Distribution of Ixodes ricinus L. ticks and prevalence of their endoparasites in Lithuania and its determinant factors ; Erkių Ixodes ricinus L. ir jų platinamų endoparazitų paplitimas Lietuvoje bei jį lemiantys veiksniai
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Distribution of Ixodes ricinus L. ticks and prevalence of their endoparasites in Lithuania and its determinant factors ; Erkių Ixodes ricinus L. ir jų platinamų endoparazitų paplitimas Lietuvoje bei jį lemiantys veiksniai

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VYTAUTAS MAGNUS UNIVERSITY LITHUANIAN FOREST RESEARCH INSTITUTE Daiva Ambrasien ė DISTRIBUTION OF IXODES RICINUS L. TICKS AND PREVALENCE OF THEIR ENDOPARASITES IN LITHUANIA AND ITS DETERMINANT FACTORS Summary of Doctoral Dissertation Biomedical Sciences, Ecology and Environmental Sciences (03 B) Kaunas, 2007 1The right of doctoral studies was granted to Vytautas Magnus University jointly with Lithuanian Forest Research Institute on July 15, 2003, by the decision No.926 of the Government of the Republic of Lithuania. Dissertation was carried out at Vytautas Magnus University in 2002-2007. Scientific Supervisor: Assoc. prof. dr. Algimantas Paulauskas (Vytautas Magnus University, Biomedical Sciences, Biology – 01 P) The Committee of the doctoral dissertation: Chairman: Prof. habil. dr. Regina Gražulevi čien ė (Vytautas Magnus University, Biomedical Sciences, Ecology and Environmental Sciences – 03 B) Members: Dr. Dalius Butkauskas (Institute of Ecology of Vilnius University, Biomedical nvironmental Sciences – 03 B) Prof. habil. dr. Romualdas Juknys (Vytautas Magnus University, Biomedical Sciences, Ecology and EnvironmJuozas Rimantas Lazutka (Vilnius University, Biomedical Sciences, Biology – 01 B) Prof. habil. dr. Remigijus Ozolin čius (Lithuanian Forest Research institute, Biomedical Sciences, Ecology and Environmental Sciences – 03 B) Opponents: Assoc. prof. dr.

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VYTAUTAS MAGNUS UNIVERSITY LITHUANIAN FOREST RESEARCH INSTITUTE Daiva AmbrasienDISTRIBUTION OFIXODES RICINUSL. TICKS AND PREVALENCE OF THEIR ENDOPARASITES IN LITHUANIA AND ITS DETERMINANT FACTORS
Summary of Doctoral Dissertation Biomedical Sciences, Ecology and Environmental Sciences (03 B)
Kaunas, 2007
The right of doctoral studies was granted to Vytautas Magnus University jointly with Lithuanian Forest Research Institute on July 15, 2003, by the decision No.926 of the Government of the Republic of Lithuania. Dissertation was carried out at Vytautas Magnus University in 2002-2007. Scientific Supervisor: Assoc. prof. dr.Algimantas Paulauskas Magnus University, Biomedical (Vytautas Sciences, Biology  01 P) The Committee of the doctoral dissertation: Chairman: Prof. habil. dr.Regina Graulevičien(Vytautas Magnus University, Biomedical Sciences, Ecology and Environmental Sciences  03 B) Members: Dr.Dalius Butkauskas (Institute of Ecology of Vilnius University, Biomedical Sciences, Ecology and Environmental Sciences  03 B) Prof. habil. dr.Romualdas Juknys (Vytautas Magnus University, Biomedical Sciences, Ecology and Environmental Sciences  03 B) Prof. habil. dr.Juozas Rimantas Lazutka(Vilnius University, Biomedical Sciences, Biology  01 B) Prof. habil. dr.Remigijus Ozolinčius(Lithuanian Forest Research institute, Biomedical Sciences, Ecology and Environmental Sciences  03 B) Opponents: Assoc. prof. dr.Artras Gedminas (Lithuanian Forest Research institute, Biomedical Sciences, Forestry  014 B) Prof. habil. dr.Aniolas Sruoga (Institute of Ecology of Vilnius University, Biomedical Sciences, Ecology and Environmental Sciences  03 B) The Doctoral Dissertation will be defended on the 30th of October 2007 at 2 p.m. in a public meeting of the Doctoral committee at Vytautas Magnus University, Faculty of Natural Science, VincasČepinskis Sciences Reading Room (No 605).  Address: Vileikos st. 8, LT-44404, Kaunas, Lithuania The Summary of Doctoral Dissertation was distributed on the 28th of September 2007. The Dissertation can be reviewed at M. Mavydas National Library of Lithuania and at the Libraries of Vytautas Magnus University and Lithuanian Forest Research Institute.
