Rickettsial Agents Research Articles

Molecular detection and identification of Rickettsia in psocids collected from herbs

The aims of this study were to assess the psocids that potentially carry Rickettsia and to characterize Rickettsia species. We collected booklice from dried fragments of Chinese medicinal herbs for retail. Species identification and Rickettsia detection were performed by using nondestructive DNA extraction method and PCR. Of the 224 booklice samples collected, the following species were identified: Liposcelis pearmani (50.89%), L. bostrychophila (25.89%) and L. entomophila (23.21%). Twenty-four samples were found to be positive for Rickettsia (10.71%, 24/224), and the number of Rickettsia-positive samples per species was as follows: L. pearmani (n = 10), L. bostrychophila (n = 8) and L. entomophila (n = 6). The phylogenetic tree revealed that the rickettsial agents found in booklice cluster along with Rickettsia felis belong to the spotted fever group (SFG). Mixed booklice contamination occurs in herbal fragments. The presence of Rickettsia-infected booklice suggests that there is a risk of louse-borne rickettsioses to humans in contact with herbs. These findings can contribute to the development of a disease control program that assists relevant practitioners and promotes public health.

Detection and characterization of a novel spotted fever group Rickettsia genotype in Haemaphysalis leporispalustris from California, USA

The rabbit tick, Haemaphysalis leporispalustris Packard, is known for its association with Rickettsia rickettsii as it harbors both virulent and avirulent strains of this pathogen. In this manuscript we report findings and preliminary characterization of a novel spotted fever group rickettsia (SFGR) in rabbit ticks from California, USA. Rickettsia sp. CA6269 (proposed “Candidatus Rickettsia lanei”) is most related to known R. rickettsii isolates but belongs to its own well-supported branch different from those of all R. rickettsii including strain Hlp2 and from Rickettsia sp. 364D (also known as R. philipii) and R. peacockii. This SFGR probably exhibits both transovarial and transstadial survival since it was found in both questing larvae and nymphs. Although this rabbit tick does not frequently bite humans, its role in maintenance of other rickettsial agents and this novel SFGR warrant further investigation.

Confirming Rickettsia rickettsii as the etiological agent of lethal spotted fever group rickettsiosis in human patients from Espírito Santo state, Brazil

Although Espírito Santo state is considered an endemic area for Brazilian spotted fever (BSF) with related lethal cases, it also constitutes the only state of southeastern Brazil that currently lacks a specific confirmation of the specific rickettsial agent. In an attempt to a species level confirmation of the etiological agent of fatal rickettsiosis cases in Espírito Santo state, in this study we tested human sera obtained between 2015 to 2017 by means of qPCR and subsequent conventional PCR protocols targeting gltA (citrate synthase) and ompA (190-kDA outer membrane protein) rickettsial genes. All samples were found to contain rickettsial DNA through the citrate synthase qPCR protocol. By conventional PCR, rickettsial gltA and ompA specific DNA fragments were detected in 25% (one sample) and 50% (2 samples) of the screened sera, respectively. Obtained consensuses for each gene partial sequences were 100% identical to Rickettsia rickettsii gltA and ompA genes. The present study confirms for the first time R. rickettsii as the etiological agent of a lethal spotted fever group rickettsiosis in human patients from Espírito Santo state.

Suspected and Confirmed Vector-Borne Rickettsioses of North America Associated with Human Diseases.

The identification of pathogenic rickettsial agents has expanded over the last two decades. In North America, the majority of human cases are caused by tick-borne rickettsioses but rickettsiae transmitted by lice, fleas, mites and other arthropods are also responsible for clinical disease. Symptoms are generally nonspecific or mimic other infectious diseases; therefore, diagnosis and treatment may be delayed. While infection with most rickettsioses is relatively mild, delayed diagnosis and treatment may lead to increased morbidity and mortality. This review will discuss the ecology, epidemiology and public health importance of suspected and confirmed vector-transmitted Rickettsia species of North America associated with human diseases.

