First isolation of Rickettsia monacensis from a patient in South Korea

A Rickettsia sp. was isolated from the blood of a patient with an acute febrile illness using the shell vial technique; the isolate was named CN45Kr and was identified by molecular assay as Rickettsia monacensis , which was first recognized as a pathogen in Spain. Sequencing analysis showed that the gltA sequence of the isolate was identical to that of Rickettsia sp. IRS3. The ompA‐ 5mp fragment sequence showed 100% identity to those of R. monacensis and Rickettsia sp. In56 and ompA‐3pA In56 and 100% identity to that of Rickettsia sp. IRS3. The ompB sequence was found to have 99.9% similarity to that of R. monacensis IrR/Munich. This study confirms the pathogenicity of this agent and provides additional information about its geographic distribution.

     

Spotted fever group Rickettsia in ticks from southeastern Spain natural parks

During an 8-years study, we collected from vegetation or domestic and wild mammals 1246 ticks (624 males, 511 females and 111 nymphs) belonging to 13 species in Jaen province (Andalusia) and we analyzed these ticks by PCR and sequencing for the presence of rickettsiae. Specific rickettsiae DNA was detected in 243 (19.5%) of the ticks tested. Sequence analysis of amplicons of gltA, ompA and ompB genes revealed that Ixodes ricinus were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 27.0% and 2.7%, respectively), while in I. ventalloi we found only this last species (12.5%). Moreover, Dermacentor marginatus presents R. slovaca (24.7%) and R. raoultii (59.9%). In Rhipicephalus sanguineus group ticks (Rh. sanguineus, Rh. turanicus and Rh. pusillus) only R. massiliae (15.2%) was found. Haemaphysalis punctata and Ha. sulcata were infected with a Rickettsia sp. near R. hoogstraalii (prevalence of 3.1% and 16.1%, respectively). In addition, Ha. punctata appeared infected with R. monacensis-like Rickettsia (1.0%) and R. raoultii (9.3%). None of I. hexagonus, Hyalomma lusitanicum, Hyalomma sp., Ha. hispanica or Rh. bursa studied ticks contained rickettsiae.     

Rickettsia monacensis sp. nov., a spotted fever group Rickettsia,from ticks (Ixodes ricinus) collected in a European city park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.     

Detection of spotted fever group Rickettsia spp. from bird ticks in the U.K.

Migratory birds are known to play a role in the long‐distance transportation of microorganisms. To investigate whether this is true for rickettsial agents, we undertook a study to characterize tick infestation in populations of the migratory passerine bird Riparia riparia (Passeriformes: Hirundinidae), the sand martin. A total of 194 birds were sampled and ticks removed from infested birds. The ticks were identified as female Ixodes lividus (Acari: Ixodidae) using standard morphological and molecular techniques. Tick DNA was assayed to detect Rickettsia spp. using polymerase chain reaction and DNA was sequenced for species identification. A single Rickettsia spp. was detected in 100% of the ticks and was designated Rickettsia sp. IXLI1. Partial sequences of 17‐kDa and ompA genes showed greatest similarity to Rickettsia sp. TCM1, an aetiological agent of Japanese spotted fever‐like illness, previously described in Thailand. Phylogenetic analysis showed that Rickettsia sp. IXLI1 fitted neatly into a group containing strains Rickettsia japonica, Rickettsia sp. strain Davousti and Rickettsia heilongjiangensis. In conclusion, this research shows that U.K. migratory passerine birds host ticks infected with Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents.     

Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil

During 2006-2008, a total of 260 adult ticks were collected from domestic and wild animals in different regions of the state of Santa Catarina (SC), Brazil, including areas where human cases of Brazilian spotted fever have been reported. Collected ticks belonging to nine species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Amblyomma longirostre, Amblyomma ovale, Amblyomma tigrinum, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus) were tested by polymerase chain reaction (PCR) for rickettsial infection. Overall, eight (3.1%) ticks were found to be infected with Rickettsia species. After sequencing the PCR products, we determined that the sequences generated from three A. aureolatum, one A. ovale and one R. sanguineus from the municipality of Blumenau, one A. ovale from the municipality of águas Mornas and one A. ovale from the municipality of Urussanga were identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia parkeri strain Atlantic rainforest. The sequence generated from one A. longirostre from Blumenau was 100% identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia amblyommii strain AL. Because R. parkeri strain Atlantic rainforest was recently shown to have caused two cases of human spotted fever in other states of Brazil, the role of this rickettsial agent as a possible etiological agent of spotted fever in SC is discussed.     

Molecular detection of Candidatus Rickettsia asembonensis in fleas collected from pets and domestic animals in Puducherry,India

Rickettsia are obligate intracellular pathogens transmitted by arthropod vectors. The re-emergence of several rickettsioses imposes severe global health burden. In addition to the well-established rickettsial pathogens, newer rickettsial species and their pathogenic potentials are being uncovered. There are many reports of spotted and typhus fever caused by rickettsiae in India. Hence, in this study we screened the ectoparasites of pet and domestic animals for the presence of rickettsia using polymerase chain reaction. Nine cat flea samples (Ctenocephalides felis felis), that tested positive for the presence of rickettsia were subjected to Multi Locus Sequence Typing. Nucleotide sequencing and Phylogenetic analysis of gltA, ompB and 16rrs genes revealed that the rickettsiae detected in cat fleas was Rickettsia asembonensis. Further studies are required to assess Rickettsia asembonensis pathogenic potential to human and its enzootic maintenance of in various hosts and vectors.     

