CiteULike: carpi's library [198 articles]

Fine-scale genetic variation and evolution of West Nile Virus in a transmission "hot spot" in suburban Chicago, USA.

L Bertolotti — 2008-07-02T14:04:15-00:00

Virology, Vol. 374, No. 2. (10 May 2008), pp. 381-389.

Mosquitoes and birds were sampled for West Nile virus (WNV) in suburban Chicago, USA, in a "hot spot" of arboviral transmission. Viral genetic diversity within this area was similar to that within Illinois and the United States. Diversity was higher among viruses from mosquitoes than from birds, higher among viruses from birds in urban "green spaces" than from birds in residential areas, but lower among viruses from mosquitoes in green spaces than from mosquitoes in residential areas. Viral transmission was distance-limited, as evidenced by decreasing autocorrelation of WNV sequences with increasing geographic separation. The evolutionary rate of WNV within the study area between 21 July and 4 October 2005 was ten times higher than that for WNV across North America between 2002 and 2005. These results indicate that WNV transmission and evolutionary dynamics can vary seasonally and in response to fine-scale environmental conditions and landscape characteristics related to urbanization.

Novel variant of tickborne encephalitis virus, Russia.

VA Ternovoi — 2008-06-27T14:03:12-00:00

Emerging infectious diseases, Vol. 13, No. 10. (October 2007), pp. 1574-1578.

We isolated a novel strain of tickborne encephalitis virus (TBEV), Glubinnoe/2004, from a patient with a fatal case in Russia. We sequenced the strain, whose landmark features included 57 amino acid substitutions and 5 modified cleavage sites. Phylogenetically, Glubinnoe/2004 is a novel variant that belongs to the Eastern type of TBEV.

Comparative genomics of emerging human ehrlichiosis agents.

JC Hotopp — 2008-06-26T22:19:23-00:00

PLoS genetics, Vol. 2, No. 2. (February 2006)

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.

Epidemiological survey of tick-borne encephalitis virus and Anaplasma phagocytophilum co-infections in patients from regions of the Czech Republic endemic for tick-borne diseases.

P Zeman — 2008-06-16T16:56:11-00:00

Wiener klinische Wochenschrift, Vol. 119, No. 17-18. (2007), pp. 538-543.

During the period 2000-2003, patients hospitalized for suspected tick-borne encephalitis in the Czech Republic were screened for possible A. phagocytophilum co-infection. Blood samples taken at admission were tested for the presence of A. phagocytophilum DNA by nested PCR using a modified target sequence as an internal control, and sera were tested for the presence of antibodies by indirect immunofluorescence and western blotting methods using cell-culture-derived antigens. To verify the assay specificity, a set of 45 sera of Patagonian residents served as a non-tick-exposed control group, and a set of 14 B. henselae-positive sera was used to check cross-reactivity. Of 809 patients hospitalized, 80 (9.9%) showed IgG antibodies reactive to A. phagocytophilum at > or =80 (reciprocal dilution factor) and 50 (6.2%) at > or =160; two (0.2%) patients showed elevated IgM titers of 40. No full blood obtained from 162 patients tested positive in PCR when false negativity was excluded. During hospitalization, the diagnosis of tick-borne encephalitis was confirmed in 536 patients, 57 (10.6%) of whom had anti-A. phagocytophilum IgG antibodies reactive at > or =80 and 41 (7.6%) at > or =160, which did not differ significantly from the whole set (P = 0.66/0.30), the maximum IgG titer registered was 5120, and no IgM titer reached the 40 cut-off. Available paired sera from 189 tick-borne encephalitis patients showed no significant shifts, but one case of slight seroconversion (IgG shift from < 80 to 320) was detected in one of the non-tick-borne encephalitis patients. The sex of the patient showed no significance for the prevalence of A. phagocytophilum antibodies; however, the seropositive patients were older on average than those who were seronegative (43.5 +/- 15.9 vs. 37.9 +/- 18.3 years, P = 0.05). Clinical manifestation of the disease did not differ noticeably between patients with and without A. phagocytophilumreactive antibodies, except for fever duration, which was significantly longer in patients with titers > or =1280. Overall, A. phagocytophilum co-infection did not seem to be a frequent and/or significant complication of tick-borne encephalitis acquired in the Czech Republic.

Anaplasma phagocytophilum Infection in Ixodes ricinus, Bavaria, Germany.

C Silaghi — 2008-06-06T16:34:08-00:00

Emerging infectious diseases, Vol. 14, No. 6. (June 2008), pp. 972-974.