Molecular analyses reveal an abundant diversity of ticks and rickettsial agents associated with wild birds in two regions of primary Brazilian Atlantic Rainforest

Brazilian wild birds are recognized as frequent and important hosts for immature stages of more than half of the 32 recognized species of Amblyomma ticks recorded in that country. Several species of Amblyomma harbor rickettsial agents, including members of the spotted fever group (SFG). Most studies on this topic relied primarily on morphological characterization and reported large portions of the collected ticks at the genus rather than species level. Clearly, this factor may have contributed to an underestimation of tick diversity and distribution and makes comparisons between studies difficult. The current investigation combined morphological and molecular analyses to assess the diversity of ticks and rickettsial agents associated with wild birds, captured in two regions of native Atlantic rainforest, in the state of Rio de Janeiro, Brazil. A total of 910 birds were captured, representing two orders, 34 families and 106 species, among which 93 specimens (10.2%), were parasitized by 138 immature ticks (60 larvae and 78 nymphs), representing 10 recognized species of the genus Amblyomma; together with two reasonably well classified haplotypes (Amblyomma sp. haplotype Nazaré and Amblyomma sp. strain USNTC 6792). Amplification by PCR and sequencing of rickettsial genes (htrA, gltA, ompA and ompB), demonstrated the presence of Rickettsia DNA in 48 (34%) of the ticks. Specifically, Rickettsia bellii was detected in a single larva and a single nymph of A. aureolatum; R. amblyomatis was found in 16 of 37 A. longirostre and was recorded for the first time in three nymphs of A. calcaratum; R. rhipicephali was detected in 9 (47%) of 19 Amblyomma sp. haplotype Nazaré ticks. The remaining ticks were infected with genetic variants of R. parkeri, namely strain ApPR in 12 A. parkeri and seven Amblyomma sp. haplotype Nazaré ticks, with the strain NOD found in two specimens of A. nodosum. Interestingly, a single larvae of A. ovale was shown to be infected with the emerging human pathogen Rickettsia sp. strain Atlantic rainforest (ARF), suggesting a possible role for birds in the dispersal of ticks infected with this variant of R. parkeri. The diversity of ticks and Rickettsia recorded in this study is, to our knowledge, the most abundant recorded to date in Brazil and highlighted the value of employing methods capable of providing species level identification of the ixodofauna of wild birds.

Comparative vertical transmission of Rickettsia by Dermacentor variabilis and Amblyomma maculatum

The geographical overlap of multiple Rickettsia and tick species coincides with the molecular detection of a variety of rickettsial agents in what may be novel tick hosts. However, little is known concerning transmissibility of rickettsial species by various tick hosts. To examine the vertical transmission potential between select tick and rickettsial species, two sympatric species of ticks, Dermacentor variabilis and Amblyomma maculatum, were exposed to five different rickettsial species, including Rickettsia rickettsii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia amblyommatis, or flea-borne Rickettsia felis. Fitness-related metrics including engorgement weight, egg production index, nutrient index, and egg hatch percentage were then assessed. Subsamples of egg clutches and unfed larvae, nymphs, and adults for each cohort were assessed for transovarial and transstadial transmission of rickettsiae by qPCR. Rickettsial exposure had a minimal fitness effect in D. variabilis and transovarial transmission was observed for all groups except R. rickettsii. In contrast, rickettsial exposure negatively influenced A. maculatum fitness and transovarial transmission of rickettsiae was demonstrated only for R. amblyommatis- and R. parkeri-exposed ticks. Sustained maintenance of rickettsiae via transstadial transmission was diminished from F1 larvae to F1 adults in both tick species. The findings of this study suggest transovarial transmission specificity may not be tick species dependent, and sustained vertical transmission is not common.

Infection-derived lipids elicit an immune deficiency circuit in arthropods

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Predictive Factors for Fatal Tick-Borne Spotted Fever in Brazil.

In Brazil, two pathogenic Rickettsia species have been identified causing tick-borne spotted fever (SF). The aetiological agent Rickettsia rickettsii causes serious illness, particularly in the south-eastern region of the country. Moreover, the Rickettsia sp. strain Atlantic Rainforest cause milder clinical manifestations in south-eastern, south and north-east regions. This study has sought to analyse predictive factors for fatal SF. A case-control study was performed using disease notification records in Brazil. The cases included were individuals with laboratory confirmation and fatal progression of SF, while the controls included individuals with SF who were cured. A total of 386 cases and 415 controls were identified (1:1.1), and the cases and controls were similar in age. The factors identified as being protective against death were reported presence of ticks (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.41-0.88), residing in urban areas (OR, 0.47, 95% CI, 0.31-0.74) and presenting lymphadenopathy (OR, 0.43; 95% CI, 0.23-0.82). Males exhibited a greater chance of death (OR, 1.57; 95% CI, 1.13-2.18), as did patients who were hospitalized (OR, 10.82; 95% CI, 6.38-18.35) and who presented hypotension or shock (OR, 10.80; 95% CI, 7.33-15.93), seizures (OR, 11.24; 95% CI, 6.49-19.45) and coma (OR of 15.16; 95% CI, 8.51-27.02). The study demonstrates the severity profile of the SF cases, defined either as the frequency of hospitalization (even in cases that were cured) or as the increased frequency of the clinical complications typically found in critical patients. Opportune clinical diagnosis, a careful evaluation of the epidemiological aspects of the disease and adequate care for patients are determining factors for reducing SF fatality rates.

