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An OspA antigen-capture enzyme-linked immunosorbent assay for detecting North American isolates of Borrelia burgdorferi in larval and nymphal Ixodes dammini.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed for detecting North American isolates of Borrelia burgdorferi in larval, nymphal, and adult ticks. The assay uses an anti-OspA monoclonal antibody (H5332) for antigen capture and biotin-labelled polyclonal sera with streptavidinhorseradish peroxidase for signal generation. The assay recognized 15 of 15 North American B. burgdorferi isolates and did not cross-react with spirochete antigens of Borrelia hermsii, Borrelia turicatae, Borrelia coriaceae, or Borrelia parkeri, or with tick antigens of Ixodes dammini, Ixodes scapularis, Ixodes pacificus, Ixodes cookei, Ixodes angustus, or Ambylomma americanum. The assay, with a sensitivity of less than 150 spirochetes, can detect infections in larval, nymphal, and adult ticks. In addition to fresh ticks, B. burgdorferi infections in ticks stored frozen, dried, or in 70% ethanol can be determined with the assay.

Animals↗

Ixodes (Haemixodes) longiscutatum Boero (new status) and I. (H.) uruguayensis Kohls & Clifford, a new synonym of I. (H.) longiscutatum (Acari: Ixodidae).

Females of Ixodes (Haemixodes) uruguayensis Kohls & Clifford, 1967, a species whose adults were unknown until the present, were obtained in the laboratory from engorged nymphs collected on rodents (Scapteromys tumidus and Oxymycterus nasutus) in the counties of Maldonado and San José, Uruguay. Morphological characters of these females were identical to those given in the description of the female of Ixodes longiscutatum Boero, 1944. I. uruguayensis is, thus, relegated to a junior subjective synonym of I. longiscutatum. However, because of the unique morphological characters of the immature stages, the validity of the subgenus Haemixodes Kohls & Clifford, 1967 is not questioned. Therefore, the new status of Ixodes (Haemixodes) uruguayensis Kohls & Clifford, 1967 is Ixodes (Haemixodes) longiscutatum Boero, 1944.

Animals↗

Host associations and seasonal occurrence of Haemaphysalis leporispalustris, Ixodes brunneus, I. cookei, I. dentatus, and I. texanus (Acari: Ixodidae) in Southeastern Missouri.

Hemaphysalis leporispalustris (Packard), Ixodes brunneus Koch, Ixodes cookei Packard, Ixodes dentatus Marx, and Ixodes texanus Banks were collected during a 3-yr study of pathogen-tick-host interactions in southeastern Missouri. H. leporispalustris was collected from the eastern cottontail rabbit, Northern bobwhite, and Carolina wren, and it was active all year. I. brunneus was collected by drag and from passerine birds during December, March, and April. I. cookei was collected from raccoons and mink during April, June, September, October, and November. I. dentatus was collected from the cottontail rabbit and Carolina wren throughout the year. I. texanus was collected from the eastern gray squirrel, Virginia opossum, and raccoon throughout the year.

Animals↗

Molecular phylogenetic analyses indicate that the Ixodes ricinus complex is a paraphyletic group.

The Ixodes ricinus species complex is a group of ticks distributed in almost all geographic regions of the world. Lyme borreliosis spirochetes are primarily transmitted by tick species within this complex. It has been hypothesized that the Lyme vector ticks around the world are closely related and represent a monophyletic group. This implies that vector competence in ixodid ticks for Lyme agents might have evolved only once. To test this hypothesis, we used a molecular phylogenetic approach. Two fragments of mitochondrial 16S ribosomal deoxyribonucleic acid were sequenced from 11 species in the I. ricinus complex and from 16 other species of Ixodes. Phylogenetic analysis using Bayesian methodology indicated that the I. ricinus complex is not a monophyletic group unless 3 additional Ixodes species are included in it. The known major vectors of Lyme disease agents in different areas of the world are not sister taxa. This suggests that acquisition of the ability to transmit borreliosis agents in species of Ixodes may have multiple origins.

