Potencijal rekombinantnih proteina pljuvačke krpelja Ixodes ricinus (Linnaeus, 1758) za detekciju specifičnih antitela kao markera uboda krpelja

Assessment of exposure to ticks is a significant data in epidemiological studies of tick-borne diseases, and confirmation of tick bite is important for their diagnosis. In this study, three saliva proteins, PA107, AV422 and calreticulin (CAL), of Ixodes ricinus tick, medically most important species in Europe, were examined in the context of their use for detection of specific antibodies in sera of hosts, as bite markers. Sequence variability analysis revealed high conservativity of all three proteins, with PA107 as the most conservative. Since CAL is ubiquitous in eukaryotes, the further focus of this study was on PA107 and AV422, as proteins that are specific for ticks. Homologues of these proteins from Ixodes ricinus were analyzed for the first time. Reactivity of sera of experimental animals, rats, which were infested separately with Ixodes ricinus or Dermacentor reticulatus larvae, with recombinant forms of IrPA107 and IrAV422 were examined. Specific antibodies were detected in sera of all infested rats using rIrAV422, which implied its usage for detection of bites of tick species that belong to different groups of ixodid ticks, with high sensitivity and specificity. Reactivity with IrPA107 was significantly lower. Hunting dogs, as good sentinels, were used for assessment of rIrAV422 usage for detection of markers in sera of hosts which are exposed to ticks in natural environment. Obtained seroreactivity in all dogs implies broad applicability of this saliva protein, in terms of assessment of exposure to different tick species and risk of infection with tick-borne pathogens.Procena izloženosti krpeljima je značajna u epidemiološkim studijama oboljenja čije uzročnike prenose krpelji, a potvrda uboda krpelja je važna za njihovu dijagnostiku. U ovom radu su ispitana tri proteina pljuvačke, PA107, AV422 i kalretikulin (CAL), krpelja Ixodes ricinus, medicinski najznačajnije vrste u Evropi, u kontekstu upotrebe za detekciju specifičnih antitela u serumu domaćina, kao markera uboda. Analiza varijabilnosti njihovih sekvenci je ukazala na visoku konzerviranost sva tri proteina, pri čemu se PA107 pokazao kao najkonzerviraniji. S obzirom na ubikvitarnu zastupljenost CAL kod eukariota, dalji fokus u ovoj studiji je bio na PA107 i AV422, kao proteinima koji su specifični za krpelje i čiji homolozi su prvi put analizirani kod vrste Ixodes ricinus. Ispitivana je reaktivnost seruma eksperimentalnih životinja, pacova, na kojima su hranjene larve Ixodes ricinus ili Dermacentor reticulatus, sa rekombinantnim formama IrPA107 i IrAV422. Korišćenjem rIrAV422, specifična antitela su detektovana kod svih infestiranih pacova, što ukazuje da se rIrAV422 može upotrebiti za detekciju markera uboda različitih grupa tvrdih krpelja uz visoku senzitivnost i specifičnost. Reaktivnost u slučaju IrPA107 je bila značajno manja. Za ispitivanje upotrebe rIrAV422 u detekciji markera uboda u serumima domaćina izloženih krpeljima u prirodnom okruženju, korišćeni su lovački psi kao adekvatna indikatorska grupa. Dobijena seroreaktivnost kod svih analiziranih pasa u studiji ukazuje na širok potencijal primene ovog proteina pljuvačke u proceni izloženosti različitim vrstama krpelja i rizika od infekcije patogenima koje oni prenose

Potential of recombinant Ixodes ricinus (Linnaeus, 1758) tick saliva proteins for detection of specific antibodies as markers of tick bite