Anaplasma phagocytophilum DNA was detected by real-time PCR, which targeted the msp2 gene, in 2.9% of questing Ixodes ricinus ticks (adults and nymphs; n = 2,862), collected systematically from selected locations in Bavaria, Germany, in 2006. Prevalence was significantly higher in urban public parks in Munich than in natural forests.

Spatial pattern of risk exposure to pathogens transmitted by Ixodes ricinus in north-eastern Italy and the Italy/Slovenia transborder territory.

Marina Cinco — 2008-06-06T16:31:01-00:00

International journal of medical microbiology : IJMM (29 May 2008)

The Friuli Venezia Giulia region (FVG) and the transborder territory Italy/Slovenia are endemic for Lyme borreliosis and, more recently, also for Anaplasma phagocytophilum infections. In the present study, we determined the density of questing Ixodes ricinus ticks and their infection rates with Borrelia burgdorferi and A. phagocytophilum in the four main biogeographical regions present in the study area. Within these regions, stations for collecting ticks were selected. The pathogens were detected by PCR methods specific for the two infectious agents. The prevalences of infection varied among different landscapes. The local potential risk of human infection through tick bite was drawn from tick abundance and prevalence of infection in ticks: The level of risk for B. burgdorferi infection was found to be highest in the transborder Karst territory. In that environment up to 45.5% of the ticks were infected. The prevalence dropped approaching the alpine environment (13.2%). Conversely, the potential risk to be bitten by ticks infected with Anaplasma was quite low: The annual average infection prevalence in ticks was 1.6%.

Phylogeny of North American Powassan virus.

GD Ebel — 2008-06-05T22:22:49-00:00

The Journal of general virology, Vol. 82, No. Pt 7. (July 2001), pp. 1657-1665.

To determine whether Powassan virus (POW) and deer tick virus (DTV) constitute distinct flaviviral populations transmitted by ixodid ticks in North America, we analysed diverse nucleotide sequences from 16 strains of these viruses. Two distinct genetic lineages are evident, which may be defined by geographical and host associations. The nucleotide and amino acid sequences of lineage one (comprising New York and Canadian POW isolates) are highly conserved across time and space, but those of lineage two (comprising isolates from deer ticks and a fox) are more variable. The divergence between lineages is much greater than the variation within either lineage, and lineage two appears to be more diverse genetically than is lineage one. Application of McDonald-Kreitman tests to the sequences of these strains indicates that adaptive evolution of the envelope protein separates lineage one from lineage two. The two POW lineages circulating in North America possess a pattern of genetic diversity suggesting that they comprise distinct subtypes that may perpetuate in separate enzootic cycles.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.

B Sukumaran — 2008-06-03T21:58:04-00:00

The Journal of experimental medicine, Vol. 203, No. 6. (12 June 2006), pp. 1507-1517.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

An application of statistics to comparative metagenomics.

Beltran Rodriguez-Brito — 2006-03-24T12:11:46-00:00

BMC Bioinformatics, Vol. 7, No. 1. (20 March 2006)

ABSTRACT: BACKGROUND: Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. RESULTS: Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that are overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. CONCLUSIONS: The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

Infection and co-infection rates of Anaplasma phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial endosymbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin.

FE Steiner — 2008-05-09T15:55:46-00:00

Journal of medical entomology, Vol. 45, No. 2. (March 2008), pp. 289-297.

In total, 394 questing adult blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), collected at four sites were analyzed by polymerase chain reaction (PCR) for five microbial species: Anaplasma phagocytophilum, Babesia microti, Babesia odocoilei, Borrelia burgdorferi, and the rickettsial I. scapularis endosymbiont. Identities of genetic variants of A. phagocytophilum were determined by sequencing a portion of the 16S DNA. In 55% of infected ticks (193/351), a single agent was detected. In 45% (158/351), two or more agents were detected; 37% harbored two agents and 8% harbored three agents. One male tick, collected from Ft. McCoy, WI, harbored all four microbial genera The highest rates of co-infection were by the Ixodes endosymbiont and B. burgdorferi (95/351). Two species of Babesia co-occurred within a single tick population in Wells National Estuarine Research Reserve, Wells, ME, whereas only B. odocoilei was found in other tick populations. Only A. phagocytophilum human anaplasmosis variant was detected in questing ticks from Tippecanoe River State Park, IN; from Wells; and Ft. McCoy, whereas a single infected tick from Presque Isle, PA, was infected by AP-Variant 1. Partially engorged ticks from deer in Tippecanoe River State Park were all infected with AP-Variant 1. Frequency of infections with each agent varied among populations. Rates and types of co-infections were not significantly different from random except for the Ixodes endosymbiont and B. burgdorferi in male ticks, which co-occurred less frequently than expected. Thus, I. scapularis hosts an array of pathogenic and symbiotic agents and potential evidence of interactions among microbial species was observed.

