Katarína Štefanidesová

Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic

Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild to severe diseases in humans are associated with arthropod vectors. Dermacentor marginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rickettsia raoultii distributed across Europe. A total of 794 D. marginatus, D. reticulatus and Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep, goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR amplifying parts of gltA, ompA and sca4 genes. Next analyses of Rickettsia-positive samples by PCR–RFLP and/or sequencing showed D. reticulatus ticks were more infected with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of R. raoultii was 8.1–8.6% and 22.3–27% in D. marginatus and D. reticulatus, respectively. The prevalence of R. slovaca was 20.6–24.3% in D. marginatus and 1.7–3.4% in D. reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was evaluated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multiplication was recorded on the 7th and 8th day postinfection in Vero and L929 cells, respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. Our study describes the detection and isolation of tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and highlight their risk for humans.

Rickettsial Agents in Slovakian Ticks (Acarina, Ixodidae) and Their Ability to Grow in Vero and L929 Cell Lines

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty‐six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA‐based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR‐positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram‐negative cell wall, and a cytoplasmic membrane.

Serologic evidence of Anaplasma phagocytophilum infections in patients with a history of tick bite in central Slovakia

ZusammenfassungZiel der Studie war es, das Risiko einer Anaplasmen-Infektion in bekannten Endemie-Gebieten der Lyme-Borreliose in der Zentral-Slowakei zu erfassen. Die Zirkulation von Anaplasma phagocytophilum in Zecken und Wildtieren wurde schon seit Jahren in Naturherden dieses Gebietes beobachtet. Aus dieser Region wurden Serumproben von Patienten gewonnen, die nach einem Zeckenstich klinische Symptome der Lyme-Borreliose entwickelt hatten. Insgesamt wurden Seren von 76 Patienten mittels eines kommerziellen Immunofluoreszenz Assays zum Nachweis von IgG Antikörpern gegen A. phagocytophilum getestet; 19 (25%) waren positiv. Eine Infektion mit A. phagocytophilum wurde bei einem Kind (4%), 12 Erwachsenen (39%) im Alter zwischen 22 und 56 Jahren und bei sechs Personen (32%) über 56 Jahre serologisch bestätigt. Ein statistisch signifikanter Unterschied in der Seroprävalenz (P < 0,01) wurde zwischen Kindern und Erwachsenen ermittelt. IgG-Antikörper gegen A. phagocytophilum wurden bei sieben Patienten mit klinischer Lyme-Borreliose, bei sieben Personen mit Antikörpern gegen Borrelia burgdorferi sensu lato und bei fünf Borrelien-seronegativen Personen ermittelt. Die häufigsten Symptome bei den A. phagocytophilum seropositiven Patienten waren Kopfschmerzen, Gelenks- und Muskelschmerzen, Fieber, Ausschlag, und Lymphknotenschwellung. Diese Patienten stammten aus den Gemeinden Vtáčnik (5/19), Štiavnické vrchy (1/19), Kremnické vrchy (10/19) und Žiarska kotlina (3/19). Die Ergebnisse zeigen, dass ein Risiko für Infektionen mit A. phagocytophilum in den Naturherden der Zentral-Slowakei besteht, sodass die humane Anaplasmose in die Differentialdiagnose mit einbezogen werden sollte, insbesondere dann, wenn eine akut fieberhafte Erkrankung nach Zeckenstich auftritt.SummaryThe aim of this study was to determine the risks of human anaplasmosis in an area of central Slovakia endemic for Lyme borreliosis. The circulation of Anaplasma phagocytophilum in ticks and wild animals has been observed in natural foci in this area for several years. Samples of human sera from patients with Lyme borreliosis and persons with a history of recent tick bite and clinical symptoms indicating Lyme borreliosis were collected in central Slovakia. A total of 76 human sera were analyzed using an indirect HGE IgG immunofluorescent assay kit. IgG antibodies against A. phagocytophilum were found in 19 (25%) sera (15 female, 4 male patients). A. phagocytophilum infection was serologically confirmed in one (3.8%) child, 12 (38.7%) persons aged 22–56 and six (31.6%) persons older than 56. A statistically significant difference in seroprevalence (P < 0.01) was observed between children (3.8%, 1/26) and adults (36%, 18/50). Antibodies against A. phagocytophilum were detected in seven patients with clinically diagnosed Lyme borreliosis and in another seven individuals with assessed antiborrelia antibodies. IgG antibodies against A. phagocytophilum were detected in five persons seronegative for borrelia. The most frequent clinical symptoms in patients with positive A. phagocytophilum serology were cephalalgia, arthralgia, myalgia, fever, exanthema, neurological symptoms and lymphadenopathy. Positive sera were obtained from patients living in villages and towns in the orographic entities Vtáčnik (5/19), Štiavnické vrchy (1/19), Kremnické vrchy (10/19) and Žiarska kotlina (3/19). Our results demonstrate the risk of acquiring A. phagocytophilum infection in natural foci in central Slovakia. Human anaplasmosis should be considered in the differential diagnosis, especially in cases of acute febrile illness with tick-bite history.

