Complement inhibitor of C5 activation from the soft tick Ornithodoros moubata

Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.     

Saliva of the soft tick, Ornithodoros moubata, contains anti-platelet and apyrase activities.

1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. The mean specific activities for ATP and ADP hydrolysis at pH 7.5 were 0.97 and 0.74 mumoles orthophosphate/min/mg protein. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.     

A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations 10(6) bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.     

Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata

The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus. The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion. In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt. The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS. The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry. The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals. The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects. Based on this evidence, we have identified the protein as the tick gut lysozyme. The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes. The pH optimum of the tick lysozyme was in the range from pH 5-7. Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut.     

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.     

Ornithodoros moubata: host immunoglobulin G in tick hemolymph

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.     

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata.

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.     

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.     

Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity

Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata. Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.     

Molecular cloning,structure and bait region splice variants of alpha2-macroglobulin from the soft tick Ornithodoros moubata

The sequence of a alpha(2)-macroglobulin (alpha(2)M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4,944 bp long and contains one open reading frame coding for a protein precursor composed of 1,494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish alpha(2)Ms. Phylogeny analysis revealed that TAM is closely related to Limulus alpha(2)M and displays the highest similarity to the partial sequence of alpha(2)M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus alpha(2)Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal.     

Identification and expression analysis of an actin gene from the soft tick, Ornithodoros moubata (Acari: Argasidae)

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.     

Transovarial transmission of African swine fever virus in the argasid tick Ornithodoros moubata

The aim of this study was to determine filial infection prevalence of experimentally infected colony Ornithodoros moubata Walton (Ixodoidea: Argasidae) ticks for African swine fever virus (ASFV). Three groups of ticks were used: an uninfected control group, one group orally infected with the VIC T90/1 isolate and another group orally infected with the LIV 13/33 isolate of ASFV. The results show that filial infection prevalences were not constant but were highly variable between egg batches from different ticks and between successive egg batches from the same tick. Filial infection prevalences ranged from 1.8% to 31.8% for ticks infected with the VICT90/1 isolate and from 1.2% to 35.5% for ticks infected with the LIV 13/33 isolate. A similar pattern was noted after the third feed. Immunohistochemisty showed that virus replicates in the developing larval cells and not in the yolk sac cells or within the outer layers of the eggs. The results show that ASFV can replicate to a high titre (10(5.1)log10HAD50) within the larval cells of the developing egg.     

In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick Ornithodoros moubata

The involvement of complement (C) in inflammatory diseases has driven the search for agents capable of inhibiting dysregulated complement activation. Many of these reagents inhibit the C3 convertases during the early stages of the cascade. However, a drawback of total systemic C inhibition, particularly in longterm treatment of chronic disease, is potentiation of infection and immune complex disease due to an inability to opsonize complexes and foreign cells and to lyse pathogens. Recent identification of a C5-binding protein in the salivary gland of the soft tick Ornithodoros moubata has enabled development of a terminal pathway-specific reagent, OmCI, with potential to ameliorate disease while leaving key physiological processes unaffected. Here we demonstrated that OmCI has broad cross-species activity. When given intravenously to rodents, OmCI totally ablated complement hemolytic activity, which gradually restored as C5 was resynthesized. The circulating half-life of OmCI was 30 h, demonstrating a much slower clearance than other small, biological agents. Using C5-sufficient and C5-deficient mice we showed that prolonged half-life was due to binding to plasma C5. Surface plasmon resonance analysis of C5 binding to OmCI confirmed a high binding affinity with a slow dissociation rate. OmCI was effective in preventing experimental autoimmune myasthenia gravis induced by passive transfer in normal Lewis rats. OmCI ablated clinical disease, reduced C3 and C9 deposition at the neuro-muscular junction, and effected a marked reduction in cellular infiltration at this site. These data offer exciting prospects for targeted treatment of complement-mediated diseases without the detrimental inhibition of the opsonic roles of complement.     

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).     

Immune responses of the argasid tick Ornithodoros moubata moubata induced by infection with the filarial worm Acanthocheilonema viteae

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.     

Effects of infection of the tick Ornithodoros moubata with African swine fever virus

The effects of infection with African swine fever virus (ASFV) on adult and nymphal Ornithodoros moubata Murray (Ixodoidea, Argasidae) ticks were examined. Three groups of ticks were used, an uninfected control group, one group infected with the VIC T90/1 isolate of ASFV and another group infected with the LIV 13/33 isolate of ASFV. Infection with ASFV did not affect the oviposition rates of infected ticks when compared with uninfected ticks. There was no difference between infected and uninfected ticks in progeny hatching rates and first nymphal stage feeding rates. Feeding rates of infected adult ticks were also unaffected. However, a significant increase in mortality rates was observed amongst the adult ticks that fed on an infective bloodmeal compared to ticks fed on an unifected bloodmeal.