Candidatus Rickettsia andeanae,a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.     

Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase-encoding gene (gltA) and 190-kDa antigen-encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT-1 and FLA-1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO-1, IO-2, IO-25, IM-1 and IP-2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT-1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT-1 and FLA-1 were placed as R. japonica, and strain AT-1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT-1 and FLA-1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT-1 should be considered as a new species among SFG rickettsiae.     

Identification of potential microRNA markers related to Crimean-Congo hemorrhagic fever disease

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the arbovirus Crimean-Congo hemorrhagic fever virus (CCHFV). The CCHFV has a single-stranded RNA genome of negative sense. MicroRNAs (miRNAs) are key players in virus-host interactions and viral pathogenesis. We investigated the miRNA gene expression profiles in patients with CCHF using microarray for the first time in the world. Microarray analysis was performed using mirBase Ver 21 (Agilent Technologies, Santa Clara, CA). All statistical analyses were performed across the case-control, fatal-control, and fatal-nonfatal case groups using Genespring (Ver 3.0). Fifteen miRNAs were statistical significant in patients with CCHF compared with the controls (5 were upregulated, 10 were downregulated). Seventy-five and sixty-six miRNAs are in fatal compared with control and nonfatal case, respectively (fold change ([FC] ≥50) were statistically significant. In this study, the target genes of important miRNAs were identified and Gene Ontology analyses were performed across all groups. As a result of this study, we propose that the detection of miRNAs in patients with CCHF will allow the determination of therapeutic targets in diseases. CCHF is an important public health problem that can often be fatal. In this study, we investigated miRNA expression in case-control, fatal-control, and fatal-nonfatal case groups. Significant miRNAs associated with fatality were detected in CCHF. This study will serve as a source of data for the development of an antagomir-based therapy against CCHF using miRNAs in the future.     

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naïve rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.     

Detection of DNA of Causative Agent of Spotted Fever Group Rickettsiosis in Japan from the Patient's Blood Sample by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was applied for the etiological diagnosis of spotted fever group (SFG) rickettsiosis in Japan. Nucleotide primers derived from the 17-kDa antigen gene of Rickettsia rickettsii primed a rickettsia-specific 246-base-pair product for all of the Katayama, Abe, Misaka and Kojima strains, which we had isolated previously. Moreover, we were able to detect the same product by PCR amplification from the peripheral blood of a patient in the acute stage of the illness. The PCR method is considered to be useful for rapid etiological diagnosis of SFG rickettsiosis in Japan.     

Survey of the vectorial competence of ticks in an endemic area of spotted fever group rickettsioses in Fukui Prefecture, Japan

The prevalence of SFGR in ixodid ticks in the Mt. Arashima-dake area in the northern part of Fukui Prefecture was surveyed, because of strong suspicions that the first case identified in this Prefecture had become infected with R. helvetica in this region. The ticks identified consisted of three genera and six species; I.ovatus, I. persulcatus, I. monospinosus, H. flava, H. japonica and D. taiwanensis. Of all 222 ticks collected, only I. monospinosus ticks (8 of 32 examined) were positive for SFGR isolates, which were genetically identified as R. helvetica. Ticks (157 of all 222) positive for SFGR-DNA fragments consisted of I. monospinosus (14 of 32), I. persulcatus (11 of 55), I. ovatus (3 of 38), H. flava (5 of 21) and H. japonica (2 of 9). Of these, thirteen I. monospinosus, eight I. persulcatus, three I. ovatus, two H. flava and one H. japonica were identified by nucleotide sequences as positive for R. helvetica. DNA fragments from three H. flava and one H. japonica showed greater homology to R. japonica than to R. helvetica or R. asiatica. The present results indicate that it is most likely that the vector tick of R. helvetica infection in Fukui Prefecture is I. monospinosus.     

Seroepidemiology of spotted fever group and typhus group rickettsioses in humans, South Korea

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1: 40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill-Zinsser disease was prevalent in Korea.     

