GENOTYPING OF TICKS AND TICK BORNE PATHOGENS ON THE TERRITORY OF THE REPUBLIC OF CROATA

Krpelji se nakon komaraca smatraju najvažnijim biološkim vektorima uzročnika bolesti u svijetu. Dosadašnja podjela se temeljila na identifikaciji vrsta temeljem specifičnih morfoloških obilježja. Učestalija primjena molekularnih metoda dovela je do promjena u sistematizacija, ali i do dokaza novih patogena u krpeljima, životinjama i ljudima. Kako bi se utvrdio značaj, vektorski potencijal i raznolikost krpelja na području RH istraživanje je provedeno na 509 arhivskih prethodno morfološki determiniranih krpelja. Krpelji su pojedinačno analizirani sekvenciranjem odsječka 16S rRNA gena. U svakog pojedinačnog krpelja istražena je prisutnost patogena iz rodova Babesia, Theileria, Anaplasma, Ehrlichia, Hepatozoon, Mycoplasma, Borrelia, Rickettsia, Francisella i Coxiella, te je provedeno mapiranje krpelja i dokazanih patogena. Sekvenciranjem je dokazano šest vrsta krpelja iz roda Ixodes: I. gibbosus, I. canisuga, I. kaiseri, I. ventalloi, I. hexagonus i I. ricinus. Unutar vrsta I. hexagonus i I. ricinus su dokazana tri i 24 različita genotipa. Rod Dermacentor je bio zastupljen sa dvije vrste, D. marginatus i D. reticulatus. Unutar roda Rhipicephalus dokazana je vrsta R. bursa i skupina R. sanguineus sensu lato (s.l.), sa četiri različita genotipa. Tri skupine sekvenci tri genotipa su bile slične sekvencama R. turanicus, a četvrta R. sanguineus sensu stricto (s.s.). Hyalomma margintum je bila jedina vrsta roda Hyalomma, dok su unutar roda Haemaphysalis dokazane: H. parva, H. inermis i H. concinna. Vrste iz rodova Babesia i Theileria su dokazane u 6,3% i 6,1% krpelja. Rod Babesia je bio zastupljen s osam skupina sekvenci koje su odgovarale B. canis, B. vulpes, B. microti, B. venatorum, Babesia sp. „Badger type A” i „Badger type B”, B. ovis, Babesia cf. crassa i Babesia sp „tavsan“. Unutar roda Theileria dokazane su četiri vrste: T. ovis, T. orientalis (buffeli/sergenti), T. equi i T. capreoli. U 18% krpelja su dokazane proteobakterije iz porodice Anaplasmataceae: Anaplasma capra, A. phagocytophilum, Ehrlichia canis, Ehrlichia sp., „Candidatus Neoehrlichia lotoris“ i endosimbionti Wolbachia sp. i Midichloria mitochondrii. Anaplasma capra je dokazana u R. bursa, te R.turanicus 2 i R. turanicus 3, a E. canis u R. bursa. Tri vrste iz roda Hepatozoon su sekvenciranjem dokazane u 6,1% krpelja: H. canis, H. felis i Hepatozoon „Badger type“. Pet vrsta iz roda Rickettsia, R. slovaca, R. raoultii, R. aeschlimannii, R. massiliae i R. monacensis su dokazane u 11,7% krpelja. U R. turanicus 3 s Pelješca dokazana je Francisella tularensis subsp. holartica, dok je endosimbiont „Francisella-sličan“ dokazan u 1,5% D.reticulatus i D. marginatus. Na području kontinentalne Hrvatske u genotipovima I. ricinus su dokazane Borrelia afzelii i B. valaisiana u 1,9% i 0,8% krpelja. Istraživanjem nisu dokazane bakterije iz roda Mycoplasma i vrsta Coxiella burnetii. Ovo istraživanje predstavlja prvo opsežno istraživanje krpelja i patogena u krpeljima u Hrvatskoj.Ticks are obligate hematophagous ectoparasites of mammals, birds, amphibians and reptiles, and currently are after mosquitoes considered as most important biological vectors of pathogens in the world.Overall 920 species of ticks have been described so far, classified within three families (Ixodidae, Argasidae and Nuttalliellidae), among which Ixodidae (hard ticks) represents the most important family. About 10% of known tick species can transmit various pathogens such as viruses, rickettsiae, bacteria and parasites. With development of molecular methods, a new species, strains or genetic variants of pathogens/microorganisms, are being detected in ticks worldwide, and the spectrum of potential tick transmitted pathogens affecting domestic animals and humans that can cause disease important not only in veterinary medicine but also within public health aspects, continues to increase. Current classification of ticks was based