection for humans. 2010 The Authors. Clinical Microbiology and Infection; 2010 European Society of Clinical Microbiology and Infectious Diseases.

  5. Native strains of Beauveria bassiana for the control of Rhipicephalus sanguineus sensu lato.

    PubMed

    Cafarchia, Claudia; Immediato, Davide; Iatta, Roberta; Ramos, Rafael Antonio Nascimento; Lia, Riccardo Paolo; Porretta, Daniele; Figueredo, Luciana Aguiar; Dantas-Torres, Filipe; Otranto, Domenico

    2015-02-05

    Rhipicephalus sanguineus sensu lato ticks are widespread worldwide due to their adaptability to survive under different environmental conditions. They may act as vectors of a wide range of pathogens to humans and animals and their control is based on the use of chemical products on dogs and in the environment. Alternative control strategies, such as the use of entomopathogenic fungi as bio-control agents have also been investigated. The ability of native strains of Beauveria bassiana sensu lato in causing mortality in different tick species (e.g., Amblyomma cajennense and Rhipicephalus microplus) has been demonstrated. However, limited studies have assessed the use of B. bassiana for the control of R. sanguineus s.l. and none of them have employed native strains of this fungus. Here we investigated the pathogenicity of a native strain of B. bassiana (CD1123) against all developmental stages of R. sanguineus s.l.. Batches of eggs, larvae, nymphs and adult ticks were immersed in a suspension of 10(7) conidia/ml of B. bassiana s.l., isolated from a R. sanguineus s.l. engorged female. All treatment and control groups were observed for 20 days, and the biological parameters (i.e., mortality, hatching, moulting percentage, pre-oviposition period, oviposition period and rate, eggs production efficiency, reproductive efficiency and fitness indexes) were assessed. The effect of the B. bassiana strain tested herein on eggs, larvae, nymphs and adults showed a significantly higher mortality than those of the control groups (p < 0.05) at 5 days post-infection. No infected eggs hatched and no infected larvae moulted. Only 15% of infected nymphs moulted into adults. All biological parameters of treated groups differed significantly (p < 0.001) from those of control groups. This study demonstrates that a suspension containing 10(7) conidia/ml of a native B. bassiana strain is highly virulent towards all life-cycle developmental stages of R. sanguineus s.l. and may be of

  6. Genetic structure and demographic history of Colletotrichum gloeosporioides sensu lato and C. truncatum isolates from Trinidad and Mexico

    PubMed Central

    2013-01-01

    Background C. gloeosporioides sensu lato is one of the most economically important post-harvest diseases affecting papaya production worldwide. There is currently no information concerning the genetic structure or demographic history of this pathogen in any of the affected countries. Knowledge of molecular demographic parameters for different populations will improve our understanding of the biogeographic history as well as the evolutionary and adaptive potential of these pathogens. In this study, sequence data for ACT, GPDH, β-TUB and ITS gene regions were analyzed for C. gloeosporioides sensu lato and C. truncatum isolates infecting papaya in Trinidad and Mexico in order to determine the genetic structure and demographic history of these populations. Results The data indicated that Mexico is the ancestral C. gloeosporioides sensu lato population with asymmetrical migration to Trinidad. Mexico also had the larger effective population size but, both Mexico and Trinidad populations exhibited population expansion. Mexico also had greater nucleotide diversity and high levels of diversity for each gene. There was significant sub-division of the Trinidad and Mexico populations and low levels of genetic divergence among populations for three of the four gene regions; β-TUB was shown to be under positive selection. There were also dissimilar haplotype characteristics for both populations. Mutation may play a role in shaping the population structure of C. gloeosporioides sensu lato isolates from Trinidad and from Mexico, especially with respect to the ACT and GPDH gene regions. There was no evidence of gene flow between the C. truncatum populations and it is possible that the Mexico and Trinidad populations emerged independently of each other. Conclusions The study revealed relevant information based on the genetic structure as well as the demographic history of two fungal pathogens infecting papaya, C. gloeosporioides sensu lato and C. truncatum, in Trinidad and Mexico

  7. Genetic structure and demographic history of Colletotrichum gloeosporioides sensu lato and C. truncatum isolates from Trinidad and Mexico.

    PubMed

    Rampersad, Sephra N; Perez-Brito, Daisy; Torres-Calzada, Claudia; Tapia-Tussell, Raul; Carrington, Christine V F

    2013-06-22

    C. gloeosporioides sensu lato is one of the most economically important post-harvest diseases affecting papaya production worldwide. There is currently no information concerning the genetic structure or demographic history of this pathogen in any of the affected countries. Knowledge of molecular demographic parameters for different populations will improve our understanding of the biogeographic history as well as the evolutionary and adaptive potential of these pathogens. In this study, sequence data for ACT, GPDH, β-TUB and ITS gene regions were analyzed for C. gloeosporioides sensu lato and C. truncatum isolates infecting papaya in Trinidad and Mexico in order to determine the genetic structure and demographic history of these populations. The data indicated that Mexico is the ancestral C. gloeosporioides sensu lato population with asymmetrical migration to Trinidad. Mexico also had the larger effective population size but, both Mexico and Trinidad populations exhibited population expansion. Mexico also had greater nucleotide diversity and high levels of diversity for each gene. There was significant sub-division of the Trinidad and Mexico populations and low levels of genetic divergence among populations for three of the four gene regions; β-TUB was shown to be under positive selection. There were also dissimilar haplotype characteristics for both populations. Mutation may play a role in shaping the population structure of C. gloeosporioides sensu lato isolates from Trinidad and from Mexico, especially with respect to the ACT and GPDH gene regions. There was no evidence of gene flow between the C. truncatum populations and it is possible that the Mexico and Trinidad populations emerged independently of each other. The study revealed relevant information based on the genetic structure as well as the demographic history of two fungal pathogens infecting papaya, C. gloeosporioides sensu lato and C. truncatum, in Trinidad and Mexico. Understanding the genetic

  8. An Invasive Mammal (the Gray Squirrel, Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato.

    PubMed

    Millins, Caroline; Magierecka, Agnieszka; Gilbert, Lucy; Edoff, Alissa; Brereton, Amelia; Kilbride, Elizabeth; Denwood, Matt; Birtles, Richard; Biek, Roman

    2015-07-01

    Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n = 679), an invasive species in the United Kingdom, as B. burgdorferi sensu lato hosts. We found that gray squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi sensu lato in the United Kingdom, and 11.9% were infected with B. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospecies Borrelia garinii was most common. The second most frequent infection was with Borrelia afzelii. Genotyping of B. garinii and B. afzelii produced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi sensu lato infection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains of B. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species in B. burgdorferi sensu lato epidemiology can be highly variable and thus difficult to predict. Copyright © 2015, Millins et al.

  9. Comparative exine ontogeny in some members of the family Zygophyllaceae sensu lato.

    PubMed

    Nasri-Ayachi, M Ben; Nabli, M A

    2006-08-01

    Exine ontogeny is studied in five taxa of the family Zygophyllaceae sensu lato (Peganum harmala L., Zygophyllum album L., Fagonia cretica L., Tribulus terrestris L., and Nitraria retusa [Forsk.] Asch.). In the beginning of the tetrad stage, the plasmalemma is attached to the callose wall, except in T. terrestris, where it describes crests and hollows. The primexine matrix is fibrillar, bilayered in T. terrestris and unilayered in the other taxa. In all species except P. harmala, the procolumellae are heterogeneous with clear zones and they become compact later. In T. terrestris, they are built on the crests. In Z. album and T. terrestris, a primordial nexinic lamella is set up. It is tripartite with a white line seen at some levels; on its external leaflet, the foot layer is observed, and on its internal leaflet, there is the endexine with numerous lamellae. This white line disappears often in the mature exine. In T. terrestris, there is a thick nexine that is coarsely lamellate inside. In the aperture zone, the columellae are lacking, the tectum and the foot layer get thinner; they unite and form the apertural membrane with the external part of the endexine. There is a granulolamellar endexinic zone well developed in P. harmala, whereas it is threelayered and weakly developed in T. terrestris.

  10. Why are there several species of Borrelia burgdorferi sensu lato detected in dogs and humans?

    PubMed

    Skotarczak, Bogumiła

    2014-04-01

    Borrelia burgdorferi sensu lato is a group of spirochete bacteria species some of which cause borreliosis in humans and dogs. Humans and dogs are susceptible to illness from many of the same tick-borne pathogens, including B. burgdorferi s.l. (Bbsl). Little is known about the pathogenic role of the species of Bbsl in canines. The molecular methods which detect and amplify the DNA of borreliae and allow differentiating borreliae species or strains have not been used in canine diagnostics yet. Until now, it has been believed that in European dogs, like in humans, at least three pathogenic species occur but the most frequently described symptoms may be associated with the infection caused by B. burgdorferi sensu stricto species. A dog as well as a human is a host for many species of Bbsl, because borreliacidal ability of serum of dogs and humans is evident only in certain genospecies of Bbsl. Therefore both a dog and a human harbor more species than in case of some wild animal species which create older phylogenetic Bbsl species-host systems and these animals may act even as a non-competent reservoir host. Apart from many genospecies of Bbsl, a dog harbors other tick-borne agents and dual or triple infections may occur.

  11. Delineation of a new species of the Borrelia burgdorferi Sensu Lato Complex, Borrelia americana sp. nov.

    PubMed

    Rudenko, Nataliia; Golovchenko, Maryna; Lin, Tao; Gao, Lihui; Grubhoffer, Libor; Oliver, James H

    2009-12-01

    Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.

  12. Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis.

    PubMed

    Zwick, Michael E; Joseph, Sandeep J; Didelot, Xavier; Chen, Peter E; Bishop-Lilly, Kimberly A; Stewart, Andrew C; Willner, Kristin; Nolan, Nichole; Lentz, Shannon; Thomason, Maureen K; Sozhamannan, Shanmuga; Mateczun, Alfred J; Du, Lei; Read, Timothy D

    2012-08-01

    The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

  13. Seroprevalence of Borrelia burgdorferi sensu lato in roe deer (Capreolus capreolus) from northwestern Spain.

    PubMed

    Pato, Francisco J; Panadero, Rosario; Vázquez, Luis; López, Ceferino M; Díaz, Pablo; Vázquez, Esther; Díez-Baños, Pablo; Morrondo, Patrocinio; Fernández, Gonzalo

    2013-09-01

    In the present study, the seroprevalence of Borrelia burgdorferi sensu lato in roe deer in relation to different parameters in northwestern Spain was investigated. A total of 154 roe deer hunted between April 2007 and October 2008 from different localities of Galicia (northwest Spain) were examined. From each animal, a blood sample and all attached ticks found were collected. All the specimens for tick stages (larva, nymph, and adult) were speciated based on reference keys. Antibodies against B. burgdorferi were detected by indirect immunofluorescence (titer > or = 1:64). The percentage of roe deer seropositive for B. burgdorferi was 68.8% (106/ 154), of which 88.7% (94/106) were parasitized by ticks. Ixodes ricinus was the only species identified and was detected in 83.1% of roe deer with a mean (standard deviation [SD]) intensity of 46 +/- 47 ticks. Individual host characteristics such as age or sex did not have any effect on the prevalence of B. burgdorferi, but significant seasonal variation was observed, with higher prevalences in April-July than in August-October. Antibodies against B. burgdorferi were related to the presence of ticks. When analyzing all the factors together, the total number of ticks parasitizing roe deer was found as the most influential factor on B. burgdorferi prevalence. The results of this study have shown that roe deer in the northwest of Spain are highly exposed to B. burgdorferi and that exposure is related to the presence of I. ricinus.

  14. A molecular phylogeny and classification of Leptochloa (Poaceae: Chloridoideae: Chlorideae) sensu lato and related genera

    PubMed Central

    Peterson, Paul M.; Romaschenko, Konstantin; Snow, Neil; Johnson, Gabriel

    2012-01-01

    Background and Aims Leptochloa (including Diplachne) sensu lato (s.l.) comprises a diverse assemblage of C4 (NAD-ME and PCK) grasses with approx. 32 annual or perennial species. Evolutionary relationships and a modern classification of Leptochloa spp. based on the study of molecular characters have only been superficially investigated in four species. The goals of this study were to reconstruct the evolutionary history of Leptochloa s.l. with molecular data and broad taxon sampling. Methods A phylogenetic analysis was conducted of 130 species (mostly Chloridoideae), of which 22 are placed in Leptochloa, using five plastid (rpL32-trn-L, ndhA intron, rps16 intron, rps16-trnK and ccsA) and the nuclear ITS 1 and 2 (ribosomal internal transcribed spacer regions) to infer evolutionary relationships and revise the classification. Key results Leptochloa s.l. is polyphyletic and strong support was found for five lineages. Embedded within the Leptochloa sensu stricto (s.s.) clade are two Trichloris spp. and embedded in Dinebra are Drake-brockmania and 19 Leptochloa spp. Conclusions The molecular results support the dissolution of Leptochloa s.l. into the following five genera: Dinebra with 23 species, Diplachne with two species, Disakisperma with three species, Leptochloa s.s. with five species and a new genus, Trigonochloa, with two species. PMID:22628365

  15. Molecular phylogenetic analysis of Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) using plastid RBCL DNA sequences.

    PubMed

    Wurdack, Kenneth J; Hoffmann, Petra; Samuel, Rosabelle; de Bruijn, Anette; van der Bank, Michelle; Chase, Mark W

    2004-11-01

    Analysis of plastid rbcL DNA sequence data of the pantropical family Phyllanthaceae (Malpighiales) and related biovulate lineages of Euphorbiaceae sensu lato is presented. Sampling for this study includes representatives of all 10 tribes and 51 of the 60 genera attributed to Euphorbiaceae-Phyllanthoideae. Centroplacus and Putranjivaceae (Phyllanthoideae-Drypeteae) containing a paraphyletic Drypetes are excluded from Phyllanthaceae. Croizatia, previously thought to be a "basal" member of Euphorbiaceae-Oldfieldioideae (Picrodendraceae), falls within Phyllanthaceae. Phyllanthaceae with the mentioned adjustments form a monophyletic group consisting of two sister clades that mostly correspond to the distribution of tanniniferous leaf epidermal cells and inflorescence structure. With the exception of bigeneric Hymenocardieae and monotypic Bischofieae, none of the current Phyllanthoideae (Phyllanthaceae) tribal circumscriptions are supported by rbcL. Antidesma, Bischofia, Hymenocardia, Martretia, and Uapaca, all of which have previously been placed in monogeneric families, are confirmed as members of Phyllanthaceae. Savia is polyphyletic, and Cleistanthus appears paraphyletic. Paraphyly of Phyllanthus is also indicated, but this pattern lacks bootstrap support. Morphological characters are discussed and mapped for inflorescence structure, tanniniferous epidermal cells, breeding system, and fruit and embryo type. A table summarizes the main characters of six euphorbiaceous lineages.

  16. Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms.

    PubMed

    Dai, Yu-Cheng; Zhou, Li-Wei; Cui, Bao-Kai; Chen, Yan-Qiu; Decock, Cony

    2010-08-01

    Twenty-six species of Phellinus sensu lato, reported as medicinal mushrooms, are enumerated in this review. The species' names were checked and revised according to contemporary taxonomy and the latest version of the International Code of Botanical Nomenclature (Vienna Code); two misapplied names of Phellinus baumii Pilát and Phellinus himalayensis Y.C. Dai in previous reports are also discussed. Of the 20 types of medicinal functions, the most shared functions are antitumor and improving immunity, both of which may be viewed as the basal functions of Phellinus s. l. In addition, alleviating septic shock, anti-inflammatory, and antioxidation are also a routine functions mentioned often. The main medicinal metabolites, including several kinds of polysaccharides and polyphenols, are introduced. Different methods and conditions could purify various polysaccharides with difference in activity level even from the same species, while all polyphenols are hispidin and its derivatives in general. Three aspects of mechanism contribute to antitumor activities of polysaccharides: (1) promoting an immune response, (2) inducing cell apoptosis, and (3) inhibiting metastasis. Other general mechanisms of the metabolites in antioxidant activity, and in treating diabetes, as well as complications are summarized. We also elaborate on potential scientific strategies for obtaining the medicinal metabolites from Phellinus s. l., such as artificial cultivation, the discoveries of more species with medicinal functions, the utilization of species growing quickly, and the optimization of culture conditions and media supplements in fermentation.

  17. Update of phylogenetic and genetic diversity of Sporothrix schenckii sensu lato.

    PubMed

    Rangel-Gamboa, Lucía; Martínez-Hernandez, Fernando; Maravilla, Pablo; Arenas-Guzmán, Roberto; Flisser, Ana

    2016-03-01

    Sporothrix schenckii sensu lato causes subcutaneous mycosis. In this article we analysed its phylogeny and genetic diversity using calmodulin DNA sequences deposited in GenBank database. Population genetics indices were calculated, plus phylogenetic and haplotype network trees were built. Five clades with high values of posterior probability, 47 haplotypes and high diversity in the complex were found. Analysis of partial calmodulin sequences alignment revealed conserved and polymorphic regions that could be used as reference for taxonomic identification. The use of population genetics analysis allowed understanding the phylogenetic proximity of S. schenckii s. str. and S. brasiliensis; scarce genetic flow among them with low migration index and high ancestry coefficient was found. Similarly, S. globosa, S. mexicana and S. pallida sequences showed highly differentiated species with no genetic exchange. The phylogenetic tree suggests that S. mexicana shared a common ancestor with S. pallida; while S. globosa and S. brasiliensis are more related to S. schenckii s. str. and showed less haplotype diversity and restrictions in geographic distribution. In the haplotype network tree S. schenckii s. str. species displayed worldwide distribution without dispersion centres; while S. brasiliensis and S. globosa, exhibited Brazil and Euro-Asia as dispersion centres, respectively. Our data suggest that S. schenckii complex has been submitted to a divergent evolution process, probably due to the pressure of the environment and of the host. In contrast, S. brasiliensis could have been submitted to purifying selection or expansion process.

  18. Identification of Bacillus anthracis spore component antigens conserved across diverse Bacillus cereus sensu lato strains.

    PubMed

    Mukhopadhyay, Sanghamitra; Akmal, Arya; Stewart, Andrew C; Hsia, Ru-Ching; Read, Timothy D

    2009-06-01

    We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.

  19. Polysynovitis in a horse due to Borrelia burgdorferi sensu lato infection--Case study.

    PubMed

    Passamonti, Fabrizio; Veronesi, Fabrizia; Cappelli, Katia; Capomaccio, Stefano; Reginato, Alice; Miglio, Arianna; Vardi, Doron M; Stefanetti, Valentina; Coletti, Mauro; Bazzica, Chiara; Pepe, Marco

    2015-01-01

    Lyme borreliosis (LB) is a multi-systemic tick-borne disease affecting both humans and animals, including horses, and is caused by a group of interrelated spirochetes classified within the Borrelia burgdorferi sensu lato (s.l.) complex. Despite the high reported seroprevalence in the European equine population for B. burgdorferi s.l., to-date no documented clinical cases have been described. A 6-year-old Paint gelding was referred with a history of three weeks of fever, intermittent lameness and digital flexor tendon sheath effusion of the right hind limb. Based on a strict diagnostic protocol, which included serological tests for infectious diseases and molecular investigations, a final diagnosis was made of polysynovitis due to B. burgdorferi s.l. infection. An unreported aspect observed in this case was the absence of the pathogen DNA in two of the affected joints. To the authors' knowledge, the case described represents the first documented clinical case of equine LB in Italy. Moreover, the absence of pathogen DNA in two of the affected joints observed in this case revealed a possible similarity with the same condition described in humans, where an immunomediated pathogenesis for arthropathy due to B. burgdorferi s.l. infection is suspected. Since humans and horses share the same habitat, this report supports the role of the horse as potential sentinel for human biological risk.

  20. Structures of xyloglucans in primary cell walls of gymnosperms, monilophytes (ferns sensu lato) and lycophytes.

    PubMed

    Hsieh, Yves S Y; Harris, Philip J

    2012-07-01

    Little is known about the structures of the xyloglucans in the primary cell walls of vascular plants (tracheophytes) other than angiosperms. Xyloglucan structures were examined in 13 species of gymnosperms, 13 species of monilophytes (ferns sensu lato), and two species of lycophytes. Wall preparations were obtained, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-(1→4)-β-glucanase preparation. The oligosaccharides released were analysed by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and by high-performance anion-exchange chromatography. The xyloglucan oligosaccharide profiles from the gymnosperm walls were similar to those from the walls of most eudicotyledons and non-commelinid monocotyledons, indicating that the xyloglucans were fucogalactoxyloglucans, containing the fucosylated units XXFG and XLFG. The xyloglucan oligosaccharide profiles for six of the monilophyte species were similar to those of the gymnosperms, indicating they were also fucogalactoxyloglucans. Phylogenetically, these monilophyte species were from both basal and more derived orders. However, the profiles for the other monilophyte species showed various significant differences, including additional oligosaccharides. In three of the species, these additional oligosaccharides contained arabinosyl residues which were most abundant in the profile of Equisetum hyemale. The two species of lycophytes examined, Selaginella kraussiana and Lycopodium cernuum, had quite different xyloglucan oligosaccharide profiles, but neither were fucogalactoxyloglucans. The S. kraussiana profile had abundant oligosaccharides containing arabinosyl residues. The L. cernuum profile indicated the xyloglucan had a very complex structure.

  1. A taxonomic review of the Neoserica (sensu lato) abnormis group (Coleoptera, Scarabaeidae, Sericini)

    PubMed Central

    Ahrens, Dirk; Liu, Wan-Gang; Fabrizi, Silvia; Bai, Ming; Yang, Xing-Ke

    2014-01-01

    Abstract The present paper revises the species belonging to the Neoserica (sensu lato) abnormis group, so far known only with two nominal species. Twenty new species are herein described from Indochina and southern China: N. abnormoides sp. n. (Vietnam, China), N. allolaotica sp. n., N. namthaensis sp. n., N. simplicissima sp. n. (Laos), N. thailandensis sp. n. (Thailand), N. alloputaoana sp. n., N. kanphantensis sp. n., N. natmatoungensis sp. n., N. putaoana sp. n., N. taunggyiana sp. n. (Myanmar), N. lamellosa sp. n., N. tonkinea sp. n. (Vietnam), N. bairailingshanica sp. n., N. euyunnanica sp. n., N. huangi sp. n., N. jiangxiensis sp. n., N. trifida sp. n., N. yaoi sp. n., N. yingjiangensis sp. n. (China), N. cardamomensis sp. n. (Indochina and southern China). One new combination is established: Neoserica ponderosa Arrow, 1946, comb. n. The lectotypes of Neoserica abnormis Moser, 1908 and the taxonomically uncertain N. inclinata Brenske, 1898, which very likely also belongs to this species group, are designated herein. A key to the species and to species groups is given, the genitalia of all species including their habitus are illustrated. Maps of species distribution are included. PMID:25317056

  2. Molecular systematics in the acanthocephalan genus Echinorhynchus (sensu lato) in northern Europe.

    PubMed

    Väinölä, R; Valtonen, E T; Gibson, D I

    1994-01-01

    New biological species and high levels of inter- and intraspecific genetic divergence were discovered in an allozyme study of some North European members of the acanthocephalan genus Echinorhynchus (sensu lato), parasites of fish and malacostracan crustaceans. (i) A strong differentiation between the marine E. gadi and the fresh- and brackish-water E. salmonis (genetic identity I congruent to 0) supports a generic distinction between these taxa; however, the subdivision would not entirely concur with the concepts of Echinorhynchus (sensu stricto) and Metechinorhynchus suggested earlier. (ii) Samples of E. gadi from the Baltic, Norwegian and North Seas included three distinct, partially sympatric biological species (spp. I-III; I congruent to 0.5). (iii) E. bothniensis, previously only known from the northern Baltic Sea, represents a complex of freshwater taxa with an intermediate host relationship to the 'glacial relict' Mysis spp. and with a distributional and host analogy to the North American E. leidyi. A population in a northern lake in the Barents Sea basin is closely related to E. bothniensis of the Baltic area, but is probably specifically distinct; the divergence between these populations (I congruent to 0.6) is similar to that between their Mysis host species. (iv) Considerable intraspecific differentiation (FST = 0.25), probably reflecting post-glacial population bottlenecks, was found between Baltic and nearby lacustrine E. bothniensis, and between Atlantic and Baltic E. gadi sp. I.

  3. Borrelia burgdorferi sensu lato diversity and its influence on pathogenicity in humans.

    PubMed

    Baranton, Guy; De Martino, Sylvie J

    2009-01-01

    Among the Spirochaetes, the Borrelia burgdorferi sensu lato complex is responsible for Lyme borreliosis. This complex comprises more than 13 Borrelia species. Four of them are clearly pathogenic for humans: B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. spielmanii. They can generate erythema migrans, an initial skin lesion, and can then spread deeply into the host to invade distant tissues, especially the nervous system, the joints or the skin. In humans, Borrelia pathogenicityseems to be linked with taxonomic position, but in vitro studies show the role of plasmids in B. burgdorferi s.l. pathogenesis. The inter- and intraspecies genetic diversity of B. burgdorferi s.l. evidences a clonal evolution of the chromosome, while plasmid genes are quite variable, suggesting their major role in Borrelia adaptability. The plasmid-encoded adhesins and vlse, crasps and osp genes determine invasiveness and host immune evasion of B. burgdorferi s.l., and select the bacterial host spectrum. The geographic distribution of B. burgdorferi s.l. is closely related to its vectors and competent hosts, and its development within these influences its diversity, taxonomy and pathogenesis, primarily via genetic lateral transfer. Copyright 2009 S. Karger AG, Basel.

  4. First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli).

    PubMed

    Schramm, Frédéric; Gauthier-Clerc, Michel; Fournier, Jean-Charles; McCoy, Karen D; Barthel, Cathy; Postic, Danièle; Handrich, Yves; Le Maho, Yvon; Jaulhac, Benoît

    2014-10-01

    The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes. Copyright © 2014 Elsevier GmbH. All rights reserved.

  5. First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico.

    PubMed

    Rodriguez-Vivas, R I; Ojeda-Chi, M M; Trinidad-Martinez, I; Bolio-González, M E

    2017-03-01

    Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose-mortality regressions, 50% lethal concentrations (LC50 ), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter-population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC50 : 1.0-13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter-population variation in the phenotypic level of resistance was evident (RRs at LC50 : 1.0-104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.

  6. Rubisco Evolution in C4 Eudicots: An Analysis of Amaranthaceae Sensu Lato

    PubMed Central

    Kapralov, Maxim V.; Smith, J. Andrew C.; Filatov, Dmitry A.

    2012-01-01

    Background Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO2. In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots. Methodology/Principal Findings We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution. Conclusions/Significance Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco. PMID:23285238

  7. Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe.

    PubMed

    Skuballa, Jasmin; Petney, Trevor; Pfäffle, Miriam; Oehme, Rainer; Hartelt, Kathrin; Fingerle, Volker; Kimmig, Peter; Taraschewski, Horst

    2012-02-01

    In order to determine whether European hedgehogs (Erinaceus europaeus and E. roumanicus) play a role in the epidemiological cycle of Borrelia burgdorferi sensu lato in Central Europe and Great Britain, tissue samples of hedgehogs from Germany (n=211), Austria (n=4), the Czech Republic (n=22), and the U.K. (n=32) were tested for the presence of these tick-borne pathogens. PCR for amplification of the B. burgdorferi s.l.-specific 5S-23S intergenic spacer region as well as the outer surface protein A (ospA) gene were used. B. burgdorferi s.l. DNA was detected in 35 of the 259 E. europaeus and in 2 of 10 E. roumanicus. B. burgdorferi prevalences in E. europaeus ranged from 0% (U.K.) to 37.5% (Czech Republic), for E. roumanicus from 0% (Czech Republic) to 50.0% (Austria). Sequencing revealed the occurrence of 3 different B. burgdorferi genospecies in E. europaeus: B. afzelii was the dominant genospecies, followed by B. bavariensis (previously B. garinii OspA serotype 4) and B. spielmanii, the latter was detected for the first time in Hamburg (Germany). B. afzelii and B. bavariensis were also found in E. roumanicus. Our results suggest that hedgehogs modulate the epidemiology of certain species of the B. burgdorferi s.l. complex, potentially affecting the distribution and abundance of individual B. burgdorferi s.l. genospecies in various habitats. We hypothesise that juvenile or individuals with low immune competence in particular, have a high reservoir potential for the 3 genospecies identified here.

  8. Established Population of Blacklegged Ticks with High Infection Prevalence for the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, on Corkscrew Island, Kenora District, Ontario.

    PubMed

    Scott, John D; Foley, Janet E; Clark, Kerry L; Anderson, John F; Durden, Lance A; Manord, Jodi M; Smith, Morgan L

    2016-01-01

    We document an established population of blacklegged ticks, Ixodes scapularis, on Corkscrew Island, Kenora District, Ontario, Canada. Primers of the outer surface protein A (OspA) gene, the flagellin (fla) gene, and the flagellin B (flaB) gene were used in the PCR assays to detect Borrelia burgdorferi sensu lato (s.l.), the Lyme disease bacterium. In all, 60 (73%) of 82 adult I. scapularis, were infected with B. burgdorferi s.l. As well, 6 (43%) of 14 unfed I. scapularis nymphs were positive for B. burgdorferi s.l. An I. scapularis larva was also collected from a deer mouse, and several unfed larvae were gathered by flagging leaf litter. Based on DNA sequencing of randomly selected Borrelia amplicons from six nymphal and adult I. scapularis ticks, primers for the flagellin (fla) and flagellin B (flaB) genes reveal the presence of B. burgdorferi sensu stricto (s.s.), a genospecies pathogenic to humans and certain domestic animals. We collected all 3 host-feeding life stages of I. scapularis in a single year, and report the northernmost established population of I. scapularis in Ontario. Corkscrew Island is hyperendemic for Lyme disease and has the highest prevalence of B. burgdorferi s.l. for any established population in Canada. Because of this very high infection prevalence, this population of I. scapularis has likely been established for decades. Of epidemiological significance, cottage owners, island visitors, outdoors enthusiasts, and medical professionals must be vigilant that B. burgdorferi s.l.-infected I. scapularis on Corkscrew Island pose a serious public health risk.

  9. Established Population of Blacklegged Ticks with High Infection Prevalence for the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, on Corkscrew Island, Kenora District, Ontario

    PubMed Central

    Scott, John D.; Foley, Janet E.; Clark, Kerry L.; Anderson, John F.; Durden, Lance A.; Manord, Jodi M.; Smith, Morgan L.

    2016-01-01

    We document an established population of blacklegged ticks, Ixodes scapularis, on Corkscrew Island, Kenora District, Ontario, Canada. Primers of the outer surface protein A (OspA) gene, the flagellin (fla) gene, and the flagellin B (flaB) gene were used in the PCR assays to detect Borrelia burgdorferi sensu lato (s.l.), the Lyme disease bacterium. In all, 60 (73%) of 82 adult I. scapularis, were infected with B. burgdorferi s.l. As well, 6 (43%) of 14 unfed I. scapularis nymphs were positive for B. burgdorferi s.l. An I. scapularis larva was also collected from a deer mouse, and several unfed larvae were gathered by flagging leaf litter. Based on DNA sequencing of randomly selected Borrelia amplicons from six nymphal and adult I. scapularis ticks, primers for the flagellin (fla) and flagellin B (flaB) genes reveal the presence of B. burgdorferi sensu stricto (s.s.), a genospecies pathogenic to humans and certain domestic animals. We collected all 3 host-feeding life stages of I. scapularis in a single year, and report the northernmost established population of I. scapularis in Ontario. Corkscrew Island is hyperendemic for Lyme disease and has the highest prevalence of B. burgdorferi s.l. for any established population in Canada. Because of this very high infection prevalence, this population of I. scapularis has likely been established for decades. Of epidemiological significance, cottage owners, island visitors, outdoors enthusiasts, and medical professionals must be vigilant that B. burgdorferi s.l.-infected I. scapularis on Corkscrew Island pose a serious public health risk. PMID:27877080

  10. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  11. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  12. Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America.