Animals↗

Parasitization of humans in West Virginia by Ixodes cookei (Acari: Ixodidae), a potential vector of Lyme borreliosis.

In 32 collections, two larvae, 33 nymphs, and one adult female Ixodes cookei Packard were collected from humans in West Virginia from August 1987 to May 1990. Most were attached. The ticks were found in 14 counties and were the most abundant Ixodes found biting humans. One nymphal I. cookei was removed from the left axilla of a 39-yr-old woman who lives and works in Monongalia and Marion counties, W. Va. The bite was the center of an expanding erythematous lesion reaching 4 cm in diameter, clearing centrally, and typical of erythema migrans. This association and the near absence of Ixodes dammini Spielman, Clifford, Piesman & Corwin from the state suggests the possibility that I. cookei may be an important vector of Lyme borreliosis in West Virginia. In five separate collections, five nymphal Ixodes dentatus Marx were removed from humans in four counties, implicating this species as a potential minor vector of Lyme borreliosis in West Virginia.

Adult↗

Seasonal correlation of sporadic schizophrenia to Ixodes ticks and Lyme borreliosis.

BACKGROUND: Being born in winter and spring is considered one of the most robust epidemiological risk factors for schizophrenia. The aetiology and exact timing of this birth excess, however, has remained elusive so far. Since during phylogeny, Borrelia DNA has led to multiple germ-line mutations within the CB1 candidate gene for schizophrenia, a meta analysis has been performed of all papers on schizophrenic birth excesses with no less than 3000 cases each. All published numerical data were then plotted against the seasonal distributions of Ixodes ticks worldwide. RESULTS: In the United States, Europe and Japan the birth excesses of those individuals who later in life develop schizophrenia mirror the seasonal distribution of Ixodes ticks nine months earlier at the time of conception. South of the Wallace Line, which limits the spread of Ixodes ticks and Borrelia burgdorferi into Australia, seasonal trends are less significant, and in Singapore, being non-endemic for Ixodes ticks and Lyme disease, schizophrenic birth excesses are absent. CONCLUSION: At present, it cannot be excluded that prenatal infection by B. burgdorferi is harmful to the implanting human blastocyst. The epidemiological clustering of sporadic schizophrenia by season and locality rather emphasises the risk to the unborn of developing a congenital, yet preventable brain disorder later in life.

Journal Article↗

The phylogenetic position of Ixodes stilesi Neumann, 1911 (Acari: Ixodidae): morphological and preliminary molecular evidences from 16S rDNA sequences.

The female of Ixodes stilesi Neumann, 1911 (Acari Ixodidae) is redescribed and the male and nymph are described from specimens collected from Pudu puda (Molina) (Artiodactyla: Cervidae) in Chile. Both sexes of I. stilesi have characteristics of the subgenera Ixodes Latreille, 1795 and Ixodiopsis Filippova, 1957. The females of I. stilesi are peculiar in having the combination of the sinuous scutum outline, rounded porose areas with distinct borders separated by the width of one area, slender and long palpi, and two subequal spurs on coxa I. The male is unique in having a combination of a posteriorly wrinkled marginal folder, a basis capituli longer than wide, a non-crenulate hypostome toothed portion, two spurs on coxa II to IV and the presence of a pseudoscutum. The nymph of I. stilesi has blunt anterior and posterior processes on palpal article I (characteristics of the subgenus Ixodiopsis and some Pholeoixodes Schulze, 1942) and a wing-shaped basis capituli with a prominent triangular cornua. Phylogenetic analyses based on 16S mitochondrial rDNA sequences of 12 Neotropical and two Australian Ixodes species, plus three argasids, were carried out to clarify the position of I. stilesi. The results of phylogenetic analyses and morphological characters indicate a close relationships between I. stilesi and two other Neotropical species of uncertain subgeneric status, I. neuquenensis Ringuelet, 1947 and I. sigelos Keirans, Clifford & Corwin, 1976.

Animals↗

Detection of Borrelia garinii, Borrelia tanukii and Borrelia sp. closely related to Borrelia valaisiana in Ixodes ticks removed from dogs and cats in Japan.

Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.