on species identification according to morphological features, but frequent use of molecular methods has led to changes in systematization. For example, sequence analysis of 12S rRNA gene, classified the genus Boophilus in the genus Rhipicephalus, while analysis of 12S rRNA, 16S rRNA and COX1showed that Rhipicephalus sanguineus is considered a complex group (sensu lato) and includes at least 17 related species. So far in Croatia, studies based on morphological determination of ticks have described 21 tick species grouped within five genera, while molecular studies are scarce. The first genetic study on ticks within R. sanguineus complex showed presence of two genetic lines Rhipicephalus sp. phylogenetically classified in group II with subgroups Rhipicephalus sp. IIa and Rhipicephalus sp. IIb. Within the genus Ixodes, two species of ticks, I. canisugaand I. hexagonus have been confirmed. Numerous studies in Croatia focused on detection of of tick transmitted pathogens in animals, while studies on pathogens in tick, and even tick species present in Croatia are lacking. The aim of the study was to investigate tick species present in Croatia based on sequencing of 16S rRNA gene fragment and to compare results with results of morphological determination. Furthermore, to investigate pathogens in individual ticks in order to obtain more precise data on ticks and pathogens in ticks they may carry for the first time in Croatia and to compare detected pathogens with tick “genetic” lines. One of the aims was to visualize ticks and detected pathogens by mapping, as well. The study was carried out on 509 archived morphologically identified ticks. Ticks from different locations were collected from domestic and wild animals, dogs, cats, humans and environment during period of 2014 to 2017. DNA was extracted manually from each tick and analyzed individually based on PCR amplification of 16S rRNA gene fragment. All extracted DNA from each tick was screened for presence of Babesia, Theileria, Anaplasma, Ehrlichia, Hepatozoon, Mycoplasma, Borrelia, Rickettsia, Francisella and Coxiella DNA using conventional PCR and subsequent sequencing. Amplified products were analysed using capillary electrophoresis, purified with ExoSAP-IT® PCR Clean-Up Reagent kitand sequenced in both directions in Macrogen Europe. Sequences were assembled using.Lasergene® software, edited with SeqmanTM , and compared with available squences in GenBank® using the BLAST® . Obtained results were visualised by mapping with QGIS® software. In the current study we have detected 15 tick species belonging to five genera. Genus Ixodes (31,2%; 159/509) was the most representative genus with six species: I.ricinus, I. canisuga, I. hexagonus, I. gibbosus, I. kaiseri and I. ventalloi. Ixodes gibbosus, I. kaiseri and I. ventalloi were confirmed molecularly for the first time in Croatia. The number of species is not surprising, because previous studies morphologically described eight species in Croatia. Out of the five known species in Europe, three species from subgenus Pholeoixodes, I. canisuga, I. hexagonus, and I. kaiseri were detected in this study. Presence of I. kaiseri from red fox in the continental region represents the first confirmation of species in Croatia, but also one of the rarest findings in Europe. So far has been detected from foxes and dogs from Germany, Romania, Hungary and Serbia. Ixodes canisuga (4,5%; 23/509) ticks were geographically limited to continental Croatia, and as other species from subgenus Pholeoixodes, werecollected from red foxes, except one tick from environment.Sequences were identical to sequences from other European countries, including neighboring countries such as Serbia, Bosnia and Herzegovina and Hungary. The last species, I. hexagonus (3,7%, 19/509) was detected in all regions, except Istria. Unlike I. canisuga, sequence analysis showed presence of three different groups of sequences grouped within three isolates I. hexagonus 1, I. hexagonus 2 