Symbiotic bacteria in oocyte and ovarian cell mitochondria of the tick Ixodes ricinus: biology and phylogenetic position

Rymaszewska — 2007-03-27T18:16:15-00:00

Parasitology Research, Vol. 100, No. 5. (April 2007), pp. 917-920.

Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes.

SG Rijpkema — 2008-05-08T20:14:17-00:00

Journal of clinical microbiology, Vol. 33, No. 12. (December 1995), pp. 3091-3095.

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.

Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis, Anaplasma phagocytophilum.

RF Massung — 2008-05-08T19:49:32-00:00

Journal of clinical microbiology, Vol. 41, No. 2. (February 2003), pp. 717-722.

Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of Anaplasma phagocytophilum-infected HL-60 cells. The specificity of the assays that were able to detect <or=2.5 infected cells was tested by the use of template DNA extracted from Ehrlichia chaffeensis, Rickettsia rickettsii, and Bartonella henselae. The assays with the lowest limits of detection were shown to be a nested assay that amplifies the 16S rRNA gene (primer pairs ge3a-ge10 [primary] and ge9-ge3 [nested]; detects 0.25 infected cell), a direct assay that amplifies the major surface protein gene msp2 (primer pair msp2-3f-msp2-3r; detects 0.25 infected cell), and a direct assay that amplifies the 16S rRNA gene (primer pair ehr521-ehr790; detects 0.25 infected cell). The specificity and limit of detection of the MSP2 and 16S rRNA direct assays were further tested by use of A. phagocytophilum template DNA from both North America and Europe and from human, tick, white-footed mouse, equine, deer, bovine, and wood rat samples and of template DNA from closely related species (Anaplasma marginale, the white-tailed deer agent, and additional E. chaffeensis-positive samples). Three manufacturers' PCR kits were tested and showed distinct variations in the limit of detection, specificity, and nonspecific background amplification. The importance of these results for the molecular diagnosis of human granulocytic ehrlichiosis is discussed.

Genome sequencing in microfabricated high-density picolitre reactors

Marcel Margulies — 2005-07-31T21:14:04-00:00

Nature (31 July 2005)

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species.

S Epis — 2008-04-29T20:21:24-00:00

Parasitology, Vol. 135, No. 4. (April 2008), pp. 485-494.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus.

T Beninati — 2008-04-29T20:12:05-00:00

Applied and environmental microbiology, Vol. 70, No. 5. (May 2004), pp. 2596-2602.

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.

Tick genomics: The Ixodes genome project and beyond

Pagel — 2008-04-29T20:09:24-00:00

International Journal for Parasitology, Vol. 37, No. 12. (October 2007), pp. 1297-1305.

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.

MEGAN analysis of metagenomic data.

DH Huson — 2008-02-14T19:21:34-00:00

Genome Res, Vol. 17, No. 3. (March 2007), pp. 377-386.

Metagenomics is the study of the genomic content of a sample of organisms obtained from a common habitat using targeted or random sequencing. Goals include understanding the extent and role of microbial diversity. The taxonomical content of such a sample is usually estimated by comparison against sequence databases of known sequences. Most published studies use the analysis of paired-end reads, complete sequences of environmental fosmid and BAC clones, or environmental assemblies. Emerging sequencing-by-synthesis technologies with very high throughput are paving the way to low-cost random "shotgun" approaches. This paper introduces MEGAN, a new computer program that allows laptop analysis of large metagenomic data sets. In a preprocessing step, the set of DNA sequences is compared against databases of known sequences using BLAST or another comparison tool. MEGAN is then used to compute and explore the taxonomical content of the data set, employing the NCBI taxonomy to summarize and order the results. A simple lowest common ancestor algorithm assigns reads to taxa such that the taxonomical level of the assigned taxon reflects the level of conservation of the sequence. The software allows large data sets to be dissected without the need for assembly or the targeting of specific phylogenetic markers. It provides graphical and statistical output for comparing different data sets. The approach is applied to several data sets, including the Sargasso Sea data set, a recently published metagenomic data set sampled from a mammoth bone, and several complete microbial genomes. Also, simulations that evaluate the performance of the approach for different read lengths are presented.

