*119621*

Efremova G.A.¹, Bychkova E.I¹., Aksenova E.A.², Jakovich M.M.¹, Fedorova I.A.¹ , Luhanina N.V.².

SPECIES COMPOSITION OF TICKS DESCRIBED ON THE TERRITORY OF BELARUS

¹SSPA « Scientific-practical center of the National Academy of Sciences of Belarus for biological resource », Minsk, Republic of Belarus

²Institute of Genetics and Cytology of the National Academy of Sciences of Belarus, Minsk, Republic of Belarus

Ticks are relatively poor in terms of species diversity, but important in the pathology of human and domestic animal group of parasites. On the taxonomy level they quite clearly differ on 2 environmental groups of species: pasture and nest- burrow (Table). Pasture tick species spend the periods between feed on the surface or in the upper layers of the soil, nest- burrow - in the nests and burrows of various vertebrates, although, as will be shown below, there are species and, obviously, of a transitional character, capable to dwell on the open areas and in the shelters of the host .

The first information about ticks - vectors of pyroplasmosis in Belarus we find in the work of the V.L. Yakimov [37], who describes 2 species of ticks: Ixodes ricinus (Linnaeus, 1758) and Dermacentor reticulatus Fabrcius, 1794. It should be noted that for a long time to refer to this species was used the name Dermacentor pictus Hermann, 1804. At the present time the priority name restored [13]. Among other parasites, on the animals 2 species of ticks (I. ricinus and Dermacentor marginatus Sulzer, 1776) and on the cut grass (June 24, 1932.) one copy of the tick Dermacentor silvarum Olenev, 1927 [38, 33] were found. From the literature it is known that before the 1940 the confusion in terms of nomenclature and taxonomy of the genus Dermacentor had a place. Later it was determined that on

Table

The species composition of ticks described on the territory of Belarus

Type of parasite	Species of ticks	Occurrences	Presence	The author, who first pointed out the species
Pasture species of parasite	Ixodes ricinus Linnaeus, 1758	on birds	+	37
		on animals, on vegetation	+
	Ixodes persulcatus Schulze, 1930	on animals, on vegetation	+	34
	Haemaphysalis concinna Koch., 1844	there is no indication		20
	Haemaphysalis punctata Canestrini. et Fanzago., 1877	there is no indication		20
	Dermacentor silviarum Olenev,1927	there is no indication		33
	Dermacentor reticulatus Fabricius, 1794 (=Dermacentor pictus Hermann,1804)	on animals, on vegetation	+	37
	Dermacentor marginatus Sulzer, 1776	there is no indication		33
Nest- burrow species of parasite	Ixodes arboricola Schulze et Schlottke,1929	on birds	+	15
		in nests	+
	Ixodes lividus Koch,1844	on birds	+	16
		in nests	+
	Ixodes apronophorus Schulze,1924	on birds	+	4
		on animals	+
	Ixodes crenulatus Koch.,1844	in burrows	+	4
	Ixodes trianguliceps Birula,1895	on animals	+	4
		in burrows	+

the territory of Europe species D. silvarum was absent. Described under this name tick, maybe should concern the species D. marginatus or D. reticulatus, and ticks described as D. marginatus, obviously, were D. reticulatus. As regards to the genus Dermacentor, we can say with certainty that the research on modern methodological level and a large faunistic material all stages of development of the ticks, gives grounds to assert that within the territory of Belarus inhabit one species of this genus - D. reticulatus. While it is possible the presence of local reproduction of this species, associated with cattle and the places of their grazing, in some areas of Belarus. This question requires further study. The message on detection of in Belarus, the tick D. silvarum is obviously a mistake.

Representatives of genus Haemaphysalis have different features of formation of the habitats [25]. From they may occur in Belarus ticks of this genus - H. concinna, the relict type, formed in humid and warm climates of the tertiary period. Settling of this species of tick is associated with birds, which have a high ability to move to the countryside. Tick H. punctata is the species of Western origin of the group species with the area of the Mediterranean type, whose ancestors lived through in the South of the Quaternary cold, began to settle, in the main, to the southern forests, providing them with the necessary moisture and heat. It belongs to the representative of the group which most spread to the North up to 50 degrees of North latitude. But its area of distribution does not fall outside the limits of Ukraine. It is possible, of course, assume that both detected in Belarus species of genus Haemaphysalis associated with the imported birds, moreover, that within the main part of the natural habitat, both in birds in all stages of development were found. In a paper devoted specifically ticks of birds [29], these species are not mentioned.