Rickettsial seropositivity in the indigenous community and animal farm workers, and vector surveillance in Peninsular Malaysia

Rickettsioses are emerging zoonotic diseases that are often neglected in many countries in Southeast Asia. Rickettsial agents are transmitted to humans through exposure to infected arthropods. Limited data are available on the exposure of indigenous community and animal farm workers to the aetiological agents and arthropod vectors of rickettsioses in Peninsular Malaysia. Serological analysis of Rickettsia conorii and Rickettsia felis was performed for 102 individuals from the indigenous community at six rural villages and 87 workers from eight animal farms in Peninsular Malaysia in a cross-sectional study. The indigenous community had significantly higher seropositivity rates for R. conorii (P<0.001) and R. felis (P<0.001), as compared to blood donors from urban (n=61). Similarly, higher seropositivity rates for R. conorii (P=0.046) and R. felis (P<0.001) were noted for animal farm workers, as compared to urban blood donors. On the basis of the sequence analysis of gltA, ompA and ompB, various spotted fever group rickettsiae closely related to R. raoultii, R. heilongjiangensis, R. felis-like organisms, R. tamurae, Rickettsia sp. TCM1, R. felis, Rickettsia sp. LON13 and R. hulinensis were identified from tick/flea samples in animal farms, indigenous villages and urban areas. This study describes rickettsial seropositivity of the Malaysian indigenous community and animal farm workers, and provides molecular evidence regarding the presence of rickettsial agents in ticks/fleas infesting domestic animals in Peninsular Malaysia.Emerging Microbes & Infections (2017) 6, e18; doi:10.1038/emi.2017.4; published online 12 April 2017

First report of Rickettsia raoultii and R. slovaca in Melophagus ovinus, the sheep ked.

Background Melophagus ovinus (Diptera: Hippoboscidae), a hematophagous ectoparasite, is mainly found in Europe, Northwestern Africa, and Asia. This wingless fly infests sheep, rabbits, and red foxes, and causes inflammation, wool loss and skin damage. Furthermore, this parasite has been shown to transmit diseases, and plays a role as a vector. Herein, we investigated the presence of various Rickettsia species in M. ovinus.MethodsIn this study, a total of 95 sheep keds were collected in Kuqa County and Alaer City southern region of Xinjiang Uygur Autonomous Region, northwestern China. First, collected sheep keds were identified on the species level using morphological keys and molecular methods based on a fragment of the 18S ribosomal DNA gene (18S rDNA). Thereafter, to assess the presence of rickettsial DNA in sheep keds, the DNA of individual samples was screened by PCR based on six Rickettsia-specific gene fragments originating from six genes: the 17-kilodalton antigen gene (17-kDa), 16S rRNA gene (rrs), surface cell antigen 4 gene (sca4), citrate synthase gene (gltA), and outer membrane protein A and B genes (ompA and ompB). The amplified products were confirmed by sequencing and BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).ResultsAccording to its morphology and results of molecular analysis, the species was identified as Melophagus ovinus, with 100% identity to M. ovinus from St. Kilda, Australia (FN666411). DNA of Rickettsia spp. were found in 12 M. ovinus samples (12.63%, 12/95). Rickettsia raoultii and R. slovaca were confirmed based on phylogenetic analysis, although the genetic markers of these two rickettsial agents amplified in this study showed molecular diversity.ConclusionsThis is the first report of R. raoultii and R. slovaca DNA in M. ovinus. Rickettsia slovaca was found for the first time around the Taklimakan Desert located in China. This finding extends the geographical range of spotted fever group rickettsiae. Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1885-7) contains supplementary material, which is available to authorized users.

