Spotted Fever Group Rickettsiae Research Articles

Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conorii-infected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses.

Turkey tick news: A molecular investigation into the presence of tick-borne pathogens in host-seeking ticks in Anatolia; Initial evidence of putative vectors and pathogens, and footsteps of a secretly rising vector tick, Haemaphysalis parva

This Turkey-based study investigated the presence of various tick-borne microorganisms in a broad-range of host-seeking ticks (n = 1019) that exhibit both hunter and ambusher characteristics. All collected ticks were analyzed individually via PCR-sequencing, resulting in the identification of 18 different microorganisms: six Babesia spp., including one putative novel species (Ba. occultans, Ba. crassa, Ba. rossi, Babesia sp. tavsan1, Babesia sp. tavsan2, and Babesia sp. nov.); six SFG rickettsiae (Ri. aeschlimannii, Ri. s. mongolitimonae, Ri. slovaca, Ri. raoultii, Ri. monacensis, and Ri. hoogstraalii); two Borrelia burgdorferi sensu lato spp. (Bo. afzelii and Bo. lusitaniae); two unnamed Hepatozoon spp.; Theileria annulata; and Hemolivia mauritanica. This provided evidence for the natural transstadial survival of these tick-borne microorganisms in adult ticks (in addition a nymph) of Turkey. Surprisingly, this study determined the presence of five different microorganisms (Ba. crassa, Ba. rossi, Babesia sp. Ucbas, Hepatozoon sp., and Ri. hoogstraalii) in host-seeking Haemaphysalis parva adults, for which poor data exist on its vectorial competence. Therefore, this study provides important data indicating the potential vectorial capacity of Ha. parva. This study also revealed the presence of the close ecological and evolutionary relationships between two important vector ticks, Hyalomma marginatum and Hy. aegyptium and determined genetic variations (distinct phylogenetic divergences inside the main clades) in some pathogenic SFG rickettsiae that are found in these ticks. Additionally, the presence of two Babesia species described very recently in hares with unknown vectors, namely Babesia sp. tavsan1 and Babesia sp. tavsan2, were detected for the first time in ticks. Finally, two unnamed Hepatozoon spp. were detected in Haemaphysalis ticks and their phylogenetic positions were demonstrated. Consequently, this study provides important data on the diversity of tick-borne microorganisms in host-seeking ticks and on potentially novel microorganisms (Babesia and Hepatozoon species) and their possible vectors (Ha. parva, Ha. sulcata, Hy. aegyptium, Hy. marginatum, and Rh. turanicus).

Molecular evidence of the spotted fever group Rickettsiae in ticks from Yunnan Province, Southwest China.

Ixodid ticks transmit many obligate intracellular Rickettsial species. Several previous studies have identified Rickettsia species in the northeastern and southern part of China, but few reports on the prevalence of infection of spotted fever group Rickettsiae (SFGR) in ticks in southwest China are available. Here, we investigated SFGR in 394 adult ticks of five species including Dermacentor nuttalli, Dermacentor silvarum, Haemaphysalis longicornis, Ixodes sinensis and Ixodes persulcatus, collected in the border region between China and Burma in Yunnan Province. PCR was used to detect the presence of the citrate synthase (gltA) gene of Rickettsia species. SFGR was found in 12.1% (15/124) of I. persulcatus ticks, which was significantly higher than the 7.2% (7/97) positive D. nuttalli, 5.4% (3/56) D. silvarum, 5.6% (4/72) H. longicornis and 4.4 (2/45) I. sinensis. A portion of the gltA and ompA gene data subjected to phylogenetic analysis revealed that the detected SFGR clustered into two species, Rickettsia raoultii and the new Rickettsia species Candidatus Rickettsia jingxinensis. Detection of both Rickettsia spp. in this region indicates a potential public health threat posed by SFGR infection in Yunnan Province.

Rickettsia spp. Infecting Lone Star Ticks (Amblyomma americanum) (Acari: Ixodidae) in Monmouth County, New Jersey.

