Molecular detection of spotted fever group rickettsiae associated with ixodid ticks in Egypt

Tick-borne diseases comprise a complex epidemiological and ecological network that connects the vectors, pathogens, and a group of host species. The aim of this study was to identify bacteria from the genus Rickettsia associated with ixodid ticks infesting camels and cows in Egypt. Ticks were collected from 6 different localities: Qina, Giza, Qalet El Nakhl, New Valley, El Arish, and Minufia, from July to October 2008. Species were identified using PCR, followed by sequencing. The gltA and rOmpA genes were used for the initial detection of Rickettsia spp. Further characterization of positive samples utilized primers targeting rOmpB, sca4, and intergenic spacers (mppA-purC, dksA-xerC, and rpmE-tRNA(fMet)). Cows were infested with Hyalomma anatolicum excavatum and Boophilus annulatus. Camels were infested with Hyalomma dromedarii, H. impeltatum, and H. marginatum marginatum. Approximately 57.1% of H. dromedarii ticks collected from Qalet El Nakhl were infected with Rickettsia africae, exhibiting 99.1-100% identity to reference strains. Within H. impeltatum, 26.7% and 73.3% of ticks from El Arish were infected with R. africae and R. aeschlimannii, with 98.3-100% and 97.9-100% identity, respectively. Furthermore, 33.3% of H. marginatum marginatum ticks in Qalet El Nakhl were infected with the same two species as H. impeltatum, demonstrating 99.1-100% and 99.3-100% identity, respectively. By comparing percent identities and phylogenetic relationships, R. africae is identified for the first time in Egypt, in addition to R. aeschlimannii, which exhibits 100% identity with the Stavropol strain in GenBank. In conclusion, the obtained data underscore the medical and veterinary importance of tick-borne rickettsioses, which necessitate further investigation by authorities in Egypt. Moreover, additional characterization of these rickettsial isolates should be performed to designate their strains, using a polyphasic strategy combining genotypic and phenotypic tests, to facilitate their deposition in the rickettsial collection of the WHO and/or ATCC.     

Endemicity of spotted fever group rickettsiae in Connecticut

To compare rickettsial infectivity and seropositivity rates against spotted fever group (SFG) rickettsiae, ticks and wild mammals were collected from three areas where Rickettsia rickettsii was thought to be enzootic in Connecticut during 1978-1979, and from four additional sites (with no reported human cases) between 1976 and 1979. Of the 1,001 Dermacentor variabilis examined by the hemolymph test, 59 (5.9%) contained rickettsia-like organisms; direct immunofluorescence tests verified the presence of SFT rickettsiae in 14 specimens. Prevalence of rickettsiae-infected ticks at Newtown, an area where human cases of Rocky Mountain spotted fever probably originated, was 2.2%. Rates for six other areas ranged between 0 and 6.3%. Isolations included Rickettsia montana from four ticks collected at Branford and Woodbridge, and R. rickettsii (R-like strain) from the blood of an acutely ill person. Microagglutination (MA) tests indicated that 15 (14.9%) of 101 Peromyscus leucopus (white-footed mice) from Newtown had agglutinins in titers greater than or equal to 1:8 against R. rickettsii, whereas five of 92 white-footed mice (5.4%) from Brandord, West Hartford, Woodbridge, and Sharon were considered MA-positive. Indirect microimmunofluorescence tests of Procyon lotor (raccoon) sera revealed antibodies to R. rickettsii in 33 of 69 (47.8%) samples from Newtown and in two of 60 (3.3%) from Guilford. Additionally, 17 raccoons had sera specific to R. montana (n = 8) or to the 369-C rickettsia strain (n = 9). Since rickettsia-positive ticks are high-titered seropositive mammals occurred at widely separated sites in Connecticut there are probably several foci of SFG rickettsiae distributed throughout the D. variabilis range.     

T cells mediate cross-protective immunity between spotted fever group rickettsiae and typhus group rickettsiae

Rickettsioses are severe infections caused by obligately intracellular bacteria that preferentially infect the endothelium lining the vasculature. The causative agents, rickettsiae, have been divided according to biological, genetic, and antigenic parameters into 2 main groups: spotted fever and typhus. They have not been thought to stimulate cross-reactive protective immune responses; however, in this study, we show that, in relevant animal models that mimic human rickettsial infections, there is reciprocal immunological cross-protection between spotted fever group and typhus group rickettsiae. Furthermore, we present evidence that T cells are responsible for this cross-immunity and that cross-stimulation of T cells also occurs in humans.     