    PubMed

    Avila, Héctor G; Santos, Guilherme B; Cucher, Marcela A; Macchiaroli, Natalia; Pérez, Matías G; Baldi, Germán; Jensen, Oscar; Pérez, Verónica; López, Raúl; Negro, Perla; Scialfa, Exequiel; Zaha, Arnaldo; Ferreira, Henrique B; Rosenzvit, Mara; Kamenetzky, Laura

    2017-06-01

    The aim of this work was to determine Echinococcus granulosus sensu lato species and genotypes in intermediate and definitive hosts and in human isolates from endemic regions of Argentina and Brazil including those where no molecular data is available by a combination of classical and alternative molecular tools. A total of 227 samples were isolated from humans, natural intermediate and definitive hosts. Amplification of cytochrome c oxidase subunit I gene fragment was performed and a combination of AluI digestion assay, High Resolution Melting analysis (HRM) assay and DNA sequencing was implemented for Echinococcus species/genotype determination. E. granulosus sensu stricto (G1) was found in sheep (n=35), cattle (n=67) and dogs (n=5); E. ortleppi (G5) in humans (n=3) and cattle (n=108); E. canadensis (G6) in humans (n=2) and E. canadensis (G7) in pigs (n=7). We reported for the first time the presence of E. ortleppi (G5) and E. canadensis (G6) in humans from San Juan and Catamarca Argentinean provinces and E. canadensis (G7) in pigs from Cordoba Argentinean province. In this work, we widened molecular epidemiology studies of E. granulosus s. l. in South America by analyzing several isolates from definitive and intermediate hosts, including humans from endemic regions were such information was scarce or unavailable. The presence of different species/genotypes in the same region and host species reinforce the need of rapid and specific techniques for accurate determination of Echinococcus species such as the ones proposed in this work.

  13. Borrelia burgdorferi Sensu Lato in Siberian chipmunks (Tamias sibiricus) introduced in suburban forests in France.

    PubMed

    Vourc'h, Gwenaël; Marmet, Julie; Chassagne, Michelle; Bord, Séverine; Chapuis, Jean-Louis

    2007-01-01

    Numerous vertebrate reservoirs have been described for Borrelia burgdorferi sensu lato (sl), which includes the etiological agents of Lyme Borreliosis (LB). The Siberian chipmunk (Tamias sibiricus) is a rodent originating from Asia, where it is suspected to be a B. burgdorferi reservoir. It has been intentionally released into the wild in Europe since the 1970s, but has not yet been subject to any study regarding its association with the LB agent. In this paper we studied Siberian chipmunk infestation with the LB vector (Ixodes ricinus) and infection prevalence by LB spirochetes in a suburban introduced population. We compared these findings with known competent reservoir hosts, the bank vole (Myodes [clethrionomys] glareolus) and wood mouse (Apodemus sylvaticus). All Siberian chipmunks were infested with larvae and larval abundance was higher in this species (mean number of larvae [95% Confidence Interval]: 73.5 [46.0, 117.2]) than in the two other rodent species (bank voles: 4.4 [3.0, 6.3] and wood mice: 10.2 [4.9, 21.2]). Significant factors affecting abundance of larvae were host species and sampling season. Nymphs were most prevalent on chipmunks (86.2%, mean: 5.1 [3.3, 8.0]), one vole carried only two nymphs, and none of the mice had any nymphs. Nymph abundance in chipmunks was affected by sampling season and sex. Furthermore, the infection prevalence of B. burgdorferi sl in the Siberian chipmunk was the highest (33.3%) and predominantly of B. afzelii. The infection prevalence was 14.1% in bank voles, but no wood mouse was found to be infected. Our results suggest that the Siberian chipmunk may be an important reservoir host for LB.

  14. Clinical characteristics associated with Borrelia burgdorferi sensu lato skin culture results in patients with erythema migrans.

    PubMed

    Strle, Franc; Lusa, Lara; Ružić-Sabljić, Eva; Maraspin, Vera; Lotrič Furlan, Stanka; Cimperman, Jože; Ogrinc, Katarina; Rojko, Tereza; Videčnik Zorman, Jerneja; Stupica, Daša

    2013-01-01

    Clinical characteristics associated with isolation of Borrelia burgdorferi sensu lato from skin have not been fully evaluated. To gain insight into predictors for a positive EM skin culture, we compared basic demographic, epidemiologic, and clinical data in 608 culture-proven and 501 culture-negative adult patients with solitary EM. A positive Borrelia spp. skin culture was associated with older age, a time interval of >2 days between tick bite and onset of the skin lesion, EM ≥ 5 cm in diameter, and location of the lesion on the extremities, whereas several other characteristics used as clinical case definition criteria for the diagnosis of EM (such as tick bite at the site of later EM, information on expansion of the skin lesion, central clearing) were not. A patient with a 15-cm EM lesion had almost 3-fold greater odds for a positive skin culture than patients with a 5-cm lesion. Patients with a free time interval between the tick bite and onset of EM had the same probability of a positive skin culture as those who did not recall a tick bite (OR=1.02); however, the two groups had >3-fold greater odds for EM positivity than patients who reported a tick bite with no interval between the bite and onset of the lesion. In conclusion, several yet not all clinical characteristics used in EM case definitions were associated with positive Borrelia spp. skin culture. The findings are limited to European patients with solitary EM caused predominantly by B. afzelii but may not be valid for other situations.

  15. Molecular Typing of Borrelia burgdorferi Sensu Lato by Randomly Amplified Polymorphic DNA Fingerprinting Analysis

    PubMed Central

    Wang, Guiqing; van Dam, Alje P.; Spanjaard, Lodewijk; Dankert, Jacob

    1998-01-01

    To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species. PMID:9508310

  16. Evolutionary trends in venom composition in the western rattlesnakes (Crotalus viridis sensu lato): toxicity vs. tenderizers.

    PubMed

    Mackessy, Stephen P

    2010-07-01

    The Western Rattlesnake (Crotalus viridis sensu lato, now including Crotalus oreganus) is broadly distributed across the western half of the United States, northwestern Mexico and southwestern Canada, and eight subspecies are currently recognized. Although some venom characteristics have been noted for most subspecies, a systematic study of venoms from all subspecies has not been reported. Venom was extracted from snakes collected from approximate geographic range centers for all subspecies and analyzed using SDS-PAGE, MALDI-TOF mass spectrometry, enzyme and toxicity assays. Electrophoretic and mass spectrometric analyses demonstrated that small myotoxins, disintegrins and PLA(2) were abundant in most venoms. PIII and PI metalloproteinases ( approximately 54 kDa and 23 kDa, respectively) were common to all venoms except C. o. concolor, C. o. caliginis and C.o. helleri. Metalloproteinase activity was highest in C. o. cerberus and lowest in C. o. concolor venoms ( approximately 100-fold difference). Conversely, C. o. concolor venom was the most toxic and C. o. cerberus venom was least toxic (15-fold difference). In general, venoms with high metalloproteinase activity were less toxic (type I venoms), while venoms which were highly toxic showed low protease activity (type II venoms). Within the C. viridis/oreganus complex, these two extremes of venom compositional phenotypes are observed, and it appears that high metalloproteinase activity and high toxicity are incompatible qualities of these venoms. The functional significance of these biochemical characteristics likely relates to characteristics of prey consumed, and venoms with low metalloproteinase activity may constrain snake prey selection or foraging activity patterns.

  17. Trematode diversity in the freshwater snail Bithynia siamensis goniomphalos sensu lato from Thailand and Lao PDR.

    PubMed

    Kiatsopit, N; Sithithaworn, P; Kopolrat, K; Namsanor, J; Andrews, R H; Petney, T N

    2016-05-01

    In order to obtain a comprehensive understanding of trematode diversity in Bithynia siamensis goniomphalos sensu lato, the first intermediate host of the liver fluke Opisthorchis viverrini s.l., the prevalence of larval trematode species was investigated in different localities in Thailand and Lao People's Democratic Republic (Lao PDR). In Thailand, snail samples were collected from 29 localities in the nine provinces: Buri Ram, Surin, Chaiya Phum, Maha Sarakham, Khon Kaen, Kalasin, Mukdahan, Sakon Nakhon and Nakhon Phanom. In Lao PDR, snail samples were collected from 21 localities in Vientiane Province and six localities in Savannakhet Province. Snails were identified by standard morphological criteria and then examined for trematode infection using the cercarial shedding method. Twenty different types of cercariae were detected and identified, based on morphological criteria. Virgulate type 1 emerged as the most common cercaria, with an average prevalence of 10.90% (range 0.26-54.22%) in Thailand and 6.58% (range 1.15-89.77%) in Lao PDR. Opisthorchis viverrini s.l. cercariae were the fourth most common in Thailand, with an average prevalence of 1.59% (0.15-6.93), while in Lao PDR their prevalence was 0.96% (0.08-8.37). The high diversity of trematode cercariae observed in this study indicates that B. s. goniomphalos s.l. is highly susceptible to infection with a variety of trematode species. However, the role of non-opisthorchiid trematodes as fish-borne parasites in human health is not fully known and further molecular identification is required.

  18. Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal.

    PubMed

    Dantas-Torres, Filipe; Maia, Carla; Latrofa, Maria Stefania; Annoscia, Giada; Cardoso, Luís; Otranto, Domenico

    2017-03-13

    The taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal. A total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens. The ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.) CONCLUSIONS: These results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this.

  19. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds

    PubMed Central

    2013-01-01

    Background The taxonomic status of the brown dog tick (Rhipicephalus sanguineus sensu stricto), which has long been regarded as the most widespread tick worldwide and a vector of many pathogens to dogs and humans, is currently under dispute. Methods We conducted a comprehensive morphological and genetic study of 278 representative specimens, which belonged to different species (i.e., Rhipicephalus bursa, R. guilhoni, R. microplus, R. muhsamae, R. pusillus, R. sanguineus sensu lato, and R. turanicus) collected from Europe, Asia, Americas, and Oceania. After detailed morphological examination, ticks were molecularly processed for the analysis of partial mitochondrial (16S rDNA, 12S rDNA, and cox1) gene sequences. Results In addition to R. sanguineus s.l. and R. turanicus, three different operational taxonomic units (namely, R. sp. I, R. sp. II, and R. sp. III) were found on dogs. These operational taxonomical units were morphologically and genetically different from R. sanguineus s.l. and R. turanicus. Ticks identified as R. sanguineus s.l., which corresponds to the so-called “tropical species” (=northern lineage), were found in all continents and genetically it represents a sister group of R. guilhoni. R. turanicus was found on a wide range of hosts in Italy and also on dogs in Greece. Conclusions The tropical species and the temperate species (=southern lineage) are paraphyletic groups. The occurrence of R. turanicus in the Mediterranean region is confirmed. A consensual re-description of R. sanguineus s.s. and R. turanicus will be necessary to solve the taxonomic problems within the so-called R. sanguineus group. PMID:23880226

  20. First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

    PubMed

    Rodriguez-Vivas, R I; Ojeda-Chi, M M; Trinidad-Martinez, I; Pérez de León, A A

    2017-01-15

    The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l. Interviews were conducted with dog owners to gather information on the history of dog treatment with conventional acaricides and IVM. The larval immersion test was used on the progeny of adult female ticks infesting dogs to test for susceptibility to IVM. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals, and slope were estimated by probit analysis. Ten tick populations (66.7%) were classified as resistant compared with the most susceptible population. A high inter-population variation in the phenotypic level of IVM resistance was evident (resistance ratio at LC50% and LC99% ranged from 1.0 to 30.5, and from 1.0 to 458.8, respectively). Tick populations classified as resistant were collected from dogs known to have been treated with IVM. To our knowledge, this is the first report of IVM resistance in R. sanguineus s.l. worldwide. Veterinary and pet owner education on integrated tick management practices is required to avoid widespread resistance and increased treatment failure with products containing IVM and other macrocyclic lactones used to control endo- and ectoparasites affecting dogs. Integrated tick management will also help mitigate the burden of brown dog tick-borne diseases on human and animal populations. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato in peri-urban forests of France.

    PubMed

    Marchant, Axelle; Le Coupanec, Alain; Joly, Claire; Perthame, Emeline; Sertour, Natacha; Garnier, Martine; Godard, Vincent; Ferquel, Elisabeth; Choumet, Valerie

    2017-01-01

    Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carry bacteria belonging to the Borrelia burgdorferi sensu lato complex. The objective of this work was to explore eco-epidemiological factors of Lyme borreliosis in peri-urban forests of France (Sénart, Notre-Dame and Rambouillet). We investigated whether the introduction of Tamias sibiricus in Sénart could alter the density of infected ticks. Moreover, the density and tick infection were investigated according to the tree species found in various patches of Sénart forest. For this purpose, ticks were sampled during 3 years. In the Sénart forest, the density of nymph and adult ticks showed no significant difference between 2008, 2009 and 2011. The nymph density varied significantly as a function of the month of collection. Regarding the nymphs, a higher rate of infection and infected density were found in 2009. Plots with chipmunks (C) presented a lower density of both nymphs and adult ticks than plots without chipmunks (NC) did. A higher rate of infection of nymphs with Borrelia was seen in C plots. The prevalence of the various species of Borrelia was also found to vary between C and NC plots with the year of the collect. The presence of chestnut trees positively influenced the density of both nymphs and adults. The infected nymph density showed a significant difference depending on the peri-urban forest studied, Sénart being higher than Rambouillet. The prevalence of Borrelia species also differed between the various forests studied. Concerning the putative role that Tamias sibiricus may play in the transmission of Borrelia, our results suggest that its presence is correlated with a higher rate of infection of questing ticks by Borrelia genospecies and if its population increases, it could play a significant role in the risk of transmission of Lyme borreliosis.

  2. Signatures of recombination in clonal lineages of the citrus brown spot pathogen, Alternaria alternata sensu lato.

    PubMed

    Stewart, Jane E; Thomas, Kalyn A; Lawrence, Christopher B; Dang, Ha; Pryor, Barry M; Timmer, L M Pete; Peever, Tobin L

    2013-07-01

    Most Alternaria spp. are considered asexual but recent molecular evolution analyses of Alternaria mating-type genes show that the mating locus is under strong purifying selection, indicating a possible role in sexual reproduction. The objective of this study was to determine the mode of reproduction of an Alternaria alternata sensu lato population causing citrus brown spot in central Florida. Mating type of each isolate was determined, and isolates were sequenced at six putatively unlinked loci. Three genetically distinct subpopulations (SH1, SH4A, and SH4B) were identified using network and Bayesian population structure analyses. Results demonstrate that most subpopulations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle or parasexual cycle. Although isolates were sampled in close physical proximity (≈2,500-m² area), we were able to reject a random mating model using multilocus gametic disequilibrium tests for two subpopulations, SH1 and SH4B, suggesting that these subpopulations were predominantly asexual. However, three recombination events were identified in SH1 and SH4B and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third subpopulation (SH4A), where only one mating type was present, extensive reticulation was evident in network analyses, and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a nonmeiotic mechanism of recombination such as the parasexual cycle may be operating in this subpopulation. The level of gene flow detected among subpopulations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these A. alternata subpopulations.

  3. Detection of Borrelia burgdorferi sensu lato in lizards and their ticks from Hungary.

    PubMed

    Földvári, Gábor; Rigó, Krisztina; Majláthová, Viktória; Majláth, Igor; Farkas, Róbert; Pet'ko, Branislav

    2009-06-01

    To investigate the involvement of lizard species in the natural cycle of Borrelia burgdorferi sensu lato (s.l.) in Hungary, a total of 186 reptiles belonging to three species--126 green lizards (Lacerta viridis), 40 Balkan wall lizards (Podarcis taurica), and 20 sand lizards (Lacerta agilis)--were captured in 2007 and 2008. All ticks removed from the lizards were Ixodes ricinus, either larvae (324/472; 68.6%) or nymphs (148/472; 31.4%). More than half (66/126; 52.4%) of L. viridis individuals were infested, and the prevalence of tick infestation on both the other two species was 35% each. All 472 I. ricinus ticks and tissue samples collected from 134 collar scales and 62 toe clips of lizards were further analyzed for the presence of B. burgdorferi s.l. with polymerase chain reaction. The amplification of B. burgdorferi s.l. DNA was successful in 8% (n = 92) of L. viridis, 9% (n = 32) of P. taurica, and 10% (n = 10) of L. agilis tissue samples. Restriction fragment length polymorphism genotyping identified the species Borrelia lusitaniae in all tested lizard samples. Prevalence of B. burgdorferi s.l. in ticks collected from L. viridis, P. taurica, and L. agilis was 8%, 2%, and 0%, respectively. Most of the infected ticks carried B. lusitaniae (74% of genotyped positives); however, Borrelia afzelii (5%) and B. burgdorferi sensu stricto (21%) were detected in ticks removed from green lizards and Balkan wall lizards, respectively. We conclude that lizards, particularly L. viridis, can be important hosts for I. ricinus larvae and nymphs; thus, they can be regarded as reservoirs of these important pathogen vectors. The role of green lizards has been confirmed, and the implication of Balkan wall lizards is suggested in the natural cycle of B. lusitaniae at our study site.

  4. Retreat and extinction of the Late Pleistocene cave bear (Ursus spelaeus sensu lato).

    PubMed

    Baca, Mateusz; Popović, Danijela; Stefaniak, Krzysztof; Marciszak, Adrian; Urbanowski, Mikołaj; Nadachowski, Adam; Mackiewicz, Paweł

    2016-12-01

    The cave bear (Ursus spelaeus sensu lato) is a typical representative of Pleistocene megafauna which became extinct at the end of the Last Glacial. Detailed knowledge of cave bear extinction could explain this spectacular ecological transformation. The paper provides a report on the youngest remains of the cave bear dated to 20,930 ± 140 (14)C years before present (BP). Ancient DNA analyses proved its affiliation to the Ursus ingressus haplotype. Using this record and 205 other dates, we determined, following eight approaches, the extinction time of this mammal at 26,100-24,300 cal. years BP. The time is only slightly earlier, i.e. 27,000-26,100 cal. years BP, when young dates without associated collagen data are excluded. The demise of cave bear falls within the coldest phase of the last glacial period, Greenland Stadial 3. This finding and the significant decrease in the cave bear records with cooling indicate that the drastic climatic changes were responsible for its extinction. Climate deterioration lowered vegetation productivity, on which the cave bear strongly depended as a strict herbivore. The distribution of the last cave bear records in Europe suggests that this animal was vanishing by fragmentation into subpopulations occupying small habitats. One of them was the Kraków-Częstochowa Upland in Poland, where we discovered the latest record of the cave bear and also two other, younger than 25,000 (14)C years BP. The relatively long survival of this bear in karst regions may result from suitable microclimate and continuous access to water provided by deep aquifers, indicating a refugial role of such regions in the Pleistocene for many species.

  5. Retreat and extinction of the Late Pleistocene cave bear ( Ursus spelaeus sensu lato)

    NASA Astrophysics Data System (ADS)

    Baca, Mateusz; Popović, Danijela; Stefaniak, Krzysztof; Marciszak, Adrian; Urbanowski, Mikołaj; Nadachowski, Adam; Mackiewicz, Paweł

    2016-12-01

    The cave bear ( Ursus spelaeus sensu lato) is a typical representative of Pleistocene megafauna which became extinct at the end of the Last Glacial. Detailed knowledge of cave bear extinction could explain this spectacular ecological transformation. The paper provides a report on the youngest remains of the cave bear dated to 20,930 ± 140 14C years before present (BP). Ancient DNA analyses proved its affiliation to the Ursus ingressus haplotype. Using this record and 205 other dates, we determined, following eight approaches, the extinction time of this mammal at 26,100-24,300 cal. years BP. The time is only slightly earlier, i.e. 27,000-26,100 cal. years BP, when young dates without associated collagen data are excluded. The demise of cave bear falls within the coldest phase of the last glacial period, Greenland Stadial 3. This finding and the significant decrease in the cave bear records with cooling indicate that the drastic climatic changes were responsible for its extinction. Climate deterioration lowered vegetation productivity, on which the cave bear strongly depended as a strict herbivore. The distribution of the last cave bear records in Europe suggests that this animal was vanishing by fragmentation into subpopulations occupying small habitats. One of them was the Kraków-Częstochowa Upland in Poland, where we discovered the latest record of the cave bear and also two other, younger than 25,000 14C years BP. The relatively long survival of this bear in karst regions may result from suitable microclimate and continuous access to water provided by deep aquifers, indicating a refugial role of such regions in the Pleistocene for many species.

  6. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds.

    PubMed

    Dantas-Torres, Filipe; Latrofa, Maria Stefania; Annoscia, Giada; Giannelli, Alessio; Parisi, Antonio; Otranto, Domenico

    2013-07-23

    The taxonomic status of the brown dog tick (Rhipicephalus sanguineus sensu stricto), which has long been regarded as the most widespread tick worldwide and a vector of many pathogens to dogs and humans, is currently under dispute. We conducted a comprehensive morphological and genetic study of 278 representative specimens, which belonged to different species (i.e., Rhipicephalus bursa, R. guilhoni, R. microplus, R. muhsamae, R. pusillus, R. sanguineus sensu lato, and R. turanicus) collected from Europe, Asia, Americas, and Oceania. After detailed morphological examination, ticks were molecularly processed for the analysis of partial mitochondrial (16S rDNA, 12S rDNA, and cox1) gene sequences. In addition to R. sanguineus s.l. and R. turanicus, three different operational taxonomic units (namely, R. sp. I, R. sp. II, and R. sp. III) were found on dogs. These operational taxonomical units were morphologically and genetically different from R. sanguineus s.l. and R. turanicus. Ticks identified as R. sanguineus s.l., which corresponds to the so-called "tropical species" (= northern lineage), were found in all continents and genetically it represents a sister group of R. guilhoni. R. turanicus was found on a wide range of hosts in Italy and also on dogs in Greece. The tropical species and the temperate species (= southern lineage) are paraphyletic groups. The occurrence of R. turanicus in the Mediterranean region is confirmed. A consensual re-description of R. sanguineus s.s. and R. turanicus will be necessary to solve the taxonomic problems within the so-called R. sanguineus group.

  7. Epidemiology and echinocandin susceptibility of Candida parapsilosis sensu lato species isolated from bloodstream infections at a Spanish university hospital

    PubMed Central

    Garcia-Effron, Guillermo; Canton, Emilia; Pemán, Javier; Dilger, Amanda; Romá, Eva; Perlin, David S.

    2012-01-01

    Objectives The aims of this work were to study the epidemiological profiles, differences in echinocandin susceptibilities and clinical relevance of the Candida parapsilosis sensu lato species isolated from proven fungaemia cases at La Fe University Hospital of Valencia (Spain) from 1995 to 2007. Results The prevalence of these species was: C. parapsilosis sensu stricto, 74.4%; Candida orthopsilosis, 23.54%; and Candida metapsilosis, 2.05%. The incidence of the species complex as agents of fungaemia remained stationary until 2005 and doubled in 2006. The incidence of C. orthopsilosis showed an increasing trend during the study period, while C. parapsilosis sensu stricto incidence diminished. Also, an important epidemiological change was observed starting in 2004, when 86.5% of the C. parapsilosis sensu lato strains were found in adult patients, while before that year only 13.5% of the isolates were found in this population. Conclusions Echinocandin drug susceptibility testing using the CLSI M27-A3 document showed a wide range of MIC values (0.015–4 mg/L), with micafungin being the most potent in vitro inhibitor followed by anidulafungin and caspofungin (MIC geometric mean of 0.68, 0.74 and 0.87 mg/L, respectively). C. metapsilosis was the most susceptible species of the complex to anidulafungin and micafungin in vitro (MIC50 for anidulafungin and micafungin: 0.06 mg/L), while there were no differences between C. parapsilosis sensu lato species when caspofungin MIC50s were compared (MIC50 1.00 mg/L). Differences in caspofungin in vitro susceptibility were observed between the different clinical service departments of La Fe Hospital. PMID:22868644

  8. Isolation of live Borrelia burgdorferi sensu lato spirochaetes from patients with undefined disorders and symptoms not typical for Lyme borreliosis.

    PubMed

    Rudenko, N; Golovchenko, M; Vancova, M; Clark, K; Grubhoffer, L; Oliver, J H

    2016-03-01

    Lyme borreliosis is a multisystem disorder with a diverse spectrum of clinical manifestations, caused by spirochaetes of the Borrelia burgdorferi sensu lato complex. It is an infectious disease that can be successfully cured by antibiotic therapy in the early stages; however, the possibility of the appearance of persistent signs and symptoms of disease following antibiotic treatment is recognized. It is known that Lyme borreliosis mimics multiple diseases that were never proven to have a spirochaete aetiology. Using complete modified Kelly-Pettenkofer medium we succeeded in cultivating live B. burgdorferi sensu lato spirochaetes from samples taken from people who suffered from undefined disorders, had symptoms not typical for Lyme borreliosis, but who had undergone antibiotic treatment due to a suspicion of having Lyme disease even though they were seronegative. We report the first recovery of live B. burgdorferi sensu stricto from residents of southeastern USA and the first successful cultivation of live Borrelia bissettii-like strain from residents of North America. Our results support the fact that B. bissettii is responsible for human Lyme borreliosis worldwide along with B. burgdorferi s.s. The involvement of new spirochaete species in Lyme borreliosis changes the understanding and recognition of clinical manifestations of this disease.

  9. The propensity of voles and mice to transmit Borrelia burgdorferi sensu lato infection to feeding ticks.

    PubMed

    Radzijevskaja, Jana; Paulauskas, Algimantas; Rosef, Olav; Petkevičius, Saulius; Mažeika, Vytautas; Rekašius, Tomas

    2013-10-18

    Lyme borreliosis (LB) caused by the spirochete Borrelia burgdorferi sensu lato is the most common tick-borne zoonosis in the Northern Hemisphere. B. burgdorferi s.l. can infect humans and wild and domestic animals. Ixodes ricinus is the main vector, and small rodents are the most important mammalian reservoirs hosts of B. burgdorferi s.l. in Europe. The prevalence of B. burgdorferi s.l. in I. ricinus ticks from captured rodents, calculated specific infectivities, and transmission coefficients were estimated in order to investigate the role of voles and mice in transmission of the LB causative agent. A total of 12.3% (53 out of 431) of immature I. ricinus ticks from rodents in Lithuania and 3.25% (21 out of 646) in Norway were infected with B. burgdorferi s.l. In Lithuania a total of 40% infested Microtus arvalis, 29% of Myodes glareolus and 4.8% of Apodemus flavicollis carried infected larvae and 67% of M. glareolus, 36% of M. arvalis but none of A. flavicollis carried infected nymphs. In Norway, 2.4% of larvae and 12.1% of nymphs feeding on A. flavicollis were infected. A total of 9% of infested A. flavicollis carried infected larvae and 13% - infected nymphs. Borrelia afzelii was the single genospecies identified in ticks feeding on rodents in Lithuania, and was predominant in ticks collected from rodents in Norway. According to calculated indices of specific infectivity and tick-to host transmission coefficient, M. arvalis and M. glareolus voles were found to be more efficient in transmitting B. burgdorferi s.l. to ticks than A. flavicollis mice. GLMM analysis showed that rodent species significantly influenced the probability of a larva being infected with B. burgdorferi s.l. The larvae feeding on M. arvalis and M. glareolus were more likely to be infected with B. burgdorferi s.l. than those feeding on A. flavicollis. This is the first study to report the quantitative roles of voles and mice in the transmission of B. burgdorferi s.l. to larval ticks in

  10. Habitat discrimination by gravid Anopheles gambiae sensu lato – a push-pull system

    PubMed Central

    2014-01-01

    Background The non-random distribution of anopheline larvae in natural habitats suggests that gravid females discriminate between habitats of different quality. Whilst physical and chemical cues used by Culex and Aedes vector mosquitoes for selecting an oviposition site have been extensively studied, those for Anopheles remain poorly explored. Here the habitat selection by Anopheles gambiae sensu lato (s.l.), the principal African malaria vector, was investigated when presented with a choice of two infusions made from rabbit food pellets, or soil. Methods Natural colonization and larval survival was evaluated in artificial ponds filled randomly with either infusion. Dual-choice, egg-count bioassays evaluated the responses of caged gravid females to (1) two- to six-day old infusions versus lake water; (2) autoclaved versus non-autoclaved soil infusions; and assessed (3) the olfactory memory of gravid females conditioned in pellet infusion as larvae. Results Wild Anopheles exclusively colonized ponds with soil infusion and avoided those with pellet infusion. When the individual infusions were tested in comparison with lake water, caged An. gambiae sensu stricto (s.s.) showed a dose response: females increasingly avoided the pellet infusion with increasing infusion age (six-day versus lake water: odds ratio (OR) 0.22; 95% confidence interval (CI) 0.1-0.5) and showed increasing preference to lay eggs as soil infusion age increased (six-day versus lake water: OR 2.1; 95% CI 1.4-3.3). Larvae survived in soil infusions equally well as in lake water but died in pellet infusions. Anopheles gambiae s.s. preferred to lay eggs in the non-autoclaved soil (OR 2.6; 95% CI 1.8-3.7) compared with autoclaved soil. There was no change in the avoidance of pellet infusion by individuals reared in the infusion compared with those reared in lake water. Conclusion Wild and caged An. gambiae s.l. females discriminate between potential aquatic habitats for oviposition. These choices benefit

  11. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge.

    PubMed

    Alvarez Rojas, Cristian A; Romig, Thomas; Lightowlers, Marshall W

    2014-01-01

    Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1-10 and the 'lion strain'), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1-3 (=E. granulosus sensu stricto) and G6-10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1-3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6-7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by

  12. Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation ofsusceptible dog hosts is reduced by slow release of semiochemicalsfrom a less susceptible host

    USDA-ARS?s Scientific Manuscript database

    Domestic dog breeds are hosts for the tick Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as tick repellents. We report th...

  13. Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer

    PubMed Central

    Chiara, Matteo; Caruso, Marta; D’Erchia, Anna Maria; Manzari, Caterina; Fraccalvieri, Rosa; Goffredo, Elisa; Latorre, Laura; Miccolupo, Angela; Padalino, Iolanda; Santagada, Gianfranco; Chiocco, Doriano; Pesole, Graziano; Horner, David S.; Parisi, Antonio

    2015-01-01

    Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu. PMID:26185097

  14. CODEHOP PCR and CODEHOP PCR primer design.

    PubMed

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  15. Identification and molecular survey of Borrelia burgdorferi sensu lato in sika deer (Cervus nippon) from Jilin Province, north-eastern China.

