Prevalence of causative agents of transmissive tick-borned ricketsious in the Crimean peninsula

Abstract

The paper presents the results of a study of the prevalence of Ixodid ticks - potential carriers of tick-borne rickettsiosis pathogens. Ectoparasites were collected in various natural and climatic zones of the Crimean Peninsula within the year 2016-2018. As a result of screening with the help of real-time PCR analysis (PCR-RT), a genetic marker (a section of the gltA gene) of the rickettsia group of tick-borne spotted fever was detected in ticks. The most common DNA marker of rickettsia was found in ticks in the eastern regions of the steppe zone - 50,6 %, in the north-western part of the steppe zone this value was 12,0 %. The least amount of rickettsia target DNA was detected in ticks collected in the mountain forest and south bank zones - 4,5 %. As a result of sequencing of positive DNA samples from fragments of the gltA, ompA, ompB, and sca4 genes, the species composition of rickettsias was established. The DNA of 8 species of rickettsia was identified: Circulation of three R. conorii, R. massiliae, R. sibirica subsp. mongolotimonae, R. slovaca, R. aeschlimannii, R. monacensis, R. helvetica, R. raoultii. R. massiliae, R. slovaca, and R. helvetica were established in the Crimean Peninsula for the first time. The peculiarities of the geographical distribution of the identified rickettsia species were determined, which was due to the spread of mites-carriers of pathogens. The revealed diversity of rickettsia species and their vectors, due to the isolation of the areas of the main feeding animals and the established routes of migratory birds, suggests the circulation of other rickettsia species on the territory of the Crimean Peninsula. The obtained results suggest that the diseases of tick-borne rickettsiosis in the Crimean Peninsula can be caused not only by R. conorii, as previously thought, but also by other types of rickettsii.

Use of eschar swab DNA to diagnose Rickettsia conorii subspecies conorii infection in Crimea: A case report

“…On day 2 of treatment (July 25) a scab was removed from the eschar and the underlying area was sampled using a dry sterile cotton swab by gentle rotation in the wound area and transferred into a sterile container containing 200 µL of sterile PBS and stored at −20 °C. Two separate DNA extractions were performed each using 100 µL of swab material using “Real Best Extraction 100” reagent kit according to the manufacturer's instructions (“Vector-Best”, Novosibirsk), purified DNA was eluted using 100 µL of the elution buffer as described previously [11 , 12] . Each DNA was first tested in triplicates for Rickettsia glt A fragment using “Real Best DNA Rickettsia species” reagent kit (“Vector-Best”) and corresponding positive and negative controls included with the kit.…”

“…Each DNA was first tested in triplicates for Rickettsia glt A fragment using “Real Best DNA Rickettsia species” reagent kit (“Vector-Best”) and corresponding positive and negative controls included with the kit. Amplification and sequencing larger portions of Rickettsia glt A, omp A, omp B and sca 4 was done according to the previously described protocols [11 , 12] . Primers used are listed in Supplementary Table S1.…”

“…Several tick-borne spotted fever group rickettsiae have been identified in Crimea; however, MSF is the only rickettsiosis that has been diagnosed in the peninsula [11 , 12 , 14] . Previously, we tested R. sanguineus collected from different parts of Crimea and identified DNA of R. conorii subspecies conorii in ticks collected in different locales including Saki district [11 , 12] .…”

“…Several tick-borne spotted fever group rickettsiae have been identified in Crimea; however, MSF is the only rickettsiosis that has been diagnosed in the peninsula [11 , 12 , 14] . Previously, we tested R. sanguineus collected from different parts of Crimea and identified DNA of R. conorii subspecies conorii in ticks collected in different locales including Saki district [11 , 12] . In this study, we molecularly confirmed a case of SFGR disease in Crimea caused by R. conorii subspecies conorii and corroborated the utility of eschar swab material as a source of DNA for PCR-based diagnostics.…”

History and Current Status of Mediterranean Spotted Fever (MSF) in the Crimean Peninsula and Neighboring Regions along the Black Sea Coast

“…Contemporary acarological studies conducted in the Crimea Peninsula during the last decade indicated the occurrence of diverse tick species in different ecological settings [ 92 , 93 , 94 ]. Nidicolous R. sanguineus is mostly present in the peridomestic environment [ 75 , 76 ].…”