Molecular detection of Rickettsia rhipicephali and other spotted fever group Rickettsia species in Amblyomma ticks infesting wild birds in the state of Minas Gerais, Brazil

The current study evaluated parasitism of wild birds by ticks in a fragment of Atlantic Forest in the state of Minas Gerais, Brazil, and examined the ticks for rickettsial agents. Birds were captured during 2014 and 2015 and ticks were identified by sequencing fragments of the 16S and 12S ribosomal DNA. Among 260 birds representing 19 families and 52 species, a total of 69 (26.5%) were found to be infested by larvae (LL) and/or nymphs (NN) of Amblyomma longirostre (Koch, 1844) (45 LL, 4 NN), Amblyomma calcaratum Neumann, 1899 (9 LL, 15 NN), Amblyomma nodosum Neumann, 1899 (2 NN), Amblyomma parkeri Fonseca and Aragão, 1952 (21 LL), Amblyomma sp. haplotype Nazaré (77 LL), and Haemaphysalis leporispalustris (Packard, 1869) (17 LL, 1 NN). The use of PCR and sequencing of the rickettsial genes gltA, htrA, ompA and ompB, revealed the presence of “Candidatus Rickettsia amblyommii” in A. longirostre (13/49; 26%) and Rickettsia parkeri (strain ApPR) in both A. parkeri (1/21; 5%) and haplotype Nazaré (42/77; 55%) ticks. In addition, we detected Rickettsia rhipicephali in 31 (40%) of the 77 haplotype Nazaré ticks. This is the first record of this rickettsial agent in a species of the genus Amblyomma. The pathogenic potential of this bacterium is undetermined, but the unprecedented association with Amblyomma ticks may represent a cause for concern for public and/or animal health.

Rickettsial infection in ticks infesting wild birds from two eco-regions of Argentina.

Several tick-borne Rickettsia species are recognized human pathogens in Argentina. Here we evaluated rickettsial infection in ticks collected on passerine birds during 2011-2012 in two eco-regions of Argentina. The ticks were processed by molecular analysis through polymerase chain reaction (PCR) detection and DNA sequencing of fragments of two rickettsial genes, gltA and ompA. A total of 594 tick specimens (532 larvae and 62 nymphs), representing at least 4 species (Amblyomma tigrinum, Ixodes pararicinus, Haemaphysalis juxtakochi, Haemaphysalis leporispalustris), were evaluated. At least one A. tigrinum larva, collected on Coryphospingus cucullatus in Chaco Seco, was infected with Rickettsia parkeri, whereas at least 12 larvae and 1 nymph of I. pararicinus, collected from Troglodytes aedon, Turdus amaurochalinus, Turdus rufiventris, C. cucullatus and Zonotrichia capensis, were infected with an undescribed Rickettsia agent, genetically related to several rickettsial endosymbionts of ticks of the Ixodes ricinus complex. R. parkeri is a recognized human pathogen in several American countries including Argentina, where a recent study incriminated A. tigrinum as the potential vector of R. parkeri to humans. Birds could play an important role in dispersing R. parkeri-infected A. tigrinum ticks. Additionally, we report for the first time a rickettsial agent infecting I. pararicinus ticks.

Isolation and characterization of a novel Rickettsia species (Rickettsia asembonensis sp. nov.) obtained from cat fleas (Ctenocephalides felis).

A novel rickettsial agent, 'Candidatus Rickettsia asembonensis' strain NMRCiiT, was isolated from cat fleas, Ctenocephalides felis, from Kenya. Genotypic characterization of the new isolate based on sequence analysis of five rickettsial genes, rrs, gltA, ompA, ompB and sca4, indicated that this isolate clustered with Rickettsia felis URRWXCal2. The degree of nucleotide similarity demonstrated that isolate NMRCiiT belongs within the genus Rickettsia and fulfils the criteria for classification as a representative of a novel species. The name Rickettsia asembonensis sp. nov. is proposed, with NMRCiiT (=DSM 100172T=CDC CRIRC RAS001T=ATCC VR-1827T) as the type strain.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

Seroepidemiology of rickettsial infections in Northeast India.

Resurgence of scrub typhus was reported in Northeast India in 2010 after a gap of 67 years since World War II. However, the presence of other rickettsial infections remained unknown from this region. A seroepidemiological investigation was undertaken in the scrub typhus affected areas from 2013-2015 in Assam, Arunachal Pradesh and Nagaland to assess the exposure to other rickettsial diseases besides scrub typhus. Samples were collected from people residing in scrub typhus reporting areas. Serology was performed by an indirect ELISA for the three rickettsial agents' viz., scrub typhus group orientiae (STGO), spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). A sample with total net absorbance ≥1.000 was considered as positive. An entomological survey was also carried out in the affected areas. Overall, 1265 human blood samples were collected, of which 30.8% (n=390), 13.8% (175) and 4.2% (53) had antibodies against STGO, SFGR and TGR respectively. Presence of antibodies against more than one of the rickettsial groups was also detected. Among the arthropods collected, chiggers of Leptotrombidium deleinse, fleas belonging to Ctenocephalides felis and Pulex irritans, ticks belonging to Rhipicephalus microplus, Haemaphysalis spp. were predominant. Candidatus Rickettsia senegalensis was detected in C. felis. Our findings confirm wide circulation of rickettsial infections and their probable vectors in the northeast region of India.Accession numbers: KU163367, KU163368, KU499847, KU499848.