    PubMed

    Zhai, Bintao; Niu, Qingli; Yang, Jifei; Liu, Zhijie; Liu, Junlong; Yin, Hong; Zeng, Qiaoying

    2017-02-01

    Lyme disease caused by Borrelia burgdorferi sensu lato (s.l.) is a common disease of domestic animals and wildlife worldwide. Sika deer is first-grade state-protected wildlife animals in China and have economic consequences for humans. It is reported that sika deer may serve as an important reservoir host for several species of B. burgdorferi s.l. and may transmit these species to humans and animals. However, little is known about the presence of Borrelia pathogens in sika deer in China. In this study, the existence and prevalence of Borrelia sp. in sika deer from four regions of Jilin Province in China was assessed. Seventy-one blood samples of sika deer were collected and tested by nested-PCRs based on 16S ribosomal RNA (16S rRNA), outer surface protein A (OspA), flagenllin (fla), and 5S-23S rRNA intergenic spacer (5S-23S rRNA) genes of B. burgdorferi s.l. Six (8.45%) samples were positive for Borrelia sp. based on sequences of 4 genes. The positive samples were detected 18 for 16S rRNA, 10 for OspA, 16 for fla and 6 for 5S-23S, with the positive rates 25.35% (95% CI=3.8-35.6), 14.08% (95% CI=3.0-21.6), 22.54% (95% CI=4.3-36.9) and 8.45% (95% CI=1.7-22.9), respectively. Sequence analysis of the positive PCR products revealed that the partial 4 genes sequences in this study were all most similar to the sequences of B. garinii and B. burgdorferi sensu stricto (s.s.), no other Borrelia genospecies were found. This is the first report of Borrelia pathogens in sika deer in China. The findings in this study indicated that sika deer as potential natural host and may spread Lyme disease pathogen to animals, ticks, and even humans.

  16. PCR und Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Konrad, Regina; Busch, Ulrich

    Die vielfältigen Anwendungsmöglichkeiten der Polymerasekettenreaktion (polymerase chain reaction, PCR) machen sie zu einer der wichtigsten und am häufigsten eingesetzten Methoden in der molekularbiologischen Forschung und Diagnostik. Für diese Technologie wurde der Erfinder der Methode, Kary Mullis, 1993 mit dem Nobelpreis ausgezeichnet. Die PCR erlaubt einen hochsensitiven und spezifischen in-vitro-Nachweis von Desoxyribonukleinsäuren (DNA), da im Zuge der Reaktion Sequenzabschnitte gezielt vermehrt werden. Innerhalb weniger Stunden können aus einem einzigen Zielmolekül 1012 identische Moleküle entstehen [1].

  17. Congruence and indifference between two molecular markers for understanding oral evolution in the Marynidae sensu lato (Ciliophora, Colpodea)

    PubMed Central

    Dunthorn, Micah; Katz, Laura A.; Stoeck, Thorsten; Foissner, Wilhelm

    2012-01-01

    Our understanding of the evolution of oral structures within the Colpodida is confounded by the low number of morphological characters that can be used in constructing hypotheses, and by the low taxon and character sampling in molecular phylogenetic analyses designed to assess these hypotheses. Here we increase character sampling by sequencing the mitochondrial SSU-rDNA locus for three isolates of the Marynidae sensu lato. We show that the inferred mitochondrial and nuclear SSU-rDNA trees, as well as concatenated and constrained analyses, are congruent in not recovering a monophyletic Marynidae. However, due to low node support, the trees are indifferent to whether the morphological characters used to unite the Marynidae are the result of retention of ancestral states or convergence. In light of this indifference and an increased amount of nuclear and mitochondrial SSU-rDNA data, alternative hypotheses of oral evolution in the Colpodida are presented. PMID:22356924

  18. Optimization of the Esperanza window trap for the collection of the African onchocerciasis vector Simulium damnosum sensu lato.

    PubMed

    Toé, Laurent D; Koala, Lassane; Burkett-Cadena, Nathan D; Traoré, Bizini M; Sanfo, Moussa; Kambiré, Sié Roger; Cupp, Eddie W; Traoré, Soungalo; Yameogo, Laurent; Boakye, Daniel; Rodríguez-Pérez, Mario A; Unnasch, Thomas R

    2014-09-01

    A simple inexpensive trap (Esperanza window trap) was shown recently to collect significant numbers of Simulium ochraceum sensu lato, a major vector of Onchocerca volvulus in Mesoamerica. Here, we report studies optimizing this trap for the collection of Simulium damnosum s.l., the major vector of O. volvulus in Africa. A shortened, blue and black striped version of the Esperanza window trap, when baited with a combination of CO2 and worn trousers, rivalled human landing collections in the number of S. damnosum s.l. females collected. Traps baited with a commercially available human skin lure and CO2 resulted in collections that were not significantly different than those obtained from traps baited with worn trousers and CO2. This suggests that the Esperanza window trap may offer a replacement for human landing collections for monitoring onchocerciasis transmission in Africa.

  19. The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato)

    PubMed Central

    Glenwright, Helen; Pohl, Susanne; Navarro, Ferran; Miro, Elisenda; Jiménez, Guillermo; Blanch, Anicet R.; Harwood, Colin R.

    2017-01-01

    Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we

  20. The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato).

    PubMed

    Glenwright, Helen; Pohl, Susanne; Navarro, Ferran; Miro, Elisenda; Jiménez, Guillermo; Blanch, Anicet R; Harwood, Colin R

    2016-01-01

    Bacillus toyonensis strain BCT-7112(T) (NCIMB 14858(T)) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context

  1. Borrelia burgdorferi sensu lato prevalence and diversity in ticks and small mammals in a Lyme borreliosis endemic Nature Reserve in North-Western Spain. Incidence in surrounding human populations.

    PubMed

    Espí, Alberto; Del Cerro, Ana; Somoano, Aitor; García, Verónica; M Prieto, José; Barandika, José F; García-Pérez, Ana L

    2016-07-18

    To determine the prevalence and diversity of Borrelia burgdorferi sensu lato (s.l.) in an endemic Nature Reserve (Sierra del Sueve) in North-Western Spain, and the risk of human exposure to infected ticks in Asturias, 1013 questing ticks and 70 small mammals were collected between 2012 and 2014. A retrospective descriptive analysis was also carried out on human Lyme borreliosis (LB) cases reported to the local hospital (Cabueñes). Samples were screened for B. burgdorferi s.l. presence by a nested PCR assay, and genospecies were confirmed by sequencing. B. burgdorferi s.l. was detected in 1.4% (12/845) of I. ricinus questing nymphs, 9.1% (2/33) of questing adults, and 12.9% (9/70) of small mammals, as well as in the other tick species. PCR positive samples of 17 questing tick and 6 small mammals were sequenced. Four genospecies were identified: B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana. Phylogenetic analyses based on the flaB gene showed the heterogeneity of B. afzelii in this area. The detection of B. burgdorferi s.l. among questing ticks and small mammals in the study area, as well as the abundance of ticks and of large wild and domestic mammals, indicate a high risk of infection by B. burgdorferi s.l. in the area. Reporting of LB cases to the local hospital support this, and shows the need of thorough monitoring of B. burgdorferi infection in ticks and hosts in the area. More investigations are needed to assess the role of different wildlife species and the risk of transmission to humans.

  2. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  3. Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer.

    PubMed

    Chiara, Matteo; Caruso, Marta; D'Erchia, Anna Maria; Manzari, Caterina; Fraccalvieri, Rosa; Goffredo, Elisa; Latorre, Laura; Miccolupo, Angela; Padalino, Iolanda; Santagada, Gianfranco; Chiocco, Doriano; Pesole, Graziano; Horner, David S; Parisi, Antonio

    2015-07-15

    Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes.

    PubMed

    Ziccardi, Mariangela; Souza, Lucieri O P; Gandra, Rafael M; Galdino, Anna Clara M; Baptista, Andréa R S; Nunes, Ana Paula F; Ribeiro, Mariceli A; Branquinha, Marta H; Santos, André L S

    2015-12-01

    Candida parapsilosis (sensu lato), which represents a fungal complex composed of three genetically related species - Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis, has emerged as an important yeast causing fungemia worldwide. The goal of the present work was to assess the prevalence, antifungal susceptibility and production of virulence traits in 53 clinical isolates previously identified as C. parapsilosis (sensu lato) obtained from hospitals located in the Southeast of Brazil. Species forming this fungal complex are physiologically/morphologically indistinguishable; however, polymerase chain reaction followed by restriction fragment length polymorphism of FKS1 gene has solved the identification inaccuracy, revealing that 43 (81.1%) isolates were identified as C. parapsilosis sensu stricto and 10 (18.9%) as C. orthopsilosis. No C. metapsilosis was found. The geographic distribution of these Candida species was uniform among the studied Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo). All C. orthopsilosis and almost all C. parapsilosis sensu stricto (95.3%) isolates were susceptible to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. Nevertheless, one C. parapsilosis sensu stricto isolate was resistant to fluconazole and another one was resistant to caspofungin. C. parapsilosis sensu stricto isolates exhibited higher MIC mean values to amphotericin B, fluconazole and caspofungin than those of C. orthopsilosis, while C. orthopsilosis isolates displayed higher MIC mean to itraconazole compared to C. parapsilosis sensu stricto. Identical MIC mean values to voriconazole were measured for these Candida species. All the isolates of both species were able to form biofilm on polystyrene surface. Impressively, biofilm-growing cells of C. parapsilosis sensu stricto and C. orthopsilosis exhibited a considerable resistance to all antifungal agents tested. Pseudohyphae were observed in 67.4% and 80

  5. Molecular Characterization of Echinococcus granulosus Sensu Lato from Farm Animals in Egypt

    PubMed Central

    Amer, Said; Helal, Ibrahim B.; Kamau, Evelyne; Feng, Yaoyu; Xiao, Lihua

    2015-01-01

    Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt. PMID:25760944

  6. Deep molecular divergence and exceptional morphological stasis in dwarf cannibal snails Nata sensu lato Watson, 1934 (Rhytididae) of southern Africa.

    PubMed

    Moussalli, Adnan; Herbert, David G

    2016-02-01

    The genus Nata Watson, 1934 is a southern African endemic belonging to the Gondwanan family of carnivorous snails, Rhytididae. We present a molecular phylogeny of the genus based on two mitochondrial (16S and COI) and two nuclear genes (ITS2 and 28S RNA), and complement this with an appraisal of morphological characters relating to both the shell and soft parts. We identify four reciprocally monophyletic lineages for which valid names are already available, plus two undescribed species restricted to the Albany Thicket Biome. We show that Nata sensu lato may not be monophyletic. Rather there exist two deep lineages within Nata s.l., one lineage potentially sister to a clade dominated by the Australian and New Zealand radiation, and the other occupying a basal position within Rhytididae. Accordingly we recommend a revision recognising two genera, namely Nata s.s. and Natella respectively. Despite deep molecular divergences within Nata s.s., phenotypic evolution has been remarkably conserved, and contrasts greatly with that exhibited across other major lineages within the Rhytididae.

  7. Borrelia burgdorferi sensu lato and co-infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg, Germany.

    PubMed

    May, K; Jordan, D; Fingerle, V; Strube, C

    2015-12-01

    To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe-based quantitative real-time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co-infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co-infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia-positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.

  8. Borrelia burgdorferi sensu lato detected in skin of Norwegian mountain hares (Lepus timidus) without signs of dissemination.

    PubMed

    Kjelland, Vivian; Ytrehus, Bjørnar; Vikørren, Turid; Stuen, Snorre; Skarpaas, Tone; Slettan, Audun

    2011-04-01

    The mountain hare (Lepus timidus) population in southern Norway appears to be in decline. Necropsy and laboratory examinations of 36 hares found dead or diseased during 2007-2009 in Vest- and Aust-Agder counties showed that disease and deaths were attributed to multiple causes, with no specific etiology emerging as a cause for population decline. To investigate whether Borrelia burgdorferi sensu lato (s.l.) infection is associated with mortality in mountain hares, tissues and ticks collected from hares were investigated for infection with the spirochete. Borrelia burgdorferi s.l. DNA was not detected in samples from internal organs, whereas Borrelia afzelii, B. burgdorferi sensu stricto (s.s.), and the not-yet-defined Borrelia sp. SV1 were found in skin samples from hares and in adult and nymphal Ixodes ricinus feeding on hares. Only B. burgdorferi s.s. and Borrelia sp. SV1 were detected in larvae feeding on hares. Our results indicate that disseminated Borrelia infection in hares rarely occurs and, presumably, does not play a central role in the suspected population decline. The results also suggest that the mountain hare to some degree functions as a transmission host for B. burgdorferi s.s. and Borrelia sp. SV1.

  9. Ecological differentiation of diploid and polyploid cytotypes of Senecio carniolicus sensu lato (Asteraceae) is stronger in areas of sympatry.

    PubMed

    Sonnleitner, Michaela; Hülber, Karl; Flatscher, Ruth; Escobar García, Pedro; Winkler, Manuela; Suda, Jan; Schönswetter, Peter; Schneeweiss, Gerald M

    2016-02-01

    Ecological differentiation is recognized as an important factor for polyploid speciation, but little is known regarding whether the ecological niches of cytotypes differ between areas of sympatry and areas where single cytotypes occur (i.e. niche displacement). Ecological niches of four groups of Senecio carniolicus sensu lato (s.l.) (western and eastern diploid lineages, tetraploids and hexaploids) were characterized via Landolt indicator values of the accompanying vascular plant species and tested using multivariate and univariate statistics. The four groups of S. carniolicus s.l. were ecologically differentiated mainly with respect to temperature, light and soil (humus content, nutrients, moisture variability). Niche breadths did not differ significantly. In areas of sympatry hexaploids shifted towards sites with higher temperature, less light and higher soil humus content as compared with homoploid sites, whereas diploids and tetraploids shifted in the opposite direction. In heteroploid sites of tetraploids and the western diploid lineage the latter shifted towards sites with lower humus content but higher aeration. Niche displacement can facilitate the formation of stable contact zones upon secondary contact of polyploids and their lower-ploid ancestors and/or lead to convergence of the cytotypes' niches after they have attained non-overlapping ranges. Niche displacement is essential for understanding ecological consequences of polyploidy. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Evaluation of a community-based trapping program to collect Simulium ochraceum sensu lato for verification of onchocerciasis elimination.

    PubMed

    Rodríguez-Pérez, Mario A; Adeleke, Monsuru A; Rodríguez-Luna, Isabel C; Cupp, Eddie W; Unnasch, Thomas R

    2014-10-01

    Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO2 had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated. Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission. These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination.

  11. Rickettsia rickettsii infecting Rhipicephalus sanguineus sensu lato (Latreille 1806), in high altitude atlantic forest fragments, Ceara State, Brazil.

    PubMed

    Silva, Arannadia Barbosa; Duarte, Myrian Morato; da Costa Cavalcante, Robson; de Oliveira, Stefan Vilges; Vizzoni, Vinicius Figueiredo; de Lima Duré, Ana Íris; de Melo Iani, Felipe Campos; Machado-Ferreira, Erik; Gazêta, Gilberto Salles

    2017-09-01

    In Brazil, Spotted Fever (SF) is caused by Rickettsia rickettsii and Rickettsia parkeri strain Atlantic Forest. In recent years, several human cases of a milder SF have been reported from the Maciço de Baturité region of Ceará State. Previous studies in this region found R. parkeri strain Atlantic Forest to be present in Rhipicephalus sanguineus sensu lato and Amblyomma ovale ticks. The present study isolated and identified the Rickettsia spp. present in this new endemic area in Brazil. In March 2015, R. sanguineus s.l. and A. ovale were collected in rural areas of the Maciço de Baturité region, and subjected to the isolation technique. A bacterium was isolated from one R. sanguineus s.l., which phylogenetic analysis clustered to the R. rickettsii group. In conclusion, R. rickettsii bacteria is circulating in the studied area and may in future have an impact on the clinical diagnoses and consequently cause changes in the profile of the disease in the region. In addition, we suggest the increase of epidemiological and environmental surveillance in the area, in order to prevent Brazilian Spotted Fever cases. Copyright © 2017. Published by Elsevier B.V.

  12. Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied.

    PubMed

    Ruiu, Luca; Falchi, Giovanni; Floris, Ignazio; Marche, Maria Giovanna; Mura, Maria Elena; Satta, Alberto

    2015-03-01

    The lethal and sub-lethal effects of sporulated cultures of a novel Bacillus cereus sensu lato strain lacking detectable cry genes and identified through morphological and genetic analyses, have been studied on the Mediterranean fruit fly Ceratitis capitata. The lethal effects on young larvae were concentration dependent, with a median lethal concentration (LC50) of 4.48 × 10(8)spores/g of diet. Sporulated cultures of this strain significantly extended development time and reduced immature survival, and the size of emerging fly adults. Besides spores, the toxicity has been associated to the insoluble extra-spore fraction characterized through a proteomic approach. The profile of the extra-spore protein fraction (ES) showed major protein bands within the 35-65 kDa range. The results of mass spectrometry analysis highlighted the presence of putative virulence factors, including members of protein families previously associated to the insecticidal action of other microbial entomopathogens. These proteins include metalloproteases, peptidases and other enzymes.

  13. Lack of insecticidal effect of mosquito coils containing either metofluthrin or esbiothrin on Anopheles gambiae sensu lato mosquitoes.

    PubMed

    Lukwa, Nzira; Chiwade, Tonderai

    2008-12-01

    Use of mosquito coils for personal protection against malaria and mosquito nuisance is advocated under mosquito and malaria control programmes. We performed field studies of mosquito coils containing either metofluthrin or esbiothrin in experimental huts situated in Kamhororo village, Gokwe district, Zimbabwe. All tests were performed on 3-5 day old reared female Anopheles gambiae sensu lato mosquitoes. The burning times were 9hr 20min for mosquito coils containing metofluthrin and 8 hr for those containing esbiothrin and the results were significantly different (p = <0.001). The mean knock down rate for mosquito coils containing metofluthrin was 90% and that for esbiothrin was 73.3% and the results were significantly different (p = 0.00). Mosquito coils containing metofluthrin had a mean repellence of 92.7% as compared to 85.4% for esbiothrin and the results were not significantly different (p=0.27). The protection time as required by EPA (1999) was 6 hr for mosquito coils containing metofluthrin and 5 hr for those containing esbiothrin. The mean insecticidal effect of mosquito coils containing metofluthrin was 84% as compared to 83% for those containing esbiothrin and the results were not significantly different (p = 0.56). Both mosquito formulations could not be classified as having insecticidal effect since none of them met the 95% mortality rate criteria.

  14. Seroprevalence of antibodies to Borrelia burgdorferi sensu lato in healthy adults from western Norway: risk factors and methodological aspects.

    PubMed

    Hjetland, Reidar; Nilsen, Roy M; Grude, Nils; Ulvestad, Elling

    2014-11-01

    The aim of this study was to assess the seroprevalence of antibodies to Borrelia burgdorferi sensu lato in a healthy adult population from Sogn and Fjordane county in western Norway by different assays. Sera from 1213 blood donors at four different blood banks were analysed in Enzygnost Lyme link VlsE/IgG (IgG), Enzygnost Borreliosis IgM (IgM), and Immunetics C6 Lyme ELISA kit (C6). Sera showing positive or grey-zone reactivities were further examined with Borrelia-EUROLine-RN-AT IgG blot and Borrelia-EUROLine-RN-AT IgM blot. The seroprevalences were 9.6%, 8.2%, 8.4%, 6.4% and 5.7%, respectively. The seroprevalence for IgG was lower in the eastern part of the county and in owners of pet animals. It was higher in men, and increased with age and number of tick bites. C6 and IgG gave comparable results. IgM only was found in 4.5%, more often in women, did not increase with age, and showed no relationship with geography, and 56.4% were positive in IgM blot. In conclusion, antibodies to B. burgdorferi s.l. are common in blood donors in western Norway. The results may be used for evaluation of predictive values of test results in patients, as well as a basis for test algorithms in the laboratory.

  15. Seasonal Transmission of Opisthorchis viverrini sensu lato and a Lecithodendriid Trematode Species in Bithynia siamensis goniomphalos Snails in Northeast Thailand

    PubMed Central

    Namsanor, Jutamas; Sithithaworn, Paiboon; Kopolrat, Kulthida; Kiatsopit, Nadda; Pitaksakulrat, Opal; Tesana, Smarn; Andrews, Ross H.; Petney, Trevor N.

    2015-01-01

    Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012–2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host–parasite interactions with important implications for the epidemiology of O. viverrini s.l. PMID:25918210

  16. The systematics and population genetics of Opisthorchis viverrini sensu lato: implications in parasite epidemiology and bile duct cancer.

    PubMed

    Sithithaworn, Paiboon; Andrews, Ross H; Petney, Trevor N; Saijuntha, Weerachai; Laoprom, Nonglak

    2012-03-01

    Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host.

  17. Seasonal transmission of Opisthorchis viverrini sensu lato and a lecithodendriid trematode species in Bithynia siamensis goniomphalos snails in northeast Thailand.

    PubMed

    Namsanor, Jutamas; Sithithaworn, Paiboon; Kopolrat, Kulthida; Kiatsopit, Nadda; Pitaksakulrat, Opal; Tesana, Smarn; Andrews, Ross H; Petney, Trevor N

    2015-07-01

    Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012-2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host-parasite interactions with important implications for the epidemiology of O. viverrini s.l.

  18. Changing patterns of prevalence in Opisthorchis viverrini sensu lato infection in children and adolescents in northeast Thailand.

    PubMed

    Khuntikeo, Narong; Sithithaworn, Paiboon; Loilom, Watcharin; Namwat, Nisana; Yongvanit, Puangrat; Thinkhamrop, Bandit; Kiatsopit, Nadda; Andrews, Ross H; Petney, Trevor N

    2016-12-01

    Infection with the liver fluke Opisthorchis viverrini sensu lato (s.l.), a group 1 carcinogen, is the most important risk factor for developing cholangiocarcinoma (CCA) in Southeast Asia. Cholangiocarcinoma is a fatal disease with the world's highest incidence being found in northeast Thailand. Liver fluke infection occurs through eating raw or partially cooked cyprinid fish containing metacercariae and, therefore, the control of O. viverrini s.l. infection should lead to a reduction in CCA incidence. In this report, we review and analyze the age-prevalence profile data of O. viverrini to reveal temporal changes in patterns of prevalence pre- and post-control programs in Thailand. The profiles of O. viverrini prevalence have transformed from high prevalence in school children prior to 1983 to low prevalences after 1994. This pattern strongly suggests the influence of the health education program on the likelihood of school children becoming infected. In conjunction with current developments in health and socioeconomic conditions, we predict that the incidence of CCA will be reduced with time as the population cohorts that experienced the education programs reach the age at which CCA is most likely to develop, i.e. >50 years. The lessons learned in Thailand may be applicable to other areas endemic for human liver flukes.

  19. Evaluation of a Community-Based Trapping Program to Collect Simulium ochraceum sensu lato for Verification of Onchocerciasis Elimination

    PubMed Central

    Rodríguez-Pérez, Mario A.; Adeleke, Monsuru A.; Rodríguez-Luna, Isabel C.; Cupp, Eddie W.; Unnasch, Thomas R.

    2014-01-01

    Background Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO2 had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated. Methodology/Principal findings Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission. Conclusions/Significance These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination. PMID:25340517

  20. Morphological and ontogenetic stratification of abyssal and hadal Eurythenes gryllus sensu lato (Amphipoda: Lysianassoidea) from the Peru-Chile Trench

    NASA Astrophysics Data System (ADS)

    Eustace, Ryan M.; Ritchie, Heather; Kilgallen, Niamh M.; Piertney, Stuart B.; Jamieson, Alan J.

    2016-03-01

    The globally ubiquitous lysianassoid amphipod, Eurythenes gryllus, has been shown to consist of multiple genetically distinct cryptic taxa, with depth considered a major driver of speciation and morphological divergence. Here we examine morphological variation of E. gryllus sensu lato through a continuous depth distribution that spans from abyssal (3000-6000 m) into hadal depths (>6000 m) in the Peru-Chile Trench (SE Pacific Ocean). Three distinct morphospecies were identified: one was confirmed as being E. magellanicus (4602-5329 m) based on DNA sequence and morphological similarity. The other two morphologically distinct species were named based upon depth of occurrence; Abyssal (4602-6173 m) and Hadal (6173-8074 m). The three Eurythenes morphospecies showed vertical ontogenetic stratification across their bathymetric range, where juveniles were found shallower in their depth range and mature females deeper. Potential ecological and evolutionary drivers that explain the observed patterns of intra and inter-specific structure, such as hydrostatic pressure and topographical isolation, are discussed.

  1. Mapping human risk of infection with Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in a periurban forest in France.

    PubMed

    Vourc'h, G; Abrial, D; Bord, S; Jacquot, M; Masséglia, S; Poux, V; Pisanu, B; Bailly, X; Chapuis, J-L

    2016-07-01

    Lyme borreliosis is a major zoonosis in Europe, with estimates of over 26,000 cases per year in France alone. The etiological agents are spirochete bacteria that belong to the Borrelia burgdorferi sensu lato (s. l.) complex and are transmitted by hard ticks among a large range of vertebrate hosts. In Europe, the tick Ixodes ricinus is the main vector. In the absence of a vaccine and given the current difficulties to diagnose and treat chronic Lyme syndromes, there is urgent need for prevention. In this context, accurate information on the spatial patterns of risk of exposure to ticks is of prime importance for public health. The objective of our study was to provide a snapshot map of the risk of human infection with B. burgdorferi s. l. pathogens in a periurban forest at a high resolution, and to analyze the factors that contribute to variation in this risk. Field monitoring took place over three weeks in May 2011 in the suburban Sénart forest (3,200ha; southeast of Paris), which receives over 3 million people annually. We sampled ticks over the entire forest area (from 220 forest stands with a total area of 35,200m(2)) and quantified the density of questing nymphs (DON), the prevalence of infection among nymphs (NIP), and the density of infected nymphs (DIN), which is the most important predictor of the human risk of Lyme borreliosis. For each of these response variables, we explored the relative roles of weather (saturation deficit), hosts (abundance indices of ungulates and Tamias sibiricus, an introduced rodent species), vegetation and forest cover, superficial soil composition, and the distance to forest roads. In total, 19,546 questing nymphs were collected and the presence of B. burgdorferi s. l. was tested in 3,903 nymphs by qPCR. The mean DON was 5.6 nymphs per 10m(2) (standard deviation=10.4) with an average NIP of 10.1% (standard deviation=0.11). The highest DIN was 8.9 infected nymphs per 10m(2), with a mean of 0.59 (standard deviation=0.6). Our

  2. De novo sequencing of the Hypericum perforatum L. flower transcriptome to identify potential genes that are related to plant reproduction sensu lato.

    PubMed

    Galla, Giulio; Vogel, Heiko; Sharbel, Timothy F; Barcaccia, Gianni

    2015-03-31

    or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum.

  3. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  4. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  5. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  6. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  7. Explanatory chapter: PCR primer design.

    PubMed

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Pcr by Thermal Convection

    NASA Astrophysics Data System (ADS)

    Braun, Dieter

    The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

  9. Large scale spatial risk and comparative prevalence of Borrelia miyamotoi and Borrelia burgdorferi sensu lato in Ixodes pacificus.

    PubMed

    Padgett, Kerry; Bonilla, Denise; Kjemtrup, Anne; Vilcins, Inger-Marie; Yoshimizu, Melissa Hardstone; Hui, Lucia; Sola, Milagros; Quintana, Miguel; Kramer, Vicki

    2014-01-01

    Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n=70) and nymphal (n=36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America.

  10. Comparison of MKP and BSK-H media for the cultivation and isolation of Borrelia burgdorferi sensu lato

    PubMed Central

    Ružić-Sabljić, Eva; Maraspin, Vera; Stupica, Daša; Rojko, Tereza; Bogovič, Petra; Strle, Franc; Cerar, Tjaša

    2017-01-01

    The isolation of B. burgdorferi sensu lato requires the use of complex cultivation media. The aim of the study was to compare the usefulness of BSK-H (a commercial medium produced by HiMedia, India) and MKP medium. MKP and BSK-H media were prepared in accordance with the relevant protocols. Borrelia strains and skin culture biopsies were simultaneously inoculated into both media, incubated and checked for growth. Borrelial growth characteristics, isolation rates and characteristics of the isolated borreliae were analysed and compared. Initially, numbers of spirochaetes were higher in BSK-H than in MKP; however, in comparison with MKP, the strains subcultured in BSK-H medium were more frequently irregular, thin and non-motile, and rapidly died. In addition, the borrelial isolation rate from erythema migrans skin samples was higher in MKP than in BSK-H medium (108/171, 63.2% versus 70/171, 40.9%; p<0.0001). The far most frequently isolated species was Borrelia afzelii (92.9% and 97.2% strains isolated from BSK-H and MKP, respectively). Comparison of strains cultured from individual patients in both media showed differences in plasmid contents in 9/46 (19.6%) strain pairs, and protein profiles differed in 30/43 (69.8%) strain pairs, most often in the expression of OspC (in 27/28 patients OspC was expressed only in strains growing in MKP). BSK-H medium supports the growth of borrelial strains but MKP is superior with regard to the isolation rate, morphology and motility of strains. BSK-H medium supports fast initial growth of borreliae but this is followed by rapid deformation and death of the spirochaetes. PMID:28170447

  11. In vitro repellency of DEET and β-citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum.

    PubMed

    Ferreira, Lorena Lopes; Oliveira Filho, Jaires Gomes de; Mascarin, Gabriel Moura; León, Adalberto A Pérez de; Borges, Lígia Miranda Ferreira

    2017-05-30

    Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repellent, versus β-citronellol in a Petri dish bioassay. A semicircle of filter paper (31.8cm(2)) was treated with 87μl of one of four concentrations (0.200, 0.100, 0.050 and 0.025mg/cm(2)) of β-citronellol, DEET or solvent (ethanol). A head-to-head test was developed treating one side with increasing concentrations of β-citronellol as above mentioned, against the highest concentration of DEET. Besides that a blank assay was performed. Three males and three females were placed in the middle of the plate and their location was evaluated 5, 10 and 30min after the test was initiated. As a result, the time had no significant effect on repellency response of the ticks exposed to both compounds and their concentrations. The repellency response raised according with the increase of concentration. Additionally, our findings indicate that the tick A. sculptum was more sensitive to the compounds tested and β-citronellol showed a higher efficacy than DEET. In addition, β-citronellol could be formulated to protect humans and other animals from R. sanguineus s. l. and A. sculptum infestation, as well as the diseases transmitted by these species. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Ferritin 1 silencing effect in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) during experimental infection with Ehrlichia canis.

    PubMed

    Ferrolho, Joana; Antunes, Sandra; Sanches, Gustavo S; Couto, Joana; Évora, Patrícia M; Rosa, Catarina; André, Marcos R; Machado, Rosângela Z; Bechara, Gervásio H; Domingos, Ana

    2017-01-01

    Rhipicephalus sanguineus sensu lato (s.l.) is a very common ectoparasite of domestic dogs able to transmit several pathogens of human and veterinary importance. Tick infestations and tick-borne diseases (TBDs) remain a serious and persistent problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to control are pursued. Whilst vaccination is recognised as a potential control method to reduce tick infestation, no anti-R. sanguineus vaccine is available. Ticks depend on their blood meals to obtain nutrients and to achieve sexual maturity, which exposes them to vast amounts of iron. Although an essential molecule for several biological processes, its excess can lead to oxidative stress. Iron homeostasis is achieved with the help of iron-binding proteins called ferritins, among others, present in several tick tissues and developmental stages. These evolutionarily conserved proteins regulate iron homeostasis by storing and releasing iron in a controlled manner. In this study the R. sanguineus ferritin 1 gene was silenced through RNA interference (RNAi) in adult females exposed to an experimental infection with Ehrlichia canis. The purpose of this study was to assess the role of this protein in tick feeding, ovary development, oogenesis, and pathogen acquisition. Our data has shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphologic and histochemical changes in the ovaries (OV) and oocytes. Furthermore, our data revealed that no E. canis DNA was found in either experimental group. Determining the function of molecules that act in key biological processes, such as blood digestion or reproduction, and that could be considered potential tick antigens will contribute towards the improvement of current control measures against these ectoparasites and the pathogens they vector.