VYTAUTO DIDIOJO UNIVERSITETAS LIETUVOS MIKINSTITUTAS Daiva AmbrasienERKIIXODES RICINUSL. IR JPLATINAMENDOPARAZITPAPLITIMAS LIETUVOJE BEI JLEMIANTYS VEIKSNIAI
Daktaro disertacijos santrauka Biomedicinos mokslai, ekologija ir aplinkotyra (03 B)
Kaunas, 2007
Disertacija rengta 2002-2007 metais Vytauto Didiojo universitete Mokslinis vadovas: Doc. dr.Algimantas Paulauskass,taioberivtesisidemonicidDtoauunoojityV(mokslai, biologija  01 B) Disertacija ginama Vytauto Didiojo universiteto Ekologijos ir aplinkotyros mokslo krypties taryboje: Pirminink: Prof. habil. dr.Regina Graulevičienauyt(ViidDtonuvijooetatreiss,biomedicinos mokslai, ekologija ir aplinkotyra  03 B) Nariai: Dr.Dalius Butkauskas(Vilniaus universiteto Ekologijos institutas, biomedicinos mokslai, ekologija ir aplinkotyra 03 B) Prof. habil. dr.Romualdas Juknys(Vytauto Didiojo universitetas, biomedicinos mokslai, ekologija ir aplinkotyra  03 B) Prof. habil. dr.Juozas Rimantas Lazutka(Vilniaus universitetas, biomedicinos mokslai, biologija  01 B) Prof. habil. dr.Remigijus Ozolinčius(Lietuvos mik biomedicinos institutas, mokslai, ekologija ir aplinkotyra  03 B) Oponentai: Doc. dr.Artras Gedminas mik (Lietuvos biomedicinos mokslai, institutas, mikotyra  014 B) Prof. habil. dr.Aniolas Sruoga (Vilniaus universiteto Ekologijos institutas, biomedicinos mokslai, ekologija ir aplinkotyra  03 B) Disertacija bus ginama vieame Ekologijos ir aplinkotyros mokslo krypties tarybos posdyje2007 m. spalio 30 d. 14 val. Vytauto Didiojo universiteto II rmuose, Vinco Čepinskio tikslijmokslskaitykloje (605 kab.)  Adresas: Vileikos g. 8, LT-44404, Kaunas, Lietuva Disertacijos santrauka isiuntinta 2007 m. rugsjo 28 d. Disertacijąigalimaperrti Lietuvos nacionalinje M. Mavydo bibliotekoje, Vytauto DidiojouniversitetoirLietuvosmikinstituto bibliotekose.