Tick-borne rickettsiae are undergoing epidemiological changes in the eastern United States while human encounters with lone star ticks (Amblyomma americanum L.) have increased substantially. We used real-time polymerase chain reaction assays to test for three species of spotted fever group rickettsiae (SFGR) (Rickettsiales: Rickettsiaceae) in 1,858 nymphal A. americanum collected from Monmouth County, New Jersey, a coastal county with endemic Lyme disease and established tick surveillance. Out of the 1,858 tested, 465 (25.0%) were infected with Rickettsia amblyommatis Karpathy, a species of undetermined pathogenicity found frequently in A. americanum, while 1/1,858 (0.05%) contained Rickettsia rickettsii Brumpt, the agent of Rocky Mountain spotted fever. No ticks tested positive for mildly pathogenic Rickettsia parkeri Lackman, and no ticks were co-infected with multiple Rickettsia spp. Our results indicate that A. americanum could be involved in transmission of R. rickettsii to humans in New Jersey, albeit rarely. The much higher rates of R. amblyommatis infection are consistent with hypotheses that human sera reacting to this species could contribute to reports of mild SFGR cases.

A retrospective study of vector-borne disease prevalence in dogs with proteinuria: Southeastern United States.

BackgroundProteinuria is a risk factor for progressive kidney injury in dogs. Enhanced understanding of potential associations between canine vector‐borne diseases (CVBD) and proteinuria is needed.ObjectivesTo determine the proportion of evaluated proteinuric dogs exposed to ≥1 CVBD, including Babesia spp., Ehrlichia spp., spotted‐fever group Rickettsia, Bartonella spp., Anaplasma spp., hemotropic Mycoplasma spp., Borrelia burgdorferi, and Dirofilaria immitis, and to determine if demographic or clinicopathologic differences exist between proteinuric dogs exposed to CVBD versus proteinuric dogs with no evidence of CVBD exposure.AnimalsTwo‐hundred nine proteinuric dogs, concurrently tested for CVBD, which were examined at a single academic veterinary hospital between January 2008 and December 2015.MethodsRetrospective cross‐sectional study. Demographic, clinicopathologic, and CVBD test results were extracted from medical records. A multivariable logistic regression model was used to assess associations between CVBD and selected variables.ResultsBased on serology and polymerase chain reaction testing, 34% of proteinuric dogs (72/209) were exposed to ≥1 CVBD. Exposure to Rickettsia spp. (19%), Ehrlichia spp. (12%), and B. burgdorferi (9%) were most common. The CVBD exposure was lower in dogs tested in autumn or spring, higher in intact dogs, and higher in dogs with lower serum albumin and higher serum creatinine concentrations.Conclusions and Clinical ImportanceExposure to CVBD, particularly exposure to Rickettsia spp., Ehrlichia spp., and B. burgdorferi was found in proteinuric dogs from the southeast United States. Additional controlled prospective studies examining a potential causal relationship between CVBD and proteinuria are warranted.

Molecular confirmation & characterization of Rickettsia conorii in north India: A report of three cases.

Background & objectives:In India, spotted fever group rickettsiae (SFGR) are an underdiagnosed cause of acute febrile illness (AFI). The non-specific Weil-Felix test is the first diagnostic modality for the diagnosis of SFGR in many laboratories due to the lack of advanced diagnostic facilities in developing countries. The aim of this study was to detect SFGR using molecular methods in the patients, presenting with AFI in a tertiary care centre in north India.Methods:Consecutive patients (>14 yr of age) with AFI were enrolled over a six month period. Standard investigations for common pathogens causing AFI in India (malaria, dengue, scrub typhus, leptospirosis and enteric fever) were carried out. In patients who were negative for all of the above investigations, blood was subjected to polymerase chain reaction (PCR) targeting outer membrane protein A (ompA) gene of Rickettsia.Results:Of the 51 patients with an undiagnosed aetiology, three were positive by ompA PCR. Two of the PCR products produced good sequences and BLAST identification confirmed them as Rickettsia conorii. The sequences of R. conorii reported from south India clustered with two previously reported novel rickettsial genotypes. The study sequences clustered in a group different from that of Rickettsia spp. of the south Indian sequences reported earlier.Interpretation & conclusions:This study showed the existence of R. conorii in north India. Testing for SFGR may be included in the diagnostic workup of AFI for better disease management.

