Ehrlichia-infected ticks on migrating birds.

During the spring of 1996, an estimated 581,395 Ehrlichia-infected ticks were imported into Sweden by migrating birds. Ehrlichia gene sequences found in ticks collected from these migrating birds were identical to those of granulocytic ehrlichiosis found in domestic animals and humans in Sweden. These findings support the idea that birds may play a role in dispersing Ehrlichia

Structure of the Expression Site Reveals Global Diversity in MSP2 (P44) Variants in Anaplasma phagocytophilum

Anaplasma phagocytophilum, a recently reclassified bacteria in the order Rickettsiales, infects many different animal species and causes an emerging tick-borne disease of humans. The genome contains a large number of related genes and gene fragments encoding partial or apparently full-length outer membrane protein MSP2 (P44). Previous data using strains isolated from humans in the United States suggest that antigenic diversity results from RecF-mediated conversion of a single MSP2 (P44) expression site by partially homologous donor sequences. However, whether similar mechanisms operate in naturally infected animal species and the extent of global diversity in MSP2 (P44) are unknown. We analyzed the structure and diversity of the MSP2 (P44) expression site in strains derived from the United States and Europe and from infections of different animal species, including wildlife reservoirs. The results show that a syntenic expression site is present in all strains of A. phagocytophilum investigated. This genomic locus contained diverse MSP2 (P44) variants in all infected animals sampled, and variants also differed at different time points during infection. Although similar variants were found among different populations of U.S. origin, there was little sequence identity between U.S. strain variants (including genomic copies from a completely sequenced U.S. strain) and expression site variants infecting sheep and dogs in Norway and Sweden. Finally, the possibility that combinatorial mechanisms can generate additional diversity beyond the basic donor sequence repertoire is supported by the observation of shared sequence blocks throughout the MSP2 (P44) hypervariable region in reservoir hosts. These data suggest similar genetic mechanisms for A. phagocytophilum variation in all hosts but worldwide diversity of the MSP2 (P44) outer membrane protein

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen

Top-Down-Assisted Bottom-Up Method for Homologous Protein Sequencing: Hemoglobin from 33 Bird Species

Ticks are vectors for disease transmission because they are indiscriminant in their feeding on multiple vertebrate hosts, transmitting pathogens between their hosts. Identifying the hosts on which ticks have fed is important for disease prevention and intervention. We have previously shown that hemoglobin (Hb) remnants from a host on which a tick fed can be used to reveal the host's identity. For the present research, blood was collected from 33 bird species that are common in the U.S. as hosts for ticks but that have unknown Hb sequences. A top-down-assisted bottom-up mass spectrometry approach with a customized searching database, based on variability in known bird hemoglobin sequences, has been devised to facilitate fast and complete sequencing of hemoglobin from birds with unknown sequences. These hemoglobin sequences will be added to a hemoglobin database and used for tick host identification. The general approach has the potential to sequence any set of homologous proteins completely in a rapid manner. Graphical Abstract ᅟ

Prevalence of <it>Borrelia burgdorferi </it>sensu lato and <it>Anaplasma phagocytophilum </it>in questing <it>Ixodes ricinus </it>ticks in relation to the density of wild cervids

<p>Abstract</p> <p>Background</p> <p><it>Borrelia burgdorferi </it>sensu lato and <it>Anaplasma phagocytophilum </it>have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing <it>Ixodes ricinus </it>ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.</p> <p>Methods</p> <p>For detection of <it>Anaplasma phagocytophilum </it>a 77-bp fragment in the <it>msp</it>2 gene was used. Detection of <it>Borrelia burgdorferi </it>sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the <it>fla </it>gene. The <it>Osp</it>A gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for <it>Borrelia burgdorferi </it>sensu stricto, <it>B. afzelii </it>and <it>B. garinii</it>.</p> <p>Results</p> <p>Infection rates with <it>Borrelia </it>spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ²= 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ²= 24.04, p < 0.0001); Strøm vs. Tjore (χ²= 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ²= 16.63, p < 0.0001). The <it>Borrelia </it>genospecies were dominated by. <it>B. afzelii </it>(82%) followed by <it>B. garinii </it>(9.7%) and <it>B. burgdorferi </it>sensu stricto (6.9%). <it>B. burgdorferi </it>s.s. was only found on the island of Jomfruland. The infection rate of <it>Anaplasma phagocytophilum </it>showed the following figures; Fjelløyvær vs Hinnebu (χ²= 16.27, p = 0.0001); Strøm vs. Tjore (χ²= 13.16, p = 0.0003); Strøm vs. Hinnebu (χ²= 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ²= 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ²= 5.06, p = 0.0245). Wild cervids may serve as a reservoir for <it>A. phagocytophilum</it>. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. <it>burgdorferi </it>s.l. and <it>A. phagocytophilum </it>in questing <it>I. ricinus </it>ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.</p> <p>Conclusion</p> <p>The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of <it>Borrelia burgdorferi </it>sensu lato in host seeking <it>Ixodes ricinus</it>, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of <it>Anaplasma phagocytophilum </it>showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.</p