Animals↗

Population genetic structure of Ixodes ricinus in Switzerland from allozymic data: no evidence of divergence between nearby sites.

Ixodes ricinus is a vector and reservoir of numerous infectious agents, especially Borrelia burgdorferi, the agent of Lyme disease. In Switzerland, its ecology and physiology have been well studied. Moreover, the foci of some infectious agents transmitted by this tick are identified. They can display relatively to extremely small geographical size depending on the diseases considered. In order to understand how the gene flows occur and to characterise the genetic structure of Ixodes ricinus populations, we used an indirect method based on genetic markers: allozymes. The sampling was carried out in 5 localities. Eighteen loci were analysed and 2 appeared polymorphic. This shows the low allozymic variability displayed by Ixodes ricinus. Based on these 2 loci, the populations appeared panmictic in Switzerland. This may be explained by the wide range of vertebrate species this tick can infest, especially birds. However, the result is surprising if we consider the extreme localisation of the foci of some infectious agents. We conclude that more powerful genetic markers could be used in order to better understand the epidemiology of tick-borne diseases in Switzerland.

Alleles↗

Description of the nymph and larva and redescription of the female of Ixodes neuquenensis Ringuelet, 1947 (Acari: Ixodidae), a parasite of the endangered Neotropical marsupial Dromiciops gliroides Thomas (Microbiotheria: Microbiotheriidae).

The female of Ixodes neuquenensis Ringuelet, 1947 (Acari: Ixodidae) is redescribed and the nymph and larva are described from specimens collected from the endangered marsupial Dromiciops gliroides Thomas (Microbiotheria: Microbiotheriidae) in Argentina. At first sight the female of I. neuquenensis resembles a member of the subgenus Ixodes Latreille, 1795. However, the female of I. neuquenensis is peculiar in having the combination of two spurs on coxae II-IV and a pair of chitinous plaques internal to coxa I. Both the nymph and larva have an anterior and posterior process on palpal article I, characteristics of the subgenus Ixodiopsis Filippova, 1957 and some representatives of the subgenus Pholeoixodes Schulze, 1942. Analysis of 16S mitochondrial rDNA sequences showed no strong relationship with any known Ixodes subgenus.

Animals↗

Mitochondrial DNA sequence variation in ixodes pacificus (Acari: ixodidae).

The western black-legged tick, Ixodes pacificus, is a primary vector of the spirochaete, Borrelia burgdorferi, that causes Lyme disease. We used variation in a 355-bp DNA portion of the mitochondrial cytochrome oxidase III gene to assess the population structure of the tick across its range from British Columbia to southern California and east to Utah. Ixodes pacificus showed considerable haplotype diversity despite low nucleotide diversity. Maximum parsimony and isolation-by-distance analyses revealed little genetic structure except between a geographically isolated Utah locality and all other localities. Loss of mtDNA polymorphism in Utah ticks is consistent with a post-Pleistocene founder event. The pattern of genetic differentiation in the continuous part of the range of Ixodes pacificus reinforces recent recognition of the difficulties involved in using genetic frequency data to infer gene flow and migration.

Animals↗

Epidemiology and impact of coinfections acquired from Ixodes ticks.

Ixodes scapularis and other ticks in the Ixodes ricinus complex may transmit multiple pathogens, but research on coinfections has been limited. Coinfections occur with varying frequency in ticks, but single infections are more common than dual infections. The proportion of I. scapularis or I. ricinus ticks coinfected with both Borrelia burgdorferi sensu lato and Anaplasma phagocytophila is generally low, ranging from < 1% to 6% in six geographic areas. A higher prevalence of tick coinfection (26%) has been reported in Westchester County, New York. Genetic variants of the human disease-causing strain of A. phagocytophila are present in some tick populations, and they may affect the risk of coinfection or clinical illness. The proportion of Ixodes ticks coinfected with B. burgdorferi and Babesia microti has ranged from 2% in New Jersey to 19% on Nantucket Island, Massachusetts. In humans, cross-sectional seroprevalence studies have found markers of dual infection in 9-26% of patients with a tick-borne infection, but such studies often fail to distinguish simultaneous coinfection from sequential infections. Several studies have prospectively assessed the occurrence of acute coinfection. Among patients with a confirmed tick-borne infection, coinfection rates as high as 39% have been reported. The most commonly recognized coinfection in most of the eastern United States is Lyme borreliosis (LB) and babesiosis, accounting for approximately 80% of coinfections. LB and human granulocytic ehrlichiosis coinfections are less common, occurring in 3-15% of patients with a tick-borne infection in Connecticut or Wisconsin. Studies of clinical outcomes suggest that patients with acute Babesia coinfection have more severe symptoms and a longer duration of illness than patients with LB alone, but the risk of spirochete dissemination is similar. Coinfections can modify the immune response and alter the severity of arthritis in animal models. Future coinfection research should focus on long-term clinical outcomes, the role of genetic variants, immunologic effects, and the potential role of Bartonella species as tick-borne pathogens.