Serotypes of tick-borne spotted fever group rickettsiae from western California

A rickettsial survey of ixodid ticks known to bite man was conducted in 1979 in four coastal counties of California to obtain isolates from tick species that might be involved in the transmission of spotted fever-like illnesses, and to examine serologic characteristics of the rickettsiae relative to defined members of the spotted fever group (SFG). One hundred and seventy (19.4%) of 877 ticks comprising three species were shown by hemolymph test to harbor rickettsia-like organisms. A total of 85 SFG rickettsia isolates was obtained by Vero-cell culture; 82 were from Dermacentor occidentalis, two were from D. variabilis, and one was from Ixodes pacificus. As determined by microimmunofluorescence, the isolates comprised four distinct serotypes. Two serotypes were obtained only from D. occidentalis, and one each only from D. variabilis and I. pacificus, respectively. Most D. occidentalis isolates possessed the serologic characteristics of Rickettsia rhipicephali, but there were similar to yet distinguishable from, R. rickettsii and are members of an unclassified serotype referred to as 364D. The two isolates from D. variabilis resembled the unclassified 369C serotype previously shown to be associated with this species and D. andersoni elsewhere in the United States. The I. pacificus isolate was similar to strains of the unclassified Tillamook serotype isolated from this tick in several localities in western Oregon. Representative strains of the four serotypes could also be distinguished on the basis of pathogenicity for Vero cells, chick embryos, guinea pigs, and/or meadow voles. The significance of these findings relative to occurrence of tick-associated illnesses in western California is briefly discussed.     

我国斑点热立克次体感染与免疫的实验研究

研究证明黑龙江立克次体能够感染人血管内皮细胞和BALB/c小鼠,引起小鼠菌血症和器官损伤;全基因组序列分析发现黑龙江立克次体毒力相关基因。表面蛋白质组分析鉴定了黑龙江立克次体和立氏立克次体的表面蛋白,用表面蛋白免疫C3H/HeN小鼠,发现某些表面蛋白为保护性抗原,并揭示这些保护性抗原均能够诱导抗原特异CD₄⁺和CD₈⁺ T细胞增殖并产生和分泌IFN-γ和/或TNF-α,以及诱导高水平特异性IgG2a产生,在这些免疫因素的协同作用下使小鼠有效抵抗立克次体感染。用黑龙江立克次体感染Tim-3高表达或低表达人血管内皮细胞以及Tim-3高表达转基因小鼠,结果有力证明Tim-3高表达能够促进人血管内皮细胞和小鼠抵抗黑龙江立克次体感染。     

Prevalence of antibodies to spotted fever group rickettsiae in dogs from southeastern Australia

Recent epidemiologic data suggests that Rickettsia australis, the cause of Queensland tick typhus, is present in southeastern Australia. In order to further confirm this observation, a canine serosurvey was undertaken to determine if naturally occurring antibodies were present in pet and farm dogs from this newly-recognized endemic area. Thirty-five of 312 surveyed dogs (11.2%) had indirect immunofluorescent antibody titers of 1:64 or greater against R. australis antigen. Positive control sera were obtained from two dogs experimentally inoculated with R. australis. One of these dogs was serially sampled and a rickettsemia could not be documented. None of 26 control sera obtained from dogs from South Australia, New Zealand, western Victoria, or North Carolina had antibody titers greater than or equal to 1:64. These results suggest that spotted fever group rickettsiae are present in Southeastern Australia.     

Injury restricted to cells infected with spotted fever group rickettsiae in parabiotic chambers

One chamber of paired parabiotic chambers separated by 0.2 micron poresized membrane filters which prevented passage of rickettsiae were infected with either Rickettsia rickettsii or R. conorii. Infected VERO cell monolayers underwent necrosis. Uninfected monolayers in adjoining chambers which shared the same extracellular milieu including soluble rickettsial products did not undergo necrosis.     

Antigenic analysis of Chinese strains of spotted fever group rickettsiae by protein immunoblotting

Protein immunoblotting demonstrated that 6 Chinese strains of spotted fever group (SFG) rickettsiae from northern China had antigenic polypeptides identical to those of Rickettsia siberica (strains 246 and 232), and dissimilar to other SFG rickettsiae. The various species of other SFG rickettsiae exhibited serologically distinct epitopes as well as many cross-reactive epitopes in protein immunoblotting. All SFG rickettsiae examined in this study had major antigenic polypeptides of 113-160 kDa.     