  13. The Heterogeneity, Distribution, and Environmental Associations of Borrelia burgdorferi Sensu Lato, the Agent of Lyme Borreliosis, in Scotland

    PubMed Central

    James, Marianne C.; Gilbert, Lucy; Bowman, Alan S.; Forbes, Ken J.

    2014-01-01

    Lyme borreliosis is an emerging infectious human disease caused by the Borrelia burgdorferi sensu lato complex of bacteria with reported cases increasing in many areas of Europe and North America. To understand the drivers of disease risk and the distribution of symptoms, which may improve mitigation and diagnostics, here we characterize the genetics, distribution, and environmental associations of B. burgdorferi s.l. genospecies across Scotland. In Scotland, reported Lyme borreliosis cases have increased almost 10-fold since 2000 but the distribution of B. burgdorferi s.l. is so far unstudied. Using a large survey of over 2200 Ixodes ricinus tick samples collected from birds, mammals, and vegetation across 25 sites we identified four genospecies: Borrelia afzelii (48%), Borrelia garinii (36%), Borrelia valaisiana (8%), and B. burgdorferi sensu stricto (7%), and one mixed genospecies infection. Surprisingly, 90% of the sequence types were novel and, importantly, up to 14% of samples were mixed intra-genospecies co-infections, suggesting tick co-feeding, feeding on multiple hosts, or multiple infections in hosts. B. garinii (hosted by birds) was considerably more genetically diverse than B. afzelii (hosted by small mammals), as predicted since there are more species of birds than small mammals and birds can import strains from mainland Europe. Higher proportions of samples contained B. garinii and B. valaisiana in the west, while B. afzelii and B. garinii were significantly more associated with mixed/deciduous than with coniferous woodlands. This may relate to the abundance of transmission hosts in different regions and habitats. These data on the genetic heterogeneity within and between Borrelia genospecies are a first step to understand pathogen spread and could help explain the distribution of patient symptoms, which may aid local diagnosis. Understanding the environmental associations of the pathogens is critical for rational policy making for disease risk

  14. Borrelia burgdorferi Sensu Lato Spirochetes in Wild Birds in Northwestern California: Associations with Ecological Factors, Bird Behavior and Tick Infestation

    PubMed Central

    Newman, Erica A.; Eisen, Lars; Eisen, Rebecca J.; Fedorova, Natalia; Hasty, Jeomhee M.; Vaughn, Charles; Lane, Robert S.

    2015-01-01

    Although Borrelia burgdorferi sensu lato (s.l.) are found in a great diversity of vertebrates, most studies in North America have focused on the role of mammals as spirochete reservoir hosts. We investigated the roles of birds as hosts for subadult Ixodes pacificus ticks and potential reservoirs of the Lyme disease spirochete B. burgdorferi sensu stricto (s.s.) in northwestern California. Overall, 623 birds representing 53 species yielded 284 I. pacificus larvae and nymphs. We used generalized linear models and zero-inflated negative binomial models to determine associations of bird behaviors, taxonomic relationships and infestation by I. pacificus with borrelial infection in the birds. Infection status in birds was best explained by taxonomic order, number of infesting nymphs, sampling year, and log-transformed average body weight. Presence and counts of larvae and nymphs could be predicted by ground- or bark-foraging behavior and contact with dense oak woodland. Molecular analysis yielded the first reported detection of Borrelia bissettii in birds. Moreover, our data suggest that the Golden-crowned Sparrow (Zonotrichia atricapilla), a non-resident species, could be an important reservoir for B. burgdorferi s.s. Of 12 individual birds (9 species) that carried B. burgdorferi s.l.-infected larvae, no birds carried the same genospecies of B. burgdorferi s.l. in their blood as were present in the infected larvae removed from them. Possible reasons for this discrepancy are discussed. Our study is the first to explicitly incorporate both taxonomic relationships and behaviors as predictor variables to identify putative avian reservoirs of B. burgdorferi s.l. Our findings underscore the importance of bird behavior to explain local tick infestation and Borrelia infection in these animals, and suggest the potential for bird-mediated geographic spread of vector ticks and spirochetes in the far-western United States. PMID:25714376

  15. Cercarial emergence patterns for Opisthorchis viverrini sensu lato infecting Bithynia siamensis goniomphalos from Sakon Nakhon Province, Thailand.

    PubMed

    Laoprom, Nonglak; Kiatsopit, Nadda; Sithithaworn, Paiboon; Kopolrat, Kulthida; Namsanor, Jutamas; Andrews, Ross H; Petney, Trevor N

    2016-09-01

    Opisthorchis viverrini sensu lato is a food-borne trematode which is classified as a class 1 carcinogen, with infection potentially leading to cholangiocarcinoma. Snails of the genus Bithynia act as the first intermediate hosts and an amplifying point in the parasite life cycle. In order to investigate seasonal effect on transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos, cercarial emergence and output profiles were monitored at different season. A total of 4533 snails originating from Sakon Nakhon Province, Thailand, collected during the three main seasons, were analyzed for O. viverrini s.l. Emergence of O. viverrini s.l. cercariae from snails was monitored daily from 06:00 to 18:00 h for seven consecutive days. The prevalence of infection in the snails was highest in the hot-dry season and declined in the rainy and cool-dry seasons. Peak cercarial emergence occurred between 08:00 and 10:00 h during the rainy and cool-dry seasons and between 10:00 and 12:00 h during the hot-dry season. The cercarial output was highest in the hot-dry season, similar to a previous study from Lao People's Democratic Republic (PDR). Average cercarial output/snail in Thailand was higher than in Lao PDR. The number of cercariae emerging from the snails was strongly related to snail size, but the relationship between prevalence of infection and snail size differed between seasons. Observed discrepancies in the emergence patterns and per capita cercarial release may reflect differences in environmental, snail, and/or parasite factors particularly biological characteristics between the cryptic species of O. viverrini s.l. and B. s. goniomphalos from Thailand and Lao PDR.

  16. Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

    PubMed Central

    2013-01-01

    Background Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1). Results Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. Conclusions Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues. PMID:24112474

  17. A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).

    PubMed

    Chen, Jin-Min; Zhou, Wei-Wei; Poyarkov, Nikolay A; Stuart, Bryan L; Brown, Rafe M; Lathrop, Amy; Wang, Ying-Yong; Yuan, Zhi-Yong; Jiang, Ke; Hou, Mian; Chen, Hong-Man; Suwannapoom, Chatmongkon; Nguyen, Sang Ngoc; Duong, Tang Van; Papenfuss, Theodore J; Murphy, Robert W; Zhang, Ya-Ping; Che, Jing

    2017-01-01

    The horned toad assemblage, genus Megophrys sensu lato, currently includes three groups previously recognized as the genera Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remain controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstruction; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA data. Analyses reveal statistically significant mito-nuclear discordance. All analyses resolve paraphyly for horned toads involving multiple strongly supported clades. These clades correspond with geography. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys s.l. and to account for the morphological, molecular and biogeographic differences among these groups, but we also provide an alternative option. Our study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. We provide an estimation of timeframe for the horned toads. The mitochondrial and nuclear trees indicate the presence of many putative undescribed species. Widespread species, such as Xenophrys major and X. minor, likely have dramatically underestimated diversity. The integration of morphological and molecular evidence can validate this discovery. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.

  18. Comparison of MKP and BSK-H media for the cultivation and isolation of Borrelia burgdorferi sensu lato.

    PubMed

    Ružić-Sabljić, Eva; Maraspin, Vera; Stupica, Daša; Rojko, Tereza; Bogovič, Petra; Strle, Franc; Cerar, Tjaša

    2017-01-01

    The isolation of B. burgdorferi sensu lato requires the use of complex cultivation media. The aim of the study was to compare the usefulness of BSK-H (a commercial medium produced by HiMedia, India) and MKP medium. MKP and BSK-H media were prepared in accordance with the relevant protocols. Borrelia strains and skin culture biopsies were simultaneously inoculated into both media, incubated and checked for growth. Borrelial growth characteristics, isolation rates and characteristics of the isolated borreliae were analysed and compared. Initially, numbers of spirochaetes were higher in BSK-H than in MKP; however, in comparison with MKP, the strains subcultured in BSK-H medium were more frequently irregular, thin and non-motile, and rapidly died. In addition, the borrelial isolation rate from erythema migrans skin samples was higher in MKP than in BSK-H medium (108/171, 63.2% versus 70/171, 40.9%; p<0.0001). The far most frequently isolated species was Borrelia afzelii (92.9% and 97.2% strains isolated from BSK-H and MKP, respectively). Comparison of strains cultured from individual patients in both media showed differences in plasmid contents in 9/46 (19.6%) strain pairs, and protein profiles differed in 30/43 (69.8%) strain pairs, most often in the expression of OspC (in 27/28 patients OspC was expressed only in strains growing in MKP). BSK-H medium supports the growth of borrelial strains but MKP is superior with regard to the isolation rate, morphology and motility of strains. BSK-H medium supports fast initial growth of borreliae but this is followed by rapid deformation and death of the spirochaetes.

  19. Large Scale Spatial Risk and Comparative Prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus

    PubMed Central

    Padgett, Kerry; Bonilla, Denise; Kjemtrup, Anne; Vilcins, Inger-Marie; Yoshimizu, Melissa Hardstone; Hui, Lucia; Sola, Milagros; Quintana, Miguel; Kramer, Vicki

    2014-01-01

    Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n = 70) and nymphal (n = 36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America. PMID:25333277

  20. Recurrent evolution of host and vector association in bacteria of the Borrelia burgdorferi sensu lato species complex.

    PubMed

    Becker, Noémie S; Margos, Gabriele; Blum, Helmut; Krebs, Stefan; Graf, Alexander; Lane, Robert S; Castillo-Ramírez, Santiago; Sing, Andreas; Fingerle, Volker

    2016-09-15

    The Borrelia burgdorferi sensu lato (s.l.) species complex consists of tick-transmitted bacteria and currently comprises approximately 20 named and proposed genospecies some of which are known to cause Lyme Borreliosis. Species have been defined via genetic distances and ecological niches they occupy. Understanding the evolutionary relationship of species of the complex is fundamental to explaining patterns of speciation. This in turn forms a crucial basis to frame testable hypotheses concerning the underlying processes including host and vector adaptations. Illumina Technology was used to obtain genome-wide sequence data for 93 strains of 14 named genospecies of the B. burgdorferi species complex and genomic data already published for 18 additional strain (including one new species) was added. Phylogenetic reconstruction based on 114 orthologous single copy genes shows that the genospecies represent clearly distinguishable taxa with recent and still ongoing speciation events apparent in Europe and Asia. The position of Borrelia species in the phylogeny is consistent with host associations constituting a major driver for speciation. Interestingly, the data also demonstrate that vector associations are an additional driver for diversification in this tick-borne species complex. This is particularly obvious in B. bavariensis, a rodent adapted species that has diverged from the bird-associated B. garinii most likely in Asia. It now consists of two populations one of which most probably invaded Europe following adaptation to a new vector (Ixodes ricinus) and currently expands its distribution range. The results imply that genotypes/species with novel properties regarding host or vector associations have evolved recurrently during the history of the species complex and may emerge at any time. We suggest that the finding of vector associations as a driver for diversification may be a general pattern for tick-borne pathogens. The core genome analysis presented here

  1. Variation in vegetative morphology tracks the complex genetic diversification of the mycoheterotrophic species Pyrola japonica sensu lato.

    PubMed

    Shutoh, Kohtaroh; Kaneko, Shingo; Suetsugu, Kenji; Naito, Yuichi I; Kurosawa, Takahide

    2016-09-01

    Although the evolution of full mycoheterotrophy has attracted many plant researchers, molecular phylogenetic studies that focus on the transition from partial to full mycoheterotrophy are limited to a few taxa. Pyrola japonica sensu lato is an ideal model for examining the evolution of mycoheterotrophy, owing to its variable leaf size, which suggests that the species comprises several transitional stages. To elucidate the molecular and morphological changes that occur during the evolutionary transition between partial and full mycoheterotrophy in P. japonica s.l. from 18 populations in Japan, we estimated a parsimony network of plastid haplotypes based on three noncoding regions, measured the leaf size and scape color of the shoots, and compared morphology among haplotypes. The seven haplotypes exhibited star-like relationships, and at least three divergent haplotypes were associated with differences in morphology. The first was mainly observed in large-leaved and green-scaped populations, whereas the second was observed in extremely small-leaved and reddish-scaped populations, which indicated a high degree of mycoheterotrophy, and the last was detected among mixed populations with both green- and reddish-scaped shoots with intermediate leaf sizes. In addition, the inconsistent association between the haplotypes and morphology suggests a complex relationship. Pyrola japonica s.l. has at least three separate genetic lineages that have different leaf morphologies. The genetic lineages and their coexistence could have led to the variable leaf size and suggest the possibility that gene flow from partial to full mycoheterotrophs could reverse the evolutionary transition to full mycoheterotrophy. © 2016 Shutoh et al. Published by the Botanical Society of America.This work is licensed under a Creative Commons Attribution License (CC-BY-NC).

  2. Borrelia burgdorferi sensu lato spirochetes in wild birds in northwestern California: associations with ecological factors, bird behavior and tick infestation.

    PubMed

    Newman, Erica A; Eisen, Lars; Eisen, Rebecca J; Fedorova, Natalia; Hasty, Jeomhee M; Vaughn, Charles; Lane, Robert S

    2015-01-01

    Although Borrelia burgdorferi sensu lato (s.l.) are found in a great diversity of vertebrates, most studies in North America have focused on the role of mammals as spirochete reservoir hosts. We investigated the roles of birds as hosts for subadult Ixodes pacificus ticks and potential reservoirs of the Lyme disease spirochete B. burgdorferi sensu stricto (s.s.) in northwestern California. Overall, 623 birds representing 53 species yielded 284 I. pacificus larvae and nymphs. We used generalized linear models and zero-inflated negative binomial models to determine associations of bird behaviors, taxonomic relationships and infestation by I. pacificus with borrelial infection in the birds. Infection status in birds was best explained by taxonomic order, number of infesting nymphs, sampling year, and log-transformed average body weight. Presence and counts of larvae and nymphs could be predicted by ground- or bark-foraging behavior and contact with dense oak woodland. Molecular analysis yielded the first reported detection of Borrelia bissettii in birds. Moreover, our data suggest that the Golden-crowned Sparrow (Zonotrichia atricapilla), a non-resident species, could be an important reservoir for B. burgdorferi s.s. Of 12 individual birds (9 species) that carried B. burgdorferi s.l.-infected larvae, no birds carried the same genospecies of B. burgdorferi s.l. in their blood as were present in the infected larvae removed from them. Possible reasons for this discrepancy are discussed. Our study is the first to explicitly incorporate both taxonomic relationships and behaviors as predictor variables to identify putative avian reservoirs of B. burgdorferi s.l. Our findings underscore the importance of bird behavior to explain local tick infestation and Borrelia infection in these animals, and suggest the potential for bird-mediated geographic spread of vector ticks and spirochetes in the far-western United States.

  3. Occurrence of Hepatozoon canis and Cercopithifilaria bainae in an off-host population of Rhipicephalus sanguineus sensu lato ticks.

    PubMed

    Ramos, Rafael Antonio Nascimento; Giannelli, Alessio; Carbone, Domenico; Baneth, Gad; Dantas-Torres, Filipe; Otranto, Domenico

    2014-04-01

    Hepatozoon canis (Eucoccidiorida, Hepatozoidae) and the filarioid Cercopithifilaria bainae (Spirurida, Onchocercidae) are tick-transmitted infectious agents of dogs, highly prevalent in the Mediterranean basin in association with Rhipicephalus sanguineus sensu lato. Ticks were collected from the environment every 25±2 days in a confined location in southern Italy where a community of dogs lives, from August 2012 to July 2013. In order to study the occurrence of H. canis and C. bainae, 1091 tick specimens (770 adults; 271 nymphs, and 50 larvae) were dissected, and oocysts of H. canis and larvae of C. bainae were morphologically identified. Out of 1091 dissected ticks, 13.47% (n=147) were positive for H. canis, with the highest prevalence recorded in unfed adults (16.4%; 126/770), followed by nymphs collected as larvae and allowed to moult (14%; 7/50), unfed nymphs dissected immediately after collection (3%; 8/271), and adults collected as nymphs and allowed to moult (2%; 6/271). The highest number of H. canis-positive ticks (35.5%; 43/121; P<0.05) was recorded during the summer months (i.e., June-July). In addition, 6% of adult ticks (n=66) were positive for third-stage larvae of C. bainae, with the highest number in June (17%; 14/84; P<0.05). Based on the results reported herein, H. canis and C. bainae infections in the study area seem to be dependent on the seasonality of vector tick populations. Hence, dogs living in these areas are more exposed to both pathogens during the warmer months. These findings provide new insights into the ecology of both H. canis and C. bainae.

  4. PCR in forensic genetics.

    PubMed

    Morling, Niels

    2009-04-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.

  5. [PCR: basics and new developments].

    PubMed

    Berchtold, M W; Hübscher, U

    1996-01-01

    Since its discovery 10 years ago PCR has been introduced for a variety of practical applications. PCR has opened new dimensions particularly in laboratory diagnostics because of its sensitivity, accuracy and speed. In spite of the availability of user friendly kits, basic knowledge is of great importance for the user especially if PCR has to be optimized for special needs or when specific problems arise. The general mechanism of the reaction and the significance of the reaction components and the PCR conditions are discussed initially. Several recent developments in PCR (new enzymes, RNA-PCR, improvements of the specificity, prevention of contamination and development of new equipment) that are critical for the user are shortly introduced. Finally, "long PCR" is discussed in order to demonstrate that even 10 years after the invention of PCR significant new breakthroughs in the PCR technology are still possible.

  6. A Novel Multivalent OspA Vaccine against Lyme Borreliosis Is Safe and Immunogenic in an Adult Population Previously Infected with Borrelia burgdorferi Sensu Lato

    PubMed Central

    Barrett, P. Noel; Pöllabauer, Eva-Maria; O'Rourke, Maria; Portsmouth, Daniel; Schwendinger, Michael G.; Crowe, Brian A.; Livey, Ian; Dvorak, Thomas; Schmitt, Bernhard; Zeitlinger, Markus; Kollaritsch, Herwig; Esen, Meral; Kremsner, Peter G.; Jelinek, Tomas; Aschoff, Roland; Weisser, Roland; Naudts, Ingomar F. K.; Aichinger, Gerald

    2014-01-01

    Lyme borreliosis (LB) patients who recover, as well as previously infected asymptomatic individuals, remain vulnerable to reinfection with Borrelia burgdorferi sensu lato. There is limited information available about the use of OspA vaccines in this population. In this study, a randomized double-blind phase I/II trial was performed to investigate the safety and immunogenicity of a novel multivalent OspA vaccine in healthy adults who were either seronegative or seropositive for previous B. burgdorferi sensu lato infection. The participants received three monthly priming immunizations with either 30 μg or 60 μg alum-adjuvanted OspA antigen and a booster vaccination either 6 months or 9 to 12 months after the first immunization. The antibody responses to the six OspA serotypes included in the vaccine were evaluated. Adverse events were predominantly mild and transient and were similar in the seronegative and seropositive populations. Substantial enzyme-linked immunosorbent assay (ELISA) and surface-binding antibody responses against all six OspA antigens were induced after the primary immunization schedule in both populations, and they were substantially increased with both booster schedules. The antibody responses induced by the two doses were similar in the seronegative population, but there was a significant dose response in the seropositive population. These data indicate that the novel multivalent OspA vaccine is well tolerated and immunogenic in individuals previously infected with B. burgdorferi sensu lato. (This study is registered at ClinicalTrials.gov under registration no. NCT01504347.) PMID:25185574

  7. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  8. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  9. Onchocerca volvulus: comparison of field collection methods for the preservation of parasite and vector samples for PCR analysis.

    PubMed Central

    Toé, L.; Back, C.; Adjami, A. G.; Tang, J. M.; Unnasch, T. R.

    1997-01-01

    In recent years, methods for the identification of the filarial worm Onchocerca volvulus and its vector, blackflies of the Simulium damnosum complex (S. damnosum sensu lato (s.l.)), based on the amplification of parasite and vector DNA sequences with the polymerase chain reaction (PCR), have been developed. Routine application of these methods requires techniques for sample collection and preservation that are compatible with the limitations of field collection, yet preserve DNA in a form suitable for PCR. Two different methods for sample preservation were evaluated by the field collection teams and the DNA probe laboratory of the Onchocerciasis Control Programme in West Africa. The most successful involved the preservation of material from O. volvulus and its associated vectors in a dried state on microscope slides. Of over 1200 parasite samples preserved in this manner, more than 93% retained DNA yielding positive results in PCR analysis (1208/1291). Vector material (malpighian tubules and ovaries) preserved in the same manner on the same microscope slides also yielded DNA that was suitable for PCR. Images Fig. 2 PMID:9447777

  10. The discovery after 94 years of the elusive female of a myrmecolacid (Strepsiptera), and the cryptic species of Caenocholax fenyesi Pierce sensu lato.

    PubMed Central

    Kathirithamby, Jeyaraney; Johnston, J Spencer

    2004-01-01

    Due to its extreme sexual dimorphism and disparate hosts, no female myrmecolacid has been matched to its conspecific male to date. Here, for the first time to our knowledge, a morphological description is given of the matched female and male myrmecolacid, Caenocholax fenyesi waloffi ssp. nov. from Veracruz, Mexico: the female parasitic in a cricket and the male parasitic in an ant. For examined segments of DNA, the male and female are identical. Male C. fenyesi Pierce sensu lato was described 94 years ago from Veracruz. The male from Texas USA, which, for the same DNA segments, shows 15% divergence from the morphologically identical male from Veracruz, is given subspecies status, and is named Caenocholax fenyesi texensis ssp. nov. The discovery of the female finally enables many interesting studies to be pursued, such as speciation in morphologically cryptic taxa, the sexes of which parasitize disparate hosts. Caenocholax fenyesi sensu lato may also be evaluated for biocontrol of the red imported fire ant, Solenopsis invicta Buren, which is a pest in the USA and Australia. PMID:15101403

  11. Copro-PCR prevalence of Echinococcus granulosus infection in dogs in Kerman, south-eastern Iran.

    PubMed

    Mirbadie, S R; Kamyabi, H; Mohammadi, M A; Shamsaddini, S; Harandi, M F

    2017-01-30

    The main objective of this study was to determine the prevalence of taeniid parasites and the specific detection of Echinococcus granulosus using copro-DNA polymerase chain reaction (PCR) analysis in the stray dogs of Kerman, south-eastern Iran. From September 2013 to May 2014, faecal samples of stray dogs were collected from different parts of the city of Kerman and its suburbs. Faecal samples from dogs were collected randomly within 24 h of defecation. All samples were transferred to the research lab and coprological examinations were conducted by the formalin-ether concentration method. In the microscopically positive samples, mitochondrial cytochrome c oxidase subunit 1 (cox1) specific primers were used to determine the taeniid identity of the infection. In addition, another set of primers was used for the specific diagnosis of E. granulosus sensu lato. In total, 307 faecal samples from stray dogs were examined for the presence of the parasites. Taeniidae eggs were detected in 34 dogs (11.07%). All 34 taeniid-positive specimens were PCR positive for cox1 (444 bp). Of all taeniid-positive specimens, 21 samples (6.8% of all dog specimens) were positive according to primers specific for E. granulosus. The findings of the present study revealed that canine echinococcosis is prevalent in the stray dogs in Kerman. The findings of the present study have important implications for hydatid control programmes in the area.

  12. Borrelia spirochetes in Russia: Genospecies differentiation by real-time PCR.

    PubMed

    Mukhacheva, T A; Kovalev, S Y

    2014-10-01

    Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis which is widespread in Russia. Nowadays, three clinically important B. burgdorferi s.l. genospecies, B. afzelii, B. garinii, B. bavariensis sp. nov., can be found in Russia, as well as B. miyamotoi, which belongs to the tick-borne relapsing fever group of spirochetes. Several techniques have been developed to differentiate Borrelia genospecies. However, most of them do not allow detection of all of these genospecies simultaneously. Also, no method based on the RT-PCR TaqMan approach has been proposed to differentiate the genetically closely related species B. bavariensis and B. garinii. In the present paper, we investigated two species of ticks, I. persulcatus and I. pavlovskyi (1343 and 92 adults, respectively). Two sets of primers and probes for RT-PCR, with uvrA, glpQ and nifS genes as targets, were designed to detect four Borrelia genospecies in positive samples. The average prevalence of Borrelia sp. was about 40%, with B. afzelii as the most prevalent genospecies. Mixed infections of B. bavariensis and B. garinii were found to be extremely rare. While B. bavariensis was predominant in I. persulcatus, I. pavlovskyi ticks were infected exclusively by B. garinii. The proposed technique proved to be efficient in selection of individual Borrelia species for further genetic analysis, in particular, for multilocus sequence typing. Also, it could be applied for the differentiation of Borrelia genospecies in clinical material.

  13. Rodent species as natural reservoirs of Borrelia burgdorferi sensu lato in different habitats of Ixodes ricinus in The Netherlands.

    PubMed

    Gassner, Fedor; Takken, Willem; Plas, Carin Lombaers-van der; Kastelein, Pieter; Hoetmer, Arno J; Holdinga, Maarten; van Overbeek, Leonard S

    2013-09-01

    Rodents are natural reservoirs for human pathogenic spirochaetes of the Borrelia burgdorferi complex [B. burgdorferi sensu lato (s.l.)], and the pathogens are transmitted by Ixodes ricinus ticks to humans in The Netherlands. B. burgdorferi s.l. infection prevalence in questing ticks, rodents, and ticks feeding on these rodents, all sampled within the same short time span of five days in three different areas in The Netherlands, were compared in order to establish the relationship between ticks, reservoir hosts, and B. burgdorferi s.l. Questing nymphs were found in all 3 areas and numbers differed per area and even per site within areas. Infection prevalence in questing nymphs ranged between 0 and 20%. Apodemus sylvaticus and Myodes glareolus were the dominant rodents captured, and their numbers differed per area. Infection prevalence, determined by ear biopsies, ranged between 0 and 33.3% for both rodent species. Larvae were most frequently found feeding on these rodents, and their Borrelia infection prevalence ranged between 0 and 6.3% (A. sylvaticus) and between 0 and 29.4% (M. glareolus). The burden of nymphs feeding on rodents was low and varied per area with only 2 of 42 nymphs infected. Comparisons made on the basis of infection prevalence indicated that there was no clear relationship between rodents and questing nymphs when sampled within the same short time span. However, a possible relationship was present when questing ticks were sampled over longer periods in time (months) within or near the same areas (range of infection prevalence between 3.7 and 39.4). Confounding factors thus play a role in the interaction between rodents, ticks, and B. burgdorferi s.l., and it is very likely that other reservoir host species are responsible for the observed fluctuations. It is concluded that the local variations in rodent-Borrelia-tick interactions only partially explain the Lyme borreliosis risk in the sites studied and that other ecological determinants, notably

  14. Comparative biology of the tropical and temperate species of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) under different laboratory conditions.

    PubMed

    Labruna, Marcelo B; Gerardi, Monize; Krawczak, Felipe S; Moraes-Filho, Jonas

    2017-01-01

    Recent studies have shown that the taxon Rhipicephalus sanguineus sensu lato (s.l.) is represented in Latin America by two distinct species, designated as 'tropical species' (distributed from Mexico to Brazil) and 'temperate species' (restricted to the southern cone of South America). Since both tropical and temperate species of R. sanguineus s.l. are parasites primarily of domestic dogs, the reasons for their distinct geographical distribution in South America could be related to particular requirements of abiotic conditions for off-host development. With the purpose to test this hypothesis, this study evaluated the off-host developmental stages (eggs, engorged larvae, nymphs and females) of both tick species simultaneously inside incubators with temperature and photoperiod regimens that simulated the summer and winter conditions of tropical Brazil (where the 'tropical species' occurs) and temperate Brazil (where the 'temperate species' occurs). Results showed that the temperate species had significantly higher survival rates than the tropical species, when engorged ticks (larvae, nymphs and females) and eggs were incubated at lower temperatures simulating winter seasons of many parts of the southern cone of South America, where the temperate species is known to occur. These results suggest that the absence of established populations of the tropical species in temperate areas of South America is related to the low overwinter capacity of the tropical species in those areas. Regarding the temperate species, unfed adults that molted from nymphs under summer conditions of either tropical or temperate Brazil remained dormant, at the state of behavioral diapause for at least 20 weeks. Contrastingly, when engorged nymphs of the temperate species were held at winter conditions for at least 3 months, and then transferred to summer conditions to complete molting, no diapause was observed in adult ticks. These results were corroborated by infestation trials, which showed

  15. [New records of ectoparasites of the eastern water bat Myotis petax Hollister, 1912 (Vespertilionidae, Chiroptera) and the revision of the previous material collected from Myotis daubentonii s. lato in Eastern Palaearctic].

    PubMed

    Orlova, M V; Orlov, O L; Zhigalin, A V

    2014-01-01

    New findings of ectoparasites from the eastern water bat Myotis petax Hollister, 1912, belonging to the Siberian-Russian Far Eastern complex and earlier attributed as a subspecies of the Daubenton's bat Myotis daubentonii s. lato, is given. Fourteen species of bloodsucking arthropods feed on M. petax; these species include 4, 3, and 7 species of gamasid mites, fleas, and bloodsucking flies, respectively.

  16. The enzootic life-cycle of Borrelia burgdorferi (sensu lato) and tick-borne rickettsiae: an epidemiological study on wild-living small mammals and their ticks from Saxony, Germany.

    PubMed

    Obiegala, Anna; Król, Nina; Oltersdorf, Carolin; Nader, Julian; Pfeffer, Martin

    2017-03-13

    Borrelia burgdorferi (sensu lato) and rickettsiae of the spotted fever group are zoonotic tick-borne pathogens. While small mammals are confirmed reservoirs for certain Borrelia spp., little is known about the reservoirs for tick-borne rickettsiae. Between 2012 and 2014, ticks were collected from the vegetation and small mammals which were trapped in Saxony, Germany. DNA extracted from ticks and the small mammals' skin was analyzed for the presence of Rickettsia spp. and B. burgdorferi (s.l.) by qPCR targeting the gltA and p41 genes, respectively. Partial sequencing of the rickettsial ompB gene and an MLST of B. burgdorferi (s.l.) were conducted for species determination. In total, 673 small mammals belonging to eight species (Apodemus agrarius, n = 7; A. flavicollis, n = 214; Microtus arvalis, n = 8; Microtus agrestis, n = 1; Mustela nivalis, n = 2; Myodes glareolus, n = 435; Sorex araneus, n = 5; and Talpa europaea, n = 1) were collected and examined. In total, 916 questing ticks belonging to three species (Ixodes ricinus, n = 741; Dermacentor reticulatus, n = 174; and I. trianguliceps, n = 1) were collected. Of these, 474 ticks were further investigated. The prevalence for Rickettsia spp. and B. burgdorferi (s.l.) in the investigated small mammals was 25.3 and 31.2%, respectively. The chance of encountering Rickettsia spp. in M. glareolus was seven times higher for specimens infested with D. reticulatus than for those which were free of D. reticulatus (OR: 7.0; 95% CI: 3.3-14.7; P < 0.001). In total, 11.4% of questing I. ricinus and 70.5% of D. reticulatus were positive for Rickettsia spp. DNA of B. burgdorferi (s.l.) was detected only in I. ricinus (5.5%). Sequence analysis revealed 9 R. helvetica, 5 R. raoultii, and 1 R. felis obtained from 15 small mammal samples. Small mammals may serve as reservoirs for Rickettsia spp. and B. burgdorferi (s.l.). While the prevalence for Rickettsia spp. in M. glareolus is most

  17. Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda).

