Brown dog ticks collected in Romania and Hungary: molecular identification and pathogens

GHODRATI, Sajjad. Brown dog ticks collected in Romania and Hungary: molecular identification and pathogens. In EMOP XIV. 2024.

Basic information

• Original name: Brown dog ticks collected in Romania and Hungary: molecular identification and pathogens
• Authors: GHODRATI, Sajjad.
• Edition: EMOP XIV, 2024.

Other information

• Type of outcome: Conference abstract
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL

Abstract

Introduction Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) refers to a group of tick species commonly called brown dog ticks. Ticks in the R. sanguineus complex are three-host parasites typically found on dogs, occasionally infesting humans and a variety of wild and domestic animals, such as rodents and birds. Some of the brown dog ticks are considered invasive species with extending distribution, mainly due to their association with domestic dogs. These ticks are mostly found in tropical and subtropical areas and their fast-extending distribution range is apparently enhanced also by climatic and environmental changes. Rhipicephalus sanguineus s.l. are well-known vectors of a range of pathogens of medical and veterinary importance. These include Anaplasma, Ehrlichia, Rickettsia, and piroplasmids such as Babesia, Theileria, and Hepatozoon. The differences in vectorial capacity of different taxa within the R. sanguineus s.l. represent a neglected part in the ecology of this group of tick-borne pathogens because many previous studies do not distinguish properly between the species of these ticks. Moreover, the asymptomatic status of infected dogs from endemic foci further increases the chance of pathogen importation into non-endemic regions with traveling dogs. The taxonomy of R. sanguineus s.l. ticks has been questioned and debated for many years. Several factors contribute to this confusion, including the limited description of the original species and the high level of morphological similarity among ticks within the complex. For instance, R. rossicus looks very similar to R. pumilio, and R. linnaei resembles R. sanguineus sensu stricto, as do R. hibericus and R. turanicus. Accurate identification of R. sanguineus s.l. species is crucial for understanding their role as disease vectors. Despite recent research on the genetic diversity of these ticks and newly described taxa, there are still gaps in our understanding of the epidemiology, vector capacity, and vector competence. Only in the last three years, three new species have been distinguished, raising questions about their roles in disease transmission. For example, R. linnaei (2022) was previously known as the "tropical lineage" of Rhipicephalus sanguineus, distinct from the "temperate lineage" now referred to as R. sanguineus s.str. Similarly, R. rutilus (2023) was formerly referred to as the Southeastern Europe lineage of R. sanguineus, and R. hibericus (2024) was previously identified as R. turanicus in Southwestern Europe. Descriptions and re-descriptions of new species over the last decade suggest that many ticks “superficially” identified as R. sanguineus may actually be other species, calling for more intense research. In Europe, this includes not only the “core” distribution range in the Mediterranean but also countries where Rhipicephalus ticks range from sporadic to endemic, including Hungary and Romania. In Hungary, canine pathogens such as A. platys, B. vogeli, and R. conorii have never been reported. Similarly, the ability of R. rossicus, the common dog tick in Eastern Romania, to transmit pathogens is unclear and likely underestimated. The genetic complexity of the brown dog tick led us to conduct genetic identification of brown dog ticks collected from domestic dogs and vegetation in Hungary and Romania. We also investigated the presence of pathogens Ehrlichia, Anaplasma, Rickettsia, and piroplasmids, including Babesia, Theileria, and Hepatozoon.

Abstract (in English)

Materials and methods A total of 169 Rhipicephalus ticks were collected from Hungary and 119 from Romania. Most samples were collected from dogs and cats, while the rest were gathered from vegetation using the flagging method. All Hungarian samples were from Paks town in central Hungary, and all Romanian samples were from Chilia Veche in the Danube Delta region. Ticks were identified morphologically using standard keys and photographed under a KEYENCE VHX-5000 digital microscope (Keyence, Belgium) to be used for molecular identification of the ticks. All the ticks were analyzed by amplification and sequencing of COI, 16S rDNA, and 12S rDNA. All the obtained sequences were assembled, edited, and analyzed using the Geneious Prime® software v. 2023.0.1. and identified by the NCBI BLASTn analysis. Only the representative sequences for each unique haplotype (selected by ALTER (Alignment Transformation Environment were used for the final analysis. Maximum-likelihood (ML) phylogenetic trees with 1000 bootstraps were constructed for each gene using IQTREE. After genetic characterization, all samples were screened for pathogens, including Ehrlichia, Anaplasma, Rickettsia, Babesia, Theileria, and Hepatozoon. Results All ticks were morphologically identified as R. sanguineus s.l. using the standard keys. All 169 Rhipicephalus ticks were collected from 13 dogs in Hungary, sourced from a single veterinary clinic, suggesting a focal occurrence. Through maximum likelihood phylogenetic analysis, all 169 ticks were conclusively identified as Rhipicephalus sanguineus s.str., supported by robust bootstrap values across all three COI, 16S rDNA, and 12S rDNA gene sequences. In the case of tested Romanian ticks (73/119), phylogenetic analysis revealed the majority (n=62) to be R. rossicus, followed by R. sanguineus s.s. (n=10), and R. rutilus (n=1) supported by high bootstrap values in all three gene sequences. Among the 169 Hungarian samples, 168 tested negative for all pathogens assessed, with only one tick testing positive for A. phagocytophilum. None of the 119 Romanian ticks tested positive for Anaplasma, with the study still in progress.