Animals↗

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks.

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.

Administration, Oral↗

Taxonomic status of Ixodes neotomae and I. spinipalpis (Acari: Ixodidae) based on mitochondrial DNA evidence.

Ixodes spinipalpis Hadwen & Nuttall and I. neotomae Cooley are enzootic vectors of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner in western North America. The taxa overlap in host preference, habitat, and morphology. Mitochondrial DNA was compared between the taxa to test for reproductive isolation. A 300-bp region of the mitochondrial 16S ribosomal DNA gene was amplified in 28 specimens of I. neotomae and 149 specimens of I. spinipalpis. These products were screened for sequence variation using single-strand conformation polymorphism analysis, and 9 haplotypes were detected. Haplotype frequencies varied between taxa; however, Shannon diversity analysis indicated that most variation arose among collections within each taxon, and no unique haplotypes characterized either one. Phylogenetic analysis of 18 sequences, representing a replicate of each of the 9 haplotypes, was performed with Ixodes pacificus Cooley & Kohls and Ixodes jellisoni Cooley & Kohls as outgroups. Strong monophyletic support was found for a clade containing I. neotomae and I spinipalpis and within this clade no lineages comprised exclusively either taxon. These results argue against treatment of I. neotomae and I. spinipalpis as distinct species.

Animals↗

Tick-borne borrelioses pathogen identification in Ixodes ticks (Acarina, Ixodidae) collected in St. Petersburg and Kaliningrad Baltic regions of Russia.

Two isolated Baltic seashore populations of Ixodes ticks were studied as vectors of different Borrelia genospecies in Russia by using darkfield microscopy and modified polymerase chain reaction (PCR). In the Kalinigrad region (Kurish Spit, forests near the settlements of Lesnoye and Rybachy), 788 Ixodes ricinus (L.) adults and nymphs were collected by flagging and studied by darkfield microscopy during 1995-1996. There were 88 darkfield microscopy positive specimens (11.2%) of which 69 were also analyzed by PCR. Borrelia afzelii and B. garinii were found individually and together in ticks. In this region, on the Kurish Spit, 7 patients with tick borrelioses were observed: 2 in the Russian part of Spit and 5 in the Lithuanian part. A significant difference was found between Borrelia prevalence during the spring and fall peaks of tick abundance. Specimens that were darkfield microscopy positive prevailed in the fall (25.15%) in comparison with the spring peak (7.3%). The number of specimens with identified genospecies prevailed in the spring: 22 out of 35 versus 4 out of 31 in the fall. Among 29 PCR positive I. ricinus, 21 contained B. afzelii, 3 had B. garinii, and 2 had dual infection. In 1995, only B. afzelii infected specimens were observed. In the vicinity of St. Petersburg (the seashore of the northern Gulf of Finland, in forests near Lisy Nos, Morskaja) during 1992-1996, 31 patients with a tick-borne borrelioses were registered. We collected 487 Ixodes persulcatus Schulze by flagging and studied them by darkfield microscopy in 1995-1996 of which 144 ticks (29.6%) were darkfield microscopy positive. Sixty darkfield-positive specimens were analyzed by PCR, and in 88.3% of cases genospecies were identified. B. afzelii and B. garinii were identified individually and together in ticks. In 1995, I. persulcatus with dual infection prevailed with 11 out of 21 (52.4% positive), whereas in 1996, most I. persulcatus ticks contained B. garinii (81.2%). Dual infection was observed in 4 of 32 (12.5%) ticks. Dual infections in I. persulcatus females increased within the seasonal peak of tick activity as was observed in 1995 and in 1996. Many patients not only had erythema migrans, but also exhibited early neurological symptoms that coincided with the number of tick vectors that had dual infections in June, indicating that these patients were bitten by female ticks that had dual infections. A significant difference existed between levels of infection in I. ricinus and I. persulcatus, with all 3 types of Borrelia infection observed 2 times more often in I. persulcatus than in I. ricinus and dual infection occurred in I. persulcatus 3.7 times more often. It appeared that I. persulcatus is a much more dangerous vector of tick-borne borrelioses than I. ricinus.

