RESUMO
Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called 'Candidatus Cryptoplasma californiense'. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.
Assuntos
Anaplasmataceae/classificação , Anaplasmataceae/isolamento & purificação , Biodiversidade , Ixodes/microbiologia , Anaplasmataceae/genética , Anaplasmataceae/fisiologia , Animais , California , Filogenia , Rickettsia/isolamento & purificação , Rickettsia/fisiologia , Análise de Sequência de DNA , SimbioseRESUMO
BACKGROUND: Lyme disease, caused by various species of Borrelia, is transmitted by Ixodes ticks in North America and Europe. Studies have shown the genotype of Borrelia burgdorferi sensu stricto (s.s.) or the species of B. burgdorferi sensu lato (s.l.) affects the ability of the bacteria to cause local or disseminated infection in humans. METHODOLOGY/PRINCIPAL FINDINGS: We used a multilocus PCR electrospray mass spectrometry assay to determine the species and genotype Borrelia from ticks collected in New York, Connecticut, Indiana, Southern Germany, and California and characterized isolates from parts of the United States and Europe. These analyses identified 53 distinct genotypes of B. burgdorferi sensu stricto with higher resolution than ospC typing. Genotypes of other members of the B. burgdorferi sensu lato complex were also identified and genotyped including B. afzelii, B. garinii, B. lusitaniae, B. spielmanii, and B. valaisiana. While each site in North America had genotypes unique to that location, we found genotypes shared between individual regions and two genotypes found across the United States. Significant B. burgdorferi s.s. genotypic diversity was observed between North America and Europe: only 6.6% of US genotypes (3 of 45) were found in Europe and 27% of the European genotypes (3 of 11) were observed in the US. Interestingly, 39% of adult Ixodes scapularis ticks from North America were infected with more than one genotype of B. burgdorferi s.s. and 22.2% of Ixodes ricinus ticks from Germany were infected with more than one genotype of B. burgdorferi s.l. CONCLUSIONS/SIGNIFICANCE: The presence of multiple Borrelia genotypes in ticks increases the probability that a person will be infected with more than one genotype of B. burgdorferi, potentially increasing the risks of disseminated Lyme disease. Our study indicates that the genotypic diversity of Borrelia in ticks in both North America and Europe is higher then previously reported and can have potential clinical consequences.
Assuntos
Borrelia/genética , Variação Genética , Ixodes/microbiologia , Doença de Lyme/microbiologia , Doença de Lyme/parasitologia , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Técnicas de Tipagem Bacteriana , Borrelia/classificação , Borrelia/isolamento & purificação , Europa (Continente) , Loci Gênicos/genética , Genótipo , Geografia , Humanos , América do Norte , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.
Assuntos
DNA/genética , RNA/genética , Carrapatos/genética , Carrapatos/microbiologia , Carrapatos/virologia , Animais , Borrelia/genética , Borrelia/isolamento & purificação , DNA/isolamento & purificação , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças Transmitidas por Carrapatos/genética , Doenças Transmitidas por Carrapatos/prevenção & controleRESUMO
OBJECTIVE: Infection with simian immunodeficiency virus (SIV), like HIV, can lead to central nervous system (CNS) abnormalities. One of the alterations observed in the brain is the accumulation of highly activated CD8 lymphocytes that, while fighting the infection, may cause tissue damage. In order to determine whether these CD8 cells in the brain comprise a distinct clonal population the expression of T-cell receptor (TCR) genes of two SIV-infected monkeys with CNS abnormalities were analyzed, comparing brain to periphery. METHODS: RNA from magnetically sorted CD8+ cells obtained from the brain, blood, lymph nodes, and spleen was analyzed for the distribution of 24 Vbeta family genes by reverse transcriptase-polymerase chain reaction followed by Southern blot. The CDR3 region of the most enriched family in each brain was sequenced in all the sites for comparison. RESULTS: The pattern of Vbeta distribution in the brain and the periphery was polyclonal, but an increase in certain Vbeta families was found in the brain, suggesting that regional mechanisms participate in the determination of the local clonal specificities. The sequence of the CDR3 domain of predominant Vbeta families in the brain revealed that approximately one-third of the CD8 cells were not identified in the periphery. CONCLUSION: CD8 cells in the brain exhibit a distinct clonal repertoire. This distinction may have implications for regional immunity, regulation, or selection of site-specific viral mutants.