Процена изложености крпељима је значајна у епидемиолошким студијама обољења чије узрочнике преносе крпељи, а потврда убода крпеља је важна за њихову дијагностику. У овом раду су испитана три протеина пљувачке, PA107, AV422 и калретикулин (CAL), крпеља Ixodes ricinus, медицински најзначајније врсте у Европи, у контексту употребе за детекцију специфичних антитела у серуму домаћина, као маркера убода. Анализа варијабилности њихових секвенци је указала на високу конзервираност сва три протеина, при чему се PA107 показао као најконзервиранији. С обзиром на убиквитарну заступљеност CAL код еукариота, даљи фокус у овој студији је био на PA107 и AV422, као протеинима који су специфични за крпеље и чији хомолози су први пут анализирани код врсте Ixodes ricinus. Испитивана је реактивност серума експерименталних животиња, пацова, на којима су храњене ларве Ixodes ricinus или Dermacentor reticulatus, са рекомбинантним формама IrPA107 и IrAV422. Коришћењем rIrAV422, специфична антитела су детектована код свих инфестираних пацова, што указује да се rIrAV422 може употребити за детекцију маркера убода различитих група тврдих крпеља уз високу сензитивност и специфичност. Реактивност у случају IrPA107 је била значајно мања. За испитивање употребе rIrAV422 у детекцији маркера убода у серумима домаћина изложених крпељима у природном окружењу, коришћени су ловачки пси као адекватна индикаторска група. Добијена серореактивност код свих анализираних паса у студији указује на широк потенцијал примене овог протеина пљувачке у процени изложености различитим врстама крпеља и ризика од инфекције патогенима које они преносе.Assessment of exposure to ticks is a significant data in epidemiological studies of tick-borne diseases, and confirmation of tick bite is important for their diagnosis. In this study, three saliva proteins, PA107, AV422 and calreticulin (CAL), of Ixodes ricinus tick, medically most important species in Europe, were examined in the context of their use for detection of specific antibodies in sera of hosts, as bite markers. Sequence variability analysis revealed high conservativity of all three proteins, with PA107 as the most conservative. Since CAL is ubiquitous in eukaryotes, the further focus of this study was on PA107 and AV422, as proteins that are specific for ticks. Homologues of these proteins from Ixodes ricinus were analyzed for the first time. Reactivity of sera of experimental animals, rats, which were infested separately with Ixodes ricinus or Dermacentor reticulatus larvae, with recombinant forms of IrPA107 and IrAV422 were examined. Specific antibodies were detected in sera of all infested rats using rIrAV422, which implied its usage for detection of bites of tick species that belong to different groups of ixodid ticks, with high sensitivity and specificity. Reactivity with IrPA107 was significantly lower. Hunting dogs, as good sentinels, were used for assessment of rIrAV422 usage for detection of markers in sera of hosts which are exposed to ticks in natural environment. Obtained seroreactivity in all dogs implies broad applicability of this saliva protein, in terms of assessment of exposure to different tick species and risk of infection with tick-borne pathogens

Divlje kanide kao domaćini za vektorima prenosive zoonozne patogene u Srbiji

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia.Divlje kanide su prepoznate kao važni domaćini rezervoari za nekoliko vektorima prenosivih zoonotskih patogena i predstavljaju dobre sentinel vrste za epidemiološki nadzor. Identifikacija domaćina rezervoara za pojedine zoonotske patogene na određenom području omogućava primenu odgovarajućih mera kako bi se sprečila pojava zoonoza kod ljudi i domaćih životinja. Cilj ove studije bio je da se predstavi uloga divljih kanida u epidemiologiji zoonoza koje se prenose vektorima u Srbiji na temelju prethodno objavljenih radova i da se dostupni podaci prodiskutuju sa epidemiološkog stanovišta. Na osnovu dosadašnjih molekularnih istraživanja u Srbiji, divlje kanide su prepoznate kao potencijalni rezervoari za nekoliko krpeljima prenosivih zoonotskih patogena. Kod šakala je potvrđeno prisustvo Anaplasma phagocytophilum, dok je prisustvo Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii i Borrelia lusitaniae molekularnim metodama potvrđeno kod lisica. Rezultati pokazuju da su divlje kanide potencijalni domaćini rezervoari i izvor zoonotskih patogena u Srbiji i da predstavljaju dobre domaćine za više vrsta krpelja koji su potvrđeni kompetentni vektori pojedinih patogena. Potrebno je kontinuirano epidemiološko praćenje bolesti uzrokovanih patogenima čije je prisustvo prethodno potvrđeno kod divljih kanida. Takođe, potrebna su dodatna istraživanja kako bi se otkrila uloga drugih divljih i domaćih životinja u epidemiologiji zoonoza koje se prenose vektorima u našoj zemlji

Borrelia lusitaniae and Borrelia valaisiana – potential for causing Lyme borreliosis in Serbia