and I. hexagonus 3. Sequences showed up to two nucleotide differences from each other and shared 99% similarity. Ixodes hexagonus 1 dominated in all areas and was present in southern Adriatic (Mljet). In central Adriatic, it appeared together with I. hexagonus 2, while in the continental region with I. hexagonus 2 and I. hexagonus 3. Detection of I. hexagonus in central and southern Adriatic represents the first finding of tick species in the coastal region. Sequences of I. hexagonus 1 and I. hexagonus 3 were identical to sequences from European countries, while I. hexagonus 2 with the same sequences shared a slightly lower similarity of 99,7%. One of the interesting findings represent genetic confirmation of I. ventalloi (1,2%; 6/509) from european hares from Vir. Besides its first molecular confirmation in Croatia, represents the first finding in Southeastern Europe. Current study confirmed presence of I. gibbosus (0,8%, 4/509) in Croatia. So far has been described only on Brač, therefore its finding on sheeps from Cres, Rab and Pag indicates its possible presence in other coastal areas.Sequences shared 99% similarity with sequences from Turkey and Greece. Sequencing confirmed 24 different groups of sequences of I. ricinus (20,8%; 106/509), similar to other European studies, for the first time in Croatia. Sequences shared 98% similarity and differed up to two nucleotides. Genotypes were geographically located in continental region, besides two ticks from Gorski kotar and central Adriatic. Ticks were mostly collected from the environment, but also from canids and wild ruminants, except for one tick collected from humans. The most frequent isolate was I. ricinus 1 (71,7%; 76/106), followed by I. ricinus 7 (4,7%; 5/106), I. ricinus 6 (2,8%; 3/106) and I ricinus 3 (1,9%; 2/106). The remaining 20 genotypes were detected each in one tick. Sequences of I. ricinus 1, I. ricinus 7, I. ricinus 15, I. ricinus 16 and I. ricinus 20 were identical to I. ricinus sequences in Europe, while sequences of 19 isolates showed similarity from 98% to 99%. Dermacentor reticulatus is considered as tick species which has the fastest expansion throughout Europe, and whose southern limits have not been determined precisely. This study confirmed its presence in all investigated areas, besides Istria and southern Adriatic. Confirmation of tick in northern (Senj, mouflon) and central Adriatic (Vir, european hare) represents the southernmost finding of the species in Croatia. All 16S rRNA sequences were identical and shared 99% to 100% similarity with D. reticulatus sequences from Europe. Another member of genus, D. marginatus has been detected southwards, on Hvar from wild boarand throughout continental Croatia. Rhipicephalus bursa and R. sanguineus sensu lato (s.l.) were confirmed within genus Rhipicephalus. Rhipicephalus bursa (11,2%; 57/509) was present in almost entire coastal area. In continental region was collected from foxes in Zagreb and Daruvar and from sheep from Slunj, which represents its first confirmation outside coastal area and evidence of its spreading from south to north. Sequences shared 99% similarity with sequences of R. bursafrom Europe, such as Turkey, France and Spain. One third of the ticks belonged to the Rhipicephalus sanguineus s.l. (32,2%; 164/509). Group of 149 sequences belonged to the R. turanicus. Differences within sequences varied up to three nucleotides and were classified into three groups: R. turanicus 1, R. turanicus 2 and R. turanicus 3. The most frequent isolate R. turanicus 3 was found in central and southern Adriatic, but also in Zagreb area. Confirmation of R. turanicus in Zagreb from fox is the first description of species in continental Croatia. Remaining two genotypes have been confirmed throughout central and southern Adriatic, as well. Sequences were identical to the sequences of R. turanicus from Turkey and R.turanicus found on Murter. Fourth group of sequences (9,1%; 15/164) shared similarity of 94% with the