Animals↗

Evolution of duplicate control regions in the mitochondrial genomes of metazoa: a case study with Australasian Ixodes ticks.

To investigate the evolution pattern and phylogenetic utility of duplicate control regions (CRs) in mitochondrial (mt) genomes, we sequenced the entire mt genomes of three Ixodes species and part of the mt genomes of another 11 species. All the species from the Australasian lineage have duplicate CRs, whereas the other species have one CR. Sequence analyses indicate that the two CRs of the Australasian Ixodes ticks have evolved in concert in each species. In addition to the Australasian Ixodes ticks, species from seven other lineages of metazoa also have mt genomes with duplicate CRs. Accumulated mtDNA sequence data from these metazoans and two recent experiments on replication of mt genomes in human cell lines with duplicate CRs allowed us to re-examine four intriguing questions about the presence of duplicate CRs in the mt genomes of metazoa: (1) Why do some mt genomes, but not others, have duplicate CRs? (2) How did mt genomes with duplicate CRs evolve? (3) How could the nucleotide sequences of duplicate CRs remain identical or very similar over evolutionary time? (4) Are duplicate CRs phylogenetic markers? It appears that mt genomes with duplicate CRs have a selective advantage in replication over mt genomes with one CR. Tandem duplication followed by deletion of genes is the most plausible mechanism for the generation of mt genomes with duplicate CRs. Once duplicate CRs occur in an mt genome, they tend to evolve in concert, probably by gene conversion. However, there are lineages where gene conversion may not always occur, and, thus, the two CRs may evolve independently in these lineages. Duplicate CRs have much potential as phylogenetic markers at low taxonomic levels, such as within genera, within families, or among families, but not at high taxonomic levels, such as among orders.

Animals↗

Determination of members of a Borrelia afzelii-related group isolated from Ixodes nipponensis in Korea as Borrelia valaisiana.

The 16S rRNA sequences of the Korean Borrelia strains 5MT and 9MT, isolated from Ixodes nipponensis, showed identities of 99.0-99.1% to that of B. afzelii. The strains were tentatively classified as belonging to the B. afzelii-related group. In this study, Korean isolates, including these strains, were characterized further and compared with recently described new species. These strains generated a RFLP pattern that has not been found previously in RFLP analysis of the 5S-23S rRNA intergenic spacer and the flagellin gene. When phylogenetic trees were constructed, based on the 5S-23S rRNA intergenic spacer, flagellin gene and 16S rRNA sequences, these Korean isolates formed a cluster with the Borrelia strain Am501 isolated from Ixodes columnae in Japan and Borrelia valaisiana strains VS116T and UK isolated from Ixodes ricinus in Europe and were distinguishable from the other species. However, these three groups of strains were divergent from each other in the molecular masses of the putative outer surface protein A (OspA) and in the sequences of the ospA gene. These findings suggest that these Korean isolates and one Japanese isolate are members of B. valaisiana and that OspA of this species is divergent, as is that of Borrelia garinii. This led to the speculation that B. valaisiana strains are adapted to the vector ticks found in each locality.

Animals↗

Ultrastructure and phylogenetic analysis of 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks.

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.

Anaplasmataceae↗