Ecology of a tick-borne spotted fever in southern Brazil.

Rio Grande do Sul is the southernmost state of Brazil, bordering Uruguay. Clinical cases of spotted fever group (SFG) rickettsiosis were recently reported in Rio Grande do Sul. None of these cases was lethal, and all were confirmed by seroconversion to R. rickettsii antigens. Because serological cross-reactions are well known to occur between different SFG agents, the SFG agent responsible for the clinical cases remains unknown in Rio Grande do Sul, where no rickettsial agent is known to infect ticks. During 2013-2014, ticks and blood sera samples were collected from domestic dogs and wild small mammals, and from the vegetation in a SFG-endemic area of Rio Grande do Sul. Dogs were infested by Amblyomma ovale adult ticks, whereas small mammals were infested by immature stages of A. ovale, Ixodes loricatus, and adults of I. loricatus. Ticks collected on vegetation were adults of A. ovale, and immature stages of A. ovale, Amblyomma dubitatum, and Amblyomma longirostre. Three Rickettsia species were detected: Rickettsia bellii in I. loricatus, Rickettsia amblyommii in A. longirostre, and a Rickettsia parkeri-like agent (Rickettsia sp. strain Atlantic rainforest) in A. ovale. Seroreactivity to SFG antigens were detected in 19.7% (27/137) canine and 37.5% (15/40) small mammal sera, with highest titers to R. parkeri. Results indicate that the R. parkeri-like agent, strain Atlantic rainforest, is circulating between A. ovale ticks, dogs and small mammals in the study area, suggesting that this SFG pathogen could be one of the etiological agents of SFG clinical cases in Rio Grande do Sul.

First detection of Candidatus Rickettsia barbariae in the flea Vermipsylla alakurt from north-western China.

BackgroundVermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Vermipsylla alakurt is mainly distributed in alpine pastoral areas of Kazakhstan, Mongolia, China and Nepal, and infests sheep, yaks and horses, causing irritation, poor condition, anaemia and even death. However, to date, no rickettsial agents have been reported in V. alakurt.FindingsA total of 133 fleas were collected directly from the tails of three sheep flocks (n = 335) in Minfeng County, Xinjiang Uygur Autonomous Region, north-western China. Of these, 55 fleas were identified by morphological examination and molecular analysis of four loci (the ribosomal 18S and 28S rDNA genes and the mitochondrial genes cytochrome c oxidase subunit II and elongation factor 1-alpha). Eight Rickettsia-specific gene fragments originated from seven genes: the 17-kilodalton antigen gene (17-kDa), citrate synthase gene (gltA), 16S rRNA gene (rrs), outer membrane protein A gene (ompA), surface cell antigen 1 gene (sca1), PS120 protein gene (gene D), and outer membrane protein B gene (ompB, two fragments), were used to identify the species of Rickettsia in 53 fleas. The amplified products were sequenced and included in a phylogenetic analysis to verify the taxonomic identification of the rickettsial agents. Based on morphological and molecular evidence, the flea was identified as Vermipsylla alakurt. Nine samples were positive (16.98 %, 9/53) for Rickettsia spp. The phylogenetic tree revealed that the rickettsial agents found in V. alakurt cluster with Candidatus Rickettsia barbariae.ConclusionsOur study suggests that: (i) V. alakurt may serve as a carrier for Candidatus R. barbariae; and (ii) Candidatus R. barbariae, previously reported in Israel, is the eighth newly discovered validated Rickettsia species in China. This finding extends our knowledge of the distribution of Candidatus R. barbariae and the profile of carriers, which not only comprise ticks but also fleas.