An example of the transition from the pasture to the nest- burrow type of parasitism is the only one the representative of the subgenus Exopalpiger Schlze - Ixodes trianguliceps Birula, 1895 - in Belarus. In according to data of N.A.Filippova [12] this species is characterized by primitive pastoral type of parasitism and expressed stenoecic, in the sense that species inhabit everywhere in the forest biocoenoses, in which, along with the understory and the grass soil litter are well-developed [8]. According to the data V. F. Sapegina [20] and a number of other authors, it refers to the nest- burrow type species. On the data of I. T. Arzamasov [5] in the conditions of Belarus larvae and nymphs of this species are found on hosts throughout the year, including the winter period. In the mouse’s burrow the active individuals in the course of the year were find [36]. Similar data obtained by us in the foci of tick-borne encephalitis in Central Belarus, where the index of abundance of I. trianguliceps on the dominant species of micromammalia - red voles - in the winter sometimes even exceeded the figures of the summer period. Females I. trianguliceps on animals only in the spring and summer time were occurred. The aforesaid gives us grounds to assert that, in any case, on the territory of Belarus, for I. trianguliceps is characterized by mixed type of parasitize, in which the larvae and nymphs, which allocate in burrows of rodents and on the animals, are active throughout the year, and imago is only in the spring-summer period, when they lead pasture way of life. On the cattle I. trianguliceps never was found [30].

Among the representatives of the nest- burrow tick complex on territory of the Republic of Belarus 3 species genus Ixodes (Ixodes arboricola Schulze et Schlottke, 1929, Ixodes apronophorus Schulze, 1924 and highly specialized parasite of the sand swallows Ixodes lividus Koch, 1844) were registered. It should be noted that, in 1981, of the larvae, as well as from the adult ticks I. lividus collected by us in the colony near the city of Minsk, where in 1974 from gamasin mites was isolated the virus of tick-borne encephalitis (TBE) [17,10], after 7 years were allocated 2 strains of viruses tick borne encephalitis (TBE) [9,18]. Detection of TBE virus in the nests of the sand swallows in the spring before the arrival of the birds testifies to the fact that the infection persisted in diapaused ticks, transovum infected with the virus.

In publication of Z.Z. Syargeeva Z.Z. [33] pointed out that, according to the literature, in Belarus meets another type of pasture ticks of the genus Ixodes - I. persulcatus P. Sch. Up to 1957 the data on displaying in Belarus tick this species is not found. But in 1957 [34] about the discovery in the focus of tularemia on the territory of the Borysov district 1 female, 2 nymphs and 1 larvae I. persulcatus (all the individuals in the fees from the plants in coniferous-broadleaf forest) were reported. Then this species was celebrated on the small rodents in the subzone of oak-dark coniferous forests in the North of Belarus [6] and in the South (the territory of the ÏÃÐÇ) [21].

The issue of the presence in biocenoses of Belarus ticks I. persulcatus demanded clarification and further study on the faunistic and genetic levels, because of its relevance in epidemiological terms. Ticks are carriers of many pathogens of various systematic groups of the animal Kingdom: viruses, bacteria, protozoa. In particular, they are the carriers of acute illness arbovirus nature - tick borne encephalitis (TBE). On the territory of the Russian Far East to Central Europe 3 genotype of the virus, the causative agent of the tick-borne encephalitis is registered. Carrier of the far Eastern and Ural-Siberian genotype is the taiga tick Ixodes persulcatus Schulze, 1930. A disease caused by these viruses, manifests itself in a severe form and in most cases ends with a fatal outcome. In the European region, including the territory of Belarus, circulates West genotype, carriers of which is species of ticks Ixodes ricinus (Linne, 1758). The disease runs in a less severe form, amenable to treatment and full recovery. Tick-borne encephalitis on the territory of Belarus is registered with 1925. In the 1990 and up to the present time marked a new period of reactivation of natural foci of tick-borne encephalitis, which is accompanied by the rise of the number and, most importantly, virus form in ticks. For the whole period of official registration in Belarus noted 2012 patients with tick-borne encephalitis. According to T.I. Samoilova [27] from 22 strains of the virus of tick-borne encephalitis in the territory of the Republic of Belarus 10 strains were genetyped. The results of molecular hybridization revealed that 7 strains belong to the Western genotype and one strain to the Far East genotype, carrier which is I. persulcatus. Thus, in the territory of the Republic of Belarus, in the main, virus Western gene variant of tick-borne encephalitis are circulate, and the basis of the virus population the strains of the average pathogenicity are formed. At the same time, the author is installed circulation on the territory of the Republic highly pathogenic strains, which allows to assume, that this strain not from ticks I.ricinus, but from ticks of the species I.persulcatus was selected.

Specific identification of the two species of ticks (I. ricinus and I. persulcatus) is difficult due to their morphological similarity. At the present time for the species diagnostics of ticks in addition to the microscopic methods researches are widely used methods of molecular-genetic analysis [7, 24]. Much of the research in this field is based on a study of the variability of the mitochondrial genome [22, 23, 31]. Also is carried out the analysis of phylogenetic relationships of species [14], including with the use of microsatellite markers [8, 26]. In connection with this, we carried out research to find a DNA-markers for species identification of morphologically similar species of ticks I. ricinus and I. persulcatus, found on the territory of Belarus. Ticks of the genus Ixodes on the territory of the Osipovicy and Mogilev districts of Mogilev region, as well as the Shchuchin district of Grodno region were collected. As a control used ticks I. persulcatus, collected on the territory of the Leningrad region, part of the area of distribution of ticks I. persulcatus, which there are dominant species among Ixodes ticks (around the city Saint-Petersburg).

