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      Molecular Detection of Borrelia burgdorferi Sensu Lato and Anaplasma phagocytophilum in Ticks Collected from Dogs in Urban Areas of North-Eastern Poland

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          Abstract

          From 2016 to 2018, ticks were collected from 272 dogs admitted to veterinary clinics in the city of Olsztyn (north-eastern Poland). Among 522 collected ticks, 423 were identified as Ixodes ricinus (413 females and 10 males) and 99 as Dermacentor reticulatus (62 females and 37 males). Non-engorged (86 individuals) and engorged (436 individuals) ticks were screened for the presence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Borrelia and A. phagocytophilum species detection was determined based on the sequence of the fla B and 16S RNA genes, respectively. DNA of B. burgdorferi s.l. was identified in 31.6% (165/522, 95% CI: 27.6–35.8%) of ticks ( I. ricinus 151/423, 35.7%, 95% CI: 31.1–40.4%; D. reticulates 14/99, 14.1%, 95% CI: 7.9–22.6%). A. phagocytophilum was identified in 0.96% (5/522, 95% CI: 0.3–2.2%) of specimens. All positive samples were engorged I. ricinus females (5/402, 1.2%, 95% CI: 0.4–2.9%). In 85.4% (141/165, 95% CI: 79.1–90.4%) of Borrelia infected ticks, the DNA of one genospecies was revealed. The DNA of at least two different genospecies was detected in 14.5% of specimens (24/165, 95% CI: 9.5–20.8). The coexistence of B. burgdorferii s.l. and A. phagocytophilum was not detected.

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          Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

          The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.
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            Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health

            Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis,” Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.
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              Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.

              Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chaffeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.
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                Author and article information

                Journal
                Pathogens
                Pathogens
                pathogens
                Pathogens
                MDPI
                2076-0817
                09 June 2020
                June 2020
                : 9
                : 6
                : 455
                Affiliations
                [1 ]Department of Parasitology and Invasive Diseases, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-719 Olsztyn, Poland; michmm@ 123456uwm.edu.pl
                [2 ]Department of Medical Biology, Collegium Medicum, School of Public Health, University of Warmia and Mazury in Olsztyn, Zolnierska 14c, 10-561 Olsztyn, Poland; katarzyna.kubiak@ 123456uwm.edu.pl
                [3 ]Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland; magdalena.szczotko@ 123456uwm.edu.pl (M.S.); marta.chajecka@ 123456student.uwm.edu.pl (M.C.)
                Author notes
                [* ]Correspondence: m.dmit@ 123456uwm.edu.pl
                Author information
                https://orcid.org/0000-0001-8515-3054
                https://orcid.org/0000-0001-6400-8958
                https://orcid.org/0000-0001-8231-4182
                https://orcid.org/0000-0002-9986-3847
                Article
                pathogens-09-00455
                10.3390/pathogens9060455
                7350305
                32526836
                652ca6e8-6843-4d5c-9524-801f7ebaf557
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 08 May 2020
                : 07 June 2020
                Categories
                Article

                borrelia burgdorferi sensu lato,anaplasma phagocytophilum,ixodes ricinus,dermacentor reticulatus,ticks,dogs,urban areas

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