Comparative value of blood and skin samples for diagnosis of spotted fever group rickettsial infection in model animals

The definitive diagnosis of spotted fever group (SFG) rickettsioses in humans is challenging due to the retrospective nature and cross reactivity of the serological methods and the absence of reliable and consistent samples for molecular diagnostics. Existing data indicate the transient character of bacteremia in experimentally infected animals. The ability of arthropod vectors to acquire rickettsial infection from the laboratory animals in the absence of systemic infection and known tropism of rickettsial agents to endothelial cells of peripheral blood vessels underline the importance of local infection and consequently the diagnostic potential of skin samples. In order to evaluate the diagnostic sensitivity of rickettsial DNA detection in blood and skin samples, we compared results of PCR testing in parallel samples collected from model laboratory animals infected with Rickettsia rickettsii, Rickettsia parkeri and Rickettsia slovaca-like agent at different time points after infection. Skin samples were collected from ears – away from the site of tick placement and without eschars. Overall, testing of skin samples resulted in a higher proportion of positive results than testing of blood samples. Presented data from model animals demonstrates that testing of skin samples from sites of rickettsial proliferation can provide definitive molecular diagnosis of up to 60–70% of tick-borne SFG rickettsial infections during the acute stage of illness. Detection of pathogen DNA in cutaneous samples is a valuable alternative to blood-PCR at least in model animals.

Isolation of the Pathogen Rickettsia sp. Strain Atlantic Rainforest From Its Presumed Tick Vector, Amblyomma ovale (Acari: Ixodidae), From Two Areas of Brazil.

In this study, Amblyomma ovale Koch ticks were collected from domestic dogs in two localities of the Atlantic rainforest biome of Brazil: 1) the Paty Valley of the Chapada Diamantina National Park, Bahia state (northeastern Brazil), and 2) Adrianópolis, Paraná state (southern Brazil). Ticks were screened for the presence of Rickettsia-like structures by the hemolymph test with Giménez staining, and then processed for isolation of rickettsiae in Vero cell culture by the shell-vial technique. Rickettsiae were isolated from one A. ovale tick of each of the two localities. The two isolates were successfully established in the laboratory with several passages, each one reaching >90% infection of the cells. The two isolates were identified as the spotted fever group (SFG) agent Rickettsia sp. strain Atlantic rainforest, as their gltA (350 bp), ompB (781 bp), and ompA (567 bp) gene fragments were 100% equal to GenBank corresponding sequences of the original strain Atlantic rainforest, reported to be infecting a human in southeastern Brazil, and also 100% equal to the available ompA sequence of strain Bahia, reported to be infecting a human in Paty Valley, the same area of the present study in Bahia state. Ten dogs from Paty Valley were serologically tested against rickettsial antigens by the indirect immunofluorescence antibody test. At least 60% of them were seroreactive to SFG rickettsiae. The role of A. ovale as vector of Rickettsia sp. strain Atlantic rainforest in the Paty Valley area, as well as in other parts of Latin America, is discussed.

Comparative evaluation of Amblyomma ovale ticks infected and noninfected by Rickettsia sp. strain Atlantic rainforest, the agent of an emerging rickettsiosis in Brazil

In 2010, a novel spotted fever group rickettsiosis was reported in the Atlantic rainforest coast of Brazil. The etiological agent was identified as Rickettsia sp. strain Atlantic rainforest, and the tick Amblyomma ovale was incriminated as the presumed vector. The present study evaluated under laboratory conditions four colonies of A. ovale: two started from engorged females that were naturally infected by Rickettsia sp. strain Atlantic rainforest (designated as infected groups); the two others started from noninfected females (designated as control groups). All colonies were reared in parallel from F0 engorged female to F2 unfed nymphs. Tick-naïve vesper mice (Calomys callosus) or domestic rabbits were used for feeding of each tick stage. Rickettsia sp. strain Atlantic rainforest was preserved by transstadial maintenance and transovarial transmission in A. ovale ticks for at least 2 generations (from F0 females to F2 nymphs), because nearly 100% of the tested larvae, nymphs, and adults from the infected groups were shown by PCR to contain rickettsial DNA. All vesper mice and rabbits infested by larvae and nymphs, and 50% of the rabbits infested by adults from the infected groups seroconverted, indicating that these tick stages were vector competent for Rickettsia sp. strain Atlantic rainforest. Expressive differences in mortality rates and reproductive performance were observed between engorged females from the infected and control groups, as indicated by 75.0% and 97.1% oviposition success, respectively, and significantly lower egg mass weight, conversion efficiency index, and percentage of egg hatching for the infected groups. Our results indicate that A. ovale can act as a natural reservoir for Rickettsia sp. strain Atlantic rainforest. However, due to deleterious effect caused by this rickettsial agent on engorged females, amplifier vertebrate hosts might be necessary for persistent perpetuation of Rickettsia sp. strain Atlantic rainforest in A. ovale under natural conditions.