Clogmia albipunctata (Williston, 1893) midgut physiology: pH control and functional relationship with Lower Diptera (nematoceran) especially with hematophagous species.

Clogmia albipunctata (Williston, 1893) is a non-hematophagous insect belonging to the order Diptera, suborder Nematocera (Lower Diptera) and family Psychodidae. In the present work, we investigated how C. albipunctata control their midgut pH under different physiological conditions, comparing their midgut physiology with some nematoceran hematophagous species. The C. albipunctata midgut pH was measured after ingestion of sugar, protein and under the effect of the alkalinizing hormone released in the hemolymph of the hematophagous sand fly Lutzomyia longipalpis obtained just after a blood meal. The midgut pH of unfed or sugar-fed C. albipunctata is 5.5-6, and its midgut underwent alkalinization after protein ingestion or under treatment with hemolymph collected from blood fed L. longipalpis. These results suggested that in nematocerans, mechanisms for pH control seem shared between hematophagous and non-hematophagous species. This kind of pH control is convenient for successful blood digestion. The independent evolution of many hematophagous groups from the Lower Diptera suggests that characteristics involved in midgut pH control were already present in non-hematophagous species and represent a readiness for adaptation to this feeding mode.

Tick Salivary Kunitz-Type Inhibitors: Targeting Host Hemostasis and Immunity to Mediate Successful Blood Feeding.

Kunitz domain-containing proteins are ubiquitous serine protease inhibitors with promising therapeutic potential. They target key proteases involved in major cellular processes such as inflammation or hemostasis through competitive inhibition in a substrate-like manner. Protease inhibitors from the Kunitz superfamily have a low molecular weight (18-24 kDa) and are characterized by the presence of one or more Kunitz motifs consisting of α-helices and antiparallel ß-sheets stabilized by three disulfide bonds. Kunitz-type inhibitors are an important fraction of the protease inhibitors found in tick saliva. Their roles in inhibiting and/or suppressing host homeostatic responses continue to be shown to be additive or synergistic with other protease inhibitors such as cystatins or serpins, ultimately mediating successful blood feeding for the tick. In this review, we discuss the biochemical features of tick salivary Kunitz-type protease inhibitors. We focus on their various effects on host hemostasis and immunity at the molecular and cellular level and their potential therapeutic applications. In doing so, we highlight that their pharmacological properties can be exploited for the development of novel therapies and vaccines.

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Inhibition of the classical pathway of the complement system by saliva of Amblyomma cajennense (Acari: Ixodidae).

Inhibition of the complement system during and after haematophagy is of utmost importance for tick success in feeding and tick development. The role of such inhibition is to minimise damage to the intestinal epithelium as well as avoiding inflammation and opsonisation of salivary molecules at the bite site. Despite its importance, the salivary anti-complement activity has been characterised only in species belonging to the Ixodes ricinus complex which saliva is able to inhibit the alternative and lectin pathways. Little is known about this activity in other species of the Ixodidae family. Thus, the aim of this study was to describe the inhibition of the classical pathway of the complement system by the saliva of Amblyomma cajennense at different stages of the haematophagy. The A. cajennense saliva and salivary gland extract (SGE) were able to inhibit the complement classical pathway through haemolytic assays with higher activity observed when saliva was used. The anti-complement activity is present in the salivary glands of starving females and also in females throughout the whole feeding process, with significant higher activity soon after tick detachment. The SGE activity from both females fed on mice or horses had no significant correlation (p > 0.05) with tick body weight. The pH found in the intestinal lumen of A. cajennense was 8.04 ± 0.08 and haemolytic assays performed at pH 8.0 showed activation of the classical pathway similarly to what occurs at pH 7.4. Consequently, inhibition could be necessary to protect the tick enterocytes. Indeed, the inhibition observed by SGE was higher in pH 8.0 in comparison to pH 7.4 reinforcing the role of saliva in protecting the intestinal cells. Further studies should be carried out in order to identify the inhibitor molecule and characterise its inhibition mechanism.

Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Poly-L-Lysine-Based αGal-Glycoconjugates for Treating Anti-αGal IgE-Mediated Diseases.