Molecular evidence of Rickettsia spp. in ixodid ticks and rodents in suburban, natural and rural habitats in Slovakia

BackgroundNatural foci of tick-borne spotted fever group (SFG) rickettsiae of public health concern have been found in Slovakia, but the role of rodents in their circulation is unclear. Ticks (Ixodes ricinus, Ixodes trianguliceps, Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna and Haemaphysalis inermis) and tissues of rodents (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus and Micromys minutus) were examined for the presence of SFG rickettsiae and Coxiella burnetii by molecular methods. Suburban, natural and rural habitats were monitored to acquire information on the role of ticks and rodents in the agents’ maintenance in various habitat types of Slovakia.ResultsThe overall prevalence of rickettsial infection in questing I. ricinus and D. marginatus was 6.6% and 21.4%, respectively. Rickettsia helvetica, R. monacensis and non-identified rickettsial species were detected in I. ricinus, whereas R. slovaca and R. raoultii were identified in D. marginatus. Rickettsia spp.-infected I. ricinus occurred during the whole tick questing period. Rickettsia helvetica dominated (80.5%) followed by R. monacensis (6.5%). The species were present in all studied habitats. Rickettsia slovaca (66.7%) and R. raoultii (33.3%) were identified in D. marginatus from the rural habitat. Apodemus flavicollis was the most infested rodent species with I. ricinus, but My. glareolus carried the highest proportion of Rickettsia-positive I. ricinus larvae. Only 0.5% of rodents (A. flavicollis) and 5.2% of engorged I. ricinus removed from My. glareolus, A. flavicollis and M. arvalis were R. helvetica- and R. monacensis-positive. Coxiella burnetii was not detected in any of the tested samples. We hypothesize that rodents could play a role as carriers of infected ticks and contribute to the maintenance of rickettsial pathogens in natural foci.ConclusionsLong-term presence of SFG Rickettsia spp. was confirmed in questing ticks from different habitat types of Slovakia. The results suggest a human risk for infection with the pathogenic R. helvetica, R. monacensis, R. slovaca and R. raoultii.

Life cycle of Rickettsia slovaca in L929 cell line studied by quantitative real‐time PCR and transmission electron microscopy

Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.

The repellent efficacy of eleven essential oils against adult Dermacentor reticulatus ticks

Dermacentor reticulatus ticks are among the most important arthropod vectors of zoonotic disease agents in Europe. Eleven essential oils, namely basil (Ocimum basilicum), bergamot (Citrus bergamia), clove bud (Syzygium aromaticum), citronella (Cymbopogon winterianus), creeping thyme (Thymus serpyllum), lavender (Lavandula angustifolia), lemon-scented gum (Corymbia citriodora), marjoram (Origanum majorana), peppermint (Mentha piperita), spearmint (M. spicata), and red thyme (Thymus vulgaris) were tested for repellency against adult D. reticulatus ticks at concentrations of 1 and 3%. Clove bud, creeping thyme and red thyme essential oils were the most efficient - repelling 83, 82 and 68% of ticks when diluted to 3%, respectively. The mixture of creeping thyme and citronella containing 1.5% of each showed higher repellency (91%) than individual essential oils at the concentration of 3%.

Ultrastructural study of the life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and vero cell lines

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Anaplasma phagocytophilum and other tick-borne bacteria in wild animals in western Slovakia

Tick-borne bacterial zoonoses of livestock and free-ranging ungulates caused by Anaplasma spp. are common in Central Europe. The aim of this study was to analyze the prevalence of Anaplasma phagocytophilum and other tick-borne bacteria in wild animals from western Slovakia. Infection with A. phagocytophilum was recorded in 62.86% of analyzed roe deer (Capreolus capreolus), in two red deer (Cervus elaphus) and two wild boars (Sus scrofa). Dermacentor reticulatus and Ixodes ricinus ticks collected on red deer were not A. phagocytophilum-infected. However, spotted fever group rickettsiae were detected in ticks collected from red deer. High prevalence of A. phagocytophilum in roe deer as well as positive red deer and wild boars suggest the occurrence of natural foci in western Slovakia.

Low-cost light-induced therapy to treat rickettsial infection

Antimicrobial photodynamic therapy uses a nontoxic photosensitizer with the assistance of harmless visible light to activate the photosensitizer. Consequently, the excited state of the photosensitizer interacts with molecular oxygen to produce reactive oxygen species, which have the antimicrobial effect. In this study, we evaluated the effect of photodynamic therapy on Vero cells infected with rickettsia using methylene blue as a photosensitizer along with red light. A significant reduction (by 96%) in the number of viable Rickettsia slovaca was determined by quantitative RT-PCR 48 h after the treatment with methylene blue followed by 30 min of red light excitation. A statistically significant reduction of R. slovaca was also recorded with pretreatment (by 99%). To the best of our knowledge, this result is the first one in the literature to confirm the effectiveness of photodynamic therapy for the elimination of R. slovaca and to suggest this technique as a good supportive treatment for rickettsial infections.