Metagenomics to Paleogenomics: Large-Scale Sequencing of Mammoth DNA.

Hendrik N Poinar — 2006-01-20T14:30:08-00:00

Science (20 December 2005)

We sequenced 28 million base pairs of DNA in a metagenomics approach using a woolly mammoth (Mammuthus primigenius) sample from Siberia. Thanks to exceptional sample preservation and use of a novel emulsion polymerase chain reaction and pyrosequencing technique, 13 million base pairs (45.4%) of the sequencing reads were identified as mammoth DNA. Sequence identity between our data and African elephant (Loxodonta africana) was 98.55%, consistent with a paleontologically based divergence date of 5 to 6 million years. The sample includes a surprisingly small diversity of environmental DNAs. The high percentage of endogenous DNA recoverable from this single mammoth would allow for completion of its genome, unleashing the field of paleogenomics.

Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae).

B Pichon — 2008-04-24T19:44:46-00:00

Journal of medical entomology, Vol. 40, No. 5. (September 2003), pp. 723-731.

In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses. The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal. For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes. Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences. Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting. For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex. The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I. ricinus nymphs that had fed on B. microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest. The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest. Few pathogens were detected but the discovery of Borrelia valaisiana and B. burgdorferi s.s. in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.

TS Gritsun — 2008-04-17T21:59:51-00:00

Virus research, Vol. 49, No. 1. (May 1997), pp. 27-39.

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

Diversity of Ixodes ricinus tick-associated bacterial communities from different forests.

Leo van Overbeek — 2008-04-17T15:12:03-00:00

FEMS microbiology ecology (18 March 2008)

Nymphal Ixodes ricinus ticks (n=180) were collected from three different areas in the Netherlands to investigate the effect of forest composition on tick-associated microbial communities. Sampled habitats differed in thickness of leaf litter and humus layers and vegetation associations and were located near Amsterdam (Beech-Oak), Ede (Birch-Oak) and Veldhoven (Birch-Oak). Analysis of nine 16S rRNA gene clone libraries made from individual ticks showed nearest matches with presumed pathogens Candidatus Neoehrlichia mikurensis and Rickettsia australis and arthropod endosymbionts Wolbachia pipientis and Candidatus Midichloria mitochondrii. Total bacterial species diversity (Shannon index) and Borrelia species infections were determined in I. ricinus by, respectively, PCR-denaturing gradient gel-electrophoresis and PCR-reverse line blot with probes specific for Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia ruski. Bacterial diversity differed significantly per area and was lowest in Ede. In contrast, Borrelia species-infected ticks were more abundant in Ede, Candidatus Neoehrlichia mikurensis-infected ticks in Ede and Veldhoven, and R. australis-infected ticks in Amsterdam. Borrelia afzelii was the most common Borrelia species found in all three areas. Bacterial tick diversity was influenced by local differences in forest structure, which is proposed to modulate animal populations that are commonly parasitized by I. ricinus.

A Metagenomic Survey of Microbes in Honey Bee Colony Collapse Disorder.

Diana L Cox-Foster — 2007-09-09T13:26:15-00:00

Science (6 September 2007)

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees (IAPV), was strongly correlated with CCD.

Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source

Olivia Champion — 2006-01-11T13:03:04-00:00

PNAS, Vol. 102, No. 44. (1 November 2005), pp. 16043-16048.

Campylobacter jejuni is the predominant cause of bacterial gastroenteritis worldwide, but traditional typing methods are unable to discriminate strains from different sources that cause disease in humans. We report the use of genomotyping (whole-genome comparisons of microbes using DNA microarrays) combined with Bayesian-based algorithms to model the phylogeny of this major food-borne pathogen. In this study 111 C. jejuni strains were examined by genomotyping isolates from humans with a spectrum of C. jejuni-associated disease (70 strains), chickens (17 strains), bovines (13 strains), ovines (5 strains), and the environment (6 strains). From these data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities (P = 1); a livestock clade comprising 31/35 (88.6%) of the livestock isolates and a "nonlivestock" clade comprising further clades of environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent was a cluster of six genes (cj1321 to cj1326) within the flagellin glycosylation locus, which were confirmed by PCR analysis as genetic markers in six additional chicken-associated strains. Surprisingly these studies show that the majority (39/70, 55.7%) of C. jejuni human isolates were found in the nonlivestock clade, suggesting that most C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. This study has provided insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease-control strategies. The comparative phylogenomics approach described provides a robust methodological prototype that should be applicable to other microbes.