Anti-αGal IgE antibodies mediate a spreading allergic condition known as αGal-syndrome (AGS). People exposed to hard tick bites are sensitized to αGal, producing elevated levels of anti-αGal IgE, which are responsible for AGS. This work presents an immunotherapy based on polymeric αGal-glycoconjugates for potentially treating allergic disorders by selectively inhibiting anti-αGal IgE antibodies. We synthesized a set of αGal-glycoconjugates, based on poly-L-lysine of different degrees of polymerization (DP1000, DP600, and DP100), to specifically inhibit in vitro the anti-αGal IgE antibodies in the serum of αGal-sensitized patients (n=13). Moreover, an animal model for αGal sensitization in GalT-KO mice was developed by intradermal administration of hard tick' salivary gland extract, mimicking the sensitization mechanism postulated in humans. The in vitro exposure to all polymeric glycoconjugates (5-10-20-50-100 µg/mL) mainly inhibited anti-αGal IgE and IgM isotypes, with a lower inhibition effect on the IgA and IgG, respectively. We demonstrated a differential anti-αGal isotype inhibition as a function of the length of the poly-L-lysine and the number of αGal residues exposed in the glycoconjugates. These results defined a minimum of 27 αGal residues to inhibit most of the induced anti-αGal IgE in vitro. Furthermore, the αGal-glycoconjugate DP1000-RA0118 (10 mg/kg sc.) showed a high capacity to remove the anti-αGal IgE antibodies (≥75% on average) induced in GalT-KO mice, together with similar inhibition for circulating anti-αGal IgG and IgM. Our study suggests the potential clinical use of poly-L-lysine-based αGal-glycoconjugates for treating allergic disorders mediated by anti-αGal IgE antibodies.

An updated catalog of lipocalins of the chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae).

The haematophagy process by arthropods has been one of the main targets of studies in the parasite-host interaction, and the kissing-bug Rhodnius prolixus, vector of the protozoan Trypanosoma cruzi, has been one of the main models for such studies. Still in the 1980s, it was identified that among the salivary proteins for disrupting vertebrate host homeostasis, lipocalins were among the most relevant proteins for this process. Since then, 30 lipocalins have been identified in the salivary glands of R. prolixus, that promotes vasodilatation, prevents inflammation, act as anticoagulants and inhibits platelet aggregation. The present work aims to identify new lipocalins from R. prolixus, combining transcriptome and genome data. Identified new genes were mapped and had their structure annotated. To infer an evolutionary relationship between lipocalins, and to support the predicted functions for each lipocalin, all amino acid sequences were used to construct phylogenetic trees. We identified a total of 29 new lipocalins, 3 new bioaminogenic-biding proteins (which act to inhibit platelet aggregation and vasodilation), 9 new inhibitors of platelet aggregation, 7 new apolipoproteins and 10 lipocalins with no putative function. In addition, we observed that several of the lipocalins are also expressed in different R. prolxius tissues, including gut, central nervous system, antennae, and reproductive organs. In addition to newly identified lipocalins and a mapping the new and old lipocalins in the genome of R. prolixus, our study also carried out a review on functional status and nomenclature of some of the already identified lipocalins. Our study reinforces that we are far from understanding the role of lipocalins in the physiology of R. prolixus, and that studies of this family are still of great relevance.

Effect of Triatoma infestans saliva on mouse immune system cells: The role of the pore-forming salivary protein trialysin.

Triatoma infestans is one of the most important vectors of Trypanosoma cruzi in the Americas. While feeding, they release large amounts of saliva that will counteract the host's responses triggered at the bite site. Despite the various activities described on T. infestans saliva, little is known about its effect on the modulation of the host's immune system. This work aimed to describe the effects of T. infestans saliva on cells of the mouse immune system and access the role in hematophagy. The effect of saliva or salivary gland extract (SGE) was evaluated in vivo and in vitro by direct T. infestans feeding on mice or using different biological assays. Mice that were submitted to four bites by three specimens of T. infestans had their anti-saliva IgG serum levels approximately 2.4 times higher than controls, but no change in serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels was observed. No macroscopic alterations were seen at the bite site, but an accumulation of mononuclear and polymorphonuclear cells shortly after the bite and 24 h later were observed in histological cuts. At low concentrations (up to ∼5 µg/well), SGE induced TNF-α production by macrophages and spleen cells, IFN-γ and IL-10 by spleen cells and NO by macrophages. However, at higher concentrations (10 and 20 µg/well), viability of macrophages and spleen cells was reduced by SGE, reducing the production of NO and cytokines (except TNF-α). The salivary trialysin was the main inducer of cell death as macrophage viability and NO production was restored in assays carried out with SGE from trialysin knockdown insects. The reduction of the salivary trialysin by RNAi affected the total ingestion rate, the weight gain, and retarded the molt from second to the fifth instar of T. infestans nymphs fed on mice. The results show that T. infestans saliva modulates the activity of cells of the host immune system and trialysin is an important salivary molecule that reduces host cells viability and impacts the feeding performance of T. infestans feeding on live hosts.

Importance and physiological effects of hemolymphagy in triatomines (Hemiptera: Reduviidae).