other sequences and corresponded to the sequences of R.sanguineus s.s. Rhipicephalus sanguineus s.s. was collected only from dogs from the northern (Rovinj, Premantura) and central Adriatic (Zadar, Obrovac Sinjski, Hvar), and in continental region (Zagreb, Vinkovci). Sequences were identical to sequences of Rhipicephalus sanguineus from dogs from Serbia and Croatia. Hyalomma marginatum (6,9%; 35/509) was the only species from the genus Hyalomma. Ticks were present in coastal region and collected mainly from cows, then horses, goats, donkeys and dogs. Sequences were identical to each other but also to other sequences from Europe but at the same time shared 99,7% similarity with the 16S rDNA sequences of Hy. rufipes from Afrika and Hy. turanicum from Irak. For the first time in Croatia three species from Haemaphysalis genus were confirmed: H. parva (1,5%; 8/509), H. inermis (1,2%; 6/509) and H. concinna (0,4%; 2/509). Ticks from eastern Croatia were collected from the environment, except H. concinna from foxes (Požega) and H. inermis from foxes and horses (Jastrebarsko, Zadar). Sequences of H. concinna were identical with sequences from Hungary, while sequences of H. inermis and H. concinna shared 99,5% similarity with sequences from Australia and Turkey. Members from family Anaplasmataceae were the most commonly detected microorganisms occuring in 18% (92/509) of ticks. Sequencing revealed several species of anaplasma Anaplasma capra, A. phagocytophilum, Ehrlichia canis, Ehrlichia sp., “Candidatus Neoehrlichia lotoris” and endosymbionts Wolbachia sp. and Midichloria mitochondrii. The most significant finding is the detection of DNA of A. capra and E. canis for the first time in Croatia. Zoonotic A. capra was detected in 5,1% of ticks belonging to R. bursa, R. turanicus, D. marginatus and H. inermis. Ticks from Rhipicephalus genus were present in central and southern Adriatic (sheeps, goats), while D. marginatus and H. inermis in continental Croatia (sheep, fox). DNA of Ehrlichia canis was detected in 0,4% of R. bursa from sheep and goats from central Adriatic. Piroplasms (12,4%; 63/509) were the second most common group of pathogens detected in 12,4% of ticks in this study. Sequencing showed almost equal prevalence of Babesia spp. (6,3%) and Theileria spp. (6,1%). Babesia genus included eight group of sequences corresponding to B. canis, B. vulpes, B. microti, B. venatorum, Babesia sp. “Badger type A” and “Badger type B”, B. ovis, Babesia cf. crassa and Babesia sp. "tavsan". Babesia canis represents well known piroplasm in dogs from Croatia. Sequencing revealed two genotypes of B. canis, B. canis 1 (1,9%) and B. canis 2 (0,4%). Babesia canis DNA was detected in D. reticulatus (fox, brown bear, dog) as expected, but also in D. marginatus (dog, human), R. bursa (goat, cattle), R. turanicus (goat), I. hexagonus (fox) and I. ricinus (fox). Zoonotic babesia B. venatorum and B. microti were detected in I. ricinus from foxes and environmentin continental Croatia. Within the genus Theileria, four species have been detected: T. ovis, T. orientalis (buffeli/ sergenti), T. equi and T. capreoli. The most common species T. ovis was detected in 3,5% of ticks: R. turanicus (sheep), D. reticulatus (foxes), R. bursa (cow, sheep), Hy. marginatum (dog) and I. ricinus (red deer). Pathogen was more abundant in central and southern Adriatic. Theileria orientalis (buffeli/sergenti) was detected in 1,9% of ticks: D. reticulatus, D. marginatus and I. gibbosus. Pathogen was geographically limited to continental Croatia, with the southernmost finding in D. reticulatus from Otočac and I. gibbosus from Cres. Theileria capreoli was so far confirmed in grey wolves but and dogs in Croatia, but never in ticks. Detection in one D. marginatus from environment represents its first detection in ticks in Europe. Three species of genus Hepatozoon were confirmed in 6,1% of ticks: H. canis, H. felis and Hepatozoon „Badger type". Sequence analysis showed two different