Phenotypic and genotypic characterization of spotted fever group Rickettsiae isolated from Catalan Rhipicephalus sanguineus ticks

Abstract

Eighty-nine Rhipicephalus sanguineus ticks and 21Rhipicephalus bursa ticks collected in Catalonia were tested by the hemolymph test to establish their infection rate with spotted fever group rickettsiae. By Giménez staining, 11.2% of the R. sanguineus isolates and 0% of the R. bursa isolates were found to contain rickettsia-like organisms. Six spotted fever group rickettsial strains (Bar29, Bar31, Gir4, Tar1, Tar2, and Tar3) were isolated from these ticks and were characterized by phenotypic and genotypic analyses. PCR followed by restriction fragment length polymorphism analysis showed that the six strains were identical and were characterized by the same restriction profiles as a strain, Mtu5, previously isolated from Rhipicephalus turanicus ticks in the South of France. Microimmunofluorescence serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified organisms, and Western blot (immunoblot) assay with mouse polyclonal sera confirmed this observation. Pulsed field gel electrophoresis of the whole genome of three of the strains showed that, although closely related, the profile of Tar1 was slightly different from that of the Bar strains. Phylogenetic analysis showed that this new rickettsial sero-and genotype, which will be named the "Catalan strain," is closely related to Rickettsia massiliae. This strain shows an unexpected resistance to rifampin. The epidemiological implications of these findings are considered.

Production of monoclonal antibodies against Rickettsia massiliae and their use in antigenic and epidemiological studies

“…These shared protein epitopes, which were located on both immunodominant proteins of R. massiliae (106-and 120-kDa proteins), were also demonstrated to exist on the surface proteins of Bar29, "R. aeschlimanni," R. rhipicephali, and R. japonica by Western immunoblotting. Among these species, Bar29 isolated from R. sanguineus ticks from the Catalan region (Spain) had the most epitopes in common with R. massiliae, concurring with previous studies on their phenotypic and genotypic characteristics (12,47,48).…”

“…However, the production of monoclonal antibodies against the rickettsial species of unknown pathogenicity toward humans has not yet been attempted. The antigenicity of these species has previously been analyzed only with mouse polyclonal antisera (11,12,43).…”

“…In recent years, with the introduction of improved techniques for their isolation and identification (22,36,44,(46)(47)(48), numerous novel SFG rickettsiae have been identified, but their pathogenicities toward humans remain unclear (5,11,12). R. massiliae is such a species which was originally isolated from Rhipicephalus turanicus ticks collected from regions near Marseille, France (11,13).…”

“…Because shell vial centrifugation isolation methods (36) and molecular biology-based identification schemes (22,44,46,47) for R. massiliae are laborious in large-scale field work, we aimed to develop a more convenient, monoclonal antibody-based methodology to facilitate the detection of R. massiliae in ticks. Additionally, phenotypic and genotypic characterization of R. massiliae has suggested that this species is closely related to the other rickettsial species, such as Bar29, "R. aeschalimanni," and R. rhipicephali (12,47,48). Therefore, we also aimed to investigate the antigenic relationships between R. massiliae and other rickettsial species, using monoclonal antibodies produced against Mtu1, the type strain of R. massiliae (13).…”

Ten Years' Experience of Isolation of Rickettsia spp. from Blood Samples Using the Shell‐Vial Cell Culture Assay

“…To identify the SFG rickettsia species seen by IFI and by Gimenez staining, polymerase chain reaction (PCR)/RFLP was used. 8 PCR amplification was performed by using oligonucleotide primer pairs Rp CS877p and Rp CS.1258n generated from the citrate synthase gene of R. prowazekii, Rr 190.70p and Rr 190.602n generated from the 190-kDa antigen gene of R. rickettsii, and BG1-21 and BG2-20 generated from the 120-kDa antigen gene of R. rickettsii. 9,10 PCRs were carried out using a Perkin-Elmer 9600 thermocycler (Roche Diagnostics, Norwalk, CT), according to the protocol described by Regnery et al 10 The amplified products were analyzed on a 1.8% agarose gel (Bio-Rad Laboratories, Hercules CA) in 0.5 × Tris-borate-EDTA (TBE) buffer (AppliChem GmbH, Darmstat, Germany).…”