Bacterial pathogenomics

Mark Pallen — 2007-10-20T17:10:38-00:00

Nature, Vol. 449, No. 7164. (17 October 2007), pp. 835-842.

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis.

A Johansson — 2008-04-13T18:32:58-00:00

Journal of bacteriology, Vol. 186, No. 17. (September 2004), pp. 5808-5818.

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.

Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis.

M Broekhuijsen — 2005-02-12T15:37:17-00:00

J Clin Microbiol, Vol. 41, No. 7. (July 2003), pp. 2924-2931.

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.

Molecular epidemiology, evolution, and ecology of Francisella.

P Keim — 2008-04-10T20:17:27-00:00

Annals of the New York Academy of Sciences, Vol. 1105 (June 2007), pp. 30-66.

Tularemia is a disease caused by several subspecies of Francisella tularensis, although the severity of the disease varies greatly from subspecies to subspecies. Currently, there are four recognized subspecies (tularensis, holarctica, mediasiatica, and novicida), in addition to a second Francisella species, F. philomiragia. It is clear from molecular sampling of the environment that these human pathogens are a mere fraction of the Francisella diversity. Taxonomic nomenclature is now being based upon several DNA-sequence-based approaches and this advance provides for robust phylogenetic models that are guiding the systematics of this genus. This in turn allows for better molecular epidemiological investigations and more precise ecological analysis. Tularemia ecology is still only partially understood, with many knowledge gaps about the disease reservoir and vectors. Molecular analysis has identified a major population split within F. tularensis subsp. tularensis that points toward distinctive ecological adaptations, vectors, and host species. Current medical practice does not rely upon subspecies or subpopulation identification, although this information may have predictive value for clinical outcome, especially in the United States. Combined molecular and epidemiological analyses suggest that the population split in F. tularensis subsp. tularensis matches two distinct human diseases in the United States with different mortality rates. DNA-sequence-based typing of F. tularensis subsp. holarctica from tularemia outbreaks in Europe and the United States proves regional identity among isolates and also demonstrates that this subspecies successfully disseminated worldwide in recent evolutionary time.

Evidence of tick-borne organisms in mule deer (Odocoileus hemionus) from the western United States.

MJ Yabsley — 2008-04-08T14:57:24-00:00

Vector borne and zoonotic diseases (Larchmont, N.Y.), Vol. 5, No. 4. (2005), pp. 351-362.

Free-ranging mule deer (MD; Odocoileus hemionus) from Arizona and California were tested for evidence of infection with several tick-borne pathogens, including species of Ehrlichia, Anaplasma, Babesia, and Borrelia. Of 125 mule deer tested from Arizona, 29 (23%) and 11 (9%) had antibodies reactive to E. chaffeensis and A. phagocytophilum by indirect immunofluorescent antibody testing, respectively; none of the six MD tested from California were seropositive. Using a commercial competitive ELISA kit, antibodies reactive to Anaplasma spp. were detected in 19 (15%) MD from Arizona and four of six (67%) MD from California. Polymerase chain reaction (PCR) testing for tick-borne pathogens was conducted on blood samples from 29 MD from Arizona and 11 MD from California. Twenty-two of 29 (75.9%) MD from Arizona had PCR evidence of infection with at least one tick-borne pathogen. We detected an Anaplasma sp. in 19 of 29 (65.5%) MD and a Babesia sp. in 10 of 29 (34%) MD. Sequencing of these amplicons indicated that the Anaplasma sp. was the same that had previously been detected in MD from California and the Babesia sp. was similar to one previously detected in a reindeer (Rangifer tarandus tarandus) from California. All of the California MD had evidence of infection with a tick-borne pathogen. Two different species of Anaplasma spp. were detected in MD from California, eight of of 11 MD were infected with an Anaplasma sp., and three of 11 MD were infected with A. ovis. This is the first report of a mule deer naturally infected with A. ovis. Ten of 11 MD from California were infected with a Babesia-like organism previously associated with human disease, and a single MD was PCR positive for Borrelia coriaceae, which has been associated with epizootic bovine abortion. Together, these data suggest that MD in northern Arizona and eastern California are exposed to several pathogens of human and veterinary importance.

Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction.

D Hulínská — 2008-04-08T14:54:16-00:00

APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, Vol. 112, No. 4-5. (y 2004), pp. 239-247.

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.

Prevalence of Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks from Lithuania and Norway.