H. canis genotypes, H. canis 1 and H. canis 2. Both genotypes were geographically limited to continental region, except one H. canis 1 detected in R. sanguineus s.s. from central Adriatic (Medviđa). Pathogen was detected in ticks collected from foxes (I. hexagonus, I. ricinus, I. canisuga, D. reticulatus, horse (I. ricinus), environment (I. ricinus) and in two ticks from dogs, I. ricinus and R. sanguineus s.s. Rhipicephalus turanicus 3 from cats from Dugi otok was found to harbour DNA of H. felis (0,4%). Five species from Rickettsia genus were detected in 11,7% of ticks namely: R. slovaca, R. raoultii, R. aeschlimannii, R. massiliae and R. monacensis. The causative agents of TIBOLA syndrome R. slovaca and R. raoultii have been detected in 0,9% and 4,7% of ticks. Rickettsia slovaca DNA was found in D. reticulatus (fox, mouflon) from northern Adriatic and D. marginatus (human, cattle, sheep) from continental Croatia. Rickettsia raoultii was expectedly frequentin D. reticulatus collected from various animal species and environment. Besides D. reticulatus, rickettsia DNA was present in D. marginatus and H. parva from environment in continental region.So far, DNA of zoonotic rickettsia from spotted fever group R.aeschlimannii was detected in Split and Sinj area. This study confirmed its DNA in Hy. marginatum throughout coastal area. Rickettsia DNA was also present in R. turanicus from southern Adriatic. Rickettsia massiliae was detected in 2,4% R. turanicus ticks from domestic ruminants from southern Adriatic. Rickettsia monacensis (0,9%) was detected in I. ricinus from continental region and in Hy. marginatum from Sinj. Francisella tularensis (0,2%) was detected in one R. turanicus 3 from Pelješac. Sequence was identical to F. tularensis subsp. holartica from D. marginatus (BIH, Portugal), H. concinna (Hungary), I. ricinus (Germany, Serbia) and european hare (Spain). In 1,5% of D. reticulatus (golden jackal, wild boar, horse, fox, environment) and D. marginatus (human) from continental Croatia sequences were identical to „Francisella-like“ endosymbiont from D. reticulatus collected from environment in Poland. Borrelia burgdorferi s.l. DNA was detected in 2,7% ticks. Sequencing revealed higher prevalence of B. afzelii (1,9%) than B. valaisiana (0,8%). Similar to previous studies, both species have been detected in various genotypes of I. ricinus in continental Croatia. Most ticks were collected from environment, except two ticks from dog and fox. Bacteria from Mycoplasma genus, as well as Coxiella burnetti were not detected. This study is one of the most comprehensive studies on genetic diversity of ticks collected from different species of domestic and wild animals, dogs, cats, humans, and the environment, not only in Croatia but also in Europe, which includes comparison of morphological determination and genotyping of 16S rDNA gene fragment. Sequencing of 16S rRNA and comparison with the BLAST® has shown to be the most suitable method for detecting tick species and at the same time the analyzed gene is sufficiently heterogeneous to enable the differentiation of potential genotypes within the species. This study confirmed presence of D. reticulatus, D. marginatus, I. ricinus, I. hexagonus, I. canisuga, R. bursa, R. turanicus sensu lato, R. sanguineus sensu strico, Hy. marginatum, but for the first time species such as I. gibbosus, I. kaiseri, I. ventalloi, H. concinna, H. inermis and H. parva, have been confirmed molecularly. The genetic approach showed genetic diversity within the population of I. ricinus and R. turanicus in Croatia, and also the presence of at least two species within the R. sanguineus sensu lato complex. The systematic approach in detection of tick transmitted pathogens in individual ticks showed presence on numerous pathogens, important not only in veterinary medicine but also within public health aspects. This research contributed to the knowledge of tick species, their distribution and tick transmitted pathogens