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Genetic diversity of ixodid ticks Ixodes ricinus (L.) and tick-borne pathogens in foci of the Republic of Moldova


Author: Movilă Alexandru
Degree:doctor of biology
Speciality: 03.00.15 - Genetics
Year:2008
Scientific adviser: Ion Toderaş
doctor habilitat, professor, Institute of Zoology of the ASM
Scientific consultant: Maria Duca
doctor habilitat, professor, Moldova State University
Institution:
Scientific council:

Status

The thesis was presented on the 19 September, 2008
Approved by NCAA on the 23 October, 2008

Abstract

Adobe PDF document0.55 Mb / in romanian
Adobe PDF document0.62 Mb / in russian

Thesis

CZU 579.895.421(478)(043.3)+595.421(478)(043.3

Adobe PDF document 1.68 Mb / in russian
121 pages


Keywords

genetic polymorphism, molecular-genetic markers, ixodid ticks, tick-borne pathogens, vertebrate hosts, molecular phylogeny.

Summary

The aim of this study was to investigate the molecular-genetic diversity of Ixodes ricinus ticks, tick-borne pathogens and vertebrate hosts in foci of the Republic of Moldova.

Altogether, 750 ticks were examined from stationary foci (Chisinau city, “Codri” and “Padurea domneasca” forest reserves, “Vadul-lui-Voda” recreation zone) in the Republic of Moldova.

The genetic diversity of I. ricinus (L.) ticks was investigated at a 420 base pair fragment of the 12S rDNA mitochondrial genetic marker. The number of invariable, variable and noninformative sites were 86,69%, 2,91% and 10,4%, respectively. The frequency of transitions (1,3%) was higer than transvertions (1,11%). Gaps were detected in 0,5% cases. Following alignment, five different haplotypes were detected. Three haplotypes are unique for Republic of Moldova, while the other two are shared with ticks from the Central and Western Europe. Haplotype diversity was lowest in the “Codri” and “Padurea domneasca” forest reserves and greatest in the urbanocenosis of Chisinau city.

B. burgdorferi s.l. was detected in 13,7±0,8% of I. ricinus ticks. Borreliae were most prevalent in female ticks collected from Chisinau city. Using PCR-RFLP and PCR-RLB at the 23S-5S rDNA genetic marker we confirmed the prevalence of five B. burgdorferi s.l. species: B. burgdorferi s.s., B. garinii, B. afzelii, B. valasiana, B. lusitaniae. The dominant species in Moldova was B. afzelii.

Members of the family of Anaplasmataceae were detected in 8,02±0,43%. BLAST analysis of partially sequenced 16S rDNA PCR products confirmed these samples as Anaplasma phagocytophilum and Сandidatus Nicolleae massilliensis with 98% - 100% sequence similarity. In the phylogenetic tree constructed on the basis of 420 bp of the 16S rDNA, A. phagocytophilum sequences from Moldova clustered with granulocytic species that are pathogenic in humans and animals in Europe and the USA with 98% bootstrap support. Сandidatus Nicolleae massilliensis, on the other hand, were clearly separated with 100 bootstrap values. These data represent the first evidence of anaplasmataceae in the ticks from Moldova.

To evaluate the importance of vertebrate species as tick hosts and reservoir hosts in foci of the Republic of Moldova, we applied PCR-RLB targeting 12S rDNA vertebrate mitochondrial gene for blood meal analysis in questing I. ricinus ticks. The most prevalent identified host DNA for nymphs were two birds species (Turdus merula and Erithacus rubecula), two small mammal species (Apodemus sylvaticus and Clethrionomys glareolus) and one lizard (Lacerta viridis). The dominant hosts for adults ticks were foxes, deers and wild boars.

B. burgdorferi s.s., A. phagocytophilum and Сandidatus N. massilliensis are considered generalist species, there is evidence that B. garinii ribotype 20047 and B. valasiana are maintained by avian hosts and that B. afzelii, B. garinii ribotype NT 43, B. lusitaniae is more commonly associated with rodents and lizards hosts, respectively.