INTRODUCTIONDuring past few decades well-marked developments are observed in communities of animals and insects due to climate change, growing migration and increased anthropogenic pressure (alakevičius, 2001; alakevičius, 2007). A change of components within a community provokes constituent changes of communities. Due to climate change, marked developments are tracked in various links of pathogen-host distribution. Markedly increased number of ectoparasites is observed in Central and Eastern Europe (Rogers and Randolph, 2006; Eisen, 2007). The World Health Organization (WHO) forecasts the continued increase of parasitosis in the forthcoming decades due to the high prevalence of parasites with various genotypes, resistant to medicine and not relevant to locality  imported forms. Growing migration and increased level of environmental pollution negatively affect human immune system, changes of societal norms, climate change, risk behaviour (drug abuse, commercial sex work, HIV and its consequences). As a result they create increased demands for diagnostics of infectious diseases parasites and prevention of diseases in national locations and worldwide. Ticks are blood feeding wide group of arthropods of utmost medical, epidemiological and veterinary significance throughout the world. They are obligatory external parasites for every class of vertebrate and may bite people. Therefore from the beginning of the 20thcentury, ticks have been described as vectors of diseases, including bacterial (spotted fever rickettsioses, recurrent fever borreliosis, tularemia, Q fever et. al.), viral and protozoan zoonoses. (Wang et al., 1999; Parola and Raoult, 2001a; Herwaldtet al.,2003;Brouqui et al., 2004). Differences among pathogenic organisms are determined by different natural conditions, influencing prevalence and biodiversity of main vectors and reservoir hosts. Climate, landscape, geo-botanic conditions are favourable to existence and development for Ixodidae ticks in Lithuania.Ixodes ricinus, Ixodes trianguliceps,Dermacentor pictus,Dermacentor marginatusticks are prevalent in Lithuanian territory (ygutien, 2000; ygutienIt is estimated, that 10 % of, 2001). prevalent ticks in Lithuania are infected with pathogens.Ixodes ricinusspecies usually participates in the transmission of Lyme borreliosis, tick-born encephalitis and other infectious diseases. The ecology of ticks, the outcome of their interactions with their natural environment, is fundamental to the spatial and temporal variation in the risk of infection by tick-borne pathogens. The zoonotic tick-born diseases form a large proportion of the emerging bacterial infectious diseases. The most prominent of these diseases are Lyme disease, tick-borne encephalitis, erlichiosis, babesiosis and etc. Three protagonists are implicated in this tick-borne disease: the pathogen, the tick vector and the reservoir host. In Europe, the main vector of these pathogens (B. burgdorferis.l., Ehrlichia sp. andBabesia divergens) is the ticks,Ixodes ricinus,and mammals such as small rodents, rats, squirrels, dormice, shrews, hares and etc., and birds such as groundforaging passerines, are all known as reservoirs. Both the populations of vectors and reservoirs are regulated by ecological factors which influence the ecology of the pathogens. Vector-borne infectious agents often have geographical variation in their ecology, prevalence, and virulence due to differences in hosts or vectors, biotic and abiotic (climatic) influences on the ecology, and heritable determinants of the disease agents virulence. Transient geographical variation in prevalence of the infectious agents occurs with changes in host and vector density (including changes in the proportion of hosts
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that are immune) and with historical events such as disease introduction and extinction. The study of tick ecology and their endoparasites should comprise three levels pathogen-vector-reservoir interactions for understanding the epidemiology of the infection diseases. The researches on ticks-borne transmissed pathogens are very complicated by their multi-dimensional aspect. Such kind of researches should entail all ecological dimensions: pathogenic organism, vector and reservoir host. Also, it should be taken into account that each pathogenic organism has both, specific vectors and reservoir hosts, and it adapts itself to certain ecology conditions. Pathogens are transmitted among mammalians and birds by vectors (ticks whose ecologic prevalence conditions and life cycle are complicated). Ticks undergo three postembryonic development phases (larva, nymph, and adult), basic food for them is blood of animals and birds. Characteristic feature of various species of ticks is self-developing during the period of changing the host. Developmental cycle of ticks when the host is changed during each single phase is known as a three hosts cycle . Only some aspects of developmental cycle of ticks, seasonal and daily activities changes as well as infections of the ticks by various pathogens and the dominance of different endoparasites in various biotopes in Lithuania have been investigated recently. Today, using molecular research methods, there is a possibility to detect and investigate the ways by which pathogenic organisms spread and their mutual relationship. Researches on ectoparasites and endoparasites prevalence depending on locality, host, climate conditions and many other factors were already initiated in few European countries. Several authors stress (Gray et al., 1999; Wang et al., 1999) the demand for more detailed researches on ecological factors influencing ticks infections by endoparasites depending on their residence as well as the use of new methods for identification of the endoparasites. Molecular-based method has been successfully introduced into ecological researches of parasite-host system. Polymerase chain reaction (hereinafter referred to as the PCR) is presently the most commonly used method in molecular ecology for description and identification of population structure of huge variety of organisms. In practical terms, the PCR is comfortable to use due to very high sensitivity of the reaction that allows detecting a pathogen even when minimum quantity of it is found. The above mentioned researches might be also beneficial for DNA analysis. Products of PCR could be cloned with the aim to detect its primary structure and to compare it with the ones available in the international gene data basis. The specificity of certain genotypes of pathogens, their prevalence and circle of hosts, peculiarities of their functioning can be possibly investigated. These above mentioned detailed researches have been started recently and successfully have extended in the different countries of the world. However in Lithuania scientific research studies dedicated to ectoparasites and endoparasites prevalence, final host, and the other aspects of epidemiology and veterinary are only in the starting point of development. The aim of the study The aim of the study was to investigate: the distribution ofIxodes ricinus ticks and their transmitted parasites prevalence in different biotopes in Lithuania; and to find out factors influencing the activity dynamics of ticks and prevalence of endoparasites.