Detection of tick-borne rickettsial pathogens in naturally infected dogs and dog-associated ticks in Medellin, Colombia

Tick-borne rickettsial pathogens (TBRP) are important causes of infections in both dogs and humans. Dogs play an important role as a biological host for several tick species and can serve as sentinels for rickettsial infections. Our aim was to determine the presence of TBRP in dogs and in dog-associated ticks and their potential risk to human diseases in Medellin, Colombia. DNA for E. canis (16S rRNA and dsb) and A. platys (groEl) was detected in 17.6% (53/300) and 2.6% (8/300) of dogs, respectively. Antibodies against Ehrlichia spp. 82 (27.3%) and Anaplasma spp. 8 (2.6%) were detected in dogs. Antibody reactivity against both agents were found in 16 dogs (5.3%). Eight dogs showed antibody for Rickettsia spp. with titers that suggest 3 of them had a probable exposure to R. parkeri. Rhipicephalus sanguineus s.l. (178/193) was the main tick in dogs, followed by R. microplus (15/193). The minimum infection rates (MIR) in R. sanguineus were 11.8% for E. canis and 3.4% for A. platys. E. canis and A. platys are the main TBRP infecting dogs and ticks and R. sanguineus s.l. is likely involved in the transmission of both agents. Interestingly, we found serological evidence of exposure in dogs for spotted fever group rickettsiae.

Distribution of <i>Haemaphysalis longicornis</i> and Its Infected Agents: A Pooled Analysis of China Field Survey and Global Published Data

Background: Haemaphysalis longicornis, a vector of various diseases with medical and veterinary importance, is native to eastern Asia, and recently invaded the United States as an emerging disease threat. Methods: We collected multi-source data from field survey, reference book, literature search and related websites. Geographic distribution of H. longicornis and its infected agents was mapped by geographical information system techniques. Hosts of H. longicornis and infection rates of H. longicornis-borne agents were estimated by meta-analysis. Ecological niche model was used to predict potential distribution of H. longicornis around the world. Findings: H. longicornis was broadly distributed in ten countries, predominantly in eastern Asia, the United States, and countries in the southwest Pacific Ocean. The tick was known to feed on a variety of domestic and wild animals. Nearly 60 agents, including spotted fever group rickettsiae, Anaplasmataceae family, Borrelia burgdorferi sensu lato, Babesia, Theileria, Francisella, Bartonella, Coxiella, Hepatozoon, Toxoplasma, and viruses were detected in H. longicornis, over half of which were pathogenic to humans. H. longicornis-borne agents were mainly reported in eastern Asia. The predictive modeling revealed that H. longicornis might infest more extensive regions, unexpectedly in Europe, South America, and Africa. Interpretation: H. longicornis is widely distributed in the world, and infected with various pathogens. Authorities and health care workers should be aware of the threat of the tick species to public health and veterinary medicine. Expanding surveillance and further investigations should be enhanced. Funding Statement: This study was supported by the Natural Science Foundation of China (81621005) and National Key Research and Development Program of China (2019YFC1200505). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Not required.

New Spotted Fever Group Rickettsia Isolate, Identified by Sequence Analysis of Conserved Genomic Regions.

The clinical features of spotted fever group (SFG) Rickettsia induced disease range from a mild to severe illness. The clinical complexity is even greater due to the fact that the disease can be caused by different species with varying degrees of virulence. Current knowledge asserts that the Israeli SFG (ISF) strain Rickettsia conorii israelensis is the only human pathogenic SFG member in Israel. Current diagnostic procedures distinguish between SFG and the typhus group rickettsiosis, assuming all SFG-positive clinical samples positive for ISF. Molecular studies on questing ticks over the past decade have uncovered the existence of other SFG strains besides ISF in Israel and the region. This study describes the first documented analysis of SFG-positive samples from Israeli patients with the goal of distinguishing between ISF and non-ISF SFG strains. We managed to identify a new Rickettsia isolate from three independent clinical samples in Israel which was shown to be an as-yet unknown SFG member, showing no absolute identity with any known Rickettsia species present in the NCBI database.

Bacterial communities in Haemaphysalis, Dermacentor and Amblyomma ticks collected from wild boar of an Orang Asli Community in Malaysia