Prevalence of antibodies against spotted fever group rickettsiae in a rural area of Colombia

We recently rediscovered Rocky Mountain spotted fever in Villeta, Colombia, near the same locality (Tobia) where it was first recognized in 1937. To have a better idea of the magnitude of this problem, sera from 392 randomly recruited healthy adults from Villeta were analyzed by indirect immunofluorescent antibody assay to detect IgG against Rickettsia rickettsii as antigen. The seropositivity rate for spotted fever group rickettsiae was 40.3%. We did not find any association between the presence of antibodies to spotted fever group rickettsiae and several demographic and epidemiologic variables, which could be a reflection of unique features of this area.     

Evidence of spotted fever group rickettsiae in state of Rio de Janeiro,Brazil

Ticks were obtained from dogs from February to September of 1999 at weekly intervals, in the County of Piraí, State of Rio de Janeiro. Four hundred seventy four ixodids were taxonomically identified, 103 Amblyomma cajennense, seven Amblyomma ovale, 209 Rhipicephalus sanguineus, and 155 Amblyomma sp. An hemolymph test associated with Giemsa's stain revealed two specimens in 163 ticks tested (R. sanguineus and Amblyomma sp), containing rickettsia-like organisms. Direct immunofluorescence verified the presence of spotted fever group rickettsia in one specimen of R. sanguineus. Considering the limited information on rickettsiosis in Brazil, principally in relation to the vectors involved in perpetuating it in foci, these preliminary results give us an idea on the importance of infection in ticks, allowing to expand our knowledge on this zoonosis.     

黑龙江逊克地区森林革蜱斑点热立克次体DNA的检测

目的 调查黑龙江逊克地区蜱传斑点热自然疫源地,发现该地区蜱携带斑点热立克次体的种类。方法 采用斑点热立克次体ompA和gltA基因特异的PCR,检测该地区森林革蜱的DNA样本,并对扩得阳性产物进行测序和聚类分析。结果 从60只森林革蜱中检测有14只扩得斑点热立克次体ompA和gltA基因片段,阳性率为23.33%。随机选择2只蜱的阳性片段进行测序,二者同源性为100%,ompA基因序列与Rickettsia sp.JL-02同源性为99.30%, 与Rickettsia raoultii为99.18%。结论 黑龙江省逊克地区森林革蜱携带与Rickettsia sp.JL-02株亲缘关系相近的斑点热群立克次体。     

First detection of spotted fever group Rickettsiae in ticks in Serbia

Ticks can transmit multiple pathogenic bacteria responsible for diseases in animals and humans such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and spotted fever group Rickettsia sp. The current study aimed to investigate the presence of Rickettsiae in ticks collected from seven localities in Serbia. One hundred thirty-one (131) questing ticks belonging to 5 tick species (Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Haemaphysalis Concinna, and Ixodes ricinus) were collected in 2007 and 2009. Ticks were tested by polymerase chain reaction, amplifying gltA, ompA, and 17-kd genes, and sequencing analysis, revealing the presence of Rickettsia helvetica and Rickettsia monacensis in I. ricinus ticks only (infection rates 7.7% and 15.4% for R. helvetica and R. monacensis, respectively). R. helvetica has been isolated from I. ricinus ticks and has been implicated in fatal perimyocarditis. R. monacensis was first identified in I. ricinus samples collected in Germany and has recently been implicated in human infection. The results of the current study make fundamental the need to evaluate the incidence of infection with R. helvetica and R. monacensis among the resident population.     

A survey for spotted fever group rickettsiae and ehrlichiae in Amblyomma variegatum from St. Kitts and Nevis

Eighty-nine Amblyomma variegatum ticks were collected from the islands of St. Kitts and Nevis in the Caribbean and preserved in 70% ethanol or local rum. After being washed in sterile water, their DNA was extracted and analyzed by a polymerase chain reaction (PCR) for DNA of spotted fever group rickettsiae and ehrlichiae. None of the tested ticks was positive in a PCR assay using the primers 16S EHRD and 16S EHRR for the 16S rRNA gene of Ehrlichia spp.. Forty-one percent of the A. variegatum (36 of 89 of which 34 [47%] of 72 were adult males, 2 (13%) of 16 were adult females, and 0 (0%) of 1 were nymphs) were positive in a PCR assay using the primer pair 190-70 and 190-701 for the outer membrane protein A (ompA) gene of spotted fever group rickettsiae. All PCR amplification products obtained had 100% sequence homology with Rickettsia africae, the agent of African tick-bite fever.     