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Main tasks of the study  To researchIxodes ricinusL ticks seasonal and daily activity changes in the permanent forestry stationary (Vilnius district, Nemenčin forestry, 146 block) and to evaluate the influence of air temperature and quantity of precipitations on activity of ticks.  To identify species of ticks and level of infection (Borrelia burgdorferis.l., Babesia divergensandEhrlichiain different biotopes in Lithuania by usingsp) molecular biology methods.  To identify level of infection of endoparasites due to developmental stages and sex of ticks.  and describe dominant genotypes of endoparasiteTo find, identify B. burgdorferis.l in Lithuania.  To estimate the prevalence of mixed infections within ticks in Lithuania by using molecular methods.The scientific novelty of the study The scientific researches on ectoparasite-endoparasite system (I. ricinusBorrelia burgdorferis.l.,Babesia divergens and Ehrlichia in different biotopes in sp.) Lithuania have been carried out for the first time. Also, it was the first investigation of prevalence of ticks (according to development phases and different sexes) in different biotopes and ticks infectious by microparasites. Influence of the main factors (air temperature, precipitations) on seasonal and daily activity changes of ticks have been described. Experiments on ectoparasites and endoparasites have been employed by the procedures on molecular level, including PCR method. As the result of the study the specificity of ticks species has been identified. Moreover, microparasites and their genotypes have been detected and identified by molecular biology procedures and described. In addition, the prevalence of microparasites in different biotopes in Lithuania has been detected. In the research study the co-infection phenomenon was confirmed for the first time in Lithuania. Volume of the work The dissertation is written in Lithuanian and consists of the Introduction, Materials and Methods, Results and Discussion, Conclusions, List of authors publications and List of references. The dissertation is comprised of 116 pages. Data of the research study are presented in the 16 tables and 59 figures. METERIALS AND METHODS Collection of ticks.The study was conducted from 2001 to 2004 and collection of ticks was carried out from early spring, when the first ticks emerge after winter, to the early frost, of each year in 18 districts (43 sites) of Lithuania with different landscapes (biotopes): agricultural land  type I of biotopes; pine forest  type II, deciduous and mixed forest  type III of landscape) (Table 1). Ixodes ricinusticks used for investigations of dynamics were collected flagging undergrowth with 1 m2white towel (Li ir Dunley, 1998; Eisen et al., 2004; Petko et al., 2006) in Nemenčin (146 block), Vilnius district (pine forest), which is in forestry distance of 16 km from Vilnius. Collection of ticks has been carried out four times a month (once in week), to establish seasonal activity ofI. ricinus.