Ticks are hematophagous vectors of arthropod-borne disease agents globally. In Malaysia, despite seroprevalence studies indicating the presence of tick-borne diseases among the indigenous people, the etiological agents of these diseases are still unclear. These indigenous people, also known as the Orang Asli, still live in forested areas with frequent contact with wildlife. Wild boar are ubiquitously found in the forested areas where the Orang Asli communities are located and are commonly hunted as a food supplement. In this study, we aim to determine the tick species parasitizing wild boar from an Orang Asli community, and explore the tick-associated bacterial communities using 16 s rRNA amplicon sequencing on the Ion Torrent PGM™ platform.A total of 72 ticks were collected from three wild boar and were morphologically identified as Haemaphysalis hystricis (n = 32), Dermacentor compactus (n = 15), Amblyomma testudinarium (n = 13), Dermacentor steini (n = 10) and Dermacentor atrosignatus (n = 2). Across all tick samples, 910 bacterial taxa were identified. Although the bacterial communities were not significantly distinct between tick species in beta-diversity analyses, Coxiella, Rickettsia and Francisella were detected at high relative abundance in H. hystricis, D. compactus and D. steini respectively. Many other bacterial genera, including those that have been described in many different tick species, were also identified, including Pseudomonas, Acinetobacter, Staphylococcus and Corynebacterium. Beta-diversity analyses also showed that the bacterial communities were separated based on the animal host from which the ticks were collected from, suggesting that the bacterial communities here may be influenced by the animal skin microflora, host blood or the environment. PCR screening confirmed the presence of Rickettsia sp. related to spotted fever group Rickettsia in some of the ticks.This study provides baseline knowledge of the microbiome of H. hystricis, D. atrosignatus, D. compactus, D. steini and A. testudinarium parasitizing wild boar in this region. The information gained in this study provides the basis to target our efforts in H. hystricis, D. compactus and D. steini for the future investigation of vector competence and the zoonotic potential for the Coxiella, Rickettsia and Francisella detected here, as well as their implications for the risks of tick-borne diseases among the Orang Asli communities.

Diversity of Rickettsia in Ticks Collected in Abruzzi and Molise Regions (Central Italy).

Rickettsiae have worldwide occurrence and rickettsiosis are widely recognized as emerging infections in several parts of the world. For decades, it was thought that a single pathogenic tick-borne spotted fever group (SFG), Rickettsia, occurred in each continent. Nowadays, thanks to molecular biology, new species of Rickettsia responsible for disease in humans are continuously identified worldwide. In a framework of diagnostic activities of the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Gaporale” and considering some reports of suspected human clinical cases of rickettsiosis, a survey on ticks collected form animals and humans was carried out with the aim to identify the Rickettsia species circulating in Abruzzi and Molise regions. A total of 603 ticks, previously identified at species level by morphology, pooled into 178 tick samples, were tested by pan-Rickettsia RealTime PCR. DNA from specimens positive for Rickettsia spp. was then sequenced in order to identify the Rickettsia species involved. The highest infection rate was detected in Dermacentor marginatus followed by Ixodes ricinus. The selected targets for this purpose were OmpA and gltA. Rickettsia slovaca, Rickettsia monacensis, Rickettsia massiliae, Rickettsia conorii, Rickettsia aeschlimannii, Rickettsia helvetica, Rickettsia raoultii, and Rickettsia felis – like organisms were identified in this study. These are the first data available in the literature for the circulation of SFG Rickettsia species in the selected geographical area. Results made evidence of high rate of infection in ticks. All Rickettsia species detected have been previously involved in human infection. The diversity of Rickettsia detected, and tick species collected reflects the biodiversity in term of wildlife and environment of the area. An association between Rickettsia species, ticks, and the relationships with vertebrate host species are discussed. Due to the peculiar eco-biology of each Rickettsia species, the use of diagnostic tools able to identify Rickettsia at the species level is thus recommended in order to assess the risk for humans and to elucidate more precise etiological diagnosis in clinical cases.

Insight into diversity of bacteria belonging to the order Rickettsiales in 9 arthropods species collected in Serbia

Rickettsiales bacteria in arthropods play a significant role in both public health and arthropod ecology. However, the extensive genetic diversity of Rickettsiales endosymbionts of arthropods is still to be discovered. In 2016, 515 arthropods belonging to 9 species of four classes (Insecta, Chilopoda, Diplopoda and Arachnida) were collected in Serbia. The presence and genetic diversity of Rickettsiales bacteria were evaluated by characterizing the 16S rRNA (rrs), citrate synthase (gltA) and heat shock protein (groEL) genes. The presence of various Rickettsiales bacteria was identified in the majority of tested arthropod species. The results revealed co-circulation of five recognized Rickettsiales species including Rickettsia, Ehrlichia and Wolbachia, as well as four tentative novel species, including one tentative novel genus named Neowolbachia. These results suggest the remarkable genetic diversity of Rickettsiales bacteria in certain arthropod species in this region. Furthermore, the high prevalence of spotted fever group Rickettsia in Ixodes ricinus ticks highlights the potential public health risk of human Rickettsia infection.

Spotted fever group rickettsiae canine serosurveillance near the US-Mexico border in California.

