Investigation of the tick-borne pathogens Rickettsia helvetica and Anaplasma phagocytophilum in the blood of the domestic goat (Capra hircus).

The bacterial species Anaplasma phagocytophilum and Rickettsia helvetica are pathogenic for humans and domestic animals and are transmitted by ticks, e.g., of the Ixodes genus. Most of the vertebrate species constituting reservoirs for anaplasmas are known, but the potential reservoirs of rickettsiae are still under discussion. This study presents an analysis of the DNA of tick-borne pathogens isolated from the whole blood of goats grazing on meadows in West Pomerania, Poland. No DNA of A. phagocytophilum was found in the blood of the goats, while the DNA of R. helvetica was detected in 5.5% of the animals. The potential role of ruminants in the circulation of R. helvetica remains unknown.

Virus detection in questing ticks is not a sensitive indicator for risk assessment of tick-borne encephalitis in humans.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted arbovirus causing human disease in Europe, but information on its endemic occurrence varies between countries because of differences in surveillance systems. Objective data are necessary to ascertain the disease risk for vaccination recommendations and other public health interventions. In two independent, separately planned projects, we used real-time RT-PCR to detect TBE virus in questing ticks. In Poland, 32 sampling sites were selected in 10 administrative districts located in regions where sporadic TBE cases were reported. In Germany, 18 sampling sites were selected in two districts located in a region with high TBE incidence. Altogether, >16,000 ticks were tested by real-time RT-PCR, with no sample testing positive for TBEV. A systematic search for published studies on TBEV prevalence in ticks in Poland and Germany also suggested that testing large numbers of collected ticks could not consistently assure virus detection in known endemic foci. Although assignment of results to administrative regions is essential for TBE risk mapping, this was possible in only 10 (investigating 22,417 ticks) of 15 published studies (>50,000 ticks) identified. We conclude that the collection and screening of ticks by real-time RT-PCR cannot be recommended for assessment of human TBE risk. Alternative methods of environmental TBEV monitoring should be considered, such as serological monitoring of rodents or other wildlife.

Variability within the msp2 gene in populations of Anaplasma phagocythopilum.

Anaplasma phagocytophilum is an obligate intracellular bacterial parasite of verterbrate granulocytes. This bacterium is the aetiologic agent of human granulocytic anaplasmosis. The msp2 gene encoding major surface protein 2 is unique for Anaplasma and displays high antigenic variation. A fragment of the msp2 gene (334 bp) of A. phagocytophilum, amplified with DNA isolated from Ixodes ricinus, Syringophilidae, Capreolus capreolus and Canis lupus familiaris, was used to determine polymorphisms of Anaplasma within Polish populations. Sequence analysis of this fragment was used for observation of five different genetic variants of the bacterium within Polish sequences. The average genetic distance in Polish sequences was 0.7 % and the majority of observed substitutions had a synonymous character. High intraspecific variability observed in the msp2 gene of A. phagocytophilum is a strong proof of the high evolutionary plasticity, adaptation abilities, and abilities for fast distribution of this parasite in various environments.

Divergence within the marker region of the groESL operon in Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an obligate intracellular bacterial parasite in human and animal granulocytes. In Europe, A. phagocytophilum is transmitted by Ixodes ticks; Ixodes ricinus is the vector of the parasite in Poland. In terms of epidemiology, the identification of pathogens in ticks increasingly relies on molecular techniques. Polymerase chain reaction (PCR) with species-specific primers is a tool that allows the quick and accurate detection of pathogens in ticks, humans, or animals. DNA was extracted from the blood of Capreolus capreolus and Cervus elaphus, and amplified using the primers HS1/HS6 (external) and HS43/HSVR (internal). For sequencing, six samples from roe deer and two samples from red deer were selected, and the resulting sequences were submitted to GenBank (accession numbers DQ779568, DQ779567, EU157919, EU157920, EU157921, EU157922). These nucleotide sequences were compared with each other and five variants were distinguished in roe deer and one in red deer. A comparison of the sequences of the author's database revealed 45 polymorphic sites of substitution character (76% transitions and 24% transversions). The homology tree revealed two groups, one with sequences only from roe deer, while the second with sequences isolated mainly from red deer, livestock animals, and humans. These strains of A. phagocytophilum are also present in Poland.

Molecular evidence of coinfection of Borrelia burgdorferi sensu lato, human granulocytic ehrlichiosis agent, and Babesia microti in ticks from northwestern Poland.

To assess the potential risk for tick-borne agents, Ixodes ricinus were collected from 2 sites in northwestern Poland. The ticks were tested by polymerase chain reaction for coinfection with Borrelia burgdorferi sensu lato (s. l.), human granulocytic ehrlichiosis (HGE) agent, and Babesia microti. Of the 533 processed ticks, 16.7% were positive for B. burgdorferi s. l., 13.3% for B. microti, and 4.5% for the HGE agent. Twenty ticks were coinfected with 2 or 3 of the pathogens.

[Prevelance of the etiological agent of human ehrlichiosis (HGE) in ticks from west-north Poland]. / Wstepne badania czynnika etiologicznego ludzkiej ehrlichiozy (HGE) w kleszczach z zachodniopólnocnej Polski.

Ehrlichias occur in ticks in the cells of their haemolymph-hematocytes. They enter the vertebrate host organism with the saliva of the tick, during a blood meal. Humans can also be the hosts for this pathogen. Two pathogens cause a humane disease-monocytic ehrlichiasis (E. chaffensis) or granulocytic ehrlichiasis (HGE factor). The above disease units are difficult to diagnose because of their non-specific symptoms. A preliminary study has been conducted on the prevalence of the HGE factor in the ticks, Ixodes ricinus in the recreational areas of the West-Pomeranian Province. All forms of I. ricinus were collected from 3 sites. All the sites are known to be frequented by hikers and gatherers of forest mushrooms and berries. The site selection involved also careful consideration of the tree- and underbrush type. The ticks were collected twice a year in spring (May/June) and in autumn (August\September), which was associated with the biological activity of the collected acarines. A total of 1159 Ixodes ricinus ticks were collected, in this number 172 females, 167 males, 597 nymphs, and 223 larvae. Using the PCR technique, the 16SrRNA-gene fragment was amplified using primers specific for the HGE factor: EHR 790 and EHR 521. The studied population contained 3.7% infected females in spring and 2.7% in autumn, 0.68% infected males in spring, no infected in autumn. The nymphs were infected in spring (2.17%) and in autumn too (0.73%), but the larvae were not infected in both seasons. Analysing the above-mentioned results it can be concluded that the decisive majority of the individuals transmitting the HGE factor are the adult forms. The present study was only a preliminary one. In the future much more sites will be monitored, in the recreational areas of both the city of Szczecin and the entire province.