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Prevalence of Tick-Borne Encephalitis in Ixodes Ricinus and Dermacentor

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Prevalence of Tick-Borne Encephalitis in Ixodes Ricinus and Dermacentor VYTAUTAS MAGNUS UNIVERSITY FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BORIS MOSSE Prevalence of Tick-Borne encephalitis in Ixodes ricinus and Dermacentor reticulatus ticks in Belarus Bachelor Thesis “Biology and Genetics” study program, state code 6121DX011 Life Sciences SUPERVISOR: ALGIMANTAS PAULAUSKAS DEAN OF THE FACULTY: PROF. DR. SAULIUS MICKEVIČIUS KAUNAS 2020 1 Experimental work was done during autumn semester 2019. Reviewer of the bachelor’s thesis is: The work is defended: at a remote public meeting of the Bachelor's Thesis Defense Commission on 18s of june, 2020, 9:00 remotely. Vytautas Magnus University, Department of Biology. Address of the department: Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos street 8, lt-44404, Kaunas, republic of Lithuania. 2 CONTENTS SUMMARY ............................................................................................................................... 4 INTRODUCTION ...................................................................................................................... 5 Main goal of the work: ................................................................................................................ 5 Tasks of the work: ................................................................................................................... 5 LITERATURE REVIEW ............................................................................................................ 6 1. General biology of Ixodes ticks ............................................................................................ 6 1.2. Classification ................................................................................................................ 6 1.2. Life cycles of ticks ........................................................................................................ 6 1.3 Geographical distribution and habitats of ticks ............................................................... 7 1.4 Pathogens transmitted by Ixodes ticks ............................................................................ 8 2. Epidemiology of tick-borne pathogens................................................................................. 9 2.1 Lyme borreliosis and tick-borne encephalitis in Europe .................................................. 9 2.2 Low prevalent pathogens distribution ........................................................................... 10 2.3. Ticks distribution and epidemiological situation of Tick-Borne Pathogens in Belarus . 11 3 Tick-borne encephalitis virus .............................................................................................. 14 3.1 Classification of tick-borne encephalitis virus .............................................................. 14 3.2 Genome and structural characteristics of tick-borne encephalitis virus ......................... 15 3.3 Tick-borne encephalitis pathogenesis and virus replication ........................................... 16 3.4 Tick-borne encephalitis virus natural cycles ................................................................. 17 3.5 Prevalence rate of tick-borne encephalitis virus in ticks ................................................ 17 MATERIALS AND METHODS .............................................................................................. 18 1. Collection of questing ticks ............................................................................................... 18 2. Identification of ticks ......................................................................................................... 18 3. RNA extraction from tick samples ..................................................................................... 20 4. Real-time reverse transcription-PCR .................................................................................. 22 5. Pooled prevalence calculations .......................................................................................... 23 RESULTS AND DISCUSSION ................................................................................................ 23 CONCLUSIONS ...................................................................................................................... 26 REFERENCES ......................................................................................................................... 28 3 SUMMARY With an annual average number of more than 2,900 cases, tick-borne encephalitis is the vector-borne diseases with largest impact on human health in Europe. Over the last decades, Belarus contributes a highly endemic area for major tick- borne diseases comparing to other European countries. To evaluate the existence of encephalitis virus endemic foci we determined the prevalence of TBEV in I. ricinus and D. reticulatus ticks collected from different locations in Belarus. A total of 52 ticks were collected at two locations of Belarus in south and north of Minsk region during April-June 2019. The number of places planned for this research was significantly reduced due to the coronavirus pandemia and border closure. Samples were screened for the presence of specific TBEV RNA by quantitative real-time PCR. Ticks positive for encephalitis virus were not detected. Parallel study held at the same period by Belarus Centre for Epidemiology and Microbiology detected seven positive samples of I. ricinus and D. reticulatus from four collection sites. The TBEV prevalence was estimated as 2.23% in I. ricinus and 1,75% in D. reticulatus. Thus, encephalitis continues to be a priority tick-borne infection in Belarus. 4 INTRODUCTION Ticks are obligate haematophagous (blood-feeding) ectoparasites that feed on a variety of vertebrate host animals, including mammals, birds, reptiles, and sometimes amphibians. They transmit a wide range of disease-causing organisms and are of great medical and veterinary importance. In Europe the diseases transmitted by ticks are considered the most important vector-borne diseases. Concerning impact on human health the two most widespread are Lyme borreliosis and tick-borne encephalitis. They also play a major role in the transmission of other tick-born ediseases, in particular anaplasmosis, babesiosis, tularemia, and rickettsiosis. The wide distribution of bacterial, virus and protozoal agents of human pathogenicity and the extreme range of their prevalence in ticks indicate the need for comprehensive studies on ticks and tick- borne pathogens in particular in Eastern Europe countries. Main goal of the work: To evaluate the existence of encephalitis virus endemic foci and determine the prevalence of TBEV in ticks collected from different locations in Belarus. Tasks of the work: (i) Collect data about distribution of I. ricinus and D. reticulatus in Belarus. (ii) Check the presence of I. persulcatus in Belarus (iii) Make statistical analysis of the prevalence of TBE in this species. 5 LITERATURE REVIEW 1. General biology of Ixodes ticks 1.2. Classification Ticks are arthropods closely related to mites, with which they form the subclass Acari within the class Arachnida. Members of the Acari are the only known parasitic arachnids. The 896 known species of ticks can be further divided into three families, the Argasidae (soft ticks, 193 species), the Ixodidae (hard ticks, 702 species), and the Nutalliellidae (which consist of only one species from Africa, Nuttalliella namaqua) (Guglielmone et al., 2010). Hard ticks can be easily identified by their visible mouthparts, and by the presence of the scutum, a plate-like shield on the dorsal surface. They normally feed only once during each of the three parasitic life stages (larva, nymph, adult female) and remain attached to the host for a long time (Gray, 2002). The name of the type species Ixodes ricinus is derived from the greek word "ixos" for the European mistletoe plant (Viscum album). Soft ticks lack a hardened upper surface and have less conspicuous mouthparts than ixodes ticks. They feed repeatedly, each time taking only small amounts of blood, and remain normally in the close vicinity of their hosts, such as in their nests and burrows (endophilic behaviour) (Gray, 2002). While some argasid species do transmit diseases, ixodes ticks are by far more important vectors for several zoonoses (infectious diseases that can be transmitted between animals and humans) and are considered the most important ectoparasites of livestock (Gray, 2002). Following morphological comparisons and molecular studies a group of 21 Ixodes species was suggested named as Ixodes ricinus-persulcatus group (Filippova, 2002). This complex contains all four Ixodes species (I. ricinus, I. persulcatus, I. scapularis, and I. pacificus) that are the main vectors for Lyme borreliosis (LB)-causing spirochaetes. Based on phylogeographic data, it was assumed that the European population of this tick species is genetically mostly homogeneous (Noureddine et al., 2011). Later, a phylogeographic genetic structure was revealed by use of mitochondrial genes multi-sequence analysis at least in the case of geographically distant populations (Dinnis et al., 2014). 1.2. Life cycles of ticks The life cycles of ixodes ticks, during which there are four stages (egg, larva, nymph, and adult), take between one and six years. Each of the three parasitic stages seeks the hosts, attaches, feeds and drops off. Most ixodes species require the new hosts for each feeding stage 6 (i.e. they are three-host ticks). Hosts may be confined to one single species
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