Triatomines are hematophagous insects and the vectors for Trypanosoma cruzi in the Americas. Although their preferred meal is blood from vertebrate hosts, unfed triatomine nymphs are often seen feeding on different arthropod species. Triatomine saliva has a wide range of activities that aid the hematophagic process. However, nothing is known about its role during hemolymphagy. In the current study, we reproduced hemolymphagy under laboratory conditions and evaluated the influence of hemolymphagy on the survival of Triatoma infestans nymphs. The effects of saliva on the activation of the prophenoloxidase cascade in the invertebrate host and the influence of the saliva on the motility of the bugs and contractions of the dorsal vessels were assessed. Hemolymphagy prolonged the survival rate of T. infestans first instars from 60 to >120 d compared with unfed nymphs. The saliva from T. infestans caused a 50% reduction in the amplitude and frequency of the dorsal vessel contractions of adult Rhodnius prolixus and induced paralysis for >10 min when the saliva was injected into second instars. T. infestans saliva was able to inhibit the activation of the prophenoloxidase cascade from a R. prolixus hemolymph, but had no effect on the phenoloxidase activity after the cascade was activated. The paralyzing molecule in the saliva was <5 kDa and probably had no proteic or lipidic characteristics. These results suggest that triatomine saliva may play an important role during hemolymphagy by inducing paralysis and suppressing immune responses in the invertebrate host. The importance of hemolymphagy for the survival of nymphs in periods when vertebrate blood is scarce is also discussed.

Soft ticks perform evaporative cooling during blood-feeding.

Feeding on the blood of warm-blooded vertebrates is associated to thermal stress in haematophagous arthropods. It has been demonstrated that blood-sucking insects protect their physiological integrity either by synthesising heat-shock proteins or by means of thermoregulatory mechanisms. In this work, we describe the first thermoregulatory mechanism in a tick species, Ornithodoros rostratus. By performing real-time infrared thermography during feeding on mice we found that this acarian eliminates big amounts of fluid (urine) through their coxal glands; this fluid quickly spreads over the cuticular surface and its evaporation cools-down the body of the tick. The spread of the fluid is possible thanks to capillary diffusion through the sculptured exoskeleton of Ornithodoros. We discuss our findings in the frame of the adaptive strategies to cope with the thermal stress experienced by blood-sucking arthropods at each feeding event on warm-blooded hosts.

Locomotion activity and its effects on the survival of Amblyomma sculptum (Acari: Ixodidae) nymphs under laboratory conditions.

The active locomotion of ticks is directly associated with the epidemiology of tick-borne diseases, as it has important implications for the interaction of ticks with their hosts and their dispersion in the environment. In an attempt to elucidate the factors involved in the dispersion of Amblyomma sculptum, the present work aimed to characterize different aspects of the active locomotion of A. sculptum nymphs under laboratory conditions. To this end, nymphs were placed on a string at a 70° inclination and their walking activity was recorded daily along with their survival period. During their lifetime, ticks walked an average of 110 m. Their locomotion was not in a straight line and nymphs changed direction 142 times throughout their lifetimes. The mean distance walked per experimental day was 1.8 m, while the average walking distance before changing direction was 52 cm. The distance walked per experimental day reduced over time. The survival of ticks was affected by walking; resting nymphs survived for over 6 months, while the survival of those that walked daily was reduced to approximately 62 days. The results showed that A. sculptum nymphs were able to cover distances of over 100 m throughout their lifetimes, but they walked short distances at a time and constantly changed direction. This behavior indicates that ticks are not able to disperse over long distances by means of active locomotion.

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation.

BACKGROUND: Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). METHODS: T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. RESULTS: Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. CONCLUSION: Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.

Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae) salivary gland cells.

In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

Amblyomma sculptum Salivary Protease Inhibitors as Potential Anti-Tick Vaccines.

Amblyomma sculptum is the main tick associated with human bites in Brazil and the main vector of Rickettsia rickettsii, the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are directly related to feeding success and vector competence. In the present study, we identified sequences of A. sculptum salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A. sculptum females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of A. sculptum.

Use of a candidate gene array to delineate gene expression patterns in cattle selected for resistance or susceptibility to intestinal nematodes.

In the present study, we use microarray technology to investigate the expression patterns of 381 genes with known association to host immune responses. Hybridization targets were derived from previously characterized bovine cDNAs. A total of 576 reporters (473 sequence-validated cDNAs and 77 controls) were spotted onto glass slides in two sets of four replicates. Two color, comparative hybridizations across both mesenteric lymph node (MLN) and small intestine mucosa (SIM) RNA samples were done between animals with previously demonstrated phenotypic differences based on natural exposure to gastrointestinal (GI) nematodes over a 6-month exposure period. A total of 138 significant hybridization differences were detected by mixed model analysis of variance. A subset of these significant differences was validated by quantitative, real-time RT-PCR to assay transcript levels for 18 genes. These results confirmed that in the SIM, susceptible animals showed significantly higher levels in the genes encoding IGHG1, CD3E, ACTB, IRF1, CCL5 and C3, while in the MLN of resistant animals, higher levels of expression were confirmed for PTPRC, CD1D and ITGA4. Combined, the results indicate that immune responses against GI nematode infections involve multiple response pathways. Higher levels of expression for IgE receptor, integrins, complement, monocyte/macrophage and tissue factors are related to resistance. In contrast, higher levels of expression for immunoglobulin chains and TCRs are related to susceptibility. Identification of these genes provides a framework to better understand the genetic variation underlying parasite resistance.