Molecular detection of Rickettsia aeschlimannii in Hyalomma spp. ticks from camels (Camelus dromedarius) in Nigeria,West Africa

Several species of the spotted fever group rickettsiae have been identified as emerging pathogens throughout the world, including in Africa. In this study, 197 Hyalomma ticks (Ixodida: Ixodidae) collected from 51 camels (Camelus dromedarius) in Kano, northern Nigeria, were screened by amplification and sequencing of the citrate synthase (gltA), outer membrane protein A (ompA) and 17‐kDa antigen gene fragments. Rickettsia sp. gltA fragments were detected in 43.3% (42/97) of the tick pools tested. Rickettsial ompA gene fragments (189 bp and 630 bp) were detected in 64.3% (n = 27) and 23.8% (n = 10) of the gltA‐positive tick pools by real‐time and conventional polymerase chain reaction (PCR), respectively. The amplicons were 99–100% identical to Rickettsia aeschlimannii TR/Orkun‐H and R. aeschlimannii strain EgyRickHimp‐El‐Arish in GenBank. Furthermore, 17‐kDa antigen gene fragments of 214 bp and 265 bp were detected in 59.5% (n = 25) and 38.1% (n = 16), respectively, of tick pools, and sequences were identical to one another and 99–100% identical to those of the R. aeschlimannii strain Ibadan A1 in GenBank. None of the Hyalomma impressum ticks collected were positive for Rickettsia sp. DNA. Rickettsia sp. gltA fragments (133 bp) were detected in 18.8% of camel blood samples, but all samples were negative for the other genes targeted. This is the first report to describe the molecular detection of R. aeschlimannii in Hyalomma spp. ticks from camels in Nigeria.     

Identification of newly isolated Babesia parasites from cattle in Korea by using the Bo-RBC-SCID mice

Attempts were made to isolate and identify Korean bovine Babesia parasite. Blood samples were collected from Holstein cows in Korea, and Babesia parasites were propagated in SCID mice with circulating bovine red blood cells for isolation. The isolate was then antigenically and genotypically compared with several Japanese isolates. The Korean parasite was found to be nearly identical to the Oshima strain isolated from Japanese cattle, which was recently designated as Babesia ovata oshimensis n. var. Haemaphysalis longicornis was the most probable tick species that transmitted the parasite.     

Characterization of an unidentified Sarcocystis falcatula-like parasite from the South American opossum, Didelphis albiventris from Brazil

An unidentified isolate of a Sarcocystis falcatula-like parasite was obtained from the lungs of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris from Brazil. Four captive budgerigars fed sporocysts from the opossum intestine died of acute sarcocystosis 8, 10, and 12 days after oral inoculation (DAI); one budgerigar was killed 12 DAI when it was lethargic. Schizonts and merozoites found in the lungs of the budgerigars reacted mildly with polyclonal S. falcatula antibody. The parasite was isolated in equine kidney cell cultures inoculated with lung tissue from a budgerigar that was killed 12 DAI. Two budgerigars inoculated subcutaneously with 100,000 culture-derived S. falcatula merozoites developed acute sarcocystosis and S. falcatula-like schizonts were found in their lungs 15 and 16 DAI. Four budgerigars kept as unfed controls in the same environment remained free of Sarcocystis infection. The parasite underwent schizogony in African green monkey kidney cells and bovine turbinate cells. Merozoites divided by endopolygeny, often leaving a residual body. Polymerase chain reaction studies using primers JNB33/JNB54 and Hinf I and Dra I digestion indicated that the isolate was not S. falcatula. Results of this study indicated that the South American opossum, D. albiventris, is a definitive host for yet another S. falcatula-like parasite.     

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

Evaluating levels of PCR efficiency and genotyping error in DNA extracted from engorged and non‐engorged female Dermacentor variabilis ticks

Polymerase chain reaction (PCR)‐based methods are increasingly used to elucidate tick biology. However, DNA extracted from ticks may provide poor PCR templates as a result of PCR inhibition by mammalian blood or contamination by male DNA (in fertilized females). In this study, the effects of removing the bloodmeal and reproductive organs were evaluated through paired DNA extractions in engorged and non‐engorged Dermacentor variabilis (Say) (Acari: Ixodidae), prior to PCR amplification at 12 microsatellites. The first extraction utilized only mouthparts and legs (‘mouthpart’ samples) and the second utilized tick bodies (‘body’ samples). The results indicated that contamination by male DNA was an unlikely source of genotyping error in mouthpart and body samples. Engorged females showed higher levels of PCR inhibition in body vs. mouthpart samples, with a 29% decrease in amplification success rates per PCR and a 10‐fold increase in levels of missing genotypes in body samples. By contrast, non‐engorged females showed little difference in amplification success rates or numbers of missing genotypes in body vs. mouthpart samples. We discuss analytical concerns related to this systematic bias in PCR problems and recommend the removal of the bloodmeal and reproductive organs prior to DNA extraction, especially in engorged female ticks.     

Detection of Mycoplasma fermentans in saliva sampled from infants, preschool and school children, adolescents and adults by a polymerase chain reaction-based assay.

Attempts were made to detect Mycoplasma fermentans in saliva sampled from 201 subjects (108 males and 93 females) aged from 4 months to 59 years by a polymerase chain reaction-based assay. M. fermentans was detected in saliva from 110 (54.7%) of 201 subjects, and 10 (28.6%) of 35 subjects aged from 4 months to 3 years. Of ten positive subjects, three were aged from 16 to 23 months and five were from 26 to 31 months. The incidence tended to increase with age up to the teens. The incidence was significantly greater in teenagers than in subjects aged from 7 to 12 years, but there was no significant difference in the incidence between the group of teenagers and each of the groups of subjects older than the teenagers. Thus, it was suggested that M. fermentans colonized the mouth at the age of about 16 months up to the age of 19 years.