Jana Radzijevskaja — 2008-04-07T21:02:08-00:00

International journal of medical microbiology : IJMM (26 March 2008)

We detected Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks collected from different locations in Lithuania and Norway by using the Taq Man based real-time PCR method. The msp2 gene of A. phagocytophilum and the 18S rRNA gene of B. divergens have been chosen as amplification targets. The overall infection rate of A. phagocytophilum in Norwegian ticks was 4.5% (10/224) and in Lithuanian ticks 3% (4/140). The prevalence varied in locations between 0% and 9% in Lithuania and in Norway. Three out of 140 (2%) ticks were infected with B. divergens in Lithuania and two out of 224 (0.9%) in Norway. The prevalence of B. divergens infection varied from 0% to 3% and from 0% to 4% in different sites in Lithuania and Norway, respectively.

Dynamic transmission of numerous Anaplasma phagocytophilum genotypes among lambs in an endemically infected sheep flock.

Georgia A F Ladbury — 2008-04-07T20:59:36-00:00

Journal of clinical microbiology (26 March 2008)

The transmission dynamics of A. phagocytophilum strains circulating within juvenile members of a sheep flock grazing on Ixodes ricinus-infested pasture in southern Norway were monitored. PCR-based detection the bacterial p44 fragments in the blood of 16 lambs sampled weekly for 16 weeks following their release into pasture revealed rickettsemia in all animals, with an increasing proportion of infected animals as the survey progressed. Comparison of partial msp4 sequences obtained from infected blood samples revealed 24 distinct genotypes, some of which were repeatedly encountered, occurring in up to six sheep over a 14 week period, whereas others were only observed once. Individual sheep were infected by up to five distinct genotypes, with a specific genotype being encountered for between one and three consecutive weeks, and in some sheep, genotypes detected early in the study were also present in later samples. In general, detection of A. phagocytophilum by PCR correlated well with the observation of infected neutrophils in blood smears. Together these results reveal a previously unrecognized diversity of A. phagocytophilum strains simultaneously circulating within an endemically-infected population and are consistent with a remarkably dynamic transmission of strains among infected animals.

Greek goat encephalitis virus strain isolated from Ixodes ricinus, Greece.

A Papa — 2008-04-07T20:54:37-00:00

Emerging infectious diseases, Vol. 14, No. 2. (February 2008), pp. 330-332.

A strain of Greek goat encephaltitis virus was isolated from engorged Ixodes ricinus ticks that had fed on goats in northern Greece. The strain was almost identical to the prototype strain isolated 35 years ago.

Succinate dehydrogenase gene arrangement and expression in Anaplasma phagocytophilum.

Robert F Massung — 2008-04-06T23:11:57-00:00

Gene (16 February 2008)

DNA sequencing of the region directly downstream of the Anaplasma phagocytophilum (strain MRK) 16S rRNA gene identified homologues of sdhC and sdhD; however, further sequencing by gene walking failed to identify additional sdh gene homologues. The sequence downstream of sdhD identified a partial gene, pep1, predicted to encode a protein >35.3 kDa with 26.3% identity to a hypothetical Ehrlichia canis protein with no known function. The recently completed sequence of the A. phagocytophilum genome confirmed our findings and indicated that the sdhA and sdhB genes are duplicated in a tandem orientation, and located distant from the sdhC and sdhD genes. The expression of the A. phagocytophilum 16S rRNA, sdhC, and sdhD genes was examined by reverse transcriptase PCR which showed that these three genes are expressed as an operon. The pep1 gene was expressed independent of the 16S-sdhCD operon from a promoter between sdhD and pep1. Further analysis of the sdhA and sdhB genes suggested the tandem duplication of the genes in conserved and may be unique to the species A. phagocytophilum. While the conservation of the A. phagocytophilum Sdh proteins, including the residues required for heme- and quinone-binding by SdhC and SdhD, suggests these subunits form an active enzymatic complex, the unusual genomic arrangement and expression pattern of these genes support previous studies (rRNA, ftsZ) indicating that gene rearrangement and operon fragmentation are common in the genomes of Anaplasma and other obligate intracellular bacteria. OMB disclaimer: the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the Department of Health and Human Services.

Unraveling the immune regulatory mechanisms imposed by Anaplasma.

WC Brown — 2008-04-06T23:08:20-00:00

Veterinary journal (London, England : 1997), Vol. 175, No. 1. (January 2008), pp. 10-11.

The Human Microbiome Project

Peter Turnbaugh — 2007-10-19T10:14:14-00:00

Nature, Vol. 449, No. 7164. (18 October 2007), pp. 804-810.