Kompleks Borrelia burgdorferi sensu lato trenutno obuhvata dvadest i jednu opisanu vrstu borelija a karakteriše ih značajna varijabilnost u pogledu geografske distribucije kao i specifičnosti na relaciji domaćini rezervoari- kompetentni vektor. Poznato je da samo određeni sojevi vrsta borelija izazivaju bolest kod ljudi. U Severnoj Americi, lajmsku boreliozu uzrokuje samo jedna vrsta borelija-Borrelia burgdorferi sensu stricto (s.s.), dok u Evropi infekciju kod ljudi može uzrokovati nekoliko različitih vrsta borelija-Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii i Borrelia burgdorferi s.s. Vrste Borrelia valaisiana, Borrelia lusitaniae i Borrelia bissettii identifikovane su samo u pojedinačnim slučajevima i prepoznate su kao potencijalni patogeni. Različite vrste borelija povezane su sa različitim kliničkim manifestacijama lajmske borelioze; B. afzelii obično ostaje lokalizovana u koži, B. garinii i B. bavariensis se obično povezuju s poremećajima nervnog sistema, dok se B. burgdorferi s.s. često povezuje sa razvojem artritisa. Iako postoje registrovani slučajevi lajmske borelioze u Srbiji, ne postoje podaci o tome koje vrste borelija izazivaju određene kliničke manifestacije lajmske borelioze. Region Srbije karakteriše visoka rasprostranjenost i raznovrsnost vrsta borelija u krpeljima i njihovim domaćinima. Istraživanja prisustva borelija u krpeljima kao i mikrobiološka izolacija sojeva borelija iz krpelja u Srbiji, ukazala su na dominaciju vrste B. lusitaniae, slede vrste B. afzelii, B. bavariensis, B. garinii, B. valaisiana i B. burgdorferi s.s. Takođe, B. lusitaniae je detektovana u krpeljima sakupljenim sa guštera i u uzorcima slezine lisica, dok je B. valaisiana detektovana u krpeljima sakupljenim sa šakala i ptica. Ispitivanje in vitro osetljivosti lokalnih sojeva borelija (izolovanih iz krpelja vrste Ixodes ricinus sakupljenih sa vegetacije) na ljudski serum, pokazalo je da su sojevi B. valaisiana otporni na komplement, dok su određeni sojevi B. lusitaniae (u prisustvu komplementa) podjednako pokretljvi kao i jedini postojeći soj B. lusitaniae izolovan iz hronične kožne lezije, poreklom iz Portugala. Iako su lokalni sojevi borelija osetljivi na antibiotike in vitro, ostaje nepoznato koje kliničke manifestacije lajmske borelioze mogu izazvati vrste B. valaisiana i B. lusitaniae. Potrebno je da se razjasne mehanizami rezistencije sojeva B. valaisiana i B. lusitaniae (koji cirkulišu u ovoj oblasti) u prisustvu komplementa.The twenty-one described Borrelia species with considerable variability in host and vector associations, and geographical distribution have been classified to the Borrelia burgdorferi sensu lato complex. Only a subset of Borrelia species is known to cause human illness. In North America, Lyme borreliosis (LB) is predominantly caused by the Borrelia burgdorferi sensu stricto (s.s.), while in Europe, the overall diversity of Borrelia species is higher and LB can be caused by Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, and Borrelia burgdorferi s.s., while Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have been identified in single cases only and are recognized as potential pathogens. Different Borrelia species are involved in distinct clinical manifestations of LB; B. afzelii usually remains localized in the skin, B. garinii and B. bavariensis are usually associated with nervous system disorders, while B. burgdorferi s.s. is commonly associated with development of the arthritis. Although there are registered cases of LB in Serbia, there is no data on which Borrelia species cause certain clinical manifestations of LB. Region of Serbia is characterized by high prevalence and diversity of Borrelia species in ticks and their vertebrate hosts. The studies on the presence and isolation of Borrelia from ticks from Serbia pointed to the domination of B. lusitaniae followed by B. afzelii, B. bavariensis, B. garinii, B. valaisiana and B. burgdorferi s.s. Also, B. lusitaniae was detected in ticks collected from lizards and in spleen samples collected from red foxes, while B. valaisiana was detected in ticks collected from golden jackals and birds. In vitro susceptibility of local Borrelia strains isolated from ticks to human complement, showed B. valaisiana strains resisted complement-mediated killing, while certain B. lusitaniae strains (in the presence of complement) were equally motile as the only existing one B. lusitaniae human isolate from Portugal. Although local Borrelia strains are susceptible to antibiotics in vitro, it remains unknown what clinical manifestations of LB can be caused by B. valaisiana and B. lusitaniae. It is needed to elucidate the mechanism behind B. valaisiana and B. lusitaniae strains (circulating in the area) survival in the presence of human complement