    PubMed

    Gvoždík, Václav; Canestrelli, Daniele; García-París, Mario; Moravec, Jiří; Nascetti, Giuseppe; Recuero, Ernesto; Teixeira, José; Kotlík, Petr

    2015-02-01

    European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these 'short-call tree frogs' based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lineages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridization were detected between them (H. arborea×H. orientalis; H. arborea×H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship between the geographically remote Iberian H. molleri and H. orientalis from around the Black Sea. Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felix arabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show

  18. Molecular survey of Dirofilaria immitis and Dirofilaria repens by direct PCR for wild caught mosquitoes in the Republic of Korea.

    PubMed

    Lee, Sang-Eun; Kim, Heung-Chul; Chong, Sung-Tae; Klein, Terry A; Lee, Won-Ja

    2007-09-01

    Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) x 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.

  19. Seasonal Variation in Biting Rates of Simulium damnosum sensu lato, Vector of Onchocerca volvulus, in Two Sudanese Foci

    PubMed Central

    Zarroug, Isam M. A.; Hashim, Kamal; Elaagip, Arwa H.; Samy, Abdallah M.; Frah, Ehab A.; ElMubarak, Wigdan A.; Mohamed, Hanan A.; Deran, Tong Chor M.; Aziz, Nabil; Higazi, Tarig B.

    2016-01-01

    Background The abundance of onchocerciasis vectors affects the epidemiology of disease in Sudan, therefore, studies of vector dynamics are crucial for onchocerciasis control/elimination programs. This study aims to compare the relative abundance, monthly biting-rates (MBR) and hourly-based distribution of onchocerciasis vectors in Abu-Hamed and Galabat foci. These seasonally-based factors can be used to structure vector control efforts to reduce fly-biting rates as a component of onchocerciasis elimination programs. Methods A cross-sectional study was conducted in four endemic villages in Abu-Hamed and Galabat foci during two non-consecutive years (2007–2008 and 2009–2010). Both adults and aquatic stages of the potential onchocerciasis vector Simulium damnosum sensu lato were collected following standard procedures during wet and dry seasons. Adult flies were collected using human landing capture for 5 days/month. The data was recorded on handheld data collection sheets to calculate the relative abundance, MBR, and hourly-based distribution associated with climatic factors. The data analysis was carried out using ANOVA and Spearman rank correlation tests. Results Data on vector surveillance revealed higher relative abundance of S. damnosum s.l. in Abu- Hamed (39,934 flies) than Galabat (8,202 flies). In Abu-Hamed, vector populations increased in January-April then declined in June-July until they disappeared in August-October. Highest black fly density and MBR were found in March 2007 (N = 9,444, MBR = 58,552.8 bites/person/month), and March 2010 (N = 2,603, MBR = 16,138.6 bites/person/month) while none of flies were collected in August-October (MBR = 0 bites/person/month). In Galabat, vectors increased in September-December, then decreased in February-June. The highest vector density and MBR were recorded in September 2007 (N = 1,138, MBR = 6,828 bites/person/month) and September 2010 (N = 1,163, MBR = 6,978 bites/person/month), whereas, none appeared in

  20. Seasonal Variation in Biting Rates of Simulium damnosum sensu lato, Vector of Onchocerca volvulus, in Two Sudanese Foci.

    PubMed

    Zarroug, Isam M A; Hashim, Kamal; Elaagip, Arwa H; Samy, Abdallah M; Frah, Ehab A; ElMubarak, Wigdan A; Mohamed, Hanan A; Deran, Tong Chor M; Aziz, Nabil; Higazi, Tarig B

    2016-01-01

    The abundance of onchocerciasis vectors affects the epidemiology of disease in Sudan, therefore, studies of vector dynamics are crucial for onchocerciasis control/elimination programs. This study aims to compare the relative abundance, monthly biting-rates (MBR) and hourly-based distribution of onchocerciasis vectors in Abu-Hamed and Galabat foci. These seasonally-based factors can be used to structure vector control efforts to reduce fly-biting rates as a component of onchocerciasis elimination programs. A cross-sectional study was conducted in four endemic villages in Abu-Hamed and Galabat foci during two non-consecutive years (2007-2008 and 2009-2010). Both adults and aquatic stages of the potential onchocerciasis vector Simulium damnosum sensu lato were collected following standard procedures during wet and dry seasons. Adult flies were collected using human landing capture for 5 days/month. The data was recorded on handheld data collection sheets to calculate the relative abundance, MBR, and hourly-based distribution associated with climatic factors. The data analysis was carried out using ANOVA and Spearman rank correlation tests. Data on vector surveillance revealed higher relative abundance of S. damnosum s.l. in Abu- Hamed (39,934 flies) than Galabat (8,202 flies). In Abu-Hamed, vector populations increased in January-April then declined in June-July until they disappeared in August-October. Highest black fly density and MBR were found in March 2007 (N = 9,444, MBR = 58,552.8 bites/person/month), and March 2010 (N = 2,603, MBR = 16,138.6 bites/person/month) while none of flies were collected in August-October (MBR = 0 bites/person/month). In Galabat, vectors increased in September-December, then decreased in February-June. The highest vector density and MBR were recorded in September 2007 (N = 1,138, MBR = 6,828 bites/person/month) and September 2010 (N = 1,163, MBR = 6,978 bites/person/month), whereas, none appeared in collection from April to June. There

  1. Molecular phylogenetic relationships among members of the family Phytolaccaceae sensu lato inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Lee, J; Kim, S Y; Park, S H; Ali, M A

    2013-02-28

    The phylogeny of a phylogenetically poorly known family, Phytolaccaceae sensu lato (s.l.), was constructed for resolving conflicts concerning taxonomic delimitations. Cladistic analyses were made based on 44 sequences of the internal transcribed spacer of nuclear ribosomal DNA from 11 families (Aizoaceae, Basellaceae, Didiereaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae s.l., Polygonaceae, Portulacaceae, Sarcobataceae, Tamaricaceae, and Nepenthaceae) of the order Caryophyllales. The maximum parsimony tree from the analysis resolved a monophyletic group of the order Caryophyllales; however, the members, Agdestis, Anisomeria, Gallesia, Gisekia, Hilleria, Ledenbergia, Microtea, Monococcus, Petiveria, Phytolacca, Rivinia, Schindleria, Seguieria, Stegnosperma, and Trichostigma, which belong to the family Phytolaccaceae s.l., did not cluster under a single clade, demonstrating that Phytolaccaceae is polyphyletic.

  2. Further studies on Contracaecum spasskii Mozgovoi, 1950 and C. rudolphii Hartwich, 1964 (sensu lato) (Ascaridida: Anisakidae) from piscivorous birds in China.

    PubMed

    Li, Liang; Xu, Zhen; Zhang, Lu-Ping

    2013-03-01

    Contracaecum spasskii Mozgovoi, 1950, collected from the great crested grebe Podiceps cristatus (Linnaeus) (Podicipediformes: Podicipedidae), is redescribed using both light and, for the first time, scanning electron microscopy. Contracaecum spasskii differs from its congeners by having marked transverse cuticular annulations, the length of the oesophagus and spicules, the ratio between the intestinal caecum and the ventricular appendix, the number and arrangement of male caudal papillae, and especially by the particular morphology of the lips and interlabia. Some previously unreported morphological features of C. spasskii are also revealed and others corrected. Contracaecum rudolphii Hartwich, 1964 (sensu lato) is also redescribed based on the specimens collected from the great cormorant Phalacrocorax carbo sinensis (Blumenbach) (Pelecaniformes: Phalacrocoracidae) from China. Based on the geographical perspective, the present Chinese material may represent the species C. rudolphii B.

  3. The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry.

    PubMed

    Soriano, S V; Pierangeli, N B; Pianciola, L A; Mazzeo, M; Lazzarini, L E; Debiaggi, M F; Bergagna, H F J; Basualdo, J A

    2015-01-01

    Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 μm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

  4. Correlation of Borrelia burgdorferi Sensu Lato Prevalence in Questing Ixodes ricinus Ticks with Specific Abiotic Traits in the Western Palearctic▿†

    PubMed Central

    Estrada-Peña, Agustín; Ortega, Carmelo; Sánchez, Nely; DeSimone, Lorenzo; Sudre, Bertrand; Suk, Jonathan E.; Semenza, Jan C.

    2011-01-01

    This meta-analysis of reports examining ticks throughout the Western Palearctic region indicates a distinct geographic pattern for Borrelia burgdorferi sensu lato prevalence in questing nymphal Ixodes ricinus ticks. The greatest prevalence was reported between the 5°E and 25°E longitudes based on an analysis of 123 collection points with 37,940 nymphal tick specimens (87.43% of total nymphs; 56.35% of total ticks in the set of reports over the target area). Climatic traits, such as temperature and vegetation stress, and their seasonality correlated with Borrelia prevalence in questing ticks. The greatest prevalence was associated with mild winter, high summer, and low seasonal amplitude of temperatures within the range of the tick vector, higher vegetation indices in the May-June period, and well-connected vegetation patches below a threshold at which rates suddenly drop. Classification of the target territory using a qualitative risk index derived from the abiotic variables produced an indicator of the probability of finding infected ticks in the Western Palearctic region. No specific temporal trends were detected in the reported prevalence. The ranges of the different B. burgdorferi sensu lato genospecies showed a pattern of high biodiversity between 4°W and 20°E, partially overlapping the area of highest prevalence in ticks. Borrelia afzelii and Borrelia garinii are the dominant species in central Europe (east of ∼25°E), but B. garinii may appear alone at southern latitudes and Borrelia lusitaniae is the main indicator species for meridional territories. PMID:21498767

  5. Isolation of Borrelia burgdorferi sensu lato from blood of adult patients with borrelial lymphocytoma, Lyme neuroborreliosis, Lyme arthritis and acrodermatitis chronica atrophicans.

    PubMed

    Maraspin, V; Ogrinc, K; Ružić-Sabljić, E; Lotrič-Furlan, S; Strle, F

    2011-02-01

    Reports on patients with European Lyme borreliosis in whom borreliae were isolated from the blood are rare and nearly exclusively limited to those with solitary or multiple erythema migrans. Here we report on patients with other manifestations of Lyme borreliosis in whom borreliae were isolated from their blood. This is a retrospective review of the medical files of patients diagnosed with borrelial lymphocytoma, Lyme neuroborreliosis, Lyme arthritis and acrodermatitis chronica atrophicans at the Department of Infectious Diseases of the UMC Ljubljana, Slovenia, for whom a borrelia blood culture was ordered. The clinical features of patients whose blood culture tested positive for Borrelia burgdorferi sensu lato were reviewed, and the association between the proportion of patients with a positive blood culture and various clinical manifestations was examined. Borrelia burgdorferi sensu lato was isolated from the blood of 1/53 (1.9%) patients with borrelial lymphocytoma, 6/176 (3.4%) patients with Lyme neuroborreliosis, 1/13 (7.7%) patients with Lyme arthritis, and 3/200 (1.5%) patients with acrodermatitis chronica atrophicans. The time interval from the onset of symptoms attributed to Lyme borreliosis and the blood culture ranged from 1 day to >2 years (median 3.5 weeks). At the time of the blood culture, erythema migrans was present in 4/11 (36.4%) borrelia blood culture-positive patients, i.e. in the patient with borrelial lymphocytoma, the patient with Lyme arthritis and the 2/6 patients with Lyme neuroborreliosis. Only two of these 11 (18.2%) patients had fever at the time of the blood culture. In European patients with Lyme borreliosis, borreliae can be cultured from the blood not only early in the course of the disease but also occasionally later during disease progression.

  6. [Rapid PCR authentication Lonicera japanica].

    PubMed

    Jiang, Chao; Hou, Jing-Yi; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Jin, Yan

    2014-10-01

    To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.

  7. Neurocryptococcosis: diagnosis by PCR method.

    PubMed

    Paschoal, Regina Célia; Hirata, Mário Hiroyuki; Hirata, Rosário Crespo; Melhem, Márcia de Souza Carvalho; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues

    2004-01-01

    Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

  8. A study of PCR inhibition mechanisms using real time PCR.

    PubMed

    Opel, Kerry L; Chung, Denise; McCord, Bruce R

    2010-01-01

    In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.

  9. Primer design versus PCR bias in methylation independent PCR amplifications.

    PubMed

    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  10. Mitochondrial PCR-RFLP Assay to Distinguish Triatoma brasiliensis macromelasoma from Triatoma brasiliensis brasiliensis Subspecies (Hemiptera: Reduviidae).

    PubMed

    Vendrami, Daniel Pagotto; Ceretti-Junior, Walter; Obara, Marcos Takashi; Marrelli, Mauro Toledo

    2013-01-01

    Triatoma brasiliensis sensu lato (s.l.), the main vector of Chagas disease in northeastern Brazil, is a species complex comprising four species, one with two subspecies (T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. sherlocki, and T. melanica), and each taxon displaying distinct ecological requirements. In order to evaluate the genetic relationships among nine T. brasiliensis s.l. populations from northeastern Brazil, we analyzed their mitochondrial cytochrome c oxidase subunit 1 sequences and suggested a PCR-RFLP assay to distinguish between T. b. macromelasoma and T. b. brasiliensis subspecies. All the specimens were morphologically identified as T. b. brasiliensis. The resulting phylogenies identified two major clades that are congruent with the geographical populations studied. Based on collection sites and in accordance with type-location, one clade was identified as the subspecies T. b. macromelasoma. The second clade grouped T. b. brasiliensis populations. Restriction endonuclease sites were observed in the sequences and used in PCR-RFLP assays, producing distinct fingerprints for T. b. macromelasoma and T. b. brasiliensis populations. The results suggest that these are different species and that gene flow occurs only among T. b. brasiliensis populations, possibly associated with human activity in the area.

  11. A new PCR assay for the detection and differentiation of Babesia canis and Babesia vogeli.

    PubMed

    Annoscia, Giada; Latrofa, Maria Stefania; Cantacessi, Cinzia; Olivieri, Emanuela; Manfredi, Maria Teresa; Dantas-Torres, Filipe; Otranto, Domenico

    2017-10-01

    Babesia spp. are globally distributed tick-borne protozoan parasites that infect the red blood cells of a wide range of vertebrate hosts, including humans. Diagnosis of babesiosis is often impeded by the transient presence of the parasites in peripheral blood, as well as by their pleomorphic nature. Given the reports of an expanding and, in some cases, sympatric geographical distribution of Babesia canis and Babesia vogeli in dogs and associated vectors, in Europe, the development of time-efficient and cost-effective diagnostic tools to detect and differentiate these two species is warranted. In this study, we designed and developed a novel polymerase chain reaction (PCR) assay targeting the parasite cytochrome c oxidase subunit 1 (cox1) gene, for the simultaneous detection and differentiation of B. canis and B. vogeli. The analytical sensitivity of the PCR was evaluated using serial dilutions of genomic DNA extracted from individual and artificially mixed canine blood samples infected by B. canis (3×10(2) infected erythrocytes/ml, ie/ml) and B. vogeli (2.1×10(1) ie/ml). The analytical specificity of the assay was assessed using blood samples positive for Hepatozoon canis, Ehrlichia canis, Anaplasma platys, Babesia microti, Babesia rossi and Theileria annae (syn. Babesia vulpes). The clinical specificity of the PCR assay was evaluated on 147 blood samples from dogs and 128 tick specimens (Dermacentor reticulatus and Rhipicephalus sanguineus sensu lato). Species-specific bands of the expected sizes (i.e., 750bp for B. canis and 450bp for B. vogeli), and two bands in the mixed blood samples were obtained. The PCR assay developed herein detected a low number of infected erythrocytes (i.e., 3×10(-2)B. canis, 2.1×10(-2)B. vogeli ie/ml). Of the 147 blood samples, nine (6.1%) were positive for B. canis and six (4.1%) for B. vogeli, whereas only one tick (D. reticulatus) was positive for B. canis. This PCR assay represents a rapid and reliable tool for the diagnosis of

  12. Taxa and names in Cynoglossum sensu lato (Boraginaceae, Cynoglosseae): an annotated, synonymic inventory, with links to the protologues and mention of original material

    PubMed Central

    Stier, Victoria

    2015-01-01

    Abstract Background An inventory is presented of all names so far validly published in Cynoglossum sensu lato and its segregate genera: Adelocaryum, Afrotysonia, Kuschakewiczia, Lindelofia, Mattiastrum, Paracaryum, Rindera, Solenanthus, Trachelanthus, and their synonyms. Names and designations that were not validly published in the cited place, and later isonyms, are accounted for when they have been included in the International Plant Name Index (IPNI). Problems with IPNI entries, including errors and omissions, are discussed, and the hope is expressed that the present inventory may be of use for fixing them. New information The inventory, generated from a list of structured data, is presented in two Supplements, as a searchable HTML document comprising a sequence of entries with internal cross-links and links to external sources, in particular to protologues accessible online or, copyright restrictions permitting, made available as scanned documents via DOIs, and as machine-readible file. With minor exceptions, all names have been verified in their original place of publication, and all were nomenclaturally assessed. Colour coding is used to distinguish between names (in green) pertaining to Cynoglossum sensu lato, for which complete synonymies are provided; and names (in orange) pertaining to other genera but published under Cynoglossum or its segregates. They are listed together with their basionym and the corresponding correct name (if it exists), but without complete synonymy. Acceptable, potentially correct names appear in bold-face type, both under a broadly defined Cynoglossum (for which purpose validation of 81 new combinations and the name of 1 new species was necessary) and under one or more of its segregates. When a name was published for a new taxon, original material is indicated, usually by direct quotation from the protologue. New type designations are exceptional (two cases), whereas former type designations are cited whenever known. Furthermore

  13. Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Romania.

    PubMed

    Dumitrache, Mirabela Oana; Paştiu, Anamaria Ioana; Kalmár, Zsuzsa; Mircean, Viorica; Sándor, Attila David; Gherman, Călin Mircea; Peştean, Cosmin; Mihalca, Andrei Daniel; Cozma, Vasile

    2013-04-01

    Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are two tick-borne pathogens of medical and/or veterinary importance which are distributed worldwide. Erinaceus roumanicus, the Northern white-breasted hedgehog, is a common synanthropic species that is known to carry not only the hedgehog tick, Ixodes hexagonus, but also I. ricinus, the most common European tick species. I. ricinus is the main vector of both mentioned pathogens. Within this framework and because only limited information is available on the role of E. roumanicus in the ecology of B. burgdorferi s.l. and Anaplasma phagocytophilum in Europe, we carried out an epidemiological surveillance on this species in Romania. From the 57 examined hedgehogs collected in 12 different counties, 24 presented tick infestation. Most ticks (n=959) were morphologically identified as larvae, nymphs, or adults of I. ricinus. The prevalence of B. burgdorferi s.l. was 0.4%, and that of A. phagocytophilum 12%. In all positive cases for B. burgdorferi s.l., restriction fragment length polymorphism revealed the genospecies B. afzelii. In Romania, only limited information is available on the epidemiology of B. burgdorferi s.l. and A. phagocytophilum. As hedgehogs commonly share the same environment with humans and other potential reservoir hosts for tick-borne pathogens, our study provides new epidemiological data of public health importance.

  14. Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment.

    PubMed

    Dréan, Paul; McAuley, Catherine M; Moore, Sean C; Fegan, Narelle; Fox, Edward M

    2015-02-19

    The Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia. Five of the seven B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms. This study showed that the B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats.

  15. First molecular evidence of the simultaneous human infection with two species of Echinococcus granulosus sensu lato: Echinococcus granulosus sensu stricto and Echinococcus canadensis.

    PubMed

    Oudni-M'rad, Myriam; M'rad, Selim; Ksia, Amine; Lamiri, Rachida; Mekki, Mongi; Nouri, Abdellatif; Mezhoud, Habib; Babba, Hamouda

    2016-03-01

    Cystic echinococcosis is a widespread zoonotic parasitic disease especially in Tunisia which is one of the most endemic countries in the Mediterranean area. The etiological agent, Echinococcus granulosus sensu lato, implies dogs and other canids as definitive hosts and different herbivore species as intermediate hosts. Human contamination occurs during the consumption of parasite eggs passed in the environment through canid feces. Hydatid cysts coming from a child operated for multiple echinococcosis were collected and analyzed in order to genotype and to obtain some epidemiological molecular information. Three targets, ribosomal DNA ITS1 fragment, NADH dehydrogenase subunit 1 (nad1), and mitochondrial cytochrome c oxydase subunit 1 (CO1) genes, were amplified and analyzed by RFLP and sequencing approach. This study presents the first worldwide report in human of a simultaneous infection with Echinococcus granulosus sensu stricto (genotype G1) and Echinococcus canadensis (genotype G6) species. This is also the first report of the presence of E. canadensis in the Tunisian population which argues in favor of a greater importance of this species in human infestation in Tunisia than previously believed.

  16. A phylogenetic re-appraisal of the family Liagoraceae sensu lato (Nemaliales, Rhodophyta) based on sequence analyses of two plastid genes and postfertilization development.

    PubMed

    Lin, Showe-Mei; Rodríguez-Prieto, Conxi; Huisman, John M; Guiry, Michael D; Payri, Claude; Nelson, Wendy A; Liu, Shao-Lun

    2015-06-01

    The marine red algal family Liagoraceae sensu lato is shown to be polyphyletic based on analyses of a combined rbcL and psaA data set and the pattern of carposporophyte development. Fifteen of eighteen genera analyzed formed a monophyletic lineage that included the genus Liagora. Nemalion did not cluster with Liagoraceae sensu stricto, and Nemaliaceae is reinstated, characterized morphologically by the formation of the primary gonimolobes by longitudinal divisions of the gonimoblast initial. Yamadaella and Liagoropsis, previously placed in the Dermonemataceae, are shown to be independent lineages and are recognized as two new families Yamadaellaceae and Liagoropsidaceae. Yamadaellaceae is characterized by two gonimoblast initials cut off bilaterally from the fertilized carpogonium and diffusely spreading gonimoblast filaments. Liagoropsidaceae is characterized by at least three gonimoblast initials cut off by longitudinal septa from the fertilized carpogonium. In contrast, Liagoraceae sensu stricto is characterized by a single gonimoblast initial cut off transversely or diagonally from the fertilized carpogonium. Reproductive features, such as diffuse gonimoblasts and unfused carpogonial branches following postfertilization, appear to have evolved on more than one occasion in the Nemaliales and are therefore not taxonomically diagnostic at the family level, although they may be useful in recognizing genera.

  17. Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato

    PubMed Central

    Meddeb, Mariam; Carpentier, Wassila; Cagnard, Nicolas; Nadaud, Sophie; Grillon, Antoine; Barthel, Cathy; De Martino, Sylvie Josiane; Jaulhac, Benoît; Boulanger, Nathalie

    2016-01-01

    In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether “species-related” inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved. PMID:27706261

  18. Spores and vegetative cells of phenotypically and genetically diverse Bacillus cereus sensu lato are common bacteria in fresh water of Northeastern Poland.

    PubMed

    Bartoszewicz, Marek; Czyzewska, Urszula

    2017-09-20

    Gram-positive rods named B. cereus sensu lato (sl), are common in natural habitats and food products. It is believed that they are restricted to spores, however their ecology in aquatic habitats is still poorly investigated. Thus, the aim of the study was to assess the rain-dependent fluctuations of the concentration of B. cereus sl vegetative cells and spores with evaluation of their phylogenetic and population structure in relation to the toxicity and psychrotolerance. We proved that vegetative cells of B. cereus sl are widely distributed in fresh water of rivers and lakes being as common as spores. Moreover, heavy rain shows huge impact on their concentration in undisturbed environments. The diversity of B. cereus sl reflects multiple sources of bacteria and differences between distinct environments. Next, their diverse genetic structure and phenotypes better fit to their ecological properties than to the taxonomic affiliation.

  19. A molecular evaluation of the Liagoraceae sensu lato (Nemaliales, Rhodophyta) in Bermuda including Liagora nesophila sp. nov. and Yamadaella grassyi sp. nov.

    PubMed

    Popolizio, Thea R; Schneider, Craig W; Lane, Christopher E

    2015-08-01

    We have undertaken a comprehensive, molecular-assisted alpha-taxonomic examination of the rhodophyte family Liagoraceae sensu lato, a group that has not previously been targeted for molecular studies in the western Atlantic. Sequence data from three molecular markers indicate that in Bermuda alone there are 10 species in nine different genera. These include the addition of three genera to the flora - Hommersandiophycus, Trichogloeopsis, and Yamadaella. Liagora pectinata, a species with a type locality in Bermuda, is phylogenetically allied with Indo-Pacific species of Hommersandiophycus, and the species historically reported as L. ceranoides for the islands is morphologically and genetically distinct from that taxon, and is herein described as L. nesophila sp. nov. Molecular sequence data have also uncovered the Indo-Pacific L. mannarensis in Bermuda, a long-distance new western Atlantic record. DNA sequences of Trichogloeopsis pedicellata from the type locality (Bahamas) match with local specimens demonstrating its presence in Bermuda. We described Yamadaella grassyi sp. nov. from Bermuda, a species phylogenetically and morphologically distinct from the generitype, Y. caenomyce of the Indo-Pacific. Our data also indicated a single species each of Ganonema, Gloiocallis, Helminthocladia, Titanophycus, and Trichogloea in the flora.

  20. Subjective health complaints are not associated with tick bites or antibodies to Borrelia burgdorferi sensu lato in blood donors in western Norway: a cross-sectional study.

    PubMed

    Hjetland, Reidar; Reiso, Harald; Ihlebæk, Camilla; Nilsen, Roy M; Grude, Nils; Ulvestad, Elling

    2015-07-14

    There is controversy about chronic health consequences of tick-borne infections, especially Lyme borreliosis. This study aims to assess whether general function, physical fitness and subjective health complaints are associated with tick bites or antibodies to Borrelia burgdorferi sensu lato in blood donors. Sera from 1,213 blood donors at four different blood banks in Sogn and Fjordane county in western Norway were obtained during January to June 2010, and analysed for specific IgG and IgM antibodies. A questionnaire including questions on tick bites, subjective health complaints, general function and physical fitness was completed. Tick bites had been experienced by 65.7% of the study population. 78 (6.4%) were positive for IgG (9.7% in men, 2.4% in women), and 69 (5.7%) for IgM (6.1% in men, 5.1% in women), verified by immunoblot. No association between number of experienced tick bites or seropositivity for Borrelia antibodies and subjective health complaints, reduced general function or reduced physical fitness was found. The results do not support any association between tick bites or Borrelia antibodies and subjective health complaints in blood donors in an endemic area for Lyme borreliosis.

  1. Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere.

    PubMed

    Ivanova, Larisa B; Tomova, Alexandra; González-Acuña, Daniel; Murúa, Roberto; Moreno, Claudia X; Hernández, Claudio; Cabello, Javier; Cabello, Carlos; Daniels, Thomas J; Godfrey, Henry P; Cabello, Felipe C

    2014-04-01

    Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by polymerase chain reaction and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group B. chilensis VA1 in honor of its country of origin. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Applications of Digital PCR for Clinical Microbiology.

    PubMed

    Kuypers, Jane; Jerome, Keith R

    2017-03-15

    Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory.

  3. Co-infection of Borrelia burgdorferi sensu lato and Rickettsia species in ticks and in an erythema migrans patient.

    PubMed

    Tijsse-Klasen, Ellen; Sprong, Hein; Pandak, Nenad

    2013-12-10

    Lyme borreliosis is the most prevalent tick-borne disease in Europe. Ixodes ricinus also carries other pathogenic bacteria, but corresponding human diseases are rarely reported. Here, we compared the exposure to Rickettsia helvetica and Rickettsia monacensis with that to Lyme borreliosis spirochetes. We assumed that their exposure corresponds to their infection rate in questing I. ricinus. Three Rickettsia species were detected in ticks with a total prevalence of 7.9%, of which the majority was R. helvetica (78%) and R. monacensis (21%). From the same geographic area, skin biopsies of erythema migrans patients were investigated for possible co-infections with Rickettsia spp.. Forty-seven out of 67 skin biopsies were PCR positive for Borrelia burgdorferi s.l. and one sample was positive for R. monacensis. The Borrelia genospecies from the R. monacensis positive patient was identified as Borrelia afzelii. The patient did not show any symptoms associated with rickettsiosis. Co-infections of I. ricinus with Rickettsia spp. and B. burgdorferi s.l. were as high as expected from the individual prevalence of both pathogens. Co-infection rate in erythema migrans patients corresponded well with tick infection rates. To our knowledge, this is the first reported co-infection of B. afzelii and R. monacensis.

  4. Borrelia burgdorferi Sensu Lato Species in Europe Induce Diverse Immune Responses against C6 Peptides in Infected Mice▿

    PubMed Central

    Krupka, Inke; Knauer, Jens; Lorentzen, Leif; O'Connor, Thomas P.; Saucier, Jill; Straubinger, Reinhard K.

    2009-01-01

    The diversity of Lyme-borreliosis-inducing Borrelia species in Europe set high standards for the use of serodiagnostic test systems in terms of specificity and sensitivity. In the United States, the one-step C6 antibody test system based on the invariable domain IR6 of the VlsE molecule has been established as a successful diagnostic tool for testing canine samples. However, only a limited set of data are available regarding the antigenicity of the C6 peptides in an experimental murine model and sensitivity of the test regarding European Borrelia species. In order to investigate antibody reactions induced by these spirochetes, a total of 142 C3H/HeN mice were inoculated with Borrelia burgdorferi sensu stricto N40, B. garinii PBi, two isolates of B. afzelii, B. spielmanii A14S, B. valaisiana Rio6, B. valaisiana VS116, or B. lusitaniae. Infection of the mice was documented utilizing tissue culture and PCR. The IR6 sequences of B. burgdorferi sensu stricto B31, B. garinii IP90, and two B. afzelii ACAI strains have been used to synthesize and test additional C6 peptides. Compared to the well-established two-tiered test system, the results indicate that single C6 peptides derived from B. burgdorferi sensu stricto and B. garinii can be used in an enzyme-linked immunosorbent assay-based technique to detect murine antibodies induced by either agent. Little is known about the prevalence or pathogenicity of the B. afzelii strains in mammalian hosts, but our experimental data indicate differences in the C6 peptide test sensitivity for the detection of antibodies induced by different strains or isolates of B. afzelii. PMID:19726618

  5. Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

    PubMed Central

    Amro, Ahmad; Mentis, Andreas; Pratlong, Francine; Dedet, Jean-Pierre; Votypka, Jan; Volf, Petr; Ozensoy Toz, Seray; Kuhls, Katrin; Schönian, Gabriele; Soteriadou, Ketty

    2012-01-01

    Background New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. Methodology/Principal Findings The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. Conclusion The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring

  6. Serological detection of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis antibodies and Dirofilaria immitis antigen in a countrywide survey in dogs in Poland.