Ticks DNA extraction after homogenization in liquid nitrogen was carried out according to the standard proteinase K DNA isolation protocol with following phenol-chloroform deproteinization [19]. PCR amplification was performed in 25-μl reaction mixture containing 1 μl DNA, 0.2 μmol of each primer/l, 250 μmol of each deoxynucleoside triphosphate (dNTP)/l, 2.5 mmol MgCl2/l, 1 U Taq polymerase, and 1× PCR buffer (Dialat Ltd., Moscow, Russia) using GeneAmp PCR System 2700 (Applied Biosystems) and MyCycler (BIORAD) thermal-cyclers with the following parameters: an initial denaturation step at 94 °C for 5 min; 30 cycles: denaturation at 94 °C for 60 s; annealing at 47 °C for 2 min and extension at 72 °C for 3 min; followed by final extension at 72 °C for 2 min. PCR-amplified products were further digested with endonuclease AluI (Fermentas AB., Lithuania) during the 12 hours and were resolved on 2% agarose gels in 1õÒÀÅ buffer at a voltage of 5V/cm. The electrophoregram was analyzed in UV light and was photographed using the digital camera Nikon 2100. During the DNA sequence alignment of the ITS2 (ribosomal internal transcribed spacer 2, localized between rRNA ribosomal subunits 5,8S and 28S genes) of I. ricinus and I. persulcatus ticks (number GenBank D88883 and D88877) the recognition site of endonuclease Alu I for I. persulcatus sequence was revealed, not typical for species I. ricinus. Using a program GENERUNR the following PCR primers were designed: Forward 5'CTC TTT GAA CGG ACA TTG 3 and Reverse 5’ GTT GTC TGG CCT GAT T 3’. 801 base pairs long PCR products of I. persulcatus were digested by AluI and two fragments of the size 638 and 163 of base pairs were identified. While for I. ricinus 801 base pairs long PCR products only were detected.

Ticks, collected on the territory of different regions of Belarus have been investigated, the DNA has been highlighted and PCR-RFLP analysis has been held. As a control used samples of I. persulcatus, which collected on the territory of its distribution range - the Leningrad region. It was found that all the investigated ticks, collected in the territory of the various districts of the country were treated to the species I.ricinus [1]. To identification the tick species in the collection from Vitebsk region 12 copies of adults (males and females), presumably on the basis of morphological signs referred to species I. persulcatus the above-mentioned set of primers for species identification of ticks were used and the correctness of definition of the detected ticks was confirmed [2].

The discovery in Belarus taiga tick from the theoretical point of view expands our knowledge of fauna of this important type of bloodsucking arthropods. The scientific findings are the basis for further research on the structure of the parasitic system of taiga tick and ways of its formation in the conditions of antropogenic impact on ecosystems. In addition, in connection with the «global warming» more and more seriously the question arises about what changes will occur in the distribution and ecology of vectors of the transmissible disease and, in particular, Ixodidae ticks.

Thus, in the list of species of the Ixodidae ticks, which were described on the territory of Belarus there are 12 species. From which are everywhere and with high quantity 2 species are founded: I. ricinus and D. reticulatus. Imaginal stages of both species prefer cattle and large wild mammals as hosts, but they are able to attack the men. Preimaginal stages of I. ricinus parasitize on almost all species of mammals, birds and reptiles. The selectivity of them to the host species is determined only by the degree of accessibility of the victims. Larvae and nymphs of species D. reticulatus, on the contrary, are found on birds very rare. In more or less congested state ticks on bird, practically, are not detected and can be attributed to a number of specific parasites mammals. Despite the differences in the phenology, biology, station confinement, these species have a number of similarities, as vectors of transmissible diseases of man and domestic animals, for which they are able to act as an obligatory or the facultative hosts. The main feature of role of these species in circulation of pathogens transmitted by them is the ability to transfer by way of ovum or phase passing of agents, regardless of their systematic position, which provides the long-term existence of the foci of infections and invasions. The main difference between the two species as vectors in the conditions of Belarus consists in the fact that the tick I. ricinus develops in 3-4 year life cycle whereas D. reticulatus – in one year. The development during one year, not for 3-4 as I. ricinus determines the fate of pathogens transmitted by them and the long years of the dynamic features of the foci. With these two species of widespread of ticks pasturable type (I. ricinus and D. reticulatus) associated pathogens of human and animal diseases of viral, borreliosis, protozoan and bacterial nature that need to be taken into account while the tick activities and the assessment of their significance for health, and veterinary medicine. Indeed, apart from the West of tick-borne encephalitis and West Nile fever in Belarus circulates yet, at least, 2 arboviruses tick-borne transmission, which with the big share of probability, may manifest itself as the emerging-reemerging infections.

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