Simple Method to Differentiate among <b><i>Rickettsia</i></b> Species

Abstract

In this work we present a new option to identify 11 rickettsial species that cause human rickettsioses, with some advantages over the previous methods described. Using rickettsial isolates from 11 Rickettsia species as a sample, we used the polymerase chain reaction to amplify a 990- to 1,000-bp DNA fragment from the ompB gene, common for the 11 Rickettsia species analyzed in this study, which were digested with AluI restriction enzyme to obtain different digestion patterns. This restriction pattern can be visualized using a polyacrylamide gel electrophoresis technique. Using this method we could differentiate between the 11 Rickettsia species analyzed regardless of the group to which the Rickettsia belonged. We developed a simple method to identify 11 Rickettsia species which cause human rickettsioses using polymerase chain reaction and restriction fragment length polymorphism techniques with the advantage that it only needs one amplicon and only one restriction enzyme to obtain the restriction pattern. The identification of the species infecting vectors, reservoirs, and humans is essential to establish the ecological and behavioral ecosystem involved in its maintenance and transmission in nature in the specific region where the pathogen is circulating. This method is very helpful to identify Rickettsia species in a short time.

Rickettsia typhi IN RODENTS AND R. felis IN FLEAS IN YUCATÁN AS A POSSIBLE CAUSAL AGENT OF UNDEFINED FEBRILE CASES

“…PCR and sequencing: Conventional Polymerase Chain Reaction (PCR) technique was selected to amplify DNA fragments from two different rickettsial genes: 17 kDa lipoprotein and rickettsial outer membrane protein B ( ompB ). A 434 bp fragment from the rickettsial gene 17 kDa was obtained using primers Fw1: 5´-GCTCTTGCAACTTCTATGTT-3´ and Rv2: 5´-CATTGTTCGTCAGGTTGGCG-3´) 22 and a 990 - 999 bp fragment of the rickettsial gene ompB using primers ompB330(1) fw (5'-ATGGCTTCAAAAACCAAATTTTCTAA-3') and ompB330(1)Rv (5'- AGCTCTACCTGCTCCATTATCTGTACC-3') 15 . PCR reaction was performed using Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and using a Multigene Thermalcycler (Labnet International, Inc).…”

Molecular screening of ticks for the presence of Rickettsia species: A public health concern

“…that are found in many European countries and the major vectors of these bacteria are Dermacentor marginatus and Dermacentor reticulatus ticks as a high percentage of these ticks have been reported to be infected with these bacteria [31][32][33][34][35][36][37][38][39][40]. In a study conducted in Spain, R. slovaca was reported to be 16% in the blood samples of sheep, over 21% in goats and cattle [6] while the bacterial DNA has also been detected in blood sample obtained from goat, thus suggesting the possibility of R. slovaca being haboured in domestic ruminants [41].…”

“…Different Rickettsia species that are pathogenic to humans have emerged as tick-borne diseases recently [1]. Members of the genus Rickettsia are pleomorphic, Gram-negative, intracellular parasites that consist of the spotted fever group (SFG) rickettsiae which are the etiologic agents of tick-borne rickettsioses of humans [4] as well as three other groups which are the typhus group (TG) rickettsiae, the transitional group (TGR) comprising Rickettsia felia and Rickettsia akari (R. akari) and the ancestral rickettsiae group (Rickettsia bellii and Rickettsia canadensis) which are not known to cause disease in human [5,6]. Numerous species of tick-borne Rickettsia that were hitherto thought as nonpathogenic are recently being implicated in human infections while many more species of unknown pathogenicity are frequently being described across the globe.…”

The Rickettsioses

“…When amplification is performed using certain primer sets, restriction fragment length polymorphism analysis can help identify the etiologic agent. 76 Real-time PCR has improved analytical sensitivity over conventional and nested PCR assays -it can detect fewer than 10 copies of genomic DNA per reaction. 74 Furthermore, with the use of species-specific probes, real-time PCR assays can diagnose to the species level without sequencing.…”