Molecular detection of Babesia spp. in ticks in northern Serbia

In order to evaluate the prevalence rate of Babesia spp. in ticks collected from vegetation at seven localities in northern Serbia, tick samples were subjected to molecular analysis. A total of 132 unfed adult ticks of five different species (Dermacentor marginatus, Dermacentor reticulatus, Ixodes ricinus, Haemaphysalis concinna and Haemaphysalis punctata), were examined by PCR for the presence of Babesia spp. Out of the analyzed ticks, 10.61% (14/132) were positive for babesial DNA. The presence of babesiae was found at the localities Pančevački Rit, Titov Gaj, Makiš, PKB and Kljajićevo. Prevalence in D. reticulatus ticks was 21.57% (11/51) and in H. concinna ticks, 8.57% (3/35). Sequencing and phylogenetic analysis showed a clustering of the obtained sequences with those of B. canis from the GenBank database. These results add to the knowledge of the distribution of babesial pathogens and their vectors in Serbia

Evolutionary dynamics and geographical dispersal of Borrelia lusitaniae

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis

Molecular characterization of coi gene of ixodes ricinus (linnaeus, 1758) from Serbia

The Ixodes ricinus tick is common in the central part of the Balkan Peninsula. It is a vector of pathogenic agents causing diseases in humans and animals. Little is known about the genetic structure of I. ricinus in this region. We have investigated intraspecific variability of the COI gene among I. ricinus ticks collected from different regions of Serbia, and the correlation between the various types of habitat and genetic variability of ticks. The obtained COI gene sequences are the first barcoding sequences of I. ricinus ticks collected at localities in Serbia. Intraspecific variability of these COI gene sequences was very low, and there was no correlation between the various types of habitat and genetic variability of ticks. Samples from isolated localities (canyon/gorge) showed no genetic differentiations from the majority of samples from open areas

First evidence of tick-borne protozoan pathogens, babesia sp. And hepatozoon canis, in red foxes (vulpes vulpes) in Serbia

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Co-infection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes

Knowledge, Attitudes, and Practices on Tick-Borne Encephalitis Virus and Tick-Borne Diseases within Professionally Tick-Exposed Persons, Health Care Workers, and General Population in Serbia: A Questionnaire-Based Study

This study assessed the level of knowledge, attitudes, and practices (KAP) regarding tick-borne encephalitis virus (TBEV) and tick-borne diseases (TBDs) among different groups of people in Serbia. Professionally tick-exposed persons (PTEPs), health care workers (HCWs), and the general population (GP) were subjected to an anonymous, voluntary, online questionnaire using Microsoft Forms. A total of 663 questionnaire responses were collected (February–March 2021), while 642 were included in the analysis. The significant difference in knowledge in TBDs existed between GP and PTEPs, and HCWs (p < 0.001). The perception of risk-to-tick exposure and TBDs was generally high (42.4 (95% CI: 33.6–51.2) within GP, 44.9 (95% CI: 35.8–53.9) within PTEPs and 46.2 (95% CI: 38.0–54.5) within HCWs), while fear was low (13.7 (95% CI: 7.9–19.5) within GP, 12.6 (95% CI: 7.3–19.9) within PTEPs, and 13.5 (95% CI: 7.4–19.5) within HCWs). Protective practices differed across groups (F (2639) = 12.920, p < 0.001, η2 = 0.039), with both PTEPs (t = 3.621, Cohen d = 0.332, p < 0.001) and HCWs (t = 4.644, Cohen d = 0.468, p < 0.001) adhering to more protective practices than the GP, without differences between PTEPs and HCWs (t = 1.256, Cohen d = 0.137, p = 0.421). Further education about TBDs in Serbia is required and critical points were identified in this study