Multimethylation of Rickettsia OmpB Catalyzed by Lysine Methyltransferases

Methylation of rickettsial OmpB (outer membrane protein B) has been implicated in bacterial virulence. Rickettsial methyltransferases RP789 and RP027-028 are the first biochemically characterized methyltransferases to catalyze methylation of outer membrane protein (OMP). Methylation in OMP remains poorly understood. Using semiquantitative integrated liquid chromatography-tandem mass spectroscopy, we characterize methylation of (i) recombinantly expressed fragments of Rickettsia typhi OmpB exposed in vitro to trimethyltransferases of Rickettsia prowazekii RP027-028 and of R. typhi RT0101 and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (ii) native OmpBs purified from R. typhi and R. prowazekii strains Breinl, RP22, and Madrid E. We found that in vitro trimethylation occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), whereas monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. Native OmpBs from highly virulent R. prowazekii strains Breinl and RP22 contain multiple clusters of trimethyllysine in contrast to a single cluster in OmpB from mildly virulent R. typhi. Furthermore, OmpB from the avirulent strain Madrid E contains mostly monomethyllysine and no trimethyllysine. The native OmpB from Madrid E was minimally trimethylated by RT0101 or RP027-028, consistent with a processive mechanism of trimethylation. This study provides the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncovering the link between OmpB methylation and rickettsial virulence.     

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.     

Neglected human Rickettsia felis infection in Taiwan: A retrospective seroepidemiological survey of patients with suspected rickettsioses

BackgroundCurrent knowledge on Rickettsia felis infection in humans is based on sporadic case reports. Here we conducted a retrospective seroepidemiological survey of R. felis infection among febrile patients visiting a medical center in Taipei.Methodology/Principal findingsA total of 122 patients with suspected rickettsioses presenting with fever of unknown origin (FUO) but tested negative for scrub typhus, murine typhus, or Q fever were retrospectively identified during 2009 to 2010. The archived serum samples were examined for the presence of antibodies against R. felis, Rickettsia japonica, and Rickettsia typhi using microimmunofluorescence (MIF) assay. Serological evidence of Rickettsia exposure was found in 23 (19%, 23/122) patients. Eight patients had antibodies reactive to R. felis, including four with current infection (a ≥4-fold increase in IgG titer between acute and convalescent sera). The clinical presentations of these four patients included fever, skin rash, lymphadenopathy, as well as more severe conditions such as pancytopenia, hepatomegaly, elevated liver enzymes/bilirubin, and life-threatening acute respiratory distress syndrome. One of the patients died after doxycycline was stopped after being tested negative for scrub typhus, Q fever, and murine typhus.ConclusionsRickettsia felis is a neglected flea-borne pathogen in Taiwan, and its infection can be life-threatening. Further prospective studies of the prevalence of R. felis among patients with FUO and compatible clinical manifestations are warranted.     

Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells

The long-standing proposal that phospholipase A₂ (PLA₂) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA₂ activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA₂ activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA₂ activity were reduced by PLA₂ inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.     

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37^oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39^oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.     

Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.     

Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P₂ at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P₂ at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.     

Review of microarray studies for host-intracellular pathogen interactions

Obligate intracellular bacteria are privileged soldiers on the battlefield that represent host-pathogen interactions. Microarrays are a powerful technology that can increase our knowledge about how bacteria respond to and interact with their hosts. This review summarizes the limitations inherent to host-pathogen interaction studies and essential strategies to improve microarray investigations of intracellular bacteria. We have compiled the comparative genomic and gene expression analyses of obligate intracellular bacteria currently available from microarrays. In this review we explore ways in which microarrays can be used to identify polymorphisms in different obligate intracellular bacteria such as Coxiella burnetii, Chlamydia trachomatis, Ehrlichia chaffeensis, Rickettsia prowazekii and Tropheryma whipplei. These microarray studies reveal that, while genomic content is highly conserved in obligate intracellular bacteria, genetic polymorphisms can potentially occur to increase bacterial pathogenesis. Additionally, changes in the gene expression of C. trachomatis throughout its life cycle, as well as changes in the gene expression profile of the pathogens R. prowazekii, Rickettsia rickettsii, Rickettsia typhi, T. whipplei and C. trachomatis following environmental changes, are discussed. Finally, an in vivo model of Rickettsia conorii within the skin is discussed. The gene expression analyses highlight the capacity of obligate intracellular bacteria to adapt to environmental changes and potentially to thwart the host response.     

Transformation of Rickettsia prowazekii to Rifampin Resistance

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rif^s) Madrid E strain and a rifampin-resistant (Rif^r) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rif^s Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rif^r region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.     

Serological Studies of the Antigenic Similarity between Typhus Group Rickettsiae and Weil-Felix Test Antigens

The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT-118 and P. vulgaris OX19 in the enzyme-linked immunosorbent assay. Sera from these patients reacted with ladder-like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.     

Application of quantitative measures of gene order similarity to phylogenetic reconstructions (exemplified by bacteria of the genus <Emphasis Type="Italic">Rickettsia</Emphasis>)

Data reflecting evolutionary changes in chromosomal gene order can be used for phylogenetic reconstructions along with the results of nucleotide sequence comparison. By the example of bacteria of the genus Rickettsia, we have shown that phylogenetic reconstructions based on quantitative estimates of the similarity and cladistic analysis of gene order data, may, in some cases, amend and fill up classical phylogenetic trees. When applied, these approaches enabled us to substantiate the hypothesis that Rickettsia felis species had split before the typhus (R. typhi, R. prowazekii) and spotted fever (R. connorii) group divergence and thus R. felis does not belong to the latter group. In general, rickettsias evolved towards increasing intracellular parasitic specialization. Five Rickettsia species whose genomes have been sequenced and annotated completely actually form an evolutionary series R. bellii—R. felis—R. conorii—R. prowazekii—R. typhi. Within this series, a reduction in genome size and rapid decrease of genome rearrangement rates (genome plasticity loss) gradually occur.     

mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP_UV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP_UV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP_UV fluorescence.     

Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils,Macrophages and NK Cells

Rickettsia (R.) typhi is the causative agent of endemic typhus, an emerging febrile disease that is associated with complications such as pneumonia, encephalitis and liver dysfunction. To elucidate how innate immune mechanisms contribute to defense and pathology we here analyzed R. typhi infection of CB17 SCID mice that are congenic to BALB/c mice but lack adaptive immunity. CB17 SCID mice succumbed to R. typhi infection within 21 days and showed high bacterial load in spleen, brain, lung, and liver. Most evident pathological changes in R. typhi-infected CB17 SCID mice were massive liver necrosis and splenomegaly due to the disproportionate accumulation of neutrophils and macrophages (MΦ). Both neutrophils and MΦ infiltrated the liver and harbored R. typhi. Both cell populations expressed iNOS and produced reactive oxygen species (ROS) and, thus, exhibited an inflammatory and bactericidal phenotype. Surprisingly, depletion of neutrophils completely prevented liver necrosis but neither altered bacterial load nor protected CB17 SCID mice from death. Furthermore, the absence of neutrophils had no impact on the overwhelming systemic inflammatory response in these mice. This response was predominantly driven by activated MΦ and NK cells both of which expressed IFNγ and is considered as the reason of death. Finally, we observed that iNOS expression by MΦ and neutrophils did not correlate with R. typhi uptake in vivo. Moreover, we demonstrate that MΦ hardly respond to R. typhi in vitro. These findings indicate that R. typhi enters MΦ and also neutrophils unrecognized and that activation of these cells is mediated by other mechanisms in the context of tissue damage in vivo.     

Transposon Mutagenesis of the Obligate Intracellular Pathogen Rickettsia prowazekii

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL^P), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL^P-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.     

Transformation Frequency of a mariner-Based Transposon in Rickettsia rickettsii

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.     

Comparative analysis of FUR regulons in gamma-proteobacteria

Iron is an essential element for the survival and pathogenesis of bacteria. The strict control of iron homeostasis is mediated by the FUR repressor, which is highly conserved among various bacterial species. Here we apply the comparative genomics approach to analyze candidate Fur-binding sites in the genomes of Escherichia coli (K12 and O157:H7), Salmonella typhi, Yersinia pestis and Vibrio cholerae. We describe a number of new loci encoding siderophore biosynthesis and transport proteins. A new regulator of iron-acquisition systems was found in S.typhi. We predict FUR regulation for several virulence systems. We also predict FUR regulation for the chemotaxis system of V.cholerae that is probably involved in the process of pathogenesis.     

Molecular Detection of Rickettsia typhi in Cats and Fleas

Background

Rickettsia typhi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R . typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R . typhi in our location analyzing its presence in cats and fleas.

Methodology/Principal Findings

221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R . typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant.Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R . typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R . typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R . felis was observed.

Conclusions

Although no clinical case has been described in this area, the presence of R . typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R . typhi is detected in naturally infected cats.     

Genomic Characterization of Burkholderia pseudomallei Isolates Selected for Medical Countermeasures Testing: Comparative Genomics Associated with Differential Virulence

Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.     

High Seroprevalence for Rickettsia rickettsii in Equines Suggests Risk of Human Infection in Silent Areas for the Brazilian Spotted Fever

Equines play a role in the epidemiology of Brazilian spotted fever (BSF) since they are a primary host for the tick Amblyomma sculptum. We studied the seroprevalence for three species of Rickettsia in equines in four endemic (with human cases) and in four non-endemic areas (no human cases) in the Piracicaba River Basin, São Paulo, Brazil. A serological survey of 504 equines was performed: around 63 animals were sampled in each area and tested through indirect immunofluorescence assay for R. rickettsii, R. parkeri, and R. bellii in 2012–2013. Blood samples were seropositive for 183 equines (36.3%) in which 73 (39.9%) were from non-endemic areas. In the studied sites equines were highly exposed to Rickettsia infection ranging from 6.1% to 54.7%, with Geometric Mean Titers greater in endemic area (p = 0.012). Results suggest that Rickettsia may be more widespread than the surveillance of BSF has detected. These results highlight the need to include data on the seroprevalence of sentinel animals to improve human diagnoses and surveillance in areas with no reported human cases.     

Clonal Strain Persistence of Candida albicans Isolates from Chronic Mucocutaneous Candidiasis Patients

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency disorder characterised by susceptibility to chronic Candida and fungal dermatophyte infections of the skin, nails and mucous membranes. Molecular epidemiology studies of CMC infection are limited in number and scope and it is not clear whether single or multiple strains inducing CMC persist stably or are exchanged and replaced. We subjected 42 C. albicans individual single colony isolates from 6 unrelated CMC patients to multilocus sequence typing (MLST). Multiple colonies were typed from swabs taken from multiple body sites across multiple time points over a 17-month period. Among isolates from each individual patient, our data show clonal and persistent diploid sequence types (DSTs) that were stable over time, identical between multiple infection sites and exhibit azole resistant phenotypes. No shared origin or common source of infection was identified among isolates from these patients. Additionally, we performed C. albicans MLST SNP genotype frequency analysis to identify signatures of past loss of heterozygosity (LOH) events among persistent and azole resistant isolates retrieved from patients with autoimmune disorders including CMC.