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      Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB

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          Abstract

          Background

          Tick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples ( n = 1979) collected from sheep ( n = 1727) and goats ( n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections.

          Results

          Overall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp.

          Conclusion

          The results indicated that pathogens causing TBHDs are highly prevalent in sheep and goats in Turkey. The diagnostic sensitivity of species-specific single PCR was generally higher than that of RLB. However, the latter approach was still capable of identifying a high proportion of individuals containing mixed-species infections. The use of species-specific single PCR is recommended to accurately estimate pathogen prevalence and to identify co-infected hosts.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13071-017-2151-3) contains supplementary material, which is available to authorized users.

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          Most cited references65

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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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            Multiplex PCR: critical parameters and step-by-step protocol.

            By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.
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              International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health.

              A general review is given of the tick-borne hemoparasitic diseases of greatest economic importance in ruminants, babesiosis, anaplasmosis, theileriosis and cowdriosis, each caused by one or more species of hemoparasites. Most affected are cattle and small ruminants, buffalo are more resistant and little is known regarding camels. The situation varies from one continent or region to another. Innate and breed susceptibility to these diseases are of tremendous importance. Disease in the field cannot be considered separated from the whole complex of tick-borne diseases and from the ticks themselves, particularly if the aim is to attain endemic stability. International coordination is needed now that research funds are scarce. An appendix contains tables with hemoparasites of various domestic animals and notes with background details.
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                Author and article information

                Contributors
                hbilgic@adu.edu.tr
                serkanbakirci@adu.edu.tr
                onrks@yahoo.com
                ahakanunlu@hotmail.com
                selin-uner@hotmail.com
                hasanerentr@yahoo.com
                Willie.Weir@glasgow.ac.uk
                tulinkaragenc@yahoo.com
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                27 April 2017
                27 April 2017
                2017
                : 10
                : 211
                Affiliations
                [1 ]ISNI 0000 0004 0595 4313, GRID grid.34517.34, Department of Parasitology, , University of Adnan Menderes, Faculty of Veterinary Medicine, ; 09016 Isıklı/Aydın, Turkey
                [2 ]GRID grid.411703.0, Department of Veterinary Medicine, , University of Yuzuncu Yil, Vocational high School of Gevas, Programme of Laboratorian and Veterinary Health, ; 65700 Van, Turkey
                [3 ]ISNI 0000 0001 2193 314X, GRID grid.8756.c, School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, , University of Glasgow, ; Bearsden Road, Glasgow, G61 1QH UK
                Article
                2151
                10.1186/s13071-017-2151-3
                5408456
                28449722
                8b68b97d-e335-4455-987f-2f71db69cfba
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 6 September 2016
                : 21 April 2017
                Funding
                Funded by: EU, FP7 Project
                Award ID: KBBE–3–245145-PIROVAC
                Award Recipient :
                Funded by: Adnan Menderes University, Scientific Research Programs (TR)
                Award ID: VTF–13001
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Parasitology
                prevalence,tick-borne pathogens,sheep,goat,rlb,species-specific pcr,turkey
                Parasitology
                prevalence, tick-borne pathogens, sheep, goat, rlb, species-specific pcr, turkey

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