Genotyping of spotted tick fever rickettsiae, detected in Russia and Kazakhstan

R. slovaca was first detected in the ticks D. marginatus gathered in the Stavropol Territory and the Voronezh Region (European Russia). The recently discovered rickettsial genotype DnS14 was first found in the ticks D. silvarum from Buryatia and D. niveus from the Karaganda Region (Central Kazakhstan). The rickettsial genotype RpA4 was most common in the ticks of the genus Dermacentor in Russia and Central Kazakhstan. An analysis of the spread of rickettsias of the STF group shows their close ecological relation to definite types of Ixodes. The rickettsias R. slovaca and RpA4 co-exist in the ticks D. marginatus and D. reticulatus (the western part of a Dermacentor area in Eurasia) and DnS14 and R. sibirica do in D. nuttalli and D. silvarum (the eastern part of the area). D. marginatus and D. reticulatus in the areas characterized by the most specific saturation of a Dermacentor area (the south of West Siberia) are carriers and reservoir of R. sibirica. The rickettsial genotype DnS28 may be now considered to be environmentally associated with one species of ticks--D. nuttalli. At least 6 genotypes of STF rickettsias--R. sibirica, R. astrahan fever (R. conorii), R. slovaca, RpA4, DnS14, DnS28--has been currently identified in Russia and Kazakhstan.     

Serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae among residents of Gag Island,Indonesia

The causative agents of scrub and murine typhus are considered endemic to Indonesia. However, the presence of spotted fever group rickettsiae and ehrlichiae have not been previously described in this country. During an investigation of arthropod-borne diseases on Gag Island, located northwest of the island of New Guinea in eastern Indonesia, the prevalence of antibody to the etiologic agents of monocytic ehrlichiosis, spotted fever rickettsiosis, and scrub and murine typhus were determined. Analysis of 55 blood samples from residents of Gag Island showed seroreactivity to antigen preparations of Ehrlichia chaffeensis (7 of 48, 14.6%), two spotted fever group rickettsiae: Rickettsia rickettsii (5 of 48, 10.4%) and R. conorii (10 of 49, 20.4%), Orientia tsutsugamushi (5 of 53, 9.4%), and R. typhi (1 of 48, 2.1% [by an indirect immunofluorescence assay] and 1 of 50, 2.0% [by an enzyme-linked immunosorbent assay]). These results show serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae for the first time in Indonesia in a location where the prevalence of antibody to O. tsutsugamushi and R. typhi was lower.     

190 kD R.rOmpA基因序列对斑点热群立克次体的检测

目的为探讨190 kD R.rOmpA基因序列对斑点热群立克次体的检测作用.方法从立氏立克次体190 kD外膜蛋白A(R.rOmpA)基因序列设计引物,利用PCR检测的方法,检测了683只蜱类标本和146个鼠类脏器标本,并随机抽取1株森林革蜱阳性扩增产物进行克隆与序列测定.结果从蜱类标本和鼠类脏器标本中检测出了斑点热立克次体DNA片段;所测序列的分型结果表明与前苏联的DnS 14株型别一致,与我国曾经检测出的斑点热立克次体株差异较大.结论190kD外膜蛋白A基因序列可用于斑点热立克次体检测,并可用于斑点热群立克次体基因型或亚型之间的鉴别.     

Molecular identification of a collection of spotted Fever group rickettsiae obtained from patients and ticks from Russia

Thirty-one rickettsial isolates from ticks or patients in North Asian tick typhus (NATT) foci from the Ural region to the Russian Far East were obtained at the Omsk Research Institute of Natural Foci Infections between 1954 and 2001. Using citrate synthase (gltA) and outermenbrane protein a (ompA) gene sequencing, we identified these isolates as Rickettsia sibirica sensu stricto (25 isolates), R. sibirica strain BJ-90 (2 isolates), R. slovaca (1 isolate), and R. heilongjiangensis (3 isolates). We demonstrate that Ixodes persulcatus ticks should be considered potential vectors of NATT. We also demonstrate the presence of R. slovaca in Ural and R. heilongjiangensis in Siberia and Russian Far East, where they may cause human infections misdiagnosed as cases of NATT. Clinicians should be aware that several spotted fever rickettsioses with different prognoses coexist in Russia in areas where NATT was the only previously recognized rickettsiosis.