Metagenomics: DNA sequencing of environmental samples

Susannah Tringe — 2005-11-01T23:03:40-00:00

Nature Reviews Genetics, Vol. 6, No. 11. (01 November 2005), pp. 805-814.

Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes.

K Kurokawa — 2008-02-24T22:20:35-00:00

DNA Res, Vol. 14, No. 4. (31 August 2007), pp. 169-181.

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes.

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

Falk Warnecke — 2007-11-23T05:36:59-00:00

Nature, Vol. 450, No. 7169., pp. 560-565.

A Virus Reveals Population Structure and Recent Demographic History of Its Carnivore Host

Roman Biek — 2006-02-11T11:40:15-00:00

Science, Vol. 311, No. 5760. (27 January 2006), pp. 538-541.

Directly transmitted parasites often provide substantial information about the temporal and spatial characteristics of host-to-host contact. Here, we demonstrate that a fast-evolving virus (feline immunodeficiency virus, FIV) can reveal details of the contemporary population structure and recent demographic history of its natural wildlife host (Puma concolor) that were not apparent from host genetic data and would be impossible to obtain by other means. We suggest that rapidly evolving pathogens may provide a complementary tool for studying population dynamics of their hosts in "shallow" time.

Recombination in feline lentiviral genomes during experimental cross-species infection.

M Poss — 2008-04-02T16:16:13-00:00

Virology, Vol. 359, No. 1. (1 March 2007), pp. 146-151.

Domestic cats develop an asymptomatic, productive infection with a feline immunodeficiency virus (PLV) derived from a naturally infected cougar (P. concolor). We previously demonstrated that there are extensive G to A substitutions, characteristic of host cytidine deaminase editing, and positive selection on reverse transcriptase in the PLV genome during this cross-species infection. In this study, we evaluated full-length viral genomes from each of four cats infected with PLV to determine if viral recombination occurred during this single source infection. Recombination rates were measurable in three of the four infected cats. In two of these animals, a single site in reverse transcriptase was under positive selection and there was significant topological incongruence among individual genes in the 3' half of the genomes. The break point was proximate to a splice site used for accessory gene expression. Our data indicate that recombination can facilitate lentivirus persistence in unfavorable environments such as a new host species.

Out of Africa: A Molecular Perspective on the Introduction of Yellow Fever Virus into the Americas

Juliet — 2005-01-20T00:29:54-00:00

PloS Pathogens (2007)

ellow fever virus (YFV) remains the cause of severe morbidity and mortality in South America and Africa. To determine the evolutionary history of this important reemerging pathogen, we performed a phylogenetic analysis of the largest YFV data set compiled to date, representing the prM/E gene region from 133 viral isolates sampled from 22 countries over a period of 76 years. We estimate that the currently circulating strains of YFV arose in Africa within the last 1,500 years and emerged in the Americas following the slave trade approximately 300–400 years ago. These viruses then spread westwards across the continent and persist there to this day in the jungles of South America. We therefore illustrate how gene sequence data can be used to test hypotheses of viral dispersal and demographics, and document the role of human migration in the spread of infectious disease.

The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

JD Thompson — 2006-09-25T16:16:48-00:00

Nucleic Acids Res, Vol. 25, No. 24. (15 December 1997), pp. 4876-4882.

CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.

Geographical information systems and bootstrap aggregation (bagging) of tree-based classifiers for Lyme disease risk prediction in Trentino, Italian Alps.

A Rizzoli — 2005-04-28T09:22:23-00:00

J Med Entomol, Vol. 39, No. 3. (May 2002), pp. 485-492.

The risk of exposure to Lyme disease in the province of Trento, Italian Alps, was predicted through the analysis of the distribution of Ixodes ricinus (L.) nymphs infected with Borrelia burgdorferi s.l. with a model based on bootstrap aggregation (bagging) of tree-based classifiers within a geographical information system (GIS). Data on L ricinus density assessed by dragging the vegetation in 438 sites during 1996 were cross-correlated with the digital cartography of a GIS, which included the variables altitude, exposure and slope, substratum, vegetation type and roe deer density. Ticks were more abundant at altitudes below 1,300 m a.s.l., in the presence of limestone and vegetation cover with thermophile deciduous forests and high densities of roe deer. A bootstrap aggregation procedure (bagging) was used to produce a model for the prediction of tick occurrence, the accuracy of which was tested on actual tick counts assessed by a further dragging campaign carried out during 1997 to determine infection prevalence and resulted in average 77%. Other tests of the model were made on additional and independent data sets. The prevalence of infection with Borrelia burgdorferi s.l, determined by polymerase chain reaction on 2,208 nymphs collected by random dragging in 245 transects selected within eight areas where the model predicted the occurrence of I. ricinus during 1997, was 17.5% and was positively correlated to tick abundance and roe deer density. These findings were used to relate the output of the bagged model (probability of tick occurrence) to the density of infected nymphs through a stepwise model selection procedure and thus to produce a GIS digital map of the probability distribution of infected nymphs in the Province of Trento at high resolution scale (50 by 50-m cell resolution). The application of the bagging procedure increased the accuracy of the prediction made by a single classification tree, a well-known classification method for the analysis of epidemiological data.

Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses

Melik — 2007-06-07T16:15:12-00:00

Archives of Virology, Vol. 152, No. 5. (May 2007), pp. 1027-1034.

A real-time RT-PCR method for the universal detection and identification of flaviviruses.

G Moureau — 2008-03-12T09:57:52-00:00

Vector Borne Zoonotic Dis, Vol. 7, No. 4. (2007), pp. 467-477.

Here we describe an optimized molecular protocol for the universal detection and identification of flaviviruses. It combines the convenient real-time polymerase chain reaction (PCR) format with a broad spectrum of flavivirus detection. This assay, based on the amplification of a 269-272 nt (depending on the flavivirus tested) region at the N terminal end of the NS5 gene, enabled the amplification of 51 flavivirus species and 3 tentative species. Sequencing of the amplicons produced by reverse transcriptase (RT)-PCR permitted the reliable taxonomic identification of flavivirus species by comparison with reference sequences available in databases, using either the BLASTN algorithm or a simple phylogenetic reconstruction. The limit of detection of the assay (2-20,500 copies/reaction depending on the virus tested) allowed the detection of different flaviviruses from a series of human sera or veterinary samples. Altogether, the characteristics of this technique make it a good candidate for the identification of previously identified flaviviruses in cell culture and the investigation of field samples, and also a promising tool for the discovery and identification of new species, including viruses distantly related to "classical" arthropod-borne flaviviruses.

The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame.

TS Gritsun — 2008-03-12T07:09:59-00:00

Virology, Vol. 354, No. 1. (10 October 2006), pp. 217-223.

Comparative alignment of the 3'untranslated regions (3'UTRs) of tick-borne flaviviruses has previously revealed short direct repeat sequences about 25-70 nucleotides long [Gritsun, T.S., Venugopal, K., Zanotto, P.M., Mikhailov, M.V., Sall, A.A., Holmes, E.C., Polkinghorne, I., Frolova, T.V., Pogodina, V.V., Lashkevich, V.A., Gould, E.A., 1997. Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs. Virus Res. 49 (1) 27-39; Wallner, G., Mandl, C.W., Kunz, C., Heinz, F.X., 1995. The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus. Virology, 213 (1) 169-178]. We now show that these short sequences appear to have originated from longer repeat sequences (LRSs) that are present both in the 3'UTR and the open reading frame of the genome. We propose that the 3'UTR, and possibly the open reading frame, evolved through multiple duplications, deletions and mutations of a primordial sequence element.

Localized deer absence leads to tick amplification.

SE Perkins — 2008-03-09T19:47:28-00:00

Ecology, Vol. 87, No. 8. (August 2006), pp. 1981-1986.

Deer support high tick intensities, perpetuating tick populations, but they do not support tick-borne pathogen transmission, so are dilution hosts. We test the hypothesis that absence of deer (loss of a dilution host) will result in either an increase or a reduction in tick density, and that the outcome is scale dependent. We use a complementary methodological approach starting with meta-analysis, followed up by a field experiment. Meta-analysis indicated that larger deer exclosures reduce questing (host-seeking) tick density, but as the exclosure becomes smaller (<2.5 ha) the questing tick density is increased (amplified). To determine the consequences for tick-borne pathogen transmission we carried out a field experiment, comparing the intensity of ticks that fed on hosts competent for tickborne pathogen transmission (rodents) in two small (<1 ha) deer exclosures and their replicated controls. Intensity of larval ticks on rodents was not significantly different between treatments, but nymph intensity, the tick stage responsible for tick-borne encephalitis (TBE) transmission, was higher in deer exclosures. TBE seropositive rodents were found in a deer exclosure but not in the controls. We propose that localized absence of deer (loss of a dilution host) increases tick feeding on rodents, leading to the potential for tick-borne disease hotspots.