    PubMed

    Krämer, Friederike; Schaper, Roland; Schunack, Bettina; Połozowski, Andrzej; Piekarska, Jolanta; Szwedko, Aleksandra; Jodies, Robert; Kowalska, Dagmara; Schüpbach, Dörte; Pantchev, Nikola

    2014-09-01

    Canine vector-borne diseases (CVBDs) have increasingly become a focus of attention in the past few years. Nevertheless, in many parts of Europe information on their occurrence is still scarce. In a large study in Poland 3,094 serum samples taken from dogs throughout all 16 Polish provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen. A total of 12.31% (381/3,094; 95% confidence interval [CI]: 11.18-13.52%) and 3.75% (116/3,094; 95% CI: 3.11-4.48%) of the dogs were positive for A. phagocytophilum and B. burgdorferi s.l. antibodies, respectively. Furthermore, 0.26% (8/3,094; 95% CI: 0.11-0.51%) were positive for E. canis antibodies and 0.16% (5/3,094; 95% CI: 0.05-0.38%) for D. immitis antigen. The highest percentages of A. phagocytophilum-positive dogs were noted in Lesser Poland, Silesia and Łódź Provinces. For B. burgdorferi s.l., the highest prevalence was recorded in Łódź Province. Co-infections with A. phagocytophilum and B. burgdorferi s.l. were recorded in 1.71% of all examined dogs (53/3,094; 95% CI: 1.29-2.23%). One dog even had a triple infection, testing positive for E. canis too. Both A. phagocytophilum and B. burgdorferi s.l. have previously been reported in Poland and were confirmed in the present study by positive samples from all 16 provinces. Concerning E. canis and D. immitis travel history or importation cannot be excluded as factors which may have determined the occurrence of these pathogens in the relevant animals. Practitioners in Poland should be aware of the above mentioned CVBDs and of prophylactic measures to protect dogs and their owners.

  7. Comparative efficacy of two oral treatments for dogs containing either afoxolaner or fluralaner against Rhipicephalus sanguineus sensu lato and Dermacentor reticulatus.

    PubMed

    Beugnet, Frederic; Liebenberg, Julian; Halos, Lenaïg

    2015-04-15

    The present study compares the efficacy of two recent oral ectoparasiticides containing isoxazolines (NexGard(®), containing afoxolaner and administered at a monthly regimen, and Bravecto™ containing fluralaner and administered at a tri-monthly regimen) against Rhipicephalus sanguineus sensu lato and Dermacentor reticulatus ticks on dogs. 24 dogs were randomly allocated to untreated control, NexGard(®) treated, and Bravecto™ treated groups. The treatments were administered on Days 0, 28 and 56 for afoxolaner and on Day 0 for fluralaner. Tick infestations were performed weekly with 50 unfed adult ticks per each species on each dog from Days 30 to 84 (with the exception of R. sanguineus on Day 63). Ticks were counted at 24h post-infestation. The dogs from both treated groups had statistically significantly (p<0.05) less R. sanguineus and D. reticulatus ticks compared to the untreated dogs on all assessment days. Percent efficacy against R. sanguineus ranged from 86.4% to 99.5% at 24h post-infestation for NexGard(®) and from 65.7% to 100% for Bravecto™. Statistically significantly (p<0.05) less R. sanguineus ticks were recorded for NexGard(®) treated dogs compared to Bravecto™ treated dogs on Day 78. Percent efficacy against D. reticulatus ranged from 85.2% to 99.6% at 24h post-infestation for NexGard(®) and from 63.4% to 99.1% for Bravecto™. Statistically significantly (p<0.05) less D. reticulatus ticks were recorded for NexGard(®) treated dogs compared to Bravecto™ treated dogs on Days 71, 78 and 85.

  8. Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host.

    PubMed

    de Oliveira Filho, Jaires Gomes; Ferreira, Lorena Lopes; Sarria, André Lucio Franceschini; Pickett, John A; Birkett, Michael A; Mascarin, Gabriel Moura; de León, Adalberto A Pérez; Borges, Lígia Miranda Ferreira

    2017-01-01

    Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs. Copyright © 2016 Elsevier GmbH. All rights reserved.

  9. Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth

    PubMed Central

    Walker-York-Moore, Laura; Moore, Sean C.; Fox, Edward M.

    2017-01-01

    Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA, hblA and entFM toxin genes, with lower prevalence of bceT and hlyII. When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log10(CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices. PMID:28714887

  10. Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

    PubMed

    Cucher, Marcela Alejandra; Macchiaroli, Natalia; Baldi, Germán; Camicia, Federico; Prada, Laura; Maldonado, Lucas; Avila, Héctor Gabriel; Fox, Adolfo; Gutiérrez, Ariana; Negro, Perla; López, Raúl; Jensen, Oscar; Rosenzvit, Mara; Kamenetzky, Laura

    2016-02-01

    To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation. © 2015 John Wiley & Sons Ltd.

  11. Serological survey of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Ehrlichia canis infections in rural and urban dogs in Central Italy.

    PubMed

    Ebani, Valentina Virginia; Bertelloni, Fabrizio; Torracca, Beatrice; Cerri, Domenico

    2014-01-01

    Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum are well known zoonotic pathogens, whereas Ehrlichia canis is usually considered to be of veterinary concern, although on the basis of recent reports it also seems to be able to infect humans. The aim of the study was to determine the seroprevalence of B. burgdorferi s.l., A. phagocytophilum and E. canis in an Italian canine population, and to verify if there are differences between dogs living in urban areas and those from a rural environment. Blood sera of 1,965 dogs, 1,235 from cities and 730 from rural areas, were tested by indirect immunofluorescent assay (IFAT). The overall seroprevalence was highest for E. canis (7.07%), followed by A. phagocytophilum (4.68%), and B. burgdorferi s.l. (1.47%). Rural dogs showed the highest seroprevalence to B. burgdorferi s.l. and A. phagocytophilum. No significant differences were observed between rural and urban E. canis-positive dogs. A low percentage (1.32%) of dogs with dual seropositivity was detected, and no triple positive reactions were observed. No significant differences were detected in the seroprevalence of the three agents in relationship to the age and gender of the dogs. Seroprevalence in the five years considered were not statistically different, except for the lowest rate for E. canis observed in 2012. The results confirm the presence of B. burgdorferi s.l., A. phagocytophilum and E. canis in Italian dogs in both urban and rural areas. Monitoring pet dogs, which share the same environment with their owners, is useful for identifying the presence of tick-borne disease agents of both veterinary and public health significance.

  12. Seroprevalence and risk factors associated with Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and D. immitis in hunting dogs from southern Italy.

    PubMed

    Piantedosi, Diego; Neola, Benedetto; D'Alessio, Nicola; Di Prisco, Francesca; Santoro, Mario; Pacifico, Laura; Sgroi, Giovanni; Auletta, Luigi; Buch, Jesse; Chandrashekar, Ramaswamy; Breitschwerdt, Edward B; Veneziano, Vincenzo

    2017-08-04

    Canine vector-borne diseases (CVBDs) are caused by a range of pathogens transmitted to dogs by arthropods. The present study investigates Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and Dirofilaria immitis seroprevalences in hunting dogs from southern Italy. Dogs (no. 1335) were tested using a commercial in-clinic enzyme-linked immunosorbent assay kit. Odds ratios (ORs) were calculated by logistic regression analysis to identify risk factors. Overall, 138/1335 dogs (10.3%) were seroreactive to at least one CVBD pathogen. E. canis, Anaplasma spp., B. burgdorferi s.l., and D. immitis seroprevalences were 7.6, 4.4, 0.3, and 0.2%, respectively. E. canis and Anaplasma spp. co-exposures were found in 30 dogs (2.2%), compared with Anaplasma spp. and B. burgdorferi s.l. co-exposures in 2 dogs (0.1%). Adult age was a risk factor for E. canis (OR 2.35) seroreactivity whereas hunting fur-bearing animals for E. canis (OR 4.75) and Anaplasma spp. (OR 1.87), respectively. The historical presence of tick infestation was identified as a risk factor for positivity to E. canis (OR 2.08) and Anaplasma spp. (OR 2.15). Finally, larger dog pack size was significantly associated with E. canis (OR 1.85) and Anaplasma spp. (OR 2.42) exposures. The results of the present survey indicated that hunting dog populations are at relative risk of CVBDs in southern Italy. Further studies are needed to evaluate the role of hunting dogs in the epidemiology of vector-borne organisms due to sharing common environments with wild, sympatric animal populations.

  13. ITS1, 5.8S and ITS2 secondary structure modelling for intra-specific differentiation among species of the Colletotrichum gloeosporioides sensu lato species complex.

    PubMed

    Rampersad, Sephra N

    2014-01-01

    The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of member species which are of quarantine importance is impacted by a number of factors that negatively affect species identification. Structural information of the rRNA marker may be considered to be a conserved marker which can be used as supplementary information for possible species identification. The difficulty in using ITS rDNA sequences for identification lies in the low level of sequence variation at the intra-specific level and the generation of artificially-induced sequence variation due to errors in polymerization of the ITS array during DNA replication. Type and query ITS sequences were subjected to sequence analyses prior to generation of predicted consensus secondary structures, including the pattern of nucleotide polymorphisms and number of indel haplotypes, GC content, and detection of artificially-induced sequence variation. Data pertaining to structure stability, the presence of conserved motifs in secondary structures and mapping of all sequences onto the consensus C. gloeosporioides sensu stricto secondary structure for ITS1, 5.8S and ITS2 markers was then carried out. Motifs that are evolutionarily conserved among eukaryotes were found for all ITS1, 5.8S and ITS2 sequences. The sequences exhibited conserved features typical of functional rRNAs. Generally, polymorphisms occurred within less conserved regions and were seen as bulges, internal and terminal loops or non-canonical G-U base-pairs within regions of the double stranded helices. Importantly, there were also taxonomic motifs and base changes that were unique to specific taxa and which may be used to support intra-specific identification of members of the C. gloeosporioides sensu lato species complex.

  14. Impact of Strain Heterogeneity on Lyme Disease Serology in Europe: Comparison of Enzyme-Linked Immunosorbent Assays Using Different Species of Borrelia burgdorferi Sensu Lato

    PubMed Central

    Hauser, Ulrike; Krahl, Heide; Peters, Helmut; Fingerle, Volker; Wilske, Bettina

    1998-01-01

    For the standardization of serological tests for Lyme borreliosis (LB) in Europe, the influence of the heterogeneity of Borrelia burgdorferi sensu lato must be assessed in detail. For this study four immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) with octyl-β-d-glucopyranoside extracts of strains PKo (Borrelia afzelii), PBi (Borrelia garinii), and PKa2 and B31 (both B. burgdorferi sensu stricto) were compared. Strains PKo, PBi, and PKa2 at the passages used for antigen preparations abundantly expressed outer surface protein C (OspC), whereas strain B31 at the passage used for antigen preparation did not express OspC. Sera (all from Germany) from 222 patients with clinically defined LB of all stages, 133 blood donors, and 458 forest workers were tested. None of the forest workers had symptoms consistent with LB at the time that the samples were collected. For IgM tests, receiver operating characteristic curves demonstrated that discrimination between sera from patients and blood donors was best with strain PKo and worst with strain B31. The discriminatory abilities of the four IgG ELISAs were similar in a diagnostically reasonable specificity range (90 to 100%). More than 20% of the sera from forest workers reacted strongly in the PKo IgG ELISA (optical density value, >1.5; other assays, less than 8%). Western blots of the sera with the most discrepant ELISA results revealed almost exclusive reactivity with p17. This highly immunogenic antigen is only expressed by strain PKo. This observation might be important for the development of assays enabling discrimination between asymptomatic or previous infection and active disease. PMID:9466753

  15. Prevalence of Borrelia burgdorferi sensu lato in synanthropic rodents in two rural communities of Yucatán, México.

    PubMed

    Solís-Hernández, Analilia; Rodríguez-Vivas, Roger Iván; Esteve-Gassent, María Dolores; Villegas-Pérez, Sandra Luz

    2016-03-22

    Lyme disease is a multisystemic zoonotic disease caused by Borrelia burgdorferi sensu lato. This spirochete circulates in an enzootic cycle between the primary vertebrate reservoir and its tick vectors. Different species of rodents are known to be efficient natural reservoirs for B. burgdorferi s.l.  To estimate the prevalence of B. burgdorferi s.l. in synanthropic rodents from two rural communities of Yucatán, México.  A total of 123 rodents (94 Mus musculus and 29 Rattus rattus) were trapped, and ear and bladder samples were collected. Flagelin B (flaB) genes and outer membrane lipoproteins ospC y p66 were amplified in order to detect B. burgdorferi s.l. presence in the samples. The obtained amplicons were sequenced.  The overall infection rates in rodents were 36.5% for flaB (45/123), 10.5% (13/123) for p66, and 3.2% (4/123) for ospC. Rattus rattus had 17.2% of infection and M. musculus, 42.5%. From all examined tissue, 11.3% (14/123) of bladders, and 17.0% (21/123) of ears were infected with the spirochete Borrelia burgdorferi s.l. No statistical differences (p>0.05) were found between the two tissue samples used for diagnosis. The ospC gen was 98% homologous to Borrelia garinii, one species of the B. burgdorferi s.l. complex.  We concluded that rodents have a high prevalence of B. burgdorferi s.l. infection, and both species of rodents, M. musculus and R. rattus, might be playing an important role in the maintenance of this bacterium in rural communities of Yucatán, México.

  16. Combining a Climatic Niche Model of an Invasive Fungus with Its Host Species Distributions to Identify Risks to Natural Assets: Puccinia psidii Sensu Lato in Australia

    PubMed Central

    Kriticos, Darren J.; Morin, Louise; Leriche, Agathe; Anderson, Robert C.; Caley, Peter

    2013-01-01

    Puccinia psidii sensu lato (s.l.) is an invasive rust fungus threatening a wide range of plant species in the family Myrtaceae. Originating from Central and South America, it has invaded mainland USA and Hawai'i, parts of Asia and Australia. We used CLIMEX to develop a semi-mechanistic global climatic niche model based on new data on the distribution and biology of P. psidii s.l. The model was validated using independent distribution data from recently invaded areas in Australia, China and Japan. We combined this model with distribution data of its potential Myrtaceae host plant species present in Australia to identify areas and ecosystems most at risk. Myrtaceaeous species richness, threatened Myrtaceae and eucalypt plantations within the climatically suitable envelope for P. psidii s.l in Australia were mapped. Globally the model identifies climatically suitable areas for P. psidii s.l. throughout the wet tropics and sub-tropics where moist conditions with moderate temperatures prevail, and also into some cool regions with a mild Mediterranean climate. In Australia, the map of species richness of Myrtaceae within the P. psidii s.l. climatic envelope shows areas where epidemics are hypothetically more likely to be frequent and severe. These hotspots for epidemics are along the eastern coast of New South Wales, including the Sydney Basin, in the Brisbane and Cairns areas in Queensland, and in the coastal region from the south of Bunbury to Esperance in Western Australia. This new climatic niche model for P. psidii s.l. indicates a higher degree of cold tolerance; and hence a potential range that extends into higher altitudes and latitudes than has been indicated previously. The methods demonstrated here provide some insight into the impacts an invasive species might have within its climatically suited range, and can help inform biosecurity policies regarding the management of its spread and protection of valued threatened assets. PMID:23704988

  17. Investigating the Host-Range of the Rust Fungus Puccinia psidii sensu lato across Tribes of the Family Myrtaceae Present in Australia

    PubMed Central

    Morin, Louise; Aveyard, Ruth; Lidbetter, Jonathan R.; Wilson, Peter G.

    2012-01-01

    The exotic rust fungus Puccinia psidii sensu lato was first detected in Australia in April 2010. This study aimed to determine the host-range potential of this accession of the rust by testing its pathogenicity on plants of 122 taxa, representative of the 15 tribes of the subfamily Myrtoideae in the family Myrtaceae. Each taxon was tested in two separate trials (unless indicated otherwise) that comprised up to five replicates per taxon and six replicates of a positive control (Syzygium jambos). No visible symptoms were observed on the following four taxa in either trial: Eucalyptus grandis×camaldulensis, E. moluccana, Lophostemon confertus and Sannantha angusta. Only small chlorotic or necrotic flecks without any uredinia (rust fruiting bodies) were observed on inoculated leaves of seven other taxa (Acca sellowiana, Corymbia calophylla ‘Rosea’, Lophostemon suaveolens, Psidium cattleyanum, P. guajava ‘Hawaiian’ and ‘Indian’, Syzygium unipunctatum). Fully-developed uredinia were observed on all replicates across both trials of 28 taxa from 8 tribes belonging to the following 17 genera: Agonis, Austromyrtus, Beaufortia, Callistemon, Calothamnus, Chamelaucium, Darwinia, Eucalyptus, Gossia, Kunzea, Leptospermum, Melaleuca, Metrosideros, Syzygium, Thryptomene, Tristania, Verticordia. In contrast, the remaining 83 taxa inoculated, including the majority of Corymbia and Eucalyptus species, developed a broad range of symptoms, often across the full spectrum, from fully-developed uredinia to no visible symptoms. These results were encouraging as they indicate that some levels of genetic resistance to the rust possibly exist in these taxa. Overall, our results indicated no apparent association between the presence or absence of disease symptoms and the phylogenetic relatedness of taxa. It is most likely that the majority of the thousands of Myrtaceae species found in Australia have the potential to become infected to some degree by the rust, although this wide

  18. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants.

    PubMed

    Martínez-Álvarez, Juan C; Castro-Martínez, Claudia; Sánchez-Peña, Pedro; Gutiérrez-Dorado, Roberto; Maldonado-Mendoza, Ignacio E

    2016-05-01

    Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10(9) c.f.u. g(-1) was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10(7) c.f.u. g(-1). Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.

  19. Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania

    PubMed Central

    Kabula, Bilali; Kisinza, William; Tungu, Patrick; Ndege, Chacha; Batengana, Benard; Kollo, Douglas; Malima, Robert; Kafuko, Jessica; Mohamed, Mahdi; Magesa, Stephen

    2014-01-01

    desarrollo de resistencias en las poblaciones de los vectores Anopheles. Evaluar dichos marcadores es crucial para los programas de monitorización de resistencias. Hemos investigado la presencia y la distribución de las mutaciones de resistencia knockdown (kdr) en Anopheles gambiae s.l. en Tanzania. Métodos Se recolectaron mosquitos Anopheles intradomiciliarios de 10 lugares diferentes y se evaluaron en busca de resistencia a insecticidas utilizando el protocolo estándar de la OMS. Mediante un diagnóstico molecular basado en la PCR se genotiparon los mosquitos y se detectaron los genotipos kdr. Resultados Los An. gambiae evaluados eran resistentes a lambdacialotrina en Muheza, Arumeru y Muleba. De 350 An. gambiae s.l. genotipados, 35% eran An. gambiae s.s. y 65% eran An. arabiensis. Se detectaron mutaciones L1014S y L1014F tanto en An. gambiae s.s. como en An. arabiensis. La mutación puntual L1014S se encontró con una frecuencia alélica de 4-33%, mientras que L1014F tenía una frecuencia alélica de 6-14%. La mutación L1014S estaba ampliamente asociada a An. gambiae s.s. (Chi-Cuadrado = 23.41; P < 0.0001) y la L1014F estaba asociada con An. arabiensis (Chi-Square = 11.21; P = 0.0008). El alelo L1014S estaba significativamente asociado con mosquitos resistentes a la lambdacialotrina (P < 0.001). Conclusión La simultaneidad de mutaciones de L1014S y L1014F junto con informes de resistencia a los insecticidas sugiere que la resistencia a piretroides se está convirtiendo en un fenómeno común entre las poblaciones del vector de la malaria en Tanzania. La presencia de la mutación L1014F en estas poblaciones del Este de África indican la diseminación del gen a lo largo del continente africano. Determinar las implicaciones potenciales a nivel operativo de estos hallazgos sobre el control de la malaria requiere de más estudios. PMID:24386946

  20. PCR hot-start using duplex primers.

    PubMed

    Kong, Deming; Shen, Hanxi; Huang, Yanping; Mi, Huaifeng

    2004-02-01

    A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.

  1. PCR screening of tick-borne agents in sensitive conservation areas, Southeast Portugal.

    PubMed

    Santos-Silva, Maria Margarida; Melo, Pedro; Santos, Nuno; Antunes, Sandra; Duarte, Luís Raposo; Ferrolho, Joana; Milhano, Natacha; Santos, Patrícia Tavares; Domingos, Ana; Santos, Ana Sofia

    2017-02-01

    The Southeast region of Portugal, particularly the Guadiana valley, is currently the reintroduction territory of Lynx pardinus (Iberian lynx), one of the most endangered felids in the world that is only found in the Iberian Peninsula. Over the last century, populations have declined, placing L. pardinus at extremely high risk of extinction in the wild and relying on reintroduction projects. Among the aspects taken into account in the establishment of new populations is the sanitary status of the selected habitats, especially concerning infectious diseases, including tick-borne pathogens (TBPs). This study presents the results of TBPs survey on ticks collected at sensitive conservation areas of Southeast Portugal. From 2012 to 2014, 231 ticks obtained from vegetation, sympatric domestic and wild animals were submitted for analysis. The presence of Babesia spp., Cytauxzoon spp., Theileria spp., Hepatozoon spp., Anaplasma spp., Ehrlichia spp., Candidatus Neoehrlichia mikurensis, among other Anaplasmataceae, and Coxiella burnetii were investigated by PCR. Six tick species were recorded, Dermacentor marginatus (n = 13/5.6%), Hyalomma lusitanicum (n = 175/75.8%), Ixodes ricinus (n = 4/1.7%), Rhipicephalus bursa (n = 7/3.0%), R. pusillus (n = 21/9.1%) and R. sanguineus sensu lato (n = 11/4.8%). The molecular screening confirmed the presence of two tick-borne pathogens, C. burnetii (N = 34) and Anaplasma platys (N = 1), and one tick-endosymbiont, Candidatus Midichloria mitochondrii (N = 45). The results obtained provide new information on the circulation of ticks and TBPs with potential veterinary importance in Iberian lynx habitat.

  2. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  3. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  4. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  5. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.

    PubMed

    Yang, Rongchang; Paparini, Andrea; Monis, Paul; Ryan, Una

    2014-12-01

    Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R(2)⩾0.999) and faecal samples (R(2)⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that

  6. [Progress in digital PCR technology and application].

    PubMed

    Lin, Jiaqi; Su, Guocheng; Su, Wenjin; Zhou, Changyi

    2017-02-25

    Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.

  7. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  8. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  9. Direct in situ rt-PCR.

    PubMed

    Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto

    2011-01-01

    In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.

  10. Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

    PubMed

    Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

    2011-07-01

    Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

  11. Multiplexed Primer Prediction for PCR

    SciTech Connect

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequences used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.

  12. Divergence of Borrelia burgdorferi sensu lato spirochetes could be driven by the host: diversity of Borrelia strains isolated from ticks feeding on a single bird

    PubMed Central

    2014-01-01

    Background The controversy surrounding the potential impact of birds in spirochete transmission dynamics and their capacity to serve as a reservoir has existed for a long time. The majority of analyzed bird species are able to infect larval ticks with Borrelia. Dispersal of infected ticks due to bird migration is a key to the establishment of new foci of Lyme borreliosis. The dynamics of infection in birds supports the mixing of different species, the horizontal exchange of genetic information, and appearance of recombinant genotypes. Methods Four Borrelia burgdorferi sensu lato strains were cultured from Ixodes minor larvae and four strains were isolated from Ixodes minor nymphs collected from a single Carolina Wren (Thryothorus ludovicianus). A multilocus sequence analysis that included 16S rRNA, a 5S-23S intergenic spacer region, a 16S-23S internal transcribed spacer, flagellin, p66, and ospC separated 8 strains into 3 distinct groups. Additional multilocus sequence typing of 8 housekeeping genes, clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA was used to resolve the taxonomic status of bird-associated strains. Results Results of analysis of 14 genes confirmed that the level of divergence among strains is significantly higher than what would be expected for strains within a single species. The presence of cross-species recombination was revealed: Borrelia burgdorferi sensu stricto housekeeping gene nifS was incorporated into homologous locus of strain, previously assigned to B. americana. Conclusions Genetically diverse Borrelia strains are often found within the same tick or same vertebrate host, presenting a wide opportunity for genetic exchange. We report the cross-species recombination that led to incorporation of a housekeeping gene from the B. burgdorferi sensu stricto strain into a homologous locus of another bird-associated strain. Our results support the hypothesis that recombination maintains a majority of sequence polymorphism within Borrelia

  13. Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

    PubMed

    Montenegro, Víctor M; Bonilla, Marta C; Kaminsky, Darwin; Romero-Zúñiga, Juan José; Siebert, Susanne; Krämer, Friederike

    2017-03-15

    In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number

  14. Geographical distribution of Amblyomma cajennense (sensu lato) ticks (Parasitiformes: Ixodidae) in Brazil, with description of the nymph of A. cajennense (sensu stricto).

    PubMed

    Martins, Thiago F; Barbieri, Amália R M; Costa, Francisco B; Terassini, Flávio A; Camargo, Luís M A; Peterka, Cássio R L; de C Pacheco, Richard; Dias, Ricardo A; Nunes, Pablo H; Marcili, Arlei; Scofield, Alessandra; Campos, Artur K; Horta, Mauricio C; Guilloux, Aline G A; Benatti, Hector R; Ramirez, Diego G; Barros-Battesti, Darci M; Labruna, Marcelo B

    2016-03-31

    Until recently, Amblyomma cajennense (Fabricius, 1787) was considered to represent a single tick species in the New World. Recent studies have split this taxon into six species. While the A. cajennense species complex or A. cajennense (sensu lato) (s.l.) is currently represented by two species in Brazil, A. cajennense (sensu stricto) (s.s.) and Amblyomma sculptum Berlese, 1888, their geographical distribution is poorly known. The distribution of the A. cajennense (s.l.) in Brazil was determined by morphological examination of all lots of A. cajennense (s.l.) in two large tick collections of Brazil, and by collecting new material during three field expeditions in the possible transition areas between the distribution ranges of A. cajennense (s.s.) and A. sculptum. Phylogenetic analysis inferred from the ITS2 rRNA gene was used to validate morphological results. Morphological description of the nymphal stage of A. cajennense (s.s.) is provided based on laboratory-reared specimens. From the tick collections, a total 12,512 adult ticks were examined and identified as 312 A. cajennense (s.s.), 6,252 A. sculptum and 5,948 A. cajennense (s.l.). A total of 1,746 ticks from 77 localities were collected during field expeditions, and were identified as 249 A. cajennense (s.s.), 443 A. sculptum, and 1,054 A. cajennense (s.l.) [these A. cajennense (s.l.) ticks were considered to be males of either A. cajennense (s.s.) or A. sculptum]. At least 23 localities contained the presence of both A. cajennense (s.s.) and A. sculptum in sympatry. DNA sequences of the ITS2 gene of 50 ticks from 30 localities confirmed the results of the morphological analyses. The nymph of A. cajennense (s.s.) is morphologically very similar to A. sculptum. Our results confirmed that A. cajennense (s.l.) is currently represented in Brazil by only two species, A. cajennense (s.s.) and A. sculptum. While these species have distinct distribution areas in the country, they are found in sympatry in some

  15. Evaluation of the acaricidal activity of thymol incorporated in two formulations for topical use against immature stages of Rhipicephalus sanguineus sensu lato (Latreille, 1806) (Acari: Ixodidae).

    PubMed

    Delmonte, Camila; Cruz, Paula Barroso; Zeringóta, Viviane; de Mello, Valéria; Ferreira, Felipe; Amaral, Maria da Penha Henriques; Daemon, Erik

    2017-09-05

    The objective of this study was to assess, for the first time, the in vitro acaricidal activity of two topical formulations containing thymol, on immature stages of Rhipicephalus sanguineus sensu lato. For this purpose, two base formulations were prepared: an oil-in-water (O/W) emulsion and a hydroalcoholic solution, containing different thymol concentrations (0.5 to 20 mg/mL). We used the larval packet test for non-engorged larvae and nymphs, and the immersion test for engorged larvae and nymphs. For emulsion, a mortality rate of 94.2% was achieved at 0.75 mg/mL in non-engorged larvae. For engorged larvae, there was 95.0% mortality at 5.0 mg/mL. Non-engorged nymphs showed 83.3% mortality at 2.5 mg/mL, and for engorged nymphs, 86.0% mortality was verified at 5.0 mg/mL. For the hydroalcoholic solution, the mortality found for non-engorged larvae was 88.1% at 2.5 mg/mL. For engorged larvae, the highest mortality was 25.0% at 20 mg/mL; non-engorged nymphs had 91.0% mortality at 1.0 mg/mL and for engorged nymphs; the maximum value verified was 18.3% mortality at 20 mg/mL. Preliminary stability tests were carried, and the hydroalcoholic solution remained stable under all the conditions analyzed. The O/W emulsion showed signs of early instability at the concentration of 5.0 mg/mL. The results obtained indicate that the acaricidal activity of thymol, when included in the proposed formulations, was enhanced against non-engorged larvae with topical treatment in comparison with data in the literature. Although there were variations in toxicity between the different stages, these formulations are promising for future therapeutic use.

  16. Life stage-related differences in density of questing ticks and infection with Borrelia burgdorferi sensu lato within a single cohort of Ixodes pacificus (Acari: Ixodidae).

    PubMed

    Eisen, Rebecca J; Mun, Jeomhee; Eisen, Lars; Lane, Robert S

    2004-07-01

    The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m2 for nymphs to 0/100 m2 for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m2 (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5-1.6%). Peak densities of adult I. pacificus were 0-0.1/100 m2 in leaf litter, 0-6.5/100 m2 in ecotonal grasslands, and 2.0-39.0/100 m2 in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.-infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This

  17. Divergence of Borrelia burgdorferi sensu lato spirochetes could be driven by the host: diversity of Borrelia strains isolated from ticks feeding on a single bird.

