Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs.

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.

Spotted fever group rickettsiae in ticks feeding on humans in northwestern Spain: is Rickettsia conorii vanishing?

During a 7-year study, we identified and analyzed by PCR 4,049 ticks removed from 3,685 asymptomatic patients in Castilla y León (northwestern Spain). A total of 320 ticks (belonging to 10 species) were PCR-positive for rickettsiae. Comparison of amplicon sequences in databases enabled us to identify eight different spotted fever group (SFG) rickettsiae: Rickettsia slovaca, Rickettsia sp. IRS3/IRS4, R. massiliae/Bar29, R. aeschlimannii, Rickettsia sp. RpA4/DnS14, R. helvetica, Rickettsia sp. DmS1, and R. conorii. Although Mediterranean spotted fever (MSF) is an endemic disease in Castilla y León, R. conorii was found in only one Rhipicephalus sanguineus tick, whereas other pathogenic SFG rickettsiae were much more prevalent in the same area. Our data suggest that in Castilla y León, many MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsiae present in ticks biting people in this region of Spain.

Antigens from the midgut membranes of Ornithodoros erraticus induce lethal anti-tick immune responses in pigs and mice.

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In southern Europe O. erraticus lives in close association with swine on free-range pig farms. Application of acaricides for the eradication of O. erraticus from pig farms is inefficient. This is the reason why we tried to develop an anti-O. erraticus vaccine as alternative method of control. Accordingly, we were prompted to investigate the protective possibilities of a midgut membrane extract from the parasite (GME) that has not been studied hitherto. Administration of the GME with Freund's adjuvants (FAs) to pigs and mice induced a protective response able to kill 80% of the immature forms of the parasite in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. The action of the vaccine is the result of damage to the midgut wall of the argasid, and, in mice, it has been shown that this damage is mediated by activation of the complement system. In pigs, the administration of GME with alum, instead of with FAs, reduced the degree of protection. The protective antigens of the GME were expressed by all the developmental stages examined and are probably proteins from the luminal membrane of midgut epithelial cells. These antigens were seen to be more abundant in recently fed parasites than in fasting specimens, suggesting that their expression is induced after blood ingestion.

Cloning and characterization of a plasminogen-binding enolase from the saliva of the argasid tick Ornithodoros moubata.

Significant amounts of enolase have recently been found in the saliva of the argasid tick Ornithodoros moubata, raising the question as to what the function of enolase in the tick-host interface is. Enolase is a multifunctional glycolytic enzyme known to act as a plasminogen receptor on cellular surfaces, promoting fibrinolysis and extracellular matrix degradation. Fibrinolysis could be important for ticks to dissolve clots that might be formed during feeding as well as to prevent clotting of the ingested blood meal in the tick midgut. Additionally, enolase-mediated extracellular matrix degradation could contribute to the tick feeding lesion. Moreover, previous observations suggested an additional antihaemostatic role for O. moubata enolase as a P-selectin antagonist ligand. Accordingly, the aim of the present study was to investigate the potential role of the O. moubata salivary enolase as a plasminogen receptor and P-selectin ligand, and to evaluate its potential as an antigen target for anti-O. moubata vaccines. The study included the cloning, sequencing and recombinant production of the O. moubata enolase, plasminogen binding and activation assays, P-selectin binding assays, animal immunization trials, and RNAi knockdown of the enolase gene. Here we confirmed that enolase is secreted to the saliva of the tick and provide convincing evidence for a role of this salivary enolase as a plasminogen receptor, most likely stimulating host fibrinolysis and maintaining blood fluidity during tick feeding. The RNAi experiments and immunization trials indicated that enolase could be also involved in the regulation of tick reproduction, suggesting new potential control strategies. Finally, the P-selectin binding experiments demonstrated that this enolase is not a P-selectin ligand.

An insight into the proteome of the saliva of the argasid tick Ornithodoros moubata reveals important differences in saliva protein composition between the sexes.

Tick saliva contains pharmacologically active molecules that allow these parasites to obtain a blood meal from the host and facilitate host infection by tick-borne pathogens. Recent transcriptomic and proteomic analyses of the salivary glands of several tick species have provided data sets that are invaluable for a better understanding of tick sialomes and tick-host-pathogen relationships. Here we performed a proteomic study of the saliva from the argasid tick Ornithodoros moubata. Saliva samples from female and male specimens were analyzed separately by LC-MS/MS before and after their equalization to facilitate the identification of the less abundant proteins. We report the array of 193 proteins identified in the saliva of O. moubata showing: (i) the broad and complex composition of the saliva of this tick, in good agreement with the complexity of the argasid and ixodid sialomes described previously; (ii) a notable difference in the saliva proteomes of females and males, since only 10 of the proteins identified appeared to be shared by both sexes; and (iii) the presence in the salivary fluid of a wide range of proteins known to be housekeeping/intracellular, which could be secreted in unconventional ways, including exosome secretion.

Purification and characterisation of a P-selectin-binding molecule from the salivary glands of Ornithodoros moubata that induces protective anti-tick immune responses in pigs.

Ornithodoros moubata is an argasid tick that lives in Africa in wild and synanthropic habitats. It feeds on warthogs, domestic swine and humans, and is able to transmit severe diseases such as human relapsing fever and African swine fever. The elimination of O. moubata from the synanthropic surroundings should improve control of the diseases it transmits. Previous attempts to develop a vaccine against O. moubata showed that a salivary gland extract (SGE) induced a protective response that inhibited the feeding of the ticks by up to 60%. Our aim in the present work was to isolate and characterise the salivary antigens responsible for the protective immune response induced with the SGE. The work reported here describes the finding and partial characterisation of a tick salivary glycoprotein of 44 kDa (Om44) that binds host P-selectin, presumably preventing the adhesion of leucocytes and platelets to vessel walls, thus allowing the ticks to complete their feeding. Preliminary analysis indicated that Om44 is not a homologue of the mammalian PSGL-1s and lacks sialyl-Lewis(X), Lewis(X) and Lewis(Y) determinants but carries heparin, which is the P-selectin-binding motif. Om44 is not recognised by the pig immune system after natural contact with O. moubata, but it can be neutralised by specific vaccine-induced antibodies, resulting in feeding inhibitions of around 50% in adults and nymphs-4, 25% in nymphs-3, 5% in nymphs-2 and 0% in nymphs-1 when they are fed on Om44-vaccinated pigs. In addition, the fecundity of females was inhibited by up to 43.8%. Om44 provides new prospects for the development of new anti-tick vaccines.