Dogs are vulnerable to pathogens transmitted by brown dog ticks. An epidemic of Rocky Mountain spotted fever (RMSF) is underway in Mexicali, a Mexican city bordering California affecting people and dogs; several human cases have been reported in California residents who travelled to Mexico. To evaluate risks of RMSF, we conducted seroprevalence surveys in Imperial County in 2016 and 2017 using dogs as sentinels. Blood was collected from 752 dogs and was tested for antibodies against R.rickettsii, E.canis and A.phagocytophilum (as a proxy for A.platys). Samples were considered seropositive to spotted fever group rickettsia (SFGR) if the R.rickettsia titre was ≥1:64 and seropositive to E.canis and A.phagocytophilum if the titre was ≥1:32. Owners provided information on dog age, exposure risks, health status and tick prevention. We assessed associations between SFGR seropositivity and driving distance to the nearest US-Mexico border crossing station, whether proximity to a border crossing increased likelihood of taking dogs across the border, and whether distance to the border was associated with seropositivity. Logistic regression was performed to assess relationships between the titre classes and other predictor variables. 12.2% of dogs were seropositive against SFGR. Dogs close to the border were significantly more likely to be taken across the border and to be seropositive. Risk factors that increased seropositivity included owners seeing ticks on the dog (OR=1.9), being an adult dog, travel to Mexico (OR=3.0) and living in a rural area (OR=4.0). There was statistically significant co-exposure to SFGR and Anaplasma spp. Surveillance for brown dog tick-vectored pathogens can help identify dogs and people at risk for RMSF. Tick prevention, particularly in dogs, and surveillance of tick-borne pathogens can help prevent the spread of rickettsioses and other diseases in this dynamic border region.

Survey on tick-borne pathogens in ticks removed from humans in Northwestern Italy.

Ticks are able to transmit several pathogens to the host while feeding, and thus are considered the most important vectors of infectious agents together with mosquitos. The global incidence of tick-borne diseases (TBDs) is rising, due to increased interactions between pathogens, hosts and vectors, linked to global changes. Given that information about the prevalence of tick-borne pathogens in ticks removed from humans in Italy are scarce, the aim of the present study was to identify the species of ticks biting humans in Northwestern Italy and tick-borne pathogens they harbour. An overall number of 128 ticks from 92 patients were collected from April to October 2018, almost 98% of which belonging to the Ixodes ricinus species. Molecular analysis showed the presence of Babesia spp. in 29 out of 93 analysed tick pools, with a Minimum Infection Rate (MIR) of 31.18% (29/93; CI95% 22.67-41.19%), while 1 out of 93 pools tested positive for SFG Rickettsiae (MIR=1.08%; CI95% 0.19-5.84%). No samples tested positive for A. phagocytophilum and Borrelia spp. Sequencing revealed the presence of Babesia venatorum (28 pools), Theileria buffeli/orientalis complex (1 pool) and Rickettsia monacensis. Among these, B. venatorum and R. monacensis are zoonotic pathogens able to cause from moderate to severe infections in humans. These data highlight the importance of passive surveillance to assess the epidemiology of TBDs that pose a threat to human health.

Molecular epidemiology and phylogeny of spotted fever group Rickettsia in camels (Camelus dromedarius) and their infesting ticks from Tunisia.

Rickettsia species are adapted to a wide range of specific animal hosts. Camels (Camelus dromedarius) have been identified as a carrier of various zoonotic pathogens and became a focus of growing public health interest. This study reported the occurrence of rickettsial infection in camels and infesting ticks from five Tunisian governorates. Based on ompB PCR, eight out of 293 camels (2.7%) were found to be infected with Rickettsia spp. Furthermore, 13 tick specimens of Hyalomma impeltatum (10.4%) and 9 of H.dromedarii (8.0%) harboured DNA of Rickettsia bacteria with an overall prevalence rate of 9.2% (22/237). Molecular prevalence of Rickettsia infection varied significantly according to tick infestation for camels and among tick genders. Five rickettsial species, showing a potential public health interest, were revealed by sequencing. Based on ompB partial sequences, five species were identified corresponding to R.aeschlimannii, R.monacensis, R.helvetica and R.massiliae in camels and to R.africae, R.aeschlimannii, R.monacensis and R.helvetica in ticks. Based on ompA typing, three species were revealed corresponding to R.africae and R.monacensis in camels and to R.africae, R.aeschlimannii and R.monacensis in ticks. This is the first report consolidating the hypothesis that camels may serve as potential hosts for Rickettsia spp. and Hyalomma spp. ticks as possible vectors in arid and Saharan areas of Tunisia. The present data highlight the importance of preventive measures and survey that must be implemented in camel herds in order to limit the spread of these vector-borne bacteria to animals and humans.