Occurrence of the Parasitic Fly Philornis torquans on Fledglings of the Rufous-Fronted Thornbird ( Phacellodomus rufifrons) in Southeast Brazil.

Philornis is a neotropical genus of muscid fly that interacts with birds and may affect the development and survival of the birds' offspring. Although Philornis is a relatively common parasite, there is a lack of information about Philornis hosts in several parts of the Americas. In this study, two nests of the Rufousfronted Thornbird ( Phacellodomus rufifrons) were collected in Pedro Leopoldo, southeast Brazil. The first contained four nestlings of advanced age (about 20 d old) and a recently emerged Philornis torquans female adult fly. The second nest contained three nestlings (less than 7 d old) and several Philornis torquans subcutaneous larvae. One of the nestlings was infested by 53 larvae, which had attacked several parts of its body and caused individual wounds containing 1 to more than 15 larvae. The length of the larvae ranged from 3 to 18 mm and only one was a second instar; the remaining 69 were third instars. The pupal period lasted 9-13 d. In total, 71 larvae were collected from the nest, with nestling parasitism varying from 7 to 53 larvae (mean- 23.7±25.5 larvae/nestling).

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Functional aspects of salivary nitric oxide synthase of Rhodnius prolixus (Hemiptera, Reduviidae) and nitric oxide trafficking at the vector-host interface.

Rhodnius prolixus expresses nitric oxide synthase (NOS) in the cytosol of the salivary gland (SG) cells. The NO produced is stored in the SG lumen bound to NO-carrier haemeproteins called nitrophorins (NPs). NPs bind tightly to NO in the acidic SG lumen, but release NO when the pH becomes high, e.g., at the host skin (pH~7.4). NO elicits potent and transient relaxation of vascular smooth muscle. Here, we investigated the role of salivary NO in the R. prolixus feeding behaviour and the salivary vasodilator activity of the host microcirculation. NOS knockdown in R. prolixus changed the SG colour, decreased the number of NO-loaded NPs and caused impairment of feeding performance. When salivary gland extracts (SGEs) were obtained from NOS- and NPs-knockdown insects and prepared in pH 5.0 solution and injected (i.v.) into mice via the tail vein, no vasodilation was observed, whereas SGEs from control insects caused long-term venodilation in the mouse skin. SGs disrupted directly in PBS (pH 7.4) containing BSA produced long-term vasodilation compared to the controls without BSA due to the possible formation of nitroso-albumin, suggesting that host serum albumin extends the NO half-life when NO is injected into the host skin by triatomine during their blood-feeding.

Isolation of Rickettsia rickettsii from the tick Amblyomma sculptum from a Brazilian spotted fever-endemic area in the Pampulha Lake region, southeastern Brazil.

Brazilian spotted fever (BSF) is a tick-borne disease caused by the bacterium Rickettsia rickettsii, the deadliest spotted fever of the world, transmitted in southeastern Brazil mainly by the tick Amblyomma sculptum, a member of the Amblyomma cajennense species complex. In the present study, over 5000 adults of A. sculptum ticks were collected by dry ice traps in the Municipal Ecological Park, alongside the Pampulha Lake region, a BSF-endemic area of Belo Horizonte city, state of Minas Gerais, southeastern Brazil. Ticks were taken alive to the laboratory, where a sample of 2100 specimens was processed for isolation of R. rickettsii. For this purpose, ticks were macerated and intraperitoneally inoculated into guinea pigs. Only one out of 21 inoculated guinea pigs presented high fever within 21days post inoculation with tick homogenates. This febrile animal was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). A spleen sample from a febrile guinea pig was used to inoculate Vero cells, resulting in a successful isolation and in vitro establishment of rickettsiae. Rickettsia-infected Vero cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB), which were all 100% identical to corresponding sequences of R. rickettsii from GenBank. The present R. rickettsii isolate was designated as strain Pampulha. A minimal infection rate of 0.05% R. rickettsii-infected ticks was estimated for A. sculptum population of the Pampulha Lake region. Our results, coupled with epidemiological evidences, suggest that R. rickettsii strain Pampulha, isolated from A. sculptum ticks in the present study, is the strain responsible for human clinical cases of BSF in the Pampulha Lake region of Belo Horizonte city.

The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B.

Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni2+ increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system.