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Table 1.Locations of ticks sampling and quantity of ticks examined by molecular procedures Biotopes N Site District 1. Klaipda 1. loc. ventoji 14 2. Giruliai loc. 24 IAtyrpice.ultural3. Juodkrantloc. 13 g 4. Kau2. Kretingačiai lake loc. 41 land3. Panevys 5. Mučinai vil. loc. 50 4. Ukmerg Pail 6.loc. 98 1. Ignalina 1. Ginuč 50iai, Almaja lake loc. 2. Girminiai, Viksvasis lake loc. 55 II type.3. Kirdeikiai 13 loc. Pine forest4. Girminiai 20 vil. loc. 2. Vilnius 5. Nemenčinloc 397 * 3. Varna 6. Puvoč 37iai vil. loc 1. Birai 1. Birai forest, Latveliai loc. 20  2. Bir 31forest, Spalvikiai loc. 3. Birforest, Tamoi 18nai loc. 4. Galvokai loc. 100 2. Jonikis 5. Jonikis loc. 8 6. Datknai forest, Daunoriks loc. 37 3. Kaunas 7. Kleboniks loc. 23 8. Panemunloc. 19 9. Zuikinloc. 27 10. Kačerginloc. 12 11. Romainiai forest, Nova loc. 5 12. Giriniai loc. 8 13. Karm 25lava loc. III type..4. Kelmuvyt.T14akra51P.ornesisfant10co.ialDeciduoust, Kuprloc. 16 16. Tytuvnai forest, Patytaukis loc. 8 faonrdmixed17. ukikloc. 35 est5. Marijampol 18. KazlRda loc. 60 6. Maeikiai 19. Jazminas" loc. 31 7. Prienai 20. Birtonas, leiviniai loc. 10 21. Birtonas, vrinč 52ius loc. 8. Radvilikis 22. Antanikiai loc. 39 23. Bulikautyni 9 loc. 24. iaulnai forest, Mrai loc. 41 9. iauliai 25. Dain 9parko loc. 26. Talos parko loc. 102 27. Kur 10nai loc. 10. ilut Kintai loc. 28. 21 11. Utena 29. Skaistailis loc. 32 30. Aliai loc. 22 31. Klovainiai loc. 27 Total 1679 N  number of examined ticks; loc.  locality; vil.  village *  1821 ticks have been collected in Nemenčine locality and used for population dynamics research. 397 ticks of all collected ones have been examined by molecular genetics methods 8
Daily activity of ticks was investigated by using ticks collected during the following four intervals per day: I: 8:00-9:00 h, II: 11:00-12:00 h, III: 13:00-14:00 h, IV: 16:00-17:00 h. Totally 1821 unfedI. ricinus ticks were collected in the Vilnius district during 2001-2003 years (269 of those were nymphs, 796  adult female and 756 adult male ticks). Attached nymphs and adults were collected into vials.I. ricinus abundance was counted separately for females, males and nymphs. Immediately after collecting the ticks they were immersed in 70 % ethanol and stored at 4 °C processed. All specimens (except for 5, which wereDermacentorsp.) were identified asI. ricinus-like by their morphological characteristics. Ticks samples used for molecular examination were collected by flagging lower vegetation at peripheral and inner parts of deciduous and mixed forests, pine forests and agricultural land. Collected ticks were stored into coded tubes with 70 % ethanol solution and stored until the process. In total, 347 ticks were examined for species identification and 1679 for presence endoparasites by using PCR procedures. Taxonomic identification of ticks and presence of endoparasites were performed according to the schemes, presented in Figure 1 and Figure 2. TICKS COLLECTION IN THE DIFFERENT SITES OF LITHUANIA
TICKS SAMPLES DELIVERY TO THE MOLECULAR BIOLOGY LABORATORY
TICKS DNA EXTRACTION
MOLECULAR IDENTIFICATION OF THE DETECTION OF
SPECIFIC PCR FOR I. ricinus IDENTIFICATION
SPECIFIC PCR (fla gene) FOR B. rgdorferiub s.l.
AGAROSE GEL ELECTROPHORESIS 
RESULTS Figure 1.The scheme of the ticks identification and detection of endoparasitesB. burgdorferis.l.
