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Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

ABSTRACT: Continuous culture of tick cell lines has proven a valuable asset in isolating and propagating several different vector-borne pathogens, making it possible to study these microorganisms under laboratory conditions and develop serological tests to benefit public health. We describe a method for effective, cost- and labor-efficient isolation and propagation of Rickettsia raoultii using generally available laboratory equipment and Rhipicephalus microplus cells, further demonstrating the usefulness of continuous tick cell lines. R. raoultii is one of the causative agents of tick-borne lymphadenopathy (TIBOLA) and is, together with its vector Dermacentor reticulatus, emerging in novel regions of Europe, giving rise to an increased threat to general public health.Dermacentor reticulatus ticks were collected in the Donau-Auen (Lobau) national park in Vienna, Austria. The hemolymph of ten collected ticks was screened by PCR-reverse line blot for the presence of rickettsial DNA. A single tick tested positive for R. raoultii DNA and was used to infect Rhipicephalus microplus BME/CTVM2 cells.Sixty-five days after infection of the tick-cell line with an extract from a R. raoultii-infected tick, we observed intracellular bacteria in the cultured cells. On the basis of microscopy we suspected that the intracellular bacteria were a species of Rickettsia; this was confirmed by several PCRs targeting different genes. Subsequent sequencing showed 99-100 % identity with R. raoultii. Cryopreservation and resuscitation of R. raoultii was successful. After 28 days identical intracellular bacteria were microscopically observed.R. raoultii was successfully isolated and propagated from D. reticulatus ticks using R. microplus BME/CTVM2 cells. The isolated strain shows significant molecular variation compared to currently known sequences. Furthermore we show for the first time the successful cryopreservation and resuscitation of R. raoultii.

SUBMITTER: Wijnveld M

PROVIDER: S-EPMC5093946 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

Wijnveld Michiel M Schötta Anna-Margarita AM Pintér Adriano A Stockinger Hannes H Stanek Gerold G

Parasites & vectors 20161103 1

<h4>Background</h4>Continuous culture of tick cell lines has proven a valuable asset in isolating and propagating several different vector-borne pathogens, making it possible to study these microorganisms under laboratory conditions and develop serological tests to benefit public health. We describe a method for effective, cost- and labor-efficient isolation and propagation of Rickettsia raoultii using generally available laboratory equipment and Rhipicephalus microplus cells, further demonstrat ...[more]

PMID: 27809928

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Project description:Tick cell lines play an important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms. In an attempt to derive continuous Dermacentor reticulatus cell lines, embryo-derived primary cell cultures were set up from eggs laid by field ticks originally collected as unfed adults in The Netherlands and maintained for up to 16 months. After several months, it became evident that cells in the primary cultures were infected with a Rickettsia-like intracellular organism. Supernatant medium containing some D. reticulatus cells was inoculated into cultures of 2 Rhipicephalus (Boophilus) microplus cell lines, BME/CTVM2 and BME/CTVM23, where abundant growth of the bacteria occurred intracellularly on transfer to both cell lines. Bacterial growth was monitored by light (live, inverted microscope, Giemsa-stained cytocentrifuge smears) and transmission electron microscopy revealing heavy infection with typical intracytoplasmic Rickettsia-like bacteria, not present in uninfected cultures. DNA was extracted from bacteria-infected and uninfected control cultures, and primers specific for Rickettsia 16S rRNA, ompB, and sca4 genes were used to generate PCR products that were subsequently sequenced. D. reticulatus primary cultures and both infected tick cell lines were positive for all 3 Rickettsia genes. Sequencing of PCR products revealed 99-100% identity with published Rickettsia raoultii sequences. The R. raoultii also grew abundantly in the D. nitens cell line ANE58, poorly in the D. albipictus cell line DALBE3, and not at all in the D. andersoni cell line DAE15. In conclusion, primary tick cell cultures and cell lines are useful systems for isolation and propagation of fastidious tick-borne microorganisms. In vitro isolation of R. raoultii from Dutch D. reticulatus confirms previous PCR-based detection in field ticks, and presence of the bacteria in the tick eggs used to initiate the primary cultures confirms that transovarial transmission of this Rickettsia occurs.

Project description:In Europe, two tick species of the genus Dermacentor occur, Dermacentor marginatus and Dermacentor reticulatus. When the spatial distribution of both species in Germany was studied comprehensively for the first time in 1976, D. marginatus populations were recorded along the Rhine and Main river valleys in southwestern Germany, while D. reticulatus was very rare. In the last 50 years, however, a considerable range expansion of D. reticulatus has been noted in several European countries. To assess the current distribution of Dermacentor spp. in Germany, citizens were asked to send in ticks suspected to belong to the genus Dermacentor or that were of "unusual" appearance. From February 2019 until February 2020, 3,902 Dermacentor ticks were received in total. Of those, 15.48% (604/3,902) were identified as D. marginatus and 84.24% (3,287/3,902) as D. reticulatus, while 11 specimens could not be identified to species level. The majority of D. reticulatus specimens was collected from dogs (1,212/2,535; 47.12%), while D. marginatus was mostly collected from horses (184/526; 34.98%). Our results confirm that the adults of both Dermacentor species are active all year round. D. reticulatus specimens were sent in from all federal states except the Free and Hanseatic City of Hamburg, while D. marginatus specimens were only received from locations in southwestern Germany. Overall, data obtained from this citizen-science study show that D. reticulatus has significantly expanded its range, especially in northern Germany. Regarding D. marginatus, new locations northwest of the previous range were detected, although the distribution has remained rather stable as compared to D. reticulatus. The spread of D. reticulatus, the vector of Babesia canis, is of major importance for veterinarians and dog owners in terms of canine babesiosis outbreaks or endemization in hitherto B. canis-free areas. Thus, veterinarians and veterinary students need to be informed about the new situation to be able to give adequate advice to dog owners on the extended D. reticulatus range and appropriate control measures.

Dataset Information

Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

Publications

Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

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