A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

DETECTION OF A Rickettsia GENOTYPE RELATED TO THE OLD WORLD INFECTING Amblyomma sculptum TICK IN AN ENDEMIC AREA OF BRAZILIAN SPOTTED FEVER

Brazilian Spotted Fever is an important tick-borne disease caused by Rickettsia rickettsii and transmitted mainly by the human-biting tick Amblyomma sculptum. During an epidemiological surveillance in Pedro Leopoldo, an endemic area of Minas Gerais State, southeastern Brazil, ectoparasites were collected from vertebrate hosts and from the environment. Rickettsial genes were obtained from a male A. sculptum and the resulting phylogenetic tree grouped this bacterium with Rickettsia sp. isolate Pampulha, a strain closely related to the pathogenic species Rickettsia tamurae and Rickettsia monacensis. This is the first report of sequencesphylogenetically related to R. tamurae and R. monacensis infecting A. sculptum in Brazil.
 KEY WORDS: Spotted Fever Group Rickettsia; Amblyomma sculptum; Spotted Fever focus;Ixodidae; Brazil.

Investigation and research on ticks carrying spotted fever group rickettsia in the border area of Tumen River Basin

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.

Epidemiological aspects of Rickettsia parkeri in the Atlantic forest biome of Espírito Santo state, Brazil

Since 2010, a new rickettsiosis caused by Rickettsia parkeri, a species included in the spotted fever group (SFG) and transmitted by the tick Amblyomma ovale, has been described in Brazil. Considering that A. ovale does occur in the Espírito Santo state (ES), the present study aimed at preliminarily describing the epidemiology of R. parkeri in the Atlantic forest of this state. Between June 2016 and September 2018, 33 villages from nine municipalities of the ES were included in a study for the molecular detection and isolation of SFG rickettsiae from adult Amblyomma ticks collected on dogs with free access to the forest. Serologic screenings against SFG rickettsiae in these animals and their owners (humans) were performed through immunofluorescence assay (IFA) using antigens of Rickettsia rickettsii and R. parkeri. Additionally, local health secretariats were informed on clinical manifestations of R. parkeri infection and told to communicate any suspected case. A total of 280 adult ticks were collected and taxonomically classified as A. ovale (n = 152), Amblyomma aureolatum (n = 127) and Amblyomma sculptum (n = 1). Overall, Rickettsia DNA was detected in 12/266 ticks. The sequencing of PCR products revealed that 0.7% (1/144) and 0.8% (1/121) of the analyzed A. ovale and A. aureolatum ticks were infected by R. parkeri strain Atlantic rainforest, respectively, and 8.3% (10/121) of the A. aureolatum ticks infected by Rickettsia bellii. Among the above PCR-positive ticks, only one isolate from one A. aureolatum tick was successfully established in the laboratory. DNA extracted from the third passage of this isolate was designated as strain M9A and molecularly characterized using primers targeting the Rickettsia gltA gene, whose sequence matched 100% the corresponding sequences of R. bellii. Seroprevalence against SFG rickettsiae in sampled dogs (n = 83) was 41% or 57%, depending on the rickettsial antigen (R. rickettsii strain Taiaçu or R. parkeri strain Atlantic rainforest, respectively). A total of 37 (45%) canine sera showed titers to R. parkeri at least fourfold higher than to R. rickettsii antigen. Among humans, 10% (4/41) of the samples reacted to at least one rickettsial antigen, with the highest endpoint titer varying from 64 to 128 for R. rickettsii and R. parkeri; no human serum showed ≥4-fold difference between the highest endpoint titers. Finally, during the study period, suspicions on cases of R. parkeri-rickettsiosis were not informed by the health secretariats. Our results confirm the presence and exposure to R. parkeri strain Atlantic rainforest, associated with two anthropophilic tick species (A. ovale and A. aureolatum) parasitizing domestic dogs with unrestrained access to forest areas. Consequently, the occurrence of R. parkeri infection in humans inhabiting the Atlantic forests of ES should not be discarded.

Minimal Duration of Tick Attachment Sufficient for Transmission of Infectious Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Its Primary Vector Dermacentor variabilis (Acari: Ixodidae): Duration of Rickettsial Reactivation in the Vector Revisited.

It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for 'reactivation' before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10-12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.