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COLLECTION OF TICKS IN THE DIFFERENT SITES OF LITHUANIA
SAMPLES DELIVERY TO THE MOLECULAR BIOLOGY LABORATORY
DNA EXTRACTION
THE DETECTION OF DIFFERENT ENDOPARASITES
PCR FOR FORMULTIPLEX PCR PCREhrlichiasp FORBorrelia ensabesi diver .a
B rgdorferi. buiiB. azeliis.s.B. garin f
REVERSE AGAROSE GEL REAL-TIME PCRHYBRIDIZATIONISESORHPORTCELE
RESULTS ANALYSIS Figure 2.The scheme of the endoparasitesB. burgdorferis.l. genospecies (B. afzelii, B. garinii andB. burgdorferis.s.),Ehrlichiasp. andBabesia divergens identification by using molecular biology methods  DNA extraction.All ticks were analysed individually. Modified procedure with the ammonium hydroxide solution (2.5 %) was used for DNA extraction from ticks (Stanczak et al., 1999). Obtained DNA lysates were stored at 4 °C until the usage of them as templates for PCR. DNA lysates were stored at -20 °C for longer periods. Molecular identification of ticks.Differences in the morphology of two species I. ricinusandI. persulcatusare very small (Филиппова, 1985). It is important to note, that tick mouthparts and adjacent structures that are usually essential for identification may become damaged during the removal of ticks from its host. Consequently, these difficulties could be resolved by using procedures based on detection of molecular genetic markers. Ticks were analysed by using PCR techniques for species identification (Fukunaga et al., 2000) with species specific primers (Table 1). The 150 bp segment of the 5.8S rRNA gene, which is specific ofI. ricinus, was amplified. Examination of ticks for the presence of B. burgdorferi s.l. and genotyping of B. burgdorferi s.l.Collected ticks were tested individually for the presence of spirochetes by using polymerase chain reaction (PCR) techniques able to identify
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B. burgdorferiPCR was performed according to Stanczak (Stanczak et al., 1999) l.  s. with specific primers FL6 and FL7 (Table 1) according to sequences of conserved regions of thefla of geneB. burgdorferis.l. Obtained specific PCR products of 276 pairs were considered as a positive result. 190 tick lysates with theB. burgdorferis.l. endoparasites (according to PCR data) were examined additionally for the presence of 3Borreliapathogenic genomic species: B. afzelii, B. garinii andB. burgdorferis.s. by using multiplex PCR, performed according to Demaerschalk et al. (Demaerschalk et al., 1995; Jenkins et al., 2001). Specific primers GI-F/GI-R; GII-F/GII-R; and GIII-F/GIII-R used in the PCR are presented in Table 1. Obtained specific PCR products of 544 bp length by agarose gel electrophoresis were considered as a positive result ofB. burgdorferis.s., 345 bp  of B. gariniiand 189 bp  ofB. afzeliise.satiporaednExamination of ticks for the presence of Ehrlichia sp. and species.The presence Ehrlichiagroup pathogen was determined by using PCR withEhrlichiaspecific primers EHR 521/747 of 16S rRNR gene (Table 1) as described before (Pancholi et al., 1995; Jenkins et al., 2001). Obtained specific PCR products of 247 bp by agarose gel electrophoresis were considered as a positive result ofEhrlichiasp. All positiveEhrlichiasp. samples were re-amplified and labelled by PCR according to Schouls et al. (Schouls et al., 1999) forEhrlichia identification. species These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. In order to identify species the biotinylatedEhrlichia PCR product was hybridized with seven different oligonucleotide probes in the reverse line blot assay. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification ofEhrlichiaspecies. The reverse line blot technique is the method for the simultaneous detection and identification of microorganisms in field samples such as ticks (Schouls et al., 1999). Examination of ticks for the presence of Babesia divergens.The pathogen Babesiadivergenswas detected by Real-time PCR method with the ABI Prism 7000 system. The specific primers BdiF/BdiR, probe BdiT, presented in Table 1, and the standard 2-steps PCR conditions (according toTaqMan®Universal PCR MasterMixprotocols andTelelab, Norway recommendations) were used in the ABI Prism 7000 (according to 7000Sequence Detection and Analyse systems protocols). Statistical methods.were estimated by using the STATSOFT calculations  All statistical package STATISTICA for WINDOWS 6 and MS Excel. Differences in the prevalence of infected ticks in different biotopes were evaluated by using statistical calculations. Parametre (differences in proportion) and non-parametre (Pirsono Chi-square test)) statistical calculations were performed. The comparison of DNA sequences of simpatric species ofI. ricinus andI. persulcatuswas done by using Gene data bank Blast program. Phylogenetic analysis was performed by using the procedure of maximal parsimony and Mega 3.1. program.
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