    PubMed

    Rudenko, Nataliia; Golovchenko, Maryna; Belfiore, Natalia M; Grubhoffer, Libor; Oliver, James H

    2014-01-02

    The controversy surrounding the potential impact of birds in spirochete transmission dynamics and their capacity to serve as a reservoir has existed for a long time. The majority of analyzed bird species are able to infect larval ticks with Borrelia. Dispersal of infected ticks due to bird migration is a key to the establishment of new foci of Lyme borreliosis. The dynamics of infection in birds supports the mixing of different species, the horizontal exchange of genetic information, and appearance of recombinant genotypes. Four Borrelia burgdorferi sensu lato strains were cultured from Ixodes minor larvae and four strains were isolated from Ixodes minor nymphs collected from a single Carolina Wren (Thryothorus ludovicianus). A multilocus sequence analysis that included 16S rRNA, a 5S-23S intergenic spacer region, a 16S-23S internal transcribed spacer, flagellin, p66, and ospC separated 8 strains into 3 distinct groups. Additional multilocus sequence typing of 8 housekeeping genes, clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA was used to resolve the taxonomic status of bird-associated strains. Results of analysis of 14 genes confirmed that the level of divergence among strains is significantly higher than what would be expected for strains within a single species. The presence of cross-species recombination was revealed: Borrelia burgdorferi sensu stricto housekeeping gene nifS was incorporated into homologous locus of strain, previously assigned to B. americana. Genetically diverse Borrelia strains are often found within the same tick or same vertebrate host, presenting a wide opportunity for genetic exchange. We report the cross-species recombination that led to incorporation of a housekeeping gene from the B. burgdorferi sensu stricto strain into a homologous locus of another bird-associated strain. Our results support the hypothesis that recombination maintains a majority of sequence polymorphism within Borrelia populations because of the re-assortment of

  18. Anaplasmataceae and Borrelia burgdorferi sensu lato in the sand lizard Lacerta agilis and co-infection of these bacteria in hosted Ixodes ricinus ticks

    PubMed Central

    2011-01-01

    Background Anaplasmataceae and Borrelia burgdorferi s.l. are important tick-borne bacteria maintained in nature by transmission between ticks and vertebrate hosts. However, the potential role of lizards as hosts has not been sufficiently studied. Results The current study showed that 23 of 171 examined sand lizards Lacerta agilis were PCR positive for Anaplasmataceae. The nucleotide sequences of the several selected PCR products showed 100% homology with Anaplasma spp. found in Ixodes ricinus collected in Tunisia and Morocco (AY672415 - AY672420). 1.2% of lizard collar scale samples were PCR positive for B. lusitaniae. In addition, 12 of 290 examined I. ricinus were PCR positive for B. burgdorferi s.l. and 82 were PCR positive for Anaplasmatacea. The number of ticks per lizard and the number of ticks PCR positive for both microorganisms per lizard were strongly correlated. Moreover, we found a significant correlation between numbers of ticks infected with Anaplasmataceae and with B. burgdorferi s.l. living on the same lizard. However, there was no significant correlation between detection of both bacteria in the same tick. Conclusions To the best of our knowledge, this is the first report of Anaplasmataceae DNA and additionally the second report of B. burgdorferi s.l DNA detection in the sand lizard. PMID:21933412

  19. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    PubMed

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug(-1). Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  1. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed Central

    Schrick, Livia; Nitsche, Andreas

    2015-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings. PMID:27077041

  2. Absolute quantification by droplet digital PCR versus analog real-time PCR

    PubMed Central

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  3. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  4. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  5. Rapid Diagnosis of Leptospirosis by Multiplex PCR

    PubMed Central

    Ahmed, Siti Aminah; Sandai, Doblin Anak; Musa, Suzana; Hoe, Chee Hock; Riadzi, Mehdi; Lau, Kwok Leong; Tang, Thean Hock

    2012-01-01

    Background: Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance. Methods: In this study, we developed a multiplex PCR (mPCR) assay for detecting Leptospira’s DNA. The mPCR assay detects both the 16S rRNA gene and the major outer membrane lipoprotein gene, which is known as LipL32. Representative serovars were tested from 10 species of Leptospira and 23 other species of bacteria. Results: A positive result was obtained from all leptospiral serovars. The amplification sensitivity for the multiplex assay was 21.8 pg and 1 × 103 leptospires/ml. This mPCR assay has the potential to facilitate a rapid and sensitive diagnosis for acute leptospirosis. Conclusion: The mPCR assay developed in this study can be used for the early detection of leptospirosis. The LipL32 gene could also serve as another target to aid in the efficient detection of leptospiral infection because using 2 sets of primers in mPCR increases the sensitivity and specificity of the test. PMID:23610544

  6. Digital PCR for detection of citrus pathogens

    USDA-ARS?s Scientific Manuscript database

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  7. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  8. Efficacy and speed of kill of a topically applied formulation of dinotefuran-permethrin-pyriproxyfen against weekly tick infestations with Rhipicephalus sanguineus (sensu lato) on dogs.

    PubMed

    Blair, Jeffrey; Fourie, Josephus J; Varloud, Marie; Horak, Ivan G

    2016-05-16

    Rhipicephalus sanguineus (sensu lato) is a vector of canine babesiosis, anaplasmosis and ehrlichiosis. In order to reduce the chance of transmission of these diseases, an ectoparasiticide should rapidly repel or kill new infestations with this tick. The primary objective of the present study was to evaluate the treatment and preventive acaricidal efficacy of Vectra® 3D (54.45 mg/ml of dinotefuran, 396.88 mg/ml of permethrin and 4.84 mg/ml of pyriproxyfen) against R. sanguineus (s.l.) measured at 2, 8, and 48 h after treatment and weekly re-infestation. Twenty-four dogs were each infested with 50 adult R. sanguineus (s.l.) on Day -7 and allocated to three groups (n = 8) based on tick counts: an untreated control group (Group 1), and two groups (Groups 2 and 3) treated with Vectra®3D. The dogs in each group were infested with 50 ticks on Day -2. Vectra®3D was administered topically to the dogs on Day 0. Ticks were counted, in situ at 2 and 8 h after treatment on dogs in Groups 1 and 3. Group 3 was then withdrawn from the study and ticks were counted and removed from the dogs in Groups 1 and 2, 48 h after treatment. On Days 7, 14, 21, 28, 35 and 42, the dogs in Groups 1 and 2 were re-infested with 50 ticks, which were then counted in situ at 2 and 8 h, and counted and removed at 48 h after re-infestation. Ticks from the initial infestation were visually unaffected by 2 and 8 h after treatment. However, by 2 h after weekly re-infestation the arithmetic mean (AM) efficacy of Vectra® 3D from Days 7 through 28 ranged from 61.1 to 78.8 %, falling to 60.1 and 47.4 % on Days 35 and 42 respectively. By 8 h after weekly re-infestation, the AM efficacy ranged from 89.1 to 97.4 % falling to 81.4 and 69.8 % on Days 35 and 42 respectively. The AM efficacy 48 h after treatment after the initial infestation was 22.9 % but after weekly re-infestation the efficacy at 48 h ranged from 89.1 to 100.0 %, falling to 86.0 and 81.1 % on Days 35 and 42 respectively

  9. Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

    PubMed Central

    Gu, Z.; Ingersoll, J.; Abdul-Ali, D.; Shi, L.; Pounds, S.; Caliendo, A. M.

    2013-01-01

    Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log10 versus 4 log10 copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R2 ≥ 0.98 in each of 6 regression models) and clinical samples (R2 = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR. PMID:23224089

  10. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    DTIC Science & Technology

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  11. Pre-PCR processing: strategies to generate PCR-compatible samples.

    PubMed

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  12. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  13. New method using quantitative PCR to follow the tick blood meal and to assess the anti-feeding effect of topical acaricide against Rhipicephalus sanguineus on dogs.

    PubMed

    Fourie, J J; Joubert, A; Labuschagné, M; Beugnet, F

    2014-05-01

    A 28-day study was conducted to assess the dynamic of blood feeding by Rhipicephalus sanguineus ticks on dogs treated or not with a novel topical combination of fipronil, amitraz and (S)-methoprene. Dogs were infested weekly through exposure to ticks in crates for 4h. Ticks were then counted in the crates at 2h and 4h post dog exposure. Ticks were also counted and removed from the dogs at 2h, 4h, 6h, 12h and 24h post tick exposure. The inhibition of blood feeding was assessed by both tick quantification and designing and performing a quantitative PCR (qPCR) to detect the canine hydroxymethylbilane synthase (HMBS) gene in ticks. The percentage of repellency sensu lato based on the ticks collected in crates at 2h varied from 4.7% at day 28 to 48.3% at day 7. The immediate mortality rate of the ticks expelled at 2h varied from 1.5% at day 21 to 31.7% at day 7. The efficacy calculation showed that the acaricidal combination started to kill ticks in as little as 2h. The average efficacy reached 90.0% at 12h post crate challenges and 100% at 24h post exposure in crates. The inclusion of an internal amplification control was used to ensure that no significant template-derived PCR inhibition (≤ 6.2%) affected the overall results. The reduction of blood feeding was significant at 4h (>80.0%) and >99.0% at 24h post tick exposure in the crate. The high repellency rate and the lethal efficacy of CERTIFECT(®) resulted in significantly fewer live attached ticks, consequently reducing blood intake and fluid exchanges. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Quantitative real-time PCR eliminates false-positives in colony screening PCR.

    PubMed

    Skarratt, Kristen K; Fuller, Stephen J

    2014-01-01

    We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.

  15. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    DTIC Science & Technology

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  16. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  17. Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR.

    PubMed Central

    Shoffner, M A; Cheng, J; Hvichia, G E; Kricka, L J; Wilding, P

    1996-01-01

    The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube. PMID:8628665

  18. Quantitative DNA Analysis Using Droplet Digital PCR.

    PubMed

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  19. Fundamentals of multiplexing with digital PCR.

    PubMed

    Whale, Alexandra S; Huggett, Jim F; Tzonev, Svilen

    2016-12-01

    Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

  20. The potential advantages of digital PCR for clinical virology diagnostics.

    PubMed

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  1. Transgene detection by digital droplet PCR.

    PubMed

    Moser, Dirk A; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  2. Recent advances in quantitative PCR (qPCR) applications in food microbiology.

    PubMed

    Postollec, Florence; Falentin, Hélène; Pavan, Sonia; Combrisson, Jérôme; Sohier, Danièle

    2011-08-01

    Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

  3. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

    PubMed Central

    Koepfli, Cristian; Nguitragool, Wang; Hofmann, Natalie E.; Robinson, Leanne J.; Ome-Kaius, Maria; Sattabongkot, Jetsumon; Felger, Ingrid; Mueller, Ivo

    2016-01-01

    Accurate quantification of parasite density in the human host is essential for understanding the biology and pathology of malaria. Semi-quantitative molecular methods are widely applied, but the need for an external standard curve makes it difficult to compare parasite density estimates across studies. Droplet digital PCR (ddPCR) allows direct quantification without the need for a standard curve. ddPCR was used to diagnose and quantify P. falciparum and P. vivax in clinical patients as well as in asymptomatic samples. ddPCR yielded highly reproducible measurements across the range of parasite densities observed in humans, and showed higher sensitivity than qPCR to diagnose P. falciparum, and equal sensitivity for P. vivax. Correspondence in quantification was very high (>0.95) between qPCR and ddPCR. Quantification between technical replicates by ddPCR differed 1.5–1.7-fold, compared to 2.4–6.2-fold by qPCR. ddPCR facilitates parasite quantification for studies where absolute densities are required, and will increase comparability of results reported from different laboratories. PMID:27982132

  4. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  5. A systematic analysis of PCR contamination.

    PubMed

    Scherczinger, C A; Ladd, C; Bourke, M T; Adamowicz, M S; Johannes, P M; Scherczinger, R; Beesley, T; Lee, H C

    1999-09-01

    In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.

  6. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  7. SASqPCR: Robust and Rapid Analysis of RT-qPCR Data in SAS

    PubMed Central

    Ling, Daijun

    2012-01-01

    Reverse transcription quantitative real-time PCR (RT-qPCR) is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at http://code.google.com/p/sasqpcr/downloads/list. PMID:22238653

  8. Evaluation of Altona Diagnostics RealStar Zika Virus RT-PCR Test Kit for Zika virus PCR testing.

    PubMed

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-03-15

    Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.

  9. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  10. Direct Chromatin PCR (DC-PCR): Hypotonic Conditions Allow Differentiation of Chromatin States during Thermal Cycling

    PubMed Central

    Vatolin, Sergei; Khan, Shahper N.; Reu, Frederic J.

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90°C, 41 of 61 tested 5′ sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR. PMID:22984542

  11. Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

    USDA-ARS?s Scientific Manuscript database

    Several species and hyphal anastomosis groups (AG) of Rhizoctonia solani (sensu lato) cause brown patch diseases of turfgrasses. Conventional methods of identification of Rhizoctonia pathogens are time consuming and often inaccurate. A rapid identification assay for Waitea circinata (anamorph: Rhizo...

  12. pcrEfficiency: a Web tool for PCR amplification efficiency prediction

    PubMed Central

    2011-01-01

    Background Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair. Results We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence. Conclusions pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license. PMID:22014212

  13. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  14. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    PubMed

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  15. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

    PubMed

    Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy H C; Marchal, Kathleen; Bertrand, Sophie; De Keersmaecker, Sigrid C J

    2017-01-01

    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  16. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  17. Enhancing the efficiency of a PCR using gold nanoparticles

    PubMed Central

    Li, Min; Lin, Yu-Cheng; Wu, Chao-Chin; Liu, Hsiao-Sheng

    2005-01-01

    We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 104-fold in a quicker PCR system (i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/µl DNA were detectable in real-time PCR with gold colloid added, however, at least 106 copies/µl of DNA were needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using non-expensive polymerases, and general PCR reagent. It is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times. PMID:16314298

  18. Prevalence of Borrelia miyamotoi and Borrelia burgdorferi sensu lato in questing ticks from a recreational coniferous forest of East Saxony, Germany.

    PubMed

    Szekeres, Sándor; Lügner, Jenny; Fingerle, Volker; Margos, Gabriele; Földvári, Gábor

    2017-10-01

    The hard tick Ixodes ricinus is the most important vector of tick-transmitted pathogens in Europe, frequently occurring in urban parks and greenbelts utilized for recreational activities. This species is the most common vector of the causative agents of Lyme borreliosis in Europe. Similarly, the species spreads Borrelia miyamotoi, causing a relapsing-fever like illness. A total of 1774 Ixodes ricinus (50 females, 68 males, 840 nymphs and 818 larvae) were collected with flagging between March and September 2014 in a coniferous forest patch in Niederkaina near the town of Bautzen in Saxony, Germany. To measure questing tick density a time-based density estimating method was utilized. From each month, a total of 100 adults and nymphal ticks and all larvae (pools of 10 individuals per tube/month) were selected for the molecular analyses. For simultaneous detection of B. burgdorferi s.l. and B. miyamotoi a duplex real-time PCR targeting the flaB locus was performed. Prevalence of B. burgdorferi s.l. was 9.4% (female: 6%, male: 2.9%, nymph: 12.2%, larva: 0%) and minimum prevalence of B. miyamotoi was 1.2% (female: 0%, male: 4.3%, nymph: 2.8%, larva: 0.1%) in the 714 samples with real-time polymerase chain reaction. A real-time PCR reaction was utilized first to target the histone-like protein gene (hbb) of B. burgdorferi s.l., a hemi-nested outer surface protein (ospA) gene conventional PCR was then performed followed by a restriction enzyme analysis to distinguish B. burgdorferi s.l. genospecies. Seven B. afzelii, one B. burgdorferi s.s., one B. bavariensis and four B. miyamotoi infections were confirmed. Prevalence of Lyme borreliosis spirochetes was significantly higher in nymphs than in adults (p<0.01, Fisher exact test) probably due to the diluting effect of the local roe deer population. Our data highlight the potential risk of human infection with the emerging pathogen B. miyamotoi within the study area. Copyright © 2017 Elsevier GmbH. All rights reserved.

  19. PCR test for Microsporum canis identification.

    PubMed

    Brillowska-Dabrowska, Anna; Michałek, Ewelina; Saunte, Ditte Marie Lindhardt; Nielsen, Sanne Søgaard; Arendrup, Maiken Cavling

    2013-08-01

    Microsporum canis, for which the natural hosts are cats and dogs, is the most prevalent zoophilic agent causing tinea capitis and tinea corporis in humans. We present here a diagnostic PCR test for M. canis, since its detection and species identification is relevant to the choice of treatment and to the understanding of a probable source of infection. An M. canis-specific PCR was evaluated using 130 clinical isolates of dermatophytes (including M. canis [n = 15] and 13 other species), 10 yeast or mold isolates, 12 hair and skin samples from animals with or without experimental M. canis infection, and 35 patient specimens, including seven specimens positive for M. canis and 15 dermatophyte negative samples. All pure cultures, animal specimens and clinical samples with M. canis were detected by the PCR test, whereas none of the other fungal isolates or samples without M. canis was negative. This study indicates that the PCR test for M. canis identification applied directly to patient specimens or animal hair, as well as to clinical isolates had 100% specificity and sensitivity.

  20. The Power of Real-Time PCR

    ERIC Educational Resources Information Center

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  1. The Power of Real-Time PCR

    ERIC Educational Resources Information Center

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  2. PCR Techniques in Next-Generation Sequencing.

    PubMed

    Goswami, Rashmi S

    2016-01-01

    With the advent of next-generation sequencing and its prolific use in the clinical realm, it would appear that techniques such as PCR would not be in high demand. This is not the case however, as PCR techniques play an important role in the success of NGS technology. Although NGS has rapidly become an important part of clinical molecular diagnostics, whole genome sequencing is still difficult to implement in a clinical laboratory due to high costs of sequencing, as well as issues surrounding data processing, analysis, and data storage, which can reduce efficiency and increase turnaround times. As a result, targeted sequencing is often used in clinical diagnostics, due to its increased efficiency. PCR techniques play an integral role in targeted NGS sequencing, allowing for the generation of multiple NGS libraries and the sequencing of multiple targeted regions simultaneously. We will outline the methods we employ in PCR amplification of targeted genomic regions for cancer mutation hotspots using the Ampliseq Cancer Hotspot v2 panel (Life Technologies, Carlsbad, CA).

  3. [Quantitative PCR in the diagnosis of Leishmania].

    PubMed

    Mortarino, M; Franceschi, A; Mancianti, F; Bazzocchi, C; Genchi, C; Bandi, C

    2004-06-01

    Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a

  4. PCR Assay Specific for Chicken Feces

    PubMed Central

    Cisar, Cindy R.; Akiyama, Tatsuya; Hatley, Jonathan; Arney, Lori; Kezunovic, Nebojsa; Owen, Daniel

    2011-01-01

    Bacteroidales are fecal anaerobic bacteria that are common in the digestive systems and feces of warm-blooded animals. Some strains of Bacteroidales have been reported to be host-specific. In this study, Bacteroidales strains from chicken feces were examined for their potential use as indicators of chicken fecal contamination. Bacteroidales 16S rRNA gene sequences from chicken feces were amplified, cloned and sequenced. Phylogenetic analysis was performed using these sequences and published Bacteroidales 16S rRNA gene sequences from human and bovine feces. Primers were designed based on putative chicken feces-specific 16S rRNA gene sequences and the primer pairs were tested for specificity in PCR assays. One set of primers, chBact F1 and chBact R16, specifically amplified DNA from chicken feces in a PCR assay, but did not amplify wild turkey, cat, bovine, or deer fecal DNAs. In addition, DNA from feces contaminated straw-based chicken litter produced a product in the PCR assay. However, DNA from feces contaminated wood shavings-based chicken litter was not amplified. The PCR assay described here may prove a useful tool for the detection of chicken feces and for source tracking in watersheds with fecal contamination. PMID:24839330

  5. PCR Assay Specific for Chicken Feces.

    PubMed

    Cisar, Cindy R; Akiyama, Tatsuya; Hatley, Jonathan; Arney, Lori; Kezunovic, Nebojsa; Owen, Daniel

    2010-01-01

    Bacteroidales are fecal anaerobic bacteria that are common in the digestive systems and feces of warm-blooded animals. Some strains of Bacteroidales have been reported to be host-specific. In this study, Bacteroidales strains from chicken feces were examined for their potential use as indicators of chicken fecal contamination. Bacteroidales 16S rRNA gene sequences from chicken feces were amplified, cloned and sequenced. Phylogenetic analysis was performed using these sequences and published Bacteroidales 16S rRNA gene sequences from human and bovine feces. Primers were designed based on putative chicken feces-specific 16S rRNA gene sequences and the primer pairs were tested for specificity in PCR assays. One set of primers, chBact F1 and chBact R16, specifically amplified DNA from chicken feces in a PCR assay, but did not amplify wild turkey, cat, bovine, or deer fecal DNAs. In addition, DNA from feces contaminated straw-based chicken litter produced a product in the PCR assay. However, DNA from feces contaminated wood shavings-based chicken litter was not amplified. The PCR assay described here may prove a useful tool for the detection of chicken feces and for source tracking in watersheds with fecal contamination.

  6. Inverse PCR for Point Mutation Introduction.

    PubMed

    Silva, Diogo; Santos, Gustavo; Barroca, Mário; Collins, Tony

    2017-01-01

    Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

  7. Handheld Real-Time PCR Device

    PubMed Central

    Ahrberg, Christian D.; Ilic, Bojan Robert; Manz, Andreas; Neužil, Pavel

    2016-01-01

    Here we report one of the smallest real-time polymerase chain reaction (PCR) system up to date with approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in form of virtual reaction chambers (VRC) where a ≈ 200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate PCR performance. The standard curve slope was (−3.02 ± 0.16) cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of (0.91 ± 0.05) per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics. PMID:26753557

  8. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  9. Real-time PCR: Advanced technologies and applications

    USDA-ARS?s Scientific Manuscript database

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  10. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Herthnek, David; Bölske, Göran

    2006-01-01

    Background Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. Results Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. Conclusion We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. PMID:17020599

  11. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water.

    PubMed

    Acosta Soto, Lucrecia; Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

    2017-01-01

    The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

  12. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    PubMed

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  13. Real-time PCR (qPCR) primer design using free online software.

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  14. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    PubMed

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  15. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    NASA Astrophysics Data System (ADS)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  16. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

    PubMed Central

    Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

    2017-01-01

    The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks. PMID:28377928

  17. 16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught Anopheles gambiae Sensu Lato and A. funestus Mosquitoes Reveals New Species Related to Known Insect Symbionts

    PubMed Central

    Lindh, Jenny M.; Terenius, Olle; Faye, Ingrid

    2005-01-01

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito. PMID:16269761

  18. 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts.

    PubMed

    Lindh, Jenny M; Terenius, Olle; Faye, Ingrid

    2005-11-01

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

  19. Identification of bacterial plant pathogens using multilocus PCR and electrospray ionization-mass spectrometry (PCR/ESI-MS)

    USDA-ARS?s Scientific Manuscript database

    PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad range primers to amplify products from wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses ...

  20. Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay.

    PubMed

    Bass, Chris; Williamson, Martin S; Wilding, Craig S; Donnelly, Martin J; Field, Linda M

    2007-11-22

    The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract

  1. Rapid PCR thermocycling using microscale thermal convection.

    PubMed

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-03-05

    Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format. Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations(2-9). Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument. One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time(10,11). Here we describe results of our efforts to probe the full 3-D velocity and

  2. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    PubMed

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  3. Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria.

    PubMed

    Abd-El-Haleem, Desouky; Kheiralla, Zeinab H; Zaki, Sahar; Rushdy, Abeer A; Abd-El-Rahiem, Walaa

    2003-12-01

    In this work we developed and optimized two molecular-based approaches to monitor rapidly, sensitively and specifically bacterial pathogens from three different genera, Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., directly in waters. To achieve this aim, firstly a multiplex-PCR assay (M-PCR) was optimized using a primer pair specific for each pathogen. Secondly, as a molecular confirmatory test after isolation of the pathogens by classical microbiological methods, PCR-RFLP of their amplified 16S rDNA genes was performed. It was observed from the results that the developed M-PCR assay has significant impact on the ability to detect sensitively, rapidly and specifically the three pathogens directly in water within a short time (5 h from sampling to obtain final results), therefore it represents a considerable advancement over other known more time-consuming and less-sensitive methods for identification and characterization of these kinds of pathogens.

  4. Replaceable Microfluidic Cartridges for a PCR Biosensor

    NASA Technical Reports Server (NTRS)

    Francis, Kevin; Sullivan, Ron

    2005-01-01

    The figure depicts a replaceable microfluidic cartridge that is a component of a miniature biosensor that detects target deoxyribonucleic acid (DNA) sequences. The biosensor utilizes (1) polymerase chain reactions (PCRs) to multiply the amount of DNA to be detected, (2) fluorogenic polynucleotide probe chemicals for labeling the target DNA sequences, and (3) a high-sensitivity epifluorescence-detection optoelectronic subsystem. Microfluidics is a relatively new field of device development in which one applies techniques for fabricating microelectromechanical systems (MEMS) to miniature systems for containing and/or moving fluids. Typically, microfluidic devices are microfabricated, variously, from silicon or polymers. The development of microfluidic devices for applications that involve PCR and fluorescence-based detection of PCR products poses special challenges

  5. Experimental PCR data on soil DNA extracts

    USGS Publications Warehouse

    Griffin, Dale W.

    2016-01-01

    Bacillus species and B. anthracis presence/absence data were determined in 4,770 soil samples collected across the contiguous United States in collaboration with the USEPA. PCR data for Bacillus species and B. anthracis rpoB gene PCR amplicon detection was reported as non-detect (n), low (l), medium (m), and high (h). Results for both pag and lef genes of the pX01 plasmid were reported by the University of South Florida's Center for Biological Defense. This data was recorded as negative or positive for each of the genes and included the following combinations: neg/neg, pos/neg, neg/pos, and pos/pos. Data for the pX02 plasmid were recorded as negative (blank) or positive (Y).

  6. A nanofluidic system for massively parallel PCR

    NASA Astrophysics Data System (ADS)

    Brenan, Colin; Morrison, Tom; Roberts, Douglas; Hurley, James

    2008-02-01

    Massively parallel nanofluidic systems are lab-on-a-chip devices where solution phase biochemical and biological analyses are implemented in high density arrays of nanoliter holes micro-machined in a thin platen. Polymer coatings make the interior surfaces of the holes hydrophilic and the exterior surface of the platen hydrophobic for precise and accurate self-metered loading of liquids into each hole without cross-contamination. We have created a "nanoplate" based on this concept, equivalent in performance to standard microtiter plates, having 3072 thirty-three nanoliter holes in a stainless steel platen the dimensions of a microscope slide. We report on the performance of this device for PCR-based single nucleotide polymorphism (SNP) genotyping or quantitative measurement of gene expression by real-time PCR in applications ranging from plant and animal diagnostics, agricultural genetics and human disease research.

  7. Miniature PCR based portable bioaerosol monitor development.

    PubMed

    Agranovski, I E; Usachev, E V; Agranovski, E; Usacheva, O V

    2017-01-01

    A portable bioaerosol monitor is greatly demanded technology in many areas including air quality control, occupational exposure assessment and health risk evaluation, environmental studies and, especially, in defence and bio-terrorism applications. Our recent groundwork allowed us to formulate the concept of a portable bioaerosol monitor, which needs to be light, user friendly, reliable and capable of detecting airborne pathogens within 1-1·5 h on the spot. Conceptually, the event of a bioaerosol concentration burst is determined by triggers to commence the representative air sampling with sequential real-time polymerase chain reaction (PCR) confirmation of the targeted micro-organism present in the air. To minimize reagent consumption and idle running of the technology, an event of a bioaerosol burst is confirmed by three parameters: aerosol particle size, concentration and composition. Only particle sizes above 200 nm attract interest in the bioaerosol. Only an elevated aerosol concentration above the threshold (background aerosol concentration) is a signal to commence the analytical procedure. The combination of our previously developed personal bioaerosol sampler, aerosol particle counter based trigger and portable real-time PCR device formed the basis of the bioaerosol monitoring technology. The portable real-time PCR device was advanced to provide internally controlled detection, significantly reducing false-positive alarms. The technique is capable of detecting selected airborne micro-organisms on the spot within 30-80 min, depending on the genome organization of the particular strain. Due to recent outbreaks of infectious airborne diseases and the continuing threat of intentionally released bioaerosol attacks, investigations into the possibility of the early and reliable detection of pathogenic micro-organisms in the air is becoming increasingly important. The proposed technology consisting of a bioaerosol sampler, technology trigger and PCR device is

  8. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date.

  9. A Disposable, Self-Contained PCR Chip

    PubMed Central

    Kim, Jitae; Byun, Doyoung; Mauk, Michael G.; Bau, Haim H.

    2009-01-01

    A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just on time releasable, paraffin-passivated, dry reagents is described. During both storage and sample preparation, the paraffin immobilizes and protects the stored reagents. Fluid flow through the reactor leaves the reagents undisturbed. Prior to the amplification step, the chamber is filled with target analyte suspended in water. Upon heating the PCR chamber to the DNA’s denaturation temperature, the paraffin melts and moves out of the way, and the reagents are released and hydrated. To better understand the reagent release process, a scaled up model of the reactor was constructed and the paraffin migration was visualized. Experiments were carried out with a 30 μl reactor demonstrating detectable amplification (with agarose gel electrophoresis) of 10 fg (~200 copies) of lambda DNA template. The in-reactor storage and on-time release of the PCR reagents reduce the number of needed operations and significantly simplify the flow control that would, otherwise, be needed in lab-on-chip devices. PMID:19190797

  10. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

    PubMed

    Maheshwari, Yogita; Selvaraj, Vijayanandraj; Hajeri, Subhas; Yokomi, Raymond

    2017-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  11. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  12. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

    PubMed Central

    Hajeri, Subhas

    2017-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR. PMID:28910375

  13. Triplet Repeat Primed PCR (TP-PCR) in Molecular Diagnostic Testing for Spinocerebellar Ataxia Type 3 (SCA3).

    PubMed

    Melo, Ana Rosa Vieira; Ramos, Amanda; Kazachkova, Nadiya; Raposo, Mafalda; Bettencourt, Bruno Filipe; Rendeiro, Ana Rita; Kay, Teresa; Vasconcelos, João; Bruges-Armas, Jácome; Lima, Manuela

    2016-12-01

    Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder for which the routine molecular testing is based on PCR and automated capillary electrophoresis. When only a normal allele is detected by standard PCR, the hypothesis of a failed amplification of the expanded allele must be raised. In such cases, complementary techniques such as Southern Blot or triplet repeat primed PCR (TP-PCR) have to be applied. For SCA3, TP-PCR is implemented in some diagnostic laboratories, but a tested protocol has yet to be published. The purpose of this study was to develop and test a TP-PCR protocol for SCA3. Sixty-five blood samples previously genotyped by standard PCR were used in the TP-PCR assay. Fourteen buccal swab samples were also analyzed to confirm the robustness of the technique. The reproducibility of the TP-PCR was evaluated by analyzing all samples in a second laboratory. The results obtained by TP-PCR confirmed the previous PCR results for 64 blood samples; in one sample an expanded allele, previously undetected by PCR, was identified. The results obtained for the buccal swab samples were totally concordant with those obtained for blood. Furthermore, the results obtained in the alternative laboratory were in full agreement with the results obtained in our study. The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.

  14. Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification.

    PubMed

    Zhang, Chunsun; Xing, Da

    2010-02-01

    This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52 degrees Celsius. By making full use of the hot (90-97 degrees Celsius) and cold (60-70 degrees Celsius) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.

  15. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  16. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  17. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.

    PubMed

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H; Haydon, Rex C; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

  18. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-10-29

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  19. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  20. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    NASA Astrophysics Data System (ADS)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  1. Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR).

    PubMed

    Tiirola, Marja A; Suvilampi, Juhani E; Kulomaa, Markku S; Rintala, Jukka A

    2003-05-01

    A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

  2. Viral diagnostics in the era of digital PCR

    PubMed Central

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  3. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  4. Optimized PCR-based detection of mycoplasma.

    PubMed

    Dobrovolny, Paige L; Bess, Dan

    2011-06-20

    The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research. The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines. Given the extreme sensitivity of the kit, great

  5. Five-month comparative efficacy evaluation of three ectoparasiticides against adult cat fleas (Ctenocephalides felis), flea egg hatch and emergence, and adult brown dog ticks (Rhipicephalus sanguineus sensu lato) on dogs housed outdoors.

    PubMed

    Varloud, Marie; Hodgkins, Elizabeth

    2015-03-01

    This study was designed to compare the efficacy of three topical combinations on dogs in outdoor conditions against adult cat fleas (Ctenocephalides felis), flea egg hatch and emergence, and against adult brown dog ticks (Rhipicephalus sanguineus sensu lato). Treatment was performed on day 0 with a placebo; dinotefuran, pyriproxifen and permethrin (DPP); fipronil and (S)-methoprene (FM) or imidacloprid and permethrin (IP). Dogs (n = 32), housed outdoors for 7 months, were treated monthly for four consecutive months (on days 0, 30, 60 and 90) and infested with ~100 unfed adult fleas on days 14, 55, 74, 115 and 150 and with ~50 unfed adult ticks on days 28, 44, 88 and 104. Adult fleas were counted and removed 24 h after infestation. Immediately after flea removal, dogs were reinfested with ~100 new adult fleas 72 h prior to egg collection for up to 48 h. Flea eggs were incubated for 32 days, and newly emerged adults were counted. Ticks were counted and removed 48 h after each infestation. FM had >90 % efficacy against fleas at each time point and variable efficacy against ticks (38.0-99.6 %). Efficacy of IP was <90 % against fleas at day 64 and against ticks at day 30 of the first post-treatment. No flea eggs were laid in the treated groups until infestation was carried out >60 days after the last treatment. Despite challenging weather conditions, DPP was highly effective, providing >90 % efficacy against adult ticks as well as adult and immature fleas at every time point of the study.

