| Abstract | 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. |
| Abstract (english) | 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. |