  6. Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips.

    PubMed Central

    Cheng, J; Shoffner, M A; Hvichia, G E; Kricka, L J; Wilding, P

    1996-01-01

    We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip. PMID:8628666

  7. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  8. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  9. Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

    PubMed

    Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

    2006-12-20

    Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the

  10. An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.

    PubMed

    Champlot, Sophie; Berthelot, Camille; Pruvost, Mélanie; Bennett, E Andrew; Grange, Thierry; Geigl, Eva-Maria

    2010-09-28

    PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.

  11. An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

    PubMed Central

    Pruvost, Mélanie; Bennett, E. Andrew; Grange, Thierry; Geigl, Eva-Maria

    2010-01-01

    Background PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Methodology/Principal Findings Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. Conclusions/Significance There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA. PMID:20927390

  12. Rapid direct PCR for ABO blood typing.

    PubMed

    Lee, Hwan Young; Park, Myung Jin; Kim, Na Young; Yang, Woo Ick; Shin, Kyoung-Jin

    2011-01-01

    Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

  13. An evaluation of direct PCR amplification

    PubMed Central

    Hall, Daniel E.; Roy, Reena

    2014-01-01

    Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

  14. In silico PCR primer designing and validation.

    PubMed

    Kumar, Anil; Chordia, Nikita

    2015-01-01

    Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.

  15. A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

    PubMed

    O'Halloran, Damien M

    2017-01-01

    Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

  16. A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer.

    PubMed

    Campomenosi, Paola; Gini, Elisabetta; Noonan, Douglas M; Poli, Albino; D'Antona, Paola; Rotolo, Nicola; Dominioni, Lorenzo; Imperatori, Andrea

    2016-08-18

    Selected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum. In the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001). To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample. Analyses of serum miRNAs performed

  17. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    PubMed

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  18. Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

    PubMed

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-09-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  19. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-04-11

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

  20. Accurate quantification of supercoiled DNA by digital PCR

    PubMed Central

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  1. Droplet digital PCR for absolute quantification of pathogens.

    PubMed

    Gutiérrez-Aguirre, Ion; Rački, Nejc; Dreo, Tanja; Ravnikar, Maja

    2015-01-01

    The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

  2. Application of real time PCR for diagnosis of Swine Dysentery.

    PubMed

    Akase, Satoru; Uchitani, Yumi; Sohmura, Yoshiko; Tatsuta, Keikichi; Sadamasu, Kenji; Adachi, Yoshikazu

    2009-03-01

    Evaluation of a genetic diagnostic technique using real time PCR of Swine Dysentery (SD) was performed using nox primers. Culture, ordinary PCR and real time PCR were compared in this experiment. Sixty-seven specimens from pigs with clinical signs of SD brought to a slaughterhouse in Shibaura, Tokyo, were used. B. hyodysenteriae was isolated from 49 of the pigs, was detected by ordinary PCR in 49 of the pigs and was detected by real time PCR in 54 of the pigs. Furthermore, we were able to determine the numbers of B. hyodysenteriae cells in all positive specimens by real time PCR. The rapid diagnostic technique established in this experiment was useful for detection of B. hyodysenteriae because it was more effective than ordinary PCR and culture.

  3. DNA polymerase preference determines PCR priming efficiency

    PubMed Central

    2014-01-01

    Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification

  4. Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR.

    PubMed

    Wacharapluesadee, Supaporn; Phumesin, Patta; Supavonwong, Pornpun; Khawplod, Pakamatz; Intarut, Nirun; Hemachudha, Thiravat

    2011-08-01

    Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition.

    PubMed

    Dietrich, Dimo; Uhl, Barbara; Sailer, Verena; Holmes, Emily Eva; Jung, Maria; Meller, Sebastian; Kristiansen, Glen

    2013-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

  6. Rapid micro-PCR system for hepatitis C virus amplification

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Huang, Ming-Yuan; Young, Kung-Chia; Chang, Ting-Tsung; Wu, Ching-Yi

    2000-08-01

    A rapid micro-polymerase chain reaction ((mu) -PCR) system was integrated to amplify the complementary DNA (cDNA) molecules of hepatitis C virus (HCV). This system consists of a rapid thermal cycling system and a (mu) PCR chip fabricated by MEMS fabrication techniques. This rapid (mu) PCR system is verified by using serum samples from patients with chronic hepatitis C. The HCV amplicon of the rapid (mu) PCR system was analyzed by slab gel electrophoresis with separation of DNA marker in parallel. The (mu) PCR chip was fabricated on silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. Using silicon material to fabricate the raction well improves the temperature uniformity of sample and helps to reach the desired temperature faster. The rapid close loop thermal cycling system comprises power supplies, a thermal generator, a computer control PID controller, and a data acquisition subsystem. The thermoelectric (T.E.) cooler is used to work as the thermal generator and a heat sink by controlling the polarity of supplied power. The (mu) PCR system was verified with traditional PCR equipment by loading the same PCR mixture with HCV cDNA and running the same cycle numbers, then comparing both HCV amplicon slab gel electrophoresis. The HCV amplicon from the (mu) PCR system shows a DNA fragment with an expected size of 145 base pairs. The background is lower with the (mu) PCR system than that with the tradional PCR equipment. Comparing the traditional PCR equipment which spends 5.5 hours for 30 cycles to gain the detectable amount of HCV amplicon in slab gel separation, this (mu) PCR system takes 30 minutes to finish the 30 thermal cycles. This work has demonstrated that this rapid (mu) PCR system can provide rapid heat generation and dissipation, improved temperature uniformity in DNA amplification.

  7. STS-102 MPLM Leonardo moves into PCR

    NASA Technical Reports Server (NTRS)

    2001-01-01

    KENNEDY SPACE CENTER, Fla. -- In the payload changeout room on the Rotating Service Structure, Launch Pad 39B, workers move the Multi-Purpose Logistics Module Leonardo out of the payload canister. From the PCR Leonardo then will be transferred into Space Shuttle Discovery'''s payload bay. One of Italy'''s major contributions to the International Space Station program, Leonardo is a reusable logistics carrier. It is the primary delivery system used to resupply and return Station cargo requiring a pressurized environment. Leonardo is the primary payload on mission STS-102 and will deliver up to 10 tons of laboratory racks filled with equipment, experiments and supplies for outfitting the newly installed U.S. Laboratory Destiny. STS-102 is scheduled to launch March 8 at 6:45 a.m. EST.

  8. ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data.

    PubMed

    Perkins, James R; Dawes, John M; McMahon, Steve B; Bennett, David L H; Orengo, Christine; Kohl, Matthias

    2012-07-02

    Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s). We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

  9. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    PubMed

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  10. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    PubMed Central

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods DNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results The sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. Conclusions Real-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

  11. Detection of Salmonella in chicken meat by insulated isothermal PCR.

    PubMed

    Tsen, Hau-Yang; Shih, Chia-Ming; Teng, Ping-Hua; Chen, Hsin-Yen; Lin, Chia-Wei; Chiou, Chien-Shun; Wang, Hwa-Tang Thomas; Chang, Hsiao-Fen Grace; Chung, Te-Yu; Lee, Pei-Yu; Chiang, Yu-Cheng

    2013-08-01

    Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10⁰ CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

  12. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    PubMed Central

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

  13. Infections and mixed infections with the selected species of Borrelia burgdorferi sensu lato complex in Ixodes ricinus ticks collected in eastern Poland: a significant increase in the course of 5 years.

    PubMed

    Wójcik-Fatla, Angelina; Zając, Violetta; Sawczyn, Anna; Sroka, Jacek; Cisak, Ewa; Dutkiewicz, Jacek

    2016-02-01

    In the years 2008-2009 and 2013-2014, 1620 and 1500 questing Ixodes ricinus ticks, respectively, were examined on the territory of the Lublin province (eastern Poland). The presence of three pathogenic species causing Lyme disease was investigated: Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii. The proportion of I. ricinus ticks infected with B. burgdorferi sensu lato showed a highly significant increase between 2008-2009 and 2013-2014, from 6.0 to 15.3%. A significant increase was noted with regard to all types of infections with individual species: single (4.7-7.8%), dual (1.2-6.6%), and triple (0.1-0.9%). When expressed as the percent of all infections, the frequency of mixed infections increased from 21.4 to 49.2%. Statistical analysis performed with two methods (by calculating of odds ratios and by Fisher's exact test) showed that the frequencies of mixed infections in most cases proved to be significantly greater than expected. The strongest associations were found between B. burgdorferi s. s. and B. afzelii, and between B. burgdorferi s. s. and B. garinii. They appeared to be highly significant (P < 0.0001) when assessed by two methods for 2013-2014, and for the sum of findings for both time periods. The proportions of the individual species detected in the mixed infections in 2008-2009 and 2013-2014 revealed highly significant increases for B. burgdorferi s. s. and B. garinii (from 33.9 to 71.1% and from 18.2 to 82.9%, respectively), and an insignificant decrease for B. afzelii (from 51.4 to 41.6%). The proportions of the species B. burgdorferi s. s., B. afzelii and B. garinii (with combined single and mixed infections) for 2008-2009 and 2013-2014 were: 51.2/44.0 %, 30.6/24.9% and 18.2/31.1%, respectively. In conclusion, our results seem to indicate the detrimental trend of the increasing infection rate of I. ricinus ticks with B. burgdorferi s. l. in eastern Poland, and dramatic enhancement of mixed infections with individual species, which

  14. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    PubMed

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  15. Specific detection of chikungunya virus using a RT-PCR/nested PCR combination.

    PubMed

    Pfeffer, M; Linssen, B; Parke, M D; Kinney, R M

    2002-02-01

    Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.

  16. Forensic implications of PCR inhibition--A review.

    PubMed

    Alaeddini, Reza

    2012-05-01

    Polymerase chain reaction (PCR) is currently the method of choice for the identification of human remains in forensic coursework. DNA samples from crime scenes often contain co-purified impurities which inhibit PCR. PCR inhibition is the most common cause of PCR failure when adequate copies of DNA are present. Inhibitors have been routinely reported in forensic investigations of DNA extracted from a variety of templates. Humic compounds, a series of substances produced during decay process have been considered as the materials contaminating DNA in soil, natural waters and recent sediments. Those compounds have been frequently assigned as PCR inhibitors. The current report reviews the characteristics of PCR inhibition, including the proposed mechanisms of inhibition, detection methods and the available technologies to remove or overcome the inhibitory activities.

  17. Microfluidics-Based PCR for Fusion Transcript Detection.

    PubMed

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  18. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

  19. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    PubMed

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-06

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

  20. Misuse of PCR assay for diagnosis of mollusc protistan infections.

    PubMed

    Burreson, Eugene M

    2008-06-19

    Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

  1. Posttreatment Follow-Up of Brucellosis by PCR Assay

    PubMed Central

    Morata, Pilar; Queipo-Ortuño, María Isabel; Reguera, José María; García-Ordoñez, Miguel Angel; Pichardo, Cristina; Colmenero, Juan de Dios

    1999-01-01

    In order to evaluate the usefulness of a peripheral blood PCR assay in the posttreatment follow-up of brucellosis, a cohort of 30 patients was studied by means of blood cultures, rose Bengal, seroagglutination, Coombs' antibrucella tests, and PCR assay at the time of diagnosis, at the end of treatment, and 2, 4, and 6 months later. Of the 29 patients whose PCR assays were initially positive, 28 (96.5%) were negative at the conclusion of the treatment. PCR was positive for the two patients who had relapses and negative for another four who had suspected but unconfirmed relapses. PCR was negative for 98.3% of the follow-up samples from those patients who had a favorable evolution. In conclusion, PCR appears to be a very useful technique, not only for the initial diagnosis of the disease, but also for posttreatment follow-up and the early detection of relapses. PMID:10565954

  2. Direct PCR Improves the Recovery of DNA from Various Substrates.

    PubMed

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Skuza, Pawel; Linacre, Adrian

    2015-11-01

    This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.

  3. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  4. Comparison of droplet digital PCR and quantitative real-time PCR in mcrA-based methanogen community analysis.

    PubMed

    Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

    2014-12-01

    Two different quantitative PCR platforms, droplet digital PCR (dd-PCR) and quantitative real-time PCR (qPCR), were compared in a mcrA-based methanogen community assay that quantifies ten methanogen sub-groups. Both technologies exhibited similar PCR efficiencies over at least four orders of magnitude and the same lower limits of detection (8 copies μL-DNA extract(-1)). The mcrA-based methanogen communities in three full-scale anaerobic digesters were examined using the two technologies. dd-PCR detected seven groups from the digesters, while qPCR did five groups, indicating that dd-PCR is more sensitive for DNA quantification. Linear regression showed quantitative agreements between both of the technologies (R(2) = 0.59-0.98) in the five groups that were concurrently detected. Principal component analysis from the two datasets consistently indicated a substantial difference in the community composition among the digesters and revealed similar levels of differentiation among the communities. The combined results suggest that dd-PCR is more promising for examining methanogenic archaeal communities in biotechnological processes.

  5. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.

    PubMed

    Li, Jin; Wang, Lilin; Mamon, Harvey; Kulke, Matthew H; Berbeco, Ross; Makrigiorgos, G Mike

    2008-05-01

    PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

  6. Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper.

    PubMed

    Jóźwik, A; Frymus, T

    2005-05-01

    Two pairs of primers were prepared, both localized within the sequences of the nucleoprotein gene (NP) of canine distemper virus (CDV). A number of experiments were done to optimize the conditions of RT-PCR and nested PCR methods. The nucleic acids of the Onderstepoort, Rockborn, Snyder Hill and Lederle strains of CDV could be detected with these primers. However, they did not react with the sequences of the Edmonston strain of the measles virus. The detection limit for RT-PCR was 10 TCID50 and for nested PCR 0.1 TCID50 of CDV. The RT-PCR was able to demonstrate the nucleic acid of CDV in the blood of all seven puppies vaccinated with a modified live virus. Blood samples of 23 dogs clinically suspected of distemper were examined by RT-PCR combined with nested PCR, and the results were compared with the detection of the CDV antigen in the smears from the mucous membranes by the direct immunofluorescence (IF) test. Of the 23 dogs, 12 were positive in nested PCR, six in the IF assay, and only two in single RT-PCR. It is concluded that nested PCR seems to be the most sensitive method for ante-mortem diagnosis of canine distemper, especially in its subacute or chronic forms.

  7. Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

    PubMed

    Dorn-In, Samart; Fahn, Carmen; Hölzel, Christina S; Wenz, Sebastian; Hartwig, Isabella; Schwaiger, Karin; Bauer, Johann

    2014-10-01

    Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.

  8. Loop-linker PCR: an advanced PCR technique for genome walking.

    PubMed

    Trinh, Quoclinh; Shi, Hui; Xu, Wentao; Hao, Junran; Luo, Yunbo; Huang, Kunlun

    2012-10-01

    In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.

  9. [PCR-ELISA technology and its application in biomedical fields].

    PubMed

    Yan, Lin; Wang, Xiaoying; Guo, Yunchang

    2011-01-01

    Polymerase chain reaction-enzyme-linked immunosorbent assay (PCR- ELISA) technology is created by the use of the high sensitivity of PCR, nucleic acid probes of the specificity, microplate reader of objectivity; since its establishment, it has received wide attention. In this paper, a basic description of the technology was introduced, and the application of PCR-ELISA in biomedical field in recent years were reviewed.

  10. Diagnosis of Oropouche virus infection by RT-nested-PCR.

    PubMed

    Moreli, Marcos Lázaro; Aquino, Victor Hugo; Cruz, Ana Cecília R; Figueiredo, Luiz Tadeu M

    2002-01-01

    Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections. Copyright 2002 Wiley-Liss, Inc.

  11. An efficient method to prepare PCR cloning vectors.

    PubMed

    Hong, Soon Gyu; Choi, Ji Young; Pryor, Barry M; Lee, Hong Kum

    2009-09-01

    An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR cloning vectors with high ligation efficiencies and low blue or false-positive colonies were obtained.

  12. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    PubMed Central

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  13. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

    PubMed

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  14. A thermodynamic approach to PCR primer design.

    PubMed

    Mann, Tobias; Humbert, Richard; Dorschner, Michael; Stamatoyannopoulos, John; Noble, William Stafford

    2009-07-01

    We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.

  15. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    SciTech Connect

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  16. A trio of human molecular genetics PCR assays.

    PubMed

    Reinking, Jeffrey L; Waldo, Jennifer T; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional PCR, b) perform PCR - Restriction Fragment Polymorphism (PCR-RFLP) analysis to genotype a Single Nucleotide Polymorphism (SNP) of the TAS2R38 gene on human chromosome 7 and c) perform duplex Allele Specific Primer-PCR (ASP-PCR) to genotype SNPs of two enzyme-encoding genes in a single biochemical pathway on human chromosomes 4 and 12. All PCR reactions have been optimized to use a single easily purified sample of the students' own DNA and run under a single thermal cycler program using inexpensive reagents to produce robust and clearly interpretable results on a single agarose gel. As presented here, the lab occupies two lab periods of 2 h, 40 min each: DNA purification followed by PCR reactions set-up on Day 1 and enzyme digestion of the PCR-RFLP and agarose gel analysis on Day 2. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  17. The inhibitory effect of pentobarbitone on reverse transcription-PCR.

    PubMed

    Hyun, Changbaig; Filippich, Lucio John; Hughes, Ian

    2005-01-31

    Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.

  18. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  19. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  20. Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

    PubMed

    Varehese, Rajani; Chauhan, Vineeta S; Misra, Arvind K

    2003-06-01

    While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

  1. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    PubMed

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  2. Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy.

    PubMed

    Xu, Ruliang; Ogino, Shuji; Lip, Va; Fang, Hong; Wu, Bai-Lin

    2003-01-01

    PCR-based methods for the detection of homozygous deletion of exon 7 of the SMN1 gene have been widely used in genetic testing for spinal muscular atrophy (SMA). We compared the most commonly used PCRrestriction fragment length polymorphism (PCR-RFLP) assay with an allele-specific PCR method, evaluating their potential application in direct testing, prenatal prediction, and preimplantation diagnosis, in terms of a range of DNA amounts used in such testing. We showed that PCR-RFLP could identify the SMN1 exon 7 by amplifying 10 pg of genomic DNA, and could differentiate SMN1 from SMN2 at the 100-pg DNA level (DraIdigested SMN2 fragments served as an internal control for PCR efficiency). In contrast, allele-specific PCR for SMN1, despite some advantages in a rapid preimplantation diagnosis, quickly lost its specificity when 100 pg of genomic DNA was used. In addition, the absence of a SMN1 fragment at the 10-pg DNA level may be due to a PCR amplification failure, and, thus, it is difficult to interpret without a proper internal control. Our data indicate that PCR-RFLP can be used for most diagnostic purposes, whereas the use of allelespecific PCR may be considered with caution under certain circumstances.

  3. Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

    PubMed

    Hein, Ingeborg; Flekna, Gabriele; Krassnig, Martina; Wagner, Martin

    2006-09-01

    A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

  4. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    PubMed

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  5. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  6. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  7. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    PubMed

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  8. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  9. Detection of Salmonella spp. in oysters by PCR.

    PubMed Central

    Bej, A K; Mahbubani, M H; Boyce, M J; Atlas, R M

    1994-01-01

    PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp. Images PMID:8117091

  10. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  11. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  12. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    PubMed

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  13. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  14. Identification of Mycobacterium bovis Isolates by a multiplex PCR

    PubMed Central

    de Souza Figueiredo, Eduardo Eustáquio; Silvestre, Flávia Galindo; Campos, Wilma Neres; Furlanetto, Leone Vinícius; Medeiros, Luciana; Lilenbaum, Walter; Fonseca, Leila Sousa; Silva, Joab Trajano; Paschoalin, Vânia Margaret Flosi

    2009-01-01

    Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates. PMID:24031349

  15. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

  16. Advanced multiplex PCR assay for differentiation of Brucella species.

    PubMed

    Kang, Sung-Il; Her, Moon; Kim, Jong Wan; Kim, Ji-Yeon; Ko, Kyung Yuk; Ha, Yun-Mi; Jung, Suk Chan

    2011-09-01

    Two new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.

  17. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew A; Weeks, Shelleen R; Epperson, William B; Singh, Shri N; Steinlicht, Gina L; Fang, Ying; Skaare, Jessica L; Larsen, Jill L; Payeur, Janet B; Nelson, Eric A

    2003-03-01

    In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

  18. A thermally baffled device for highly stabilized convective PCR

    PubMed Central

    Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung

    2012-01-01

    Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field. PMID:22241586

  19. [Detection of influenza virus (RT-PCR assay and others)].

    PubMed

    Matsuzaki, Yoko

    2003-11-01

    Viral isolation is the conventional method for influenza virus diagnosis but it is less useful for immediate patient management. RT-PCR is the sensitive and rapid assay for the detection of respiratory viruses. Single step and multiplex RT-PCR is able to detect several viruses simultaneously in a single reaction. Real time PCR(TaqMan method) is able to detect the amplicon directly by release of a fluorescent reporter of the probe during the amplification reactions. This procedure can save time since it eliminates post-PCR processing steps. These RT-PCR methods should be useful for the accurate and rapid diagnosis of influenza virus infection, especially severe cases such as pneumonia and encephalopathy.

  20. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    PubMed

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  1. Rapid diagnosis of poliovirus infection by PCR amplification.

    PubMed Central

    Chezzi, C

    1996-01-01

    A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections. PMID:8784577

  2. Teaching PCR Through Inquiry in an Undergraduate Biology Laboratory Course

    NASA Astrophysics Data System (ADS)

    Dorighi, K. M.; Betancourt, J.; Sapp, J.; Quan, T. K.; Lee, J.

    2010-12-01

    In this paper, we describe the design and implementation of an inquiry-based laboratory unit on the Polymerase Chain Reaction (PCR). This unit was designed and taught for the undergraduate Eukaryotic Genetics Laboratory class (Bio105L) at the University of California, Santa Cruz. Our activity utilizes an authentic molecular biology research question to teach the underlying molecular mechanisms and experimental technique of PCR, as well as fundamental scientific process skills such as planning experiments, making predictions and interpreting data. In particular, the activity prompts students to use PCR to determine which gene has been deleted in a region of the Drosophila genome. During this activity, students also gained technical experience in common molecular biology techniques, learned about additional applications of PCR and used a hands-on approach to model each step of PCR.

  3. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  4. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  5. Detection of episomal banana streak badnavirus by IC-PCR.

    PubMed

    Harper, G; Dahal, G; Thottappilly, G; Hull, R

    1999-04-01

    A polymerase chain reaction (PCR) based strategy to detect episomal banana streak badnavirus (BSV) in banana and plantain plants that carry integrated BSV sequences was developed. Antisera used in immuno-capture polymerase chain reaction (IC-PCR) are capable of binding a large number of BSV serotypes. The primers used for PCR are capable of annealing to and amplifying across the aspartic protease-reverse transcriptase domain boundaries of both episomal and integrated BSV sequences and result in similar or identical sequence size fragments from either template. However, we show that under the conditions selected for IC-PCR, nuclear, mitochondrial or chloroplast genomic sequences are not amplified and thus only captured episomal BSV is amplified. IC-PCR is suitable for the large-scale screening of Musa for episomal BSV which is necessary for germplasm movement.

  6. Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy

    PubMed Central

    Fan, H. Christina; Blumenfeld, Yair J.; El-Sayed, Yasser Y.; Chueh, Jane; Quake, Stephen R.

    2014-01-01

    OBJECTIVE The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. STUDY DESIGN Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. RESULTS Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. CONCLUSION Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours. PMID:19375573

  7. PCR thermal management in an integrated Lab on Chip

    NASA Astrophysics Data System (ADS)

    Singh, Janak; Ekaputri, Mayang

    2006-04-01

    Thermal management modelling and simulations of a polymerase chain reaction (PCR) device to be integrated on a lab on chip (LOC) have been carried out and presented. A typical MEMS PCR in symmetrical configuration is the base model for this study. When the PCR device is integrated on a fluidic chip with many other bio-analysis components such as DNA extraction, RNA extraction, electro-chemical sensor, flow through components and channels etc., thermal symmetry required for uniform temperature across the PCR chamber is normally lost. In this paper, ANSYS 8.0 simulations in varying conditions and corresponding physical basis have been investigated and presented. Model optimizations are carried out when PCR chamber is placed, one, in the centre (symmetry) and two, in the corner (asymmetry) of the integrated chip. In both cases, temperature uniformity within ±0.5 °C variation is obtained.

  8. Biofilm formation by Borrelia burgdorferi sensu lato.

    PubMed

    Timmaraju, Venkata Arun; Theophilus, Priyanka A S; Balasubramanian, Kunthavai; Shakih, Shafiq; Luecke, David F; Sapi, Eva

    2015-08-01

    Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology.

  9. PCR detection of Babesia ovata from questing ticks in Japan.

    PubMed

    Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Okubo, Kazuhiro; Suganuma, Keisuke; Hayashida, Kyoko; Igarashi, Ikuo; Zakimi, Satoshi; Matsumoto, Kotaro; Inokuma, Hisashi; Yokoyama, Naoaki

    2014-04-01

    Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. Field effect sensors for PCR applications

    NASA Astrophysics Data System (ADS)

    Taing, Meng-Houit; Sweatman, Denis R.

    2004-03-01

    The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This causes an n-type depletion layer substrate to thicken and a p-type to thin. The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of pathogens we"ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode. Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with "floating" biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors

  11. An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

    PubMed

    Thompson, Robyn E; Duncan, George; McCord, Bruce R

    2014-11-01

    A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

  12. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    PubMed Central

    2011-01-01

    Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection. PMID:21831278

  13. Use of Repetitive Element Palindromic-PCR (rep-PCR) for the Epidemiologic Discrimination of Food-Borne Pathogens

    USDA-ARS?s Scientific Manuscript database

    The use of defined primers for polymerase chain reactions (PCR) amplicifcations of interspersed repetitive DNA elements present at distinct locations in prokaryotic genomes is referred to as Repetitive Element Palindromic Sequences Based-Polymerase Chain Reactions, rep-PCR. The initial discovery of...

  14. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.

    PubMed

    Svec, David; Tichopad, Ales; Novosadova, Vendula; Pfaffl, Michael W; Kubista, Mikael

    2015-03-01

    We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

  15. Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.

    PubMed

    Rački, Nejc; Dreo, Tanja; Gutierrez-Aguirre, Ion; Blejec, Andrej; Ravnikar, Maja

    2014-01-01

    Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR. Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR. This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.

  16. Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay.

    PubMed

    Lawson, A J; Logan, J M; O'neill, G L; Desai, M; Stanley, J

    1999-12-01

    A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured "Campylobacter spp." from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context.

  17. A comparison of four methods for PCR inhibitor removal.

    PubMed

    Hu, Qingqing; Liu, Yuxuan; Yi, Shaohua; Huang, Daixin

    2015-05-01

    Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean® DNA Clean-Up kit, DNA IQ™ System, Phenol-Chloroform extraction and Chelex®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol-Chloroform extraction and Chelex®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples.

  18. PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

    PubMed Central

    Schmidt, B L

    1997-01-01

    The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented. PMID:8993863

  19. Real time PCR in childhood tuberculosis: a valuable diagnostic tool.

    PubMed

    Dayal, Rajeshwar; Kashyap, Haripal; Pounikar, Gajanand; Kamal, Raj; Yadav, Neeraj Kumar; Singh, Manoj Kumar; Chauhan, Devendra Singh; Goyal, Ankur

    2015-02-01

    The present study was conducted to detect and quantitate Mycobacterium tuberculosis from various body fluid specimens of cases of tuberculosis by real time PCR technique and compare results with conventional PCR technique and culture. One hundred fifteen children (<18 y) with tuberculosis (diagnosed as per IAP guidelines) and 32 disease matched controls from the Department of Pediatrics, S.N. Medical College, Agra, were included in the study. Different body fluids (CSF, gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate) were subjected to culture, conventional PCR targeting insertion sequence 1S6110 and Real time PCR targeting 16srRNA of Mycobacterium tuberculosis. Real time PCR showed significantly better results than culture in all body fluids (p < 0.05). It was superior to conventional PCR in CSF (p < 0.05) but showed comparable results in gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate (p > 0.05). Hence, real time PCR is a promising diagnostic tool for childhood tuberculosis, particularly tubercular meningitis.

  20. PCR assays for detection of Baylisascaris procyonis eggs and larvae.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Kazacos, Kevin R

    2009-06-01

    The objective of this study was to develop polymerase chain reaction (PCR) assays for detection of Baylisascaris procyonis eggs and larvae in fecal, environmental, and tissue samples. We have optimized conventional and real-time PCR assays for B. procyonis using the mitochondrial cytochrome oxidase 2 gene as the target for amplification. The lower limit of detection of the parasite genomic DNA was 10 pg in the conventional PCR and 100 fg in the real-time PCR. In both PCR assays, specific amplification of a 146 bp product was achieved with DNA extracted from a single in vitro hatched B. procyonis larva and also from canine fecal samples spiked with as few as 20 unembryonated B. procyonis eggs per gram of feces. The PCR assays were successfully used for detection of B. procyonis eggs and larvae in fecal, environmental, and tissue samples. No DNA amplification was seen when the genomic DNA of related ascarids (including B. transfuga) and a hookworm was used as template in the PCR; however, amplification was seen with the very closely related B. columnaris.

  1. Rapid and simple method of qPCR primer design.

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2015-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.

  2. Multiplex PCR to detect four different tomato-infecting pathogens.

    PubMed

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  3. Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.

    PubMed

    Guimarães, Alessandro de Sá; Dorneles, Elaine Maria Seles; Andrade, Giovanna Ivo; Lage, Andrey Pereira; Miyoshi, Anderson; Azevedo, Vasco; Gouveia, Aurora Maria Guimarães; Heinemann, Marcos Bryan

    2011-12-15

    Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

  4. Development of a PCR test to diagnose Haemophilus parasuis infections.

    PubMed

    Oliveira, S; Galina, L; Pijoan, C

    2001-11-01

    A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.

  5. A multiplex PCR for detection of six viruses in ducks.

    PubMed

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10(2) copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  6. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

    NASA Astrophysics Data System (ADS)

    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  7. PCR diagnosis of Pneumocystis pneumonia: a bivariate meta-analysis.

    PubMed

    Lu, Yuan; Ling, Guoya; Qiang, Chenyi; Ming, Qinshou; Wu, Cong; Wang, Ke; Ying, Zouxiao

    2011-12-01

    We undertook a bivariate meta-analysis to assess the overall accuracy of respiratory specimen PCR assays for diagnosing Pneumocystis pneumonia. The summary sensitivity and specificity were 0.99 (95% confidence interval, 0.96 to 1.00) and 0.90 (0.87 to 0.93). Subgroup analyses showed that quantitative PCR analysis and the major surface glycoprotein gene target had the highest specificity value (0.93). Respiratory specimen PCR results are sufficient to confirm or exclude the disease for at-risk patients suspected of having Pneumocystis pneumonia.

  8. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  9. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  10. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  11. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    PubMed

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-05

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detect